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Drug efflux pumps in rabbit conjunctival epithelial cells

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Drug efflux pumps in rabbit conjunctival epithelial cells

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Content DRUG EFFLUX PUMPS IN RABBIT CONJUNCTIVAL EPITHELIAL CELLS Copyright 2002 by Jun (Johnny) Yang A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment o f the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) August 2002 Yang, Jun (Johnny) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3094389 Copyright 2002 by Yang, Jun (Johnny) All rights reserved. ® UMI UMI Microform 3094389 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA T he G raduate School U n iversity Park LOS ANGELES, CALIFORNIA 900894695 This dissertation, w ritten b y YANG, JUN (JOHNNY) Under th e direction o f h.i.s... D issertation Com m ittee, and approved b y a ll its m em bers, has been p resen ted to and accepted b y The Graduate School, in p a rtia l fulfillm ent o f requirem ents fo r th e degree o f DOCTOR OF PHILOSOPHY Augus t 6 . 2002 DISSER TA TION COMMITTEE C hairperson Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TO MY PARENTS AND MY FAM ILY Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my advisor, Dr. Vincent H.L. Lee, for his excellent scientific guidance, support, and encouragement throughout my graduate studies at USC. The extensive training in various multidisciplinary projects, coupled with the scientific freedom, have contributed to my independent thinking and enabled me to communicate effectively with scientists in various fields. They have made my graduate education stimulating and challenging. I appreciate Dr. Lee’s patience and assistance in polishing my writing and presentation skills, while his coaching and advice encouraged me to face up to the challenges in the real world. I want to thank my committee members: Dr. Kwang-Jin Kim, Dr. Wei- Chiang Shen, Dr. Sarah Hamm-Alvarez, and Dr. Robert Koda for their help and guidance in my project. My gratitude also goes to Dr. Melvin D. Trousdale and his assistant Douglas Stevenson for their supervision on the viral infection techniques; Dr. Tatyana Gurlo for helpful discussion of immunology; Dr. Curtis Okamoto for insightful comments on cell biology; and Dr. David K. Ann for his helpful suggestions on molecular biology. In addition, I would like to express thank to Dr. Ram Kannan for his help with the glutathione measurement and to all the faculty members and researchers who provided help and suggestions during my graduate study. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I wish to thank my labmates, including previous graduate students, post docs, and visiting scholars for their help and friendship over the years. I would also like to thank my wife, Cindy, for her understanding, support and inspiration. I thank her standing by me through both tough and happy times. Our mutual scientific exchange and technical support were valuable and enjoyable. Much appreciation also goes to my parents-in-law, uncle, sister-in-law, and our lovely daughter Alicia for their love and support. Finally, I would like to express my deep appreciation to my parents for their love, inspiration and sacrifice. 1 could not have finished this work without their support. I believe they will feel a deep happiness and pride that I have fulfilled my Ph.D. studies. This work was supported by NIH grants EY12578, EY10421, and EY12356. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS DEDICATION ACKNOWLEDGEMENTS LIST OF SCHEMES LIST OF TABLES LIST OF FIGURES ABBREVIATIONS ABSTRACT CHAPTERS I. INTRODUCTION 1 1. Drug efflux pumps 1 1.1. P-glycoprotein 3 1.1.1. Topology of P-gp 3 1.1.2. Localization and drug efflux functions 5 1.1.3. Proposed roles in immunology, cell apoptosis and cell differentiation 7 1.1.4. Proposed roles in lipid transport and intracellular cholesterol trafficking 8 1.1.5. Transport mechanisms of P-gp 8 1.1.6. Substrate specificity of P-gp 9 1.1.7. Modulation of P-gp 13 1.2. MRP 18 1.2.1. Topology of MRP 18 1.2.2. Localization and drug efflux functions o f MRP 20 1.2.3. Transport mechanisms 21 1.2.4. Physiological roles of MRP 24 1.2.5. Substrate specificity 25 1.2.6. Modulation of MRP 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.3. Other efflux pumps 31 1.3.1. MRP family 31 1.3.2. LRP 39 1.3.3. Antibiotic efflux pumps in bacteria 40 2. Other membrane transporters 41 2.1. Dipeptide transporters 41 2.2. Nucleoside transporters 43 2.3. Organic cation transporters 44 2.4. Monocarboxylate transporters 45 3. Conjunctiva 46 3.1. General anatomy and physiology of the conjunctiva 46 3.2. Ocular drug transport routes 49 4. General pharmacokinetics in disease states 52 5. Ocular infection and immunity 55 5.1. Adenoviral infection 5 5 5.2. Immunology o f the eye 56 6 . Cell culture models 59 6.1. General rationale of cell cultures 59 6.2. Air-interfaced epithelial cell culture 60 6.3. Virally-infected cell culture 62 II. STATEMENT OF THE PROBLEM AND SPECIFIC AIMS 65 1. Specific Aim #1. To develop rabbit conjunctival epithelial cell culture models in normal (air-interfaced condition) and disease (adenoviral infection) states 67 2. Specific Aim #2. To evaluate the expression, localization, function, and modulation of P-glycoprotein in normal and Ad5-infected states o f RCEC 70 3. Specific Aim #3. To evaluate the expression, localization, function, and modulation of MRP in normal and Ad5-infected states of RCEC 72 III. MATERIALS AND METHODS 74 1. Materials 74 1.1. Animal 74 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2. Materials used in cell culture development 74 1.2.1. Air-interfaced culture o f RCEC 74 1.2.2. Adenovirally-infected culture of RCEC 75 1.3. Materials used in P-gp study 76 1.4. Materials used in MRP study 77 2. Methods 78 2.1 .Development of rabbit conjunctival epithelial cell culture models 78 2.1.1. Air-interfaced culture of rabbit conjunctival epithelial cells 78 2.1.1a. Primary culture 7 8 2.1.1b. Transmission electron microscopy 79 2.1.1c. Periodic Acid Schiff (PAS) staining 80 2.1.1 d. Ion transport 80 2.1.1 e. Solute transport 81 2.1.1 f. Uridine transport 8 3 2.1.1 g. L-Camosine uptake 83 2.1. lh. Valacyclovir uptake 84 2.1.1i. Data analysis 84 2.1.2. Adenovirally-infected culture 8 6 2.1.2a. Viral infectivity 8 6 2.1. 2b. Viral growth curve 8 8 2.1,2c. Morphological changes of RCEC 8 8 2.1.2d. Bioelectrical parameters and chloride flux in RCEC 8 8 2.2. Evaluation of expression, localization, function, and modulation o f P-gp 89 2.2.1. Western blot analysis 89 2.2.2. Immunostaining 90 2.2.3. P-Blocker transport 91 2.2.4. Propranolol uptake 92 2.2.5. The effect o f Ad5 infection on P-gp expression and function 93 2.2.6. Data analysis 93 2.3. Evaluation of expression, localization, function, and modulation of MRP 95 2.3.1. Reverse transcription-polymerase chain reactions of MRP 95 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.2. Immunohistochemical detection of MRP 97 2.3.3. Fluorescein uptake and transport 98 2.3.4. Vincristine uptake 100 2.3.5. Leukotriene C4 (LTC4 ) uptake and transport 100 2.3.6. The effect of Ad5 infection and cytokine treatment on MRP expression and function 101 2.3.7. The effect of anti-cytokine antibodies on blocking MRP-mediated LTC4 uptake 103 2.3.8. ELISA for quantitation o f cytokine secretion in RCEC 104 IV. RESULTS 106 1. Development of rabbit conjunctival epithelial cell culture models 106 1.1. Air-interfaced culture of RCEC 106 1.1.1. The effect of medium composition on cell polarity 106 1.1.2. The effect of air-interface condition on cell polarity 107 1.1.3. Transmission electron microscopy (TEM) and periodic acid schiff (PAS) staining 111 1.1.4. Ion transport properties 114 1.1.5. Solute transport properties 114 1.2. Adenovirus type 5 (Ad5)-infected RCEC culture 129 1.2.1. Infectivity 129 1.2.2. Viral growth curve 130 1.2.3. Morphological changes o f cultured cells 130 1.2.4. The effect of Ad5 infection on ion transport 134 2. Evaluation o f expression, localization, function, and modulation o f P-gp 139 2.1. Existence and localization 139 2.2. [3-Blocker transport in RCEC 140 2.2.1. Transport o f (3-blockers 140 2.2.2. Propranolol uptake 146 2.3. P-gp expression and function in Ad5-infected cells 146 3. Evaluation of expression, localization, function, and modulation of MRP 150 3.1. Reverse transcription-polymerase chain reactions o f MRP 150 3.2. Existence and localization 154 3.3. MRP-mediated transport of fluorescein 157 3.4. The effect of glutathione on MRP-mediated fluorescein transport 163 3.5. The contribution o f P-gp and MRP to vincristine uptake 163 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.6. MRP-mediated transport of LTC4 166 3.7. MRP regulation by adenoviral infection 166 3.8. The effect of adenoviral infection and cytokine secretion on expression and function of MRP 169 3.9. Secretion of cytokine in normal and Ad5-infected cells 174 3.10. Anti-cytokine antibody treatments on MRP-mediated LTC4 uptake 174 V. DISCUSSION 180 1. Development of rabbit conjunctival epithelial cell culture models 182 1.1. Air-interfaced conjunctival epithelial cell culture 182 1.1.1. Optimal culture conditions 182 1.1.2. Morphological characterization 184 1.1.3. Ion transport properties 185 1.1.4. Barrier properties 186 1.1.5. Active transport processes 188 1.2. Adenovirally-infected cell culture 194 1.2.1. Infectivity of Ad5 to RCEC culture 194 1.2.2. The effect of Ad5-infection on ion transport 198 2. Expression of drug efflux pumps 203 2.1. Protein expression of P-gp 203 2.2. Molecular evidence of MRP 204 3. Localization o f drug efflux pumps 205 3.1. Localization of P-gp 205 3.2. Localization of MRP 206 4. The effect of efflux pumps on drug efflux 207 4.1. The effect o f P-gp on drug transport 207 4.1.1. The effect of P-gp on propranolol transport 207 4.1.2. The effect of P-gp on P-blocker absorption 212 4.2. The effect of MRP on transport of exogenous and endogenous molecules 215 4.2.1. The effect of MRP on fluorescein transport 215 4.2.2. Efflux mechanisms of MRP 216 4.2.3. Fluorescein influx mechanism 218 4.2.4. The effect of MRP on transport o f ophthalmic drugs 218 4.2.5. MRP-mediated LTC4 transport in the conjunctiva 2 2 1 4.3. Contribution of P-gp and MRP to drug efflux 222 ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. The effect of Ad5 infection and cytokines on expression and function of drug efflux pumps 224 5.1. P-gp expression and function in Ad5-infected RCEC 224 5.2. Up-regulation o f MRP by viral infection 224 5.3. Regulation o f MRP by cytokines 226 6 . Overall indications o f efflux pumps in the conjunctiva 230 7. Summary of drug efflux pumps in the conjunctiva 232 VI. CONCLUSIONS 235 1. Evaluation of expression, localization, function, and modulation o f P-gp 235 2. Evaluation of expression, localization, function, and modulation of MRP 237 3. Development of rabbit conjunctival epithelial cell culture models 239 4. Overall conclusions 241 VII. FUTURE DIRECTIONS 244 VIII. REFERENCES 252 x Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF SCHEMES Scheme 1 Ocular penetration routes for topically applied drugs......................... 51 Scheme 2 Outline of studies performed in Specific Aim # 1 ................................69 Scheme 3 Outline o f studies performed in Specific Aim # 2 ................................71 Scheme 4 Outline of studies performed in Specific Aim # 3 ................................73 xi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1-1 Representative substrates o f P -gp............................................................ 12 Table 1-2 Representative substrates and modulators of M R P .............................. 26 Table 1-3 The human multidrug resistance protein family and some of the alternative names used in the literature for individual family m em bers......................................................................34 Table 1-4 Tissue distribution and physiological functions o f human multidrug resistance proteins .................................................................35 Table 1-5 Percent amino acid identity between fully sequenced human multidrug resistance proteins..................................................... 36 Table 1-6 Similarity analyses and functions of human MRP and its most closely related ABC proteins..........................................................37 Table 1-7 Comparison o f P-gp and M RP................................................................. 38 Table 2-1 Peak transepithelial electrical resistance, potential difference, and equivalent short circuit current of the excised tissue, liquid covered culture, and air-interfaced culture of RCEC.........................................................................................108 Table 2-2 Molecular weight, molecular radius, and apparent permeability coefficient o f FITC-dextrans in the apical-to-basolateral direction in air-interfaced cultures of pigmented rabbit conjunctival epithelial cells and excised tissues..........................................................117 Table 2-3 HPLC conditions for assaying P-blockers..............................................119 Table 2-4 Logarithm of n-octanol/pH 7.4 buffer partition coefficient and apparent permeability coefficient of p- blockers in the apical-to-basolateral direction in air- interface cultured pigmented rabbit conjunctival epithelial cell layers and excised tissue.................................................. 1 2 0 Table 2-5 Permeability of L-valacycovir, acyclovir, and mannitol across cultured rabbit conjunctival epithelial cell layers.....................125 xii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3-1 Apparent permeability coefficients o f propranolol and metoprolol across cultured rabbit conjunctival epithelial cells............................................................................................................. 144 Table 4-1 Permeability o f fluorescein, LTC4 , and mannitol across cultured rabbit conjunctival epithelial cell layers........................................................................................................... 158 Table 4-2 One-hour fluorescein uptake and cell viability with pretreatment o f BSO.........................................................................159 Table 4-3 Uptake of succinate and lactate in the presence of fluorescein or unlabeled lactate............................................................... 159 Table 5-1 Comparison o f the amino acid sequence of human cMRP/MRP2 with transporters of the ATP-binding cassette superfamily..................................................................................247 xiii Reproduced with permission of the copyright owner. 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LIST OF FIGURES Figure 1-1 Relationship between lipid solubility and the rate of absorption from the lumen of rat small bowel for a variety of different compounds................................................................................ 2 Figure 1-2 Topological model o f P-glycoprotein.....................................................4 Figure 1 -3 Proposed mechanisms of P-gp-mediated transport............................... 11 Figure 1-4 Topological model o f multidrug resistant-associated protein..............19 Figure 1 -5 Proposed mechanisms of MRP-mediated transport..............................23 Figure 1-6 Sectional view o f the anterior segment o f the eye.................................48 Figure 2-1 The effect of epidermal growth factor on the primary culture o f rabbit conjunctival epithelial cells......................................................109 Figure 2-2 Mean transepithelial electrical resistance, potential difference, and equivalent short circuit current of cultures grown on Transwell filter in air-interfaced and liquid-covered cultures........................................................................................................1 1 0 Figure 2-3 Electron microscopy of air-interfaced and liquid-covered cultures of rabbit conjunctival epithelial cells on day- 6 culture post-seeding.................................................................................. 1 1 2 Figure 2-4 Periodic Acid Schiff staining of AIC and LCC cells.............................113 Figure 2-5 Influence of pharmacological modulators of ion transport on the equivalent short circuit current of AIC and the excised conjunctiva................................................................................................. 116 Figure 2-6 Relationship between the apparent permeability coefficient and molecular weight o f mannitol, FITC, FD4, FD10, FD20, and FD70 in AIC of RCEC......................................................................118 Figure 2-7 Influence o f drug lipophilicity on (3-blocker transport across AIC, LCC of RCEC, and excised conjunctiva...................................... 121 xiv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2-8 Time course o f uridine transport measured with 10 pM donor concentration across the air-interface cultured conjunctival epithelial cells............................................................................................ 1 2 2 Figure 2-9 Ten-minute uptake of L-camosine at 10 pM donor concentration in the presence of various agents.................................... 123 Figure 2-10 pH effect on 10-min L-valacyclovir uptake at 10 pM in cultured RCEC...........................................................................................126 Figure 2-11 Ten-minute uptake o f L-valacyclovir in the presence of various agents............................................................................................. 127 Figure 2-12 Concentration dependency of L-valacyclovir uptake in cultured RCEC...........................................................................................128 Figure 2-13 Immunostaining for Ad5 in cultured rabbit conjunctival epithelial cells at day 0, 1, and 2 post-seeding.......................................131 Figure 2-14 Immunostaining for Ad5 in cultured rabbit conjunctival epithelial cells at day 3 and 5 post-seeding............................................132 Figure 2-15 Growth kinetics o f Ad5 in the cultured rabbit conjunctival epithelial cell layers.................................................................................. 133 Figure 2-16 Morphology of Ad5-inoculated and mock-inoculated conjunctival epithelial cells 1 0 days post-inoculation......................... 136 Figure 2-17 Bioelectric parameters at different post-inoculation time intervals in cultured RCEC.......................................................................137 Figure 2-18 Unidirectional chloride flux and net flux across the normal and Ad5-infected RCEC layers in the absence and presence o f 8 Br-cAMP.............................................................................................. 138 Figure 3-1 Western blot analysis o f P-glycoprotein expression in cultured conjunctival epithelial cells......................................................141 Figure 3-2 Immunolocalization of P-glycoprotein in cultured RCEC layers by confocal laser-scanning microscopy...................................... 142 Figure 3-3 Immunohistochemistry o f P-glycoprotein on frozen tissue sections of excised rabbit conjunctiva................................................... 143 xv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-4 Unidirectional fluxes and net flux of propranolol at various concentrations............................................................................................145 Figure 3-5 Ten-min apical uptake o f propranolol in the presence of various agents............................................................................................. 147 Figure 3-6 Western blot analysis o f P-glycoprotein expression in rabbit conjunctival epithelial cells under various culture conditions and Ad5 challenge..................................................................................... 148 Figure 3-7 Ten-min uptake of propranolol at a donor concentration of 0.05 pM in the apical fluid in the air-interfaced culture, liquid-covered culture and Ad5-infected culture...................................149 Figure 4-1 Reverse transcription-polymerase chain reaction analysis of messenger RNA expression by genes encoding MRP in the rabbit conjunctival epithelial cells...........................................................151 Figure 4-2 Nucleotide sequence alignment o f a RT-PCR product of rabbit MRP with human M R P.................................................................152 Figure 4-3 Amino acid sequence alignment of a RT-PCR product of rabbit MRP with human M R P.................................................................153 Figure 4-4 hnmunolocalization o f MRP in cultured rabbit conjunctival epithelial cells............................................................................................ 155 Figure 4-5 hnmunolocalization o f MRP in frozen conjunctiva sections...............156 Figure 4-6 Time course of fluorescein uptake in cultured rabbit conjunctival epithelial cells......................................................................160 Figure 4-7 One-hour uptake of fluorescein from basolateral dosing in the presence of probenecid at various concentrations.................................161 Figure 4-8 One-hour uptake o f fluorescein in the presence o f MRP inhibitors and anion drugs........................................................................ 162 Figure 4-9 Time course of glutathione in rabbit conjunctival epithelial cells after treatment with 100 pM buthionine sulfoximine................. 164 xvi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-10 One-hour uptake of 200 nM vincristine in the presence o f P- gp substrates, MRP inhibitors, organic cations, or organic anions.......................................................................................................... 165 Figure 4-11 The effect o f Ad5 infection on uptake of LTC4 .................................. 167 Figure 4-12 Immunostaining of MRP in Ad5-infected RCECs..............................168 Figure 4-13 Western blot analysis of MRP in membrane proteins of various cells................................................................................................ 170 Figure 4-14 Western blot analysis of MRP in membrane proteins of rabbit conjunctival epithelial cells with various cytokine treatments............171 Figure 4-15 One-hour uptake o f LTC4 in the presence of probenecid, or with a pretreatment of NaN3/glucose free medium for 30 min, Ad5-infection, IL-1, IL-6 , and TNF-a 48 hours before uptake experiments................................................................................................ 172 Figure 4-16 Correlation o f MRP expression and one-hour uptake o f LTC4 at 10 nM in the cultured rabbit conjunctival epithelial cells control or with Ad5-infection, IL-1, IL-6 , and TNF-a treatments....................................................................................................173 Figure 4-17 Time course of IL- 6 secretion in normal and Ad5-infected rabbit conjunctival epithelial cell culture...............................................176 Figure 4-18 Time course of TNF-a secretion in normal and Ad5-infected rabbit conjunctival epithelial cell culture...............................................177 Figure 4-19 One-hour LTC4 uptake in Ad5-infected cells with various treatments of cytokine antibodies...........................................................178 Figure 4-20 One-hour LTC4 uptake in RCECs with the treatments of TNF-a alone or the combination o f TNF-a and anti-TNF-a mAh............................................................................................................. 179 Figure 5-1 Summary o f transport processes in air-interfaced culture of rabbit conjunctival epithelial cells..........................................................192 Figure 5-2 Structures of uridine, L-camosine, and L-valacyclovir....................... 193 xvii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-3 Summary o f the effect o f adenovirus on ion movement and tight junction properties o f rabbit conjunctival epithelial cells.......... 2 0 2 Figure 5-4 Structures and LogP of (3-blockers and P-gp substrates....................... 214 Figure 5-5 Compounds used in MRP study...............................................................220 Figure 5-6 Putative model o f P-gp-mediated propranolol efflux in the conjunctiva.................................................................................................233 Figure 5-7 Expression, localization, function, and modulation of MRP in the rabbit conjunctival epithelial cells...................................................234 Figure 5-8 Summary of the overall research conclusion o f drug efflux pumps in the rabbit conjunctival epithelial cells.................................. 243 xviii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABBREVIATIONS Ad5 adenovirus type 5 AIC air-interfaced culture ACV acyclovir a-to-b apical-to-basolateral BRS bicarbonate Ringer’s solution b-to-a basolateral-to-apical Caco-2 human colon carcinoma cell line cAMP 3’,5’-cyclic adenosine monophosphate CMV cytomegalovirus CPE cytopathic effect CsA cyclosporine A DMSO dimethylsulfoxide DNP 2,4-dinitrophenol ELISA enzyme-linked immunosorbent assay FBS fetal bovine serum FITC fluorescein isothiocyanate Gly-Sar glycyl-sarcosine GM-CSF granulocyte macrophage colony-stimulating factor HBSS Hank’s balanced salt solution Ieq equivalent short-circuit current Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IFN interferon IL interleukin Km Michaelis-Menten constant LCC liquid-covered culture LogP logarithm of n-octanol/water partition coefficient NK cells natural killer cells mAb monoclonal antibody MRP multidrug resistance protein MOI multiplicity o f infection MDCK Madin-Darby canine kidney cells pfu plaque-forming units PD potential difference PKC protein kinase C P-gP P-glycoprotein QSAR quantitative structure-activity relationships RCEC rabbit conjunctival epithelial cells RT-PCR reverse transcription-polymerase chain reaction TEER transepithelial electrical resistance TNF-a tumor necrosis factor a TGF transforming growth factor VACV valacyclovir Vmax maximal velocity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Purpose. To evaluate the expression, localization, function, and modulation o f the efflux pumps - P-glycoprotein (P-gp) and multidrug resistance protein (MRP) in cultured rabbit conjunctival epithelial cells (RCEC) in normal and virally-infected states. Methods. The morphology, electrophysiology, and barrier properties o f air- interfaced culture o f RCEC were evaluated. The effects of adenovirus type 5 (Ad5) infection (MOI of 10) on morphology and ion movement o f the cultured RCEC were investigated. Protein expression and localization o f P-gp and MRP in normal and Ad5-infected RCEC were determined by Western blot and immunostaining, respectively. Efflux function of P-gp on propranolol and that of MRP on fluorescein was evaluated. Moreover, MRP expression and MRP- mediated efflux of leukotriene C4 (LTC4) were investigated in normal, Ad5- infected, and cytokine-treated RCEC. MRP expression at gene level was determined by reverse transcription-polymerase chain reaction (RT-PCR). Results. The ion and solute transport properties of the air-interfaced RCEC culture (6 - 8 days) resembled the native conjunctiva. RCEC at early culture was susceptible to Ad5 infection and developed cytopathic effect. Ad5 infection decreased bioelectrical parameters by 20-70% on day 2 post-inoculation and net chloride flux o f RCEC by 60%. Western blot analysis revealed distinct bands for P-gp and MRP in RCEC. Immunofluorescence showed positive apical staining for P-gp and basolateral staining for MRP. P-gp favored basolateral-to-apical xxi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transport of propranolol which was inhibited by cyclosporine A and verapamil, while MRP favored apical-to-basolateral transport o f fluorescein which was inhibited by indomethacin and probenecid. Both Ad5 and cytokines (IL-1, IL-6 , and TNF-a) up-regulated MRP expression, thereby reducing LTC4 uptake. A RT- PCR product with a size of 652 bp from rabbit conjunctiva was 87% identical to human MRP in amino acid sequence. Conclusions. P-gp is primarily located on the apical membrane o f RCEC, playing an important role in restricting the absorption of lipophilic drugs. MRP is primarily localized on the basolateral membrane and is able to efflux organic anions and LTC4 out of the basolateral membrane. The expression of MRP was up-regulated by Ad5-infection and cytokines, which may be associated with its role in the transport of inflammatory factors, like LTC4 in ocular inflammation. The air-interfaced and adenovirally- infected cultures o f RCEC partially resemble the native conjunctiva in normal and disease states, and could be useful as in vitro models for evaluating drug transport mechanisms. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I. INTRODUCTION Drug resistance is a frequent impediment to the effective therapy for infectious and malignant diseases. Many different resistance mechanisms have been described, but those that involve proteins belong to the ATP-binding cassette (ABC) transporter superfamily have been o f particular interest because o f the increasingly prominent role these proteins play in governing drug absorption in various tissues, such as intestine. For ocular drug design, it is important for us to understand the transport mechanisms in the eye. The focus of our research is to evaluate the drug efflux mechanisms in the conjunctiva. 1. Drug efflux pumps Drug efflux pumps have drawn much attention due to their significant impact on chemotherapy and oral drug bioavailability. Multidrug resistance (MDR) was initially reported as a major obstacle in the chemotherapeutic treatment of cancer, but it was subsequently observed in normal human tissues to restrict drug absorption. As shown in Fig. 1-1, some drugs have lower intestinal permeability compared to other drugs with similar lipophilicity (Tsujit and Tamai, 1996). P-glycoprotein and multidrug resistance protein (MRP) are two widely studied drug efflux pumps associated with restricted drug absorption. Drug efflux pumps are an important part o f the defense of cells against drugs. They belong to ABC transporter family, which have more than 200 proteins now. ABC transporters have received considerable attention recently Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. because they are associated with many important biological processes in both prokaryotes and eukaryokes, as well as with clinical problems such as cystic fibrosis, antigen presentation, and multidrug resistance (Higgins, 1992). Most ABC transporters have no role in multidrug resistance and are not glycoprotein. ATP-binding domains of ABC transporters are their most characteristic features. The sequences conserved between the ATP-binding domains include two short motifs (Walker motifs), but not all proteins with the Walker motifs are ABC proteins. 4 6 •2 2 0 *4 log D Fig. 1-1. Relationship between lipid solubility and the rate of absorption from the lumen of rat small bowel o f a variety o f different compounds. The results shown with the squares represent the relationship between intestinal absorption clearance (ka) observed from the in situ jejunal loop in the presence (solid) and absence (hollow) o f cyclosporin A and octanol-buffer (pH 7.0) partition coefficients (log D) that were determined. 1, atenolol; 2, nadolol; 3, acetamide; 4, celiprolol; 5, acebutolol; 6 , doxorubicin; 7, timolol; 8 , sulfathiazole; 9, quinidine; 10, sulfamethoxazole; 11, digoxin; 12, cyclosporin A; 13, vinblastine; 14, P-estradiol; and 15, verapamil. (Adapted from Tsuji and Tamai, 1996). 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1. P-glycoprotein 1.1.1. Topology o f P-glycoprotein As early as the 1960s, numerous investigators described the resistance o f tumor cell lines to anticancer drugs, in which decreased intracellular drug accumulation limits the effectiveness of many clinically important drugs (Bellamy, 1996). A common feature o f many of these drug-resistant cell lines is the overexpression of the P-glycoprotein (P-gp). In 1976, Juliano and Ling (1976) identified a 170 kD plasma membrane glycoprotein in drug-resistant Chinese hamster ovary cells. The amount of this glycoprotein correlated with the degree of resistance. The protein was named P-glycoprotein with the P indicating its relationship to membrane permeability. In humans, MDR1 located at Chromosome 7 encodes P-glycoprotein. Fig. 1-2 illustrates the topology o f P-gp containing 1 2 transmembrane domains with 6 extracellular loops and 2 cytoplasmic ATP-binding domains based on its deduced protein sequence (upper panel), and the 1 2 -transmembrane domain model is supported by antibody localization data. An alternative topological model (lower panel) was revealed by the murine m drl or human MDR1 gene products translated in a cell-free system or expressed in Xenopus oocytes, revealing a glycosylation site in the second half of the P-gp. This raises the possibility that the different topology of P-gp may impact its function, but the function of this isoform is unknown (Bellamy, 1996). 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. coo coo Fig. 1-2. Topological model of P-gp (Adapted from Bellamy, 1996). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.2. Localization and drug efflux functions P-gp appears to typically localize in the apical membrane o f cells (Thiebaut et al., 1987): the biliary canalicular front of hepatocytes, the apical surface o f epithelial cells in small biliary ductules in liver; small ductules but not larger ducts in pancreas; the apical surface o f epithelial cells of the proximal tubules in kidney; the apical surface o f superficial columnar epithelial cells in colon and small intestine in humans (Thiebaut et al., 1987). In all these tissues, the protein is present in a highly polarized fashion. This suggests that the protein has a role in the normal secretion o f metabolites and the efflux of certain drugs such as anticancer agents, into bile, urine, and the lumen o f the gastrointestinal tract (Thiebaut et al., 1987). It seems likely that direct excretion o f drugs may represent a major route o f detoxification. However, in adrenal, P-gp is not present in a polarized distribution. It was detected in both the adrenal cortex and the medulla. This diffusive distribution suggests that P-gp may pump substances into the interstitial space in adrenal (Thiebaut et al., 1987). In the brain, P-gp was found in normal brain capillary endothelial cells, preventing toxic or nonessential compounds from entering into brain, or eliminating them from brain. P-gp is a significant component o f the blood brain barrier (BBB) (Stein et al., 1997). In the eye, the P-gp has been found located in the cornea (Kawazu et al., 1999), iris (Holash and Stewart, 1993; Schlingemann etal., 1998), and retina (Esser et al., 1998; Holash and Stewart, 1993). In the cornea, the tear-to-stroma transport o f cyclosporin A (CsA) was 7 times higher than that o f stroma-to-tear 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. direction, and this difference was decreased to 2 -fold by adding verapamil and vincristine, indicating that P-gp might be localized on the apical side o f corneal epithelial cells and restrict drug entry into the eye (Kawazu et al., 1999). In the iris, iris capillaries were stained positively for P-gp, indicating P-gp’s role o f a blood-tissue barrier function (Schlingemann et al., 1998). In the retina, P-gp expression was weak in patients with proliferative vitreoretinopathy, but became strong after daunomycin treatment, indicating certain dmgs can induce P-gp expression and this might be related to enhanced drug resistance (Esser et al., 1998). In the conjunctiva, the favorable secretion of cyclosporine A (CsA), verapamil, and dexamethasone over absorption indicated the possible existence of drug efflux pumps in the conjunctiva (Saha et al., 1998). P-gp efflux function against drug accumulation was confirmed in P-gp knockout mice. In the P-gp-deficient m drla/lb (-/-) mice, there were no physiological abnormalities in terms of viability and fertility, and a range of histological, hematological, serum-chemical, and immunological parameters were not abnormal (Borst and Schinkel, 1996; Schinkel et al., 1997a). Pharmacologically, m drla/lb (-/-) mice showed increased brain penetration and reduced elimination o f digoxin. In contrast, both m drla and m drlb P-gps contributed to the extrusion o f rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdrl-type P-gps in protecting the bone marrow against cytotoxic anticancer drugs. 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.3. Proposed roles in immunology, cell apoptosis and cell differentiation In host immune response, natural killer cells (NK) and CD 8 + T cells bind to their target cells and induce death through either the Fas/Fas ligand system, or in the majority o f cases, via release of cytotoxic granule components such as perforin and granzymes into the target cells. The relatively high constitutive expression o f P-gp on NK cells and CD 8 + T cells is intriguing, and functional P- gp may be necessary for optimal binding to the target cell or induction o f cytolytic process, or both (Chong et al., 1993; Gupta et al., 1992). In vitro cytolytic function of the NK cells from mdrl knockout mice was abolished (Schinkel et al., 1997b). Functional P-gp may serve additionally to protect these important immune cells in the hostile environment of an immune response. There is now evidence that the P-gp might be involved in the transport of cytokines, such as IL-1 p, IL-2, IL-4, IFN-y, and TNF-a out of activated lymphocytes into the surrounding medium (Johnstone et al., 2000). Bunting et al. (1998) demonstrated that mice bone-marrow stem cells transduced with a retroviral vector overexpressed the P-gp and expanded progressively. The mice developed a myeloproliferative syndrome, indicating that P-gp might influence self-renewal decisions in repopulating stem cells. Functional P-gp might play a fundamental role in regulating apoptosis (programmed cell death) (Johnstone et al., 2000). Transfected P-gp in Chinese 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hamster ovary fibroblast conferred a MDR phenotype and resistance to apoptosis induced by serum starvation, which could be reversed by addition of verapamil, indicating that the transporter function of P-gp is necessary to protect cells from death induced by growth-factor withdrawal (Robinson et a l, 1997). 1.1.4. Proposed roles in lipid transport and intracellular cholesterol trafficking MDR1 P-gp has been proposed to play a role in phospholipid translocation and cholesterol esterification (Johnstone et al., 2000). Mouse MDR2 P-gp and human MDR3 P-gp can function as phosphatidylcholine (PC) flippases, while MDR1 P-gp has been reported to regulate the translocation of a range of short- chain phospholipid analogs and endogenous phospholipids such as sphingomyelin (SM). This could explain the putative role of P-gp in modulating cholesterol esterification, because depletion o f plasma membrane SM can induce cholesterol esterification by releasing cholesterol from the plasma membrane into the intracellular pool. 1.1.5. Transport mechanisms of P-gp For many years, the model o f drug resistance conferred by P-gp has been a quite simple one. Cytotoxic drugs are actively transported out o f cells that express P-gp against a concentration gradient, thereby reducing intracellular drug accumulation and inhibiting drug-mediated cell death. Wigler and Patterson (1993) described a four-step process of substrate efflux through the MDR pump. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The drug substrate is bound to P-gp on the cytoplasmic side o f the cell membrane. The P-gp-substrate complex reacts with ATP, and subsequently produces an inorganic phosphate and undergoes a protein conformational change that pumps the substrate out of the cell (Fig. l-3a). The current model proposes “flippase” function, or “hydrophobic vacuum cleaner”. In this model, Gottesman and Pastan (1993) hypothesized that extracellular hydrophobic substrate molecules that enter the membrane lipid bilayer are extracted directly back to the extracellular medium by the P-glycoprotein before they can reach the cytoplasm (Fig. 1 -3b). The “hydrophobic vacuum cleaner” model may better explain how P-gp restricts drug entry into the cells. The question of how P-gp manages to remove such a broad range of structurally diverse compounds is still not fully understood. There is evidence for at least two allosterically coupled drug-binding sites, but the exact number o f acceptor sites is still uncertain. 1.1.6. Substrate specificity of P-gp Zamora et al. (1988) concluded that lipid solubility at physiological pH, cationic charge, and molar refractivity are important properties for modulators of MDR. Similarly, Beck et al. (1991) demonstrated that the compound should be hydrophobic, have two planar aromatic rings, and a tertiary nitrogen that would be charged at physiological pH. P-gp appears to prefer neutral and/or positively charged lipophilic molecules (Pearce et al., 1989; Zamora et al., 1988), including drugs such as 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cyclosporin A (CsA) (Augustijns et al., 1993), verapamil (Lautier et al., 1994), rhodamine 123 (Fontaine et al., 1996), vinblastine (Terao et al., 1996), etoposide (Leu and Huang, 1995), cimetidine (Dudley and Brown, 1996), celiprolol (Karlsson et al., 1993), ivermectin (Alvinerie et al., 1999), daunomycin (Kwon et al., 1996), digoxin (Cavet et al., 1996), taxol (Sparreboom et al., 1997), and progesterone (Christen et al., 1993). P-gp has been implicated in the secretion o f a p-blocker (celiprolol) in an intestinal epithelial cell line (Caco-2) (Karlsson et al., 1993). In addition, uptake of P-blockers (acebutolol, celiprolol, nadolol, and timolol) by multidrug-resistant leukemic cell line variant K562/ADM was 37- 63% lower than that by drug-sensitive K562 cells, indicating that the transport of P-blockers may be affected by P-gp (Terao et al., 1996). The common P-gp substrates are summarized in Table 1-1. 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. <•) Medium Cytosol Energy- dependent efflux Passive diffusion ii i M (b ) Medium Cytosol Membrane insertion \ Membrane release ’ Flip-flop' Outer leaflet Inner leaflet Membrane \ s release (slow) Fig. 1-3. Proposed mechanisms of P-gp-mediated transport (Adapted from Johnstone, 2000). 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-1. Representative substrates of P-gp (Aungst, 1999; Volm, 1998; Zamora et al., 1988). Anthracyclines: Immunosuppressants: Daunorubicin, Doxorubicin Cyclosporin A, PSC-833 Anti-estrogens: Indo Akaloids Tamoxifen, Toremifene Physostigimine, Reserpine Anti-HIV nrotease inhibitors Ouinolines: Nelfinavir, Ritonavir, Saquinavir Chloroquine, Quinidine, Quinine Calcium channel blockers: Diltiazem, Nifedipine, Verapamil Vinca Alkaloids Vinblastine, Vincristine Calmodulin antagonists: Chlorpromazine, Clomipramin P-blockers: Thioridazine Celiprolol, Propranolol Trifluoperazine Hormones: Progesterone 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.7. Modulation of P-gp Modulation of P-gp by pharmacological agents Extensive efforts to modulate MDR have been focused on substances that could modify the fluidity of membranes, and on drugs that have specific actions on the drug efflux pump. Tsuruo et al. (1981) first reported that a noncytotoxic dose (2.2 to 6 . 6 pM in vitro and 50 to 100 mg/kg in vivo) of verapamil enhanced the cytotoxicity of vincristine and vinblastine in P388 leukemia and its VCR- resistant subline, P3 8 8 /VCR, through competitively inhibiting the transport of anticancer drugs by P-gp. CsA was reported to improve the bioavailability of various drugs, such as atenolol, celiprolol, digoxin, vinblastine, and verapamil (Tsuji and Tamai, 1996). PSC 833, a non-immunosuppressive analogue o f CsA, is another potent MDR-reversing drug (Song et al., 1999). Its MDR-reversing activity was enhanced in vitro and in vivo by MRK-16, a monoclonal antibody that recognizes an extracellular epitope o f P-gp (Naito and Tsuruo, 1997). The inhibitory potency of PSC 833 varies for different compounds. One-tenth mg/kg PSC 833 was sufficient to significantly reduce the biliary secretion clearance of digoxin from 3.0 to 0.5 ml/min/kg, whereas 3 mg/kg PSC 833 was needed to significantly reduce the biliary excretion clearance of vincristine from 36 to 9 ml/min/kg. Three milligrams per kilogram PSC 833 significantly reduced the renal clearance o f vincristine by 30% but did not affect that o f digoxin significantly. The differential effect o f PSC 833 on the disposition o f vincristine 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and digoxin may be ascribed to the different degree of contribution of P-gp to the disposition o f these ligands (Song et al., 1999). Modulation o f P-gp by formulation Some polymers and surfactants are capable of circumventing drug efflux transporter proteins. Batrakova et al. (1998) reported that the pluronic block copolymers (poly(ethylene oxide)-6 /ock-poly(propylene oxid €)-block- poly(ethylene oxide), ECW-POn-EOm^) could inhibit P-gp in Caco-2 cells, thereby increasing drug transport. Ellis et al. (1996) reported that Cremophor EL (polyoxyethylene castor oil) and Tween 80 also inhibited etoposide elimination in isolated perfused rat-liver model. For example, they found that Cremophor at 800 mg increased the AUC (from 334 ± 23 to 1,540 ± 490 pg.min/ml), while decreasing the total clearance (from 4.8 ± 0.3 to 1.1 ± 0.3 ml/min) and biliary clearance (from 2.6 ± 1.1 to 0.5 ± 0.2 ml/min). The elimination half-life (from 62 ± 17 to 40 ± 5 min) and volume of distribution (from 424 ± 85 to 65 ± 19 ml) were also decreased. Modulation o f P-gp by oligonucleotides Since the 1980’s, the molecular basis of oligodeoxynucleotide (ODNs) modulation of specific gene expression has been studied. These strategies include antisense oligonucleotides and ribozymes. ODNs enter cells with the help of 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. specific receptors or through passive diffusion. Then they form duplices with the target mRNA and interrupt protein translation. The mRNA/ODNs duplices may also be cleaved by RNAase which leads to an inhibition o f protein synthesis (Volm, 1998). The main problem is the instability o f ODNs. However, a variety of chemical modifications can stabilize the ODNs. Thierry and coworkers (1993) also showed that the cellular uptake o f ODNs was facilitated by the encapsulation of the ODNs in liposomes, which led to a nearly complete inhibition of P-gp expression. The problems encountered in antisense-mediated modulation in general were attributed to poor uptake o f the oligonucleotides and their rapid degradation, but antisense oligonucleotides can provide for an attractive and potentially highly specific treatment. In vivo studies are underway to evaluate the efficacy of antisense oligonucleotides for inhibiting gene expression (Phillips and Zhang, 2000). Modulation o f P-gp by antibodies. Because o f their high specificity against well-defined epitopes, antibodies might be useful in the attenuation o f multidrug resistance involvement or in targeting drug to the specific tissue that bears the drug efflux pumps. MRK-16 (an antibody against the extracellular epitope of P-gp), alone did not enhance the sensitivity o f the moderately resistant KB-8-5 cells to vincristine, but its synergistic effect with CsA resulted in a two-fold increase in the P-gp reversing 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. effect of 1 pM CsA. Since the MRK-16 also induces an immune response, the combined use of MRK-16, CsA and antitumor agents should provide therapeutic benefits for the treatment o f resistant tumors (Naito et al., 1993). Furthermore, using an immunotoxin composed of MRK-16 coupled to Pseudomonas exotoxin (MRK-16-PE), multidrug-resistant human KB cells were destroyed, whereas cells not expressing the P-gp were not affected (Fitzgerald et al., 1987). Although antibody-directed approaches might be a promising strategy for circumventing MDR, several problems preempt their clinical realization. Since monoclonal antibodies are commonly of murine origin, they would induce an immune response in human. Hamada et al. (1990) demonstrated that a recombinant chimeric mouse-human antibody, MH-162, in which the antigen- recognizing variable regions of the MRK-16 are joined with the constant regions of the human antibody was less immunogenic and more effective in killing drug- resistant tumor cells than MRK-16 in human effector cells. Other problems include the attainment o f therapeutically effective antibody concentration in solid tumors, and the targeting o f specific tissue since P-gp is present in diverse tissues (Volm, 1998). Food effect on P-gp function and expression Food-drug interactions can be associated with alterations in pharmacokinetic profiles o f various drugs with respect to drug absorption, bioavailability, and metabolism (Singh, 1999). There is growing evidence that 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. some o f these effects could be partially attributed to modulation of drug efflux pumps. Grapefruit juice has been shown to significantly increase the oral availability of commonly used medications including felodipine, nifedipine, verapamil, terfenadine, saquinavir, ethinylestradiol, midazolam, and CsA (Poirson-Bichat et al., 2000). Takanaga et al. (1998) have shown that the existence o f inhibitory components in grapefruit juice for the P-gp function would inhibit vinblastine efflux in Caco-2 cells. Their group (Takanaga et al., 2000) also found that the polymethoxylated flavones (3,3',4',5,6,7,8-heptamethoxyflavone (HMF), 4',5,6 ,7,8 - pentamethoxyflavone (tangeretin), and 3',4',5,6,7,8- hexamethoxyflavone (nobiletin)), in orange juice, are inhibitors o f P-gp but not cytochrome P450 3A4, and all increased the steady-state uptake o f vinblastine by Caco-2 cells in a concentration-dependent manner. The order o f potency o f these compounds at the concentration o f 50 pM was tangeretin > HMF > nobiletin. On the other hand, the grapefruit juice improves drug bioavailability via a mechanism besides inhibition of the P-gp. The grapefruit juice components 6 ',7'- dihydroxybergamottin and bergamottin inhibited the metabolism o f drugs such as CsA (Edwards et al., 1999) and saquinavir (Eagling et al., 1999) by down- regulating the metabolic enzyme, CYP3A4. Given at a dose o f 30 pM 6 ',7'- dihydroxybergamottin to healthy subjects concomitantly dosed with 7.5 mg/kg CsA, they caused the AUC for CsA to increase by 50% and 3-fold o f apical-to- basolateral transport in Caco-2 cells. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, Soldner et al. (1999) demonstrated that grapefruit juice significantly activates P-gp-mediated efflux o f drugs that are substrates o f P-gp and potentially partially counteracting the CYP3 A-inhibitory effects o f grapefruit juice. They found that, while both apical-to-basal (A-B) and basal-to-apical (B-A) fluxes in MDCK cells of all CYP3A/P-gp substrates tested were increased in the presence of grapefruit juice, the resulting net efflux (B-A/A-B) was in all cases significantly greater with grapefruit juice compared with control (vinblastine, 28.0 vs. 5.1; CsA, 9.9 vs. 2.8; digoxin, 22. 9 vs. 14.7, fexofenadine, 22.3 vs. 11.1, losartan, 39.6 vs. 26). In contrast, no such effect of grapefruit juice was observed with felodipine, nifedipine, substrates o f CYP3A only (2 vs. 1.7 and 1.2 vs. 1.3). 1.2. Multidrug resistance protein (MRP) 1.2.1. Topology of MRP Multidrug resistance represents an obstacle in the successful application of chemotherapy. Besides P-gp which is a 170 kDa protein encoded by the MDR1 gene (Bellamy, 1996), the cloning in 1992 of the cDNA encoding the 190 kD MRP from a doxorubicin-selected lung cancer cell line provided unequivocal evidence that overexpression of a second ABC protein could cause MDR in mammalian cells (Cole et al., 1992). MRP belongs to the ATP-binding cassette (ABC) superfamily of membrane transporter proteins and functions to pump xenobiotics or their conjugates out o f cells (Wigler, 1996). Fig. 1-4 illustrates the 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. topology of MRP containing 17 transmembrane domains and 2 cytoplasmic ATP- binding domains. MRP has the extra N-terminal domain (TMDo, the region containing first five transmembrane domains) that is lacking in P-gp, and Bakos et al. (1998) have shown that a truncated MRP mutant lacking this entire TMD0 region but still containing other regions behaved like wild-type MRP in vesicle uptake and nucleotide trapping experiments. In addition, like wild-type MRP1, the MRP protein without TMDo routed to the lateral plasma membrane and transported dinitropheyl glutathione and daunorubicin. It indicates that the TMD0 region is neither required for the transport function o f MRP nor for its proper routing to the plasma membrane. Lumen NH OOH Cytoplasm Fig. 1-4. Topological model o f multidrug resistance protein (MRP) (Adapted from Cole and Deeley, 1998). 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2. Localization and drug efflux functions o f MRP MRP is commonly overexpressed in cancer cells (Wigler, 1996) causing decreased efficiency o f chemotherapeutic drugs. MRP is also expressed in normal tissues such as the intestinal, hepatic, and renal epithelial cells, and the blood brain barrier (Flens et al., 1996; Thiebaut et al., 1987), where it may play a protective role against various xenobiotics. Compared to the typical apically localized P-gp (Thiebaut et al., 1987), MRP can be found in either the apical or basolateral membranes, as well as in the cytoplasm of the kidney (Evers et al., 1996), brain (Huai-Yun et al., 1998), lung, spleen, liver, heart, skeletal muscle, and adrenal gland (Flens et al., 1996) cells. In the lung, MRP was found in apical cytoplasm below the cilia in bronchial epithelial cells (Flens et al., 1996). In the liver, MRP appears to be localized to the basolateral membrane o f the hepatocyte (Keppler et al., 1998; Muller et al., 1996), but the expression is low. However, the expression o f MRP is increased in immortalized hepatocytes, indicating its role during cell proliferation (Roelofsen et al., 1997). MRP expression in rat hepatocytes is related to the cell cycle. The protein expression switch from MRP2 to MRP occurs in the G1 phase of the cell cycle and is associated with a decreased cell polarity. Given that MRP contributes to cellular glutathione S-conjugate efflux and protects against oxidative stress-inducing quinones, this switch in expression from the apically located MRP2 to the basolaterally located MRP preserves glutathione S-conjugate transport in hepatocytes entering the cell cycle and 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. protects against certain cytotoxic agents (Roelofsen et al., 1999). In the kidney, MRP is present in the basolateral plasma membrane and could be associated with the re-absorption of organic anions (Evers et al., 1996). In Sertoli cells, MRP is located in the basolateral membrane, allowing this pump to protect the contents of the testicular tubules, the germline cells, against drug damage (Borst et al., 2000). A similar situation appears to exist in the choroid plexus, where many substances enter the cerebrospinal fluid from the epithelial cells covering the plexus. The high level of MRP in these cells plays a crucial role in preventing the entry of a drug, such as etoposide, into the cerebrospinal fluid (Wijnholds et al., 2000). In the brain, various anionic drugs have been demonstrated to be effluxed from the brain via specific mechanisms other than P-gp; they include tolbutamide, p- aminohippuric acid, taurocholic acid, valproic acid, probenecid, and leukotriene C4. Most of these compounds are transported by MRP family members, therefore, MRP may be important as a part of the BBB (Stein et al., 1997). In the eye, MRP was found in retinal pigmented epithelial cells, participating in inner blood-retinal barrier to reduce the accumulation of anionic drugs (Aukunuru et al., 2001). 1.2.3. Transport mechanisms of MRP Cole and Deeley (1998) suggested that the MRP transports the anion conjugates of drugs rather than the unmodified drugs themselves, or cotransports the unmodified drugs with glutathione (GSH) across the plasma membrane of various cells (Fig. 1-5). Indirect evidence for this hypothesis is that leukotriene C4 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (LTC4) transport in MRP-transfected HeLa cells was not affected by GSH, but was inhibited by vincristine only in the presence o f GSH. This effect of GSH was concentration dependent and inhibitable by MRP-specific monoclonal antibodies. It was shown not to be the consequence o f activating MRP by altering its redox state, since the nonreducing methyl-GSH conjugate partially substituted for GSH but other sulfhydryl-reducing agents did not (Loe et al., 1996). The observation that vincristine and etoposide stimulate ATP-dependent GSH uptake lends further support to the notion that MRP-mediated efflux of these drugs is associated with an efflux o f GSH (Cole and Deeley, 1998). In addition, Chuman et al. (1998) demonstrated that MRP-mediated vincristine accumulation in C-A120 cells was enhanced by 100 pM of buthinonine sulfoximine (BSO), a GSH synthetase inhibitor. Feller et al. (1995) reported that ATP-dependent efflux o f calcein, an anion, by MRP was not inhibited by intracellular GSH depletion in MRP MDR cell lines. The different effects of GSH depletion on the transport of negatively charged (calcein) and positively charged molecules (daunorubicin, vincristine, and rhodamine) strengthen the hypothesis that MRP transports negatively charged molecules. They suggested that GSH could be needed as co-substrate or indirect stimulant for the transport o f positively charged drugs, but not for the transport of negatively charged molecules by MRP. 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LTC GSH Vincristine ATP ADP LTC4 Cytosol ATP ADP GSH Vincristine Fig. 1-5. Proposed mechanism of MRP-mediated transport, depicting ATP- dependent transport o f leukotriene C4 and co-transport of GSH and vincristine across the plasma membrane (Adapted from Cole and Deeley, 1998). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.4. Physiological roles of MRP MRP-mediated export of LTC4 , an inflammatory mediator, suggests that the MRP may play an important role in host immune response. In MRP-deficient mrp(-/-) mice , the response to an inflammatory stimulus was impaired. This was attributed to a decreased LTC4 secretion, which was demonstrated in bone marrow derived mast cells obtained from the mice, thus supporting the hypothesis that MRP has a physiological role as a transporter of LTC4 (Wijnholds et al., 1997). This research also demonstrated that the knock-out mice is a useful model system to further evaluate the pharmacological roles of the drug-transporting MRP and to analyze the specificity and effectivity o f MRP inhibitors. A unique feature of the mechanism o f translocation of some substrates by MRP is that MRP-mediated transport is inhibited by depletion o f intracellular GSH (Renes et a l, 1999). The glutathione tripeptide plays a pivotal role in cellular detoxification processes through its capacity to conjugate to a broad range of potentially toxic substrates. Transmembrane transport of glutathione S- conjugates is essential for the elimination of such conjugates (e.g., GS-cisplatin) from the cell. This indicates that MRP functions as a detoxification protein. The fact that this protein actively pumps out oxidized GSH or GSSG as a physiological substrate raises the possibility of a role for MRP in cellular defenses against oxidative stress and perhaps also the maintenance of intracellular redox potential. The physiological function of MRP in kidney and liver is not clear yet. However, hereditary deficiencies of the canalicular MRP2 cause the Dubin in Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Johnson syndrome, which is characterized by a defect in the secretion of amphiphilic anionic conjugates from hepatocytes into bile (Keppler et al., 1997). 1.2.5. Substrate specificity o f MRP Although P-gp and MRP share less than 20% amino acid identity (Cole and Deeley, 1998), they have broad substrate specificities which partly overlap. Both proteins transport several anticancer drugs, such as daunorubicin, doxorubicin, etoposide, and vincristine. It is difficult to predict substrates from structural considerations o f the compounds. However, P-gp appears to prefer neutral and/or positively charged lipophilic molecules (Pearce et al., 1989; Zamora et al., 1988), whereas MRP has been identified as a transporter o f organic anions (Broxterman et al., 1995; Paul et al., 1996). Indeed, negatively charged conjugates such as leukotriene C4 (LTC4) (Leier et al., 1994), dinitrophenyl glutathione (Paul et al., 1996), and aflatoxin B1 exo-8,9-epoxide-GSH (Cole and Deeley, 1998) are substrates of MRP. Table 1-2 illustrates the representative substrates, and modulators, which are able to modulate MRP efflux function. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-2. Representative substrates and modulators o f MRP (Broxterman et al., 1995; Cole and Deeley, 1998; Miller et al., 1999). Substrates Modulators Bis carboxyethylcarboxyfluorescein (BCECF) Buthionine sulfoximine Calcein GSH ethyl ester Daunorubicin MK571 Dinitrophenyl gluthathione Doxorubicin Estradiol-17(3-glucuronide Fluorescein Indomethacin Leukotriene C4 Probenecid Vincristine Vinblastine 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.6. Modulations of MRP Modulation of MRP by pharmacological agents GSH is involved in the MRP-mediated transport of some substrates (Cole and Deeley, 1998); therefore, by modulating intracellular GSH level, MRP- induced MDR can be reversed. Chunman et al. (1998) reported that, by treating KB cells with 100 pM buthionine sulfoximine (BSO), the sensitivity o f the cells to vincristine resistance was increased 3-fold. Loe et al. (1996) also reported that the inhibition o f ATP-dependent LTC4 uptake by vincristine and vinblastine decreased 2-5 fold after depletion of GSH by BSO in MRP-overexpressed membrane vesicle. In addition, the multispecific organic anion transporter inhibitors, indomethacin and probenecid, are capable o f blocking MRP at 10-100 pM and increasing fluorescence accumulation in brain microvessol endothelial cells (Huai-Yun et al., 1998). The precise mechanisms by which probenecid and indomethacin reverse MDR remain unclear, but both compounds reverse MDR in MRP overexpressing HL60/AR and H69/AR cells, but not in P-gp-overexpressing HL60/Tax and P388/ADR cells (Gollapudi et al., 1997). MK-571, a leukotriene LTD4 receptor antagonist and specific MRP blocker, decreased excretion of glutathione methylfluorescein by 30% in Caco-2 cells (Gutmann et al., 1999). Gekeler et al. (1995a) demonstrated that MRP-associated vincristine resistance was reversed completely by 30 pM of MK571, a sodium salt o f a lipophilic 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. organic acid which contrasts with the structures o f modulators of P-gp-associated MDR. Annamycin (Ann) is a highly lipophilic anthracycline antibiotic that has been shown to circumvent P-gp-mediated MDR both in vitro in MDR1 cell lines, and in vivo in nude mice bearing the KB and KB-V1 xenografts (Zou et al., 1994), and MRP-mediated MDR in breast carcinoma MCF7 and small-cell lung cancer UMCC-1, and their MRP-expressing counterparts, MCF- 7/VP and UMCC-1/VP (Perez-Soler et al., 1997). The mechanisms by which Ann circumvents MRP are far from clear but appear to be related to its preferential partition into lipidic structures, thus avoiding its interaction with the efflux pump. Protein kinase C (PKC) causes the phosphorylation of P-gp and MRP, thereby regulating the functions o f these drug efflux pumps. PKC inhibitors, like chlorpromazine, trifluoperazine, tamoxifen and staurosporine, have been shown to overcome MDR phenotypes (Sato et al., 1990; Volm, 1998). Gekeler et al. (1995b) also observed a reversal o f vincristine and adriamycin resistance in MRP overexpressing human MDR sublines HL60/AR and GLC4/ADR, that was modulated by the highly selective bisindolylmaleimide PKC inhibitor GF 109203X. Since the PKC inhibitor did not significantly influence the MRP gene expression at mRNA level, the authors suggested a direct interaction of the compound with MRP or/and an indirect influence on MRP activity by altering the phosphorylation status of the transporter. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Modulation o f MRP by formulation Miller et al. (1999) reported that 22 pM o f the pluronic block copolymers (poly(ethylene oxide)-6 /ocA:-poly(propylene oxide)-Woc&-poly(ethylene oxide), EOm/2-POn-EOm/2) could inhibit MRP in Panc-1 cells (a MRP overexpressing cell line), thereby increasing fluorescein accumulation by 40-fold compared to the control. Together with the P-gp inhibition effect mentioned before, it is tempting to suggest that there is a singular mechanism to explain the inhibitory effects of pluronic on various drug efflux pumps. There are also indications that the molecules of the pluronic block copolymers must first undergo endocytic transport into the cells to reach the site o f action within the membrane for the efflux pump inhibition. Modulation o f MRP by oligonucleotides Stewart and coworkers (1996) described a series o f phosphorothioate antisense oligonucleotides that could reduce MRP mRNA and protein expression in MRP-transfected HeLa cells. The decrease in MRP mRNA by one o f these oligonucleotides, ISIS7597, was shown to be very rapid and complete, and likely to result from RNase-mediated cleavage of the mRNA. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Modulation o f MRP by antibodies A monoclonal antibody, QCRL-3 inhibited LTC4 uptake in membrane vesicles from MRP-transfected HeLa cells (Loe et al., 1996). However, QCRL-3 targets the intracellular conformational epitope o f MRP, therefore, antibodies against an extracellular component would have to be identified to serve as potential functional inhibitors of MRP. Food effect on MRP function The growing use o f herbal extracts and phytopharmaceuticals raises a new challenge in improving the pharmacokinetics o f some drugs (Evans, 2000). Different classes o f flavonoids constitute a new class of modulators of MDR (Conseil et a l, 1998; Perez-Victoria et al., 1999). For example, isoflavones such as genistein, a protein tyrosine kinase inhibitor (PTK), and daidzein, an genistein analogue with no effect on PTK, both at 20 pM inhibited anion (bilirubin-, bromsulphthalein- and rhodamine-conjugates) secretion in the isolated perfused liver of Wistar rats expressing MRP2. Because genistein and its glucuronide metabolites are substrates of MRP2, the inhibition is partially explained by competition for this transporter. Genistein at 200 pM inhibited daunorubicin (DNR) efflux in five non-P-gp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR). In contrast, genistein did not increase DNR accumulation in three P-gp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) (Versantvoort et al., 1993). 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In reducing ATP pools and/or glutathione synthesis, it might be possible to enhance the efficacy of drugs affected by such resistance mechanisms. Reduction of the ATP pool and glutathione content is achievable in cancer cells by depleting the exogenous methionine (Met) and ethionine supply. Poirson-Bichat et al. (2 0 0 0 ) observed that when methionine depletion was achieved by feeding the nude mice xenografted with human solid cancers (TC71-MA (a colon cancer), SCLC6 (a small cell lung cancer), and SNB19 (a glioma), a methionine-free diet supplemented with homocysteine, treatment with cisplatin, doxorubicin, and carmustine prolonged the survival o f mice by 2-4 fold. Doxorubicin did not affect the growth of SCLC6 , a MDRl-MRP-expressing tumor, while a methionine and ethionine deprived diet slightly decreased SCLC6 growth and, in combination with doxorubicin, an inhibition of 51% o f cell growth was obtained. 1.3. Other drug efflux pumps 1.3.1. MRP family At least six MRP homologues, MRP1 (MRP), MRP2 (also called cMOAT), MRP3, MRP4, MRP5, and MRP6 have been identified in human (Table 1-3). MRP2, MRP3, MRP4, MRP5, and MRP6 are mainly expressed in the liver (Table 1-4). Based on the complete sequences, two subgroups can be recognized (Table 1-5). Group I comprises MRP1, MRP2, MRP3, and MRP6 , which share 45-58% amino acid identity to MRP1, and is characterized by the 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. presence o f an NEb-terminal membrane-bound extension of about 200 amino acids. This adds five putative transmembrane segments to a P-gp-like core. MRP4 and MRP5 have less homology to MRP1 (34-39% amino acid identity), and their predicted structure is more similar to CFTR and P-gp (Kool et al., 1999a). MRP2 has 48.7% identity and 66.9% similarity compared with MRP1 (Cole and Deeley, 1998). MRP2 could mediate cisplatin resistance by effluxing GS-cisplatin out of the cells. Whether there is any association between clinical cisplatin resistance and raised MRP2 levels in tumors remains to be studied (Borst et al., 2000). The amino acid identity o f MRP3 cloned from human liver with MRP1 and MRP2, is 58% and 48%, respectively (Konig et al., 1999a). MDCK cells overexpressing MRP3 did not show an increase in glutathione export or a decrease in the level of intracellular glutathione, in contrast to cells overexpressing MRP1 and MRP2 (Kool et al., 1999a). The up-regulated MRP3 in the basolateral hepatocyte membrane o f patients with the Dubin-Johnson syndrome who are deficient in MRP2, indicated that MRP3 may play a role in the transport o f bile salts (Konig et al., 1999b; Kool et al., 1999a). The transport characteristics of MRP3 differ from those of MRP 1 and MRP2 in that glutathione conjugates are poor substrates for MRP3 (Kool et al., 1999b). MRP6 were found to be overexpressed only in those cell lines with high overexpression and amplification o f the MRP1 gene. MRP6 did not play a role in canalicular organic anion excretion, suggesting that it may be coamplified with MRP1 only because of the proximity of its gene location next to it on chromosome 16 (Kool et al., 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1999b). MRP6 is postulated to fulfill a "housekeeping" transport function (Ringpfeil et al., 2000). The pathogenic mutations in MRP6 may be associated with Pseudoxanthoma elasticum (PXE), the prototypic heritable connective tissue disorder (Suzuki et al., 2000). MRP4 is directly linked to the efflux o f nucleoside monophosphate analogs, such as dideoxynucleosides, potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases (Hirohashi et al., 1999). MRP5 gene expression levels were stimulated in clinical specimens with exposure to platinum drugs and found coexpressed with MRP1, indicating 2 or more MRP family proteins may contribute multifactorial clinical drug resistance to platinum drugs (Oguri et al., 2000). But MRP5 did not confer resistance to various classes of cytotoxic drugs, e.g., 2',7'- bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (McAleer et al., 1999). Similarity analyses and functions of human MRP and its most closely related ABC proteins are listed in Table 1-6. Table 1-7 summarizes the properties of P-gp and MRP. 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-3. The human multidrag resistance protein (MRP) family and some of the alternative names used in the literature for individual family members (Borst et al., 2000). Members Other names used MRP1 ABCC1 and MRP MRP2 ABCC2, cMOAT, and cMRP MRP3 ABCC3, MOAT-D, and cMOAT-2 MRP4 ABCC4 and MOAT-B MRP5 ABCC5, MOAT-C, and pABCl 1 MRP6 ABCC6, MOAT-E, MLP-1, and ARA * ABC: adenosine triphosphate-binding cassette; MOAT: multispecific organic anion transporter; cMOAT: the canalicular MOAT; ABCC: the C group; MLP-1 stands for MRP-like protein 1; ARA stands for anthracycline resistance associated. 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-4. Tissue distribution and physiologic functions o f human multidrug resistance proteins (MRPs) (Borst et al., 2000). Main location in the body Major physiological function MRP1 Ubiquitous (low in liver) Major leukotriene C4 transporter MRP2 Liver, kidney, and gut Major transporter of bilirubin Glucuronides and other organic anion: From liver into bile MRP3 Liver, adrenals, pancreas, kidney, and gut Unknown MRP4 Prostate, lung, muscle, pancreas, testis, ovary, bladder, and gallbladder Unknown MRP 5 Ubiquitous Unknown MRP6 Liver and kidney Unknown 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-5. Percent amino acid identity between fully sequenced human multidrug resistance proteins (MRPs)* (Borst et al., 2000) MRP1 MRP2/cMOAT MRP3 MRP4 MRP5 MRP6 1531 aa 1545 aa 1527 aa 1325 aa 1437 aa 1503 aa MRP1 100 MRP2 49 100 MRP3 58 48 100 MRP4 39 37 36 100 MRP5 34 35 33 36 100 MRP6 45 38 43 34 31 100 * Homology between human MRPs, expressed as percent amino acid (aa) identity. For the multiple sequence alignment the PILEUP program from the University o f Wisconsin Genetics Group (GCG) package (Version 9.1) was used. The following accession numbers were used: MRP, L05628; MRP2, U49248; MRP3, AF009670; MRP4, AF071202; MRP5, AF104942; and MRP6, AF076622. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-6. Similarity analyses and functions of human MRP and its most closely related ABC proteins (Cole and Deeley, 1998) Protein/gene (species) Function % Identity %Siirilarity MRP (human) GS-X pump, anionic conjugate transporter, multidmg resistance 100 100 mrp (mouse) QS-Xpump, anionic conjugate transporter, multidmg resistance 89.9 96.1 MOAT (human)/MRP2 GS-X pump, anionic conjugate transporter (hepatocanaliculi) 48.7 66.9 EBCR (rabbit) Probable MOAT ortholog 48.7 66.6 C. elegans mrpl (nematode) Heavy metal resistance 46.3 64.2 n CD * 1 1 1 Unknown 46.6 63.6 MRP6 (human) Unknown 42.1 60.6 YCF1 (yeast) Cadmium resistance, vacuolar GS-X pump 40.2 59.9 AtMRPl (Arabidopsis) GS-X conjugate pump 36.0 55.0 SUR1 (human) Sulfonylurea receptor, K+ channel regulate (pancreas) 33.1 53.2 sur2 (rat, mouse) Sulfonylurea receptor, K+ channel regulator (brain, heart) 32.5 53.1 YOR1/YRS1 (yeast) Oligomycin resistance 30.3 50.0 LtpgpA (leishmania) Resistance to antimonial and arsenical oxyanions 30.0 47.9 Sequences were aligned along their entire length with MRP using CLUSTAL W(1.6) multiple sequence alignment. Sequence data were obtained using the following accession numbers: MRP, L05628/P33527; trap, AF022908/1488428; MOAT, U49248/U63970; EBCR, 1430907/Z49144; C. elegans mrpl, U66260; C. elegans mrp2, U66261; MRP6, U91318; YCF1, L35327/Z48179; AtMRPl, AF008124; SUR1, L78207/U63421; sur2, D83598/D86037; YOR1/YRS1, Z73066; LtpgtA, X17154. 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-7. Comparison o f P-gp and MRP P-gP MRP First clone 1976 1992 Gene MDR1 MRP M.W. 170 kDa 190 kDa Protein size 1280 amino acids 1531 amino acids Nucleoside 2 2 Transmembrane domains 12 12 Expression Tumor Normal tissues: adrenal gland, kidney, liver, small and large intestine, pancreas, brain, testis, placenta, lung, prostate, stomach, NK cells, CD34, and bone marrow cells Tumor Normal tissues: adrenal gland, lung, heart, skeletal muscle, intestine, brain, testis, liver, kidney, spleen, and erythrocyte Typical localization Apical Apical, basolateral, and intracellular membranes Substrates Lipophilic cations Anions and GSH conjugates Modulation CsA, PSC833 pluronic block copolymers MRK-16 Probenecid, indomethacin pluronic block copolymers ISIS7597 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.3.2. LRP Lung Resistance-related Protein (LRP), a 110 kD protein, is overexpressed in several non-P-gp MDR cells lines of different histogenetic origin (Izquierdo et al., 1996a). LRP does not belong to ABC superfamily and is not a plasma membrane protein. LRP is one o f four vault proteins which form barrel-like structures localized in the perinuclear area. Vaults localize to nuclear pore complexes, playing a role in drug resistance by regulating the nucleocytoplasmic transport of drugs (Volm, 1998). LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types, indicating that LRP play a role in inherent drug resistance. In a panel o f 61 cancer cell lines which are not subjected to drug selection, LRP was a good predictor for in vitro resistance to MDR-related drugs when compared to P-gp and MRP, even for non-MDR related drugs, such as platinum compounds. LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Furthermore, reversal o f MDR parallels a decrease in LRP expression (Izquierdo et al., 1996b). 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.3.3. Antibiotic efflux pumps in bacteria Efflux pumps are not only present in eukaryotic cells, but also prokaryotic cells. Active efflux pumps from prokaryotic as well as eukaryotic cells strongly modulate the activity o f a large number o f antibiotics. Compared to the ATP- binding cassette superfamily, including P-glycoprotein and MRP, which is responsible for modulating the accumulation of antimicrobials in eukaryotes, the secondary active transporters (antiports, symports, and uniports) are responsible for the efflux of antibiotics in prokaryotes. They include five superfamilies: SMR (Small Multidrug Resistance), MET (Multidrug Endosomal Transporter), MAR (Multi Antimicrobial Resistance), RND (Resistance Nodulation Division), and MFS (Major Facilitator Superfamily) (Van Bambeke et al., 2000). These bacterial efflux pumps are believed to contribute significantly to acquired bacterial resistance due to their broad substrate specificity, their expression in important pathogens, and their cooperation with other mechanisms of resistance. In 1999, the Centers for Disease Control and Prevention (CDC) reported that of 1,600 Streptococcus pneumoniae isolates, 12-65% were partially resistance to at least one o f penicillin, tetracycline, and ceftriaxone, therefore, new antibiotics must be discovered to overcome drug resistance (Longworth, 2001). Phenotypes of irreversible multidrug resistance explains high-level intrinsic resistances found in specific organisms. The existence of antibiotic efflux pumps and their impact on therapy is taken fully into account for the selection of novel antimicrobials. The study of inhibitors (such as MC-207,110) o f efflux 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pumps as novel agents for combination therapy has been undertook in some laboratory (Lomovskaya and Watkins, 2001). The design o f specific, potent inhibitors appears to be an important goal for the improved control of infectious diseases. 2. Membrane drug transporters Besides efflux pumps, other carrier-mediated transport processes play an important role in drug transport. Endogenous substances and drugs can be actively transported by membrane transporters such as the nucleoside transporter, monocarboxylate transporter, dipeptide transporter, organic cation transporter, organic ion transporter, monosaccharide transporter, amino acid transporter, phosphate transporter, and bile acid transporter (Tsuji and Tamai, 1996). In the following, we will elaborate more about dipeptide, nucleoside, organic cation, and monocarboxylate transporters that are relevant to our research. 2.1. Dipeptide transporters Dipeptide transporters are H+ -coupled, energy-dependent transporters that are known to play an essential role in the oral absorption of natural di/tripeptides and (3-lactam antibiotics (Dantzig and Bergin, 1990). Dipeptide transporter has been successfully used as a platform for improving the oral bioavailability of 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. drugs such as methyldopa (Bai et al., 1992) and acyclovir (Han et al., 1998; Lee et al., 1997) through dipeptide prodrug derivatization. Thus far, at least four proton-coupled dipeptide transporters have been cloned. The first one cloned was PepTl from the rabbit small intestine (Fei et al., 1994). Consisting of 707 amino acid residues, this transporter is also found in the kidney, liver, and brain of the human. Its human homolog, hPepTl, shows an 81% amino acid sequence identity (identical sequence) and a 92% similarity (conserved sequence) with rabbit PepTl (Liang et al., 1995). In addition to the tissues mentioned, hPepTl mRNA is found in the placenta and pancreas. PepT2, cloned from human kidney, exhibits 50% identity and 70% similarity to PepTl (Liu et al., 1995). PepT2 is not found in the intestine. Thus far, PepTl and PepT2 have received far more attention than the other two dipeptide transporters rhaPT (cloned from rat kidney cortex) (Daniel et al., 1995) and HPT-1 (cloned from Caco-2 cells) (Dantzig et al., 1994;Liang et al., 1995) based on their wide distribution and role in drug delivery. In the conjunctiva, a proton-driven dipeptide transporter was found to mediate the absorption of dipeptides and structurally similar drugs such as bestatin, (3-lactam antibiotics, and ACE inhibitors. Results from computer modeling suggested that an appropriate dipeptide N-terminal to C-terminal distance and a favorable orientation of the side chains may be important for substrate interaction with the conjunctival dipeptide transporter (Basu et al., 1998). 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2. Nucleoside transporters Nucleosides are hydrophilic molecules, which require transporter proteins for their permeation across cell membrane. The uptake of nucleosides (or nucleobases) by nucleoside transporters is essential for nucleic acid synthesis, especially for many human cell types and in parasitic organisms that cannot synthesize nucleotides de novo. Nucleoside transporters are responsible for the entry of many cytotoxic nucleoside analogues used in cancer and viral chemotherapy (Cass et al., 1999). Two types o f nucleoside transport processes exist including equilibrative bi-directional processes (by equilibrative nucleoside transporter (ENT)) driven by chemical gradients and inwardly directed concentrative processes (by concentrative nucleoside transporter (CNT) families) driven by the sodium electrochemical gradient (Baldwin et al., 1999; Cass et al., 1998). Among the equilibrative nucleoside transport processes (es and ei, in most mammalian cell types) and the concentrative nucleoside transport processes (cit, cif, cib, csg, and cs, primarily in specialized cell types, usually epithelia), es, ei, cit, and cif are four major nucleoside transport processes of mammalian. In the conjunctiva, both Na+ - dependent (cif) and Na+-independent nucleoside transport processes are present, indicated by the pattern o f uridine transport (Hosoya et al., 1998). 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3. Organic cation transporters Organic cations have many important biological functions. Endogenous bioactive organic cations include dopamine and choline, while clinical drugs include the antihistamines, skeletal muscle relaxants, antiarrhythmics, and P- adrenoceptor blocking agents. They are transported into cells where they act as essential nutrients or have other influences on cell metabolism. In the small intestine, liver, and kidney, the organic cation transporters mediate both the absorption and excretion of organic cations (Koepsell, 1998). Two distinct processes are known to exist: (a) a facilitative carrier- mediated system that is driven by an inside-negative membrane potential difference, as exemplified by OCT1, OCT2, and OCT3; and (b) an energy- dependent secondary active OC+ /H+ exchange mechanism that is driven by an inwardly directed proton gradient generated by H+ efflux via Na+ /H+ antiport and/or H+-ATPase. Two new OCTs have recently been identified, OCTN1 and OCTN2. OCTN1 appears to exhibit H+-dependent transport and to be widely distributed in the body. OCTN2 appears to be a Na+ -dependent, carnitine-specific transport system that exists in the kidney, skeletal muscle, heart, and placenta in human (Ueda et al., 2000). In the kidney, several types of OCTs are present in the epithelium of the proximal tubule involving various functions: basolateral potential dependent OCT for secretion, apical potential-dependent OCT for reabsorption; apical ATP- 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dependent OCT for reabsorption; OC+ /H+ exchanger for secretion. In the liver, OC+ /H+ exchanger in canalicular membrane functions to excrete OC+ to the bile canaliculus. In the intestine, apical potential-dependent OCT is for reabsorption and apical OC+ /H+ exchanger functions for secretion (Zhang et a l, 1998). In the conjunctiva, an inside-negative membrane potential-dependent type on the mucosal aspect facilitate cation reaborption (Ueda et a l, 2000). 2.4. Monocarboxylate transporters (MCT) Transport o f L-lactate, pyruvate, and the ketone bodies, acetoacetate and beta-hydroxybutyrate, across the plasma membrane is of considerable importance to almost all mammalian cells. In most cells, a specific H+ - monocarboxylate cotransporter is largely responsible for this process. The capacity o f this carrier is usually very high, to support the high rates o f production or utilization o f L- lactate (Poole and Halestrap, 1993). A H+ -coupled monocarboxylate transport process appears to exist on erythrocyte membrane, cardiac muscle, and tumor cells (Poole and Halestrap, 1993), as well as on the basolateral membrane of corneal epithelium, the apical and basolateral membrane of corneal endothelium and intestinal epithelium, and the apical membrane o f retinal epithelium (Horibe e ta l, 1998; Tsuji and Tamai, 1996). In addition, a Na+ -coupled lactate transport process appears to exist on the basolateral membrane of the corneal endothelium, and retinal pigment epithelium, and the apical membrane of the conjunctival epithelium (Horibe et al., 1998). In brain, MCT1 is expressed on both the luminal 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and antiluminal membranes o f brain capillary endothelial cells and transports substrates bidirectionally (Stein et al., 1997). In the intestine, MCT1 is expressed on both apical and basolateral membranes for absorption o f substrates. In the conjunctiva, MCT also favors reabsorption o f substrates (Horibe et al., 1998). 3. Conjunctiva 3.1. General anatomy and physiology of the conjunctiva The conjunctiva is a thin, transparent mucous membrane covering the inner surface of the eyelids and exterior part of the sclera. The portions of the conjunctiva covering the posterior surface of the eyelids and the anterior surface of sclera are the palpebral and bulbar conjunctiva, respectively. The area where palpebral and bulbar conjunctiva meet is the fornix conjunctiva (Fig. 1-6). The palpebral conjunctiva is an area where reactive pathology o f the conjunctiva may be seen clinically, particularly those changes that accompany viral and bacterial infections as well as allergy and inflammation. Fornix cells have a much greater proliferative potential than bulbar and palpebral cells (Wei et a l, 1993). The conjunctiva is composed o f three layers: epithelium, substantia and submucosa. The epithelium consists of several layers of nonkeratinized and stratified surface cells. There are three major epithelial cell types in the conjunctiva: superficial, wing, and basal cells. The superficial cells are further distinguished into five different types, among which the goblet cells and Langerhans have significant 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. physiological functions. The goblet cells secret mucus and the Langerhans cells play a pivotal role in antigen recognition and processing. Epithelial cells are attached to one another by means of desmosomes and tight junctions and they communicate with each other via gap junctions (Diehold et al., 1997). The conjunctival epithelium is capable o f proliferation and transdifferentiation, which play an important role in comeal wound healing. Other major functions o f the conjunctiva include barrier function to protect the entry of foreign compounds and mucin secretion to maintain the moistness on the eye surface (Steuhl and Roden, 1984; Wei et al., 1993). Previous work in our laboratory has revealed that Cl' appears to enter the conjunctiva from the serosal fluid via Na+ /K+ /2C1‘ cotransport process and exit to the mucosal fluid via channels, resulting in active C f secretion (Kompella et al., 1993). This Cl' secretion is subject to nucleoside stimulation (e.g., UTP) of purinergic receptors, possibly the P2Y(2) and/or the P2Y(4) receptor on the mucosal side of the tissue (Hosoya et al., 1999). The C f secretion is also subject to cAMP, Ca2+ , and protein kinase C (PKC) modulation (Shiue et al., 1998). Because of the coupling of fluid flow with C1‘ secretion (Shiue et al., 2000), stimulating Cl' secretion pathway may be considered for stimulating transconjunctival fluid flow in the dry-eye state. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lacrimal gland Fornix _ _ _ _ _ conjunctiva Sclera Bulbar __ conjunctiva Cornea Palpebral__ conjunctiva Fig. 1-6. Sectional view of the anterior segment of the eye. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2. Ocular drug absorption routes Drug absorption across the conjunctiva and the sclera is generally regarded as non-productive, based on the assumption that drug entering these tissues is ending up by the general circulation and does not contribute to intraocular drug level. Hence, most efforts o f improving ocular drug delivery are aimed at enhancing corneal permeability. Miller et al. (1981) demonstrated that drug (such as pilocarpine) concentration in the aqueous humor did not always reflect that in the surrounding intraocular tissues in rabbit by topical administration. It suggested that topically applied drugs can reach the intraocular tissues by either the comeal and/or the noncomeal (conjunctival-scleral) pathway (Ahmed and Patton, 1985). Conjunctiva plays an important role in both ocular and systemic absorption of topically applied ophthalmic drugs. For example, when comeal access was blocked, although aqueous humor and comeal timolol levels dropped drastically to < 5% of its value compared to that observed when comeal access was permitted, significant timolol levels were still observed in iris-ciliary body, lens and vitreous levels, and there was no dramatic drop compared to that in the absence of comeal access (Ahmed and Patton, 1985). Conjunctiva also contributes to systemic timolol absorption (13% of the instilled dose) (Chang and Lee, 1987), as well as atenolol, levobunolol, and betaxolol (Lee et al., 1993). The bulbar conjunctiva is the first tissue across which a topically applied dmg must pass in order to reach the underlying tissues in the uveal tract via the non-comeal route (Ahmed and Patton, 1985) (Scheme 1). Compared to the 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cornea, the conjunctiva occupies a 9 and 17 times larger surface area in rabbits and humans, respectively (Watsky et al., 1988). In addition, the conjunctiva is leakier tissue compared to cornea. Conjunctival transepithelial electrical resistance is about 4 times lower than that of cornea, which permits markedly higher conjunctival-scleral penetration o f hydrophilic drugs (Horibe et al., 1997). For example, the topical instillation in the presence and absence of corneal access did not make significant difference in the concentration o f inulin, a large hydrophilic molecule, in various ocular tissues (e.g., lens, iris-ciliary body, sclera, and bulbar conjunctiva, but not aqueous humor). It indicated that much o f its intraocular access was via the noncomeal route (Ahmed and Patton, 1985). Further efforts at maximizing noncomeal absorption for similarly poorly penetrating compounds o f therapeutic interest may prove productive. Conjunctiva is commensurately permeable to both hydrophobic P- adrenergic antagonists (Saha et al., 1996a) and hydrophilic FITC-labeled dextrans (FD) up to -4 0 kDa (Horibe et al., 1997) by passive diffusion. Moreover, this tissue is capable of active transport of D-glucose by Na+ -coupled glucose transporter, uridine and antivirals (2’-deoxyuridine, 5-iodo-2’-deoxyuridine) by Na+ -dependent and -independent nucleoside transporters (Hosoya et al., 1998), lactate and anti-inflammatory drags (flurbiprofen and diclofenac) by monocarboxylate transporters (Horibe et al., 1998), dipeptides (L-camosine) and peptidomimetic antibiotics (cephalosporins) by dipeptide transporters (Basu et al., 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1998; Lee et al., 1995), and guanidine and anti glaucoma drugs (dipiverfrine, brimonidine, and carbacol) by organic cation transporters (Ueda et al., 2000). SCLERA CORNEA CONJUNCTIVA ANTERIOR CHAMBER PRECORNEAL AREA CONTRALATERAL EYE OCULAR CIRCULATION SYSTEMIC CIRCULATION INTRAOCULAR TISSUES Scheme 1. Ocular penetration routes for topically applied drugs. Key: 1 = transcomeal pathway, 2 = noncomeal pathway, 3 = systemic return pathway, 4 = lateral diffusion. From Ahmed and Patton (1985). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4. General pharmacokinetics of drugs in disease states A pharmacokinetic profile o f a drug including drug absorption, metabolism, distribution, and elimination may change in a disease state compared to that in a normal state (Williams and Benet, 1980). Most research focuses on studying the relationship o f pathological changes o f tissues and alteration of drug metabolism and pharmacokinetics. Little is known yet about alteration of transporters in disease states. Profiles of pharmacokinetics and pharmacodynamics in patients with liver disease are well documented. The permeability of the capillarized sinusoid was decreased as the critical feature in cirrhosis. Altered receptor sensitivity has been observed for some drugs in cirrhosis, while for other drugs there is no change in pharmacodynamics. An awareness o f the possibility o f multiple interactions between changes in hepatic and renal disposition and pharmacodynamics is required for a safe drug (Morgan and McLean, 1995). In patients with cystic fibrosis, the extent o f drug absorption varies widely and the rate o f absorption is slower; yet bioavailability is not altered. Despite hepatic dysfunction, patients with CF have enhanced clearance of many, but not all, drugs due to enhanced activity of cytochrome P450 in the intestine (Rey et al., 1998). Stress could affect the permeability of the blood-brain barrier. One example is that during Gulf War, Israeli troops treated with pyridostigimine to protect against organophosphate type nerve gas exhibited pronounced CNS effects, which were not apparent in earlier testing under peaceful conditions. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Studies on mice confirmed that cholinesterase inhibition by this drug was almost entirely in peripheral tissues in control animals, whereas mice stressed by forced swimming exhibited pronounced cholinerase inhibition in the CNS (Sinton et al., 2000). Some diseases can alter gastric secretion, intestinal motility, and surface area available for absorption, thereby altering drug absorption from the gastrointestinal tract (Gubbins and Bertch,1989). Many HIV-infected patients have elevated gastric pH values that may lead to alteration in drug absorption (Welage et al., 1995). In rats with pancreatitis, gut permeability to polyethylene glycol (M.W. 3350) was increased (5.4 ± 1.2%) when compared with control (1.8 ± 0.2%). Increased gut permeability can be correlated with pancreatitis severity. This increased intestinal macromolecular permeability could allow absorption of substances from the bowel into the systemic circulation and contribute to multiple organ failure (Ryan et al., 1993). In contrast, in patients with Crohn's disease, alteration of paracellular transport determined by absorption o f polyethylene glycols (M.W. 458-810) was not observed (Lindberg etal., 1995). Interstitial cystitis, a lower urinary tract disease which may involve inflammation of the bladder, increased the apparent bladder absorption rate and the peak plasma concentration of salicylate and antipyrine in feline interstitial cystitis cats were 7 to 8 times that in healthy cats. It is due to increase o f bladder permeability to drugs and slower drug elimination (Gao et al., 1994). 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In the case of nasal inflammation, drug absorption is not increased. The number o f ciliated cells, and with that mucociliary transport, is reduced during a common cold, but little data was reported on the effect of a common cold on the pharmacokinetics o f intranasally administered drugs. Probably, drug absorption is not increased, similar to the finding in allergic rhinitis. On the contrary, blockage, sneezing and rhinorrhoea might preclude the absorption (Mygind and Dahl, 1998). The comeal epithelial permeability of the dry eye patients was found to be 2.7 times that of a control group measured by computerized objective fluorophotometry (Gobbels and Spitznas, 1992) which may be partially due to markedly decreased mucin secretion (Marriott, 1989). When the eye is damaged by ocular infection, it allows pathogens to overcome the natural defenses of the eye, or in immunosuppressed patients, normal flora may become opportunistic. In deciding on appropriate treatment, both the causative pathogen and the stracture(s) affected must be considered. Differences in drug absorption, penetration, and availability to the various structures of the eye affect treatment decisions. Treatment of ocular infections depends on knowledge o f the pathophysiology and drug disposition at the site of infection (Thielen et al., 2000). In addition, understanding alteration of transport mechanisms is useful for designing successful drug therapy. Levy (1997) described a spectrum of adverse drug reactions that are caused by the combined action of dmgs and vimses, including ampicillin rash in 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. acute infectious mononucleosis; hypersensitivity reactions to sulphonamides in patients with HIV infection; Epstein-Barr virus-associated lymphoma and methotrexate; and AIDS-related Kaposi's sarcoma and nitrite use. Altered immunological function, metabolism pattern, and drug pharmacokinetics by a disease state could cause these phenomena. 5. Ocular infection and immunity A number o f viruses are known to be pathogenic in the anterior and posterior segments of the eye. Those that are the most frequent causes o f eye disease include members o f the Adenoviridae and the Herpesviridae species (Hendricks, 1999). Adenoviral infection o f the eye has been referred to as “shipyard eye” and “red eye”. This ocular infection is typically resolved by the host immune system (Hendricks, 1999). Cytomegalovirus (CMV), a member of the Herpesvirodae family, causes CMV retinitis, which is the most common ocular opportunistic infection and the leading cause of blindness in patients with AIDS. CMV infection further leads to CMV chorioretinitis, gastrointestinal disease, or other systemic disease in 20%-40% o f patients (Lalezari and Kuppermann, 1997). 5.1. Adenoviral infection Adenoviruses can infect the eye, respiratory tract, gastrointestinal tract and bladder. Ocular adenoviral infections occur worldwide. Clinically, they cause 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. three basic syndromes: epidemic keratoconjunctivitis (EKC), pharyngoconjunctival fever, and nonspecific follicular conjunctivitis. Adenovial infections are usually self-limiting, and permanent visual loss is very rare, but they are highly contagious and can become chronic conditions lasting for months to years (Viswalingam, 1993). Adenoviruses were first isolated in 1953 (Rowe et a l, 1953). Today well over 100 members o f the adenovirus group have been identified which infect a wide range o f mammalian and avian hosts. Human adenoviruses are divided into six subgenera (A, B, C, D, E, and F) on the basis o f DNA restriction enzyme analysis o f genome typing. So far, 47 human adenovirus serotypes have been distinguished on the basis o f their resistance to neutralization by antisera to known adenovirus serotypes (Shenk, 1996). Clinical isolates of adenovirus type 5 (Ad5 McEwen) have received a lot of attention, since in vivo Ad5-infected rabbit model produces a disease that mimics, in part, the human disease (Gordon et al., 1992). 5.2. Immunology of the eye The need for immune protection against a broad range of pathogenic agents exists for all tissues of the body, including the eye, airway, and GI tract. Ocular tissues are involved in sophisticated immune responses to pathophysiological conditions. HLA (human leukocyte antigen)-DR, ICAM-1 (intercellular adhesion molecule-1), IL (interleukin)-3, and GM-CSF (granulocyte macrophage colony-stimulating factor) are not expressed in normal conjunctival 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. epithelial cells, but are induced by atopic keratoconjunctivitis, vernal keratoconjunctivitis, and giant papillary conjunctivitis. IL-6, IL-8, RANTES, and TNF-a are up-regulated by conjunctivitis (Hingorani et al., 1998). Tear concentrations of the TH (Helper T-lymphocytes) 2-like cytokines IL-5 (Uchio et al., 2000) and IL-4 (Leonardi et al., 1999) increased in ocular allergic conditions such as atopic keratoconjunctivitis and vernal keratoconjunctivitis. In patients with Sjogren’s Syndrome, the expression of HLA-DR, ICAM-1, IL -la , IL-6, IL- 8, TNF-a, and TGF (transforming growth factor)-pi are significantly elevated (Jones et al., 1994; Leonardi et al., 1999), with IFN y implicated in the up- regulation of the HLA-DR in the conjunctival epithelium (Tsubota et al., 1999). Viral infection is known to induce host immune response. Ocular herpes simplex virus type 1 (HSV-1) infection increased IFN-y production and extent of inflammation in BALB/c- transgenic mice (Geiger et al., 1994). The Influenza A virus elevated IFN a /p and HLA in A549 cells (Ronni et al., 1997), and increases IL-6, IL-8, and RANTES release in human bronchial epithelial cells (Adachi et a l, 1997). Respiratory syncytical virus (RSV) induced the release o f IL-6, IL-8, and RANTES in RSV-infected bronchial epithelial cell cultures (Tristram et al., 1998) and IL-8 and IL-6 in A549 cells (Arnold et al., 1994), while IL-8 secretion in freshly isolated nasal epithelial cells is increased (Becker et al., 1993). Human rhino viral (HRV-2 and HRV-14) infection increases the production o f IL -lp, IL- 6, IL-8, TNF-a, and expression of ICAM-1, in human tracheal epithelium 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Terajima et al., 1997). Infection with the human T cell lymphotropic vims type I (HTLV) results in active production of IL -la, IL-6, G-CSF, and GM-CSF in human umbilical vein endothelial cells (Adachi et al., 1997). Recombinant adenoviruses are used as vectors for gene transfer to a wide variety o f cells and tissues, including the eye and airway. Recombinant adenovimses with reporter genes (e.g., LacZ) were evident in comeal endothelium and iris anterior surface. Infected comeal endothelial cells are swollen and partly detached even showing a severe inflammatory response in anterior uvea and limbal conjunctiva. It reveals that adenoviral vectors could evoke inflammatory responses (Borras et al., 1996a). Modulation of cytokine signaling pathway could be utilized as a potential therapy. IL-1 receptor (IL-1R) antagonist suppresses allergic eye disease by a down-regulation of the recruitment o f eosinophils and other inflammatory cells (Keane-Myers et al., 1999). Human fibroblast IFN(3 7.5 x 105IU/ml could be used to treat acute epidemic conjunctivitis (Wilhelmus et al., 1987). Viral infection has been reported to modulate the expression of drug efflux pumps. Ikeda et al. (1999) found that human T-cell-leukemia-virus-I increased MRP expression in adult T-cell leukemia (ATL) cells and it might be associated with MDR of ATL cells in vivo. Andreana et al. (1996) reported that HIV increased P-gp expression in CD4 and CD8 T cells in HIV patients. 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6. Cell culture models 6.1. General rationale o f cell cultures Studies using in vitro cell cultures have many advantages over those o f in vivo setting, including (a) rapid assessment o f the permeability and metabolism of a drug; (b) the opportunity to elucidate the molecular mechanisms of drug transport or the pathways o f drug degradation (or activation); (c) rapid evaluation of strategies for achieving drug targeting, enhancing drug transport, and minimizing drug metabolism; (d) the opportunity to use human rather than animal tissue; and (e) the opportunity to minimize time-consuming, expensive, and sometimes controversial animal studies (Audus et al., 1990). In addition, from the basic research perspective, cell culture models permit mechanistic analysis of transport and metabolism by allowing manipulation and precise control of experimental conditions, such as drug concentration and pH. Moreover, the level of expression o f specific transport proteins or metabolic enzymes could be modulated by genetic manipulation of cell population or selection o f specific cell population, thereby allowing more detailed mechanistic analysis. From a drug development standpoint, cell culture models permit the simultaneous analysis of drug transport and metabolism in relatively large numbers of samples simultaneously with relatively small amounts o f a drug. This allows more efficiency in screening the compounds generated by combinatorial synthesis (Quaroni and Hochman, 1996). 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Established and validated cell cultures include epithelial cell cultures (e.g., intestinal, rectal, buccal, sublingual, nasal, and ophthalmic mucosa as well as epidermis o f the skin) and the endothelial barriers (e.g., brain microvessal endothelial cells) (Audus et al., 1990). The intestinal epithelial cell culture model (Caco-2 cells) is currently the best-characterized and most commonly used cell culture system for absorption studies (Artursson and Karlsson, 1991; Hidalogo et a l, 1989). A primary culture o f rabbit conjunctival epithelial cells in conventional liquid-covered condition was developed (Saha et al., 1996b; Saha et al., 1996a), exhibiting tight cell layers. This model was used to study the permeability of P- blockers (Saha et al., 1996b) and uptake mechanisms of dipeptides (Basu et al., 1998). 6.2. Air-interfaced cell culture Normal epithelial function depends upon the development of cellular junctions and the polarity of the epithelium on the connective-tissue substratum. Loss of the three-dimensional organization can affect secretion and other physiologic response (Jackson et al., 1996). Cell cultures are usually maintained by immersing cells in medium, and therefore lack an air-mucosal interface. This represents a significant departure from the normal physiology of tissues like the airway and ocular epithelia. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The requirement of air-interfaced culture conditions depends on the nature of the epithelia. For example, it appears that the presence o f excess liquid on the apical surface of the cell layers may lower the capacity of active ion transport in tracheal epithelial cell cultures o f the rabbit (Mathias et al., 1995), guinea pig (Robison et al., 1993), cow (Kondo et al., 1993), and dog (Kondo et al., 1991). In contrast, active ion transport was significantly higher (159% and 163% o f that found in corresponding liquid-covered culture) in tracheobronchial epithelium cultures in an air-interface on days 4 and 5 (Robison et al., 1993). The central feature of these tracheobronchial epithelium cultures was an apical surface in direct contact with the air environment. It led to formation of tight monolayers, polarized differentiation with the formation of a pseudo-stratified ciliated columnar epithelium similar in structure and function to that in vivo. This feature was found to be well suited for studies of airway function, such as mucin secretion. Air-interfaced cultures of human bronchial epithelium (Tristram et al., 1998) and nasal turbinate tissue (Jackson et al., 1996) also exhibited morphological and electrophysiological traits in vitro comparable to the native tissues in vivo. At the air-interface, mucus coverage o f the nasal turbinate tissue remained high throughout the experimental period and remained active for at least 20 days in the presence o f adequate nutrition (Jackson et al., 1996). Moreover, air-interfaced culture condition facilitates the development of stratified cell layers, such as human buccal mucosa (Izumi et al., 2 0 0 0 ), and rabbit corneal epithelial 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell layers with peak transepithelial electrical resistance o f up to 5 kQ.cm2, 35 times higher than the value of 144 Q.cm 2 in liquid-covered cultures (Chang et al., 2000). The mechanism by which air-interface improves differentiation o f cultured cells is still unclear. Based on the observation that lactate production was less in air-interfaced culture of tracheal epithelial cells, whereas cellular adenosine triphosphate content and basal 0 2 consumption, ouabain-sensitive and -insensitive 0 2 consumption, and the uncoupled 0 2 consumption were greater, Kondo et al. (Kondo et al., 1997) contented that air-interface promotes oxidative metabolism in the growth phase by the provision o f adequate oxygenation, and apical feeding may be responsible for the disturbance o f the development of the oxidative metabolism. 6.3. Virally-infected cell culture Suitable in vivo and in vitro virally-infected models are useful for studies of the development of antiviral therapy. So far, a rabbit model (Trousdale et al., 1995a) and a cotton rat model (Tsai et al., 1992) have been developed as in vivo models for studying the adenovirus-induced eye disease in human. Adenovirus type 5 (Ad5) is capable o f replicating in the eye of these animals and elicits immunopathologic response comparable to that o f adenovirus-induced ocular disease in humans. In vitro viral cultures were conducted in A549, a lung cancer 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell line (Trousdale et al., 1994), and MRC-5 (Gordon et al., 1996), a diploid fibroblast cell line. Cytopathic effect was seen as early as day 1 following inoculation and was characterized by the rounding up and sloughing of infected cells, producing plaques in the cell monolayer (Gordon et al., 1994). In addition, the performance characteristics of human embryonic kidney (HEK), human diploid fibroblasts (HDF), and monkey kidney (MK) cells for adenovirus isolation were examined for eye and respiratory tract specimens. HEK cells were superior to HDF and MK cells in terms of both speed o f virus detection and sensitivity (Krisher and Menegus, 1987). These models were used for definitive diagnosis of adenoviral infection. In addition, both the in vitro cell culture (A549 cells) (Trousdale et al., 1994) and animal models (cotton rat and rabbit models) (Gordon et a l, 1994; Trousdale et al., 1994) have been used to evaluate the antiviral inhibitory activity of drugs such as ganciclovir and cidofovir. Gene transfer to the ocular surface epithelium is o f potential therapeutic value. Recombinant adenoviruses are used frequently as vectors for gene transfer to a wide variety o f cells and tissues, and it is hoped that they may prove useful for gene therapy. Recombinant adenoviruses as vectors are widely used to investigate transfer o f cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelium (Zabner et al., 1996). In addition, the reporter gene lacZ with a recombinant adenovirus type 5 was studied in human comeal and conjunctival cell lines. The maximum lacZ expression in vitro was 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. demonstrated in the corneal epithelial cell line at 7 days and conjunctival epithelial cell line at 2 days. These data indicate that adenovirus vector is capable of directly delivering gene to the corneal and conjunctival epithelium, suggesting a variety of possible gene therapy uses (Tsubota et al., 1998). Immunomodulating cytokine IL-10 and LacZ carried by recombinant adenoviral vectors were also able to be transfected in human conjunctival cells in culture. LacZ expression could be detected for at least 50 days after infection with multiple of infection (MOI) 200. IL-10 protein expression occurred between 4-6 days post transduction, and was maintained at a detectable level for at least 1 month. These results show human conjunctival cells can secrete biologically active IL-10 that could suppress immune mediated disorders. It opens avenues to developing a multiple target gene therapy locally (Shen et al., 2001). 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II. STATEMENT OF THE PROBLEM AND SPECIFIC AIMS The absorption efficiency of topically administered ocular drugs in the conjunctiva is an important determinant o f drug availability to the back o f the eye, i.e., to the retina. Active transport processes play an important role in the absorption o f drugs, including antibacterial, antiviral, antihypertensive, and antineoplastic agents (Tsuji and Tamai, 1996; Volm, 1998). Membrane drug transporters are membrane-bound proteins that serve a vital role in driving either drug absorption into or drug efflux out of epithelial cells. Among drug efflux pumps, P-glycoprotein (P-gp) and multidrug resistance protein (MRP), originally found in cancer cells leading multidrug resistance, are widely distributed in normal tissues and significantly influence drug absorption and elimination. In addition, these two proteins are known to be involved in other pathophysiological functions, such as in human immune responses in various tissues. Similarly, the possible efflux mechanisms in the conjunctiva could influence ocular drug delivery. In addition, given that drug administration mostly occurs in a disease state, such as viral infection, it is necessary to understand the functions of these efflux pumps in a disease state and their possible pathophysiological roles in the conjunctiva. The evidence that viral infection can modulate the expression o f drug efflux pumps includes: human T-cell-leukemia-virus-I increases MRP expression in adult T-cell leukemia (ATL) cells and it might be associated with MDR of ATL cells in vivo (Ikeda et al., 1999), and HIV increased P-gp expression in CD4 T 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells in HIV patients (Andreana et al., 1996). All these reports suggest that efflux pumps may play a role in immune responses in a disease state. The central hypothesis of my research is that P-gp and MRP may play a role in the efflux of xenobiotics and endogenous molecules in the conjunctiva in normal and adenoviral-infection states, thereby affecting ocular drug absorption and participating host immune response. The significance of this research is that it is the first integrated study o f efflux pumps in the conjunctiva which provides the in-depth understanding o f the expression, localization, function, and modulation of efflux pumps in the conjunctiva. This may provide useful information for the rational formulation and design of drugs for ocular drug delivery. This research has also set the stage for determination of the expression and function of other transporters (e.g., dipeptide and nucleotide transporters) in both normal and virally-infected states and lays the foundation for designing agents for the ocular drug delivery. This research was built upon the encouraging preliminary findings by Saha et al. (1998) that the transport of CsA and verapamil in the conjunctival epithelial cells showed asymmetry, favoring secretion to the mucosal side o f the conjunctiva. It indicated a possible existence of P-gp in this tissue. The key questions to be addressed were (a) is there evidence of P-gp protein expression and what is the localization of P-gp in the conjunctival epithelial cells? Does P-gp affect other ophthalmic drug absorption, such as P-blockers? Are P-gp’s 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression and function affected in a disease state, such as adenoviral infection? (b) Besides P-gp, does another efflux pump, MRP, exist in the conjunctiva and what could be its functions in drug transport and physiological roles? Are the expression and function of MRP modulated by adenoviral infection and what could be the underlying mechanisms? To answer these questions, the following three specific aims were proposed for this research: Specific Aim #1. To develop rabbit conjunctival epithelial cell culture models in normal (air-interfaced condition) and disease (Adenoviral infection) states Cell cultures have been proven to be a useful tool for evaluating transport mechanisms as well as studies of formulation and physiological factors influencing drug transport (Artursson and Borchardt, 1997; Borchardt, 1995). A functional primary culture of rabbit conjunctival epithelial cells (RCEC) under the conventional liquid-covered condition (LCC) was developed by Saha et al. (1996b). Air-interfaced cultures (AIC) were demonstrated to be a better culture condition to mimic tissues in vivo (e.g., trachea (Mathias et al., 1995)) in terms of morphology and barrier properties, we undertook the present study to determine whether this would also be the case in the conjunctiva on the basis of passive and active transport characteristics for ions and drugs. Given that there is no in vitro virally-infected conjunctival cell model, we attempted to establish an adenovirally-infected cell culture as a disease model for 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. studying the alteration o f transporter proteins and their drug transport properties. We characterized an in vitro Ad5-infected cell model in terms o f infectivity, electrophysiological and morphological changes, and alterations in ion transport characteristics. The air-interfaced culture and Ad5-infected culture o f RCEC were attempted to mimic the biochemical and pathophysiological aspects of normal and adenoviral infection states o f the conjunctiva. They allow further systematical dissection of the expression of efflux pumps (P-gp and MRP) under changes in environmental conditions (e.g., inflammation, viral infection, and presence of exogenous growth factors (e.g., cytokines)). The direct accessibility to both apical and basolateral membranes o f such cultures would facilitate the elucidation of drug and ion transport pathways, detailed permeation mechanisms for targeting and enhancement of drug transport. Therefore, it sets the stage for using these in vitro cell culture models to evaluate the expression and functions of efflux pumps (P-gp and MRP) in healthy and disease states. Scheme 2 summarizes the studies performed in this Specific Aim. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Specific Aim #1. Cell culture development and characterization Normal state Disease state Air-interfaced culture (IV. 1.1) AIC developm ent (1.1.1 -2) • Medium com position • Air-interface condition Adenoviral infected culture (IV. 1.2) Virus infectivity at various age (1.2.1) AIC characterization (1.1.1 -3) • Bioelectric properties • M orphology & Histology (TEM, PAS staining) Virus proliferation (1.2.2) * Morphological change o f cells (1.2.3) Ion transport (1.1.4) • Cl' channel • K+ channel • Na+/K+ ATPase • N a7K 72C l' cotransporter Passive diffusion (1.1.5) • mannitol • FDs • P-blockers Active transport (1.1.5) • Nucleoside transporter: Uridine • Dipeptide transporter: L-Camosine, Valacyclovir Bioelectrical properties (1.2.4) Passive diffusion (1.2.4) • mannitol Ion transport (1.2.4) • Baseline Cl' flux • cAM P-activation o f Cl" secretion Scheme 2. Outline of the studies performed in Specific Aim #1 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Specific Aim #2. To evaluate expression, localization, function, and modulation of P-glycoprotein in normal and Ad5-infected states of cultured rabbit conjunctival epithelial cells Based on the preliminary finding of that asymmetric transport of cyclosporin A (CsA), a P-gp substrate, in the rabbit conjunctiva (Saha et al., 1998), the main emphasis o f the experiments under this Specific Aim sought to (a) determine the expression and subcellular localization o f P-gp in the rabbit conjunctival epithelial cells, and (b) evaluate the effect of P-gp on conjunctival epithelial transport o f a lipophilic, ocular hypotensive P-adrenergic antagonist (P- blocker), propranolol. Propranolol was chosen as a model drug for probing the role o f P-gp in conjunctival P-blocker transport, since it is a prototype o f P- blockers that are used for treating glaucoma. There is evidence for transport of p- blockers being mediated by efflux pumps, including that the uptake of p-blockers (acebutolol, celiprolol, nadolol, and timolol) by multidrug-resistant leukemic cell line variant K562/ADM was 37-63% lower than that by drug-sensitive K562 cells (Terao et al., 1996). P-gp expression and function in Ad5-infected culture was further investigated in order to study the possible regulation o f P-gp in a disease state of the conjunctiva. It was anticipated that the experiments preformed in this Specific Aim will lead to dissection of the P-gp’s function in healthy and disease states and lay the platform o f research strategy for studying other efflux pumps 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (e.g., MRP) in Specific Aim #3. Scheme 3 summarizes the studies performed in this Specific Aim. Specific Aim #2. Studies of expression and functions of P-gp P-gp efflux pump in the conjunctiva (IV.2) _____________________________ i ______________________________ Expression of P-gp in the RCECs: Western blot analysis (2.1) 4 Localized P-gp: immunostaining (2.1) P-gp function on drug transport (2.2) • Propanolol transport: directionality • Metoprolol • Betaxolol P-gp function on apical uptake of propranolol uptake (2.2) • Effect of P-gp substrates; • Effect of 4E3 mAh; • Effect of metabolite inhibitors; • Effect of other P-blockers; • Effect of organic cations; 4 The effect of Ad5 infection on the expression and function o f P-gp (2.3) • Protein expression: Western blot • Function: propranolol uptake Scheme 3. Outline of the studies performed in Specific Aim #2 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Specific Aim #3. To evaluate the expression, localization, function, and modulation of MRP in normal and Ad5-infected states of cultured rabbit conjunctival epithelial cells Following investigation of P-gp (Specific Aim #2) in the in vitro cell culture models of normal and Ad5-infected RCEC (Specific Aim #1), the expression, localization, and normal pathophysiological function of MRP were studied in this Aim. The physiological roles o f MRP in various tissues include the transport of GSH conjugates, provision of cellular defenses against oxidative stress and, perhaps, also the maintenance of an intracellular redox potential. In addition, MRP is responsible for the transport of leukotriene C4 (LTC4 ), an inflammatory mediator. In this study, we attempted to evaluate the existence of MRP by Western blot analysis and RT-PCR and its localization in rabbit conjunctival epithelial cells by using immunostaining, and to investigate the effect o f MRP on drug transport by using a model MRP substrate, fluorescein. We determined whether the transport o f ophthalmic anion drugs such as diclofenac, ofloxacin, cromolyn, cidofovir, flurbiprofen, and prednisolone, is mediated by MRP. Furthermore, we evaluated the possible patho-physiological role of MRP in the transport of inflammatory mediator LTC4 in the conjunctiva. The expression of MRP and its transport function of LTC4 were determined in the cultured RCEC with Ad5 infection or following the treatment of cytokines (Interleukin 1 (IL-1), 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IL-6, and tumor necrosis factor alpha (TNF-a)). Scheme 4 summarizes the studies in this Specific Aim. Specific Aim #3. Studies of expression and functions of MRP MRP efflux pump in the conjunctiva (IV.3) Localization o f MRP: immunostaining (3.2) Molecular evidence o f MRP (3.1) • RT-PCR Contribution o f P-gp and MRP to drug efflux (3.5) Vincristine uptake: • In the presence o f P-gp substrates • In the presence o f MRP substrates Pathophysiological indication o f MRP in the conjunctiva (3.6): The transport o f LTC4, an inflammatory mediator • Directionality • Inhibition with probenecid MRP regulation and function in a disease state (3.7) • Adenoviral infection on MRP expression and function • The effect o f cytokines on MRP expression and function • The effect o f cytokine antibodies on recovery o f cytokine-induced MRP function MRP function on drug transport (3.3&4) • Fluorescein uptake: • Time course o f transport • Directional uptake • Inhibition: probenecid, indomethacin • Energy dependency: NaN3 • Efflux mechanism association with GSH • Ocular anion drugs: • Fluorescein inhibition uptake Scheme 4. Outline of the studies performed in Specific Aim #3 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. III. MATERIALS AND METHODS 1. Materials 1.1. Animal Male Dutch-belted pigmented rabbits, weighing 2.0 - 2.5 kg, were obtained from American Rabbitry (Los Angeles, CA), and all animals were handled in accordance with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23). 1.2. Development of rabbit conjunctival epithelial cell culture models 1.2.1. Air-interfaced culture o f rabbit conjunctival epithelial cells Fluorescein isothiocyanate (FITC)-labeled dextrans (FD) with average molecular weights o f 4,400 (FD4), 9,400 (FD10), 21,200 (FD20), and 71,200 (FD70), a series of p-adrenergic blockers (atenolol, timolol maleate, metoprolol tartrate, and propranolol hydrochloride), amiloride, bumetanide, ouabain, N- phenylathranilic acid (NPAA), barium chloride, uridine, acyclovir, glycine- sarcosine, and L-camosine were all purchased from Sigma Chemical Co. (St. Louis, MO). Sotalol was obtained from Bristol-Myers (Evansville, IN). [3H] Propranolol (specific activity, 19.3 Ci/mmol) and [3H] betaxolol (specific activity, 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51 Ci/mmol) were purchased from Amersham Corporation (Arlington Heights, IL). [3H] Metoprolol (specific activity, 1.05 Ci/mmol) was a gift from Astra Hassle (Sweden). [1 4C]-Mannitol (56 mCi/mmol), [3H] L-camosine (5 Ci/mmol) and [5,6-3H] uridine (42.7 Ci/mmol) were purchased from Moravek Biochemical (Brea, CA). L-Valacyclovir was a gift o f Dr. Philip Smith o f SmithKline Beecham Pharmaceuticals (Collegeville, PA). [3H]VACV (1 mCi/ml, specific activity, 2.7 Ci/mmol) was kindly provided by Dr. Patrik J. Sinko (Rutgers University, NJ). Cell culture reagents and supplies were purchased from Life Technologies (Grand Island, NY). PC-1 culture medium was purchased from Bio Whittaker (Walkersville, MD). Transwell filters (6.5 mm diameter, 0.4 pm pore size), were obtained from Costar (Cambridge, MA). 1.2.2. Adenovirally-infected culture o f rabbit conjunctival epithelial cells Ad5 McEwen strain was provided by Dr. Melvin D. Trousdale (University of Southern California, CA). Ad5 antiserum was purchased from ATCC (Rockville, MD). A549 cells were derived from a human lung carcinoma (CCL- 185; American Type Culture Collection, Rockville, MD). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-rabbit IgG was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). 3 6C1 was purchased from Amersham Co. (Arlington Heights, IL). 8-Bromoadenosine-3’,5’- cyclic monophosphate (8 Br-cAMP) was purchased from Sigma Chemical Co. (St. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Louis, MO). Eagles’s minimum essential medium (EMEM) was purchased from Life Technologies (Grand Island, NY). Agar was purchased from Sigma Chemical Co. (St. Louis, MO). 1.3. Evaluation o f expression, localization, function, and modulation o f P-gp -5 [ H] Propranolol (specific activity, 19.3 Ci/mmol) was purchased from Amersham Corporation (Arlington Heights, IL). [3H] Metoprolol (specific activity, 1.05 Ci/mmol) and unlabeled metoprolol were a gift from Astra Hassle -5 (Molndal, Sweden). [ H] Betaxolol (specific activity, 51 Ci/mmol) was purchased from Amersham Corporation (Arlington Heights, IL). Unlabeled propranolol, alprenolol, atenolol, progesterone, verapamil, rhodaminel23, and 2,4- dinitrophenol (2,4-DNP), tetraethylammonium (TEA) bromide, and guanidine were purchased from Sigma Chemical Co. (St. Louis, MO). CsA was obtained from Sandoz Pharmaceuticals (Basel, Switzerland). C219 and 4E3 murine monoclonal antibodies (mAh) were purchased from Signet (Bedham, MA). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG was obtained from ICN Pharmaceuticals, Inc. (Aurora, OH). Peroxidase-conjugated AffiniPure donkey anti-mouse IgG was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4. Evaluation o f expression, localization, function, and modulation o f MRP [ H]-Leukotriene C4 (specific activity, 115 Ci/mmol) was purchased from NEN Life Science Products, Inc (Boston, MA). [14C]-D-Mannitol (specific activity, 56 Ci/mmol), [1 4C]-lactate (specific activity, 152 mCi/mmol), and [G-3H] vincristine sulphate (specific activity, 5.5 Ci/mmol) were purchased from Amersham Corporation (Arlington Heights, IL). [3H]-Succinate (specific activity, 40 Ci/mmol) was purchased from Moravek Biochemicals, Inc (Brea, CA). Fluorescein sodium, indomethacin, probenecid, sodium azide, buthionine sulfoximine (BSO), diclofenac, ofloxacin, flubiprofen, cromolyn, prednisolone, lactate, succinate, and GSH were purchased from Sigma Chemical Co. (St. Louis, MO). Cidofovir was a gift from Gilead Sciences (Foster City, CA). M RPrl mAb was purchased from Kamiya Biomedical Company (Seattle, WA). FITC- conjugated donkey anti-rat IgG and peroxidase-conjugated AffmiPure donkey anti-rat IgG were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). IL-1, IL-6 , TNF-a, anti-mouse IL- 6 and anti-mouse TNF-a monoclonal antibodies were purchased from PharMingen (San Diego, CA). Anti mouse IFN-a and anti-mouse IFN-P polyclonal antibodies were purchased from CalBiochem (San Diego, CA). Quantikine human TNF-a immunoassay Kit was purchased from R&D Systems (Minneapolis, MN). Predicta IL- 6 ELISA Kit was kindly provided by Dr. Mmsy at Genetech (South San Francisco, CA). 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. Methods 2.1. Development o f rabbit conjunctival epithelial cell culture models 2.1.1. Air-interfaced cell culture o f rabbit conjunctival epithelial cells 2.1.1a. Air-interfaced cell culture protocol Rabbit conjunctival epithelial cells (RCEC) were harvested using a protocol modified from that developed by Saha et al. (Saha et al., 1996b). Briefly, following excision, the conjunctiva was washed in ice-cold Ca2 + /Mg2+ - free Hanks’ balanced salt solution and treated with 0.2% protease (type XIV) for 60 min at 37 °C in 95% air/5% CO2 to dissociate the cells. The isolated cells were treated with S-MEM containing 10% FBS and 1 mg/ml deoxyribonuclease (DNAase I) to stop protease reaction. The cell pellet was washed, centrifuged at lOOx g for 10 min at room temperature and filtered through a 40 pm cell strainer. The final cell pellet was re-suspended in Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (DMEM/F12) medium supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin , 0.5% gentamicin (10 mg/ml), 0.4% fungizone (2 mg/ml), 2 mM L-glutamine, 1% ITS+ (insulin 6.5 pg/ml, transferrin 6.5 pg/ml, selenious acid 6.5 ng/ml, BSA 1.25 mg/ml, and linoleic acid 5.35 mg/ml), 30 pg/ml bovine pituitary extract (BPE), 1 pM hydrocortisone, and 1 ng/ml epidermal growth factor (EGF). These cells were seeded at a density of 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2xl0 6 cells/cm2 on Transwell inserts pre-coated with collagen, and cultured in 5% CO2 and 95% air at 37°C. From day 2 onward, the growth medium was changed to PC-1 growth medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 0.5% gentamicin (10 mg/ml), and 0.4% fungizone (2 mg/ml). Cells were then switched to an air-interface (i.e., nominally fluid-free on the apical surface of the cell layers on day 4 onward, unless otherwise indicated. 2.1.1b. Transmission electron microscopy Confluent RCEC layers on day 7 post-seeding were examined by transmission electron microscopy (TEM). The cultures were washed three times with PBS at pH 7.4 and fixed in 2.5% glutaraldehyde in PBS at 4 °C for 2 hr. Fixed cells were rinsed in chilled PBS five times, and further fixed in 1.5% OSO4 in PBS at 4 °C overnight. Osmicated cells were rinsed twice in PBS, further rinsed three times with distilled water and stained with 1.5% uranyl acetate in 50% ethyl alcohol for 30 min. Stained cells were dehydraded in graded ethyl alcohol of 70%, 85%, 95%, and 100% for 10 min, respectively. The cells were then embedded with Spurr resin and sectioned for TEM with a JEOL 100C (peabody, MA) at 80 keV. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.1.1c. Periodic Acid Schiff (PAS) staining RCEC were fixed for PAS staining to estimate the proportion o f secretory cell population on day 0 and day 6 post-seeding. Day 0 cells in suspension were fixed with 3.7% formaldehyde in PBS, incubated with 0.5% periodic acid for 10 min, and then with a Schiff reagent for 15 min. The cells were subsequently rinsed with sodium carbonate working solution for 5 min and Light Green SF Yellowish for 10 sec, and finally dehydrated in absolute alcohol and toluene (Saha et al., 1996b). Day 6 cell layers were incubated with 0.5% tyrosine-EDTA for 15 min at 37 °C to detach cells from the Transwell filter, and the resultant cell suspension was fixed and stained in the same manner as for day 0 cells. The percentage o f positively stained cells in the entire population was estimated under light microscope accordingly. Day 6 cell layers were also directly stained on Transwell filter in the same way and examined under light microscopy. 2.1.1 d. Evaluation of ion transport properties Confluent cell layers (TEER ^ 1 kQ.cm ) were equilibrated in a pH 7.4 bicarbonate Ringer’s solution (BRS) containing 1.8 mM CaCL, 5.6 mM KC1, 0.8 mM M gS04, 0.8 mM NaH2 P 0 4, 116 mM NaCl, 25 mM N aH C 03, 15 mM HEPES, and 5.5 mM D-glucose (37 °C) for 30 min. Ion transport inhibitors were then added separately to either the apical or basolateral fluid. These inhibitors were: basolateral 100 pM ouabain, a Na+,K+-ATPase inhibitor; basolateral 10 pM 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bumetanide, a Na+ /K+ /2C1' cotransport blocker; basolateral 2 mM barium chloride, a potassium channel blocker; apical 300 pM NPAA, a chloride channel blocker; and apical 10 pM amiloride, aN a+ channel blocker. Potential difference (PD) and transepithelial electrical resistance (TEER) were measured at pre determined time points throughout the experiment by EVOM (World Precision Instruments, Sarasota, FL). Equivalent short circuit current (Ieq) was calculated by Ohm’s law, i.e., Ieq = PD/TEER. 2.l.le . Solute transport studies Confluent RCEC layers grown on Transwell inserts were washed once with BRS buffer. After equilibration for 30 min, transport studies were initiated by adding the dosing solution to the apical compartment. The final concentrations used were: 1 mg/ml for FITC, FD4, FD10, FD20, and FD70; 1 mM for atenolol and sotalol; 100 pM for timolol; and 10 pM and 1 pCi/ml for metoprolol, propranolol and betaxolol. In the case o f sotalol, atenolol, metoprolol, timolol, propranolol and betaxolol, their transport in freshly excised conjunctiva was also measured. We have previously reported the detailed procedure for preparing the excised rabbit conjunctiva for transport studies in the modified Ussing chamber (Kompella et al., 1993). Briefly, rabbits were euthanized with an injection of 85 mg/kg sodium pentobarbital solution into a marginal ear vein, and the entire eye ball was removed from the orbit. After trimming, the excised conjunctiva was mounted in 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the tissue adapter with a circular aperture of 1.0 cm2, which was then placed in a modified Ussing chamber. The bathing solutions (6 ml each) were bubbled with 95% air/5% CO2 to maintain the pH at 7.4 and to provide adequate agitation. The Ussing chamber assembly was maintained at 36 ± 1 °C with a circulating water bath. At predetermined times for up to 240 min, a 200 pi aliquot was removed from the basolateral side and was replaced with same volume o f fresh BRS. For FITC-dextran experiments, the fluorescence intensity of the samples was measured in a spectrofluorometer F-2000 (Hitachi, Tokyo, Japan) at an excitation wavelength of 490 nm and an emission wavelength of 515 nm. In the case of atenolol, sotalol and timolol, 200 pi was sampled from the receiver compartment, mixed with 200 pi o f acetonitrile to precipitate the proteins, and centrifuged at 3,000x g for 10 min. The supernatant was evaporated and re-dissolved in 200 pi water containing an appropriate internal standard. One hundred pi o f these reconstituted samples was then injected into an HPLC equipped with a reverse phase C l8 Microsorb column (4.6 x 250 mm, particle size 5 pm, Rainin, Woburn, MA). The HPLC conditions for each compound are listed in Table 1-3. For metoprolol, propranolol, and betaxolol, the samples were mixed with 5 ml of EconoSafe scintillation cocktail (Research Products International Corp., Mount Prospect, IL) for assay of radioactivity in a liquid scintillation spectrometer (Beckman, Fullerton, CA). 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.1.1 f. Uridine transport Uridine transport with pH 7.4 and 300 mOsm/kg BRS solution across cultured cell layers was measured by dosing a solution containing 1 pCi/ml [3 H] uridine and 10 pM unlabelled uridine into either apical or basolateral chambers. Apical-to-basolateral transport was also studied at 4 °C. 2.1.1 g. L-Camosine uptake All the uptake experiments were performed in a humidified atmosphere of 5% CO2 and 95% air at 37°C. An inwardly directed proton gradient (apical pH 6.0, basolateral pH 7.4) from the apical fluid to the cell interior was imposed (Basu et al., 1998). Prior to each experiment, the cell layers were washed with BRS and preequilibrated for 30 min. Uptake was initiated by spiking the apical or basolateral fluid with 20 pCi/ml [3 H]L-camosine and an appropriate amount of unlabeled L-camosine. After 10 min, uptake was terminated by suctioning off the dosing solution and washing the cell layers with ice-cold BRS (pH 7.4). The cell layers were then solubilized in 0.5 ml of 0.5% Triton X-100 solution. Twenty microliters of the cell lysate were taken for protein assay by the method of Bradford (Bradford, 1976) with bovine serum albumin as a standard. The rest of the cell lysate sample was mixed with 5 ml of EconoSafe scintillation cocktail for measurement o f radioactivity in a liquid scintillation counter. Drug uptake was 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expressed as amount of drug accumulated per mg of cellular protein over the duration o f measurement. L-Camosine uptake at 10 pM was preformed in the presence o f 10 mM Gly-Sar and 1 mM valacyclovir. 2.1.lh. L-Valacyclovir uptake Confluent cell layers whose transepithelial electrical resistance around 1 kQ.cm were used in this study. After equilibrating the cells in a pH 7.4 bicarbonate Ringer’s solution at 37 °C for 30 min, the optimal pH for uptake was determined by adding [3 H]VACV (10 pM and 3 pCi/ml) in the donor chamber (apical) at pH 5.5, 6.0, 7.4, or 8.0. The inhibition studies o f VACV transport and uptake were performed under optimal pH. Concentration dependent uptake was performed with 1 pCi/ml [3 H]VACV and 1,10, 50, 100, 500, 1000, 2000, and 10,000 pM non-radiolabelled VACV respectively. The transport o f VACV at 10 pM (1 pCi/ml) was performed. The radioactivity was measured in a scintillation counter, and cellular protein was estimated by a dye-binding protein assay. 2.1.1i. Data analysis Data were presented as mean ± s.e.m. Flux was derived from a plot of the steady-state slope of a plot of the cumulative amount appearing in the receiver 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fluid vs. time. The Papp of unidirectional fluxes for solutes was estimated by normalizing the flux dQ/dt (mol/sec), against the nominal surface area (A = 0.33 cm ) and initial solute concentration in the donor fluid Co (mol/ml), or Papp = (dQ/dt)/(AxC0). The FD transport data was subjected to equivalent pore analysis. The permeability-to-diffusion coefficient ratio at 37 °C o f a test solute with a Stokes- Einstein radius (re) for a single homogeneous population of equivalent pores (with a radius of rp) is shown as follows (Horibe et al., 1997): Papp/D = (Ap/dx)(l - p)2(l - 2.104p + 2.809p3 + 0.948p5 - 1.372p6 ) where Ap is the total pore area, dx is the mean thickness of the barrier, and p = re/rp. The diffusivity o f FD was estimated from the relationship: D(MW)1 /3 = constant, where the constant was calculated using bovine serum albumin as a reference (MW = 67,000 daltons, D = 0.88 x 10‘6 cm2 /sec) (Mathias et a l, 1996). From the experimental Papp’s and the calculated Stokes-Einstein radii of molecules, the equivalent pore radius (rp) and the ratio (Ap/dx) between total pore area and pore length were estimated on the basis of a nonlinear curve fitting software TableCurve (Jandel Scintific Inc., San Rafael, CA), assuming a dx o f 10 pm for the conjunctival epithelial cell layers (Saha et al., 1996b). The Papp value of (3-blockers was plotted against the logarithm o f the n- octanol.water partition coefficient (logP), and fitted to the sigmodial model as defined by the following equation using TableCurve (Saha et al., 1996a): 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Papp = m + n/(l + exp((o-logP)/q)) where m is the minimal Papp observed, n is the maximal Papp observed, o is the half maximal log P (where Papp = (m+n)/2), and q is the slope o f the linear increase in Papp vs. log P. One-way analysis of variance (ANOVA) was used to determine the significant difference among group means, p < 0.05 was considered as statistically significant. 2.1.2. Adenovirus-infected culture o f rabbit conjunctival epithelial cells 2.1.2a. Viral infectivity To determine the Ad5 infectivity to conjunctival epithelial cells at various culture age, freshly isolated RCEC were inoculated with Ad5 at a multiplicity of infection (MOI) of 10 for 3 hr, followed by washing and then seeding onto Transwell filters. In addition, RCEC plated onto Transwell filters were inoculated 3 hr with Ad5 at MOI of 10 from apical chamber for three hours at 37 °C on day 1, 2, 3, 4, 5, or 11. Then, the inoculum was removed, and the cell layers were rinsed three times with medium, after which regular culture medium was added and the cells were incubated at 37 °C. Virus titer was determined by plaque assay 24 hours post-inoculation. Briefly, post-inoculated cells were lysed by three freeze-thaw cycles and diluted serially (1:10) for four dilutions. Each dilution (0.1 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ml/ml) was then inoculated onto A549 monolayers in duplicate wells o f a 24-well plate. After 1-h adsorption at 37 °C in 5% CO2 incubator, inoculum was then removed, followed by overlaying the cells by 0.5 ml Eagles’s minimum essential medium (EMEM) with 10% FBS and 0.4% agar gel. After solidification o f the agar, 0.5 ml of MEM with 10% FBS was added into each well. After 7 days of culture at 37 °C and 5% CO2 incubator, A549 cells were fixed with 95% methanol and stained with 1% crystal violet. Virus titers were calculated according to the plaque count and expressed as plaque-forming units per milliliter (pfu/ml) (Yang etal., 1998). In the meantime, immunofluorescence staining was determined by using rabbit anti-Ad5 serum. RCEC grown on Transwell filters were washed with phosphate-buffered saline (PBS) and fixed with 95% methanol in PBS for 15 min. PBS supplemented with 1% BSA was used to block nonspecific binding site for 1 hour. The cells were incubated with rabbit anti-Ad5 serum (1 TOO dilution), followed by reaction with FITC-conjugated donkey anti-rabbit antibody (1:100), all at room temperature. After washing again with PBS, the cells-on-filter were cut out and mounted in the coverslip with Vectashield®. Presence o f Ad5 in the cells was assessed by using fluorescence microscopy at an excitation wavelength of 488 nm (Zeiss, Germany). 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.1.2b. Viral growth Viral yields were determined by plaque assay at 0, 24, 48, and 72 hours post-inoculation as shown in 2.1.2a (Yang et al., 1998). 2.1.2c. Morphological changes of RCEC RCEC grown on Transwell filters were washed with PBS and fixed with 95% methanol in PBS for 15 min. The morphology of cells was examined by light microscopy daily (Nikon Diaphot 300, Japan) up to 12 days post-inoculation. 2.1.2d. Bioelectrical parameters and chloride flux Bioelectrical parameters in Ad5-inoculated cells were monitored daily with EVOM voltagemeter. RCEC grown for 4 days were inoculated with Ad5 at MOI of 10 on the apical side for 3 hr, followed by washing with medium and then incubated at 37 °C incubator. Two days post-inoculation, cells were used in chloride flux study. Cell layers were first equilibrated in a pH 7.4 BRS for 30 min. Unidirectional C f fluxes in the absence and presence of 1 mM 8Br-cAMP were measured under open circuit condition in apical-to-basolateral (a-to-b) or basolateral-to-apical (b- to-a) direction using 36C1 (0.1 pCi/ml) as a tracer. The receiver solution was sampled at pre-designated time-intervals, and the same amount o f fresh BRS was replenished immediately. The samples were mixed with 5 ml o f EconoSafe scintillation cocktail (Research Products International Corp., Mount Prospect, IL) 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for assay o f radioactivity in a liquid scintillation spectrometer (Beckman, Fullerton, CA). The cumulative Cl appearing in the receiver fluid was plotted as t 'y a function o f time. The steady-state flux o f Cl" (J, pEq/h/cm ) was estimated from i / - the linear slope o f the cumulative Cl vs time and known bathing volumes and specific activity o f Cl. Net Cl" flux was determined from the difference between unidirectional a-to-b and b-to-a fluxes. 2.2. Evaluation of expression, localization, function, and modulation o f P-gp 2.2.1. Western blot analysis Western blot was performed with C219 mouse monoclonal antibody for P- gp. Freshly isolated and cultured RCEC were homogenized in MSEP (125 mM Mannitol, 40 mM sucrose, 1 mM EDTA-Tris, and 5 mM PIPES-Tris, pH 6.7) buffer containing protease inhibitors (83 pM antipain, 73 pM pepstatin A, and 0.1 mM leupeptin) in the ice bath. The suspension was centrifuged at 1,000 x g for 10 min and the supernatant was transferred into a new tube and centrifuged for 15 min at 367,000 x g. The pellet was resuspended in MSEP and membrane protein was measured by the DC protein assay (Bio-Rad, Hercules, CA), using bovine serum albumin as a standard. Twenty pg o f cell proteins were electrophoresed on 8 % SDS-polyacrylamide gel, and subsequently electrotransferred to a nitrocellulose membrane (Amersham, Downers Grove, IL). The sheet was incubated for 1 hour in phosphate-buffered saline (PBS) containing 1% bovine 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. serum albumin (BSA), 1% nonfat dry milk, and 0.05% Tween-20, to prevent nonspecific binding o f antibodies. Incubation with C219 (5 pg/ml) was at 4°C overnight in the same buffer. Immunoreactivity was visualized with peroxidase- conjugated donkey anti-mouse immunoglobulins (1:20,000). Immunoblot procedure based on the enhanced chemiluminescence method (ECL) was performed by exposing the protein side of the nitrocellulose membrane to the ECL detection reagent (Amersham, Downers Grove, IL) for 1 min, followed by immediate exposure to X-ray film. 2.2.2. Immunohistochemical detection of P-gp in cultured RCEC layers and excised conjunctiva Confluent cell layers on Transwell filters were directly fixed in 4% paraformaldhyde for 2 0 min at room temperature, followed by permeation by 0.5% Triton X-100 for five min. One percent bovine serum albumin (BSA) in PBS was used to block non-specific binding sites for 1 hr at room temperature. The cell layers were reacted with primary monoclonal antibody (4E3 mAb at 5 pg/ml) and FITC-conjugated rabbit anti-mouse secondary antibody (0.12 mg/ml) sequentially for 1 hr at room temperature, on either the apical or basolateral side of separate RCEC layers (Arceci et al., 1993). After washing the cells with PBS, the cell layers on the permeable support were cut out and mounted on a coverslip 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with Vectashield®. Immunofluorescence was observed using confocal microscopy at an excitation wavelength o f 488 nm (Zeiss, Germany). The excised conjunctiva was fixed with 4% paraformaldehyde and infiltrated with 30% sucrose solution. The tissue was snap frozen with Tissue- Tek® O.C.T. Compound in liquid nitrogen and cut into 8 pm sections on cryodisc and collected on slides. After permeabilizing the tissue section with 0.5% Triton X-100 in PBS for 15 min, 1% BSA was used to block non-specific binding sites for 1 hr, all at room temperature. The tissue sections were incubated with primary and secondary antibodies at room temperature using the same method described above. After washing, immunofluorescence was visualized under confocal microscopy (Evers et al., 1996). 2.2.3. (3 -Blocker transport Prior to each experiment, the confluent cell layers on Transwell inserts were washed and equilibrated with a pH 7.4 BRS (300 mOsm/kg water) for 30 min. Transport study was initiated by adding a dosing solution containing the radiolabeled and unlabeled forms of a given drug (0.3 pCi/ml [3H] propranolol and unlabeled propranolol at varying concentrations; 0.05 pCi/ml [3H] metoprolol at 0.05 pM or 0.5 pCi/ml [3H] metoprolol at 0.5 pM, or 1 pCi/ml [3H] betaxolol at 0.02 pM) to the apical or basolateral compartment. The apical and basolateral volumes were 0.2 and 0.8 ml, respectively. At predetermined times for up to 3 hr, 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 . 2 ml receiver solution was sampled from the basolateral side (for apical-to- basolateral transport) or 0 . 1 ml receiver solution from the apical side (for basolateral-to-apical transport), placed in a scintillation vial, and replaced immediately with an equal amount o f fresh BRS. The samples were mixed with 5 ml of EconoSafe scintillation cocktail (Research Products International Corp., Mount Prospect, IL) for assay of radioactivity in a liquid scintillation spectrometer (Beckman, Fullerton, CA). The time course o f accumulation of radioactivity was analyzed for steady-state flux and, in turn, apparent permeability coefficient, as described subsequently. 2.2.4. Propranolol uptake Prior to each uptake experiment, the cell layers were washed with BRS and preequilibrated for 30 min. Ten-min uptake was initiated by spiking the apical •> fluid with a dosing solution of propranolol ([ H] propranolol (0.3 pCi/ml) in 0.05 pM total propranolol). Propranolol (0.05 pM) uptake in the presence o f 100 pM P-gp inhibitors (CsA, progesterone, rhodaminel23, and verapamil; all dissolved in 0.1% dimethyl sulfoxide (DMSO)) was carried out in essentially the same manner. In the 4E3 mAb experiment, the cell layers were pre-incubated with 5 pg/ml 4E3 mAb in the apical bathing fluid only. In the 2,4-DNP experiment, 0.2 mM 2,4-DNP was added to both apical and basolateral bathing fluids in glucose- free BRS for 1 hr before dosing. In those experiments involving (3-blockers 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (hydrophilic atenolol, moderately lipophilic metoprolol, and highly lipophilic alprenolol) or organic cation transporter substrates (TEA and guanidine), 100 pM of each compound was present in the final donor solution. 2.2.5. Effect o f culture conditions and Ad5 infection on P-gp expression and function To evaluate the effect o f air-interfaced and liquid-covered conditions, and Ad5 infection on the expression and function o f P-gp, RCEC were cultured under air-interfaced, liquid covered conditions or with Ad5 apical inoculation at MOI of 10 on day 4 post-seeding (1.2.2.). Membrane protein of RCEC was prepared on day 6 post-seeding (2 days post-inoculation), and Western blot was conducted as mentioned above. Ten-minute apical uptake o f propranolol at 0.05 pM was conducted on day 6 post-seeding ( 2 days post-inoculation). 2.2.6. Data analysis The steady-state flux was estimated from the slope of the linear portion of a plot o f a cumulative amount of drug (radioactivity) appearing in the receiver fluid as a function o f time. The apparent permeability coefficient (Papp) of unidirectional fluxes for solutes was estimated by normalizing the flux, dQ/dt (mol/sec), against the nominal surface area (A = 0.33 cm2 ) and initial solute concentration in the donor fluid, Co (mol/ml), or Papp = (dQ/dt)/(AxCo). The 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. kinetic parameters for propranolol transport in cultured RCEC were estimated by fitting the flux against donor propranolol concentration using a nonlinear least- square curve fitting program (Table Curve 2D, Jandel Scientific Inc., San Rafael, CA). The assumption is that propranolol transport is comprised of two components: influx via simple diffusion and efflux (that is saturable) via P-gp: Jab = Kd[C] - Jmax[C]/(Km + [C]) Jba = K d[ C ] + J max[C ]/(K m + [C]) Jnet = Jba “ Jab = 2 Jm ax[C]/(Km + [C]) where Jab is the unidirectional flux in the apical-to-basolateral direction and Jba is that in the opposite direction, Jn et is the net propranolol flux in the basolateral-to- apical direction, K d is the non-saturable (i.e., passive diffusion) permeation rate, Jm ax is the maximum saturable flux, K m is the corresponding Michaelis-Menten constant, and [C] is the initial donor propranolol concentration. Two-tailed Student’s t-test for unpaired data or one-way analysis of variance was used to determine the significance of difference between means of more than two data groups. Post hoc comparisons were made using Tukey’s multiple comparison test to contrast statistical significance among group (> 3) means. P < 0.05 was considered as statistically significant. 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3. Evaluation o f expression, localization, function, and modulation o f MRP 2.3.1. Reverse transcription-polymerase chain reaction Total RNA extracted from freshly isolated rabbit conjunctival epithelial cells and cultured Caco-2 cells by Trizol ® Reagent (Gibco BRL, Grand Island, NY) was used for RT-PCR. First strand cDNA was synthesized with oligo(dT) primer and Superscript IIR T (Gibco BRL, Grand Island, NY). For the PCR step, Three paired restricted primers were designed from the highly conserved amino acid sequences identified by multiple sequence alignment o f human MRP1 and mouse m rpl, which were to amplify a 652-base pair (bp) (the sense primer corresponded to human MRP1 cDNA residues 889-915, 5’-GCT ACC GCC AGC CCC TGG-3’; the antisense primer corresponded to residues 1522-1539, 5’-GGG GCT GAC CAG ATC ATG-3’); a 538 bp (the sense primer corresponded to human MRP1 cDNA residues 1786-1803, 5’-CAT CAG GCA GGA GGA GCT- 3’; the antisense primer corresponded to residues 2307-2323, 5’-GGC CAC GGA GCC CTT GA-3’), and a 351 bp (the sense primer corresponded to human MRP1 cDNA residues 4267-4284, 5’-CAT CGC CAA GAT CGG CCT-3’; the antisense primer corresponded to residues 4601-4617, 5’-GTC CGG ATG GTG GAC TG- 3’) products. PCR was performed in a thermal cycler (Perkin-Elmer, Norwalk, CT) set to the following conditions: 94 °C for 4 min, 1 cycle; 94 °C for 1 min, 55 °C for 2 min, 72 °C for 3 min, 30 cycles; 72 °C for 10 min, 1 cycle. The PCR products by using Primer 652 were electrophoresed at 80 mV through a 1.5% 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. agarose gel, and size-selected DNA fragments were extracted and subcloned into the pGEM®-T Easy Vector System (Promega, Madison, WI) and sent for sequencing at Genemed Synthesis, Inc. (South San Francisco, CA) Primer 652; sense888-915, antisense 1522-1539 Human CCTGTCGAGGATCACCTTCTGGTGGATCACAGGGTTGATTGTCCGGGGCTACCGCCAGCC 900 Mouse CCTTTCCAGGATTACTTTCTGGTGGATTACAGGGATGATGGTGCACGGCTACCGCCAGCC 704 Human CCJGGAGGGCAGTGACCTCTGGTCCTTAAACAAGGAGGACACGTCGGAACAAGTCGTGCC960 Mouse CCJGGAGAGCAGTGACCTCTGGTCATTGAATAAGGAGGACACATCAGAAGAAGTGGTACC 764 H u m a n CATGGACTTGGCCACGTACATTAACATGATCTGGTCAGCCCCCCTGCAAGTCATCCTTGC 1557 M o u s e CATGGACTTGGCCACGTACATTAACATGATCTGGTCAGCCCCTCTGCAAGTCATCGTAGC 1364 Primer 538: sense1786-1803. antisense2307-2323 H u m a n GCTAAAGCTTTATGCCTGGGAGCTGGCATTCAAGGACAAGGTGCTGGCCATCAGGCAGGA 1797 M o u s e CCTCAAGCTGTACGCCTGGGAGCTGGCCTTCCAGGACAAAGTCATGAGCATCAGGCAGGA 1604 Human GGAGCTGAAGGTGCTGAAGAAGTCTGCCTACCTGTCAGCCGTGGGCACCTTCACCTGGGT 1857 Mouse GGAGCTCAAGGTGCTGAAGAAATCTGCCTACCTGGCAGCTGTAGGCACATTCACGTGGGT 1664 H u m a n GATGGACAAAGTGGAGGGGCACGTGGCTATCAAGGGCTCCGTGGCCTATGTGCCACAGCA 2337 M o u s e GATGGACAAGGTGGAGGGACATGTGACTCTCAAGGGCTCCGTGGCCTACGTGCCCCAGCA 2141 Primer 351: sense4267-4284. antisense4601-4617 Human CGATGGCATCAACATCGCCAAGATCGGCCTGCACGACCTCGGCTTCAAGATCACCATCAT 4314 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M o u s e TGATGGGGTCAACATCGCCAAGATCGGCCTGCACAACCTGCGCTTCAAGATCACCATCAT 4109 Human GGATGAGGCCACGGCAGCCGTGGACCTOGAAACGGACGACCTCATCCAGTCCACCATCCG 4614 Mouse GGACGAGGCTACCGCAGCTGTGGACCTAGAGACAGATAACCTTATCCAGTCCACCATCCG 4409 Human GACACAGTTCGAGGACTGCACCGTCCTCACCATCGCCCACCGGCTCAACACCATCATGGA 4674 Mouse GACGCAGTTTGAAGACTGTACTGTGCTCACGATTGCTCATCGGCTTAACACCATAATGGA 4469 2.3.2. Immunohistochemical detection o f MRP in cultured RCEC and excised conjunctiva The excised conjunctiva was fixed with 4% paraformaldehyde and infiltrated with 30% sucrose solution. The tissue was then frozen in liquid nitrogen, cut into 8 pm sections on cryodisc, and collected on “superfrost plus” charged slides. After tissue section was permeabilized with 0.5% Triton X-100 in PBS for 15 min, one percent of bovine serum albumin (BSA) was used to block non-specific binding sites for 1 hour at room temperature. The section was reacted with primary antibody (M RPrl, 2 pg/ml) and FITC-conjugated donkey anti-rat IgG (1:50) sequentially for 1 hour each at room temperature. After washing, the immunofluorescence staining was visualized under confocal microscopy (Zeiss, Germany) (Evers et al., 1996). The confluent RCEC grown on the filter were directly fixed, permeablized, blocked, and incubated with primary and secondary antibodies essentially the same way as the treatment for the tissue. Then, the cells with the filter was cut out, mounted on the slide, and viewed under confocal 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. microscopy. In Ad5-infected culture, co-staining o f MRP and Ad5 antigen was preformed in the presence of rabbit anti-Ad5 serum (1:100 dilution) and MRPrl (2 pg/ml), and then rhodamine-conjugated donkey anti-rabbit IgG (1:100) and FITC-conjugated donkey anti-rat IgG (1:50). 2.3.3. Fluorescein transport and uptake in the presence of various reagents To evaluate MRP function, fluorescein transport at 100 pM was conducted by dosing the drug from either apical or basolateral side. Fluorescein uptake at 100 pM was performed by dosing the drug from either apical or basolateral side for predetermined time points. To examine whether the fluorescein uptake is mediated by MRP, one-hour uptake o f fluorescein in the presence of prebenecid at various concentrations (10,100, and 1000 pM) or indomethacin at 10 pM was performed. To determine whether the transport o f ocular drugs is mediated by MRP, uptake of fluorescein at 100 pM was preformed in the absence or presence of ocular organic anion and steroid drugs, including cidofovir, diclofenac, ofloxacin, flubiprofen, cromolyn, and prednisolone, all at 100 and 1000 pM. After the pre incubation o f cells in glucose-free BRS containing 10 mM sodium azide and 5 mM mannitol for 30 min, fluorescein uptake was determined in order to evaluate the ATP-dependent transport process. Since one-hour uptake gave sufficient detection by spectrofluorometer, one-hour time period was chosen in later-on fluorecein uptake study. One-hour uptake was terminated by washing the cell 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. layers successively three times with 100 ml ice-cold BRS each. The washed cell layers were solubilized in 1 ml of 0.5% Triton X-100 solution and transferred to 4.5 ml cuvettes. Twenty til o f the cell lysate was taken for protein assay using the method o f Bradford (Bradford, 1976), with bovine serum albumin as a standard. Drug uptake was expressed as amount of drug accumulated per mg of cellular protein over the duration o f measurement. The fluorescence intensity of the samples was measured in a spectrofluorometer F-2000 (Hitachi, Tokyo, Japan) at an excitation wavelength o f 488 nm and an emission wavelength o f 510 nm. To examine the effect o f GSH on MRP-mediated fluorescein transport, we depleted intracellular glutathione concentration by BSO. Cells were treated with 100 pM BSO for various time, and intracellular non-protein thiol (largely GSH) was measured with 5,5’-dithiobis(-2-nirobenzoic acid) (Ellman’s reagent; Boehringer Mannheim) in the supernatant of trichloride acid (TCA) precipitated cells (Feller et al., 1995). One-hour fluorescein uptake was evaluated for the cells with 18 hr treatment o f 100 pM BSO since cellular GSH level reduced 90% after 18 hr treatment. To examine the presence o f active uptake processes for organic anions, five-min apical uptake of [14C] L-lactate at 5 pM (0.76 pCi/ml) was preformed in the presence of ImM non-radiolabeled lactate. In addition, five-min uptake of [2,3- H] succinic acid at 5 pM (1 pCi/ml) was preformed in the presence of 1 mM 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fluorescein in order to examine whether they share the same transport mechanisms. 2.3.4. Vincristine uptake To understand the relative contribution o f P-gp and MRP to total drug efflux in the conjunctiva, one-hour uptake of 200 nM [3H]vincrinstine, a known substrate of both efflux pumps, was studied under various conditions. Both apical and basolateral compartments o f the cell layers were dosed with 200 nM 1 • • • [ HJvincrinstine and one of other agent including P-gp substrates: CsA (10 pM), quinidine (100 pM), and rhodamine (100 pM), MRP substrates: indomethacin (10 pM) and probenecid (100 pM), organic cations: TEA (100 pM) and guinidine (100 pM), or organic anions: lactic acid (100 pM), nicotinic acid (100 pM), and cidofovir (100 pM). For BSO treatment group, BSO at 100 pM was used to treat the cells overnight (18 hr) before uptake experiment. 2.3.5. LTC4 uptake and transport One-hour uptake of LTC4 was performed by adding a dosing solution containing [3H] LTC4 (1.15 pCi/ml, 10 nM) to the basolateral compartment. The washing steps and protein assay was essentially the same as in fluorescein uptake experiment. The cell lysates were mixed with 5 ml of EconoSafe scintillation 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cocktail (Research Products International Corp., Mount Prospect, IL) for assay o f radioactivity in a liquid scintillation spectrometer (Beckman, Fullerton, CA). Transport study was initiated by adding a dosing solution containing the -3 radiolabeled [ H] LTC4 (1.15 pCi/ml, 10 nM) in the absence or presence of 1 mM probenecid or [14C] mannitol (1 pCi/ml, 18 pM) to the apical or basolateral compartment. In fluorescein transport experiments, the sample was collected the same way as LTC4 , but put into 4.5 ml cuvettes, and then added BRS up to 1 ml. Fluorescence intensity of the samples was measured in a spectrofluorometer F- 2000 at an excitation wavelength of 488 nm and an emission wavelength o f 510 nm (Beckman, Fullerton, CA). 2.3.6. The effect of Ad5 infection and cytokine treatment on MRP expression and function On day 4 post-seeding, rabbit conjunctival epithelial cells were inoculated with Ad5 at a multiplicity o f infection o f 10 from apical chamber for three hours at 37 °C. Then, the inoculum was removed, and the cell layers were rinsed three times with medium, after which growth medium was added and the cells were incubated at 37 °C and 5% CO2 incubator (Yang et al., 1998). Other sets of RCEC were treated with IL-1 (10 ng/ml), IL- 6 (100 U/ml), TNF-a (100 U/ml), or the combination of these three cytokines. Forty-eight hours after Ad5 inoculation or cytokine treatment, membrane protein of RCEC was prepared for Western blot 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. analysis o f MRP. In parallel, LTC4 uptake at 10 nM was preformed in other sets cells with the same treatments in order to study the function of MRP. Western blot was performed with the M RPrl, a rat monoclonal antibody for MRP (Flens et al., 1996). Cells from small intestinal epithelial cells, kidney cortex, freshly isolated-, cultured-, Ad5-infected-, and cytokine- treated RCEC were homogenized individually, in MSEP (125 mM Mannitol, 40 mM sucrose, 1 mM EDTA-Tris, and 5 mM PIPES-Tris, pH 6.7) buffer containing protease inhibitors (83 pM antipain, 73 pM pepstatin A, and 0.1 mM leupeptin) in the ice bath. Membrane preparation was the same as previously mentioned (2.2.1). Twenty pg o f cell proteins were electrophoresed on 8 % SDS-polyacrylamide gel, and subsequently electrotransferred to a nitrocellulose membrane (Amersham, Downers Grove, IL). The sheet was incubated for 1 hour in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), 1% nonfat dry milk, and 0.05% Tween-20, to prevent nonspecific binding of antibodies. Incubation with M RPrl (2 pg/ml) was at 4°C overnight in the same buffer. Immunoreactivity was visualized with peroxidase-conjugated donkey anti-rat immunoglobulins (1:20,000). Immunoblot procedure based on the enhanced chemiluminescence method (ECL) was performed by exposing the protein side of the nitrocellulose membrane to the ECL detection reagent (Amersham, Downers Grove, IL) for 1 min, followed by immediate exposure to X-ray film. The band density was measured by a program NIH image 1.52. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.7. Effect o f anti-cytokine antibodies on blocking MRP-mediated LTC4 uptake Cultured RCEC grown on the Transwell filters were inoculated with Ad5 (MOI of 10) as reported previously (2.1.2a). After Ad5 inoculation, cells were washed and fresh media with various anti-cytokine antibodies were added. Different combinations o f the five anti-cytokine antibodies were used to treat the cells. Total nine experimental groups were in the test, including (1) mock-infected cells (2) Ad-5-infected control; (3) cells treated with anti-IL6 mAb; (4) cells treated with anti-TNF-a mAb; (5) cells treated with anti-IFN-a Ab; (6 ) cells treated with anti-IFN-(3 Ab; (7) cells treated with anti-IL6 Ab and anti-TNF-a Ab; (8 ) cells treated with anti-IFN-a Ab and anti-IFN-(3 Ab; (9) cells treated with anti- IL- 6 mAb, anti-TNF-a mAb, anti-IFN-a Ab, and anti-IFN-p Ab. Antibody concentrations used were as the following: anti-mouse-IL- 6 mAb, 2 pg/ml; anti- mouse-TNF-a mAb, 10 pg/ml; anti-mouse-IFN-a polyclonal Ab, 100 U/ml; and anti-mouse-IFN-P polyclonal Ab, 100 U/ml. Fresh media with corresponding anti-cytokine antibodies were used for daily media change. LTC4 uptake at 10 nM was performed 48 hrs after Ad5 inoculation and cytokine treatments. To evaluate the neutralizing ability of anti-mouse TNF-a antibody on TNF-a-stimulated MRP-mediated LTC4 efflux, the cultured RCEC on day 4 post- seeding were treated with both 10 U/ml of mouse TNF-a and 10 pg/ml o f anti- 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mouse TNF-a antibody. After 48 hr treatment, the uptake of LTC4 at 10 nM was evaluated. 2.3.8. ELISA for quantitation o f cytokine secretion in cultured cells To evaluate cytokine secretion in normal and Ad5-infected cultures, human IL- 6 ELISA Kit was used to determine IL - 6 concentration in the supernatant o f normal and Ad5-infected cell cultures. In brief, the supernatant collected daily from day 0 to day 5 post-inoculation were stored in -7 0 °C. After thawing, 1 0 0 pi of each sample was added to the microtiter plate and incubated for 30 min at 37 °C. Test wells were washed extensively followed by addition of 100 pi biotinylated antibody for the next 30-min incubation. Avidin-peroxidase (100 pi) was added for 15 min at 37 °C to attach to biotin. At last, working substrate reagent (100 pi) was added. The absorbency o f each well was read at 450 nm. A standard curve was constructed to quantitate IL- 6 concentrations in the controls and samples. Human TNF-a ELISA Kit purchased from R&D Systems (Minneapolis, MN) was used to determine TNF-a concentration in the supernatant of normal and Ad5-infected culture. In brief, 200 pi o f supernatant collected daily post inoculation was added to a microtiter plate for 2-hr incubation, after 50 pi assay dilute was added at room temperature. Test wells were washed extensively and then 200 pi TNF-a conjugate was added into the well for 1-hr incubation. After 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. washing, 2 0 0 jal substrate solution (hydrogen peroxide and tetramethylbenzidine) was added for 20 min incubation. The absorbance of each well was read at 450 nm. The amount of TNF-a in the controls and samples was then quantitated by using standard TNF-a in the Kit. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV. RESULTS 1. Development of rabbit conjunctival epithelial cell culture models 1.1. Air-interfaced culture o f rabbit conjunctival epithelial cells 1.1.1. The role of media composition in cell polarity Approximately 15-20 million conjunctival epithelial cells were obtained from each animal, with a viability > 90% as assessed by trypan blue exclusion assay. In PC-1 medium, conjunctival epithelial cells formed confluent monolayers within 3 days, but were not able to form tight junctions and they subsequently detached from the substrata. It indicated that PC-1 might not be a good medium for cell attachment in early stages o f the culture. Thereafter, Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (DMEM/F12) base medium with antibiotics and various nutrients was tested. The cells formed tight conjunctions in DMEM/F12 medium supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin, 0.5% gentamicin (10 mg/ml), 0.4% fungizone (2 mg/ml), 2 mM L- glutamine, 1% ITS+ (insulin 6.5 pg/ml, transferrin 6.5 pg/ml, selenious acid 6.5 ng/ml, BSA 1.25 mg/ml, and linoleic acid 5.35 mg/ml), 30 fig/ml bovine pituitary extract (BPE), 1 pM hydrocortisone, and 1 ng/ml EGF. Among the components tested, EGF was found to be a critical factor for cell proliferation and differentiation. Cell cultured in the DMEM/F12 medium supplemented with EGF 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of 1 ng/ml exhibited the best bioelectrical parameters with a peak TEER o f ~1.5 kQ.cm 2 and PD o f 6 mV (Fig. 2-1). In addition, a seeding density of 1.2 x 106 /cm2 was found to be the optimum for obtaining a functional epithelial barrier (Data not shown). However, the cells developed tight junction after 12 days in DMEM/F12 medium. To shorten culture time, the DMEM/F12 medium was replaced with PC-1 which promotes cell proliferation and differentiation after the cell had attached on the substrata on day 2. Significant improvement in shortening culture time was observed, with tight junction formation occurring on day 5-8. Electrophysiological parameters composing a peak TEER of 1.5 ± 0.1 kQ.cm2, PD of 13.1 ± 0.7 mV and a Ieq of 8.9 ± 0.6 pA/cm 2 (Fig. 2-2 and Table 2-1) were obtained. Thereafter, the RCEC were cultured using this combination medium recipe of DMEM/F12 and PC-1. 1.1.2. The effect o f air-interface condition on cell polarity When air-interface conditions were instituted from day 4 o f culture, a peak TEER o f 1.06 ± 0.06 kQ.cm 2 and PD o f 17.0 ± 0.5 mV were observed (Fig. 2-2 and Table 2-1). In contrast, when air-interface conditions were imposed on day 2 or 3, a lower peak TEER of 0.68 ± 0.03 and 0.67 ± 0.03 kQ.cm 2 and a lower PD of 7.6 ± 0.6 and 11.0 ± 0.2 mV, respectively, were found. Therefore, we used air- interfaced cultures (AIC) starting on day 4 for the remaining studies. Compared with LCC, the air-interface day 4 culture developed a higher Ieq (181%) and PD 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (130%) but a lower TEER (73%), mimicking the native tissue better (Kompella et a l, 1993). Table 2-1. Peak transepithelial electrical resistance (TEER), potential difference (PD), and equivalent short circuit current (Ieq) o f the excised tissue, liquid covered culture, and air-interfaced culture. Conditions TEER (kQ.cm2 ) PD (mV) Isc or Ieq (pA/cm ) Excised tissue3 1.3 ±0.1 17.7 ±0.8 14.5 ±0.7 Liquid-covered cultureb 1.9 ±0.2 14.2 ± 1.6 8.0 ± 0.4 Liquid-covered culture0 1.5 ± 0.1 13.1 ±0.7 8.9 ± 0.6 Air-interfaced culture0 1.1 ±0.1 17.0 ±0.5 16.1 ± 0.4 3 From Ref. (Kompella et al., 1993). b From Ref. (Saha et al., 1996b). c From this study. Mean ± s.e.m. (n = 12). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.5 E o E £ o 0.5 DC 1 1 1 LU I- - 0.5 Time (day) Q o. - • — 1 ng/ml | -■— 5 ng/ml j i -0— 0 ng/ml j 8 6 4 2 0 2 - • — 1 ng/ml H i — 5 ng/ml -O— 0 ng/ml Time (day) 6.0 4.0 CM « — 1 ng/ml ■ — 5 ng/ml O— 0 ng/ml E o < 3. C (D 0.0 - 2.0 Time (day) Fig. 2-1. The effect of epidermal growth factor (EGF) on the primary culture of rabbit conjunctival epithelial cells. Each data point represents mean ± s.e.m. for n = 4. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.0 -i t M E o d j* a: U J w 0.5 0.0 3 6 9 12 15 Time (day) 20.0 15.0 > E 10.0 o Q . 5.0 0.0 9 12 15 3 6 Time (day) 30.0 20.0 C T 10.0 0.0 15 3 6 9 12 Time (day) Fig. 2-2. Mean transepithelial electrical resistance (TEER), potential difference (PD), and equivalent short circuit current (Ieq) of cultures grown on Transwell filter in LCC ( • ) and AIC (AIC day 2 (A), 3 (□), or 4 (O) onward). Key: Values observed in the excised tissue (Saha, 1996a). Each data point represents mean ± s.e.m. for n = 12. 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.3. Transmission electron microscopy (TEM) and Periodic Acid Schiff (PAS) Staining o f cultured RCEC Under the TEM, microvilli were observed in both the air-interfaced and liquid-covered cultures. Compared to liquid-covered culture, air-interfaced culture developed more cell layers (4-5 cell layers in AIC vs 2-3 cell layers in LCC (Fig. 2-3). A sporadic PAS positive staining pattern (3-4%) was seen in AIC cells, suggesting the presence o f mucin-secreting goblet cells (Fig. 2-4A). In contrast, PAS positive cells in LCC cells were fewer (~1%) (Fig. 2-4B). I l l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-3. Transmission electron microscopy of air-interfaced (A) and liquid-covered (B) cultures of rabbit conjunctival epithelial cells on day- 6 culture after seeding. Bar equals 10 pm. 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-4. Periodic Acid Schiff staining o f day 6 AIC (panel A) and LCC cells (panel B). The black dots correspond to PAS positive cells (magnification 90X). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.4. Ion transport properties Fig. 2-5 summarizes the effect of ion transport inhibitors on Ieq as percent inhibition compared to control Ieq o f AIC cells on day 6-8. Ieq was reduced by 66% by 0.1 mM ouabain (basolateral), 35% by 0.01 mM bumetanide (basolateral), 63% by 0.3 mM NPAA (apical), and 46% by 2 mM BaCh (basolateral). In contrast, 0.01 mM amiloride (apical) did not affect the Ieq. 1.1.5. Solute transport and uptake FDs transport The cumulative appearance of fluorescent label in the receiver fluid was linear with time for all FDs for the duration of the transport experiment. The Papp for FDs decreased sharply from 3.26 x 10'8 cm/s for FD4 to 0.68 x 10'8 cm/s for FD70 (Table 2-2 and Fig. 2-6). The relationship shown in Fig. 2-6 is consistent with a single pore population with an equivalent pore radius of about 8.0 nm and a pore density of 5 x 107 pores per cm2. [3-Blocker transport The PD and TEER of both the cell layers and excised tissue remained relatively constant throughout these experiments. The lag time was brief (<10 min) for all solutes studied. Table 2-3 summarizes the HPLC conditions for assaying sotalol, atenolol, and timolol. Papp o f (3-blockers appeared to follow an 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. apparent sigmoidal relationship (Table 2-4 and Fig. 2-7) with log P, with a half- maximal log P o f 1.2 for AIC and 1.0 for the excised tissue. Uridine transport The Papp of uridine transport at 10 pM in AIC yielded Papp values of 1.77 ± 0.03 xlO'5 cm/s for the apical-to-basolateral direction and 3.6 ± 0.0 x 10'7 cm/s for the basolateral-to-apical direction at 37 °C. At 4 °C, the Papp was 2.2 ± 0.2 x 10‘7 cm/s for the apical-to-basolateral direction (Fig. 2-8). L-Camosine uptake L-Camosine uptake was direction-dependent, being 4.4 times higher from the apical than from the basolateral fluid. Lowering the temperature to 4 °C and abolishing the pH gradient reduced apical L-camosine uptake 5 and 2 times, respectively. Furthermore, uptake was inhibited by 73% in the presence o f apical 10 mM glycyl sarcosine (a known substrate for the dipeptide transporter) and by 60% in the presence of 1 mM L-valacyclovir (an amino acid ester prodrug) (Fig. 2-9). 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Amiloride (a) NPAA (a) Bumetanide (b) Ouabain (b) Barium chloride (b) % Control Ieq Fig. 2-5. Influence o f pharmacological modulators of ion transport on the equivalent short circuit current (Ieq) of air-interfaced conjunctival epithelial cell layers (□) and the excised tissue (B ) (data from Kompella, 1993). Key: a, apical; b, basolateral; mean ± s.e.m. (n = 4); * and +, p < 0.05 compared to corresponding AIC and tissue controls, respectively. Tissue data for amiloride treatment are not available. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2-2. Molecular weight (MW), molecular radius, and apparent permeability coefficient (Papp) of FITC-dextrans at 1 mg/ml donor concentration in the apical- to-basolateral direction in air-interfaced cultures o f pigmented rabbit conjunctival epithelial cells and excised tissues. Solutes MW Radius (nm) Papp (xlO'8 cm /s)a AIC Excised Tissue Mannitol 182 0.43 21.78 ± 1.46 27.70 ± 4.33 6-CF 376 0.68 n.d.c 15.70 + 3.30 FITC 389 0.68 15.20 + 2.80 n.d. FD4 4,400 1.44 3.26 + 0.13 3.41+0.79 FD10 9,400 1.91 2.40 + 0.14 1.91+0.26 FD20 21,200 2.44 1.22 + 0.03 0.71 ± 0.06 FD70 71,200 3.81 0.68 ± 0.05 0.30 ± 0.02 a Mean ± s.e.m. (n = 4) bFrom Ref. (Floribe et al., 1997) c Not determined 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 tn E o 0 0 O 40000 60000 20000 80000 MW (daltons) Fig. 2-6. Relationship between the apparent permeability coefficient (Papp) and molecular weight (MW) of mannitol, FITC, FD4, FD10, FD20, and FD70 in AIC (n = 4 - 5). Key: O, AIC; • , tissue (data from Horibe et al., 1997). 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2-3. HPLC conditions for assaying (3-blockers: detection wavelength, internal standard, composition o f the mobile phase, and retention time. Drug Wavelength (nm) Internal standard Mobile phasea RTb (min) Drug RT (min) ISC Sotalol 224 Atenolol 3.5% AcN 16.2 19.1 Atenolol 224 Sotalol 3.5% AcN 19.1 16.2 Timolol 280 Metoprolol 18% AcN 14.2 17.2 a The aqueous portion of the mobile phase was 0.2% triethylamine at pH 3.0. Key: AcN, acetonitrile Retention time 0 Internal standard at 5 pg/ml 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2-4. Logarithm of n-octanol/pH 7.4 buffer partition coefficient (Log P) and apparent permeability coefficient (Papp) of p-blockers in the apical-to-basolateral direction in air-interfaced cultured pigmented rabbit conjunctival epithelial cell layers and excised tissues. Solutes Log P Papp (xl0‘ cm/s)a Air-interfaced culture Liquid-covered Culture Excised Tissue Sotalol -0.62 0.03 ± 0.01 0.02 ± 0.01 0.11 ±0.04 Atenolol 0.16 0.01 ± 0.00 0.03 ± 0.01 0.21 ±0.07 Metoprolol 1.88 1.34 ±0.05 3.09 ± 0.49 0.94 ±0.15 Timolol 1.91 0.53 ± 0.03 1.68 ±0.17 0.95 ± 0.06 Propranolol 3.21 0.98 ± 0.02 2.36 ±0.16 0.79 ± 0.08 Betaxolol 3.44 1.06 ±0.03 3.16 ±0.40 0.69 ±0.14 a Mean ± s.e.m. (n = 4-7) bFrom Ref. (Saha et al., 1996a) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. < 0 3 E ° 2 in » ■ O ^ 1 Q . Q . 1 0 Q - n -1 -4 -2 0 2 Log P Fig. 2-7. Influence o f drug lipophilicity on |3-blocker transport across AIC (O), LCC ( • ) (data from Saha et al., 1996a), and excised conjunctiva (H ). Error bars represent s.e.m. for n = 4-7. 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CM E o o E E T J £ O Q . « c re L _ h 0.5 - re c 3 1 2 3 0 Tim e (hr) Fig. 2-8. Time course of uridine transport measured with 10 pM donor concentration across the air-interfaced cultured conjunctival epithelial cells. Each data point represents mean ± s.e.m. (n = 5). Key: o, apical-to-basolaterial; • , basolateral-to-apical; A, 4°C. 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H - I * -H * ]H * _ H * H * T ---------1 ---------1 ---------1 ---------1 ---------1 - 0 20 40 60 80 100 120 % C ontrol Fig. 2-9. Ten-minute uptake o f L-camosine at 10 pM donor concentration in the presence of an inwardly directed proton gradient (apical pH = 6.0, basolateral pH = 7.4) under various conditions, including abolishing the pH gradient, pre-incubating cells at 4 °C, and in the presence of 10 mM glycyl- sarcosine or 1 mM L-valacyclovir in the donor chamber. Values are mean ± s.e.m., n = 4. Asterisks indicate a statistically significant difference from apical uptake at 37 °C. Apical Basolateral Apical pH 7.4 4°C Glycyl-Sarcosine (10 mM) L-Valacyclovir (1mM) 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L-Valacvclovir (VACV) uptake and transport in cultured RCEC p H dependency o f L-valacyclovir uptake in cultured RCEC Ten-minute apical uptake o f L-valacyclovir at 10 pM was performed in the cultured RCEC under various pH conditions (pH = 5.5, 6.0, 7.4, and 8.0). It revealed that the cells had a maximal uptake at pH 7.4, which was significantly higher than that at pH 5.5 (p < 0.05) (Fig. 2-10). Inhibition study o f L-valacyclovir uptake in cultured RCEC The apical uptake of L-valacyclovir at 10 pM was 2.8 times higher than the basolateral uptake, and was inhibited by 10 mM glycyl sarcosine (54%), a substrate for the dipeptide transporter, and unlabelled VACV (41%), but not by L- valine (83%) and acyclovir (ACV) (99%) (Fig. 2-11). Concentration dependency o f L-valacyclovir uptake in cultured RCEC VACV uptake at concentrations ranging from 1 pM to 2 mM was saturable and can be described by the Michaelis-Menten equation. The calculated Km was 469 ±178 pM and Vmax was 210 ± 27 pmol/mg protein/10 min (Fig. 2- 12). 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Directionality o f L-valacyclovir transport across cultured RCEC VACV transport at 10 pM across the cultured RCEC layers was asymmetrical, favoring the apical-to-basolateral direction by 2.4 times (Table 2- 5). Table 2-5. Permeability o f L-valacyclovir, acyclovir, and mannitol across cultured rabbit conjunctival epithelial cell layers. Values are mean ± s.e.m., n = 4-8. Papp (x 10’ cm/s) a-to-b b-to-a VACV (10 pM) 15.1 ±0.7 6.3 ± 0.2* A C V (lO pM ) 5.2 ± 0.2 nd Mannitol (18 pM) 2.2 ± 0.2 2.5 ± 0.2 *, significantly different compared to a-to-b transport (p < 0.05) nd, not determined 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 > i 8 li° T T 4 -J ° o 4 ^ Q. 4 (0 o z ' o . E 2 3 = A 0 1 o X . X X pH 5.5 pH 6.0 pH 7.4 pH 8.0 Fig. 2-10. pH effect on 10-min apical uptake of L-valacyclovir at 10 pM in cultured RCEC. Transport was preformed at pH 5.5, 6.0, 7.5, and 8.0 (apical). Values are mean ± s.e.m., n = 3. 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-11. Ten-min apical uptake of L-valacyclovir (VACV) at 10 pM donor concentration in the presence of 10 mM glycyl- sarcosine, L-valacyclovir, valine, or acyclovir in cultured RCEC. Values are mean ± s.e.m., n = 5-6. Asterisks indicate a statistically significant difference from the apical uptake. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 800 0 5 E 3 600 E a. 400 a > n s + - > a. > 200 O < > 1500 2000 500 1000 0 Concentration (pM) Fig. 2-12. Concentration dependency o f L-valacyclovir uptake in cultured RCEC at • 37 °C; O at 4 °C; — saturable component. Each point represents mean ± s.e.m., n = 4. 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2. Adenovirus type 5 (Ad5) infected conjunctival epithelial cell culture 1.2.1. Infectivity To assess the effect of culture age on the susceptibility of RCEC to Ad5 infection, subconfluent cells at the early culture ages o f 0, 1, 2, 3, 4, and 5-day post-seeding versus confluent cells at the later culture age o f 11-day post-seeding were used in the adenoviral infection studies. All the cells were grown on Transwell filters. As shown in Figs. 2-13 & 2-14, cells inoculated apically on day 0-5, but not on day 11 post-seeding, with MOI of 10, were positively stained for viral particles mainly in the nuclei, as assessed by co-staining with propidium iodide and FITC-conjugated secondary antibody. Ad5-infection in the early-aged cultures (day 0-2) adversely affected attachment o f the RCEC onto the substrata, as indicated by the retention of only a few cells on the filters on day 1 post infection. For cells infected on day 3-5 which had developed tight junction at some extent, the cells could form confluent cell layers. However, the infectivity o f these cells was low (< 5%). MOI of 50 and basolateral inoculation gave a slightly better infection (< 2-fold, data not shown) than MOI o f 10 and apical inoculation, whereas infected cell with MOI of 50 from the apical side did not have significant difference compared to that with MOI o f 10 from the apical side. 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2. Kinetics o f viral growth Ad5 growth curve (Fig. 2-15) showed that the infectious titers from media of RCEC inoculated on day 3 post-seeding, increased by about 10-fold within 24 to 72 hours post-inoculation, but the overall viral titer was low in the RCEC. 1.2.3. Morphological change o f Ad5-infected cells On day-7 post-inoculation, the cells started to develop significant morphological change. Ten days after Ad5 inoculation, the cells inoculated on day 4 post-seeding became rounded and detached from Transwell filters (Fig. 2- 16A). In contrast, cells inoculated with Ad5 on day 11 post-seeding did not show significant morphological changes (Fig. 2-16C). The control cells mock inoculated on 4th or 11th day post-seeding did not exhibit significant morphological changes (Fig. 2-16B and 2-16D). 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-13. Immunostaining for Ad5 in cultured rabbit conjunctival epithelial cells. The cells were inoculated with MOI o f 10 on day 0 (A), 1 (B), and 2 (C) post-seeding, and the immunostaining was performed 1 3 1 24 hours post-inoculation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ' iJ M lg W H P ' A 4 B Fig. 2-14. Immunostaining o f RCEC 24 hr post-inoculation. The cells were inoculated with Ad5 at MOI o f 10 on day 3 (Panel A) and day 5 post-seeding (Panel B) (Original magnification 400x). 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Virus titer x 103 pfu/ml 24 48 Post-inoculation (hr) 72 Fig. 2-15. Growth kinetics of Ad5 in the cultured rabbit conjunctival epithelial cell layers. ( • , cells inoculated on day 3; O, cells inoculated on day 11). Each data represents duplicate samples. 133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.4. The effect of Ad5 infection on ion transport of cultured RCEC Changes in bioelectric parameters Bioelectric parameters including transepithelial electric resistance (TEER) and potential difference (PD) were measured for the cells inoculated on day 4 and 11 post-seeding. The cells inoculated on day 4 post-seeding exhibited a more rapid decrease in PD and TEER 2-3 days post-inoculation than the respective controls. On day 3 post-inoculation, the TEER, PD, and Ieq o f infected cells were 27%, 20%, and 68% o f the corresponding parameters of the control. The values of electrophysiological parameters further dropped to zero on day 5 post-inoculation. In contrast, the bioelectric parameters o f cells inoculated on day 11 post-seeding were indistinguishable from those o f the control (Fig. 2-17). CT flux across cultured rabbit conjunctival epithelial cells Active Cl" transport was assessed by measuring unidirectional a-to-b and b-to-a 3 6 C1 fluxes under open circuit conditions. Under baseline conditions, a-to-b flux o f 0.44 ± 0.08 pEq/h/cm2 and b-to-a flux of 0.64 ± 0.02 pEq/h/cm2 were observed for the control RCEC (n = 4), giving a net b-to-a CT flux o f 0.20 ± 0.03 pEq/h/cm2. For the Ad5-infected cells on day 2 post-inoculation, a-to-b CT flux of 0.42 ± 0.04 pEq/h/cm2 was not significantly different from the control a-to-b flux, while the b-to-a Cl" flux of 0.51 ± 0.03 pEq/h/cm2 was significantly less than the 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. control b-to-a flux, leading to a 50% reduction in the net b-to-a Cl" flux, the value of which was 0.09 ± 0.02 pEq/h/cm2 (Fig. 2-18A). The Papp of a-to-b transport of mannitol, a paracellular marker, was 2.39 ± 0.20 x 10"7 cm/s and that of b-to-a n was 2.91 ± 0.12 xlO" cm/s, exhibiting symmetry transport. In the presence o f 1 mM 8Br-cAMP, an analog of cyclic AMP (cAMP), the a-to-b Cl" flux of 0.48 ± 0.02 pEq/h/cm2 in the non-infected RCEC was not different from the baseline value, while the b-to-a Cl' flux was stimulated by 131% to 0.84 ± 0.07 pEq/h/cm2 for the uninfected RCEC. The stimulated net Cl" flux in response to 8Br-cAMP for the non-infected RCEC was 0.36 ± 0.02 pEq/h/cm2, a 180% increase from the baseline value. In contrast, 8Br-cAMP failed to stimulate Cl" secretion in the Ad5-infected cells, the a-to-b flux was 0.43 ± 0.04 pEq/h/cm2 and the b-to-a flux was 0.58 ± 0.04 pEq/h/cm2, leading to a net b-to-a Cl" flux of 0.14 ± 0.03 pEq/h/cm2 (Fig. 2-18B). 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-16. Morphology of Ad5-inoculated and mock-inoculated (control) conjunctival epithelial cells on day 10 post-inoculation. Inoculation on day 4 post-seeding led to cell rounding and detachment (panel A), while the cells inoculated on day 11 (panel C) did not show morphological difference compared to mock- infected cells. Panels B and D were the corresponding controls (Original magnification lOOx). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 .0 L U 0.5 0.0 0 1 2 3 4 5 2.0 E o E O ' LU 0.5 UJ I- 0.0 2 3 4 5 0 1 P ost-inoculation time (day) P o s t - i n o c u l a t i o n t i m e ( d a y ) 20 > E o 1 2 3 4 5 Post-inoculation time (day) 20 > E P ost-inoculation time (day) E o < w 16 12 0 0 1 2 3 4 5 E < 16 12 8 4 0 •4 •8 P ost-inoculation time (day) P ost-inoculation time (day) Fig. 2-17. Bioelectric parameters of RCEC at various time intervals after Ad5 inoculation ( • ,B ) or mock inoculation (O, □). The RCEC were inoculated on day 4 ( • ,0) or day 11 ( 1 , □ ) post-seeding. Data are mean ± s.e.m. (n = 3). 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o 0.6 ab ba net E o JT L U X 3 o ab ba net Fig. 2-18. Unidirectional chloride flux and net flux across the Ad5-infected cell layers ) and control cell layers (□ ) in the absence (A) and presence (B) o f 8Br-cAMP. ab, apical-to-basolateral; ba, basolateral-to-apical; net = (b-to-a) - (a-to-b). 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. Evaluation of expression, localization, function, and modulation of P-gp 2.1. Existence and localization o f P-gp Existence o f P-gp As shown in Fig. 3-1, a distinct major immunoreactive band at ~ 170 kDa was evident for both the cultured and freshly isolated conjunctival epithelial cells. There exist a number of other minor bands, suggesting that the C219 mAh might recognize more than one epitope and/or signifying the presence o f truncated P-gp fractions in the conjunctival epithelial cell. The absence of any positive band in cultured conjunctival epithelial cells and freshly isolated conjunctival epithelial cells in the presence of a mouse IgG2a isotype-matched negative control antibody served as a negative control. Localization of P-gp Apical (Fig. 3-2A), but not basolateral (Fig. 3-2C), aspect o f the cultured conjunctival epithelial cell layers showed positive staining in the presence o f 4E3 mAh under confocal laser-scanning microscopy. The absence of any staining with FITC-conjugated secondary antibody alone (i.e., without 4E3 mAb pre incubation) served as a negative control (Fig. 3-2B). Immunohistochemistry of P-gp in frozen tissue sections o f rabbit conjunctiva showed positive staining o f the outermost cell layer of the epithelium, 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. weak staining in the cytoplasm of the other portions of the cell layers, and no staining in the endothelium (Fig. 3-3A). Fig. 3-3B is the corresponding phase- contrast photomicrograph. The absence of any specific staining in the tissue sections incubated with FITC-conjugated secondary antibody alone (i.e., without 4E3 mAh pre-incubation) served as a negative control (Fig. 3-3C). Fig. 3-3D shows the corresponding phase-contrast photomicrograph. 2.2. P-Blocker transport in RCEC 2.2.1. The transport o f propranolol, metoprolol, and bexaxolol The unidirectional fluxes of propranolol at varying concentrations in either the apical-to-basolateral or basolateral-to-apical direction across cultured RCEC are shown in Fig. 3-4. Basolateral-to-apical fluxes were significantly greater than apical-to-basolateral fluxes at all concentrations except 0.5 pM. The ratios of fluxes estimated from the basolateral-to-apical transport over that from the apical- to-basolateral transport were 1.9 (0.0125 pM), 2.2 (0.025 pM), 2.4 (0.05 pM), 1.8 (0.1 pM), and 1.4 (0.2 pM). The net flux, occurring in the basolateral-to-apical direction, is in concordance with the presence o f a saturable kinetic process with a Km of 71.5 ± 24.0 nM and Jm ax of 1.45 ±0.17 pmol/cm2 /hr (Fig. 3-4). In contrast, metoprolol transport at 0.05 and 0.5 pM and betaxolol transport at 0.02 pM across RCEC layers showed no directional preference (Table 3-1). 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. C219 mAb + + 170kDa) Fig. 3-1. Western blot analysis of P-glycoprotein expression in cultured conjunctival epithelial cells. The numbers to the left o f the blot indicate the positions o f molecular mass markers in kilodaltons (kD) electrophoresed in parallel. The proteins used as molecular mass standards were myosin (203 kD), P-galactosidase (118 kD), SDS-PAGE standards (Bio-Rad, Hercules, CA). Key: Lane 1 = mouse IgG2a isotype- matched negative control in cultured conjunctival epithelial cells, Lanes 2 = mouse C219 monoclonal antibody to P-gp in cultured conjunctival epithelial cells, Lane 3 = mouse C219 monoclonal antibody to P-gp in freshly isolated conjunctival epithelial cells, and Lane 4 = mouse IgG2a isotype-matched negative control in freshly isolated conjunctival epithelial cells. 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B C Fig. 3-2. Immunolocalization o f P-glycoprotein in cultured RCEC layers by confocal laser-scanning microscopy. Panels A and B are the immunofluorescence images observed with and without 4E3 mAb treatment on the apical side, respectively. Panel C is the immunofluorescence picture found with 4E3 mAb added to the basolateral fluid (original magnification 780x). Bar lengths are 10 pm. 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-3. Immunohistochemistry of P-glycoprotein on frozen tissue sections o f excised rabbit conjunctiva. Panel A: immunofluorescence with 4E3 mAb treatment, panel B: corresponding image obtained with phase contrast (original magnification 2200x); panel C: immunofluorescence without 4E3 mAb treatment, and panel D: corresponding image acquired with phase contrast (original magnification 4400x). Bar lengths are 10 pm. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3-1. Apparent permeability coefficients (Papp) of propranolol, metoprolol, and betaxolol across cultured rabbit conjunctival epithelial cells. Drug Log P a Papp (x io -5 a-to-b b cm/s) b-to-ac Propranolol (0.05 pM) 3.21 0.42 ± 0.02 1.00 ±0.07* Metoprolol (0.05 pM) 1.88 1.23 ±0.01 1.31 ±0.04 Metoprolol (0.5 pM) 1.88 1.31 ±0.07 1.41 ±0.04 Betaxolol (0.02 pM) 3.44 1.15 ±0.05 1.28 ±0.03 Data represent mean ± s.e.m. (n = 4-6); a from Ref. (Hansch et al., 1995); b apical-to-basolateral direction; c basolateral-to-apical direction; * significantly different from that in ab direction (p < 0.05). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o E o. 3 2 1 0 0 0.1 0.2 0.3 0.4 0.5 Propranolol o o E Q . X 3 20 15 10 5 0 0 0.1 0.2 0.3 0.4 0.5 Propranolol Fig. 3-4. Panel A: Net flux o f propranolol across cultured rabbit conjunctival epithelial cell layers as a function of propranolol concentration. Panel B: Unidirectional fluxes and net flux o f propranolol at 0.0125 to 0.5 pM. The data are mean ± s.e.m. (n = 4 - 6) (* p < 0.05). Key: A, Net secretory flux (Jn et = Jb a - Jab); o, Ja b (flux in the apical-to- basolateral direction); •, Jba (flux in the basolateral-to-apical direction). 145 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.2. Apical propranolol uptake in cultured conjunctival epithelial cell layers As shown in Fig. 3-5, apical uptake o f propranolol at 50 nM was significantly increased (p < 0.05) in the presence o f P-gp substrates, all at 100 pM: CsA (66%), progesterone (43%), rhodaminel23 (49%), and verapamil (52%). Apical presence of 4E3 mAb at 5 pg/ml and apical and basolateral presence o f 2,4-DNP at 0.2 mM increased the uptake of 50 nM apical propranolol by 55% and 53%, respectively. In contrast, neither the P-blockers (atenolol, metoprolol, and alprenolol) nor the organic cation transporter substrates (TEA and guanidine), all at 100 pM, affected apical 50 nM propranolol uptake (Fig. 3-5). Lowering the temperature to 4°C reduced apical propranolol uptake by 76%. 2.3. The effect o f culture conditions and Ad5 infection on P-gp expression and function As shown in Fig. 3-6, protein expression of P-gp in AIC, LCC, and Ad5- infected cells was not significantly different. Moreover, apical uptake of propranolol at 50 nM was not significantly different in these three culture systems (Fig. 3-7). 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Control DMSO Cyclosporin A Progesterone Rhodamine123 Verapamil 4E3 mAb H * 2,4-DNP Atenolol Metoprolol Alprenolol TEA Guanidine 4 degree 0 50 100 150 200 % Control Fig. 3-5. Ten-min uptake of propranolol at a donor concentration of 0.05 pM in the apical fluid in the presence of competing substrates (CsA, progesterone, rhodaminel23, and verapamil), P-blockers (atenolol, metoprolol, and alprenolol) and organic cation transporter substrates (TEA and guanidine), all at 100 pM, except for 5 pg/ml 4E3 mAb and 0.2 mM 2,4-DNP. DSMO at 0.1% was used to dissolve some lipophilic drugs. The data are mean ± s.e.m. (n = 4 - 6) (*p < 0.05). 147 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. + C219 mAb - C219 mAb -170 kD < | | | r >5° co> *; G* G ® ^ G* C ® ^ 200 kD 100 kD + C219 mAb r > C f v Pgp -► -170 kD - C219 mAb S c c a : G* V ^ 200 kD 100 kD Fig. 3-6. Western blot analysis of P-glycoprotein expression in cultured rabbit conjunctival epithelial cells under various culture conditions (AIC and LCC) and Ad5 challenge. The upper panel is the liquid-covered culture control, Ad5-infected cells was apically inoculated with Ad5 at MOI on day 4 post-seeding. Membrane protein was prepared 2 days after viral inoculation. The lower panel is the liquid-covered culture and air- interfaced culture. C219 mAb was used in Western blot, and mouse IgG2a was used as an isotype-matched negative control. 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. £ f f l iM o Q . D ) | O E a Q ) re a. 3 O o £ re i_ Q . O 12 10 8 6 4 2 0 AIC LCC Ad 5 Fig. 3-7. Ten-min uptake o f propranolol at a donor concentration of 0.05 pM in the apical fluid in the air-interfaced cutlure, liquid- covered culture and Ad5-infected culture. The RCEC were apically inoculated with Ad5 at MOI o f 10 for 3 hr on day 4 post-seeding. Propranolol uptake was performed on day 2 post-inoculation. The data are mean ± s.e.m. (n = 12) (*p < 0.05). 149 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. Evaluation of expression, localization, function, and modulation of MRP 3.1. Reverse transcription-polymerase chain reaction To evaluate MRP expression at gene level in the rabbit conjunctival epithelial cells, reverse transcription-polymerase chain reaction (RT-PCR) was performed by using restricted primers designed according to the highly conserved regions of the early, middle, and late portions o f cloned human MRP1 and mouse m rpl cDNAs. As shown in Fig. 4-1, this approach yielded products o f about 652, 538, and 351 bp, which were o f the desired size. The PCR product o f -652 bp was then subcloned and sequenced. Sequence comparison showed that the this RT-PCR product was 84% identical to human MRP in nucleotide sequence (Fig. 4-2) and 87% identical in amino acid sequence (Fig. 4-3). 150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-1. Reverse transcription-polymerase chain reaction (RT-PCR) analysis o f messenger RNA expression by genes encoding MRP in the rabbit conjunctival epithelial cells and Caco-2 cells. Lanes 1, 3, 5 are Caco-2 cells, and lanes 2, 4 ,6 are rabbit conjunctival epithelial cells. Lanes 1 and 2 are MRP specific RT-PCR product o f 652 bp, lanes 3 and 4 are MRP specific RT-PCR products o f 538 bp, and lanes 5 and 6 are MRP specific RT-PCR product o f 351 bp. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hMRP: 8 8 9 c t a c c q c c a q c c c c t g g a g g g c a g t g a c c t c t g g t c c t t a a a c a a g g a g g a c a c g t c g g a I I I 1 1 1 I I 1 1 1 1 1 1 1 I I I I I I M i l l M i l l I I I I I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 rM RP: 1 c t a c c g c c a g c c c c t g g c g a g c a a c g a c c t g t g g t c t c t g a a c a a g g a g g a c a c g t c g g a hMRP: 94 9 a c a a g t c g t g c c t g t t t t g g t a a a g a a c t g g a a g a a g g a a t g c g c c a a g a c t a g g a a g c a I I I I I I 1 1 1 1 1 1 1 I I 1 1 1 I I 1 1 1 I I 1 1 1 1 1 I I I I I 1 1 M i l l I 1 1 1 1 I I I rM RP: 6 1 g c a g g t g g t a c c t g t t t t g g t a a a g a a c t g g g a g a a g g a a t g t g c c a a a t c c a g g a g g c a hMRP : 1 0 0 9 g c c g g t g a a g g t t g t g t a c t c c t c c a a g g a t c c t g c c c a g c c g a a a g a g a g t t c c a a I I I 1 1 1 1 1 1 I 1 1 1 1 I 1 1 1 I I I I I I I I I I I I 1 1 1 1 1 1 1 1 I I I 1 1 I I 1 1 rM RP: 1 2 1 g c c c g t g a a g a t c a t g t a c t c c t c c t c c a a g g a g c c c a c c a a g c c a a a a g g g a g t t c c a a hMRP: 1 0 6 6 g g t g g a t g c g a a t g a g g a g g t g g a g g c t t t g a t c g t c a a g t c c c c a c a g a a g g a g t g g a a I I I I I I I I I I 1 1 1 1 1 1 1 1 1 1 1 M i l l 1 1 1 1 I I I I I I I I 1 1 1 1 1 1 1 1 1 1 1 rMRP: 1 8 1 g a c g g a c g t g a a c g a g g a g g c g g a g g c c t t g a t t g t c a a g t c g c c c c a g a a g g a g c g g a a hMRP: 1 1 2 6 c c c c t c t c t g t t t a a g g t g t t a t a c a a g a c c t t t g g g c c c t a c t t c c t c a t g a g c t t c t t M i l l M i l l M i l l I 1 1 1 1 1 1 1 1 1 I I 1 1 1 1 1 1 1 I I 1 1 1 1 1 1 I I 1 1 I I I I I 1 1 rM RP: 2 4 1 g c c c t c c c t g t t c a a g g t c c t g t a c a a g a c c t t c g g g c c c t a c t t c c t c a t g a g c t t c t t hMRP: 1 1 8 6 c t t c a a g g c c a t c c a c g a c c t g a t g a t g t t t t c c g g g c c g c a g a t c t t a a a g t t g c t c a t I I 1 1 I I I I 1 1 I I I I I I 1 1 I I 1 1 1 I I 1 1 1 M i l I I M i l l M i l l I I 1 1 1 1 1 rM RP: 3 0 1 c t t c a a g g c c g t g c a t g a c c t g a t g a t g t t t g c c g g c c c c g a g a t c c t a a a g c t g c t c a t hMRP: 1 2 4 6 c a a q t t c q t q a a t g a c a c q a a q q c c c c a q a c t g g c a g g g c t a c t t c t a c a c c g t g c t g c t I I I I I 1 1 1 1 1 1 1 1 1 1 I I 1 1 1 I I I 1 1 1 1 1 I I 1 1 I I I I 1 1 1 1 1 1 1 1 1 I I I 1 1 1 1 rM RP: 3 6 1 c a a c t t c g t g a a c g a c a a g a c g g c c c c g g a c t g g c a g g g g t a c t t c t a c a c g g c g c t g c t hMRP: 13 0 6 g t t t g t c a c t g c c t g c c t g c a g a c c c t c g t g c t g c a c c a g t a c t t c c a c a t c t g c t t c g t I I M M I I I 1 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 1 I I I I I I I I I I rM RP: 4 2 1 c t t c g t c a g c g c c t g c c t g c a g a c c c t g g t c c t g c a c c a g t a c t t c c a c a t c t g c t t c g t hMRP: 1 3 6 6 c a g t g g c a t g a g g a t c a a g a c c g c t g t c a t t g g g g c t g t c t a t c g g a a g g c c c t g g t g a t I I I I I M I I 1 1 1 1 1 1 1 1 1 1 1 M i l l I I I I M i l l I I M i l l 1 1 1 1 1 1 1 1 rMRP: 4 8 1 g a g c g g c a t g c g c a t c a a g a c c g c c g t c a t c g g c g c c g t c t a c c g c a a g g c g c t g g t g a t hMRP: 1 4 2 6 c a c c a a t t c a q c c a g a a a a t c c t c c a c q q t c q g g g a g a t t q t c a a c c t c a t q t c t g t g g a I I I I I I I 1 1 I I I I I I M i l l M i l l I I M i l l I I I I I I I I I I I I I I rM RP: 5 4 1 c a c g a a c t c a g c c a g g a a g t c c t c g a c g g t g g g c g a g a t c g t g a a c c t c a t g t t c t g t g a hMRP: 1 4 8 6 c g c t c a g a g g t t c a t g g a c t t g g c c a c g t a c a t t a a c a t g a t c t g g t c a g c c c c 1 5 3 9 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I rMRP: 6 0 1 c g c g c a g c g g t t c a t g g a c c t g g c c a c g t a c a t t a a c a t g a t c t g g t c a g c c c c 6 5 4 Fig. 4-2. Nucleotide sequence alignment o f a RT-PCR product targeting rabbit MRP with human MRP (NM_004996). hMRP: Human MRP; rMRP: Rabbit MRP. Identities = 554/654 (84%). 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hMRP: 2 3 3 RQPLEGSDLWSLNKEDTSEQWPVLVKNWKKECAKTRKQPVKWYSS-KDPAQPKESSKV RQPL +DLWSLNKEDTSEQWPVLVKNW+KECAK+R+QPVK++YSS K+P +PK SSK rMRP: 1 RQPLASNDLWSLNKEDTSEQWPVLVKNWEKECAKSRRQPVKIMYSSSKEPTKPKGSSKT hMRP: 2 9 2 DANEEVEALIVKSPQKEWNPSLFKVLYKTFGPYFLMSFFFKAIHDLMMFSGPQILKLLIK D NEE EALIVKSPQKE PSLFKVLYKTFGPYFLMS FFFKA+HDLMMF+GP+1LKLLI rMRP: 6 1 DVNEEAEALIVKSPQKERKPSLFKVLYKTFGPYFLMSFFFKAVHDLMMFAGPEILKLLIN hMRP: 3 5 2 FVNDTKAPDWQGYFYTVLLFVTACLQTLVLHQYFHICFVSGMRIKTAVIGAVYRKALVIT FVND APDWQGYFYT LLFV+ACLQTLVLHQYFHICFVSGMRIKTAVIGAVYRKALVIT rMRP: 1 2 1 FVNDKTAPDWQGYFYTALLFVSACLQTLVLHQYFHICFVSGMRIKTAVIGAVYRKALVIT hMRP: 4 1 2 NSARKSSTVGEIVNLMSVDAQRFMDLATYINMIWSA 4 4 7 NSARKSSTVGEIVNLM DAQRFMDLATYINMIWSA rMRP: 1 8 1 NSARKSSTVGEIVNLMFCDAQRFMDLATYINMIWSA 2 1 6 Fig. 4-3. Amino acid sequence alignment o f a RT-PCR product targeting rabbit MRP with human MRP (NP_004987). hMRP = Human MRP, rMRP = Rabbit MRP. Identities = 188/216 (87%). 153 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2. Existence and localization o f MRP Positive staining was observed primarily on the basolateral membrane (Fig. 4-4A), but not the apical membrane (Fig. 4-4B) of cultured RCEC in the presence of M RPrl mAb under confocal laser-scanning microscopy. Membrane staining pattern was not observed in the cells stained with the secondary antibody alone (Fig. 4-4C), serving as a negative control. Similarly, the apical surface of the superficial layer of rabbit conjunctiva tissue showed no staining, whereas the basolateral membrane was positively stained (Fig. 4-5 A). Without primary antibody treatment, no staining was observed in the tissue section (Fig. 4-5B). In both cultured RCEC and conjunctiva tissue, besides the predominant membrane staining pattern, limited intracellular staining was also observed. 154 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A B C Fig. 4-4. Immunolocalization o f MRP in cultured rabbit conjunctival epithelial cells. Panels A and B are the immunofluorescence images observed on the basolateral and apical membrane, respectively, with M RPrl mAb treatment by confocal laser-scanning microscopy. Panel C is the immunofluorescence picture found without M RPrl mAb, but with secondary antibody alone, (original magnification 600x). Bar lengths are 10 pm. 155 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mucosal i A B Fig. 4-5. Immunolocalization o f MRP in frozen conjunctiva sections. Panel A is the immunofluorescence image observed with M RPrl by confocal laser- scanning microscopy. Panel B is the immunofluorescence picture obtained without M RPrl mAb, but with staining o f secondary antibody alone, (original magnification 600x). Arrows point to the apical surface o f the outmost superficial cell layer. Bar lengths are 10 pm. 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.3. MRP-mediated transport and uptake o f fluorescein in cultured RCEC Papp o f a-to-b transport of fluorescein at 100 pM was 2.02 ± 0.16 xlO'7 cm/s, that of b-to-a transport was 1.41 ± 0.03 x 10'7 cm/s, indicating a preferred a- to-b transport in cultured RCEC (p < 0.05) (Table 4-1). Fluorescein uptake by the cells was time dependent, with the uptake leveling off after 1 hour. There was no significant difference in fluorescein uptake between apical and basolateral dosing (Fig. 4-6). Probenecid increased fluorescein uptake in a dose dependent manner (151% at 10 pM, 256% at 100 pM, and 290% at 1000 pM, respectively, compared to the control) (Fig. 4-7). It suggests that the inhibition o f MRP by probenecid was concentration dependent. In the presence o f 100 pM probenecid or 10 pM indomethacin, fluorescein uptake from basolateral dosing was increased by 183% (p < 0.05) and 140% (p < 0.05), respectively. Among an ocular antiviral (cidofovir), NSAIDs (diclofenac, ofloxacin, flubiprofen, and cromolyn), and a steroid anti-inflammatory drug (prednisolone), only diclofenac, ofloxacin, and flubiprofen at 1 mM increased fluorescein uptake by 103%, 129%, and 113%, respectively (Fig. 4-8), but not in low concentration o f 100 pM (data not shown). By pre-treating the cells with glucose-free BRS containing 10 mM NaN 3 for 30 min, the fluorescein uptake was increased by 133% (p < 0.05) (Fig. 4-8). To assess possible fluorescein intake mechanisms, basolateral uptake of succinate was preformed in the presence o f fluorescein. Fluorescein at 1 mM 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. significant inhibit basolateral succinate uptake at 5 pM by 91% (Table 4-3). In addition, excess non-radiolabeled lactate at 1 mM significantly inhibited 5 pM apical [1 4 C] lactate uptake by 63% (Table 4-3). Table 4-1. Apparent permeability coefficient (Papp) o f fluorescein, LTC4, and mannitol in cultured rabbit conjunctival epithelial cell layers. Solute Condition Ratio3 Papp (xlO' a-to-b cm/s) b-to-a Fluorescein (100 pM) 1.4* 2.02 ±0.16 1.41 ±0.03 LTC4 (10 nM) Control 1.4* 3.32 ± 0.44 2.33 ± 0.09 probenecid (100 pM) 1.1 2.86 ± 0.08 2.56 ± 0.08 Mannitol (18 pM) 0.9 2.18 ±0.15 2.45 ±0.15 a a-to-b/b-to-a Papp; mean ± s.e.m. (n = 3-6); * p < 0.05 between a-to-b and b-to-a. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4-2. One-hour fluorescein uptake and RCEC viability with a pretreatment of 100 pM BSO for 18 hr. Values are mean ± s.e.m., n = 3-4. Fluorescein uptake Cellular GSH Cell pmol/mg protein nmol/million viability Without treatment 55.80 ±3.22 8.97 ± 0.06 96% With BSO treatment 60.84 ±6.10 1.15 ±0.39 94% Table 4-3. Five-min basolateral uptake o f succinate in the presence o f 1 mM fluorescein and apical lactate uptake in 1 mM unlabelled lactate, respectively. Values are mean ± s.e.m., n = 3. Uptake + 1 mM + 1 mM pmol/mg protein Fluorescein lactate Succinate 0.79 ±0.19 0.15 ±0.09 (19%)* Lactate 7.12 ±0.67 3.32 ± 1.17(47%)* * p < 0.05 compared to the control. 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 0 c o o L. 80 Q. O ) E 60 o E Q. 40 a > ( Q 20 Q. D 2 3 0 1 Tim e (hr) Fig. 4-6. Time course of fluorescein uptake at 100 pM in cultured rabbit conjunctival epithelial cells. Data are mean ± s.e.m. (n =3). Keys: o, dosing from apical side; •, dosing from basolateral side. 160 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 350 300 c 0 ■ 5 250 JO 1 |2 0 0 o + 3 & §150 c o < D v P ^ 100 o (0 d ) k_ o 3 U_ Control 10 100 1000 Probenecid Cone. ( pM) Fig. 4-7. One-hour uptake of fluorescein at 100 pM from basolateral dosing in the presence o f probenecid at various concentrations. Data are mean ± s.e.m. (n = 4). 161 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n I — I * I — I * I 1 * H H I — i * i — I * I 1 * 0 100 200 300 400 F lu o re s c e in u p ta k e (% c o n tro l) Fig. 4-8. One-hour uptake of fluorescein at 100 pM from basolateral dosing in the presence of MRP inhibitors: indomethacin (10 pM) and probenecid (100 pM), organic anions (1 mM): cidofovir, ofloxacin, flurbiprofen, diclofenac and cromolyn, and steroid prednisolone, or with a pretreatment with 10 mM NaNa/glucose free medium for 30 min before uptake study. Values are mean ± s.e.m., n = 3-4. Asterisks indicate a statistically significant difference from basolateral uptake at 37 °C. Prednisolone O floxacin Flurbiprofen Diclofenac Cromolyn Cidofovir NaN3 Probenecid Indom ethacin Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.4. The effect of GSH on fluorescein uptake To evaluate whether fluorescein uptake is affected by intracellular GSH level, fluorescein uptake was investigated in GSH depletion condition. GSH concentration in the RCEC was 8.97 ± 0.06 nmol/million cells or 19.84 ± 0.02 nmol/mg protein. Overnight treatment of the RCEC with BSO at 100 pM lowered the GSH by 90% (Fig. 4-9), but cell integrity was maintained, indicated by trypan blue exclusion assay, and the fluorescein uptake was not modified (p > 0.05) (Table 4-2). 3.5. Contribution o f P-gp and MRP to vincristine uptake To understand the relative contribution of P-gp and MRP to total drug efflux, one-hour uptake o f 200 nM [3 H]vincristine, a known substrate for both efflux pumps, was studied under various conditions. Vincristine uptake was increased by adding P-gp substrates - 10 pM CsA (194%), 100 pM quinidine (154%), and 100 pM rhodamine (132%), and MRP substrates - 10 pM indomethacin (175%) and 100 pM probenecid (169%), but not by adding 100 pM organic cations - TEA (87%) and guinidine (90%), and 100 pM organic anions - lactic acid (86%), nicotinic acid (106%), or cidofovir (126%). BSO overnight treatment (18 hr) also caused significant increase in vincristine uptake (131%) (Fig. 4-10). 163 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 n 20 X </) O 15 k. i S = 10 a > O 5 0 0 4 8 12 16 20 24 Time after BSO treatment (hr) Fig. 4-9. Time course of glutathione (GSH) content in cultured rabbit conjunctival epithelial cells after treatment with 100 pM buthionine sulfoximine (BSO). Data are mean ± s.e.m. (n =3). Key: •, nmol/mg protein; o, nmol/million cells. 164 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cidofovir Nicotinic acid Lactic acid Guanidine I ----- 1 TEA I ----------- I ---- 1 BSO Probenecid I I ------1 * Indomethacin I ---- 1 * Rhodamine123 H * Quinidine I I — i * CsA 50 100 150 200 250 Vincristine uptake (% control) Fig. 4-10. One-hour uptake of 200 nM [3 H] vincristine from both apical and basolateral dosing in the presence of P-gp substrates (10 pM CsA, 100 pM quinidine, and 100 pM rhodamine), MRP inhibitors (10 pM indomethacin and 100 pM probenecid), organic cations (100 pM TEA and guinidine), or organic anions (100 pM lactic acid, 100 pM nicotinic acid, and 100 pM cidofovir). For BSO treatment group, the cells were pretreated with 100 pM BSO over night (18 hr). Values are mean ± s.e.m., n = 4-5. Asterisks indicate a statistically significant difference from control uptake at 37 °C. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.6. MRP-mediated transport and uptake o f leukotriene C4 (LTC4) in cultured RCEC Papp o f a-to-b transport of LTC4 at 10 nM across the cultured RCEC was 3.32 ± 0.44 x 10‘7 cm/s, that of b-to-a transport was 2.33 ± 0.09 x 10' 7 cm/s (p < 0.05). In the presence of 1 mM probenecid, the directionality was abolished, giving a Papp o f a-to-b transport of 2.86 ± 0.08 x 10"7 cm/s and that of b-to-a transport of 2.56 + 0.08 x 10' cm/s (p > 0.05). In contrast, the transport of mannitol, a paracellular marker, did not show directionality with Papp o f a-to-b 'j transport of 2.18 ± 0.15 x 10" cm/s and that o f b-to-a transport of 2.45 + 0.12 x 1 O ' 7 cm/s (p > 0.05) (Table 4-1). 3.7. MRP-regulation by adenoviral infection To verify that the MRP function is enhanced in inflammation, one-hour basolateral LTC4 uptake at 10 nM was conducted in normal and Ad5-infected RCEC at 37 °C. RCEC were inoculated on day 4 post-seeding and the uptake experiment was conducted 48 post-inoculation. LTC4 uptake was significantly lower (76%) in Ad5-infected cells compared to normal cells (Fig. 4-11). As shown in Fig. 4-12, MRP expression was up-regulated in Ad5-infected cells compared to non-infected cells. In addition, the Ad5-infected cell detached from the other cells. 166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LTC4 uptake c '3 o Q . U ) | o E Q . 0.012 0.008 0.004 0.000 Norm al A d5-infected Fig. 4-11. The effect of Ad5 infection on one-hour uptake of LTC4 at 10 nM from basolateral dosing. Data are mean ± sem (n = 4-6). 167 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-12. Co-localization of MRP and Ad5 in Ad5-inoculated RCEC culture by confocal laser-scanning microscopy. The upper panel is the immunofluorescence image showing the staining by FITC-conjugated donkey anti-mouse antibody and M RPrl mAb, the middle panel shows the staining by rhodamine-conjugated donkey anti-rabbit antibody and Ad5 antiserum. The lower panel shows the co-staining by two pairs of primary and secondary antibodies. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.8. Effect o f adenoviral infection and cytokines on the expression and function of MRP Western blot analysis with M RPrl monoclonal antibody revealed a distinct band at -190 kDa for freshly isolated rabbit conjunctival epithelial cells (61%), cultured RCEC (69%), adenovirus-infected RCEC (89%), as well as for the positive controls rabbit small intestinal epithelial cells (67%), kidney cortex (100%), where figures in parenthesis represent % of band intensity relative to that for the kidney (Fig. 4-13). The band density at -190 kDa for the membrane protein o f cultured RCEC was increased by pretreatment o f the cells with IL-1 at 10 ng/ml (149%), IL-6 at 100 U/ml (201%), and TNF-a at 100 U/ml (216%) compared to the control (Fig. 4-14). In the presence o f 100 pM probenecid, LTC4 uptake from basolateral dosing in cultured RCEC was increased by 44% (p < 0.05). By treating the cells with glucose-free BRS containinglO mM NaN 3, LTC4 uptake was increased by 54% (p < 0.05). LTC4 uptake was decreased by Ad5 infection (24%) (p < 0.05), by pretreatment with IL-1 (13%) (p > 0.05), IL-6 (28%) (p < 0.05), TNF-a (41%) (p < 0.05), and a combination o f three cytokines (43%) (p < 0.05) (Fig. 4-15). The MRP protein expression had a good correlation with LTC4 uptake (R2 of 0.96) (Fig. 4-16). 169 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M — 212 -M R P r l < - 122 Fig. 4-13. Western blot analysis o f MRP in membrane proteins of various cells. Upper panel was with M RPrl mAb treatment and the lower panel comprises negative controls without M RPrl mAb treatment. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■ ‘ 122 A - 212 -M RPrl 122 Fig. 4-14. Western blot analysis of MRP in membrane proteins of rabbit conjunctival epithelial cells following various cytokine treatments (IL-1 at 10 ng/ml, IL-6 at 100 U/ml, and TNF-a at 100 U/ml)). Upper panel shows the results obtained with M RPrl mAb treatment, while lower panel comprises negative controls without MRPrl mAb treatment. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 .0 2 0 o> E 0.015 o E ~ 0.010 © 8 Q. 3 o 0.005 0.000 * * X X X, * x . # Z ' ^ v - 6 ^ O ' ^ i >° G“ y, ® ' ' A " ^ f y Fig. 4-15. One-hour uptake of LTC4 at 10 nM in cultured RCEC from basolateral dosing in the presence of 100 pM probenecid, or following a pretreatment with 10 mM NaN 3/glucose free medium for 30 min, Ad5-inoculation (MOI of 10), IL-1 (10 ng/ml), IL- 6 (100 U/ml), TNF-a (100 U/ml), and the combination of IL-1, IL- 6 and TNF-a, 48 hours before uptake experiments. Values are mean ± s.e.m., n = 3-4. Asterisks indicate a statistically significant difference from basolateral uptake at 37 °C. 172 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 Control c 100 O O IL-1 IL-6 Ad5 0) a (0 TNF-a Q. 3 T fr o y = -0.32x + 133.76 R2 = 0.96 I - _l 120 100 140 160 180 200 220 MRP expression (% of Control) Fig. 4-16. The correlation o f MRP expression and one-hour basolateral uptake o f LTC4 at 10 nM in the cultured rabbit conjunctival epithelial cells (control) and following Ad5-inoculation (MOI of 10), or a pretreatment with IL-1 (10 ng/ml), IL- 6 (100 U/ml), or TNF-a (100 U/ml). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.9. Secretion o f cytokines in normal and Ad5-infected RCEC To evaluate whether Ad5 could stimulate cytokine secretion in virally- infected cells, IL - 6 Predicta and TNF-a ELISA Kits were used to determine the amounts of IL - 6 and TNF-a secreted into the extracellular media o f normal and virally-infected cultures. Fig. 4-17 shows that the IL- 6 content in Ad5-infected cells was elevated as early as 1 day after virus inoculation, rising to 2-9 times higher than that in corresponding normal cells on day 1-3 post-inoculation. Fig. 4- 18 shows that the TNF-a content in the Ad5-infected cells was consistently higher than that in the normal cells. 3.10. The effect of anti-cytokine antibody treatments on MRP-mediated LTC4 uptake To determine whether MRP overexpression and enhanced MRP-mediated LTC4 efflux in Ad5-infected cells is caused by cytokine secretion, anti-cytokine antibodies were used to block cytokine secretion in the cell cultures, thereby inhibiting MRP-overexpression by Ad5 infection. None o f the treatments (1. anti- IL6 mAb; 2. anti-TNF-a mAb; 3. anti-IFN-a polyclonal Ab; 4. anti-IFN-P polyclonal Ab; 5. anti-IL6 mAb and anti-TNF-a mAb; 6 . anti-IFN-a polyclonal Ab and anti-IFN-P polyclonal Ab; 7. anti-IL- 6 mAb, anti-TNF-a mAb, anti-IFN- a polyclonal Ab, and anti-IFN-P polyclonal Ab) were able to totally recover LTC4 uptake in Ad5-infected cells compared to that in normal cells. However, 174 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. anti-TNF-a mAb treatment groups increased LTC4 uptake about 15% in Ad5- infected cells, although those increases were not significant (4-19). TNF-a at 10 U/ml significantly decreased LTC4 uptake by 28% and anti- TNF-a mAb at 10 pg/ml was able to recover LTC4 uptake to -90% of the control (Fig. 4-20). 175 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 0 0 0 1500 E 1000 o> 500 1 1 1 1 -500 Time (days) [ ? Fig. 4-17. Time course o f IL-6 secretion in normal and virally- infected culture of rabbit conjunctival epithelial cells. Hollow bars are normal culture and solid bars are virally-infected culture. Data are mean ± sem (n = 3). 176 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 _ 100 o 80 • 60 t 40 20 0 1 2 3 4 5 Time (days) Fig. 4-18. Time course of TNF-a secretion in normal and virally- infected culture of rabbit conjunctival epithelial cell culture. Hollow bars are normal culture and solid bars are virally-infected culture. Data are mean ± sem (n = 3). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 .0 2 5 £ 0 O i_ Q. O ) | O E a 0 n s Q. 3 o I- m 0.020 0.015 0.010 0.005 T i x X 0.000 B H Fig. 4-19. One-hour LTC4 uptake from basolateral dosing in Ad5- infected cells with various treatments of anti-cytokine antibodies. Antibody concentration used was as following: anti-mouse-IL- 6 mAb, 2 pg/ml; anti-mouse-TNF-a mAb, 10 pg/ml; anti-mouse-IFN-a ployclonal Ab, 100 U/ml anti-mouse-IFN-P ployclonal Ab, 100 U/ml. Treatment groups tried were as following: (A) normal cells; (B) Ad-5 control; (C) anti-IL6 mAb; (D) anti-TNF-a mAb; (E) anti-IFN-a polyclonal Ab; (F) anti-IFN-P polyclonal Ab; (G) anti-IL6 mAb and anti-TNF-a mAb; (H) anti-IFN-a polyclonal Ab and anti-IFN-P polyclonal Ab; and (I) anti-IL- 6 mAb, anti-TNF-a mAb, anti-IFN-a polyclonal Ab, and anti-IFN-P polyclonal Ab. Data are mean ± sem (n = 4). 178 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 .0 3 0 |> 0.025 | 0.020 •f 0.015 ~ 0.010 3 2 0.005 ^ 0.000 Control TNF-alpha TNF-alpha & mAb Fig. 4-20. One-hour LTC4 uptake from basolateral dosing in RCEC with treatment with 10 U/ml of mouse TNF-a, or 10 U/ml of mouse TNF-a and 10 pg/ml of anti-mouse TNF-a antibody. After 48 hr treatment, the uptake of LTC4 at 10 nM was evaluated. Data are mean ± sem (n = 8 ). 179 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V. DISCUSSION Active transport processes play an important role in the absorption and secretion of some drugs. P-glycoprotein (P-gp) and multidrug resistance protein (MRP), originally found in cancer cells leading multidrug resistance, are widely distributed in normal tissues and significantly influence drug absorption and elimination. Viral infection has been reported to modulate the expression o f drug efflux pumps, e.g., human T-cell-leukemia-virus-I increases MRP expression in adult T- cell leukemia (ATL) cells which might be associated with MDR o f ATL cells in vivo (Ikeda et al., 1999); HIV increases P-gp expression in CD4 T cells by 2.5- fold in HIV patients (Andreana et a l, 1996). In addition, the increased expression of P-gp in NK cells and T cells may be necessary for optimal binding o f NK/T cells to the target cell or induction of cytolytic process, or both in host immune responses (Chong et al., 1993; Gupta et al., 1992). It is supported by the observation that in vitro cytolytic function of NK cells obtained from m drl knockout mice was abolished (Schinkel et al., 1997b). Functional P-gp may serve additionally to protect these important immune cells. There is now evidence that the P-gp might be involved in the transport o f cytokines out of activated lymphocytes into the surrounding medium (Johnstone et al., 2000). In MRP- deficient mrp(-/-) mice , the response to an inflammatory stimulus was impaired 180 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Wijnholds et al., 1997). This was attributed to a decreased LTC4 secretion, which was demonstrated in bone marrow derived mast cells obtained from the mice. Conjunctiva is susceptible to adenoviral infection, causing a common ocular infection disease. Conjunctiva is a promising ocular drug delivery route. On this basis, our research focused on understanding drug efflux mechanisms in cultured rabbit conjunctival epithelial cells in healthy and virally-infected states. The key questions addressed were (a) are P-gp and MRP expressed in the conjunctiva; (b) what are their subcellualr localization? (c) what are the roles drug efflux pumps play in the absorption and secretion of ophthalmic drugs, such as (3- blockers and anti-inflammatory drugs? (d) how to modulate drug efflux by P-gp and MRP? (e) are the expression and function o f efflux pumps regulated in a disease state, such as adenoviral infection, and what could be the underlying mechanisms? Three Specific Aims were designed to test the central hypothesis: P-gp and MRP may play a role in the efflux of xenobiotics and endogenous molecules in the conjunctiva in normal and adenoviral-infected states, thereby affecting ocular drug absorption and participating in host immune response. We first developed an air-interfaced conjunctival epithelial cell culture and adenovirally- infected culture as normal and disease models, respectively, in Specific Aim #1. By utilizing these two in vitro cell culture models, the expression, localization, function, and modulation o f the efflux pumps, P-gp (Specific Aim #2) and MRP 181 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Specific Aim #3) were investigated. In this section, we will discuss the findings of P-gp and MPR in the conjunctiva, as well as the relevant cell culture model development. 1. Development of rabbit conjunctival epithelial cell culture models An air-interfaced culture and the Ad5-infected culture o f RCEC were developed and validated to mimic the conjunctiva in normal and disease states, serving as in vitro cell culture models for studying drug transport mechanisms. For the air-interfaced culture, the culture conditions were optimized. The active and passive transport processes were evaluated. For the Ad5-infected culture, the infectivity o f virus, and viral infection on cellular morphology, barrier properties and bioelectrical parameters were investigated. 1.1. Air-interfaced cell culture 1.1.1. Optimization o f cell culture conditions The present study demonstrated that media composition is important for in vitro RCEC cell culture (Fig. 2-1). The DMEM/F12 base medium supplemented with insulin, transferrin, selenious acid (ITS+), and bovine pituitary extract (BPE) was recommended for other epithelial cell cultures (Ilio et al., 1995). The presence o f epidermal growth factor (EGF) at 1 to 5 and 10 ng/ml has been shown to enhance the clonal growth of rabbit meibomian gland epithelial cells in serum- 182 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. free culture (Maskin and Tseng, 1992) and to result in a concentration-dependent proliferation o f rat ventral prostatic epithelial cells (Ilio et a l, 1995). For the rabbit conjunctival epithelial cell culture, 1 ng/ml EGF was optimal for cell growth, giving rise to TEER > 1 kQ.cm2, PD > 1 0 mV and Ieq > 6 pA/cm2. In contrast, 5 ng/ml EGF resulted in cells with lower TEER, PD and Ieq value, while 10 ng/ml EGF hindered the formation o f tight junctions between cells. Although the optimal EGF concentration o f 1 ng/ml in DMEM/12 medium could facilitate tight junction formation, the length o f culture to develop significant TEER was over 10 days. Subsequently, PC-1 medium was used after 2-day culture to boost cell differentiation and shorten the peaking time of TEER to about 6-8 days. To further promote the electrophysiological characteristics of the RCEC culture, air-interfaced culture (AIC) condition more akin to those of the native tissue, as compared to the conventional liquid-covered culture (LCC) condition, was used. Air-interfaced culture o f RCEC day 4 onward exhibited peak TEER of 1.1 ±0.1 kQ.cm which was not significantly different from the excised tissue TEER value o f 1.3 ± 0.1 kQ.cm2 (Kompella et al., 1993), whereas cells in LCC had higher TEER (1.9 ± 0.2 kQ.cm2 ) (Saha et al., 1996b). Moreover, the PD (17.0 ± 0.5 mV) and Ieq (16.1 ± 0.4 pA/cm2) o f the AIC cells were similar to those of the excised tissue, while their values for the LCC cells were lower (Table 2-1, Fig. 2-2). It appears that the presence o f excess liquid on the apical surface of the cell layers may lower the capacity of active ion transport. Such a phenomenon 183 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. has been observed in tracheal epithelial cell cultures o f the rabbit (Mathias et al., 1995), guinea pig (Robison et al., 1993), cow (Kondo et al., 1993), and dog (Kondo et al., 1991). In addition, air interfaced cultures o f human bronchial epithelium (Tristram et al., 1998) and nasal turbinate tissue (Jackson et al., 1996) also exhibit better morphological and electrophysiological traits in vitro compared to immersion cultures. The conjunctival epithelial cell layers cultured on permeable filters maintained their barrier properties for approximately 4 days (days 6 through 10) (Fig. 2-2), a suitable time frame for transepithelial drug and ion transport studies. 1.1.2. Morphological characterization Both AIC and LCC showed stratified cell layers and microvilli lining on the apical surface of the cell layers. However, the LCC was flatter and had fewer cell layers, while the AIC appeared to have more differentiated cell layers (Fig. 2- 3). PAS staining pattern revealed that 3-4% of the superficial cell population were the secretory type (goblet cells). This percent goblet cell population agrees with the 3-5% value seen in freshly isolated RCEC. Goblet cells occupy 4-22% in different regions of the rabbit conjunctival epithelium (Steuhl and Roden, 1984). Interestingly, cells in liquid-covered culture exhibited a reduced secretory cell population o f approximately 1% (Fig. 2-4). Thus, the presence o f excess liquid on the apical cell surface may suppress secretory cell differentiation and mucin secretion. Although the underlying mechanism is not immediately forthcoming, 184 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. one possibility might be the improved oxygenation of, or removal o f CO2 from, the AIC cells (Kondo et al., 1991; Kondo et al., 1993). 1.1.3. Ion transport properties The overall ion transport properties o f AIC are comparable to those observed in the excised tissue (Fig. 2-5). Thus, Ieq was reduced 66% by basolateral 0.1 mM ouabain, suggesting that Na+ /K+ -ATPase is present basolaterally. The resultant inwardly directed Na+ gradient provides the electrochemical driving force for Cl' uptake by the basolaterally located Na+ /K+ /2C1’ cotransporter, whose presence is suggested by the 35% reduction in Ieq by 0.01 mM bumetanide given to basolateral fluid. The 46% inhibition of Ieq by basolaterally given Ba2 + reflects reduced K+ conductance (i.e., raising intracellular [K+ ]), thereby reducing apical Cl' efflux. NPAA effect in AIC indicates the presence o f Cl' conductive pathways in the apical membranes. The lack of an amiloride effect on Ieq suggests the absence of amiloride-sensitive Na+ channels. Thus, Cl’ enters the conjunctival epithelial cell via the basolaterally located Na+ /K+ /2C1’ cotransporter and exits the cell via the apically localized Cl' channels. A counter ion, Na+, is likely to move passively via paracellular routes between conjunctival cells, resulting in net secretion of NaCl with water following passively from the basolateral to apical side to maintain osmotic equilibrium (Kompella et al., 1993). 185 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.4. Barrier properties Drug absorption is governed by the physicochemical characteristics o f the drug, the nature o f the cell membrane a drug must pass through, and the presence of active/passive transporters expressed in the cell membranes. Mechanisms for drug transport fall into two categories: passive transport and active transport. Passive transport comprises paracellular and transcellular transport, while active transport comprises carrier-mediated transport and endo-/transcytosis. We further characterize the cell culture with respect to passive diffusion and active transport. The permeability o f the air-interfaced rabbit conjunctival epithelial cell layers to hydrophilic solutes was dependent on molecular size, where the molecular weight cut-off of the cell layers was about 20,000 daltons (Fig. 2-6). Therefore, passive diffusion of hydrophilic solutes near and above the threshold of 20,000 daltons may experience severely restricted transport when considered for topical conjunctival drug delivery. Molecules larger than FD40 may have to rely on fluid-phase and other types of endocytosis for transport (Horibe et al., 1997). In fact, the molecular weight greater than 500 has restricted absorption (Lipinski et al., 1997). Our results showed the dramatic drop of drug permeation of molecules larger than FITC (M.W. 389) across RCEC. The relationship shown in Fig. 2-6 is consistent with a single population of equivalent pores 8.0 nm in radius at a density o f 5 x 107 pores per cm2, as compared with a 5.5 nm pore size at a density o f 1.9 x 10 pores per cm in the excised conjunctiva (Honbe et al., 186 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1997). The 1.5-fold larger pore size in the cultured epithelial cells may partly explain the 2-fold larger Papp seen in FD20 and FD70 when compared with the excised tissue, although a more active endocytotic process in the cultured epithelial cells cannot be ruled out. In addition, the connective tissue and endothelial cells, present in conjunctiva but not in cultured cells, may contribute additional barriers against the entry of molecules. In the case o f P-blockers, the Papp o f the lipophilic solutes was about 3- fold lower in AIC than in LCC, but in the same range as in the excised tissue (Table 2-4), indicating that the permeability o f AIC in general better reflects that of the excised tissue than LCC. The normally secreted mucin in air-interfaced culture may provide an additional barrier against drug entry (Meaney and O'Driscoll, 1999). However, the hydrophilic p-blockers exhibited significantly higher (p < 0.05) permeability in the excised tissue than in AIC and LCC. The reason for this phenomenon is unclear. If sotalol and atenolol permeate the cultured epithelial cells via the paracellular route, their Papp should be in the same range as mannitol in AIC. Since P-blockers are organic cations at physiological pH, this difference could be caused by the participation of other transport processes such as organic cation transporters (OCT), which may exist in the tissue (Ueda et al., 2000) but might not be expressed in the cultured epithelial cells (Data not shown). Since the lipophilic beta-blockers can go through cells by simple passive diffusion, the active transport component in their overall 187 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. permeability may be minimal. In contrast, for the hydrophilic compounds, their transcellular permeability could be significantly increased by OCT. Whether OCT plays a role in the conjunctival drug absorption remains to be explored (Ueda et al., 2000). In any event, the influence o f drug lipophilicity on drug transport across AIC cell layers is described by a sigmoidial relationship (Fig. 2-7). An effective half-maximal Papp is seen at a log P value of 1.2, as compared with 1.3 for LCC and 1.0 for the excised tissue. However, lipophilicity is not the only determinant to permeability, hydrogen bond, molecular weight, and transport mechanisms should be taken into consideration. 1.1.5. Active transport properties As in the intact tissue (Basu et al., 1998; Hosoya et al., 1998), the AIC RCEC do possess the capacity for active nucleoside and dipeptide transport, as suggested by the directionality- and temperature-dependent transport /uptake of their corresponding substrates (uridine and L-camosine) (Figs. 2-8 and 2-9). Thus, uridine transport at 10 pM in AIC was 49 times higher in the apical-to-basolateral than in the opposite direction (Fig. 2-8), and was reduced 81 times upon lowering the temperature to 4°C, suggesting the possible existence o f nucleoside transporters in AIC which was shown in the intact tissue (Hosoya et al., 1998). L- Camosine uptake also showed apical preference, being 4.4 times higher than that from the basolateral side. Moreover, apical uptake was temperature- and pH- 188 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dependent. Lowering the temperature to 4 °C and abolishing the inwardly directed pH gradient by using pH 7.4 buffer reduced apical L-camosine uptake 5 and 2 times, respectively, from the corresponding control. Furthermore, uptake o f L- camosine was inhibited 73% and 60% by 10 mM glycyl sarcosine (a dipeptide substrate) and 1 mM L-valacyclovir (an amino acid ester prodrug (Han et al., 1998; Sinko and Balimane, 1998)), respectively, suggesting the possible presence of H+-coupled dipeptide transporters in AIC as shown in the intact tissue (Basu et al., 1998) (Fig. 2-9). Given that L-valacyclovir (VACV) transport is mediated by dipeptide transporters in the intestine (Ganapathy et al., 1998) and dipeptide transporters also exist in the conjunctiva (Lee et al., 1995), we tested whether the model prodrug, VACV absorption in the conjunctiva is enhanced via the dipeptide transporter. If so, it could provide a strategy to develop prodrugs o f nucleosides or nucleoside analogs for better prevention and treatment of virally-infected eye diseases. The optimal pH for uptake of VACV was 7.4 in the cultured rabbit conjunctival epithelial cells. This is consistent with the observation in hPepTl- transfected CHO cells (Guo et al., 1999). Although dipeptide transporters are known as proton-coupled transporters, H+ /peptide substrate coupling ratio was shown to depend on the net charge o f substrates (i.e., the H+ /peptide coupling ratio is 1 for zwitterionic peptides, 2 for anionic peptides, and 0 for cationic 189 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. peptides) (Temple et al., 1995). In intestine, the transport of negatively charged dipeptides was stimulated by lowering the luminal pH to 6.8, whereas increasing the luminal pH to 8.0 strongly stimulated the transport of positively charged dipeptide Phe-Lys, but inhibited that o f negatively charged dipeptide Phe-Ala (Stein et al., 1997). VACV has three pKa values 1.90, 7.47, and 9.43 (Fig. 5-2). At low pH (e.g., < 6) conditions, VACV would exist primarily as a cationic moiety, whose transport is not favored in the acidic pH. Physiological pH is favorable for the transport o f neutral and cationic species o f VACV. The decreased uptake of VACV at pH 8.0 may be explained by the instability of VACV at this pH. VACV degrades to ACV and valine, both of which are not substrates for PepTl (Guo et al., 1999). Under the optimal pH o f 7.4, apical uptake of VACV was 3-fold greater than basolateral uptake, and this apical uptake was inhibited by glycyl sarcosine and unlabeled VACV, but not by L- valine or acyclovir alone. These data indicate that VACV uptake may be mediated by dipeptide transporter in the conjunctiva. Moreover, VACV uptake studied from 1 pM to 2 mM indicates a saturable profile and fitted well with the Michaelis- Menten equation with a Km of 469 ±178 pM. This result was comparable to that reported in Caco-2 cells (Km = 0.3 mM) (Han et al., 1998), but lower than that in hPepTl/CHO cells (1.6 mM) (Guo et al., 1999) and hPepTl/XLO (5.94 mM) (Balimane et al., 1998). At 2 mM, the active transport contributed only about 30% of total uptake o f VACV in the conjunctiva. The saturation could lead to non- 190 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. directional transport o f VACV at 1 mM observed in the conjunctiva (data not shown). However, at low concentration o f 10 pM, a-to-b transport o f VACV was favorable. This provides a rationale for drug design to target specific transporters. In conclusion, air-interfaced primary cultures of rabbit conjunctival epithelial cells resemble excised tissue in healthy state and display ion and drug transport characteristics similar to those o f the excised tissue (Fig. 5-1). It may prove to be a suitable model for rapidly screening the transport properties of topical ophthalmic drug candidates as well as formulation factors influencing their transport, a future challenge in topical ocular drug delivery. Moreover, active transporter processes are present in the cultured cells, which enables this culture model to serve as an excellent model for evaluating transporters and their influence on ocular drug delivery. For the studies later on, this air-interfaced culture o f rabbit conjunctival epithelial cells was used as a culture model for the conjunctiva in a normal state. In addition, this successful culture enabled the further development o f Ad5-infected culture of RCEC. 191 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Nucleoside D ipeptide N on-polar drugs < 4 ^ - apical basolateral Fig. 5-1. Summary o f the transport processes in the air-interfaced culture of rabbit conjunctival epithelial cells. 192 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HN HO OH OH pKa 1.9 HN pka 7.5 H2N q .0 •— o Uridine L-Valacyclovir h 2 h3n+ c h 2 NH HoC C H L-Carnosine Fig. 5-2. Structures o f uridine, L-camosine, and L-valacyclovir. 193 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2. Adenovirally-infected cell culture 1.2.1. Infectivity of Ad5 to the RCEC culture Adenovirus-sensitive cell lines such as HeLa and A549 cells are commonly used to study adenovirus-induced diseases (Horwitz, 1996). Corneal and conjunctival cell cultures grown on plastic dishes have also been used to evaluate the pathogenesis of ocular diseases and the potency o f drugs against adenoviral infections (Trousdale et al., 1994; Trousdale et al., 1995b). To the best of our knowledge, there has been no report on an ocular cell culture model suitable for studying the effect o f adenoviral infection on the barrier properties, ion transport, and active transport processes in the conjunctival epithelium. Based on our previous successful primary culture of the rabbit conjunctival epithelial cells, we went on to further validate an adenovirally-infected conjunctival epithelial cell culture model that would be applied to the evaluation of effects of viral infection and inflammation on normal barrier properties, ion transport, and drug transport functions. In addition, an adenovirally-infected culture model may become a useful tool for the investigation of ocular infection mechanisms. Gordon et al. (1992) pioneered the use of the adenovirus type 5 McEwen strain, a clinical adenoviral isolate cultured from a patient with typical adenoviral keratoconjunctivitis, in the ocular animal model of adenoviral infection using the New Zealand rabbit. The same adenovirus strain was adopted in this study. 194 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The degree o f adenoviral infectivity to cultured rabbit conjunctival epithelial cells was first assessed by inoculating RCEC at different post-seeding time intervals o f the RCEC. As indicated by immunofluorescence staining (Fig. 2- 13), cells inoculated in the early stages (< 2 days) were susceptible to the adenoviral infection with MOI o f 10. However, the infection affected subsequent cell attachment and the formation o f confluent cell layers, contrary to the observations o f Hashimoto et al. (1993) that KB cell adhesion onto fibronectin was promoted at intermediate times post-infection. Cell detachment from the substrate in this case could be due to the cytopathic effects o f the viruses. The failure o f these infected cells to reach confluence rendered them unsuitable for use in studies that require intact barrier properties of the conjunctival epithelial cell layers. On the other hand, subconfluent day 3 cells (TEER <0.1 kQ.cm ) and day 5 cells (TEER of 0.2 - 0.5 kQ.cm2 ) could attain cell layer integrity after infection with Ad5 (Fig. 2-14). However, the infectivity of these cells was < 5%, much lower than those infected on days 0-2. Adenovirus was also able to replicate in these cultures (Fig. 2-15). In contrast, confluent cells on day 11 post-seeding exhibiting TEER of 1 kQ.cm2, were resistant to Ad5 infection from the apical side, suggesting that cell age affects viral infectivity. Wilson et al. (1997) observed that herpes simplex virus type-1 (HSV-1) was detected in comeal cell mechanical wounded by scraping at 12, 18, 24, 48 and 120 hr after infection, but not in intact corneas. In addition, immature neurons have been reported to be 195 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. more susceptible to infection by the arboviruses and to produce more o f the viruses when infected compared to mature neurons (Griffin, 1995). The association o f viral infectivity with incomplete formation o f tight junction in cells, as implicated in age-dependent mumps virus-induced hydrocephalus (Uno et al., 1997), could be similarly applied to the wounded cornea and immature neurons, as well as to the subconfluent conjunctival epithelial cell layers. Membrane receptors are known to play an important role in facilitating viral entry into cells. One possible reason for less adenovirus-mediated gene transfer in ciliated airway epithelia o f 6 day culture than cultured epithelia of 3 days would be the absence of substantial receptors required for efficient infection on the apical surface o f mature ocular epithelia (Zabner et al., 1996). Indeed, in polarized human retinal pigment epithelial cells (RPE), human cytomegalovirus (CMV) infection was 20-30-fold more from the apical membrane than the basolateral membrane, due to lack of receptor on the latter (Tugizov et al., 1996). However, infectivity o f day 4 RCEC was not significantly increased from basolateral inoculation, indicating that lack of the receptors on the apical membrane may not be the issue. The lack o f difference between apical and basolateral membrane susceptibility to viral entry was also seen in RPE inoculated with herpes simplex virus type 1 (Bedard et al., 1999). Viral receptors in rabbit conjunctival epithelium remain to be investigated. Since maximum infection of the ciliated airway epithelia was attained with 6-hour inoculation with MOI o f 50 (Zabner et al., 1996), the infectivity of the 196 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RCEC with Ad5 o f MOI of 50 was also evaluated. Compared with MOI of 10, a 2-fold increase in infectivity from basolateral inoculation was observed, but there was no significant difference compared to that from apical inoculation. Cell secretion o f mucin into the apical fluid may have played a role against viral entry and led to the low overall infectivity o f < 5%. Bergey et al. (1994) demonstrated that human salivary mucins may have a role in inhibiting the HIV-1 infectivity in HeLa CD4+ cell monolayers by interacting with HIV particles. Mucin has been shown by Superti et al. (1997) to markedly inhibit the adsorption o f rotavirus by, and the rotavirus antigen synthesis in, the human enterocyte-like cell line HT-29, suggesting that this protein interferes with the early phases o f rotavirus infection. Viral infectivity may also depend on the nature of the host cells. While 1-h inoculation of A549 cells with Ad5 MOI of 2 infected majority o f the cells, causing the production of 10,000-fold Ad5 titers within 24 to 72 hours (Trousdale et al., 1995a), a 3-h inoculation o f RCEC with MOI of 10 infected < 5% of the cells, causing a moderate 10-fold increase in virus titer within 24 to 72 hours. Whether this low infectivity of Ad5 in conjunctiva occurs in vivo is unknown. Despite the low infectivity of the RCEC with Ad5, it is plausible that low infectivity could still lead to pathological symptoms. Therefore, the barrier properties and ion transport of the Ad5-infected RCEC layers infected with Ad5 on day 4 post-seeding from apical inoculation, which could retain the cell integrity for 2-4 days, were further investigated. 197 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2. The effect of Ad5 infection on barrier properties and ion transport As shown in Fig. 2-17, barrier properties o f Ad5-infected RCEC layers day 3 after inoculation dropped dramatically, as indicated by a TEER value that was only 30% that of the non-infected control. Similar changes in barrier properties, indicated by the greater permeability to inulin and a striking alteration of ZO-1 pattern two-week post-inoculation, were noted in CMV-infected RPE cells (Pereira et al., 1995). The tight junction o f the blood-brain-barrier (BBB), was disrupted by HIV, creating the main route o f entry for HIV-infected monocyte into the CNS (Dallasta et al., 1999). Rotavirus infection o f Caco-2 cells also caused the disruption of tight junctions (proteins claudin-1, occludin, and ZO-1), loss of transepithelial resistance (TEER declined from 300 to 22 ohm.cm2 between 8 and 24 h after inoculation) and was accompanied by increased transepithelial permeability to macromolecules in the absence of cell death. The infection was further associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% o f control at 24 h after infection) (Dickman et al., 2000). In the case o f the Ad5-infected RCEC, they became rounded and gradually detached from the substrata starting from day 7 post-inoculation (Fig. 2-16A), unlike the mock-infected RCEC, which retained their morphological integrity even after 14 days mock-inoculation (Fig. 2-16C). This is consistent with the typical cytopathic effect o f adenovirus on fluid cultures, where large grapelike clumps of degenerated cells were observed 198 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Knipe, 1996). Swollen cells also characterized in HSV1 and HSV2-infected astrocytes (Ecob-Johnston and Whetsell, Jr., 1979). Compared to the TEER value which started dropping on day 2 post inoculation, Ieq o f the infected RCEC significantly decreased by 50% as early as 1 day post-inoculation, indicating that Ad5 may affect active ion transport earlier than tight junction disruption. As previously reported, Cl' secretion contributed about 70% of the Ieq of the conjunctiva (Kompella et al., 1993), and was associated with water secretion (Shiue et al., 1998). The lower Ieq o f Ad5- infected cells implied a malfunction of Cl' secretion and water secretion, which may provide one of the underlying mechanisms for the dry eye syndrome in adenovirally-infected patients (Trauzettel-Klosinski et al., 1980). Normal cultured RCEC exhibited a net secretory Cl' flux o f 0.20 ± 0.03 qEq/h/cm2 against an electrical gradient (apical negative) (Fig. 2-18 A), a value similar to those measured in excised rabbit conjunctiva (Kompella et al., 1993) and frog comeal epithelia (Candia, 1972), and consistent with the presence of active Cl' transport. A decreased CT flux in b-to-a direction primarily, caused 50% decreased net secretory Cl' flux o f 0.09 ± 0.02 pEq/h/cm2 in the infected RCEC. It is consistent with the drop of Ieq about 60%. It suggested that Ad5 infection inhibited active Cl’ secretion. In the presence o f 8Br-cAMP, which could increase cAMP- dependent active Cl' secretion in conjunctiva (Shiue et al., 1998) and cornea (Klyce et al., 1973), net Cl' secretion in normal RCEC was stimulated by 180% to 199 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.36 ± 0.02 pEq/h/cm2 in non-infected cells, comparable to the 111% stimulation observed in excised conjunctiva tissue (Shiue et al., 1998). In contrast, the stimulation of net C1‘ flux by 8Br-cAMP was not significant in Ad5-infected cells, implying that Ad5 could affect the cAMP-dependent Cl' secretion pathway. Since cAMP can stimulate both the C f channel and the Na+ /K+ /2C f cotransporter (Matthews et al., 1992), further investigation is required to delineate whether Ad5 influences the entry of C f into RCEC by the Na+ /K+ /2C1' cotransporter, or by the cAMP-dependent Cl' secretion pathway. Ion movement is essential for the development of cytopathic effects in virally infected cells. Voss et al. (1996) demonstrated that HIV infection caused an increased cell volume in CD4+ cells which was attributed to a HIV-induced enhancement of intracellular monovalent ion concentration. Na+ /K+ /2C1' cotransporter probably acts as the conduit for such intracellular ion accumulation since loop diuretics can block this HIV-induced cytopathic effect. Human cytomegalovirus infection was seen to increase the number of ouabain-binding sites in human fibroblasts, which may be associated with cell volume increase (Altamirano et al., 1994). In the conjunctiva, less chloride secretion accompanied by higher intracellular ion concentration in Ad5-infected RCEC might be associated with adenoviral-induced swelling of conjunctival epithelial cells. In summary, we demonstrated that Ad5 was able to infect and proliferate in non-confluent rabbit conjunctival epithelial cells grown on a permeable 200 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. support, but not confluent cultured cells. Ad5 infection led to cellular morphological changes, decreased transepithelial electrical resistance, and decreased active C f secretion in cultured rabbit conjunctival epithelial cells (Fig. 5-3). Therefore, the Ad5-infected conjunctival epithelial cell culture might be a useful in vitro disease model for studying the alteration o f cellular pathophysiology, ion and fluid movement, and barrier properties. Moreover, this model could be used to study the effect of viral infection on drug transporters, such as P-gp and MRP. 201 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. G T P ~''^ ATP GDP PHOSPHODIESTERASE Y .<------------------ CA M P > k > k > k Na+K+2C1- 0 Adenovirus Fig. 5-3. Summary of possible effects of adenovirus on ion movement and tight junction of rabbit conjunctival epithelial cells. Ad5 was able to infect and proliferate in cultured rabbit conjunctival epithelial cells, affecting cellular morphology, tight junction properties, and ion transport (CF secretion) across conjunctival epithelium. 202 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. Expression of drug efflux pumps (P-gp and MRP) Due to the important role o f P-gp and MRP in drug transport and the promising ocular delivery route via the conjunctiva, the expression and regulation of P-gp and MRP and their function on drug transport in normal and Ad5-infeced RCEC were investigated. At first, we provided biochemical and molecular evidence o f the existence of drug efflux pumps in the conjunctiva. 2.1. Protein expression o f P-gp Western blot analysis showed a distinct band for P-gp in the conjunctiva (Fig. 3-1). Despite equal loading of amounts of protein, the density of the 170kD band in freshly isolated conjunctival epithelial cells was less than that afforded by the cultured cells. A possible explanation may be dilution of epithelial cell protein, hence P-gp, by non-epithelial cell protein in the case o f the freshly isolated cells. Another possible explanation may be overexpression of P-gp in cultured cells that was induced by hormones and/or xenobiotics in the culture medium. This is indeed the case for rat hepatocyte primary cultures (Bailly et al., 1995; Stein et al., 1996) and Caco-2 cells (Schmiedlin et al., 1997). In addition, Hosoya et al. (1996) reported that P-gp expression is age- dependent in Caco-2 cells grown on Transwells. The rank order of P-gp expression level was 4 weeks « 1 week > 3 weeks > 2 weeks according to Western blot analysis, but P-gp does not become fully functional until about day 203 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 according to CsA and verapamil transport study. However, Wils et al. (1994) observed the same level o f expression o f P-gp in Caco-2 cells grown in plastic flasks from day 7 through day 22. The cell passage numbers in Hosoya's (30-50) and those in Wils' (>100) experiments were different, which indicates that P-gp expression may be associated with cell polarization and cell age. In the human ovarian carcinoma 2780 cells, CsA transport in the basolateral-to-apical direction was enhanced at the late stage o f culture (-27 days). This was due to the increased P-gp expression in the cells following full development o f cell polarity (Chu et a l, 1994). 2.2. Molecular evidence o f MRP in RCEC To verify the expression of MRP at gene level, RT-PCR was performed. Given that rabbit MRP is not cloned yet, primers designed by Miller’s group (Huai-Yun et al., 1998), which are based on a highly conserved region between human MRP and mouse mrp, were adopted in this study. These primers could successfully fish out MRP in bovine micro vessel endothelial cells (Huai-Yun et al., 1998), but not in the rabbit conjunctival epithelial cells. Less homology of rabbit MRP in this region compared to human and mouse MRP could cause the failure of detection by these primers. Consequently, three pairs o f primers were designed based on the highly conserved regions in the early, middle, and late portions of the human (5927 bp) and mouse MRP cDNA (4587 bp). By optimizing annealing temperature, duration, and cycle numbers in the RT-PCR, 204 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the expected bands with molecular sizes o f 652, 538, and 361 bp were observed in rabbit conjunctival epithelial cells (Fig. 4-1). Sequence comparison determined that the 652 bp RT-PCR product was 87% identical to human MRP in amino acid sequence (Fig. 4-3) and 84% identical in nucleotide sequence (Fig. 4-2), confirming that MRP exists in the rabbit conjunctiva. So far, no rabbit MRP is fully cloned yet and mouse MRP is 90% identical to human MRP (Cole and Deeley, 1998). 3. Localization of drug efflux pumps (P-gp and MRP) Given that specified location of membrane protein is relevant to their function in polarized cells, we went onto explore the subcellular localization of P- gp and MRP in the rabbit conjunctival epithelial cells. 3.1. Localization of P-gp Our study provides immunohistochemical evidence for the apical localization of a P-gp efflux pump in cultured RCEC layers (Fig. 3-2) and excised conjunctival epithelial tissues (Fig. 3-3). The apically localized P-gp in hepatocytes, small biliary ductules in liver, small ductules in pancreas, proximal tubules in kidney, and columnar epithelial cells in colon and small intestine in human (Thiebaut et al., 1987), along with our results, all suggest that P-gp may play the similar role in restricting apical drug entry into these tissues or limiting drug out to mucosal fluid. The localization o f P-gp may be different in epithelium 205 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and endothelium due to their anatomy difference. P-gp may be present on the basolateral side o f normal capillary endothelial cells in brain. The basolateral localization of P-gp provides the same function to prevent drug entry into the CNS from the blood circulation and contribute to blood-brain barrier (Bellamy, 1996). 3.2. Localization of MRP Positive staining of cultured RCEC with MRPrl mAh was observed primarily in the basolateral membrane and to some extent in the cytosol (Fig. 4- 4A), but not in the apical membrane (Fig. 4-4B). Similarly, strong immunoreactivity was detected in the basolateral membrane o f rabbit conjunctival epithelial tissue cryosection, but not in the apical membrane o f the superficial cell layer (Fig. 4-5A). No staining was observed in the stroma and endothelium. This pattern was comparable to the basolateral localization of MRP in kidney epithelial cells (Evers et al., 1996) and hepatocytes (Keppler et al., 1998) and sertoli cells (Borst et al., 2000). Functionally, MRP in kidney and liver probably is responsible for re-absorption of molecules, while MRP in testis is to prevent drug entry to damage the germline cells. On the other hand, MRP was found in the apical membrane o f respiratory epithelial cells, which may attribute to secretion of drugs into the lumen (Flens et al., 1996). In the intestine, epithelial cells demonstrated a supranuclear cytoplasmic staining, but the brush border and goblet cells were negative (Flens et al., 1996). Gutmann et al. (1999) demonstrated the 206 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. existence o f MRP in Caco-2 cells, but Walgren et al. (2000) were unable to locate the protein in these cells. The localization o f MRP in intestinal epithelium is unclear, presumably on basolateral side. This apparent discrepancy may result from differences in cell culture conditions. Compared to the apically localized P- gp, which serves as an efflux pump against lipophilic drugs (Yang et al., 2000), the basolaterally localized MRP would not function as an efflux pump against the apical entry o f xenobiotics into the conjunctiva. The MRP may therefore be associated with other pathophysiological functions. 4. The effect of efflux pumps on drug efflux To understand the efflux function o f apically-localized P-gp and basolaterally-localized MRP, we further evaluated transport properties of some model substrates of P-gp and MRP in RCEC. 4.1. The effect of P-gp on drug efflux 4.1.1. The effect o f P-gp on propranolol transport Probably due to the P-gp drug efflux pump, the transport of propranolol across the cultured cell layers was asymmetric, favoring secretion which was saturable (Fig. 3-4). The observed Km for conjunctival propranolol efflux was 71.5 nM. This is similar to the Km o f calcein-acetoxymethylester (50 nM) in MRP+ GLC4/ADR tumor cells (Essodaigui et al., 1998) and rhodaminel23 (34 207 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nM) in CEM/VLB100 cells (Wigler, 1996), but is much smaller than that for idarubicin (1 pM) in multidrug-resistant K562 cells (Mankhetkom et al., 1996). At low propranolol concentrations (0.0125 - 0.5 pM), the contribution o f active transport to the overall flux in the basolateral-to-apical direction ranges from 58% to 14%. Since we did observe apical localization of P-gp in the conjunctival epithelial cells (Fig. 3-2 and 3-3), uptake over a short period from the apical side may be more appropriate for determining P-gp function than the prolonged flux studies that may be fraught with non-specific effects (e.g., toxicity) caused by these inhibitors/modulators. Apical propranolol uptake in cultured conjunctival epithelial cell layers was increased by 43% to 66% in the presence of P-gp substrates and modulators - - CsA, progesterone, rhodaminel23, and verapamil, further suggesting the involvement o f P-gp in restricting apical conjunctival propranolol uptake. The uncoupling agent (2,4-DNP) increased apical uptake o f propranolol by 53%, suggesting that by blocking mitochondrial ATP synthesis, the cellular energy was depleted to lead to decreased in P-gp pump activity. 4E3 mAh, which recognizes the external portion of P-gp, increased propranolol uptake by 55%, indicating that antibody binding decreased P-gp activity (Fig. 3-5). This finding also suggests that this antibody might be useful in the attenuation of multidrug resistance involvement or targeting drugs to the specific tissue that bears P-gp. For example, using an immunotoxin composed o f MRK16 (a P-gp antibody) coupled to 208 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pseudomonas exotoxin (MRK16-PE), multidrug-resistant cells were destroyed, whereas cells not expressing the P-gp were not affected (Fitzgerald et al., 1987). It is o f interest to know whether or not the transport o f other |3-blockers is similarly affected by P-gp on the basis of competitive inhibition of propranolol uptake. Not only hydrophilic atenolol (log P = 0.16) but also moderately lipophilic metoprolol (log P = 1.88) and highly lipophilic alprenolol (log P = 3.1) and betaxolol (log P = 3.44) all failed to affect propranolol uptake (Fig. 3-4). In addition, metoprolol and betaxolol transport across cultured RCEC did not show directional dependence (Table 3-1), strongly suggesting that these particular [3- blockers may not be a P-gp substrate. Nevertheless, Karlsson et al. (1993) did find that, in addition to propranolol, atenolol and metoprolol also inhibited basolateral- to-apical secretion o f celiprolol in Caco-2 cells, where two distinct transport systems (i.e., P-gp and organic cation-H+ exchanger) were tested. Although inhibition of P-gp by atenolol and metoprolol could not be completely ruled out by these investigators, the inhibition effect on celiprolol by P-blockers may be due to organic cation-H+ exchanger. O f the p-blockers evaluated, only propranolol has a naphthalene ring (Fig. 5-4). Although P-gp substrates encompass a wide range of chemical structures and different classes o f drugs (Beck, 1991; Zamora et al., 1988), including detergents, antibiotics, antimalarials, antihypertensives, and immunosuppressives, a typical P-gp substrate is likely to be hydrophobic with two planar aromatic rings 209 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. as well as a tertiary nitrogen that confers a positive charge at physiological pH (Beck, 1991). Many efforts are directed to finding out the relationships between the structure and MDR reversal effect of these drugs (Ecker and Chiba, 1995). Structure-activity relationships (SAR), and quantitative SAR (QSAR), of different MDR reversing drugs showed a good correlation of MDR-modulating activity with the lipophilicity, hydrogen bond acceptor strength, and steric parameters of propanolamine. Additionally, ortho substitution of the side chain and the acyl moiety appears to be favorable for P-gp recognition (Chiba et al., 1998). The results of the Free-Wilson analysis show that modifications on the central aromatic ring and molar refractivity o f propafenone-type modulators generally influence pharmacological activity, indicating that both non-polar and polar interactions contribute to protein binding (Tmej et al., 1998). Wiese and Pajeva (2001) concluded that the careful selection of relevant structural and biological data processed with appropriate QSAR and especially 3D-QSAR methods is a promising approach to structure-activity studies o f MDR reversers. To date, the P- gp “pharmacophore” has not been well defined and remains a topic to be explored further. At 4 °C, the activity o f P-gp efflux pump system should be low and therefore the uptake o f propranolol by conjunctival epithelial cells would be expected to be increased, commensurately. That the uptake o f propranolol at 4 °C was only 24% of that at 37 °C suggests a non-passive component in its uptake 210 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that, in turn, serves to rate-limit its efflux mediated by P-gp. Indeed, Kurihara et al. (1987) reported that propranolol transported into the brain micro vessel endothelial cells occurred via facilitated diffusion. The organic cation transporter (OCT), which has been demonstrated in the rabbit conjunctiva (Ueda et al., 2000), did not appear to be involved. This is because neither TEA nor guanidine, which are known substrates of OCT (Urakami et al., 1998), affected propranolol uptake in conjunctival epithelial cells at 37 °C (Fig. 3-5). Besides decreased diffusion at 4 °C, another possible reason for the uptake reduction is decreased propranolol binding. In fact, Kawazu et al. (1999) observed significantly lower uptake of CsA in cultured corneal epithelial cells at 4 °C and 25 °C than that at 37 °C. They concluded CsA uptake was energy dependent and postulated the presence o f a depot for CsA binding in the form o f cyclophilin. Kurihara et al. (1987) also suggested the existence of a propranolol binding or trapping compartment in bovine brain endothelial cells. In addition, due to the high lipophilicity, propranolol may easily bind to membrane as well as receptors such as the p-adrenergic receptor (Marullo et al., 1999). At 4 °C, recycling of membrane and receptors shuts down, thereby decreasing propranolol uptake. In short, the reduction of propranolol uptake at 4°C may be contributed to by reduction of passive diffusion, binding ability, facilitated diffusion (if any), and membrane-associated uptake. Further work is required to characterize carrier- mediated conjunctival propranolol uptake. 211 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.1.2. The effect of P-gp on P-blocker absorption and bioavailability Evidence for P-gp mediated propranolol transport includes: (a) propranolol at 12 pM inhibited daunomycin uptake in liver canalicular membrane vesicle by 22% (Kwon et al., 1996); (b) R- and S-propranolol at 125 pM decreased rhodaminel23 efflux by P-gp in CEM/VLB100 cells to 43% and 28%, respectively, of the control (Wigler, 1996); (c) propranolol increased Vinca alkaloid toxicity in CEM/VLB 100 cells as indicated by a 3.5-fold reduction in its IC5 0 (Zamora et al., 1988); and (d) propranolol at 200 pM inhibited 48% of celiprolol secretion in Caco-2 cells (Karlsson et al., 1993). All these observation indicated that P-gp affect propranolol absorption. Although metoprolol, betaxolol, atenolol, alprenolol, betaxolol tested in this study may not be good substrate o f P- gp, the possibility of being substrate o f P-gp for other P-blockers can not be ruled out. On the other hand, clinical data showed that orally-administered p- blockers have good pharmacokinetic profiles and oral absorption: 100% for propranolol, > 95% for metoprolol, > 95% for alprenolol, > 80% for betaxolol, and 46-62% for atenolol (Nace and Wood, 1987). The question raised is what is the significance o f P-gp’s effect on drug absorption. In fact, good absorption and systemic availability of a drug are affected by not only permeability profile, but also other factors, such as solubility and metabolism of a drug. Good solubility of 212 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. P-blockers attribute high drug concentrations in the GI tract. The component of passive diffusion is large at high concentrations. The contribution of efflux of propranolol decreased in a dose-dependent manner. Propranolol transport at 0.5 pM across cultured rabbit conjunctival epithelium did not exhibit asymmetry (Fig. 3-4), indicating that the contribution of efflux to total transport at high concentrations is not significant. In contrast, saquinavir, an HIV protease inhibitor, with poor solubility (0.22 g/100 ml) has low bioavailability. This is generally attributed to the combined effects o f limited absorption (about 30%) (which is restricted by P-gp) and first-pass hepatic metabolism (Vella and Floridia, 1998). Therefore, it is necessary to evaluate a drug candidate by comprehensive considerations of drug efflux and influx mechanisms, drug solubility, protein binding, log D, metabolism, and elimination. In drug discovery, we should not determine the fate of a drug only by being a substrate of P-gp or not, the overall pharmacokinetics and drug metabolism need to be taken into consideration. 213 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OCH2CHCH2NHCH(CH3)2 OH OCH2CHCH2NHCH(CH3)2 OH Propranolol, logP = 3.20 (CH3)2CHNHCH2CHCH20 OH CH2CH2OCH3 Metoprolol, logP = 1.88 CH2CH2OCH2- Betaxolol, logP = 3.44 / ^ . O C H 2CHCH2NHCH(CH3)2 "OCH2C H =C H 2 Alprenolol, logP = 3.10 9CH2CHCH2NHCH(CH3)2 OH H,CO H ,CO CN CH3 _ ------- C (C H 2)3-NCH2CH2\x a CH(CH3)2 Verapamil, logP =1.83 H 3 c. .o c h 3 OCH-, CH2CONH2 Atenolol, logP = 0.16 (H3C)2HCH2C c h o I I h 3c ^ N' A Y ^ n o I c h 3 o c h 2c h 3[ c h ( c h 3)2 v ^ ° (H n HCH r / V , CH 2CH (C H 3)2 I (H3C)2HCH2C ^ / ^ O c h 3 o o n — c h 3 T ^ H I U I O 'C ^ C H < C H CH(CH 3)2 3)2 COOCH3 Rhodamine 123, logP = 2.93 c h 3 c = o CH Progesterone, logP = 3.26 Cyclosporin A, logP = 3.85 Fig. 5-4. Structures and LogP of P-blockers and P-gp substrates used in this study. 214 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2. The effect o f MRP on transport of exogenous and endogenous molecules 4.2.1. The effect of MRP on fluorescein transport Transport o f fluorescein, a MRP substrate (Huai-Yun et al., 1998), across the cultured RCEC layers was asymmetric, favoring the a-to-b transport (1.4-fold of b-to-a transport) (Table 4-1). In contrast, the paracellular marker, mannitol, did not exhibit asymmetric transport. Fluorescein uptake was time-dependent and reached a plateau after 1 hour (Fig. 4-6), but had no apical and basolateral preference. It implies that MRP would not affect drug entry but could pump intracellular drugs out of the cells. This mechanism is quite different from the “vacuum cleaner” mechanism of P-gp, which directly pumps the substrate out of the cell membrane before its entry into the cytoplasm (Gottesman and Pastan, 1993; Lee and Yang, 2000). Hence, fluorescein uptake at 1 hour following basolateral dosing was adopted for subsequent inhibition study. In the presence of the MRP inhibitors in the basolateral side, probenecid and indomethacin, fluorescein uptake was increased by 183% and 140%, respectively (Fig. 4-8), and the effect of probenecid was dose dependent (Fig. 4-7). It was comparable to the 74% and 195% increases observed in retinal epithelium (Aukunuru et al., 2001), but greater than the 25% and 50% increases in brain microvessel endothelial cells (Huai-Yun et al., 1998), and 30% and 80% increases in rat astrocyte (Decleves et al., 2000). By pre-treating the RCEC with glucose-free BRS containing 10 mM NaN 3 for 30-min to abolish intracellular ATP, fluorescein uptake was increased 215 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by 133% (Fig. 4-8), suggesting that the decreased MRP activity by ATP depletion resulted in increased fluorescein accumulation. 4.2.2. Efflux mechanisms of MRP Cole and Deeley (1998) have proposed two transport mechanisms for the MRP pump: ATP-dependent export o f substrates alone, or co-transport of substrates with GSH. To investigate whether fluorescein transport in RCEC is associated with GSH co-transport, the cells were treated with BSO, a GSH synthetase inhibitor (Kannan et al., 1999). GSH concentration in normal RCEC is 8.97 ± 0.06 (nmol/million cells), comparable with that in HepG2 (25 nmol/million cells), Hela (22 nmol/million cells), Caco-2 (32 nmol/million cells), MDCK (13 nmol/million cells) (Lu et al., 1996), and retinal Mueller (10 nmol/million cells) cells (Kannan et al., 1999). With 100 pM BSO treatment, GSH concentration in the RCEC decreased in a time-dependent manner. Overnight treatment led to depletion of GSH by 90% (Fig. 4-9), but did not affect either cell integrity indicated by typan-blue exclusion assay or fluorescein uptake (Table 4-2), suggesting that GSH might not participate in fluorescein efflux by MRP. The structural requirement for substrates to be transported alone or co-transported with GSH by MRP is not yet clear. However, MRP substrates like the glutathione conjugates, e.g., LTC4 (Cole and Deeley, 1998), and simple organic anion, e.g., calcein (Feller et al., 1995), appear to be transported alone. In contrast, 216 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. unmodified vincristine, aflatoxin B l, VP1, either cationic or neutral compounds are co-transported with GSH (Chuman et al., 1998; Cole and Deeley, 1998). MRP functions as a GS-X pump for detoxification of drugs, which are inactivated by oxidation and/or conjugation. The conjugation by itself is not enough to get rid of the drug. The conjugated drug is now more hydrophilic because o f the GSH attached to it and cannot leave the cell by passive diffusion. As drug continues to enter the cells, the GSH conjugate will accumulate to excessive concentrations (that will be toxic in themselves) unless exported by a dedicated export pump. MRP plays a role in cellular defense against oxidative stress (Cole and Deeley, 1998). However, our results o f GSH depletion counter the argument that oxidative stress affects MRP-mediated drug transport. A rank order o f substrate specificity for MRP according to the V m a x / K m ratios was reported as follows: LTC4 > LTD4 > S-(2,4-dinitrophenyl)glutathione > 1 7 p-glucuonosyl estradiol > monoglucuronosyl bilirubin > 3a-sulfatolithocholyl taurine > GSSG (Keppler et al., 1998). Together with our observations, this order suggests that the export of GSH/GSSG might not be a major function of MRP in the conjunctival epithelial cells. 217 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2.3. Fluorescein influx mechanism Fluorescein is a polar compound that has been used for evaluating corneal integrity (Macri and Pflugfelder, 2000). Although fluorescein staining is reported to be due to cellular accumulation, but not to intercellular trapping (Wilson et al., 1995), its influx mechanisms are of interest to be investigated. Fluorescein significantly inhibited basolateral uptake o f succinate, a dicarboxylate transporter (DCT) substrate, in RCEC, suggesting that they might share the same transporter (e.g., DCT) at the basolateral membrane. Moreover, 1 mM lactate significantly inhibited apical [1 4 C] lactate uptake in RCEC, suggesting the existence of monocarboxylate transporters (MCT). Therefore, the possibility of MCT/DCT mediated fluorescein influx could not be ruled out. In fact, Horibe et al. (1998) demonstrated that Na+-dependent monocarboxylate transport processes may exist in the excised conjunctiva. The detailed transport mechanisms remain to be further explored. 4.2.4. The effect o f MRP on transport of ophthalmic drugs Non-steroid anti-inflammatory drugs (NSAIDs) are among the most frequently used medicinal drugs. It is of interest to know whether or not ocular NSAIDs, sterodial anti-inflammatory drugs and antivirals would be affected by MRP. Non-steroidal anti-inflammatory ocular drugs, diclofenac, ofloxacin and flubiprofen, (Fig. 4-8), all at 1 mM increased fluorescein uptake by 103%, 129%, 218 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and 113%, respectively, but not at a lower concentration o f 100 pM. None of the NSAID, cromolyn; the steroidal anti-inflammatory drug, prednisolone; and the antiviral, cidofovir, at either 100 pM or 1 mM, affected fluorescein accumulation (Fig. 4-8), suggesting that some anion drugs might not be good substrates for MRP. Among the drugs tested, cidofovir and other acyclic nucleoside phosphonate analogs are substrates for the human and rat renal organic anion transporter 1, which is associated with the dose-dependent nephrotoxicity o f these drugs (Cihlar et al., 1999); and the transport of diclofenac and ofloxacin are also mediated by monocarboxylate transporter in the conjunctiva (Horibe et al., 1998). NSAIDs including indomethacin, sulindac, tolmetin, ofloxacin, and antimicrobial erythromycin, all have been reported to interact with MRP, thereby enhancing chemotherapeutic drug toxicity to human tumor cells (Duffy et al., 1998) and leukemia cells (Terashi et al., 2000). However, the chemosensitizing effects of MRP inhibitors vary according to the type of resistant cells as shown by the marked chemosensitizing effect of probenecid in MRP-overexpressing cell of AML-2/DX100, but not in MRP-overexpressing cells HL60/Adr (Kim et al., 2001). Together, these reports suggest that MRP exhibits substrate specificity, where organic anion is not the only requirement for its recognition. To the best of our knowledge, no SAR or QSAR studies of MRP have been reported, and the MRP pharmacophore remains an interesting topic to be explored. 219 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fluorescein .coo- HN coo- OH coo- ,CH (CH3CH2CH2)2NS02 COOH Leukotriene C4 Probenecid ■C H . COOH C l COOH Indomethacin Diclofenac COOH HOOC. OH H3C Cromolyn CH N F COOH Ofloxacin .COOH CH3 Flurbiprofen Fig. 5-5. Compounds used in MRP study. OH CH HO, Prednisolone 220 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2.5. MRP-mediated LTC4 transport in the conjunctiva The distribution of MRP at the basolateral membrane in the conjunctiva appears to be distinct from apically-localized P-gp. It implies that the physiological roles of MRP could be quite different from those o f the P-gp functions in guarding against the entry of certain xenobiotics. Indeed, MRP is known to have a high affinity for certain endogenous substances, like LTC4 and LTD4 (Cole and Deeley, 1998). The physiological role of MRP as a transporter o f cysteinyl leukotrienes was supported by the evidence that mrp knockout mice had impaired response to an inflammatory stimulus attributable to decreased LTC4 secretion (Wijnholds et al., 1997). In the cultured RCEC, the a-to-b transport of LTC4 was 1.4 times that o f the opposite direction, and this directionality was abolished in the presence of 1 mM probenecid. The paracellular marker, mannitol, did not exhibit asymmetric transport (Table 4-2). This led us to hypothesize that MRP may have a pathophysiological role in facilitating the delivery o f LTC4 , an inflammatory factor, in conjunctival epithelial cells, particularly in the disease states like inflammation and viral infection. LTC4 is known to play an important role in inducing the migration of neutrophils and microphage in host defense (Bhattacherjee, 1989). 221 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3. Contribution o f P-gp and MRP to drug efflux P-gp and MRP contribute to decreased intracellular drug concentration. It will be an important task to sort out which pump contributes to resistance, and to what extent in different tissues. Towards this goal, uptake o f vincristine, a known substrate for both drug efflux pumps (Chuman et al., 1998; Song et al., 1999) was evaluated to understand the relative contribution o f P-gp and MRP to drug efflux in RCEC. The evidence of P-gp- and MRP-mediated vincristine transport includes that (1) the sensitivity of the P-gp expressing KB-8-5 cells to vincristine increased 2-fold of the reversing effect of cyclosporin A at 1 pM (Naito et al. 1993); (2) an enhanced rate o f vincristine loss — 2.3-fold from the PC-V40 cell line and 3.9- fold from the PC-V160 cell line, two MRP expressing cell lines (Slapak et al., 1996) Vincristine uptake was increased by 132-194% by co-administration o f P- gp substrates (i.e., CsA, quinidine, and rhodamine), it is consistent with the observation that 2.2 to 6 . 6 pM verapamil overcome the resistance of P388/VCR to vincristine (Tsuruo et al., 1981). Vincristine uptake was enhanced by 169- 175% by adding MRP substrates (i.e., indomethacin and probenecid). Uptake was not affected by the presence of organic cations (e.g., TEA and guanidine), or organic anions (e.g., lactic acid, nicotinic acid, and cidofovir) (Fig. 4-10). In primary culture o f rat astrocytes, similar enhancement of 30-min vincristine accumulation was observed by 10 pM CsA (140%), 10 pM verapamil (160), 1 222 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mM probenecid (115%), and 2 mM sulfinpyrazone (180%) (Decleves et al., 2000). Overnight treatment with BSO caused a significant increase in vincristine uptake (131%) (Fig. 4-10). This is comparable with the observation by Chunman et al. (1998) that with the 48-hr treatment of 100 pM BSO, the sensitivity o f C- A120, a MRP overexpressing cells to vincristine, was 9.2-fold increased, while BSO alone did not growth o f the cell line over 4-day incubation. Due to the difficulty to assess the full inhibition of P-gp and MRP by an inhibitor, the estimation of precise contribution of P-gp and MRP to vincristine is difficult. However, the enhanced uptake in the presence o f these inhibitors of P-gp and MRP under this condition suggests that P-gp and MRP may play equally important roles in the efflux o f vincristine in the conjunctiva. The precise measurement of the contribution of P-gp and MRP to drug efflux could be achieved by using P-gp/MRP (single and double) knock-out mice (Schinkel et al., 1997a.; Wijnholds et al., 1997; Terasaki and Hosoya, 1999). This result was quite striking, given that the efflux activity of MRP has been reported to be weaker than that of P-gp in general (Cole and Deeley, 1998). The significant efflux function o f MRP in the conjunctiva may be an indication of its important pathophysiological role of transporting LTC4 in the conjunctiva. 223 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. The effect of Ad5 infection on expression and function of drug efflux pumps Based on the observation of human T-cell-leukemia-viras-I increased MRP expression in adult T-cell leukemia (ATL) cells (Ikeda et al., 1999) and HIV increased P-gp expression in CD4 T cells in HIV patients (Andreana et al., 1996), we further evaluate whether Ad5 infection, a common ocular infection disease, could regulate drug efflux pumps in the conjunctiva. 5.1. P-gp expression and function in Ad5-infected RCEC All those evidence prompted us to investigate the P-gp expression and function in RCEC with Ad5 infection, an ocular disease state. However, the protein expression of P-gp and its function on propranolol uptake in Ad5- infected cells were not significantly different (Figs. 3-6 and 3-7), implying that the major function of P-gp is against drug entry, but not pathology induced by Ad5 infection in the conjunctiva. 5.2. Up-regulation o f MRP by viral infection Previously, we demonstrated that cultured RCEC layers showed swelling and detachment after Ad5 inoculation, indicating a typical in vitro inflammation phenomenon (Yang et al., 1998). By using this Ad5-infected culture as a disease Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. model, we further investigated MRP expression, function, and modulation in the conjunctiva. Western blot analysis showed the MRP band at -190 kDa in freshly isolated RCEC (61%), cultured RCEC (69%), Ad5-infected RCEC (89%), as well as for the positive controls o f rabbit intestinal epithelium (67%), kidney cortex (100%), suggesting the existence of MRP in the conjunctiva. MRP expression in Ad5-infected RCEC was 1.3 times higher than the mock-infected control as indicated by the band density (Fig. 4-13), indicating that MRP might be overexpressed during a viral challenge. The MRP band was not observed in rabbit comeal epithelium and liver (data not shown), implying that MRP expression in comeal epithelium and liver is low. It is consistent with the observation o f a low expression of basolateral MRP in rat hepatocytes (Roelofsen et al., 1999). As shown in Fig. 4-11, the RCEC MRP efflux activity, indicated by LTC4 uptake, was enhanced. Replication-deficient adenovims, which is used as a vector in gene delivery, is less antigenic compared to the wild type adenovims which evokes prominent inflammatory responses. Our data support the hypothesis that adenovims up-regulated MRP expression that may be associated with host immune response. Whether viral replication is required for MRP up-regulation is unknown. Replication-deficient adenovims could induce protein expression, e.g., up-regulation of interferon y expression in a mouse model of airway eosinophilic 225 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inflammation (Suzuki et al., 2000), the relationship o f MRP up-regulation and viral replication remains an interesting topic to be addressed. Recent reports (Kipp et al., 2001) supports a cAMP- and a taurocholate- sensitive ABC transporter pools governing membrane ABC transporter trafficking in rat liver. Whether the up-regulation o f MRP by adenoviral infection is associated with changing trafficking pathway, increasing protein synthesis, or decreasing protein degradation is not clear. Our observation of increased MRP expression in terms o f total protein (Fig. 4-13) and membrane expression (Fig. 4- 12) suggests that the last two pathways might be the case. By measuring time- course MRP expression in normal and Ad5-infected cells treated with cycloheximide, a protein synthesis inhibitor (Kubitz et al., 2001), increasing MRP synthesis (if there is no difference o f MRP expression in these two conditions) and decreasing MRP metabolism (if MRP expression is higher in Ad5-infected cells) can be distinguished. 5.3. Regulation of MRP by cytokines Viral infection is known to induce a host immune response. In vivo, adenovirus (adenoviral vector) could induce immune response (Borras et al., 1996b). IL- 6 and IL - 8 concentration in the airway epithelium were elevated by influenza A virus (Ronni et al., 1997), respiratory syncytical virus (RSV) (Tristram et al., 1998), and human rhinovirus (Terajima et al., 1997). IL-1 ( 3 and 226 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TNF-a level in the airway epithelium were increased by the human rhinovirus (HRV-2 and HRV-14) (Terajima et a l, 1997). IFN a /p content in A549 cells was elevated by the influenza A virus (Ronni et a l, 1997), while those o f IL -la, IL-6 , G-CSF, and GM-CSF in the human umbilical vein endothelial cells were increased by the human lymphotropic virus type I (Adachi et a l, 1997). Ginsberg et al. (1991) reported that intranasal inoculation o f Ad5 in mice induced TNF-a, IL-1, and IL- 6 secretions which mediated early inflammatory reactions to the viral infection and stimulated immune responses. We further investigated MRP expression in the cultured RCEC by treatment of the cells with these cytokines. As shown in Fig. 4-15, MRP expression in the cells was up- regulated by treatment with TNF-a, IL-1, and IL-6 , indicating that MRP function may be relevant to host immune defense, and that up-regulation of MRP by Ad5 infection could be induced by cytokines. Actually, the up-regulated cytokine secretion (IL- 6 and TNF-a) was observed in Ad5-infected RCEC (Figs. 4-17 & 4-18). Elevated secretion of the proinflammatory cytokines TNF-a and IL- 6 was also observed in a primary culture of human conjunctival epithelial cells in response to stimuli (lipopolysaccharide, IL -ip, calcium ionophore A23187, or phorbol myristate acetate) (Gamache et al., 1997). Moreover, cytokine secretion was reported in other epithelial tissues, including nasal epithelium (Becker et a l, 1993), airway epithelium (Arnold et al., 1994), and intestine epithelium (McGee et al., 1996). 227 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. D'Alessandr et al. (1998) demonstrated that TNF-a exerts more cytotoxicity in a MDR variant (FLC/DOX) of Friend leukemia than in its parental, drug-sensitive, counterpart and this effect is related to the induction of apoptosis. We observed swollen and detached cells in Ad5-infected culture (Figs. 2-16 & 4-12), this could also be apoptosis o f infected RCEC induced by TNF-a. Ocular tissues have evolved to provide sophisticated immune responses in pathophysiological conditions. Various cytokines and endogenous molecules (IL- la , IFN y, IL-3, IL-4, IL-5, IL-6 , IL-8 , RANTES, TNF-a, TG F-pi, HLD-AR, ICAM-1, and GM-CSF) are found to be expressed in normal conjunctival epithelial cells or up-regulated in disease states (e.g., 8 -fold IL - 6 mRNA expression in Sjogren’s syndrome patients compared to normal people determined by RT-PCR) (Hingorani et al., 1998; Jones et al., 1994; Leonardi et al., 1999; Tsubota et al., 1999; Uchio et al., 2000). Modulation of cytokines could be a potential therapy for ocular diseases. This is exemplified by the use o f IL-1 receptor antagonist (IL-1R) to suppress allergic eye disease by a down-regulation of the recruitment of eosinophils and other inflammatory cells (Keane-Myers et al., 1999) and by the use o f human fibroblast IFNp (7.5 x 105 IU/ml) to alleviate acute epidemic conjunctivitis (Wilhelmus et al., 1987). Cytokine signaling for up- regulation of MRP expression in RCEC under inflammatory conditions needs to be further investigated. 228 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To evaluate the hypothesis that MRP up-regulation in adenovirally- infected RCEC is to provide more effective transporter function to facilitate the transport of inflammatory factors, we determined LTC4 uptake in cultured RCEC, following Ad5 challenge or treatments with cytokines. Fig. 4-15 shows that, in the presence of probenecid or following depletion of intracellular ATP, LTC4 uptake was increased by 44% and 54%, respectively, indicating a MRP-mediated transport process. Ad5 infection and the treatments with cytokines decreased LTC4 accumulation in RCEC by 13% to 43%, indirectly confirming that this inflammatory factor was more efficiently pumped out of the cells by up- regulation of MRP in inflammation. MRP expression and LTC4 uptake had a good correlation (R2 = 0.96) as shown in Fig. 4-16, suggesting that the capacity of MRP pump activity is associated with its expression. Stein et al. (1997) also reported that transfected TNF-a stimulated MRP expression at both the gene and protein levels in the colon carcinoma cell lines, HCT15 and HCT116. In this study, anti-cytokine antibodies were used in an attempt to block the cytokines (IL-6 , TNF-a, and IFN-a,p) secreted in Ad5-infected RCEC in order to evaluate whether or not MRP-mediated LTC4 efflux was related to the cytokine concentration. This could provide indirect evidence of a cytokine pathway for Ad5-induced MRP expression. However, none o f the seven treatments caused a total recovery of LTC4 uptake in Ad5-infected cells compared to normal cells. Although some antibody treatments (anti-TNF-a mAb) recovered the LTC4 229 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. uptake to some extent, none of the effects were significantly different from those without antibody treatment. Several possibilities could contribute to the inconclusive results: (a) the mouse antibody could not efficiently neutralize the rabbit cytokines; (b) there were other cytokine pathways for Ad5-induced MRP overexpression besides those tested (e.g., GM-CSF and IL - 8 are secreted in the conjunctiva (Gamache et al., 1997); IL-1 a /(3 and TNF a /p induced IL- 8 release in non-infected A549 cells (Arnold et al., 1994)). To further test the neutralization ability of an antibody to a certain cytokine, anti-mouse TNF-a mAb (Abrams et al., 1992) at 10 pg/ml was used to neutralize TNF-a at 10 U/ml (-20 pg/ml). The result showed anti-TNF-a antibody could partially inhibit TNF-a effect (recovered to 90% o f the control LTC4 uptake, Fig. 4-20), indicating that this high concentration of 10 pg/ml was not able to totally neutralize TNF-a. Whether this antibody can fully block TNF- a is not clear. 6. Overall indications of efflux pumps in the conjunctiva In most tissues, the drug efflux pumps are expressed in a highly polarized fashion. Similarly, in this study, we demonstrated that P-gp and MRP are apically and basolaterally localized in the conjunctival epithelial cells, respectively. Their efflux functions in health and disease states indicated the roles of P-gp as an efflux pump against the entry o f xenobiotics and that of MRP as a carrier o f an 230 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inflammatory mediator in a virally-infected state in the conjunctiva. These findings are comparable to the localization and functions of these two proteins in other tissues, but indicate their own characteristics due to their diverse physiological roles in different tissues. In the liver, P-gp is found in the biliary canalicular front o f hepatocytes, the apical surface of epithelial cells in small biliary ductules (Thiebaut et al., 1987), and MRP is on the basolateral membrane of the hepatocyte (Keppler et al., 1998; Muller et al., 1996). This is related to the strong secretion o f drugs and their metabolites into the bile and re-absorption of bile salt. In addition, the expression of MRP is highly increased in immortalized hepatocytes, indicating its role during cell proliferation (Roelofsen et al., 1997). In the kidney, P-gp is expressed at the apical membrane of epithelial cells of proximal tubules (Thiebaut et al., 1987) and MRP is localized in the basolateral membrane, where they are associated with secretion o f drugs into the urine and probably re-absorption o f organic anions (Evers et al., 1996). In the intestine, P- gp is expressed at the apical membrane o f superficial columnar epithelial cells, restricting drug entry, but the function of basolaterally localized MRP is not clear. In the lung and brain, both P-gp and MRP might be localized at the apical membrane o f respiratory columnar epithelial cells and basolateral membrane of brain capillary endothelial cells (Flens et al., 1996; Stein et al., 1997), respectively, contributing to the secretion o f drugs to the airway and the restricted drug permeability across blood brain barrier (BBB). 231 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. From the point o f view o f drug delivery, we probably should overcome P- gp-mediated transport and utilize MRP-mediated transport in order to improve drug absorption. To minimize the P-gp’s efflux, we could design drugs without a two-planar-ring system to avoid P-gp recognition. On the other hand, MRP in the conjunctiva could be considered to facilitate the transport o f NSAIDs such as diclofenac, ofloxacin and flurbiprofen. The up-regulation o f MRP by Ad5- infection shed a light on utilizing MRP for improving drug absorption in the ocular inflammation. 7. Summary of drug efflux pumps in the conjunctiva In summary, the energy-dependent efflux pump P-gp appears to be predominantly located on the apical plasma membrane of the conjunctival epithelium. It may play an important role in restricting the conjunctival absorption of some lipophilic drugs. We probably could add P-gp inhibitors or P-gp antibodies in the drug formulation to enhance topical ocular drug absorption (Fig. 5-6). Alternatively, a drug could be designed to retain its pharmacological action without recognition by P-gp to facilitate the absorption of topical ocular drugs, we have demonstrated the basolateral localization o f MRP in the rabbit conjunctival epithelial cells and that MRP could pump some organic anions out of the cells. The physiological function o f MRP may be associated with host immune response by playing an important role in the transport of substrates including the inflammatory factor, LTC4, to the underlying tissues (Fig. 5-7). 232 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Propranolol CsA Verapamil Progesterone R hodam inel23 4E3 mAb Propranolol Apical 2,4-DNP ADP Propranolol Propranolol Propranolol Basolateral Fig. 5-6. Putative model o f P-gp-mediated propranolol efflux in the conjunctiva. 233 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cytokines (IL-1, IL-6, TNF-a) \ ATP LTC MRP Ad5, ADP Indomethacin ADP Probenecid \ MRP, Fluorescein ATP NaN Apical Basolateral Fig. 5-7. Expression, localization, function, and modulation of MRP in the rabbit conjunctival epithelial cells. MRP is localized at the basolateral membrane of the rabbit conjunctival epitheial cells. The expression and function of MRP could be modulated by cytokines and Ad5-infection. 234 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VI. CONCLUSIONS Our research was aimed at achieving a better understanding o f drug efflux mechanisms in the conjunctiva and o f the association of drug efflux pumps with physiology and pathology of the conjunctiva. A multitude of laboratory research techniques were used, including biopharmaceutical approaches (in vitro cell culture) and cell biology (Western blot, immunostaining, and confocal microscopy). Energy-dependent efflux pump P-gp appears to be predominantly located at the apical plasma membrane, while MRP is probably localized at the basolateral membrane in rabbit conjunctival epithelium. P-gp may play an important role in restricting the conjunctival absorption o f some lipophilic drugs such as propranolol. In contrast, MRP can efflux organic anions such as fluorescein out o f the cells from basolateral membrane. MRP may be regulated by Ad5 infection and cytokines, and play a role in host immune defense by mediating transport o f inflammatory factors like LTC4. Fig. 5-8 summarizes the overall findings of drug efflux pumps in the rabbit conjunctival epithelial cells. 1. Evaluation of expression, localization, function, and modulation of P-gp The energy-dependent efflux pump P-gp appears to be predominantly located at the apical plasma membrane o f the conjunctival epithelium. It may play an important role in restricting the conjunctival absorption of some lipophilic drugs, such as propranolol and CsA. 235 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Apically localized P-gp was observed by direct immunostaining o f P-gp under confocal microscopy. Asymmetric propranolol transport was further evidence o f P-gp efflux function against drug entry into the cells. Apical propranolol uptake was increased in the presence of P-gp substrates and modulators, further suggesting the involvement o f P-gp in restricting conjunctival propranolol absorption. The uncoupling agent (2,4-DNP) increased apical uptake of propranolol, suggesting that P-gp function needs cellular energy. 4E3 mAb, which recognizes the external portion of P-gp, increased propranolol uptake, indicating that antibody binding decreased P-gp activity. This finding also suggests that this antibody might be useful in the attenuation of multidrug resistance involvement or in targeting drug to the specific tissue that expresses P- gP- O f the p-blockers evaluated, the agents without a naphthalene ring did not affect propranolol uptake. This indicated that structure requirement, such as two planar aromatic rings, for the binding of P-gp. P-gp “pharmacophore” remains a topic to be explored further. Ad5-infection did not affect P-gp expression and function, which indicated this protein may not be directly involved in the pathology of Ad5 infection of the host. 236 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. Evaluation of expression, localization, function, and modulation of MRP We demonstrated the basolateral localization of MRP in the rabbit conjunctival epithelial cells. MRP could pump some organic anions such as fluorescein and certain anion drugs out o f the cells. Viral infection and cytokine treatments could up-regulate MRP expression. The physiological function of MRP may be associated with host immune response by playing an important role in the transport of the inflammatory factor, LTC4 to the underlying tissues. The MRP in rabbit conjunctiva has 87% identity in amino acid sequence compared to human MRP. By using primers designed based on the highly conserved regions o f the human and mouse MRP cDNA, RT-PCR showed the bands with desired molecular sizes. Sequence comparison determined that the 652 bp RT-PCR product was 87% identical to human MRP in amino acid sequence and 84% identical in nucleotide sequence, confirming that MRP exists in the rabbit conjunctiva. Positive staining of cultured RCEC with MRPrl mAh was observed primarily in the basolateral membrane and to some extent in the cytosol, but not at the apical membrane of rabbit conjunctival epithelial cells. Transport of fluorescein, a MRP substrate, across the cultured RCEC layers was asymmetric, favoring the a-to-b transport. Uptake of fluorescein was enhanced by adding indomethacin and probenecid, or abolishing intracellular ATP by NaN 3 . Non steroidal anti-inflammatory ocular drugs (NSAIDs), diclofenac, ofloxacin and 237 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. flubiprofen also increased fluorescein uptake, but cromolyn, prednisolone, cidofovir did not. This suggests that some anion drugs might not be good substrates for MRP. Depletion of GSH did not affect fluorescein uptake, suggesting that GSH might not participate in fluorescein efflux by MRP. The structure requirement for MRP substrates to be transported alone or co transported with GSH is not yet clear. Vincristine accumulation in the cultured RCEC was increased at a similar range by adding P-gp substrates (CsA, quinidine, rhodamine), and by adding MRP substrates (indomethacin and probenecid). This suggests that P-gp and MRP may play equally important roles in the efflux o f vincristine. In the cultured RCEC, the a-to-b transport o f LTC4 was higher than the opposite direction, and this directionality was abolished in the presence of probenecid. MRP expression in Ad5-infected RCEC was significantly higher than the mock-infected control as indicated by the band intensity in Western blot analysis, indicating that MRP might be up-regulated during a viral challenge. MRP efflux activity in Ad5-infected cells was enhanced as indicated by LTC4 uptake. The up-regulated cytokine secretions (IL- 6 and TNF-a) were observed in Ad5-infected RCEC. RCEC with the treatments o f cytokines (IL-1, IL- 6 and TNF-a) showed up-regulation of MRP expression by Western blot analysis and decreased LTC4 accumulation, indirectly confirming that this inflammatory factor was more efficiently pumped out of the cells to the underlying tissues under 238 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inflammatory conditions by up-regulation of MRP. MRP expression and LTC4 uptake had a good correlation (R2 = 0.96). In summary, we have demonstrated the basolateral localization of MRP in the rabbit conjunctival epithelial cells. MRP could pump some organic anions out of the cells. The physiological function o f MRP may be associated with host immune response by playing an important role in the transport of substrates including an inflammatory factor, LTC4, to the underlying tissues. 3. Development of rabbit conjunctival epithelial cell culture models Air-interfaced culture of rabbit conjunctival epithelial cells In this study, we developed and characterized a functional primary air- interfaced culture o f pigmented rabbit conjunctival epithelial cells resembling the normal native tissue in vitro. These cell layers display ion and drug transport characteristics more akin to those o f the native tissue compared to previously established liquid-covered culture in terms of morphology (differentiated cell layers and goblet cell population), electrophysiology (TEER, PD, and Ieq), and barrier properties (permeability of molecules of various sizes and hydrophobicity). It may prove to be a suitable model for rapidly screening the transport properties of topical ophthalmic drug candidates as well as formulation factors influencing their transport, a future challenge in topical ocular drug delivery. 239 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The present model o f ion transport across the cultured cells suggests basolaterally localized Na+,K+-ATPase, Na+ /K+ /2C1" cotransporter, and K+ channel, and apically localized Cl' channel, which replicates the ion transporter properties in native tissue (Kompella et al., 1993). Morphologically, the air- interfaced culture has more differentiated cell layers and a larger population (-3%) of mucin-secreting goblet cells compared to liquid-covered culture. The permeation of hydrophilic and lipophilic compounds through the cultured RECE resembles that o f the native conjunctiva. Moreover, active transport processes (dipeptide transporter and nucleotide transporter) exist in the cultured RCEC, allowing us to further study active transporters and corresponding transport mechanisms in this model. Taken together, this air-interfaced cell culture model may better mimic the native conjunctiva in a healthy state, enabling it to be a useful tool for drug delivery study. Ad5-infected culture o f rabbit conjunctival epithelial cells We demonstrated that Ad5 can infect non-confluent culture of rabbit conjunctival epithelial cells grown on a permeable support. Ad5 infection caused changes in cellular morphology (swelling and detachment), decreased bioelectrical parameter (Ieq and TEER), and decreased active C f secretion in the RCEC layers. It partially resembled an inflammation state of adenoviral ocular infection in vivo. The Ad5-infected conjunctival epithelial cell culture model may 240 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. be useful for studying the pathophysiology of infected RCEC and attendant modifications in the expression and function of active transporters. 4. Overall conclusions Overall, efflux pumps exist in the conjunctiva and play important roles in ocular physiology and drug absorption. These active transport processes should be taken into consideration in developing ocular drug delivery systems (Fig. 5-8). The work has identified a good in vitro cell culture models that can be used in normal and disease states for studying ocular drug delivery and the physiology and pathology o f the conjunctiva. The functional cultures will also be useful tools for evaluating ocular drug absorption, metabolism and toxicity. Drug efflux pumps may affect the absorption and disposition of topical ocular drugs in the market, such as an immunosuppressive agent CsA, an antibiotics erythromycin, and a nonsteroidal antiinflammatory agent ofloxacin. In addition, other transporters (e.g., organic cation transporters, monocarboxylate transporters, and organic anion transporters) may influence the ocular drug delivery of hypertensive drugs (P-b lockers) and NSAIDs (ketorolac, cromolyn, and cirpofloxacin). Current treatment for macular degeneration and cytomegalovirus retinitis are limited to intravenously administered verteporfin and ganciclovir, and future efforts include understanding of transporter systems in 241 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the eye and develop novel strategies for drug delivery to the back o f the eye (e.g., retina) via conjunctiva-sclera route. The interesting findings in this study raise an equally interesting possibility that other ocular infection (e.g., by herpes virus and cytomegalovirus viruses, or bacteria) might indirectly or directly regulate certain drug efflux pumps in the conjunctiva, which could potentially affect cellular environment and drug delivery. Therefore, a strategy to improve therapeutic efficiency o f anti- virals and antibiotics will be to consider the effect of altering drug efflux pumps on these therapeutic agents. Such an undertaking would require efforts to explore new in vitro models corresponding to various disease states. Finally, our studies on P-gp and MRP have led to more fundamental inquiries utilizing molecular biology (e.g., molecular cloning), for the discovery of new gene homologies, thereby allowing further delineation o f their transport properties and regulatory mechanisms. The existence of P-gp and MRP implies that other efflux pumps may also be present in conjunctiva. The comprehensive understanding of the overall efflux mechanisms along with the influx mechanisms would be essential for the rational design o f therapy. 242 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Polar drugs ^ ----- Non-Polar drugs * ^ CsA Verapamil Progesterone Rhodaminel23 4E3 mAb s & . Propranolol —I P -gp Nucleoside Dipeptide CP < Cytokines f A d5< i+) Indomethacin C- J A D P^ ___ Probenecid \ Y MRP y - > Diclofenac 2,4-DNP ofloxacin 4 flubiprofen / , > cA M P. ^ V Fig 5-8. Summary of the overall research conclusions of the drug efflux pumps in the rabbit conjunctival epithelial cells. LTC4 Fluorescein 243 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VII. FUTURE DIRECTIONS The current project has laid the foundation for further studies to address some interesting questions: 1. Cloning o f MRP and P-gp from rabbit conjunctiva In this study, the RT-PCR product shows 87% identity in amino acid sequence compared to human MRP, indicating an isoform o f MRP in rabbit conjunctival cells. This positive RT-PCR results make the cloning o f rabbit MRP a possible reality. MRP from human, rat, and mouse (Cole et al., 1992; Cole and Deeley, 1998) have been cloned but not that the rabbit. In order to clone the whole sequence of the transporter, RACE using the Marathon™ cDNA Amplification Kit (CLONTECH, Palo Alto, CA) could be used. This system begins with double stranded cDNA synthesis from poly(A)+ RNA and ligation to the Marathon cDNA adapter. The cDNA can then be used as a template in RACE PCR to obtain the 5’ or 3’ ends or a full-length cDNA. The cDNA-deduced primary structure o f the transporter will be analyzed on the basis o f four criteria: the prediction of transmembrane domains; the position o f a-helix in the predicted secondary structure; the position of hydrophobicity peaks in hydropathy studies; the surface probability and flexibility index plot. Once primary and secondary structures of the transporter are deduced, the sequence identity can be defined by the percent identical amino acid residues 244 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and common structure features shared by its closely related homologies under comparison, which allows an understanding of the similarity of gene and function in diverse species (Garcia et al., 1995). The cDNA o f rabbit MRP could be transfected into human cells (e.g., MDCK, HEK (Braun et al., 2000)) and Xenopus oocyte (Hediger, 1994) to develop cleaner systems to further evaluate the transport properties and underlining modulation mechanisms o f the transporter. Drug transport and uptake, Western blot analysis, immunostaining, and electrophysiology methodologies applied in the current study could be adopted. P-gp has been cloned in man (Riordan et al., 1985), therefore, P-gp in rabbit conjunctiva can be cloned by using the primers deigned by the conservative regions across diverse species. 2. Identification of other MRP isoforms in the conjunctiva At least six MRP homologues, MRP1 (MRP), MRP2 (also called cMOAT), MRP3, MRP4, MRP5, and MRP6 have been identified. It will be interesting to determine if these different efflux pumps also exist in the conjunctiva, since they have distinct transport characteristics and may play separate important roles in eye physiology and ocular drug absorption. The transport characteristics of MRP3 differ from those of MRP 1 and MRP2 in that glutathione conjugates are poor substrates for MRP3 (Kool et al., 1999b). MRP4 is directly linked to the efflux o f nucleoside monophosphate 245 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. analogs, such as dideoxynucleosides (Hirohashi et al., 1999). MRP5 gene expression levels were stimulated in clinical specimens with exposure to platinum drugs, but MRP5 did not confer resistance to various classes o f cytotoxic drugs, such as BCECF (McAleer et al., 1999). MRP7 is a recent addition to the family and has not yet been characterized (Borst et al., 2000). In the MRP family, MRP2/cMOAT is known to have broad substrate specificities and play important roles in secreting anion drugs and metabolites in the kidney and liver. Table 5-1 compares the amino acid sequence of human cMRP/MRP2 with transporters o f the ATP-binding cassette superfamily. It is of interest to know whether MRP2 exists in the conjunctiva. Apical MRP2 in Caco-2 cells was observed by using anti-MRP2 antibodies M2III6 and M2I-4 (Walgren et al., 2000). GP8 antibody was used to capture MRP2 on the brush-border membrane of villi in rabbit intestine (van Aubel et al., 2000a). Therefore, these antibodies could be used for determining the expression and localization of MRP2 in the conjunctiva. The rabbit MRP2 has been cloned (van Aubel et al., 1998), and MRP2 gene expression in the conjunctiva can be evaluated by using primers designed based on known rabbit MRP2 sequence. Although MRP1 and MRP2 share similar substrate specificity including leukotriene C4, 2,4-dinitrophenyl-S- glutathione, 17(3-estradiol, 17(3-D-glucuronide (Cole and Deeley, 1998; Hirohashi et al., 2000; Keppler et al., 1997; Keppler et al., 1998), p-Aminohippurate (PAH) is widely used as a model substrate of MRP2/multispecific organic anion 246 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transporter in kidney proximal tubules (Sekine et al., 2000; Sweet et al., 1997; Uwai et al., 1998; van Aubel et al., 2000b). Some monophosphate antivirals such as adefovir and cidofovir, are reported to be transported by hOATl (Ho et al., 2000). Given that cidofovir appears to be not mediated by MRP1 in the present study, these drugs are potential candidates for evaluation o f MRP2/cMOAT function in the conjunctiva. Upon understanding the existence of various drug efflux pumps in the conjunctiva, it would be interesting to study the cooperation o f various drug efflux pumps in drug transport in the conjunctiva. Table 5-1. Comparison o f the amino acid sequence of human cMRP/MRP2 with transporters o f the ATP-binding cassette superfamily (Keppler et al., 1997). ABC Transporter Amino acids % Similarity % Identity cMRP/MRP2 human 1545 100 100 EBCR (rabbit) 1564 91 82 cMrp rat 1541 89 78 MRP (human) 1531 69 49 Mrp mouse 1528 68 49 MRP1 C. elegans 1540 65 43 MRP2 C. elegans 1525 64 43 YCF1 yeast 1515 62 41 YHD5 yeast 1592 57 32 SUR human 1581 56 33 CFTR human 1481 55 31 MDR1 human 1280 51 25 MDR3 human 1279 49 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. Evaluation o f the underlying mechanisms o f Ad5-induced MRP overexpression The Ad5-induced MRP expression in the cultured RCEC provides an impetus for a further evaluation of the underlying immunology. The current inhibition study using anti-cytokine antibodies is inconclusive. One possible reason could be antibody mismatch (anti-mouse anti-cytokine antibodies were used to neutralize rabbit cytokines). One solution would be to clone the rabbit MRP first, then to use transfected human cells to study the immunology since human cytokines and anti-human anti-cytokine antibodies are commercially available. An alternative method is to use a primary culture of human conjunctival epithelial cells. Secretion o f the proinflammatory cytokines, TNF-a, IL- 6 , and GM-CSF, was observed in the human conjunctival cell culture (Gamache et al., 1997). Therefore, the result would be more convincing if human cytokines, antibodies, and ELISA Kit were used to test any immunology-related hypotheses in the human cells. Receptor antagonist (De Benedetti et al., 2001; Mao et al., 2000; Renzetti and Gater, 1997) could be used to block the cytokine receptors, thereby abolishing a certain cytokine pathway. This could provide a rational design for evaluating the effects of a cytokine on transporters. In addition, since cytokines have various effects on cell signaling, including activating mitogen-activated protein kinase (MAPK) activity, inducing apoptosis, inducing nitric oxide synthesis (Chin et al., 1998; Dumitru et al., 2000; Ip et al., 1992; Lamas et al., 1991; Malinin et al., 248 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1997), it would be o f interest to further deduce the downstream signal transduction pathway of cytokine-induced MRP up-regulation. 4. Modification o f transporter expression and function in adenovirally-infected RCEC Given that MRP was up-regulated in Ad5-infected RCEC, it is pertinent to explore the expression and underlying mechanisms o f regulation o f other transporters, such as glucose transporter, monocarboxylate transporter, and nucleoside transporter in the Ad5-infected conjunctiva. 5. Other viral infection models Herpes virus infection in the lacrimal gland, conjunctiva and cornea is a chronic disease (Kaufman et al., 1968). Cytomegalovirus (CMV) retinitis has been a major cause o f morbidity associated with the AIDS epidemic. CMV retinitis is the most common ocular opportunistic infection and the leading cause of blindness in patients with AIDS (Sharom et al., 1995). Therefore, an optimal therapy against herpes and CMV infection is a necessity. The findings in our research raise an equally interesting possibility for development of in vitro virally-infected cell cultures using herpes and CMV. These allow us to study the effect of diverse viral infections on cellular changes (including regulation of drug efflux pumps), which could potentially affect cellular environment and drug delivery in the conjunctiva. Therefore, the drug efflux pumps should be 249 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. considered for successful therapy in viral and bacterial infections. Such an undertaking would require efforts to explore new in vitro models corresponding to various disease states. 6 . Anion influx mechanisms Our research has demonstrated that some anionic drugs, such as diclofenac, ofloxacin, and flubiprofen, may be mediated by MRP efflux. The question raised is what their influx mechanisms could be. The model compound used in the MRP study, fluorescein, is a hydrophilic compound, which should not be taken up readily by passive diffusion. Fluorescein has been used for evaluating comeal integrity (Macri and Pflugfelder, 2000). Fluorescein staining was shown to be due to staining o f individual cells. No evidence was found to support the contention that fluorescein resides in areas of cell drop out, or that staining was due to filling of intercellular spaces (Wilson et al., 1995). This indicated that this anion could be absorbed by some mechanisms. Horibe et al. (1998) demonstrated that an Na+ -dependent monocarboxylate transporter exists in the conjunctiva and mediates the transport of monocarboxylates such as lactate. In addition, Na+ - dicarboxylate transporter is known to mediate dicarboxylate compounds, such as succinic acid (Chen et al., 1998; Kekuda et al., 1999). Although these transporters favor the transport o f various carboxylate compounds, the transport of other anion compounds by these transporters can not be ruled out. 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Asset Metadata

Creator Yang, Jun (Johnny) (author)

Core Title Drug efflux pumps in rabbit conjunctival epithelial cells

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Health Sciences, Pharmacology,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Lee, Vincent H.L. (committee chair), Hamm-Alvarez, Sarah (committee member), Kim, Kwang-Jin (committee member), Koda, Robert T. (committee member), Shen, Wei-Chiang (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-547949

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Identifier 3094389.pdf (filename),usctheses-c16-547949 (legacy record id)

Legacy Identifier 3094389.pdf

Dmrecord 547949

Document Type Dissertation

Rights Yang, Jun (Johnny)

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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