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Pharmacological regulation of chloride, fluid, and solute transport in the pigmented rabbit conjunctiva

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Pharmacological regulation of chloride, fluid, and solute transport in the pigmented rabbit conjunctiva

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Content Pharmacological Regulation of Chloride, Fluid, and Solute
Transport in the Pigmented Rabbit Conjunctiva
by
Michael Hung-I Shiue
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
in Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
May 2001
Copyright 2001 Michael H. I. Shiue
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UM1 N umber: 3027778
Copyright 2000 by
Shiue, Michael Hung-I
All rights reserved.
UMI
UMI Microform 3027778
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
U niversity Park
LOS ANGELES, CALIFORNIA 900894695
This dissertation, w ritten b y
ftunt, - X . svG____________
U nder the direction o f h..).5.. D issertation
Com m ittee, and approved b y a ll its members,
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 OFPHILOSOPHY
Dean o f Graduate S tu dies
D ate May 1 1 , 2001
DISSER TA TION COMMITTEE
n Clm m erscm C \
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Michael Hung-I Shiue Vincent H. L. Lee, Ph.D.
ABSTRACT
PHARMACOLOGICAL REGULATION OF CHLORIDE, FLUID, AND SOLUTE
TRANSPORT IN THE PIGMENTED RABBIT CONJUNCTIVA
The physiological roles of conjunctiva are not fully understood. It was
hypothesized that in addition to being a passive protective barrier, conjunctiva can also
regulate electrolyte and fluid balance in the microenvironment of its mucosal surface, and
to serve as a potential route for drug delivery to the posterior segment of the eye
following topical drug instillation. The purpose of this project was three-fold: (1) to
identify and characterize the conjunctival active CF secretory properties; (2) to determine
conjunctival net fluid secretion and whether or not it was driven by active Cl“ secretion;
and (3) to examine the possibility of using conjunctival fluid flow as a vehicle for the
delivery of hydrophilic compounds. Utilizing various electrophysiological techniques
such as Ussing-type chambers, patch clamp, and double-capacitance probes, the net
serosal-to-mucosal CF secretion across the conjunctiva and its regulatory factors (cAMP,
Ca2 + , and PKC) were demonstrated, and respective CF channel types (CFTR, Ca2 + -, and
PKC-regulated CF channels) were identified in the conjunctival epithelium. The
conjunctiva was found to be fluid secreting under normal physiological conditions, and
such fluid movement was proven to be driven by active Cl" secretion. Finally, the effects
of osmotically-driven fluid flow on transconjunctival permeability to lipophilic and
hydrophilic compounds of various sizes was tested. The results revealed that the
permeability of hydrophilic compounds was significantly influenced by the fluid flow,
while the lipophilic compound permeability was not affected by the osmotic gradient. In
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summary, the findings in this project have provided deeper understanding for the
conjunctival physiology, and established the foundation for future development of
pharmacological treatment methods to utilize conjunctival fluid secretion as an alternative
means of increasing moisture and mucus hydration in the anterior segment of the eye.
(Supported by NIH grants EY10421, HL38658, HL46943, and University of Southern
California Charles and Charlotte Krown Fellowship)
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ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to my advisor, Dr. Vincent H. L. Lee,
for his tireless guidance and constant support during my graduate career. I thank him
for believing in me and training me how to confront challenges with fearlessness, and
turn obstacles into stepping stones.
I would like to thank Dr. Kwang-Jin Kim for his invaluable scientific inputs and
lending me an ear when I was overwhelmed with frustration. I will truly miss those
enlightening conversations during our Saturday lunch meetings.
This project would not have been possible without the technical assistance and
constructive feedbacks provided by my guidance and dissertation committee
members: Dr. Michael B. Bolger, Dr. Louis Byerly, and Dr. Curtis T. Okamoto.
My sincere appreciation goes to my lab-mates in the past and the present with whom
I had the pleasure to work with. They provided enthusiastic scientific discussions
and comic relief during hard times which made my graduate life a memorable one.
I am truly thankful to Andrea, who stood by me over the years and offered me
emotional support and encouragement along my long and winding journey.
Finally, I am forever grateful to my parents and sister for their sacrifice,
understanding, and unconditional love and support. Their encouragement for me to
strive for higher achievements has always been my major source of momentum.
This dissertation project was supported in part by NEI grant EY10421 (VHLL), NIH
grants HL38658 and HL46943 (KJK), and University of Southern California Charles
and Charlotte Krown Fellowship (MHIS).
ii
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF SCHEMES xiv
I. INTRODUCTION 1
1.The conjunctiva 2
2. Evolution of the conjunctival ion transport studies 4
3. Various Cl" channels found in ocular epithelia 9
3.1. cAMP-regulated Cl" channels 10
3.1.1. Cystic fibrosis transmembrane regulator
(CFTR) 12
3.1.2. Biophysical properties of CFTR 15
3.2. Ca2 + -regulated Cl" channels 16
3.2.1. The CLCA (CaCC) family 17
3.2.2. Biophysical properties and detection of
Ca2 + -regulated Cl" channels 19
3.2.3. Mechanism of Ca2 + -regulated Cl' channel
activation 20
3.3. PKC-regulated Cl" channels 21
3.3.1. Conjunctival PKC isoforms 22
3.3.2. Biophysical characteristics of PKC-dependent
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Cl' channels 23
3.3.3. Transient stimulation of Cl" secretion by Ca2 + and
PKC stimulating agents 23
4. Possible etiological involvement of conjunctiva in dry eye syndrome 26
4.1. Potential importance of conjunctival active CF secretion in
fluid transport 26
4.2. Possible connection between conjunctival Cl" secretion and
dry eye syndrome 27
4.3. Possible role of conjunctival fluid transport in drug delivery 27
II. STATEMENT OF THE PROBLEM 32
Central Hypothesis 33
Specific Aim #1: To determine the CF secretory property of
conjunctival epithelium, and to identify and characterize different CF
channel types in the conjunctival epithelium (i.e., cAMP-, Ca2 + -, and
PKC-modulated Cl" channels) 33
Specific Aim #2: To determine the fluid secreting nature of the
conjunctival epithelium, and to verify its link to active Cl" secretion 37
Specific Aim #3: To explore the feasibility of utilizing conjunctival
fluid transport as a means of ocular drug delivery 40
HI. MATERIALS AND METHODS 42
1. Animal model 43
1.1. Intact tissues 43
1.2. Conjunctival epithelial cell culture 43
iv
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2. Chemicals 44
2.1. Active ion transport modulators 44
2.2. Cellular transport tracers 44
3. Buffer ingredients 51
3.1. Bicarbonate Ringer’s solution (BR) 51
3.2. Patch clamp solutions 51
3.2.1. Whole-cell patch clamp recording 51
3.2.2. Single-channel patch clamp recording 52
4. Methods 52
4.1. Voltage clamp/Ussing-type chamber settings 52
4.1.1. Bioelectric parameter measurements 52
4.2. Measurement of 3 6 C 1 fluxes 54
4.2.1. Determination of baseline net CP secretion and
cAMP-, Ca2 + -, and PKC-sensitive CP secretion 54
4.2.2. Flux ratio analysis 55
4.3. Patch clamp recordings 56
4.3.1. Electronic setup 56
4.3.2. Microelectrode fabrication 58
4.3.3. Whole-cell recording 59
4.3.4. Reversal potential 60
4.3.5. Single channel recording 61
4.3.6. Signal conversion and data analysis 62
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4.3.6.1.1-V curve and single channel conductance 62
4.3.6.2. Open channel probability 62
4.4. Western blot analysis 63
4.4.1. Conjunctival epithelial cell lysate preparation 63
4.4.2. Detection of conjunctival CFTR 64
4.5. Intracellular Ca2 + measurement 64
4.6. Measurement of fluid transport 66
4.6.1. Epithelial fluid flux detection techniques 66
4.6.2. Determination of transconjunctival fluid transport 69
4.6.3. Osmotic water permeability (Pf) 74
4.7. Influence of conjunctival fluid flux on solute transport 74
4.8. Statistical analysis 75
IV. RESULTS 76
1. Regulation of conjunctival active ion transport processes by cAMP-,
Ca2 + -, and PKC 77
1.1. Stimulation of Isc 77
1.2. Additive stimulation of Isc by 8-Br cAMP, A23187, and
PMA 79
2. Evidence of conjunctival net CF secretion and its modulation by
cAMP, Ca2 + , and PKC 81
2.1. Baseline net Cl' secretion across isolated conjunctiva 81
2.2. Stimulation of net Cl' secretion by cAMP-, Ca2 + -, and PKC-
activating agents 81
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2.3. Inhibition of net CF secretion by CF channel blocker and
Na+-K+ -2CF cotransport inhibitor 85
2.4. Hill analysis of 8-Br cAMP dose-dependent changes in
conjunctival Isc and CF flux 85
2.5. Evidence for active CF transport - flux ratio analysis 87
3. Detection of specific conjunctival CF channel types 87
3.1. Demonstration of cAMP-sensitive whole-cell CF current 87
3.2. Stimulation of whole-cell conductance by A23187, PMA 90
3.3. Determination of conjunctival whole-cell reversal potential 92
3.4. Detection of single cAMP-, Ca2 + -, and PKC-regulated CF
channel activities 93
3.4.1. cAMP-activated single-channel conductance and
open probability 93
3.4.2. Ca2 + -activated single-channel conductance and
open probability 95
3.5. Conjunctival CFTR detection 99
4. Effect of forskolin on conjunctival fCa2 + ], 100
5. Conjunctival fluid secretion 101
5.1. Baseline fluid secretion and its stimulation 101
5.2. Demonstration of the link between fluid and CF transport 104
5.3. Effect of active Na+ absorption 106
5.4. Effect of osmolality 108
6. Effect of conjunctival fluid flux on solute transport 108
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6.1. Baseline Pa p p 108
6.2. Effect of hypotonicity 108
6.3. Effect of hypertonicity 111
6.4. Correlation between solute and fluid fluxes 111
V. DISCUSSION 113
1. Cyclic AMP-, Ca2 + -, and PKC-regulation of the conjunctival active
CF secretion 114
1.1. Evidence of baseline active Cl" secretion across intact
conjunctiva 114
1.2. Conjunctival Cl' entry mechanisms 116
1.3. Stimulation of cAMP-, Ca2 + -, and PKC-sensitive 3 6 C 1
secretion 117
1.4. Evidence for the existence of three types of Cl" channels in
the conjunctival epithelium 121
2. Detection and characterization of specific conjunctival cAMP-,
Ca2 + -, and PKC-regulated Cl" channels 122
2.1. cAMP-regulated Cl" channels 124
2.1.1. Existence of conjunctival CFTR 127
2.2. Ca2 + -regulated Cl" channels 128
2.3. Possible cross-talk between cAMP- and Ca2 + -dependent
cellular responses 131
2.4. PKC-regulated Cl" channels 132
3. Modulation of conjunctival fluid transport 136
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3.1. Baseline fluid secretion 137
3.2. Cf driven conjunctival fluid secretion 137
3.3. Effect of active Na+ absorption on conjunctival fluid flow 140
3.4. Osmotically driven conjunctival fluid flow 141
3.5. Possible role of conjunctival fluid secretion in the tear film
maintenance 143
4. Enhancement of conjunctival solute transport utilizing fluid
absorption 144
VI. CONCLUSIONS 148
1. Summary of Findings 149
2. Significance of the Findings 151
3. Future Considerations 152
3.1. Conjunctival CF channel physiology 152
3.2. In-depth investigations on conjunctival fluid transport
processes 153
3.3. Contribution of conjunctival fluid secretion in tear film
dynamics 154
3.4. Ion and fluid transport physiology during the dry eye state 155
VII. REFERENCES 158
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LIST OF TABLES
Table
4-1 Effect of serosal addition of bumetanide and mucosal addition of
NPAA, 8-Br cAMP, A23187, and PMA on PD, Pa p p of mannitol,
C f fluxes, and flux ratios for Cf fluxes between s-to-m (Jsm ) and
m-to-s (Jm s) directions
4-2 Single Ca2 + -activated Cf channel amplitude and channel open
probability and their corresponding pipette holding potentials
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LIST OF FIGURES
Figure Page
1-1 Sectional view of the anterior segment of the eye 3
1-2 Active ion and water transport processes in the pigmented rabbit
. conjunctiva 8
1-3 Signal transduction pathways for cAMP-regulated CF channel
activation, and intracellular Ca2 + release and Ca2 + - and PKC-regulated
CF channel activation 11
1-4 Proposed domain structure of CFTR 14
1-5 Protein kinase C isoforms found in the rat conjunctival tissue 25
3-1 Structures and molecular weights of cAMP-dependent CF transport
activators used - forskolin, 1,9-dideoxyforskolin, and 8-Br cAMP 45
3-2 Structures and molecular weights of Ca2 + - and PKC-dependent Cl"
transport activators used - calcimycin, PMA, and UTP 46
3-3 Structure and molecular weight of D-glucose 47
3-4 Structure and molecular weights of ion transport inhibitors used -
bumetanide, NPAA, glibenclamide, and ouabain 48
3-5 Structures and molecular weights of paracellular (3 H-HPMPC,
1 4 C-mannitol, and 3 H-mannitol), and transcellular (3 H-betaxolol)
transport markers used 49
3-6 Structures and molecular weights of fluorescent dyes used - FITC-
dextran and Indo-1 AM 50
3-7 Voltage clamp/Ussing-type chamber settings 53
3-8 Electronic setup of the patch clamp system 57
3-9 Dual capacitance piooc semp used to measure fluid transport across an
isolated rabbit conju cnval tissue 71
xi
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3-10 Water bridge used to balance the fluid level prior to initiation of the
capacitance probe experiment
3-11 Relation between fluid displacement and conjunctival fluid transport
properties
4-1 Time courses of Is c changes in freshly isolated pigmented rabbit
conjunctiva treated with 8-Br cAMP, A23187, and PMA
4-2 Time courses of Is c changes in freshly isolated pigmented rabbit
conjunctiva treated with various combinations of Br cAMP, A23187,
and PMA
4-3 Effects of mucosal A23187 and PMA on Jm s and Jsm at baseline, at the
maximal stimulation, and during post-maximal stimulation
4-4 Dose-dependent changes in Is c and CF flux measured under open-circuit
conditions in the s-to-m direction in the pigmented rabbit conjunctiva
treated with up to 3 mM 8-Br cAMP
4-5 Effect of forskolin and 1,9-dideoxyforskolin on conjunctival whole-cell
currents, and the effect of glibenclamide on forskolin-induced whole-cell
current
4-6 Conjunctival whole-cell CF conductance under baseline, 1,9-
dideoxyforskolin, forskolin, and forskolin + glibenclamide treated
conditions
4-7 Stimulation of conjunctival whole-cell currents by A23187 and PMA
4-8 Reversal potentials of baseline, forskolin-, A23187-, and PMA-
Stimulated I-V curves under symmetrical intra- and extracellular CF
concentrations and reduced extracellular C1‘ concentration
4-9 Typical conjunctival single-channel activities recorded under the cell-
attached configuration
4-10 (a) Conjunctival Ca2 + -activated single CF channel activities at various
pipette holding potentials recorded from the same patch, with a
symmetrical NaCl-based solutions in pipette and bath, under the inside-
out patch clamp configuration, (b) Corresponding all-point histograms
of the tracings in (a).
72
73
78
80
83
86
88
89
91
92
94
96
xii
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4-11 I-V relationship of the single Ca2 + -activated CF channel detected in the
conjunctival epithelium under inside-out patch clamp configuration
4-12 Western blot analysis of CFTR expression in T84 and conjunctival
epithelial cells probed with a C-terminus specific CFTR monoclonal
antibody
4-13 Effect of forskolin on [Ca2+]i in confluent conjunctival epithelial cells
grown on cover slips in a representative experiment
4-14 Typical voltage vs. time tracings demonstrating the effect of
8-Br cAMP, UTP, ouabain, and D-glucose on transconjunctival fluid
secretion
4-15 Conjunctival water flux at baseline and in response to application of
mucosal 8-Br cAMP, mucosal UTP, serosal ouabain, mucosal D-
glucose, mucosal D-mannitol, CF-free, mucosal hypertonic, and
mucosal hypotonic conditions
4-16 Changes in net transconjunctival fluid secretion rate as a function
of mucosal 8-Br cAMP concentration
4-17 Relationship between changes in net fluid secretion and the
corresponding AIS C observed by adding agents known to affect
active C1‘ secretion, Na+ absorption, and osmolality
4-18 Mucosal-to-serosal apparent permeability of mannitol, betaxolol,
HPMPC, and FD-4 under mucosal isotonic, hypertonic, and hypotonic
conditions
4-19 Relationship between mucosal-to-serosal Pa p p for mannitol, betaxolol,
HPMPC, and FD-4 and fluid flux across freshly excised conjunctival
tissue under mucosal isotonic, hypertonic, and hypotonic conditions
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LIST OF SCHEMES
Scheme
2-1 Logistic experimental sequence of Specific Aim #1
2-2 Logistic experimental sequence of Specific Aim #2
2-3 Logistic experimental sequence of Specific Aim #3
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I. INTRODUCTION
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1. The Conjunctiva
The conjunctiva is a thin, mucus secreting tissue lining the inside of the
eyelids and part of the anterior sclera, and terminates where the cornea begins. The
conjunctival epithelium can be divided into three regions: (1) bulbar conjunctival
epithelium, which is contiguous with the limbal zone of corneal epithelium; (2)
fornix epithelium, which is lining the conjunctival sac, or the “folding region” of the
inner eyelid; and (3) palpebral epithelium, which is contiguous with the epidermis of
the eyelid (202). The conjunctiva has long been thought to serve two primary
functions: (a) as a passive physical protective barrier and (b) in the maintenance of
tear film stability by the mucus secreted by the resident goblet cells (170,202).
Efforts have been made in recent years in our laboratory to investigate the feasibility
of utilizing conjunctiva as a route for drug delivery to the posterior segment of the
eye following topical drug instillation, and to further understand the physiology of
this tissue to gain the fundamental knowledge necessary to ultimately regulate
electrolyte and fluid balance in the microenvironment of its mucosal surface.
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Lacrimal gland
Fornix
conjunctiva
Bulbar _
conjunctiva
Sclera
Cornea
Palpebral
conjunctiva
Figure 1-1. Sectional view of the anterior segment of the eye.
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2. Evolution of the conjunctival ion transport studies
In 1973, Maurice measured the in vivo exchange of 2 2 Na and 3 6 C1 between the
blood and fluid across the albino rabbit conjunctiva (130). He also measured the
conjunctival potential difference in the rabbit and the human eye, although the liquid
junction potentials were not properly controlled. Surprisingly, for the next 20 years
there have been few in-depth studies on the conjunctival ion transport processes or
the potential roles played by the conjunctiva in fluid and electrolyte balance. In
1993, Kompella et al. (107,109), using the voltage-clamp technique, demonstrated
that the pigmented rabbit conjunctival epithelium is capable of active ion transport.
The baseline bioelectric parameters were as follows: transepithelial resistance (Rt),
which is an indicator of the tissue integrity, was 1.3 ± 0.08 kO.cm ; potential
difference (PD), the voltage difference between the two sides of the tissue, was 17.7
± 0.8 mV (lumen negative); and short-circuit current (Isc), which represents the sum
of all active ion transport processes, was 14.5 ± 0.8 pA/cm2. The investigation
concluded that approximately 70% of the conjunctival short-circuit current (Isc ) is
contributed by active CF transport, based on a 70% inhibition of Isc in the presence of
mucosal NPAA (a CF channel blocker), and under mucosal (m) and serosal (s) CF-
free condition.
Since then, the ion transport properties of the conjunctiva have been extensively
investigated in our laboratory (83,108,165). The conjunctival ion transport
4
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mechanisms identified to date were found to be similar to that in the comeal
epithelium (Fig. 1-2). Prior to the current project, Kompella et al (61) reported an
inhibition of Is c by mucosally applied NPAA and Cl" removal from both the s and m
reservoirs and hypothesized that conjunctiva is capable of net Cl" secretion from the
basolateral to apical side through apically located Cl' channels. Furthermore, they
observed an abolishment of conjunctival Is c in the presence of serosally instilled
ouabain and bumetanide, indicating the possible existence of a basolaterally located
Na+ -K+ -ATPase and a Na+ -K+ -2C1" cotransporter, respectively (109,172). In
addition, a K+ channel was suggested to reside on the basolateral side based on the
observed inhibition of Isc by BaCl2, a K+ channel inhibitor. However, the amiloride-
sensitive Na+ -conductive pathway, a commonly found epithelial Na+ absorption
pathway, was not detected in the conjunctival epithelium (107).
Several Na+ -coupled solute transport processes were found to be located on the
apical side of the conjunctival epithelium (Fig 1-2). Kompella et al.( 108) observed a
dose-dependent increase in conjunctival Is c when L-arginine (up to 44%, 0-0.5 mM),
D-arginine (up to 35%, 0-0.3 mM), glycine (up to 33%, 0-5 mM), and L-glutamic
acid (up to 4%, 0-5 mM) were applied to the mucosal side of the isolated rabbit
conjunctival tissue. The stimulation induced by these amino acids was not observed
when Na+ was omitted from the mucosal reservoir. These results suggested that a
Na+ -dependent amino acid transporter resides on the apical surface of the
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conjunctival epithelial cell. The apparent existence of a conjunctival Na+ -amino acid
cotransporter was further supported by Hosoya et al. (82). They observed a Na+ and
• 5
temperature dependent, saturable (over 0.01-10 mM) m-to-s transport of H-L-
arginine, and stimulation of m-to-s 2 2 Na flux in the presence of 1 mM L-arginine.
Their study further predicted a 1:1 coupling between Na+ and L-arginine based on the
Hill analysis of L-arginine transport at 0.1 mM in the presence of varying Na+
concentrations in the mucosal bathing fluid. In addition, a Na+-dependent glucose
transporter was also suggested to be present on the apical side of the conjunctival
epithelium. The first evidence was provided by Hosoya et al. (83), when a dose-
dependent elevation of conjunctival Is c was observed in the presence of mucosal D-
glucose, and a reduction of Is c was observed when a Na+ -glucose cotransport
inhibitor, phlorizin, was mucosally applied. These observations were not made when
these compounds were added serosally, or in the absence of Na+ in the bathing
solution. In-depth investigation conducted by Horibe et al. (79) demonstrated the
existence of a conjunctival Na+ -glucose cotransporter by directly measuring 2 2 Na and
3 H-3-0-methyl-D-glucose (3-O-MG) fluxes across intact rabbit conjunctiva. They
observed a net m-to-s 2 2 Na absorption in the presence of 5 mM glucose in the buffer,
and such an absorption was abolished by serosally instilled ouabain. Moreover, net
Na+ absorption was reduced by 40-60% under glucose-free condition or in the
mucosal presence of 0.5 mM phlorizin.
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The above findings on conjunctival active ion transport processes raised the question
of whether or not movement of these ions are capable of creating an osmotic gradient
to drive fluid across the conjunctival epithelium. A recent study conducted by
Hamann et al. (73) indicated that one of the water channel isoforms, aquaporin type
III (AQP3) does exist in both the human and rat conjunctival epithelium. However,
the localization of AQP3s, their regulation, and their actual involvement in fluid
movement across conjunctiva remain to be determined.
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Apical
^ f lig h t junction J
Basolateral
Aquaporin
Amino Acid
Na+
Glucose
T T
Solute — .
Na+^ -
r
h2 o
K+
Aquaporin?
H,0
Figure 1-2. Active ion and water transport processes in the pigmented
rabbit conjunctiva.
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3. Various types of epithelial Cl' channels
Chloride channels residing in the apical membrane of the epithelial cells play
an essential role in the secretion of fluid and electrolytes (61). The intracellular CF
was accumulated through basolateral membrane active ion transport processes such
as Na+ -K+ -2CF cotransports driven by electrochemical gradient (203). The
intracellular CF exits through various families of CF channels that respond to
different regulatory “cues” such as changes in concentrations of intracellular second
messengers (e.g., cAMP and Ca2 + ), changes in cell volume, phosphorylation of
protein kinases (e.g., PKA and PKC), to name a few. Although different types of CF
channels have been identified and characterized molecularly and biophysically, some
of the most dynamic questions regarding these CF channels remain unanswered: what
is the level of cross-talk between different families of CF channel? Is there any kind
of functional compensatory effect among CF channels responding to different
triggering signals? If so, to what extent? under baseline conditions, are all CF
channel types involved in the exit of CF ions? Nevertheless, these questions are
beyond the scopes of this study. The current investigation focuses on the detection
and characterization of some of the major types of epithelial CF channels (i.e.,
cAMP-, Ca2 + -, and PKC-regulated) in the conjunctiva. The combined activities of
these CF channels can be a major driving force for the fluid secretion across this
tissue.
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3.1. cAMP-regulated C l" channels
The c AMP-regulated CF channel is one of the first Cl" channels cloned and
perhaps the most commonly investigated Cl" channel in epithelial cells (61), and its
activation is dependent on protein kinase A (PKA) phosphorylation (183) (Fig. 1-
3A). Cyclic AMP-regulated Cl" channels have been detected in various cell types
including corneal epithelial cells (24,104), bronchial epithelial cells (113), T84 cells
(35), and HT-29 cells (188). Investigators have observed an increase in either net Cl
fluxes or Cl" currents utilizing patch clamp techniques under cAMP stimulating
conditions (38,132,184). Furthermore, studies conducted by Kompella et al. have
also demonstrated the stimulatory effects induced by forskolin and (3-adrenergic
agonists such as epinephrine and terbutaline on rabbit conjunctival active ion
transport processes (as indicated by Isc ) (109).
10
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A.
Extracellular Space
P-ADRENERG1C
RECEPTOR
Cl-
i i
In
Q
iiifimwniiw « ififT T m T iT iT iiT ifi
m i
G T P^ A T P
G D P
PH O SPH OD IESTERA SE . -
5 -A M P --------------- , ------ cAMP
A
m u Hi
PKA
y
Cytosol
A
y
5000
B.
Extracellular Space C l"
ill
O1
activated receptor
* 1
PIP.
GTP
GDP
Cl-
Cytosol
C a ^ -c h a rm e !
Figure 1-3. Signal transduction pathways for cAMP-regulated Cl' channel
activation (A), and intracellular Ca2 + release and Ca2 + - and PKC-regulated
Cl' channel activation (B).
11
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3.1.1. Cystic fibrosis transmembrane regulator (CFTR)
Cystic fibrosis (CF) is clinically observed to be a genetic disease that
primarily affects organs lined by epithelia, such as the airways, intestine, pancreas
and sweat gland (105,208), resulting an abnormal salt absorption and fluid secretion
in these tissues due to impaired active Cl" secretion. The link between CF and
epithelial Cl" secretion was initially established when abnormal concentration of Cl"
was detected in the sweat of patients with CF (208). This finding led to a series of
investigations on both normal and CF airway Cl" conductance via patch clamp
techniques (62,204), and identification and cloning of the gene that encodes CFTR
(154). The loss of cAMP-regulated Cl" permeability was observed in the epithelia
mentioned above, which are most significantly affected by CF (148,204). Riordan et
al. (154) later speculated that CFTR is likely to be an ion channel, based on its
primary amino acid structural properties, and it is the dysfunction of CFTR that
causes CF.
Although it is not clear whether there are different families of cAMP-regulated Cl"
channel, CFTR is the only cAMP-regulated Cl" channel cloned to date. It is a
polypeptide of 1,480 amino acids, with a molecular weight of 168,138 daltons (154).
It is organized into five domains: (a) two membrane-spanning domains, each having
six putative transmembrane segments (TM); (b) two nucleotide-binding domains,
NBD1 and NBD2 regions that are thought to form an integral part of the nucleotide-
12
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binding pockets and controls channel activity through interaction with cytosolic
nucleotides (197); (c) a regulatory (R) domain, possessing many charged amino acid
residues (lysine, arginine, histidine, aspartic acid, and glutamic acid), which can be
phosphorylated, usually by cAMP-dependent protein kinase, and it is essential for
channel opening (9,31,154,210) (Fig. 1-4).
13
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MSD1
MSD2
oooooooc
l l l l l l l l l l l l
A U M
>
• ^ v 'YV
TM12
m m i
Figure 1-4. Proposed domain structure of CFTR (modified from Welsh et al.
(208)). TM = transmembrane segment; MSD = membrane-spanning domain;
NBD = nucleotide-binding domain; R = regulatory domain.
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3.1.2. Biophysical properties of CFTR
The biophysical properties of CFTR reported by different laboratories are
fairly consistent (9). In brief, the CF current measured from wide spectrum of cell
types, under different patch clamp configurations (i.e., whole-cell, cell-attached,
inside-out, and outside-out), indicated that: (1) CFTR is a CF channel with low
conductance; (2) has a linear I-V relationship; (3) it is more selective for anions than
for cations; and (4) the channel open-state probability is neither voltage nor time
dependent (9,113,168,191). Single channel recording under cell-attached or excised,
inside-out patches using recombinant CFTR in either cells or lipid bilayers revealed
that the single channel conductance is relatively low, averaged from 7 to 10 pS, and
with an anion permeability of CF over F (9,16,46,168). Both endogenous and
recombinant CFTR were found to be stimulatory by cAMP (8,9,113,159,168).
Application of cAMP elevating agents such as forskolin, IBMX, and cAMP analogs
like 8 -BrcAMP and CPT-cAMP significantly stimulated the CFTR CF conductance
(9,168). For the inhibition of CFTR conducting CF currents, one characteristic
observation is that CFTR can be blocked by high concentration (1 mM) of
diphenylamine-2-carboxylate (DPC), but not TS-TM-Calix-4 or DIDS, a substance
known to be inhibitory to several types of epithelial CF channels, including volume-,
cAMP-, and Ca2 + -regulated CF channels (2,21,36,37,95,100,168). Furthermore,
CFTR was blocked by 5-nitro-2-(3-phenylpropyiamino) benzoic acid (NPPB), and
sensitive to other Cl" channel blockers such as Znz+ (117) and IAA-94 (115). Also,
15
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micromolar of glibenclamide has recently been reported to have non-specific
inhibitory effect on CFTR as well (149).
3.2. Ca2 + -regulated Cl' channels
Another type of CF channel, which is modulated either directly or indirectly
by Ca2 + , has been observed in numerous cell types such as rat lacrimal gland acinar
cells (53,143), T84 cells (35), human airway epithelial cells (63,93), human colonic
cells (HT-29) (135), parotid acinar cells (11,59), gallbladder epithelia (70), sweat
gland cells (167), guinea pig and feline ventricular myocytes (39), and Xenopus
oocytes (endogenous Cl' channels) (12,211), to name a few. The activation of these
CF channels was detected when intracellular Ca2 + concentration was elevated by
agents such as A23187 (Ca2 + ionophore) at <10 |iM and thapsigargin (Ca2 + -ATPase
inhibitor) at <2 pM. Cl conductance was shown to be stimulated by muscarinic
agonist, and it was further induced by intracellular dialysis, under the whole-cell
patch clamp configuration, with nonhydrolyzable GTP analogs or inositol-1,4,5-
triphosphate (IP3). These effects were postulated to be the result of increased Ca2 +
release from intracellular stores such as endoplasmic reticulum, as well as increased
Ca2 + permeability through the plasma membrane (54,121).
In CF-affected airway and sweat gland cells, the Ca2 + -regulated CF channel
remains fully functional (6,9,10,63,167). This suggests that Ca2 + - and cAMP-
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regulated Cl" channels, at least in airway and sweat gland cells, are independent
channel types. However, there are still uncertainties about the relationship between
cAMP- and Ca2 + -regulated Cl" channels, since in CF intestinal cells, both pathways
were found to be defective (9,18).
3.2.1. The CLCA (CaCC) family
Several members of the Ca2 + -regulated Cl" channel (CLCA or CaCC) family
have been cloned and functionally studied (3,45,66,71,90). hCLCAl was cloned and
characterized both molecularly and functionally in human intestinal basal crypt
epithelia and goblet cells by Grubers et al. in 1998 (71). hCLCAl is a polypeptide of
914 amino acids, with a molecular weight of 125,000 daltons. Biophysical properties
were determined utilizing hCLCA1 -transfected HEK 293 cells via various patch
clamp configurations. Under the presence of extracellular 2 pM ionomycin and 2
mM Ca2 + , an outwardly rectifying, 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid
(DIDS), dithiothreitol (DTT), and niflumic acid (NFA) inhibitable whole-cell current
was activated. Single-channel analysis performed under the cell-attached
configuration revealed a slope conductance of 13.4 pS in the presence of
extracellular 2 pM ionomycin and 1 mM Ca2 + . In addition to hCLCAl, two other
members of the human CLCA family have been cloned by Agnel et al. (3): hCLCA2
and hCLCA3. Similar to hCLCAl, both hCLCA2 and hCLCA3 were found
primarily in the digestive tract. However, hCLCA2 was also found to be distributed
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throughout various regions of the brain and the spinal cord, and both hCLCA2 and
hCLCA3 were expressed in the airway epithelia (3). Functional characteristics of
hCLCA2 and hCLCA3, however, have not been investigated yet.
Members of the CLCA family have also been cloned in other species such as bovines
and mice (45,66,90). In fact, the first CLCA protein was isolated and cloned from a
bovine tracheal cDNA expression library using an antibody probe (45). bCLCA (or
referred to bCaCC by the founding group) is similar to hCLCA family both
molecularly and biophysically. It consists of 903 amino acids with a molecular
weight of 140,000 daltons under reduced conditions. The presence of extracellular
ionomycin and high Ca2 + concentration activated a DEDS and DTT-sensitive whole
cell anion current (45). The single-channel conductance of the planar lipid bilayer
incorporated bCLCA, however, exhibited a larger, linear conductance of 25-30 pS as
compared to hCLCAl (71,90). CLCA has also been cloned from a mouse lung
cDNA library (mCLCAl) with high sequence homology with hCLCAl and bCLCA 1
(6 6 ). The size (125,000 daltons at the glycosylated state) and the sensitivity to
various inhibitors all resembled those observed in hCLCAl and bCLCA1 (66,71,90).
In additional to the molecular and functional similarities of CLCA family among
different species mentioned above, the Kyte-Doolittle (hydrophobicity) analyses
predicted four transmembrane domains for hCLCAl, bCLCA 1, and mCLCA 1
(45,66,71).
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3.2.2. Biophysical properties and detection of Ca2 + -regulated Cl" channels
In addition to the known members of the CLCA family that have been well
characterized, Ca2 + -regulated CF channels were detected electrophysiologically on
the apical membrane of numerous cell types. The biophysical properties of these
channels were often examined by different patch clamp configurations, under
increasing intracellular or cytosolic surface Ca2 + concentration, or in combination
with channel blockers (9,11,33,135). One method of determining the existence of
Ca2 + -activated Cl' channels on the apical membrane is to permeabilize the basolateral
side of the polarized cell monolayer with (ig/ml range of nystatin (9), and study the
apical membrane in isolation, similar to the technique described earlier for the
detection of apical CFTR conductance.
The characteristics and the modulation pathways of Ca2 + -mediated Cl' conductance
could vary between cell types. For example, Alton et al. (6 ) reported a rectifying
profile of Ca2 + -regulated CF channels found in sheep tracheal epithelial cells, with a
single-channel conductance of 107 pS at the negative potentials and 67 pS at the
positive potentials. On the other hand, Welsh (205) detected a single Ca2 + -sensitive
Cl" channel conductance of 29 pS at negative voltages in canine tracheal epithelial
cells. It is possible that this difference in observation was either due to species
variations or detection of different types of Ca2 + -regulated CF channel in these
investigations.
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Despite these differences, a few biophysical parameters remain common among these
CF channels in different cells. First, Ca2 + -activated CF channels were determined to
be more permeable to F than to CF (9,39) (also in the case of CLCA family). This
was determined by either whole-cell or excised membrane patch with inner
(cytosolic) anion substitution, then compared the conductances of the anions.
Second, the whole-cell I-V relationship of Ca2 + -mediated conductance is generally
linear briefly at the beginning ( - 1 0 ms), then becomes outwardly rectifying towards
the end of the pulse (191). This outwardly rectifying current was observed under
standard whole-cell patch clamp studies (9,10,63,167,191) and perforated patch
clamp studies (81), using different Ca2 + concentrations in the pipette, under
increasing intracellular Ca2 + concentration by superfusing Ca2 + ionophore, or by
acetylcholine (ACh) treatment to release Ca2 + from intracellular stores (196). This
9-+-
current was not observed when intracellular Ca was chelated with EGTA (61). As
mentioned earlier, DPC, but not DIDS, was able to block CFTR. However, Ca2 + -
activated CF current was observed to be blocked by both DIDS and DPC (9,168).
3.2.3. Mechanism of Ca2 + -regulated Cl' channel activation
For most cell types, there are two sources of increasing intracellular Ca2 + : (1) Ca2 +
release from intracellular stores (endoplasmic reticulum), and (2) Ca2 + entry across
the plasma membrane (61). The triggering of Ca2 + release from the endoplasmic
reticulum (ER) starts from the binding of an activated membrane receptor to the G
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protein, releasing the a subunit of the G protein to activate the phospholipase C that
cleaves PIP2 into B P 3 and diacylglycerol (DAG). IP3 is directly responsible for the
releasing of Ca2 + ions from the ER, and DAG activates PKC in the presence of Ca2 +
to activate other cellular events (4). Whole-cell patch clamp studies performed on
isolated lacrimal gland acinar cells showed that intracellular dialysis with non-
hydrolyzable GTP analogs or IP3 enhances the C1‘ conductance generated by
muscarinic agonists (53). It is not clear, however, whether Ca2 + ions activate Cl"
channels via a direct ligand-type interaction, or through other Ca2 + -dependant
cytoplasmic components such as calmodulin.
3.3. PKC-regulated CF channels
PKC-modulated CF transport (Fig. 1-3B) has been reported in rat comeal
epithelial cells (44), in addition to rat epididymal epithelial cells (29), guinea-pig
ventricular myocytes (174), and guinea-pig hepatocytes (112). In these studies, the
application of PKC stimulator, 0.1-1.0 pM phorbol 12-myristate 13-acetate (PMA),
stimulated both whole-cell CF current (29,112) and tsc (29;44). PMA stimulates
PKC by mimicking the effect of diacylglycerol (DAG) in the signal transduction
pathway (4). A recent report indicated that multiple PKC isoforms were found to
exist in the conjunctiva (47). However, no information is available on whether or not
all PKC isoforms are capable of regulating CF channels, or if a PKC-regulated CF
channel can be triggered by multiple PKC isoforms.
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3.3.1. Conjunctival PKC isoforms
Three categories of PKC isoforms have been identified in the rat conjunctiva
(47): classical (a, J3 I, J3 1 I, and y), novel (8 , e, r), 0, and jit), and atypical (£ and X).
Classical PKC isoforms consist of two DAG binding sites, one Ca2 + binding site, and
two ATP and protein substrate binding sites (Fig. 1-5). The structure of novel PKC
2 i
isoforms is similar to that of the classic PKC isoforms, but lacking Ca binding site.
Atypical PKC isoforms, on the other hand, contain only one DAG binding site, and
two ATP and protein substrate binding sites. The activation of the majority of the
PKC isoforms mentioned above appears to be Ca2 + independent, based on the lack of
a Ca2 + binding site in their structure (i.e., novel and atypical isoforms). Furthermore,
it is not known whether or not the type(s) of PKC isoform that is regulating the CP
channels is the same in all epithelia. Several studies have investigated the
involvement of intracellular Ca2 + on PKC-dependent CF channel activation.
Donowitz et al. (50) reported an increase in s-to-m net CF flux when the rat
descending colon mounted in the Ussing chamber was treated with 0.1 pM phorbol
dibutyrate (PDB). Such stimulation, however, was abolished when the tissue was
serosally treated with verapamil and Ca2 + -free bathing solution but not by datrolene.
Based on these observations they concluded that extracellular Ca2 + but not
intracellular stored Ca2 + is essential for the PDB-induced CF secretion.
2 2
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3.3.2. Biophysical characteristics of PKC-dependent Cl" channels
The electrophysiological properties of PKC-dependent Cl" channels have
been determined via techniques such as Isc measurements (13,38,112) and patch
clamp recordings (174;181). The (3-phorbol esters such as 12-Otetradecanoyl-
phorbol-13 -acetate (TP A), PM A, and PDB are commonly applied in the nanomolar
to micromolar range to study PKC-dependent cellular activities. The reported single
channel conductances of the PKC-dependent CF channels were generally small (-10
pS) (13,38,112). The anion selectivity of the PKC-dependent CF channels has been
investigated by several groups (156,174,198). Although different groups tested
different anions, their findings all seemed to reveal an anion selectivity sequence of F
> Br" > CF.
3.3.3. Transient stimulation of C l" secretion by Ca2 + and PKC stimulating agents
Ca2 + and PKC activation induce transient stimulation of Is c in rat epididymal
epithelium (29), rabbit comeal epithelium (44), and equine sweat gland secretory
epithelium (106). Such transient stimulation of ion transport induced by Ca2 + and
PKC activating agents has frequently been reported (29,89,189,206). The
mechanisms for the decrease in ion transport by Ca2 + and PKC stimulating agents
following maximal stimulation are not fully understood. Recent studies conducted
by Barrett’s group in T84 cells suggested D-myoinositol 3,4,5,6 -tetrakisphosphate
(Ins(3 ,4 ,5 ,6 )P4) as a negative messenger in blocking Ca2 + -regulated CF channels
23
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following the rise in intracellular Ca2 + level (89,189). Furthermore, 0.1 pM PMA
has also been shown to inhibit the basolateral Na+ -K+ -ATPase (17) and Na+ -K+ -2C1
cotransporter (34) in A6 cell and alveolar epithelium, respectively. Calcium and
PKC have also been reported to inhibit K+ channels (176). These factors may also
contribute to the reduction in CF secretion after maximal stimulation induced by
PMA.
2 4
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Ca2 + binding site
ATP and protein substrate
binding sites
DAG binding sites
Classical PKC: a, (3 1 , (311, y
A /
\w
Novel PKC: 5, e, ri, 0, p
-a
Atypical PKC:
Figure 1-5. Protein kinase C (PKC) isoforms found in the rat conjunctival
tissue (47).
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4. Possible etiological involvement of conjunctiva in dry eye syndrome
4.1. Potential importance of conjunctival active Cl' secretion in fluid transport
In salt-secreting epithelial cells, Cl' is considered to be the “driving ion” (61).
Actively transported Cl" electrically couples with passively diffused Na+ through
paracellular route creates an osmotic gradient and a driving force for water transport
through paracellular (102) and transcellular (through water channels) (60,74)
pathways. For example, the diffusional water flow across the frog cornea measured
3 4 -
by H-water fluxes was reported to be approximately 1.8 x 10' cm/sec and the rate
was further increased with increasing Cl" flux (accounting for 50% of total active ion
transport at baseline) (26). Furthermore, active Cl" transport is the primary factor in
regulating the cell volume via transcellular water movement (20,61,76). It is likely
that next to the lacrimal glands, the conjunctiva rather than the cornea may play a
more prominent role than the cornea in regulating fluid balance in the tear film for
the following three reasons: (1) The higher contribution of active Cl" (driving ion)
transport (70% in conjunctiva and 50% in cornea) to the overall active ion movement
than the corneal epithelium (Is c = 14.5 ± 0.8 and 12.3 ± 1.1 pA/cm2 in conjunctiva
and cornea, respectively) (26,107); (2) Much leakier nature of conjunctival tissue
compared to the cornea (~ 6 times lower in transmembrane resistance); and (3) The 9
and 17 times greater surface area afforded by the conjunctiva than the cornea in the
rabbit and the human, respectively (2 0 1 ).
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4.2. Possible connection between conjunctival Cl' secretion and dry eye
syndrome
The dry eye syndrome is characteristic of several chronic ocular diseases such
as keratoconjunctivitis sicca, Sjogren’s syndrome, pharyngoconjunctival fever, and
nonspecific follicular conjunctivitis (22). It is characterized by abnormal tear film
production and maintenance on the ocular surface, due to the combined defects of
tear aqueous, oil, mucin and protein secretion (22). Insufficient tear production by
the main and accessory lacrimal glands is believed to be the main etiology (127,145).
However, regardless of the etiology, conjunctival surface alteration is always
observed in dry eye patients (92). These conjunctival abnormalities include change
in conjunctival epithelial cell morphology (1,158), reduction in goblet cell population
(which contributes to the mucin deficiency) (1), and overall lusterless conjunctival
surface (22), Conceivably, the ion transport properties of this tissue are also altered,
thereby affecting transconjunctival fluid transport. Sheppard et al. (171) reported a
significant decrease in tear film volume (measured by the Schirmer test), abnormal
tear composition, and lusterless conjunctiva in patients suffering from CF.
4.3. Possible role of conjunctival fluid transport in drug delivery
Mayersohn and Gibaldi (131) first proposed in the early 70’s that the
osmotically driven fluid flow (i.e., solvent drag) could play a roll in the transport of
hydrophilic compounds across cell layers. Since then, the significance of solvent
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drag on drug transport is under constant debate. Mayersohn and Gibaldi (131)
measured the mucosal to serosal transfer of riboflavin, salicylate, and sulfanilamide
across the everted rat intestine under the influence of isootonic (300 mOsm),
hypotonic (100 mOsm), and hypertonic (500 mOsm) solutions. The results indicated
a significant inhibition (-30-65%) of transfer for all drugs, especially for riboflavin
(63%), in the presence of serosal hypotonic solution. Hypertonic solution (500
mOsm), on the other hand, increased the amount of drug transferred as compared to
the control values. However, such an increase was statistically significant only in the
case of riboflavin (50%).
To better correlate the fluid flow with drug transport under the influence of
osmolality, Ochsenfahrt and Winne (141) studied the simultaneous transport of
tritiated water and urea across rat jejunum. A positive water net flux increased the
absorption rate of tritiated water and of urea by 22 and 41%, whereas a negative
water net flux decreased it by 12 and 32%. Thus far, the transport enhancement of
drugs via the solvent drag effect appears to be restricted to hydrophilic compounds
such as riboflavin (131) and urea (141). Hollander et al. (77) investigated the rat
intestinal and rabbit brush border membrane vesicle transport of a hydrophilic
compound, polyethylene glycol 900 (PEG 900, lipid solubility <0.00079%). They
observed that the net absorption of PEG 900 followed changes in water transport.
Furthermore, based on the kinetic compatibility between PEG 900 intestinal
' ■ - 28
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absorption and simple passive diffusion, and the minimal PEG 900 transport across
the rabbit brush border membrane vesicle, they concluded that PEG 900 transport
was through the paracellular junction with minimal transcellular transport.
While the enhancement of drug transport by the solvent drag effect was widely
reported, the actual contribution of the transepithelial fluid movement in drug
transport was questioned by Nakashima et al. (139). Using a kinetic model based on
the hydrodynamic pore theory of transcapillary exchange that is designed to describe
the changes in volume, osmolality, and drug concentration in the dialysate during
peritoneal dialysis, the peritoneal transport of sulfisoxazole and benzoic acid in rat
was analyzed. The results indicated that the solvent drag effect played a limited role
in the transport of these two compounds. The transport of sulfisoxazole and benzoic
acid was found to be limited by diffusion and blood flow, respectively.
The concept of paracellular drug transport enhancement via a solvent drag effect was
again challenged recently. Karlsson et al. (94) measured the transport of small
hydrophilic compounds (creatinine, erythritol, and foscamet) with different charge
properties across Caco-2 monolayers and rat ileal mucosa in vitro under the influence
of various osmolalities (205, 310, and 510 mOsm). Although an increase in drug
transport was observed in the presence of apical hypotonic solution, the authors
found such transport enhancement coincided with a decrease in transepithelial
29
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resistance. They further provided evidence by utilizing fluorescence and
transmission electron microscopy to demonstrate the dilatation of the paracellular
spaces but no damage to the cell membranes. Based on these results, the authors
suggested that a more pronounced disruption of the epithelial tight junction rather
than stimulation of epithelial water flow is required for efficient enhancement of the
paracellular drug transport.
Among those reports where an increase in drug transport was observed in parallel to
an increase in osmotic-driven fluid transport, different degrees of the solvent drag
phenomenon was involved in the transport enhancement of different compounds. It
seems logical to hypothesize that there may be some type of correlation between
various physical chemical properties of the drugs (e.g., log P value, molecular
weight, charge... etc.) and the contribution of the solvent drag on the paracellular
transport of these dmgs. A recent study by Fagerholm et al. (55) investigated the
sensitivity of the size of hydrophilic compounds (MW 19-4000) to solvent drag (170
and 290 mOsm solution tested) across human {in vivo) and rat {in situ) jejunum.
They reported that in human jejunum, the molecular weight cutoff for the hydrophilic
compounds that can be affected by the solvent drag was approximately 180. In
contrast, such solvent drag-induced increase in small solute flux was not observed in
rats. The low molecular weight cutoff for the solvent drag effect in jejunum seems
dramatically different from the observation made in the rabbit conjunctival tissue m
30
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the current study, where a good correlation was made between transconjunctival fluid
flow and the permeability of solutes up to 4,400 (FD-4) in molecular weight. As
mentioned by Fagerholm et al. that specie variability may be a factor in the observed
discrepancy between humans and rats. The authors also reported different degrees of
solvent drag effect among individual humans. It is still unclear whether or not the
solvent drag can only influence paracellular transport. Should solvent drag affect
transcellular solute transport (e.g., urea transport through aquaporins), the
consideration would need to go beyond relationship between size of the solute and
intercellular spaces.
31
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II. STATEMENT OF THE PROBLEM
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The long-term goal of this project was to demonstrate and to characterize the
regulation of conjunctival active Cl' and fluid transport processes and their functional
relationships, thereby establishing the foundation for future development for dry eye
treatments via the pharmacological approach. Furthermore, the efficacy of utilizing
conjunctival fluid transport processes as a means of drug delivery was investigated.
Central Hypothesis
The underlying hypothesis of this project is that active Cl' secretion in
conjunctiva can be regulated by factors such as intracellular cAMP, Ca2 + , and PKC,
and it can be stimulated by agents that elevate the level/activity of these factors.
Conjunctival fluid transport can be driven by active Cl' secretion and by osmotic
gradient, which may be utilized as means of carrying hydrophilic therapeutic agents
across this tissue.
The specific aims of this project are as follows:
S p e c if ic A im #1: T o d e te r m in e th e C l s e c r e to r y p r o p e r ty o f c o n ju n c tiv a l
e p ith e liu m , a n d to identify a n d c h a r a c te r iz e d if f e r e n t C l c h a n n e l ty p e s in th e
c o n ju n c tiv a l e p ith e liu m ( i.e ., c A M P - , C a 2 \ a n d P K C - m o d u la te d C l c h a n n e ls ) .
The goal of this specific aim is two-fold. First, to determine whether
conjunctival epithelium is capable of active Cl' secretion. Second, to see if CF
channels that can be regulated by cAMP, Ca2 + , and PKC exist in the conjunctival
33
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epithelium and actively contribute to the unidirectional net CF secretion. Additional
efforts was also made to verify whether or not the cAMP-sensitive Cl" channels, if
found in the conjunctival epithelium, are indeed the same as the cystic fibrosis
transmembrane regulator (CFTR).
The first objective of this project was to determine whether conjunctival epithelium,
like many of its neighboring epithelia such as comeal epithelium ( 1 0 2 ), pigmented
ciliary epithelium (195), nonpigmented ciliary epithelium (51), lens epithelium
(336), and retinal pigmented epithelium (133), is capable of active Cl' secretion.
Cyclic AMP-, Ca2 + -, and PKC-sensitive Cl' channels are three principle types of Cl'
channels found in epithelial cells (24,29,35,53,112,113) and non-epithelial cells such
as pig and canine ventricular myocytes (38,39), leech P neurons (5), and human
fibroblasts (119). Once the Cl' secretory property of conjunctiva was verified on the
tissue level, the existence of conjunctival Cl' channels that are sensitive to
intracellular cAMP, Ca2 + concentrations, and PKC activity were investigated.
Furthermore, in order for net CF secretion to occur from the s-to-m direction, CF
channels must be residing mainly on the apical surface of the epithelium (61). For
this reason, apical CF channel detection and characterization utilizing patch clamp
techniques were performed in this project.
Following the identification of each CF channel, experiments were carried out to
further elucidate whether or not these CF channels are separate channel types, and an
34
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in-depth investigation of the possible overlapping of the regulatory signal
transduction pathways of the CF channels was conducted.
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Determination of conjunctival C l' secretion at the intact tissue level.
•Baseline net Cl" secretion under open circuit condition.
•Sensitivity to cAMP-, Ca2 + -, and PKC-stimulating agents.
•Sensitivity to CP channel and Na+ -K+ -2CP transporter inhibitors.
•Identification of CP secretion driven by active transport processes instead of tissue
potential gradient.
Determination of conjunctival cAMP-, Ca2 + -, and PKC-sensitive CP conductance at
the cellular level.
•Baseline whole-cell conductance.
•Stimulation of whole-cell conductance by cAMP-, Ca2 + -, and PKC-modulating agents.
•I-V relationship.
•Whole-cell conductance.
•Verification of CP conductance by determining the reversal potential.
_ _
, vr
Detection of cAMP-, Ca2 + -, and PKC-regulated Cl' channel activities at the single
channel level.
•Baseline single-channel conductance.
•Stimulation of single-channel conductance by cAMP-, Ca2 + -, and PKC-modulating
agents.
•I-V relationship.
•Single-channel conductance.
•Channel open probability.
______________________________________v /_____________________________________
Verification of the existence of conjunctival CFTR.
Western blot analysis.
Effect of CFTR inhibitor on conjunctival whole-cell conductance.
Scheme 2-1. Logistic experimental sequence of Specific Aim #1.
36
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Specific A im # 2 : T o d e te r m in e th e flu id s e c r e tin g n a tu r e o f th e c o n ju n c tiv a l
e p ith e liu m , a n d to v e r if y its lin k to a c tiv e C T s e c r e tio n .
The logic behind the hypothesized conjunctival fluid secretion was explained
in detail previously in the Introduction section. This aim is perhaps the most critical
link between the identification and characterization of conjunctival ion transport
processes and the development of potential therapeutic methods in treating dry eye-
related ocular diseases.
The first step of this aim was to determine whether or not conjunctiva is capable of
secreting fluid (Scheme 2-2), a phenomenon which has been observed in epithelia
such as human tracheal epithelium (91), rabbit corneal epithelium (103), and rat
salivary acinar cells (59). Furthermore, these reports have also demonstrated that the
fluid secretion in these cell types are driven by various active CF secreting
mechanisms such as cAMP- (91) and Ca2 + - (59) dependent pathways. Upon
confirmation of such phenomenon in the pigmented rabbit conjunctiva, efforts were
made to investigate the effects of various regulatory factors of conjunctival active Cl'
secretion found in Specific Aim #1 on baseline conjunctival fluid secretion. The
goal was to identify a pharmacological means of inducing fluid secretion across
conjunctiva, hence relieving the discomfort caused by the dry eye conditions.
3 7
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The second part of this aim was to test the degree of influence osmolality has on fluid
transport across conjunctival epithelium. By artificially creating an osmotic gradient
across the conjunctival tissue by exposing the mucosal side of the tissue to either
hypertonic or hypotonic solution, the direction of the fluid transport can be altered
(163). The significance of the results obtained from these experiments is two-fold.
First, the effect of mucosal osmolality, particularly hypotonicity, would set the stage
for the Specific Aim #3 to examine the feasibility of utilizing such fluid transport
mechanism as a vehicle for drug delivery across the conjunctiva. Second, the
osmotic water permeability (Pf), which mainly describes transcellular fluid transport,
can be determined based on the rate of fluid flow under a certain osmotic gradient.
Many epithelia have been reported to be transporting water transcellularly mainly
through aquaporins (212) under an osmotic challenge (49,57,155). Determination of
conjunctival Pf may indicate the occurrence of transcellular fluid permeability hence
shed light on the possible existence of a water channel (i.e., aquaporin), or other
transcellular fluid transport mechanisms.
38
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Determination of conjunctival fluid flux
Effect of G ' flux
modulating agents
E ffect of a -
free condition
Correlation
withl^
Effect of Na+flux
modulating agents
Connection to Q" secretion
Baseline fluid secretion
H ypotonicity
Osmotic water permeability (Pt )
Osmolality induced fluid flow
Hypertonidty
Confirmation of solvent-drag effect
-------------------------
Transcellular fluid flow
(existence of AQP?)
Scheme 2-2. Logistic experimental sequence of Specific Aim #2.
3 9
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S p e c if ic A im #3: T o e x p lo r e th e f e a s ib ility o f u tiliz in g c o n ju n c tiv a l flu id tr a n s p o r t
a s a m e a n s o f o c u la r d r u g d e liv e r y .
Depending on the direction, the therapeutic values of the conjunctival fluid
flow vary. As mentioned in Specific Aim #2, stimulation of conjunctival fluid
secretion may reduce the severity of the dry eye symptom. On the other hand, if fluid
absorption can be artificially and temporarily induced to a certain degree with a
physiologically tolerable hypotonicity, this may have a potential to be a vehicle for
delivering hydrophilic drugs across conjunctiva through paracellular pathways.
Based on the osmotic gradient-driven fluid flow measured in Specific Aim #2 via the
capacitance probes technique, the transport of various hydrophilic and lipophilic of
different molecular weight was monitored in the modified Ussing-type chambers
under similar osmotic conditions.
4 0
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Hypertonic (480 mOsm) Isotonic (300 m Osm) Hypotonic (100 m O sm )
Effect of various on conjunctival fluid flux
Solute transport under various mucosal osmotic gradient
Hydrophilic FD-4
Mannitol
HPMPC
Lipophilic Betaxolol
Scheme 2-3. Logistic experimental sequence of Specific Aim #3.
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III. MATERIALS AND METHODS
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1. Animal model
1.1. Intact tissues
Male, Dutch-belted pigmented rabbits (2.5-3.0 kg) were purchased from Irish
Farms (Norco, CA), and were treated in compliance with The Guiding Principles in
the Care and Use of Animals (DHEW Publication, NIH 80-23). The detailed
procedure for conjunctival tissue preparation was reported previously (107). In brief,
rabbits were euthanized with an overdose of sodium pentobarbital solution (325
mg/kg) via a marginal ear vein. Both eyeballs were excised, and the conjunctival
tissues were trimmed for mounting as flat sheets between two Lucite half chambers.
Tissue manipulations were kept to a minimum during the procedure to maintain
tissue integrity.
1.2. Conjunctival epithelial cell culture
Isolated cultured conjunctival epithelial cells were used for all patch clamp
studies and Western blot analyses. The cell isolation procedure and culture
conditions have been previously reported by Saha et al. (165). The isolated cells were
seeded at a density of 1.0 x 106 cells/cm2 (day 0) onto 35 x 10 mm tissue culture
petri dishes (Becton Dickinson, VA). Cells were grown and allowed to recover from
the isolation/culture process for 2 to 3 days prior to patch clamp analysis.
43
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2 . Chemicals
2.1. Active ion transport modulators
8-bromoadenosine-3’ ,5 ’ -cyclic monophosphate (8-Br cAMP), bumetanide,
calcimycin (A23187), D-glucose, 1,9-dideoxyforskolin, forskolin, glibenclamide,
ouabain, adenosine 5’-triphosphate (ATP), uridine 5’-triphosphate (UTP), and
phorbol 12-myri state-13 - acetate (PMA) were purchased from Sigma Chemicals Co.
(St. Louis, MO), and N-phenylanthranilic acid (NPAA) was obtained from Aldrich
Chemicals (Milwaukee, WI).
2.2. Cellular transport tracers
Fluorescein isothiocyanate-dextrans (ED-4, MW 4,400) was purchased from
Sigma Chemicals Co. (St. Louis, MO). Acetoxymethyl ester of Indo-1, Indo-1 AM,
was purchased from Calbiochem (La Jolla, CA). D-[ 1 -3 H(N)]-mannitol (26.4
Ci/mmol) was purchased from DuPont NEN (Wilmington, DE), Na3 6 Cl (0.52
mCi/mmol) was purchased from Amersham (Arlington Hts., II), [5- H]-(S)-l-[3-
Hydroxy-2-(phosphonyl-methoxy)propyl]-cytosine (HPMPC, 29 Ci/mmol) and D-[l-
1 4 C]-mannitol (50 mCi/mmol) were obtained from Moravek Biochemicals, Inc. (Brea,
CA), and 3 H-betaxolol (51 Ci/mmol) was acquired from Alcon Laboratories (Fort
Worth, TX).
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
OH
OH
OH
Forskolin
MW = 410.50
Adenylate cyclase activator
OH
1,9-dideoxyforskolin
MW = 378.51
Inactive analog of forskolin
OH
8-Br cAMP
MW = 408.10
Membrane permeant analog of cAMP
F i g u r e 3-1. Structures and molecular weights of cAMP-dependent C F transport
activators used - forskolin, 1,9-dideoxyforskolin (negative control of forskolin
effect), and 8-Br cAMP.
45
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r \
c h 3
Calcimvcin (A23187)
MW = 523.63
Ca2 + ionophore
OH
OH
■OH
Phorbol 12-mvristate 13-acetate (PMA)
MW = 616.84
PKG activator
HN '
- P 1
OH OH
Uridine 5’-trinhospfaate (UTP)
MW = 480.11
P2 -purinergic receptor agonist
F i g u r e 3-2. Structures and molecular w e i g h t s o f Ca2+ - a n d PKC-dependent C F
t r a n s p o r t a c t i v a t o r s u s e d - c a l c i m y c i n , P M A , a n d U T P .
46
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CHO
H-
HO-
H-
H-
-OH
-H
-OH
-OH
CH2OH
D-glucose
M W = 180.16
Na+ /glucose cotransport substrate
Figure 3-3. Structure and molecular weight of D-glucose, a Na+ absorption
(Na+ /glucose cotransport) stimulator.
4 7
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H O O C .
.NH
Bumetanide
CO O H
HN'
N-phen ylanthranillic acid (NPAA)
MW = 213.23
MW = 364.42
Na+/K+ /2C1- cotransport inhibitor Q channel inhibitor
C NH(CH2)2'
OCH3
o o
I I I I
S NHCNH-
I I
o
Glibenclamide
MW = 494.00
CFTR inhibitor
OH
OH
O H
OH
OH
O H O H
Ouabain
MW = 584.65
Na+ /K+-ATPase inhibitor
Figure 3-4. Structures and molecular weights of ion transport inhibitors used -
bumetanide, NPAA, glibenclamide, and ouabain.
48
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CH2CHOCH2-P OH
I I
c h 2o h o h
3 H - C i d o f o v i r ( H P M P C )
MW = 279.19
14 c h 2o h
HO H
HO H
H OH
H— -O H
CH2OH
HC-mannitol
MW = 184.16
HO-
H 0 -
H-
H-
CH2OH
-H
-H
-OH
-OH
H— C H
I
OH
3H-mannitol
M W = 1 8 4 . 1 8
h 3c
h 3c
\ H H2 H h 2
C H -n — C — C — C - 0 — \ \
/ J A
h 2 h 2 h 2
~c— c— o— c
2H-betaxolol
M W = 3 0 7 . 4 3
Figure 3-5. Structures and molecular w e i g h t s of model paracellular (3 H-
HPMPC, 1 4 C-mannitol, and 3 H-mannitol), and transcellular (3 H-betaxolol)
transport c o m p o u n d s used.
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•c h 2
•c h 2
•c h 2
FITC-dextran
MW = 4,400
* The site of attachment of FITC
is assumed to be randomly
associated with any free hydroxyl
group
Indo-1 AM CH3
MW = 1009.93
'NH
Figure 3-6. Structures and molecular weights of fluorescent dyes used - FITC-
dextran (paracellular marker), and lndo-1 AM (intracellular Ca2 + indicator).
50
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3. Buffer ingredients
3.1. Bicarbonated Ringer’s solution (BR): Unless otherwise specified, all
experiments were performed using excised conjunctiva in the Ussing-type chambers
and Lucite chambers buffered in regular BR containing 111.55 mM NaCl, 4.82 mM
KC1, 0.86 mM NaH2 P 0 4, 29.20 mM NaHC03, 1.04 mM CaCl2, 0.74 mM MgCl2,
and 5.00 mM D-glucose. The buffer was maintained at 37 °C with a circulating
water bath and pH at 7.4 by bubbling with 5% C 0 2 in air.
3.2. Patch clamp solutions
3.2.1. Whole-cell patch clamp recording: The bathing solution contained the
following: 145 mM NaCl, 10 mM HEPES, 1 mM CaCl2, and 2 mM MgCl2. The
pipette solution contained 145 mM NaCl, 10 mM HEPES, 5 mM EGTA, 0.5 mM
CaCl2, and 1 mM MgCl2. Potassium was excluded from all whole-cell patch clamp
solutions to eliminate possible K+ currents contributed by K+ channels (107) and
other K+-dependent ion transport processes such as Na+ -K+ ATPase (109) (indirectly
inhibits Na+ currents as well). ATP (1 mM) was added to the pipette solution 2 min
prior to the experiment. For the reduction of extracellular CF concentration, NaCl
concentration in the bathing solution was partially substituted by equal concentration
of Na-isethionate.
51
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3.2.2. Single-channel patch clamp recording: To detect single-CP channel
activities, symmetrical pipette and bathing solutions were used during cell-attached
and inside-out single-channel recordings. They contained: 145 mM NaCl, 10 mM
HEPES, 1 mM CaCl2, and 2 mM MgCl2.
4. Methods
4.1. Voltage clamp/Ussing-type chamber settings (Scheme 3-2)
4.1.1. Bioelectric parameter measurements
The mucosa] and the serosal sides of the tissue (0.97 cm2 ) were each bathed
with 5 ml of BR solution. Transepithelial bioelectric parameters (Isc, Rt, and PD)
were measured as reported by Kompella et al. (107). An electrical pulse of 2 mV of
3 sec duration was generated every 60 sec by a voltage clamp apparatus (Model
558C-5, University of Iowa, Bioengineering Department, Iowa City, IA) (Fig. 3-7).
The potential difference (PD) was continually measured by two matched calomel
electrodes. The Is c was measured by a pair of matched Ag/AgCl electrodes. The
transepithelial resistance (Rt) was calculated from the measured PD divided by Isc.
These bioelectric parameters were measured for all mounted conjunctival tissues
during the flux experiments as a marker for tissue integrity (Rt) and active ion
transport (Isc ).
52
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□
[01
Calomel
Electrode
Ag/AgCI
Electrode
1/
Voltage Clamp
Agar Bridges^
% ^
Conjunctiva BR Solution
s . t -
V V
3 M KCI
Figure 3-7. Voltage clamp/Ussing-type chamber settings.
53
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4.2. Measurement of 3 6 C 1 fluxes
4.2.1. Determination of baseline net Cl' secretion and cAMP-, Ca2+ -, and PKC-
sensitive Cl' secretion
The mucosal-to-serosal (ms) flux (Jm s) and the serosal-to-mucosal (sm) flux
(Jsm ) of j6Cl across the excised pigmented rabbit conjunctiva were measured in the
Us sing-type chambers (Fig. 3-7) under open-circuit conditions. 3 6 C1 fluxes were
measured both in the presence and absence of various pharmacological agents:
mucosal 1 mM NPAA, serosal 10 pM bumetanide, mucosal 1 mM 8-Br cAMP,
mucosal 10 pM A23187, or mucosal 1 pM PMA. Concentrations of these
compounds were previously reported to significantly alter the Isc in either
conjunctival (107,109), corneal (25), or epididymal epithelia (29). 3 H-Mannitol flux
was also measured as an index of paracellular passive diffusion. During the
measurement of the effect of increasing 8-Br cAMP concentrations on CF fluxes, the
tissues Is c was recorded at 30 min intervals.
Ten min following the introduction of 3 6 C 1 (0.5 pCi/ml) and 3 H-mannitol (1.0 p
Ci/ml) to the donor reservoir, 150 pi was sampled from the donor solution and 500 p
1 was sampled from the receiver solution every 15 min for 3 hr and was immediately
replaced with an equal volume of fresh BR solution containing appropriate
pharmacologic agents. In the A23187 and PMA experiments, CF fluxes were
measured every 10 min for 40 min without either compound, followed by spiking the
. 54
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mucosal donor solution with either compound to achieve a final concentration of 10
pM A23187 or 1 pM PMA. The receiver fluid was then sampled at 5 min intervals
for the next 45 min. Finally, the sampling schedule was returned to 10 min intervals
for the remaining 40 min.
4.2.2. Flux ratio analysis
To confirm the presence of active Cl" transport process in the conjunctiva, the
observed Cl" fluxes in the s-to-m (Jsm ) and m-to-s (Jm s) directions were subjected to
Ussing’s flux ratio analysis (187):
W Jm s = exp [ [ (A V)zF]/RT].................. Equation 1
where AV is the transconjunctival electrical potential difference (volt =
joule/coulomb), z is the valence (-1 for Cl"), F is the Faraday’s constant (96,485
coul/mol), R is the gas constant (8.31 J/mol/K), and T is the absolute temperature
(310 K). Significantly higher observed flux ratio than the value predicted by
Equation 1 was taken as evidence for active Cl" transport.
55
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4.3. Patch clamp recordings
4.3.1. Electronic setup
The entire system was properly grounded, and the microscope and the head
stage assembly was contained within a Faraday cage. The cellular electrical
measurements were performed with an Axopatch 200B amplifier, and all currents
were recorded into a computer through a analog-to-digital interface box, Digidata
1200A (Axon Instruments, Foster City, CA) (Fig. 3-8).
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Oscilloscope
Amplifier
L ow -pass B e sse l filter 01 - j
A out
D out
A: Analog
D: Digital
A out
0 0 0 0 0 0 0 0 0 0 0 0 .
0 0 0 0 0 0 0 0 0 0 0 0 A
D out | ^
A/D in terface b ox
1 1 11 .1 i t 3
Digital au d io ta p e re c o rd e r
I
Com puter
Figure 3-8. Electronic setup of the patch clamp system.
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4.3.2. Microelectrode fabrication
Patch pipettes were pulled from borosilicate glass (O.D. 1.2 mm, I.D. 0.69
mm, Sutter Instrument Co., Novato, CA) using Flaming/Brown micropipette puller,
Model P-97 (Sutter Instrument Co., Novato, CA), coated with Sylgard resin (Dow
Coming 184), and fire polished with Micro Forge, MF-90 (Narishige, Tokyo, Japan).
The tapering of the electrode shaft and the tip opening diameter were fabricated by
adjusting the following programmable parameters within the micropipette puller:
HEAT, PULL, VELOCITY, and TIME, where HEAT controls the level of current
applied to the platinum heating filament positioned around the center of the glass
pipette; PULL indicates the force of the hard pull applied to either end of the glass
pipette; VELOCITY is the speed of the glass carriages pulling apart under a constant
load when the glass softens by the heated filament; and TIME is the length of the
cooling period between the time when the HEAT turns off and the time when the
hard pull begins. The settings of these parameters for achieving certain pipette tip
shapes and sizes varied based on the types of micropipette used (e.g., glass wall
thickness, filamented or non-filamented pipettes... etc.). Puller inconsistency was
frequently experienced. In other words, parameter adjustments were often necessary
between individual pipette (of the same kind) fabrications by trial and error.
58
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4.3.3. Whole-cell recording
Conjunctival epithelial cells used for patch recording were grown on 35 x 10
mm tissue culture dishes for 1 to 3 days. Gigaohm seal formation against the
conjunctival epithelial cell surface was extremely difficult but often can be
established during earlier stages of the cell culture. It generally became unachievable
(or achievable but unstable) beyond 3 days after the cells were seeded. Prior to the
experiments, the culture medium was aspirated from the dish, and cells were rinsed 3
times with 2 ml bathing solution. All patch clamp recordings were conducted at room
temperature (22-25 °C) (140). Upon seal formation (>5 GO) between the
microelectrode tip and the cell surface, the whole-cell configuration was achieved by
rupturing the cell membrane by applying a gentle suction. The stepping voltage was
from -140 to +60 in 20 mV intervals (pipette holding potential at -40 m V ), with 100
ms duration. All treatment compounds were prepared to their proper final
concentrations in the bathing solution. Drugs were perfused to the cell by a
peristaltic pump at a rate of 2 ml/min.
59
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Pipette Tip
Ruptured Membrane
Gigaohm Seal
Conjunctival
Epithelial Cell
W hole-cell
4.3.4 Reversal potential
To verify whether the induced current was indeed contributed by Cl' ion, the
whole-cell reversal potential was estimated by reducing the external total Cl'
concentration from 151 to 56 mM by partial replacement of NaCl with Na-
isethionate. The reversal potential was calculated based on the Nemst Equation:
Eci = RT/F In [Cl]i/[C1]0 ................. Equation 2
where R is the gas constant (8.31 joule mol'1 K'1 ), T is the absolute temperature (298
K), F is the Faraday’s constant (9.648 x 104 m ol1 ), [Cl]i is the Cl' concentration in
the microelectrode (151 mM), and [Cl]0 is the Cl' concentration in the bath (56 mM).
60
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The theoretical Cl' reversal potential predicted by the Nemst Equation is
approximately 26 mV.
4.3.5. Single channel recording
To monitor the activity of individual Cl' channels while maintaining
intracellular integrity, currents were measured under the cell-attached single-channel
patch clamp configuration. Symmetrical buffer composition was used between the
bath and the pipette. Once the seal resistance reached >50 GO, the cell was held at
various pipette potentials, typically at ± 60 mV, ± 40 mV, and 0 mV, in order to
obtain sufficient data points to generate a single-channel I-V curve, and to estimate
channel conductance. An additional low-pass Bessel filter (Model LPF-8, Warner
Instruments Co., Hamden, CT) was installed and set at 100 Hz to reduce background
electromagnetic noise level. Cyclic AMP-, Ca2 + -, and PKC-dependent Cl' channel
conductances were monitored in the presence of extracellular 10 pM forskolin, 10
pM A23187, and 1 pM PMA, respectively. In addition, the activities of conjunctival
Ca2 + -dependent Cl" channels were also monitored under the inside-out patch clamp
configuration in the absence of A23187, exposing the cytoplasmic surface of the
patched cell membrane to higher Ca2 + concentration in the bathing solution (1 mM).
The high concentration of Ca2 + in the bathing solution was necessary to achieve
gigaohm seals.
61
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Pipette Tip
Intact Membrane
Gigaohm Seal
Conjunctival
Epithelial Cell
Cell-attached
4.3.6. Signal conversion and data analysis
4.3.6.1. I-V curve and single channel conductance
The current-to-voltage (I-V) relationship was determined by plotting single
channel current amplitude (in pA) against the respective pipette potential (in mV)
under which that particular amplitude was observed. Single channel conductance (in
pS) was estimated from the slope of the I-V curve.
4.3.6.2. Channel open probability
Channel open probability (P0 ) provides a quantitative description of the
activity of the channel vs. time, which can be calculated by the following equation:
62
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P0 = I/iN ........ Equation 3
Where I is the mean current over a certain time interval, i is the single channel
current amplitude, and N is the number of channels in the patch. P0 was determined
for channel activities recorded in the presence and absence of an agonist specific for
a particular type of Cl" channel.
4.4. Western blot analysis
4.4.1. Conjunctival epithelial cell lysate preparation
Conjunctival epithelial cells seeded at 1.0 x 106 cells/cm2 on 60 x 15 mm
tissue culture petri dishes were cultured to confluence (~7 days, by visual inspection)
prior to cell lysate preparation. Culture medium was aspirated from the dish, and
cells were washed twice with ice-cold PBS (pH 7.4). The washed cells were
detached with a cell scraper and lysed with 0.5% sodium dodecyl sulfate (SDS) in the
presence of a protease inhibitor cocktail (5 pg/ml pepstatin, 10 pg/ml chymostatin, 5
pg/ml leupeptin, and 78.3 pg/ml benzamidine) (Sigma Chemicals, St. Louis, MO).
The cell lysate was first vortexed for 10 min in the presence of Sepharose CL-2B
(Pharmacia Biothech, Piscataway, NJ) to reduce viscosity, followed by centrifugation
at 14,000x g for 10 min. The resultant supernatant was collected and protein
concentration was determined by the DC protein assay (Bio-Rad, Hercules, CA),
using bovine serum albumin as the standard.
63
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4.4.2. Detection of conjunctival CFTR
To determine the existence of CFTR in the conjunctival epithelial cell,
Western blot analysis was performed using a mouse monoclonal antibody that
recognizes the C-terminus of the human CFTR (Genzyme Diagnostics, Cambridge,
MA), following the method similar to that described by Faller et al. (56). Cell lysate
of T84 cells was also prepared the same way as for the conjunctival epithelial cell
lysate and used as a positive control for the CFTR presence (126). In addition, a
mouse IgG2a K isotype was used as a negative control. Sixty micrograms of the
protein from each cell type was electrophoresed on 8% SDS-polyacrylamide gel
(Sigma Chemicals, St. Louis, MO), and electro transferred to a nitrocellulose
membrane (Scientific Specialties Group, Mt. Holly Springs, PA). Immunoblot
procedure utilizing the enhanced chemiluminescence method (ECL) was performed
according to the manufacture’s protocol (Amersham, Downers Groves, IL).
4.5. Intracellular Ca2 + measurement
Conjunctival epithelial cells at a seeding density of 1.0 x IQ6 cells/cm2 were
grown on 1 x 2 cm glass coverslips (Hitachi Instruments, Inc., San Jose, CA ) for 7-8
days until reaching confluence. This is to maximize the cell density on the cover
slip, hence the amount of Ca2 + -sensitive fluorescent dye absorbed by the conjunctival
epithelial cells. After aspirating the culture media, the cells were washed once with
Ca2 + -containing Hanks’ balanced salt solution (HBSS) (GIBCO BRL, Grand Island,
64
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NY) at 37°C. The cells were incubated in a mixture of 10 (iM Indo-1 AM, 3
mg/ml pluronic F-127, and 0.5% DMSO for 30 min at 37°C. After washing off
excess Indo-1 AM with fresh HBSS, the cover slip was mounted to a holder and
placed inside the cuvette with Ca2 + -containing HBSS for fluorescence measurement
in a fluorometer (Model F-2000, Hitachi Instruments, Inc., San Jose, CA). The
excitation was measured at 330 and 346 nm, and the emission was at 405 and 485
nm. Fluorescence was monitored continuously and [Ca2+]j was estimated using
following equation (72):
[Ca2+]i = Kd x Fm in / Fm a x x (R - R J / (R m ax - R ) Equation 4
where Kd is the dissociation constant for Ca2+ binding to Indo-1 (230 nM); R m in is the
ratio of minimum fluorescence intensity of the two wavelengths under Ca2 + -free
condition, and R m ax is the ratio of maximum fluorescence intensity of the two wave
lengths at saturating Ca2 + ; Fm in is the fluorescence intensity of the free dye measured
at 485 nm, and Fm ax is the fluorescence intensity under Ca2 + -saturating condition at
485 nm. At the end of each experiment, 250 mM of digitonin and 10 mM of EGTA
were applied to determine the maximum (Fm a x ) and minimum (Fm in) fluorescence
intensity.
65
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4.6. Measurement of fluid transport
4.6.1. Epithelial fluid flux detection techniques
Gravimetric measurement
Several methods have been documented for the measurement of
transepithelial fluid transport. The simplest method is the classical gravimetric
measurement, which is being widely used for estimating osmotically induced fluid
transport across tissues that can be handled as a sac (e.g., bladder) or a tube (e.g.,
intestine) (40). The rate of fluid transport is indicated directly by the changes in the
weight of the fluid-filled sac or tube either at baseline or drug-treated conditions.
Stefansson et al. (180) utilized this method to quantify retinal edema in rat. They
measured the water content in the retinal tissue, which is increased in during retinal
edema. However, the major disadvantages of this technique are poor temporal
resolution (i.e., real-time display of the physiological function), uncertainty in the
amount of fluid adhering to the outside of the tissue when removing the sac from the
bathing medium, and inability to perform other types of simultaneous measurements
(e.g., transepithelial voltage, resistance, and short-circuit current).
Unidirectional 3 H-water fluxes
Direct measurement of unidirectional 3 H-water fluxes is also used for
determination of net fluid movement across cornea (23) and other epithelia such as
skin (179), vaginal (192,193), and buccal (192) tissues. One major technical
66
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difficulty of this method is that it requires a completely enclosed chamber system
(i.e., continuous flow-through perfusion system) to prevent possible loss of radio-
labeled water either through evaporation or systemic condensation, thereby skewing
the measurement of actual rate of water flow across the epithelium.
Indicator-Dilution Principle
Measurement of changes in the concentration of a substance serving as a
water-volume marker in the bathing solution can be another approach to measure
water permeability (151). Such a method is based on the principle stating that the
volume of a solution (V) can be determined under the conditions that the amount (QO
and concentration (Q) of an indicator (i) are known:
V = Qi / Q ..................... Equation 5
Therefore, if the amount of the indicator remains constant, changes in fluid volume
on either side of the epithelium would result in changes in the concentration of the
indicator. In order for this principle to work experimentally, the properties of the
indicator such as homogenous distribution in the bathing solution, impermeability or
low permeability relative to water permeability, and the existence of an accurate
detection method are required (40). Also, the size of the reservoirs containing the
indicator have to be very small in order for epithelial fluid transport to cause a
67
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detectable change in the indicator concentration. This approach can be applied for
the measurement of fluid transport across cell layers cultured on a permeable support.
Capacitance Probes
A method was developed by van Os et al. (194,213) to directly measure the
fluid volume displacement from either/both sides of the epithelium, utilizing
vertically positioned probes to detect the electrical capacitance of an air/solution
interface. This capacitance is an inverse function of the distance between the probe
and fluid surfaces (Figs. 3-9 and 3-10). The net fluid movement across the
epithelium would then result in a change in fluid displacement under the probes, thus
reflected by a change in capacitance. This method provides a much higher temporal
resolution (with a limit of detection as low as 1 nl/min) as compared to other fluid
transport measuring approaches mentioned previously, and it also allows
simultaneous measurements of transepithelial bioelectric parameters. Due to its high
resolution, the capacitance probe method has been widely used to measure fluid flow
across various epithelia such as frog (86) and bovine (52) retinal pigmented epithelia,
human tracheal epithelium (91), rabbit gallbladder epithelium (175,194), to name a
few. Although this method has overcome many of the technical shortcomings of
other detection methods for fluid flow, it is not without disadvantages. One concern
is the possible fluid absorption by the chamber material, which may alter the fluid
volume inside the chamber. Therefore, the design of the chamber requires extra
68
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considerations (e.g., built with hydrophobic material and kept in a high humidity
environment).
4.6.2. Determination of transconjunctival fluid transport
Based on the detection resolution and the potential capability of concomitant
bioelectrical measurements (bioelectric parameters were not measured during
conjunctival fluid flow detection in the present investigation due to apparatus
limitations), we chose the capacitance probe method (ASP-10-CTA/SP, Mechanical
Technology, Inc., Latham, NY) for the detection of fluid flow across an excised
pigmented rabbit conjunctiva (Fig. 3-9), similar to that described by Edelman and
Miller (52). The entire apparatus was placed in an enclosed cabinet maintained at 37
°C and at a relative humidity of 70%. In pilot experiments, we found that a relative
humidity of at least 60% was required to prevent condensation on the probe surface.
To prevent hydraulic pressure generated by uneven fluid level against either side of
the mounted conjunctival tissue at the beginning of the experiment, a water bridge
(Fig. 3-10) was utilized to balance the fluid level of the mucosal and serosal
reservoirs. One mM 8-Br cAMP (mucosal), 10 pM UTP (mucosal), 20 mM D-
glucose (mucosal), 20 mM D-mannitol (mucosal), or 0.5 mM ouabain (serosal) was
applied separately to the reservoir using the concurrent feed and drain method with a
pair of 10 pi Hamilton syringes, thus keeping a constant fluid volume during
application of the compounds in the reservoir. The concentration used was based on
69
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the Isc measurements reported previously (83,109,207). To demonstrate a role for
active Cl' secretion in transconjunctival fluid flow, water flux was measured under
the Cl - free condition, whereby a Cl'-free solution was used in both the serosal and
mucosal reservoirs.
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37°C
70% Humidity
Capacitance probes
Tissue adapter
Reference electrode
Conjunctiva
Water
jacket
Figure 3-9. Dual capacitance probe setup used to measure f l u i d transport across an
isolated rabbit conjunctival tissue. The entire assembly was kept within a closed
environment maintained at 37 °C and at a relative humidity of 70% 1 hour prior
and during the course of the experiment.
71
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F i g u r e 3 - 1 0 . W a t e r b r i d g e u s e d t o b a l a n c e t h e f l u i d l e v e l prior to t h e i n i t i a t i o n
o f t h e c a p a c i t a n c e p r o b e e x p e r i m e n t .
72
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The capacitance of the air gap between the probe and the fluid surface on each side
of the chamber was measured (Fig. 3-11). The voltage output from the capacitance
probe indicates the loss of fluid from one side of the chamber, while gain of fluid on
the opposite side. The fluid transport rate (pl/hr/cm ) was calculated based on the
slope of the voltage vs. time and conversion of voltage changes to fluid volume
displacement based on a calibration table provided by the manufacturer.
S ecretio n
B aseline
A bsorption
F i g u r e 3-11. Relation between f l u i d displacement
and conjunctival fluid transport properties.
MUCOSAL
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4.6.3. Osmotic water permeability (Pf)
To measure the effect of osmolality changes on transconjunctival fluid flux so
as to determine osmotic water permeability ( P f ) , the osmolality of the mucosal
solution was either reduced from 300 to 100 mOsm or increased to 480 mOsm by
varying NaCl concentrations. The conjunctival osmotic water permeability was
calculated in accordance with on the Kedem-Katchalsky equation (97):
where Jv is the observed water flow rate per unit surface area, R is the gas constant
(8.31 joule m ol1 K '^ T is the absolute temperature (310 K), Vw is the partial molar
volume of water (18 ml/mole), and An is the transconjunctival osmotic pressure
gradient (Ati = crRTAC; AC = difference in the molarity of NaCl). Assuming a unity
invariant reflection coefficient (a) for NaCl (163), Eq. 6 becomes:
4.7. Influence of conjunctival fluid flux on solute transport
To estimate the effect of osmotically-driven transconjunctival fluid flux on
solute permeability, the excised rabbit conjunctivas were mounted in the modified
Ussing-type chambers and the m-to-s FD-4,3H-cidofovir (HPMPC), 14C-mannitol,
Pf = Jv • RT / (Vw • An) Equation 6
Pf = Jv / (Vw • AC) Equation 7
74
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and 3H-betaxolol transport was measured under different osmotic gradients. Solute
transport was measured in the presence of mucosal isotonic (300 mOsm), hypotonic
(100 mOsm), or hypertonic (480 mOsm) conditions. Furthermore, m-to-s transport
of 3H-HPMPC was measured in the presence of mucosally applied 20 mM D-glucose
which is known to stimulate active Na+ absorption in the conjunctiva (83). Solute
transport was estimated from the cumulative amount of tracers collected from the
serosal compartment over 180 min.
4.8. Statistical analysis
Results are expressed as mean ± s.e.m. Comparisons between two sets of
data means were analyzed with Student’s unpaired t-test or one-way ANOVA. One
way analysis of variance and post-hoc comparisons based on Fisher's LSD approach
were used to determine statistical significance among more than two group means. A
p value of less than 0.05 was considered significant.
75
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IV. RESULTS
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1. Regulation of conjunctival active ion transport processes by cAMP-, Ca2 + -,
and PKC
1.1. Stimulation of Ist
Figure 4-1 shows the general trends of the time course of Is c stimulation under
mucosal addition of 1 mM 8 -Br cAMP, 10 fiM A23187, and 1 pM PM A. 8-Br
cAMP induced a sustained Is c elevation by 64%. A23187 and PM A, on the other
hand, both stimulated Isc in a transient manner, allowing a peak Is c stimulation of
6 8 % and 31%, respectively, approximately 5 min after the instillation of the drug,
followed by a gradual decline below the initial baseline values. Isc stimulation was
observed in all tissues treated with one of these compounds.
77
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20 -
- m — 8-Br cAMP
m — A23187
-h— PMA
5 --
35 70 0
T i m e ( m i n )
Figure 4-1. Time courses o f Isc changes in freshly isolated pigmented rabbit
conjunctival tissues treated with 1 mM 8-Br cAMP, 10 j i M A23187, and 1 nM
PMA. The a r r o w represents the point of drug application. Error bars
represent s.e.m. for three to five measurements from excised tissues.
78
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1.2. Additive stimulation of Isc by 8-Br cAMP, A23187, and PMA
In the serial treatment of the conjunctiva with 1 mM 8 -Br cAMP and 10 pM
A23187, 8 -Br cAMP elevated Isc by 49% above the baseline. At the maximal 8 -Br
cAMP effect, A23187 elicited another 37% increase in Isc (Fig. 4-2a). For the
reversed treatment sequence, Is c was stimulated by 32% following A23187
instillation. At the maximal A23187 effect, 8 -Br cAMP stimulated Isc by an
additional 29%. Is c gradually approached the value observed for a maximal effect by
A23187 alone (Fig. 4-2b).
The sequential treatment of the conjunctiva with 10 pM A23187 first and then 1 pM
PMA also elicited an additive stimulation. Mucosal application of A23187 increased
Isc by 42% (Fig. 4-2c). At the maximal A23187 effect, PMA further stimulated Isc by
27% (Fig. 4-2c). The reverse order of treatments stimulated Isc by 27% (PMA) and
25% (A23187), respectively (Fig. 4-2d). In both treatment sequences, Is c decreased
gradually below the baseline (i.e., value prior to the initiation of the treatment) after
the stimulation by the second drug.
The serial treatment of 1 mM 8 -Br cAMP first and then 1 pM PMA stimulated Isc by
47% and 30%, respectively (Fig. 4-2e). PMA alone increased the Is c by 31%. The
subsequent application of 8 -Br cAMP, however, did not elicit any significant change
in Is c (Fig. 4-2f).
79
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21
1 m M 8-B rcA M P
12
10 mM A 23187
3
140 70
T im e (min)
b.
21
10 mM A23187
12
1 mM 8 -B rcA M P
3
140 70
T im e (min)
c.
21
mM PMA
I
5 12
10 mM A 23187
0 35
Time (min)
70
21
10 mM A 23187
£
I
a
1 mM PM A
70 35
Tim e (min)
21
1 m M 8 -B rcA M P
E
I
g
1 mM PM A
50
T im e (min)
21
1 m M 8-B rcA M P
1 mM PMA
£
8
80 40
Tim e (min)
Figure 4-2. Time courses of Isc changes in the excised pigmented rabbit
conjunctiva treated w i t h various c o m b i n a t i o n s of 1 m M 8-Br c A M P , 10 p M
A23187, and 1 jiM PMA. Panels a and b represent t h e 8-Br c A M P / A 2 3 1 8 7
combination; panels c and d represent the A23187/PM A combination; panels e
and f represent the 8-Br cAMP/PMA combination. Error bars represent s.e.m.
for four to five measurements observed from excised tissues.
8 0
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2. Evidence for conjunctival net O ' secretion and its modulation by cAMP,
Ca2 + , and PKC under open-circuit conditions.
2.1. Baseline net O ' secretion across the excised conjunctiva
S-to-m Cl' flux ( J s m ) was 54% higher than m-to-s flux (Jm s) (p < 0.01, Table
4-1), yielding a net Cf flux of 0.15 pEq/hr/cm in the s-to-m direction. The H-
mannitol fluxes in the s-to-m and the m-to-s directions showed no significant
difference (p > 0.05).
2.2. Stimulation of net O' secretion by cAMP-, C a 2 + - , and PKC-activating
agents
The mucosal application of 1 mM 8 -Br cAMP increased J s m by 58% (p <
0.01) without altering Jm s (p > 0.05). The resultant net secretion of Cf in the s-to-m
direction was increased by 133% (p < 0.05) (Table 4-1). 3H-mannitol flux was
increased in both directions to a similar degree (~110%) (p < 0.05), in parallel with a
32% drop in the Rt. There existed, however, no significant difference in H-mannitol
fluxes in the presence of 8 -Br cAMP between the two directions (p > 0.05) (Table 4-
1).
Cf Jm s was not significantly (p > 0.05) affected by the mucosal addition of either 10
pM A23187 or 1 p M PMA (Table 4-1 and Fig. 4-3). Cf J s m , on the other hand, was
stimulated in a transient manner, being increased by 58% by A23187 and 49% by
81
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PMA at maximal stimulation (p < 0.01) and returning to baseline level thereafter (p >
0.05) (Fig. 4-3).
8 2
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a
0.8
0.6
f 0.4
U i
> ^ 5
X
0.2
_T
Qnrtrd
stimMion
fte t-
rraxiiral
stim&icn
0.8
0.6
0.4
f
Ui
3
g 0.2
5 =
J * .
o
G driroS
i
M a d m d
sflmiaticn
Rist-
Figure 4-3. Effects of mucosal 10 pM A23187 (panel a) and 1 pM PMA (panel b)
on Jm s (□ ) and Jsm (■) at baseline, at the maximal stimulation, and during post-
maximal stimulation. Columns are mean ± s.e.m. for n = 5-6. Key: * indicates a
significant difference from Jm s (p<0 .0 1 ); f indicates a significant difference from
control Jsm (p<0.05).
83
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Table 4-L Effect of serosal addition of 10 pM bumetanide and mucosal addition of 1 mM NPAA, 1 mM 8-Br cAMP, 10
pM A23187, and 1 pM PMA on potential difference (PD), apparent permeability coefficient (Papp) of mannitol, O'
fluxes, and flux ratios for O ' fluxes between s-to-m (Jsm ) and m-to-s (J^) directions.
Control Bumetanide NPAA 8-Br cAMP A23187 PMA
PD(m V) 13.8 + 1.0 (25) 4 ,3 + 0 .8 (8) 3 .8+ 0.5 (6) 21.3 + 2.2 (6) 17.4 + 2.5 (6) 15.9 ±2.1 (5)
Mannitol In K 1.23 + 0.16 (30) 1.84 ± 0.32 (6) 0.88 ± 0.24 (6) 2 .7 0 ± 0 .5 3 f (6) n.d. n.d.
(xl0~7cm/sec) Jsm 1.57 ± 0.27 (26) 2.26 ± 0.37 (9) 1.76 ± 0.50 (6) 3 .2 4 ± 0 .7 2 f (6) n.d. n.d.
CP J„K 0.28 ± 0.03 (30) 0.29 ± 0.05 (6) 0.28 ± 0.06 (6) 0.33 ± 0.04 (8) 0.37 ± 0.05 (6) 0.36 ± 0.06 (5)
(pEq/hr/cm2) Jsm 0.43 ± 0.04* (26) 0.28 ± 0.05** (9) 0.26 ± 0.03** (6) 0.68 ± 0.07t (6) 0.68 ± 0.04t (6) 0.64 ± 0.07t (5)
Jsn/Jm s (Observed) 1.54 1.03 0.93 2.06 1.84 1.78
WJms (Predicted) 0.60 0.85 0.87 0.44 0.51 0.54
All values are mean ± s.e.m. The number in the parentheses represents the number of tissues utilized.
* Significantly greater (p<0.01) than that in the m-to-s direction.
** Significantly smaller (p<0.05) than control,
t Significantly greater (p<0.05) than control,
n.d. Not determined.
2.3. Inhibition of net Cl' secretion by a CF channel blocker and a Na+ -K+ -2C1'
cotransport inhibitor.
NPAA at 1 mM decreased CF Jsm by 40% (p < 0.05) without significantly
affecting Jm s (p > 0.05) (Table 4-1). Thus, net Cl" secretion was abolished. There
was no significant change in JH-mannitol fluxes in either direction (p > 0.05)
following NPAA treatment (Table 4-1). The serosal addition of 10 pM bumetanide
decreased Jsm by 30% (Table 4-1), while not affecting Cl" Jm s (p > 0.05). Therefore,
net Cl’ secretion was also abolished. 3H-mannitol flux was not affected in either
direction by bumetanide treatment and was not significantly different from the
corresponding control values by ANOVA (Table 4-1).
2.4. Hill analysis of 8-Br cAMP dose-dependent changes in conjunctival Isc and
Cl flux
As shown in Figures 4-4a and 4-4b, both Is c and Cl" flux under open-circuit
conditions increased with 8 -Br cAMP concentration over the 0.001 to 3 mM range.
Hill analysis revealed a maximal Is c of 26.8 pA/cm2 at a half-maximal effect of
0.28 mM and a maximal Cl" Jsm of 0.97 pEq/hr/cm2 at an EC50 of 0.30 mM 8 -Br
cAMP. There exists a good correlation between observed Isc and Cl" Jsm (Fig. 4-4c).
85
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h
o
(ft
1 2 3 0
C M
|
| 0 8
w
A
X
3
o
E
1 2 3 e
E
o
U J
a 065
X
3
o
E
s
U 1
1800 1200
{ 8 Q c W P |( r r i J } [Sa-cflVpHnU/)
Figure 4-4. Dose-dependent changes in short-circuit current (Isc) ( p a n e l a) and C l "
flux measured under open-circuit conditions in the serosal-to-mucosal (s-to-m)
direction (panel b) in the pigmented rabbit c o n j u n c t i v a treated with up to 3 mM 8-
Br cAMP. Error bars denote s.e.m. for n = 6. Panel c shows the correlation
between s-to-m C l" flux and the corresponding Isc observed in the presence of
varying concentrations of 8-Br cAMP.
86
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2.5. Evidence for active Cl' transport - by flux ratio analyses
Table 4-1 also summarizes the results of 3 6 C 1 flux ratio calculations for
baseline, inhibition and stimulation of net Cl' fluxes. The observed Jsm /Jm s ratios for
bumetanide- or NPAA-treated conditions were close to the predicted values for
passive diffusion. The observed Jsm /Jm s ratios for all stimulators were 2-3 times
higher than the predicted values assumed for passive diffusion.
3. Detection of specific Cl' channel types present in conjunctiva
3.1. Demonstration of cAMP-sensitive whole-cell CF currents.
The estimated conjunctival whole-cell capacitance was 8.9 ± 1.0 pF (n = 14).
A typical tracings of whole-cell currents at baseline and those in response to 10 pM
forskolin are shown in Fig 4-5. Forskolin significantly increased conjunctival whole-
cell currents above baselines in 5 out of 7 successful upholding of the gigaohm seal
upon perfusion of the stimulant (Fig 4-5). The resultant whole-cell conductance was
elevated from the baseline of 0.42 ± 0.10 (n = 6) to 2.71 ± 0.13 nS (Fig. 4-6, n = 5) in
approximately 3-5 min. Such a stimulation of the whole-cell currents by forskolin
was not observed when an inactive analog of forskolin, 1,9-dideoxyforskolin (10
pM), was perfused to the bath (0.52 ± 0.13 nS after treatment) (n = 5; p>0.05) (Fig.
4-6). Preincubation with 100 pM glibenclamide, a CFTR inhibitor (149), hindered
the forskolin stimulation of the whole-cell conductance to 0.41 ± 0.13 nS (Figs. 4-5
and 4-6, n = 6).
87
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Baseline Baseline Glibenclamide
+60 mV
0 mV
-140 mV
+60 mV
«v0mV 0 pA—
-140 mV
O pA - 0 pA— ■
+60 mV
- OmV
-140 mV
Forskolin
Q pA -
1 0 ° pA |____
20 m s
1,9-dideoxyforskolin
OmV 0 PA—
140 mV
Forskolin + glibenclamide
+60 mV
* n m\/ n n A _ _
pw“ *w w u mv u p A “
-140 mV
+60 mV
■ 0 mV
-140 mV
Figure 4-5. Effect of 10 pM forskolin and 1,9-dideoxyforskolin on conjunctival
whole-cell currents, and the effect of 100 pM glibenclamide on forskolin-induced
whole-cell current. The cell was held at -40 mV and voltage-clamped from -140 to
+60 mV in 20 mV steps (duration of 100 ms).
8 8
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Figure 4-6. Conjunctival whole-cell C F conductance under baseline, 10 | i M 1,9-
dideoxyforskolin-, 10 pM forskolin-, and 10 pM forskolin + 100 pM glibenclamide
treated conditions. Error bars represent s.e.m. for n = 6. ^ S i g n i f i c a n t l y (p<0.01) larger
than the baseline value.
89
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3.2. Stimulation of whole-cell conductance by A23187 and PMA
When 10 pM A23187 or 1 |iM PMA was infused to the bath, whole-cell
currents of conjunctival epithelial cells were significantly elevated in an outwardly-
rectifying fashion (Figs 4-7), approximately 3 min after the perfusion of the
stimulants. The A23187-stimulated whole-cell conductance in 5 out of 8 successful
patches ranged from 1.67 ± 0.33 nS at negative potentials (-140 to 0 mV) to 2.52 ±
0.26 nS at positive potentials (0 to +60 mV, n = 5) (Fig 4-8). The PMA-induced
whole-cell conductance, on the other hand, was observed in 6 out of 10 successful
patches, ranged from 2.00 ± 0.28 nS at negative potentials (-140 to 0 mV) to 2.76 ±
0.32 nS towards positive potentials (0 to +60 mV, n = 6) (Fig 4-8).
9 0
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+60 m V
OmV
-140 m V
10 fiM A23187
+60 mV
0 m V
-140 mV
100 pA
Baseline
B aseline
1 jiM PMA
20 ms
Figure 4-7. Stimulation of conjunctival whole-cell currents by 10 jiM A23187 and
1 (xM PMA. The cells were held at -40 mV and stepped from -140 mV to +60 mV
in 20 mV intervals.
91
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3.3. Determination of conjunctival whole-cell reversal potential
The reduction of extracellular Cl" concentration from 151 to 56 mM resulted
in the rightward shift of the reversal potential from 0 to about 27 mV, closely
following the predicted change in Nemst potential for Cl". Such a phenomenon was
observed under baseline and forskolin-, A23187-, and PMA-stimulated conditions.
V (mV)
0
1
V (mV)
Baseline 10 pM forskolin
r - 1 4 0 -120 -100 -J
V (mV)
I
-140 -120 -100 -SO
V (mV)
10 pM A23187 1 (L iM PMA
Figure 4-8. Reversal potentials of baseline, forskolin (10 pM)-, A23187 (10 pM)-,
and PMA (1 pM)-stimulated I-V curves under symmetrical intra- and
extracellular Cl' concentrations (A), and reduced extracellular Cl' concentration
(from 151 to 56 mM) (■).
92
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3.4. Detection of single-channel activities under cAMP-, Ca2 + -, and PKC
stimulation
3.4.1. cAMP-activated single-channel conductance and open probability
Figure 4-9 illustrates single-channel tracings obtained before and after the
instillation of 10 jiM forskolin under the cell-attached configuration. Forskolin-
induced single-channel activity was observed in 7 out of 35 successful patches (i.e.,
seal formation). The forskolin-induced current amplitude was 0.40 ± 0.02 pA (n = 7)
at a pipette potential of -40 mV (Fig 4-9). A significant (p < 0.05) increased in
channel open probability (P0 ) was observed approximately 10 min after the forskolin
application (from 0.00 to approximately 0.30). The I-V curve appeared to be linear,
and the single-channel slope conductance was estimated to be 10 pS. The reversal
potential of the single forskolin-stimulated CF channel was at around 0 mV.
93
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Baseline (-40 mV)
5000
< 0
a
2
1
0
-1
-2
10 riNI forskolin (-40 m V)
10000
I
15000
5000
I
10000 15000
10 jiM forskolin (0 m V)
• I .... ..... .... 1 ....... i.......................- r " " I
5000 10000 15000
m s e c
Figure 4-9. Typical conjunctival single-channel activities recorded under the cell-
attached configuration. Upon seal formation (> 50 GQ) between the pipette and cell
surface, forskolin w a s perfused to the bath at 10 jxM. Increases in channel activities
were observed approximately 10 min after drug perfusion at a holding potential of -
40 mV, but not at 0 mV. Both baseline and forskolin-treated conditions were
recorded for at least 1 min ( 1 5 sec of representative tracings shown here).
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3.4.2. Ca2 + -activated single-channel conductance and open probability
Unlike forskolin-treated cells, increased channel activities were not observed
following the application o f 10 pM A23187 under cell-attached mode even after 15
min (n = 3). However, in the absence of A23187, Ca2 + -sensitive single-channel
activities were detected in 3 out of 15 successful patches by forming an inside-out
patch configuration and exposing the cytoplasmic surface of the cell membrane to
high Ca2 + concentration (1 mM) in the bathing solution. Figure 4-10a illustrates the
15 sec continuous recordings of Ca2 + -sensitive single-channel activities under -60,
± 40, ± 20, and 0 mV pipette potentials. The tracing from +60 mV was not available
due to frequent loss of tight seals at higher hyperpoladzing potentials. Larger current
amplitudes were observed under higher depolarizing/hyperpolarizing potentials (i.e.,
-60 mV > ± 40 mV > ± 20 mV). No channel activities were observed at 0 mV. The
corresponding all-point histograms (Fig 4-10b), which summarize the number of
events (i.e., channel opening and closing) within a certain observation period (15
sec), showed distinctive peaks (i.e., clear channel open/close stages), indicating
channel presence. The similar distance between first peak (channel closed state) to
second peak (1st channel), and second peak to third peak (2n d channel), suggests that
the patch may contain two channels of similar amplitude. The stimulated P0
appeared to be independent of the pipette holding potentials (Table 4-2). The single
channel slope conductance calculated from the linear 1-V curve was about 25 pS
(Fig.. 4-11).
■ 95
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2 1 o
- 6 0 m V
-2 0 m V
OmV
b.
5 0 0 0 1 0 0 0 0
msec
2 0 m V
W-, + 4 0 m V
• 60 m V
C lose d state
1 s t c h a n n e
2nd c h a n n e l
C l o s e d st at e
-2 0 m V
1 s t c h
60
0
C losed te s ta
+ 2 0 m V
C l o s e d state
1st c h a n n e l
■ 40 m V
Cl os ed state
1st c h a n n e l
P A
Figure 4-10. (a) Conjunctival Ca2 + -activated single Cl' channel activities at various
pipette holding potentials recorded from the same patch, with a symmetrical NaCl-
based solutions in pipette and bath, under the inside-out patch clamp configuration,
(b) Corresponding all-point histograms of the tracings in (a). These histograms
indicate the number of stages (i.e., opening and closing) and the current amplitude of
each stage. These distinctive peaks and similar individual current amplitudes
illustrate the existence of at least two channels within the patch.
96
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51
Table 4-2. Single C a 2 + - a c t l v a t e d C F channel amplitude and channel open
probability and their corresponding pipette holding potentials. The recordings
were made under the cell-attached configuration, and approximately 10 min
after the perfusion of 10 pM A23187 to the conjunctival epithelial cell.
Potential (mV) Amplitude (pA) J T o
-60 1.54 0.27
-40 0.89 0.29
-20 0.38 0.29
0 0.00 0.00
+20 0.52 0.29
+40 1.12 0.27
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
60 mV 40 20 -40 -20 -60
-0.5 -
-2 1
pA
Figure 4-11. I-V relationship of the single Ca2 + -activated Cl' channel detected in the
conjunctival epithelium under inside-out patch clamp configuration. The slope
conductance was estimated to be 25 pS.
98
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3.5. Conjunctival CFTR detection
Western blot analysis utilizing a C-terminus specific anti-CFTR monoclonal
antibody revealed a band of approximately 170 kD in T84 and cultured conjunctival
epithelial cell lysates (Fig. 4-12).
/
^ M.W.(kD)
202
(170 kD)
116
84
Figure 4-12. Western blot analysis of CFTR expression in T84 (positive control)
and conjunctival epithelial cells probed with a C-terminus specific CFTR
monoclonal antibody. Both T84 and conjunctival epithelial cell lysates were
prepared from confluent cells grown on tissue culture-treated petri dishes.
99
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4. Effect of forskolin on conjunctival [Ca2 + ]i
The baseline conjunctival [Ca2+]j estimated from experiments using Indo-1
AM as a C a2+ indicator was 181 ± 17 nM (n = 7). This level was not significantly
(p>0.05) altered by the application of 10 pM forskolin. Fig. 4-13 shows a typical
time course of the [Ca2 + ]; measurement.
10 mM EGTA
400
350
300
250
10 p.M forskolin
2
C 200
+ " " 1
10 0
50
250 J J .M digitonin
400 300 100 200 0
Time (sec)
Figure 4-13. Effect of 10 pM forskolin on [C a2+]j in confluent conjunctival
epithelial cells g ro w n on cover slips in a representative experiment. Digitonin
(250 pM) and EGTA (10 mM) were applied to establish maximum (Fmax) and
minimum (Fm in ) fluorescence intensity, respectively.
100
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5. Conjunctival fluid secretion
5.1. Baseline fluid secretion and its stimulation
A net s-to-m fluid secretion rate (Jv ) of 4.3 ± 0.2 fll/hr/cm2 (n = 27) was
observed in the freshly excised conjunctiva under open-circuit, baseline condition
(Figs. 4-14 and 4-15). Mucosal application of 1 mM 8-Br cAMP approximately
doubled the baseline fluid secretion (p < 0.05) to 8.4 ± 0.4 pl/hr/cm2 (n = 5) (Figs. 4-
14 and 4-15). By contrast, 10 pM UTP increased fluid secretion to 9.8 ± 0.6
pl/hr/cm2 (n = 6) (p < 0.05) transiently for 10 min (Figs. 4-14 and 4-15). The values
of 8-Br cAMP- and UTP-induced fluid secretion are not statistically different (p >
0.05). Effects of Ca2 + and PKC modulating agents, A23187 and PMA, respectively,
on conjunctival baseline fluid secretion were not recorded due to the observed
sensitivity of fluid secretion to low concentration (0.1%) of the vehicle (DMSO) that
was required to dissolve these compounds.
101
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a. b.
0.7 t
1 mM 8Br-cAMP
>
0 J
5 0.35
o
>
<
0 60 20 40
Time (min)
0.7 T
10 |iM UTP
>
< o
S’ 0.35
o
>
< 3
60 0 40 20
c. d.
Time (min)
0.7
0.5 mM Ouabain
>
o >
J? 0.35
o
>
<
0 60 40 20
0.7 t
20 mM D-glucose
>
4 )
g > 0.35
o
>
<
60 0 20 40
Time (min) Time (min)
Figure 4-14. Typical voltage vs. time tracings demonstrating the effect of (a) 1 mM
8-Br cAMP, (b) 10 pM UTP, (c) 0.5 mM ouabain, and (d) 20 mM D-glucose on
transconjunctival fluid secretion. One volt (V) deflection is equivalent to 127 (im of
fluid displacement according to the calibration table provided by the probe
manufacturer (Mechanical Technology, Inc., Latham, NY). The positive slope of the
linear segment denotes the transconjunctival fluid secretion rate.
102
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2 0 .0
15.0
10.0 -
5.0 --
x
8 0.0
a >
-5.0 --
- 10.0
-15.0
SEC R ETO R Y
a )
c
«
m
a
m
<
o
B
Q Q
C O
a.
h-
D
S
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c
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si
< Q
3
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s
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in
6
t
r!i
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m
o
o
3
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s
E
o
CM
c
c
( 0
E
o
CM
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eo
< 5 f
E
m
O
E
8
o
a.
> • .
X
A BSO R PT IV E
I
!
T
t
Figure 4-15. Conjunctival water flux at baseline (n = 35) and in r e s p o n s e to
application of mucosal 1 mM 8-Br cAMP (n = 5), mucosal 10 pM U T P (n = 6),
serosal 0.5 mM ouabain (n = 4), mucosal 20 mM D-glucose (n = 4), mucosal 20 mM
D-mannitol, Cl'-free (both sides, n = 4), mucosal hypertonic (480 mOsm, n = 4), and
mucosal hypotonic (100 mOsm, n = 4 ) conditions. The steady-state fluid f l o w was
recorded for all treatment conditions except for UTP (fluid secretion rate measured
at the peak of the transient stimulation). Error bars represent s.e.m. * indicates a
significantly higher fluid flow (p < 0.05) than baseline, f indicates a significantly
lower (p < 0.05) fluid flow than baseline.
103
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5.2. Demonstration of the link between fluid and Cl' transport
The increase in conjunctival fluid secretion afforded by 8 -Br cAMP was
concentration-dependent up to 3 mM range studied (Fig. 4-16). The observed
maximal change in fluid secretion rate (AJV ) was 5.1 ± 0.3 pl/hr/cm and the half-
maximal 8 -Br cAMP concentration was 0.32 ± 0.06 mM. This latter value is
comparable to the previously reported 8 -Br cAMP effective half-maximal
concentration (EC50) values for stimulating Isc (0.28 mM) and net CF secretion (0.30
mM) (Fig. 4-4) (173). A good correlation (r2 = 0.98) was attained between the
changes in net fluid flux and the observed changes in Isc induced by agents known to
affect active conjunctival CF secretion (i.e., excluding D-glucose) and Cl -free
condition (Fig. 4-17). In addition, net fluid transport was abolished when CF was
removed from both sides of the reservoir (0.06 ± 0.04 pl/hr/cm2, n = 4) (Fig. 4-15)
(p < 0.05). Indirect interruption of the basolateral CF uptake mechanism (i.e., Na+ -
K+ -2CF cotransport) by inhibiting the Na+-K+ ATPase via 0.5 mM ouabain also
significantly (p < 0.05) reduced the net Jv to 0.3 ± 0.1 pl/hr/cm2 (n = 4), not
significantly different from zero (p > 0.05) (Figs. 4-14 and 4-15).
104
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( g U io /j q /ir i) , a u A p v
2.7
0.0
3.0 0.0 1.5
[8-Br cAMP] (mM)
Figure 4-16. Changes in net transconjunctival fluid secretion rate as a function
of mucosal 8-Br cAMP concentration. Error bars denote s.e.m. for n = 4-6.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
5.3. Effect of active Na+ -absorption
Twenty mM D-glucose was previously determined to induce a maximal
increase in transconjunctival Is c (83) due to stimulated Na+ -dependent glucose
absorption across conjunctiva in addition to net Cl" secretion. Mucosal addition of
20 mM D-glucose to BR significantly reduced (p < 0.05) the baseline Jv by 77% to
1.0 ± 0.5 pl/hr/cm2 (n = 4) (Figs 4-14 and 4-15). Mucosally applied 20 mM D-
mannitol (which elevated the osmolality by 22 ± 1.2 mOsm) did not affect the
baseline Jv significantly (p > 0.05).
106
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SECRETORY
Hypertonic (480 mOsm)
10 -
UTP
- j l - 3 m M 8-Br cAMP
^ — im M 8-Br cAMP
* 0.5 mM 8-Br cAMP
| 0.1 mM 8-Br cABp
10.0 A Ijc (pA/cm2 )
CM
E
o
Baseline S ..
f
5.0 - 10.0
>
“ 3
<3
D-glucose Cl-free Ouabain
-10 - -
Hypotonic (100 mOsm)
ABSORPTIVE
-20
Figure 4-17. Relation between changes in net fluid secretion ( A J v n e t ) and the
c o r r e s p o n d i n g A I SC o b s e r v e d b y a d d i n g a g e n t s ( t o r e s p e c t i v e b a t h i n g f l u i d )
k n o w n to a f f e c t a c t i v e Cl' s e c r e t i o n , N a + a b s o r p t i o n , and osmolality. Error b a r s
denote s.e.m. for n = 4 - 6 . The A I SC values for 8-Br c A M P (173), Cl'-free (107),
o u a b a i n ( 1 0 7 ) , D - g l u c o s e ( 8 3 ) , a n d U T P ( 2 0 7 ) w e r e f r o m o u r p r e v i o u s r e p o r t s .
The linear correlation (r2 = 0.98) was determined only for conditions that a f f e c t
active C l" secretion (i.e., D-glucose, hypotonicity, and hypertonicity were exempt).
107
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5.4. Effect of osmolality
As shown in Fig. 4-15, lowering mucosal osmolality from 290 to 100 mOsm
resulted in a m-to-s fluid flow of 12.1 ± 1.5 pl/hr/cm2 (n = 4). Mucosal hypertonic
challenge (480 mOsm), on the other hand, elevated the baseline water flow to 14.1 ±
2.2 (il/hr/cm2 (n = 4). The transconjunctival osmotic water permeability ( P f ) was
determined to be 13.1 ± 1.9 and 13.4 ± 1.6 pm/sec with mucosal osmolality of 480
and 100 mOsm, respectively. These two P f values are not significantly different (p >
0.05).
6. Effect of conjunctival fluid flux on solute transport
6.1. Baseline Pa p p
Under baseline conditions (isotonic solutions on both sides of the
o
conjunctival tissue), the m-to-s Pa p p of FD-4 was determined to be 3.59 ± 0.18 x 10"
cm/s (n = 4), 14C-mannitol was 2.68 ± 0.48 x 10' 7 cm/s (n = 3), 3H-HPMPC was 4.90
± 0.69 x 10 7 cm/s (n = 5), and 3H-betaxolol was 4.92 ± 0.51 x 10"6 cm/s (n = 4) (Fig.
4-17). The R t of the tissues used in these transport studies was 1216.2 ± 102.2
£ 2 .cm2.
6.2 Effect of hypotonicity
Reduction of mucosal osmolality to 100 mOsm significantly (p<0.05)
increased the m-to-s Pa p p of FD-4 by 106%, 14C-mannitol by 226%, and 3H-HPMPC
108
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by 31%. 3H-betaxolol flux, on the other hand, was not significantly (p>0.05) affected
by the mucosal hypotonicity (Fig. 4-17).
109
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m to s Mannitol flux
Hyper
Baseline
*
H ypo ;
Hyper
Baseline
Hypo
3 6
Papp (x1 O '7 cm/s)
m to s HPMPC flux
3 6
Papp (X1 O'7 cm/s)
H * *
H yper
Baseline
Hypo
1
Hyper
Baseline
Hypo
m to s Betaxolol flux
3 6
Papp (x10-7 cm/s)
m to s FD-4 flux
Papp (X10-8 COT/S)
Figure 4-18. Mucosal-to-serosal apparent permeability of mannitol, betaxolol,
HPMPC, and FD-4 under mucosal isotonic (300 mOsm), hypertonic (480 mOsm),
and hypotonic (100 mOsm) conditions. * indicates a significantly higher (p < 0,05)
Pa p p value than the baseline. ** indicates a significantly lower (p < 0.05) Papp value
than the baseline. Error bars denote s.e.m. for 3-6 tissues.
110
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6.3. Effect of hypertonicity
Mucosal hypertonic condition (480 mOsm H20 ) significantly (p<0.05)
reduced the m-to-s transport of FD-4 by 50%, 14C-mannitol by 63%, and 3H-HPMPC
by 69% (Fig. 4-17). The m-to-s Pa p p of 3H-betaxolol, however, was not significantly
affected (p>0.05) (Fig. 4-17).
6.4. Correlation between solute and fluid fluxes
When m-to-s Pa p p values of 14C-mannitol, 3H-betaxolol, 3H-HPMPC, and FD-
4 were analyzed for correlation against m-to-s transconjunctival fluid flux under
isotonic, hypertonic, and hypotonic conditions, an r value ranging from 0.95 to 1.00
was determined for 14C-mannitol, 3H-HPMPC, and FD-4 (Fig. 4-18). 3H-betaxolol
on the other hand, demonstrated no correlation between the m-to-s Pa p p and fluid
fluxes (r2 ~ 0 .0 0 ).
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
0 )
£
o
r^.
O
m
6
9t Mannitol
h h Hypo
3
-16
r2 = 0.96
Baseline
HyperK^—i
16
Water flux (^l/hr/cm 2)
1
u
©
T “
6
Hypo
a
-16
HPMPC
Baseline
I— # — i
Hyper
16
Water flux (ul/hr/cm2 )
1
0
1
£
m
6
+
Hypo
-16
Betaxolol
Baseline
Water flux (nl/hr/cm 2)
Hyper
16
FD-4
9 t
®" Hypo
" 5 “
X
m _
6 3
Baseline
£
Hyper
16 -16 0
Water flux (jxl/hr/cm2 )
Figure 4-19. Relation between m-to-s Pa p p for mannitol, betaxolol, HPMPC, and
FD-4 and fluid flux across freshly excised conjunctival tissue under mucosal
isotonic ( 3 0 0 mOsm), hypertonic (480 mOsm), and hypotonic (100 mOsm)
conditions. Error bars denote s.e.m. for n = 3-6.
112
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V. DISCUSSION
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Conjunctival active ion and fluid transport processes had not been well
characterized prior to this investigation. As the cellular and physiological
characteristics of the conjunctival epithelium slowly unveiled by our laboratory and
other investigators (47,74,130,172,200), the essential functions of the conjunctiva in
maintaining fluid balance and mucus hydration in the anterior segment of the eye
have been experimentally supported. In this project, three major goals were
achieved: (1) Cl' secretion and different types of Cl' channels have been detected and
characterized in the conjunctival epithelium; (2 ) conjunctiva was demonstrated to be
fluid secreting and such a phenomenon was shown to be driven by active Cl
secretion; and (3) fluid flow can be utilized to enhance the permeability of
hydrophilic compounds across the conjunctival tissue.
1. Cyclic AMP-, Ca2 + -, and PKC-regulation of the conjunctival active Cf
secretion
1.1. Evidence of baseline active Cl' secretion across intact conjunctiva
Kompella et al. (107) and Shi and Candia (172) suggested that conjunctival
epithelium is capable of active Cl" secretion based the observed sensitivities of Is c
across isolated conjunctival tissue to different Cl" transport modulating agents and
the presence and absence of Cl' in the buffer. Such a hypothesis was confirmed in
the current investigation by demonstrating the baseline net Cl' secretory property of
the pigmented rabbit conjunctiva by direct measurements of unidirectional 36C 1
114
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fluxes. This is indicated by the 2-3 times higher observed j6Cl ratios than
those predicted from passive diffusion with the Cl" secretion stimulators, and by the
similarity in observed and predicted 36C1 JS n/Jms ratios afforded by Cl" transport
inhibitors (Table 4-1). This result, together with the excellent correlation between
the measured changes in net Cl" flux and the observed changes in Isc induced by
various pharmacological agents (Fig. 4-4), confirm the suggested active Cl" transport
in the rabbit conjunctiva (107,172). This demonstration of the baseline conjunctival
Cl" secretion was also the first indication of the existence of a s-to-m ionic driving
force for fluid across the conjunctival epithelium.
The arrest in Cl" secretion due to a significant decrease in Cl" Jsm induced by
bumetanide suggests the involvement of the Na+-(K+ )-2Cl" cotransporter in Cl" entry
from the serosal side of the conjunctiva. Furthermore, the abolition of net Cl"
secretion by the mucosal addition of NPAA (Table 4-1) suggests the involvement of
Cl' channels in Cl" exit from the mucosal side of the tissue. These data demonstrated
that the previously reported inhibition by bumetanide and NPAA on conjunctival Is c
(107) was due to the abolishment of Cl' movement through these transport pathways,
and provided first direct indications of the conjunctival Cl" entry and exit
mechanisms.
115
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1.2. Conjunctival CF entry mechanisms
Although the bumetanide-sensitive Na+ -(K+ )-2CF cotransporter is the only
active CF entry mechanism investigated in the conjunctival epithelium thus far, we
do not exclude the possibility of the existence of other CF uptake mechanisms such
as CF/HCO3 exchangers and Na+ -CF cotransporters in this system. The CF/HCO3"
exchanger has been detected on both the apical and basolateral membranes (even
within the same cell type (177) of the bovine lens epithelium (7), bovine pigmented
ciliary epithelium (75), and frog corneal epithelium (153). The function of the CF
/HCO3 exchanger is often coupled with a parallel but independent electroneutral
Na+ /H+ exchanger which involves respective counter-ions (42,177). Since these ion
transport processes are electroneutral, their activity can only be detected via non-
electrophysiological methods such as intracellular pH measurements in the presence
and absence of CF, Na+ , and HCO3', or a Na+ /H+ exchange inhibitor, amiloride, in
the extracellular bathing solution (153). Since the focus of the current investigation
was to characterize conjunctival CF exit mechanisms, these experiments were not
performed. The results from these methods would provide some insight on the
existence of CF/HCO3" and Na+ /H+ exchange processes in the conjunctival
epithelium.
Na+ -CF cotransport has been reported to be one of the CF uptake mechanisms in
several epithelia such as human (118) and bovine (137) tracheal epithelia, rabbit
116
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gallbladder epithelium (41), and corneal epithelium (19). There are two types of
Na+ -Cl" cotransporters known to date: (1) thiazide-sensitive Na+ -Cl" cotransporter
(TSC), and (2) K+ -independent, bumetanide-sensitive Na+-Q" cotransporter. Like Cl"
/HCO3" exchangers, Na+ -G" cotransporters have been found on either the apical or
basolateral side of the epithelium. Since the Na+-Cl" cotransport process can be
inhibited by bumetanide, our observation of the abolishment of net Cl" secretion
across the conjunctiva in the presence of serosally applied bumetanide does not
eliminate the possible existence of a Na+ -Cl" cotransporter on the serosal side.
Furthermore, our findings to date suggest that conjunctival active ion transport
processes resemble that in the corneal epithelium. Detection of the Na+ -Cl"
cotransport process in the corneal epithelium (19) may indicate the possible existence
of such a transport mechanism in the conjunctival epithelium. In order to determine
the possible existence of the conjunctival Na+ -Cl" cotransporter and to gain a better
understanding for the conjunctival Cl" uptake processes, it would be necessary to
measure the effect of bumetanide and thiazide on unidirectional 2 2 Na and j6C1 fluxes,
under K+ -containing and K+ -free conditions.
1.3. Stimulation of cAMP-, Ca2 + -, and PKC-sensitive 36C1 secretion
Consistent with the transconjunctival Is c results we reported previously (107),
the mucosal addition of 8 -Br cAMP significantly increased the Cl" Jsm without
affecting the Jm s (Table 4-1), yielding a 133% increase in net Cl" secretion. This
117
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observation suggests that the Cl" secretion in the pigmented rabbit conjunctiva is
regulated by the level of intracellular cAMP. The Jsm is probably responsible for the
bulk of the Isc, as suggested by the excellent agreement of the half-maximal 8-Br
cAMP concentration of 0.28 mM for Is c (Fig. 4-4a) and 0.30 mM for CF Jsm (Fig. 4-
4b) and by the excellent correlation between Isc and Cl" Jsm in the presence of varying
8-Br cAMP concentrations (Fig. 4-4c). These observations indicate the stimulation
of the overall Cl" secretion across the conjunctiva by the elevating intracellular
cAMP concentration.
The observed cAMP-induced Cl" secretion could be the result of the direct up-
regulation of the Cl" exit pathway such as Cl" channels, Cl" entry mechanisms such as
Na+ -K+ -2C1" cotransporter and Na+ -K+ -ATPase (secondary effect on Cl" entry and
exit), or both Cl" entry and exit routes. Tanimura et al. (182) demonstrated the direct
phosphorylation and functional up-regulation of the rat parotid Na+-K+ -2CF protein
in the presence of cAMP analogues and forskolin. In addition, Carranza et al. (28)
reported the stimulatory effect of PKA phosphorylation on Na+-K+ -ATPase activity
by inducing recruitment of active Na+ -K+ -ATPase units to the plasma membrane of
the proximal convoluted tubule cells. Such an up-regulatory effect of cAMP on Na+ -
K+ -ATPase activity was supported by an earlier study by Gao et al. (67) utilizing the
whole-cell patch clamp technique to detect the Na+ -K+ pump current in guinea-pig
ventricular myocytes in the presence of extracellular isoprenaline. The study also
118
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indicated the inhibition of the isoprenaline-induced current in the presence of H-7 (a
non-specific protein kinase inhibitor). Current investigation and previous
observations made in conjunctival tissues by Kompella et al. (107) and in
conjunctival epithelial cells by Saha et al (unpublished data), confirmed the existence
of conjunctival Na+-K+ -2CF cotransporter and Na+ -K+ -ATPase. In order to shed
some light on the effects of elevating conjunctival intracellular cAMP concentration
on Na+-K+ -2CF cotransporter and Na+ -K+ -ATPase activities, and their contribution to
overall increase in net CF secretion, it would be important to measure the level of
phosphorylation of conjunctival Na+ -K+ -2CF cotransporter and Na+ -K+ -ATPase
proteins directly under such a state.
The net 3 6 C 1 flux was elevated transiently by A23187 (Table 4-1 and Fig. 4-3a). This
finding suggests that a Ca2 + -modulated CF transport mechanism (e.g., Ca2 + -activated
CF channel) possibly exists in the pigmented rabbit conjunctiva. This raises the
question of whether the Ca2 + ~activated CF efflux in the conjunctival epithelium
occurs as a result of (a) direct ligand-type interaction of the Ca2 + ions with the CF
channels on the cytoplasmic domain or (b) other Ca2 + -dependent cytoplasmic events
such as Ca2 + /calmodulin-dependent phosphorylation induced by protein kinases (e.g.,
Ca-kinase) (4). These possibilities have not been demonstrated convincingly to date.
Studies by Frizzell et al. (61) using the patch clamp technique suggest that the effect
of Ca2 + on the secretory CF channels is likely to be mediated by a Ca2 + -dependent
' 119
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regulator, instead of direct Ca2 + binding to the Cl' channel. The evidence is that Cl’
channel activities, once activated by Ca2 + ionophore in the cell-attached patch
configuration, could not be altered in an excised patch whose cytoplasmic side was
exposed to various Ca2 + concentrations. Similarly, Cliff et al. (35) observed that the
whole-cell conductance of T84 cells was activated by either Ca2 + ionophores or
buffering the cellular Ca2 + levels to 300-400 nM. These results suggest a critical role
for certain cytosolic component(s) in the regulation of these Cl' channels. On the
contrary, Collier et al. (39) and Koumi et al. ( I ll) both observed dose-dependent
activation of Cl' current by Ca2 + in the inside-out patch clamp configuration in canine
ventricular myocytes and guinea-pig hepatocytes, respectively, suggesting a direct
ligand-type interaction of the Ca2 + ions with the Cl' channels.
Recent studies utilizing either the short-circuit current or the patch-clamp technique
demonstrated the PMA stimulated CF current in a dose-dependent manner
(29,112,174). Our observed transient stimulation of Is c induced by PMA (Fig. 4-1)
was similar to that described by Chan et al. (29) in the rat epididymal epithelium.
The stimulated net 3 6 C1 flux by PMA in the conjunctiva (Table 4-1), therefore,
supports the notion that PKC-sensitive CF channels are residing in the conjunctival
epithelium. The existence of PKC-sensitive CF channels has also been reported by
Crosson et al. (44) in the rabbit cornea, as indicated by the stimulation of
transcomeal Isc and membrane PKC activity by 12-O-tetradecanoylphorbol-13 acetate
120
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(TPA), a phorbol ester. The above demonstrations of cAMP-, Ca2 + -, and PKC-
induced stimulatory effects on conjunctival tissue Is c and net Cl' secretion would lead
into following in-depth detection of individual Cl" channel types.
1.4. Evidence for the existence of three types of Cl' channels in the conjunctival
epithelium
An additive stimulation of Is c that was independent of treatment sequence was
observed when the conjunctival tissues were treated serially with 1 mM 8-Br cAMP
and 10 pM A23187, or with 10 pM A23187 and 1 pM PMA (Fig. 4-2).
Interestingly, compared to the 8-Br cAMP-A23187 treatment sequence, a lesser
degree of stimulation by A23187 was observed when PMA was applied first (42% in
Fig. 4-2a vs. 25% in Fig. 4-2c). It appears that the degree of stimulation induced by
A23187 was somehow influenced by PMA. Such a phenomenon was observed again
in the 8-Br cAMP- PMA serial treatment combination, in that 8-Br cAMP failed to
stimulate Is c significantly after the PMA treatment (Fig. 4-2f). Furthermore, all
tissues that were stimulated with PMA regardless of the treatment sequence appeared
to show a gradual decline of Is c to below the initial baseline (Figs. 4-2c-f), suggesting
the possible down-regulation of Cl' secretion by PMA subsequent to its initial
stimulatory effect. A similar observation in T84 cells was made by Matthews et al.
(128), in that forskolin-stimulated Cl" efflux was gradually inhibited by prolonged
exposure to PMA. Such a trend was not observed when combined treatments of 8-Br
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cAMP and A23187 were instituted (Figs. 4-2a and b). Cliff and Frizzell (36)
concluded that cAMP and Ca2 + activated separate Cl" channels in T84 cells based on
the additive stimulation of CF currents induced by forskolin and Ca2 + ionophore.
To summarize, Cl" appears to enter the pigmented rabbit conjunctiva from the serosal
fluid via a Na+ -(K+ )-2Cl" cotransport process and exit to the mucosal fluid via channels,
resulting in active CF secretion. Active Cl" secretion in the pigmented rabbit
conjunctiva appears to be modulated by cAMP, Ca2 + , and PKC. The seemingly
independent effects induced by 8-Br cAMP, A23187, and PMA suggest the existence
of three corresponding types of CF transport mechanisms (cAMP-, Ca2 + , and PKC-
regulated CF channels) in the pigmented rabbit conjunctiva. It is possible, however,
that these stimulants may also activate the CF influx mechanism via Na+ -(K+ )-2CF
cotransport, as they do in rabbit colonocytes (166), fetal human nonpigmented ciliary
epithelium (43), and rat cerebral capillary endothelium (96).
2. D e t e c t i o n and characterization of s p e c i f i c conjunctival c A M P - , Ca2 + -, and
PKC-regulated C l" channels
The above observations demonstrated that the conjunctival tissue is capable
of secreting CF. However, in order to determine whether or not the sensitivity of net
CF secretion to cAMP-, Ca2 + -, and PKC-stimulating agents across the conjunctiva
was due to combined activities of specific CF channel types required in-depth
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examinations. To probe for specific Cl" channels in the conjunctival epithelium,
patch clamping, a high-resolution biophysical technique that allows activities of
either a single, or a group of, ion channels to be measured in isolation (140), was
used in the present investigation.
One major obstacle encountered during the patch clamping of conjunctival epithelial
cells was the extreme difficulty in forming a gigaohm seal between patch electrode
and cell membrane, and maintaining the seal following the perfusion of the drug to
the cell. This was particularly true during the cell-attached single-channel patch
recordings where a much higher seal resistance was required. For this reason,
experiments were performed within three days after the seeding of the cells, as the
difficulty of seal formation increases through time.
The baseline whole-cell conductance of 0.42 ± 0.10 nS detected in conjunctival
epithelial cells is comparable to that reported for rat lacrimal acinar cells (~1 nS)
(110). The whole-cell capacitance of conjunctival epithelial cells of 8.9 ± 1.0 pF (n =
14) measured in this study was comparable to that estimated by Watsky (~7 pF)
(200). Cell membrane behaves like a capacitor and its capacitance is an indicator for
the cell surface area (124). It was possible that fusion of small granules, which might
contain ion channels themselves, with the plasma membrane (i.e., exocytosis and
endocytosis) might occur during the course of patch clamp recordings. Lollike et al.
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(123) have reported that in human neutrophils, the diameter of the vesicles ranged
from 60-165 nm, and could fuse with the plasma membrane and contribute 0.1-5 fF
in capacitance within a cell-attached patch.
2.1. cAMP-regulated Cl' channels
Based on the 5-fold increase in the whole-cell conductance by forskolin, and
a lack of stimulation by 1,9-dideoxyforskolin (Fig. 4-6), cAMP-regulated CF
channel activities may be responsible for the observed whole-cell currents.
Furthermore, the observed rightward shift of the reversal potential of the forskolin-
induced I-V curve upon external Cl' reduction from 151 to 56 mM indicates that the
stimulated whole-cell conductance was contributed by the activation of anion
selective channels (Fig. 4-8). The agreement between the observed shifted reversal
potential and the Nemst equation-predicted reversal potential further confirms the
stimulation of Cl' channels in the presence of forskolin.
The observed cAMP-stimulated Cl" secretion across the excised conjunctiva could
involve either direct or indirect activation of cAMP-sensitive Cl' uptake mechanisms.
Although the determination of these possibilities is beyond the scope of the current
investigation, the detection of the conjunctival cAMP-sensitive Cl' channels suggests
that the observed net Cl' secretion across the conjunctival tissue was at least in part
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due to activation of apically located Cl' channels that are triggered by the elevated
intracellular cAMP level.
The existence of cAMP-sensitive Cl" channels on the conjunctival epithelial cell
surface was further suggested by a significant increase in single-channel P0 in the
presence of 10 pM forskolin (Fig. 4-9). The rabbit conjunctival cAMP-regulated C1‘
channels demonstrated similar biophysical profile to that reported in guinea-pig
myocytes (138), human pancreatic epithelial cells (37), and rat bile duct epithelial
cells (132): linear I-V relationship and estimated single channel conductance of 10
pS. These results suggest that the detected conjunctival Cl' channels could be similar
to those observed in epithelia mentioned above. These results obtained from both the
whole-cell and single-channel patch clamp recordings indicated that the previously
reported stimulation of conjunctival Is c and net Cl" secretion (107,173) by cAMP
modulating agents were due to activation of cAMP-regulated Cl' channels residing
on the conjunctival epithelial cell surface. Nevertheless, the effect of forskolin on
conjunctival whole-cell and single-channel conductances would have been more
convincing if the forskolin dose-dependency of channel conductances (whole-cell
and single-channel) and single-channel P0 were examined.
To obtain a more accurate estimate of the conjunctival single-cAMP-regulated Cl’
channel conductance, however, requires measurements of single-channel current
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amplitudes under a wider range of pipette holding potentials (only 0 and -40 mV
were used in the present study). Furthermore, an assumption of a linear I-V
relationship was made by generating the plot with only two holding voltages. This
could jeopardize the accuracy of the single-channel conductance estimation. In
addition, we cannot eliminate the possibility that the detected conjunctival single
channel activities in the presence of forskolin could include something other than CF
channel activities. Under the cell-attached patch clamp configuration where the
patched cell remained intact, residual intracellular K+ may induce K+ conductance
through conjunctival K+ channels. For this reason, conjunctival single-CF channel
conductance measured under the cell-attached configuration should have been
performed in the presence of 1-5 mM range of BaC^ (a K+ channel blocker (107)) in
the bathing solution, in order to exclude possible K+ conductances.
A low frequency of detection of the cell-attached single-channel activities was
observed in the conjunctival epithelium (7 out of 35 successful patches). A recent
study by Moyer et al. (136) investigating the trafficking of CFTR in MDCK type I
epithelial cells grown on different surfaces may provide partial explanation to this
observation. In their study, a green fluorescent protein (GFP) was conjugated to the
N-terminus (located on the cytoplasmic side) of the CFTR protein without altering its
normal functional characteristics. Interestingly, GFP-CFTR localization was found
to be substratum-dependent. In cells grown on permeable supports (5-7 days post
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seeding), GFP-CFTR was recruited to the apical surface. In cells that were grown on
glass cover slips, on the other hand, GFP-CFTR was found to be recruited mainly to
the basolateral membrane. These results may support the low detection frequency of
single conjunctival cAMP-sensitive CF channels, since the patch clamp recordings in
the current investigation were performed on cells seeded on tissue culture-treated
petri dishes. In other words, the detection rate of cAMP-dependent single-CF
channel activities in the conjunctiva may improve by growing cells on a permeable
support.
2.1.1 Existence of conjunctival CFTR
Although CFTR is the only cAMP-sensitive CF channel cloned to date, it is
not known whether this is the only type of cAMP-sensitive CF channel. It is worth
noting that the conductance of all detected single cAMP-dependent CF channels in
the conjunctival epithelium was consistent, which may suggest the existence of only
one type of cAMP-dependent CF channels in this epithelium. However, the
confirmation of this hypothesis can only be made by the molecular identification of a
cAMP-dependent CF channel protein (e.g., contains a PKA phosphorylation site) that
is structurally different from CFTR (e.g., isoforms). The 170 kD band revealed by
the C-terminus specific CFTR antibody in the Western blot analysis positively
identified the existence of conjunctival CFTR for the first time (Fig. 4-9). The
position of the CFTR band was in agreement with the size of CFTR originally
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reported by Riordan et al. (154). Two additional observations support the existence
of conjunctival CFTR: (1) the observed linear I-V relationship from the forskolin-
induced whole-cell current was consistent with the reported I-V pattern of CFTR in
the airway and colonic epithelia (10,36,113,188); and (2) the small forskolin-
stimulated single-channel conductance (12 pS) resembles that reported for the human
airway and intestinal epithelial CFTR (9,168). The presence of CFTR in the
conjunctival epithelium may indicate a pathological relevance to the dry eye
syndrome. In CF cells, CFTR was not detected on the cell surface, due to its inability
to progress through the normal biosynthetic pathway and to traffic to the apical
plasma membrane (209,210). It is possible, therefore, that dysfunctional CFTR
contributed to the reported conjunctival abnormalities observed in CF patients.
2.2. C a 2 + - r e g u l a t e d Cl' channels
The existence of other type of conjunctival Cl' channel, that can be triggered
by increasing intracellular Ca2 + concentration was confirmed by the stimulation of
the baseline conjunctival whole-cell conductance in the presence of A23187.
A232187 induced CF conductance by 4-fold at the negative potentials and 6-fold at
positive potentials. Such type of outwardly rectifying I-V relationship (Fig. 4-7 and
4-9), with the whole-cell conductance increases towards hyperpolarizing potentials,
was similar to that reported in rat lacrimal acinar cells (110), human placental
cytouophobiast cells (99), and sheep parotid secretory cel is (88). The observed
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sensitivity of whole-cell conductance to A23187 indicates that a group of channels
residing on the conjunctival epithelial cell surface was activating in response to an
elevation in the intracellular Ca2 + concentration. The Cl" conductivity of the
A23187-activated channels was also confirmed by the rightward shift of the reversal
potential when the extracellular Cf concentration was reduced from 151 mM to 56
mM (Fig. 4-8).
In-depth investigation of the Ca2 + -sensitive CF channels utilizing inside-out single
channel patch clamp configuration provided convincing evidence that this type of CF
channel does exist in the conjunctival epithelium (Fig. 4-10). Interestingly,
contradictory to the outwardly rectifying I-V pattern observed under during the
whole-cell measurement, the single Ca2 + -sensitive Cl" channel I-V relationship
appeared linear (Fig. 4-11). Such a linear CF channel conductance, with a single
channel conductance of approximately 25 pS, has been reported in the bovine
tracheal epithelial cells (90). Gruber et al (71) cloned and functionally characterized
the first human member of the family of Ca2 + -activated Cl" channel proteins. In this
study, an ionomycin-induced outwardly rectifying I- V pattern was observed under the
whole-cell configuration. However, when the single-channel activities were
measured in the presence of extracellular ionomycin under the cell-attached
configuration, the J-V relationship was linear. The authors did not elaborate on
whether or not such an observation was expected, nor did they postulate on the
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possible cause(s) of this discrepancy between the whole-cell and the single-channel I-
V patterns. Several members of the CLCA family have been detected either
molecularly or electrophysiologically in different species (45,66,90). It is possible
that these isoforms of the Ca2 + -activated Cl" channels may behave differently
biophysically, hence the detected individual CLCA characteristics may not be
consistent with the overall CLCA activities observed during the whole-cell
recordings.
The detection of Ca2 + -regulated CF channels indicates that the observed increase in
conjunctival Is c and net 3 6 C 1 fluxes in the presence of A23187 was due to activation
of specific CF channels that can be triggered by the elevation of intracellular Ca2 +
concentration. As suggested earlier in this discussion, the specific molecular
mechanisms for Ca2 + -activated CF channel activities (e.g., possible involvement of
calmodulin) in the conjunctiva are not clear at this point. Further investigation on
these CF channels would be necessary utilizing the inside-out single-channel patch
clamp configuration while exposing intracellular surface to various Ca2 +
concentrations in order to examine possible calmodulin involvement. This
experiment was not performed since the focus of the current project was on the
detection and biophysical characterization of conjunctival CF channels, instead of the
CF channel regulatory pathways. Exposure of the cytoplasmic surface of the inside-
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out patch to different Ca2 + concentration could also determine the conjunctival
single-Cl' channel conductance in a Ca2 + concentration-dependent manner.
2.3. Possible cross-talk between cAMP- and Ca2 + -dependent cellular responses
It is possible that triggering of one signal transduction pathway could have a
secondary effect on other signal transduction pathways. Several reports have
suggested the possible linkage between intracellular levels of cAMP and Ca2 + in
signal transduction mechanisms. For example, Vajanaphanich et al. (190) reported
the cross-talk between Ca2 + and cAMP-dependent signaling pathways at the level of
second messenger generation. They found that in T84 and rat pancreatic acinar cells,
a membrane permeant analog of cAMP and other cAMP inducing agents reduced the
overall Ca2 + mobilization, in which case may down-regulate CT channels that are
regulated by intracellular Ca2 + . Also, Holz et al. (78) observed a significant
elevation in [Ca2 + ]i by 10 pM forskolin and 1 mM 8-Br cAMP in pancreatic (3-cells.
Contrary to Vajanaphanich and Holz’s results (78,190), Ding et al. (48) reported that
the muscarinic-dependent Ca2 + mobilization in cat iris sphincter smooth muscle cells
was inhibited by 5 pM forskolin or isoproterenol. Although these reports have found
opposite relationships between intracellular cAMP and Ca2 + levels in different cell
types, which could be attributed by cell-specific variation in signal transduction
pathways, they demonstrated possible cross-talk between these second messengers
(e.g., cAMP-dependent protein kinase phosphorylation and activation of Ca2 + -
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ATPase (157)). For these reasons, in addition to detecting various types of Cl"
channels in the conjunctival epithelium, we also examined the possible cAMP-
induced alteration of intracellular Ca2 + level, which could subsequently affect the
activities of Ca2 + -regulated Cl" channels thought to exist in the conjunctival
epithelium. As demonstrated in Fig. 4-12, the intracellular Ca2 + concentration in the
confluent conjunctival epithelial cell layers was not affected by the application of 10
pM forskolin (from the baseline concentration of 120-130 nM). On the other hand,
0.5 pM of A23187 was found to significantly elevate the baseline Ca2 + concentration
in conjunctival epithelial cells by approximately 240% (unpublished data by Hideo
Ueda and Vincent Lee). This finding indicated that the stimulation of conjunctival
whole-cell and single-channel Cl" conductance in the presence of forskolin was solely
due to an increase in intracellular cAMP concentration, and not by the activation of
Ca2 + -dependent ion transport processes triggered by the possible elevation of Ca2 +
level, as suggested by Holz et al. (78).
2.4. PKC-regulated Cl' channels
The existence of PKC-regulated Cl" channels in the conjunctival epithelium
was demonstrated by the observed increased in whole-cell Cl" conductance in the
presence of PMA (Fig. 4-7). PMA stimulated the baseline whole-cell Cl" current in
an outwardly-rectifying fashion, and elevated the conductance by approximately 4-
fold at depolarizing potentials and 8-fold at hyperpolarizing potentials. In addition,
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the degree of the rightward shift of the reversal potential of the PMA-stimulated I-V
curve upon reduction of extracellular CF concentration from 151 to 56 mM was
similar to that predicted by the Nemst equation, which demonstrated that the PMA-
induced current was mainly contributed by CF ion (Fig. 4-8). These findings
demonstrated the existence of PKC-regulated CF channels on the surface of
conjunctival epithelial cells for the first time. These data showed that the previously
observed increase in Is c and net s-to-m 3 6 C1 fluxes in the presence of PMA was the
result of PKC-regulated CF channel activation.
As mentioned previously, different families of PKC isoforms have been found in the
conjunctiva (47). Among these families of conjunctival PKC isoforms, novel and
atypical PKC lack a Ca2 + binding site and their activities are independent of
intracellular Ca2 + concentration. The stimulation of whole-cell conductance by PMA
was not observed when Ca2 + was omitted from the pipette solution. In addition,
many investigations indicated that PMA stimulates CF channel activities that are
regulated by Ca2 + /phospholipid-dependent PKC (44,147). These observations,
however, do not conclude that CF channels can only be regulated by Ca2 + -dependent
PKC isoforms (i.e., classical PKC’s) for the following reasons: (1) CF channels that
are regulated by Ca2 + -independent PKC isoforms (should they exist) could be a small
fraction of the total PKC-regulated CF channel population; (2) novel and atypical
PKC (i.e., non-Ca -dependent PKC isoforms)-regulated CF channels may have very
' ' . " . ' 133
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low conductance; and (3) both (1) and (2). The above possibilities could all lead to
insignificant contribution to the overall PKC-dependent C f conductance in the
absence of intracellular Ca2 + . Under the assumption that novel and atypical PKC-
regulated Cl" channels do not carry significant conductance (i.e., electrophysiological
techniques cannot be used), one possible method of detecting these PKC isoforms
would be to perform Western blot analyses utilizing specific antibodies (65).
The above findings acquired through patch clamp recordings positively identified the
existence of cAMP-, Ca2 + -, and PKC-regulated C f in the rabbit conjunctival
epithelium for the first time. Although attempting to determine the population and
relative contribution in the overall conjunctival C f secretion of each type of Cf
channel may be premature and oversimplifying at this point, the patch clamp data
collected thus far may provide some preliminary indications. Conjunctival whole
cell conductance of all three types of Cf channel (cAMP-, Ca2 + -, and PKC-regulated)
mentioned earlier were similar. Furthermore, the detected single-cAMP- and Ca2 + -
regulated Cf channel conductance (under identical stimulant concentrations) were 10
and 25 pS, respectively. The detection of single-channel PKC-regulated C f channel
conductance was unsuccessful in this study due to constant inability to maintain high
gigaohm seals (>50 GQ) during the perfusion of PMA to the conjunctival epithelial
cells. However, the value reported by Koumi et al. (112) in the guinea-pig
hepatocytes was approximately 10 pS. With comparable whole-cei! conductances
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and single-channel detection frequencies (7 out of 35 for cAMP-regulated and 3 out
of 15 for Ca2 + -regulated) and under the assumption that these two channel types have
similar distribution pattern on the cell surface, it may be reasonable to hypothesize
that the population ratio between cAMP- and Ca2 + -regulated CF channels are 2:1.
This is based on comparable whole-cell conductances between the two Cl' channel
types and the 1:2 single-channel conductance ratio between cAMP (10 pS)- and Ca2 +
(25 pS)-regulated CF channels. The population of PKC-regulated CF channels, on
the other hand, assuming similar single-channel conductance to that reported for the
guinea-pig hepatocytes (10 pS), may be 80 and 40% of that of cAMP- and Ca2 + -
regulated CF channels, respectively.
It is also unknown at this stage whether different families of CF channels detected in
the rabbit conjunctival epithelium are cross-talking among each other, and capable of
compensating the dysfunction of other CF channel types to a certain degree, in an
attempt to maintain the cellular ionic balance. Chen et al. (30) examined the
muscular CF channel C1C-1 in mice carrying defective Clcadr allele of the
corresponding Clcl gene. The defective Clcadr allele causes loss of the 4.5 kb C1C-1
mRNA by 50%. Despite a 50% reduction in functional gene, the CF conductance in
these muscle cells was not significantly different from that in the wild-type muscle
cells. These results indicated a compensatory mechanism at the post-transcnptional
level by either the remaining population of CiC-1 or other CF channel types.
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In summary, conjunctival Cl" secretory processes have been demonstrated and
characterized (Specific Aim #1). The baseline Cl" secretion can be stimulated by
increasing intracellular cAMP, Ca2 + concentrations, and PKC activity, as the result of
respective cAMP-, Ca2 + -, and PKC-regulated Cl" channel activation. Based on these
findings, we may postulate that conjunctival Cl' secretion could create a serosal-to-
mucosal osmotic gradient and generate sufficient driving force to move fluid across
this tissue. Should such a hypothesis be true, the physiological role of the
conjunctiva would be far more significant than just being a passive protective layer
of tissue, which would include maintaining hydration of the air-exposing anterior
surface of the eyeball.
3. Modulation of conjunctival fluid transport
The possibility of regulating conjunctival fluid flow via C f secretion
modulating agents may be of therapeutic value in improving the dry eye conditions
via pharmacological means. During the next stage of this investigation, the
conjunctival baseline fluid secretory properties were determined, and the possibilities
of regulating such a secretory process was examined.
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3.1. Baseline fluid secretion
The pigmented rabbit conjunctival fluid secretory property has been
characterized in the present investigation. The measured baseline Jv is 4.3 ± 0.2
pl/hr/cm2. This value is on the same order of magnitude reported for frog retinal
pigment epithelium (3.9 pl/hr/cm2 ) (86), bovine lens epithelium (4.7 pl/hr/cm2 ) (58),
and human tracheal gland cells (2.0 pl/hr/cm2 ) (91). By contrast, the fluid secretion
rate in the rabbit cornea is 0.2 pl/hr/cm2 (102), only 5% of that in the pigmented
rabbit conjunctiva. The much higher fluid secretion rate in the rabbit conjunctiva as
compare to the rabbit cornea supports our hypotheses that the conjunctiva may play a
more prominent role than the cornea in maintaining the moisture in the anterior
surface of the eye.
3.2. Cl* driven conjunctival fluid secretion
The first indication of the Cr-driven conjunctival fluid secretion was the
abolishment of fluid secretion in the presence of serosal 0.5 mM ouabain and upon
removal of O ' from the bathing solutions (Fig. 4-12). It is, therefore, not surprising
that compounds known to stimulate O ' secretion in the conjunctiva, such as 8-Br
cAMP (173) and UTP (207), also stimulate conjunctival fluid secretion (Figs. 4-13
and 4-14). Indeed, there exists an excellent correlation between the changes in fluid
secretion and those in Isc in the conjunctiva under conditions that affect active O '
secretion (Fig. 4-14). The 128% enhancement of fluid secretion by 10 pM UTP may
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indicate a potential alternative route for fluid secretion should cAMP-sensitive Cl"
secretion become compromised. In fact, Jiang et al. (91) found that human CF
tracheal gland cells maintained normal fluid secretory response to UTP, despite
impairment of the cAMP-dependent Cl" secretion. Although a good correlation was
observed between the changes in conjunctival fluid secretion and Is c by various
agents, these experiments were performed using two separate groups of tissues (i.e.,
fluid secretion measurements and Is c measurements). In order to generate more
convincing data, a new chamber system that allows simultaneous measurements of
fluid flow (using capacitance probes) and bioelectric parameters (electrode
connections to the inner chamber) would be necessary. Furthermore, the
conjunctival tissue integrity can also be monitored throughout the course of the
experiment (i.e., Rt measurement).
The concept of stimulating fluid secretion via increasing intracellular second
messenger concentration was also tested in vivo. Gilbard et al. (68) developed an in
vivo rabbit model for keratoconjunctivitis sicca (KCS) by surgically cauterizing the
lacrimal gland excretory duct and removing the nictitating membrane and Harderian
gland. The same group later used this model to investigate the stimulation of tear
secretion by the accessory lacrimal glands lining inside the eyelids via agents that
elevate intracellular cyclic nucleotide levels (69). In this study, 8-Br cAMP, 8-Br
cGMP, l-isobutyl-3-methyl xanthine (a cyclic nucleotide phosphodiesterase
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inhibitor), and forskolin all found to increase tear volume significantly.
Unfortunately, although the authors acknowledged that they could not eliminate the
possible contribution of conjunctival and corneal epithelial fluid transport, they
claimed that such a possibility was unlikely without providing a reasonable
explanation. They assumed that the increased in tear volume was solely contributed
by the accessory lacrimal gland secretion . Looking from another angle, we also do
not have sufficient evidence in the current study to exclude the possible stimulation
of fluid secretion by accessory lacrimal glands when the excised conjunctiva was
exposed to various stimulatory agents. While it may not make a significant
therapeutic impact, same types of fluid transport measurement would need to be
validated utilizing a simpler system that minimizes the involvement of non-
conjunctival epithelial cells (i.e., conjunctival epithelial culture model) in order to
differentiate the possible contribution of the accessory lacrimal gland secretion to the
overall conjunctival fluid secretion. Utilizing both light and electron microscopy,
Seifert et al. (169) observed that human accessory lacrimal gland nodules are
connected to the secretory glandular epithelia, suggestive of s regulated mechanism
of secretion. However, no information is available on the exclusive measurements of
the accessory lacrimal gland secretion, and an accessory lacrimal gland cell culture
model has not been developed to date.
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3.3. Effect of active Na+ absorption on conjunctival fluid flow
Active Na+ absorption, on the other hand, has also been demonstrated to be a
driving force for fluid flow by coupling with transepithelial passive Cf diffusion in
rat alveolar epithelial cells (152) and rabbit salivary glands (116). Although the
rabbit conjunctiva was determined to be fluid secreting in the current investigation,
presumably due to a much higher Cl" contribution in the overall active ion transport
processes, it would be reasonable to assume that net fluid secretion across the
conjunctiva was the sum of fluid secretion driven by active Cl" secretion and fluid
absorption driven by active Na+ absorption. Such an assumption was confirmed by a
significant reduction in the baseline conjunctival fluid secretion (77%, Fig. 4-12), by
increasing mucosal D-glucose concentration from 5 to 25 mM, presumably
secondary to the stimulation of Na+ uptake via the Na+ -coupled glucose transporter
(83). Simultaneous stimulation of other mucosally located Na+ -uptake processes
(e.g., Na+ -amino acid and Na+ -solute cotransport) may result in a stronger m-to-s
osmotic driving force, consequently a larger reduction in the baseline s-to-m fluid
secretion. The lack of significant influence by 20 mM D-mannitol on Jv indicates
that the osmotic contribution of 20 mM D-glucose on the baseline Jv was negligible.
Recently, Wright et al. (214) reported the capability of the Na+ -glucose cotransporter
to actively transport water molecules into the cell either in the presence or absence of
a substrate. Therefore, it is likely that conjunctival Na+ ~glucose cotransporters are
involved in fluid absorption in two ways: (1) generating an osmotic gradient in the
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m-to-s direction by transcellular active Na+ absorption electrically coupled with
paracellular passive Cf diffusion, and (2) active absorption of water molecules by the
Na+-glucose cotransporter.
3.4. Osmotically driven c o n j u n c t i v a l fluid f l o w
There may exist two possible pathways for conjunctival fluid flow:
paracellular (122) and transcellular (27). Although the route(s) and mechanism(s) of
the fluid movement across leaky epithelia have been under constant dispute (178),
both pathways are probably utilized by fluid flow driven either by active CX secretion
or an osmotic gradient. Transcellular fluid flow, in addition, is probably mediated by
aquaporins (AQP). Aquaporin type 3 (AQP3) has been demonstrated in the human
and rat conjunctival epithelium, using immunoblotting of membrane proteins, high-
resolution immunocytochemistry, and immunoelectron microscopy (73). The
determination of the osmotic water permeability of 12.1 across conjunctiva indicates
the existence of transcellular fluid flow across conjunctival epithelium. Such a
finding may suggest the existence of an apically located water channel on the
conjunctival epithelial cell surface.
While reducing the osmolality of the mucosal bathing fluid from 300 to 100 mOsm
expectedly reversed the direction of conjunctival fluid flow, increasing, it to 480
mOsm stimulated conjunctiva'! fluid flow by 228%. Since tear osmolality in dry eye
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patients was reported to rise to 331 mOsm (15), conjunctival fluid flow may rise by
approximately 0.4 pl/hr/cm2. This small degree of increase may nonetheless be
insufficient to reverse the preexisting fluid deficit. Despite several reports of
symptomatic relief afforded by artificial tear substitutes in dry eye patients (125,215),
the therapeutic rationale as well as long-term therapeutic benefit of this palliative
measure needs to be reexamined in the context of direction of conjunctival fluid
flow.
The mucosal hypotonic condition was shown to induce transconjunctival fluid
movement, presumably driven by the artificially created s-to-m osmotic gradient. It
is possible, however, that the extracellular hypotonic environment could induce
conjunctival epithelial cell swelling, consequently triggering cell volume-sensitive
Cf channels, should they exist in the conjunctival epithelium. Such activation of the
possibly existing conjunctival volume-sensitive Cl' channels during hypotonic
challenge could contribute to the overall fluid secretion by active CT secretion.
Therefore, mucosal hypotonicity could induce fluid movement via two separate
mechanisms: (1) s-to-m osmotic gradient created by mucosal hypotonicity, and (2)
active Cl' secretion by volume-regulated Cf channel activation.
142
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3.5. Possible role of conjunctival fluid secretion in the tear film maintenance
The relative proportion of the tear volume attributed to the fluid secreted by
the conjunctiva is not known. Given that the surface area of the rabbit conjunctiva is
13 cm2 and assuming that all of it participates in fluid secretion at the observed rate,
the transconjunctival fluid secretion rate would amount to 56 pl/hr, 175% of the tear
turnover rate reported by Chrai et al. (32). This seemingly contradictory observation
may not be so, however, considering that the majority of the tear fluid is held under
both the upper and lower eyelids (32). Conceivably, the fluid volume may not have
been accounted for in the radioactive technetium dilution technique used to estimate
tear turnover rate. An alternative scenario is that not all of the conjunctiva
participates in fluid secretion or that fluid secretion rate varies from one region of the
conjunctiva to another.
Whether the maintenance of fluid balance in the conjunctival sac is the primary
function of the conjunctiva remains to be seen. It is tempting, however, to speculate
that conjunctival fluid flow may play an important role in hydrating the mucus
secreted by goblet cells (98). Such a possibility has been reported in the nasal
mucociliary system (144), whereby fluid secreted by the nasal epithelial cells played
a key role in hydrating mucus secreted by goblet cells. A significant decrease in
goblet cell density was reported in patients with various dry eye syndromes (150).
This pathology was hypothesized to be the result, rather than the cause, of dryness in
143
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the eye (120). It would be interesting to determine whether transconjunctival fluid
flow is affected in the dry eye state and to determine whether the formation of mucus
threads at the conjunctival surface of dry eye patients (120) is a manifestation of
reduced transconjunctival fluid secretion.
In summary, pigmented rabbit conjunctiva is capable of secreting fluid that is subject
to cAMP and purinergic modulation via active CF secretion (Specific Aim #2). The
stage is set for addressing the physiological function of transconjunctival fluid flow;
its possible alteration in pathological conditions affecting the conjunctiva, such as
inflammation, infections, and dry eye; and its restoration by pharmacologic
intervention.
4. Enhancement of conjunctival solute transport utilizing osmotically-driven
fluid absorption
The objective of this part of the investigation was to utilize the observed
osmotically-driven m-to-s fluid absorption to carry hydrophilic compounds across
conjunctiva - the solvent drag effect (Specific Aim #3). The control m-to-s transport
values of the compounds tested, FD-4 and mannitol, were similar to those previously
reported for the conjunctival epithelium (80,173). The baseline m-to-s betaxolol
flux, on the other hand, was 10 times lower than that reported previously in the rabbit
excised conjunctiva (199). Such a marked difference in the betaxolol permeability
144
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between the two studies was possibly due to the difference in tissue integrity
(particularly cellular, instead of tight-junctional integrity), as a voltage clamp unit
was utilized only in the current investigation to monitor the tissue conditions.
HPMPC showed a baseline m-to-s Pa p p (4.90 ± 0.69 x 10'7 cm/s), nearly 2.5 times the
value reported by Hosoya et al. (2.00 ± 0.30 x 10~7 cm/s) (84). We postulated that
this discrepancy was due to the difference in experimental conditions, mainly, the
current HPMPC transport experiment was performed under the open-circuit, instead
of short-circuit condition. In both studies, HPMPC should carry a net charge of -2 (at
pH 7.4). Under the open-circuit condition, the voltage potential difference across the
conjunctival tissue (mucosal side negative) would create a charge gradient that would
drive a negatively charged molecule in the m-to-s direction through paracellular
route. On the other hand, such a charge gradient would not exist under the short-
circuit conditions, as the tissue membrane potential difference was neutralized (i.e.,
voltage clamped) and created no external driving force to the paracellular flux of
HPMPC.
The baseline m-to-s fluxes of hydrophilic compounds (i.e., FD-4, mannitol, and
HPMPC) were significantly increased when the osmolality of the mucosal bathing
solution was reduced (Fig. 4-17), and it appeared to be enhanced due to the
previously observed increase in the conjunctival fluid absorption under such a
condition. On the other hand, the baseline m-to-s transport of these hydrophilic
145
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compounds was significantly reduced in the presence of mucosal hypertonic solution.
Such a reduction in the baseline m-to-s transport could be caused by an increase in
fluid flow at the reversed direction (i.e., s-to-m secretion) as the result of a s-to-m
osmotic gradient. The baseline m-to-s transport of betaxolol, a lipophilic compound,
was not significantly altered by the changes in the osmolality (Fig. 4-17). The direct
influence of osmotically-driven fluid flow on the transport of hydrophilic compounds
across conjunctiva was further supported by an excellent correlation (r2 = 0.95-1.00)
between the fluid flow values and the hydrophilic solute flux rates measured under
various mucosal osmolalities, and the lack of such a correlation for the lipophilic
betaxolol (Fig. 4-18). The link between solute and fluid fluxes under the influence of
osmolality would have been further strengthen if solute and fluid fluxes were
measured simultaneously. For example, it is possible to measure the fluxes of 1 4 C-
labled and fluorescent model solutes in conjunction with 3 H-water within a closed
chamber system, similar to that utilized by Candia and Zamudio (26). In this case,
3 H-HPMPC would need to be replaced with 1 4 C-HPMPC (to prevent signal overlap
with 3 H-water). Since a 1 4 C-labled version of betaxolol is not commercially
available, a different lipophilic model compound may be necessary. The capacitance
probe technique may not be ideal for this experiment due to its sensitivity to potential
disturbance of the reservoir fluid level from frequent solute sampling.
'' 146
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There were at least two possible factors which could contribute to the enhancement
of the paracellular conjunctival solute transport in the m-to-s direction under the
influence of mucosal hypotonicity: (1) osmotically-driven fluid transport; and (2)
possible decrease in the conjunctival tissue integrity (indicated by the electrical
resistance of the tissue, TEER or Rt). Hosoya et al. (84) have reported a reversible
43% decrease in conjunctival TEER after exposing the mucosal side of the tissue to
80 mOsm buffer for 3 hours. While the transconjunctival fluid flow was not
measured, they concluded that the increased m-to-s HPMPC flux under this
condition was due largely in part by the loosening of the tight-junctional integrity.
147
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VI. CONCLUSIONS
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1. Summary of findings
The conjunctival Cl" and fluid secretions were demonstrated and
characterized in this investigation. During the first stage of this study, the
conjunctival CF secretory property and its regulation by intracellular cAMP and Ca2 +
concentrations, and PKC activity was identified. The current investigation further
suggested that CF appears to enter the pigmented rabbit conjunctiva from the serosal
fluid via Na+ -K+ -2CF cotransport process, and exit through mucosally located channels.
The existence of three separate types of CF channel (i.e., cAMP-, Ca2 + -, and PKC-
regulated) was first suggested by the additive stimulation of the conjunctival Is c by
various agents that modulate intracellular cAMP and Ca2 + concentrations, and PKC
activity. Utilizing patch clamp techniques, the existence of cAMP-, Ca2 + -, and PKC-
regulated CF channels residing on the conjunctival epithelial cell surface was
positively confirmed. In addition, the identity of at least some, if not all, of the
conjunctival cAMP-regulated Cl" channels were positively identified to be CFTR.
Following the identification of some major regulatory factors of conjunctival CF
secretion and corresponding CF channel types, the conjunctival fluid secretory
properties were determined in the second phase of the investigation. The
conjunctival epithelium was also found to be secreting fluid from the s-to-m
direction under physiological conditions, similar to many other epithelia.
149
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Furthermore, such fluid secretion appeared to be driven by the Cl" secretory process,
which was supported by the observed modulation of the net fluid flow by factors that
were known to regulate conjunctival CF secretion. Whether or not fluid transport
driven by the transepithelial osmotic gradient created by active ion transport
processes go through the paracellular route, transcellular route, or both remains
controversial. Measurements of the osmotically driven fluid flow and the calculation
of osmotic water permeability across excised conjunctiva led to the conclusion that
transcellular fluid transport does occur across the conjunctiva. Such a finding
supports the molecularly determined existence of conjunctival aquaporin (AQP3)
reported previously by Hamann et al. (73). However, the functional involvement of
aquaporins (e.g., effects of osmolality and second messenger modulating agents on
AQP3 activities) in the observed transconjunctival fluid transport process remains to
be demonstrated.
The final stage of this research utilized the hypotonically induced fluid absorption to
carry hydrophilic compounds across excised conjunctiva. Utilizing conjunctival
fluid absorption driven by mucosal hypotonicity, the m-to-s transport of FD-4,
HPMPC, and mannitol was significantly elevated as compare to the baseline (i.e.,
mucosal and serosal isotonic condition), while no difference was observed in the
transport of lipophilic betaxolol among various osmolalities. Such a phenomenon
was parallel to the increased fluid movement caused by the mucosal hypotonicity,
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thus indicating that the solvent-drag effect may play a role in the transport
enhancement of hydrophilic compounds under mucosal hypotonic challenge.
2. Significance of the findings
The findings in the current research would create an impact at two levels: (1)
on the level of ocular physiology, and (2) the advancement of ocular disease
treatment and drug delivery. Epithelial active ion transport processes play an
essential role in cell volume regulation, extracellular fluid and electrolyte
homeostasis (129,130), and hormonogenesis (142). Since the first conjunctival ion
transport investigations (107,130), understanding of conjunctival physiology was
elevated to a new level. The present studies provided direct evidence for
conjunctival CT secretory capability, identified the CF exit and entry mechanisms in
the conjunctival epithelium, and demonstrated the existence of at least three types of
CF channels in the conjunctival epithelial cell surface that are commonly found in
other epithelia. Furthermore, the conjunctiva has been demonstrated for the first
time that it constantly secrets fluid under normal physiological conditions, and the
conjunctival CF secretory mechanisms appeared to be an important driving force for
such a phenomenon.
The establishment of the link between conjunctival CF and fluid secretions and the
significant effects of active CF transport modulating agents on fluid secretion laid the
151
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foundation for the development of pharmacological treatment methods for the dry
eye syndrome. The current findings will help achieve the ultimate goal of utilizing
conjunctival fluid secretion as an alternative means of increasing moisture and mucus
hydration in the conjunctiva and cornea. In addition, the demonstration of the
solvent-drag effect across conjunctiva provided another possibility of ocular
therapeutic transport enhancement, in addition to other widely examined means of
ocular drug delivery enhancement methods, such as the use of penetration enhancers
(e.g., surfactants (164), bile acids (134), and preservatives (146)), and iontophoresis
(14,87).
3. Future considerations
3.1. Conjunctival CF channel physiology
Despite significant findings made in this project, there are still important
questions to be answered. From the stand point of conjunctival active ion transport,
the relative contribution of each type of Cf channel is still unknown; which type of
the conjunctival C f channels is the major contributor of the overall Cf conductance
under normal physiological conditions? Are there other types of Cf channels other
than cAMP-, Ca2 + -, and PKC-regulated ones in the conjunctival epithelium (e.g.,
volume-regulated)9 Do different families of conjunctival C f channel cross-talk? If
the activities of a certain type(s) of Cl' channel was hindered for any reason, can
other families of Cf provide functional compensation in an attempt to maintain the
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normal overall net CF and fluid secretion across the conjunctiva? If so, to what
extent? This question is particularly important for developing treatments for ocular
diseases that are caused by dysfunctional ion channels (e.g., cystic fibrosis (171) and
non-cystic fibrosis related dry eye conditions (105,208)). For instance, in the case of
cystic fibrosis where CFTR proteins are dysfunctional due to their inability to
localize in the plasma membrane, to what degree can the stimulation of other types of
CF channel (e.g., Ca2 + - and PKC-activating) compensate for the role of CFTR in
active CF transport? Should this be a possible alternative, would it result in some
types of physiological side effect after a prolonged period of treatment (e.g., receptor
or channel desensitization)? A treatment concept was developed recently to decrease
the severity of the pathological dryness in the lumen by applying purinergic or
cholinergic receptor agonists (85,161,162). Both pathways are known to activate
both the Ca2 + - and PKC-regulated CF secretion to a very significant extend.
However, the major obstacle of this strategy is that the stimulation through both of
these signal transduction pathways are transient (~5 min maximal effect), and the
post-maximal stimulation often falls below the initial baseline level.
3.2. In-depth investigations on conjunctival fluid transport processes
In addition to identifying and characterizing the conjunctival CF transport
properties, two critical findings were made in this research that would redefine the
physiological roles of conjunctiva: (1) conjunctival epithelium is capable of active
153
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Cl'-driven fluid secretion; and (2) transcellular fluid transport does take place across
conjunctival epithelial cells. These two findings demonstrated the secondary, passive
transport of fluid movement across the conjunctiva. Nevertheless, it is unclear at this
point whether or not the direct regulation of the conjunctival fluid transport does take
place. Although little information is available on the direct regulation (e.g., second
messenger level, cell volume... etc.) of AQP activities, it would be intriguing to
speculate that there may be common regulatory factors for both ion and water
channel activities. To support such a hypothesis, a recent study conducted by
Klussmann et al. (101) indicated that intracellular cAMP level and PKA activity are
essential for the translocation of AQP2 into cell membranes of renal principal cells.
Recently, a novel aquaporin isoform (type 9) was cloned in the human adipose tissue
(114). Its structure contains a cAMP protein kinase phosphorylation consensus site
in the NH3 -terminal domain. These findings all indicate that certain AQP isoforms
can potentially be regulated directly by cAMP and PKA phosphorylation.
3.3. Contribution of conjunctival fluid secretion in tear film d y n a m i c s
As mentioned earlier, the observed rate of conjunctival fluid secretion was far
higher (175%) than the overall tear turnover rate reported by Chrai et al. (32). Such
an inconsistency was likely to be partially contributed by several assumptions made
in the current investigation: (1) all areas of the conjunctiva, both upper and lower
eyelids, were presumed to be secreting fluid and secreting at the same rate; (2) 100%
154
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of the fluid secreted by the conjunctiva was assumed to be contributed to the tear
volume. In other words, the possible role of conjunctival fluid secretion in mucus
hydration was neglected in this case; (3) Fluid evaporation from the surface of the
anterior surface of the eye was not taken into consideration.
It is necessary, therefore, to determine the relative contributions of conjunctival fluid
(by epithelium) and mucin secretion in maintaining normal physiological conditions
of the ocular surface. Furthermore, in addition to exploring the possibilities of
manipulating transconjunctival fluid secretion by active CF secretion stimulating
agents, it would be clinically beneficial to determine the limit of stimulating
conjunctival fluid secretion in compensating the deficient lacrimal glad tear
production.
3.4. Ion and fluid transport physiology during the dry eye state
Although conjunctival ion and fluid transport processes have been
extensively examined in this study, it is still uncertain, however, whether or not the
machinery of these transport mechanisms remained intact during the diseased state.
Therefore, the next phase of this project should be focusing on identifying and
characterizing ion and fluid transport physiology during the dry eye condition. This
step is particularly important for establishing a foundation for the development of dry
eye treatments. There are several key questions to addressed: (1) Is the overall
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conjunctival fluid secretion affected during a particular dry eye-related ocular
disease? (2) If so, what are the conjunctival physiological abnormalities involved
(e.g., various levels of ion and water channel dysfunctionalities)? (3) Should any
transport mechanisms be affected, can their function be restored via pharmacological
or genetic means, or be compensated by other alternative transport mechanisms that
are unaffected by the disease? In-depth experiments must be performed using an
artificially created dry eye model to address the above questions.
Several dry eye models are available (64,160). In these models, dry eye symptoms
were created in rabbit eyes by either mechanical restriction of blinking (64) or
surgical cannulation of orbital lacrimal gland excretory duct (160). These models
were only designed to create temporary dryness in the eye by reducing the aqueous
tear film via external means. Thus, these methods are unlikely to produce the
morphological alterations of corneal and conjunctival epithelial cells observed
clinically in the dry eye patients. An adenovirus type 5 (Ad5)-induced dry eye model
has recently been developed by Trousdale et al. (127), by intrastromal and topical
inoculation of the rabbit eye with 106 plaque-forming units of the virus. Preliminary
data demonstrated defects of lacrimal gland and a significant decrease in tear film
production three days after the rabbit eyes were infected with Ad5 (personal
communication with Dr. Trousdale). Various Ad serotypes, including Ad5, Ad8,
Ad 12, Ad 14, Adl9, and Ad37, have been implicated in the etiology of several ocular
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diseases that involve dry eye: keratoconjunctivitis, pharyngoconjunctival fever, and
nonspecific follicular conjunctivitis (185,186). Common symptoms observed in
these viral-induced ocular diseases include surface alteration within the conjunctival
epithelium (92), aqueous tear deficiency (145), and abnormal lacrimal gland function
(127; 145). These Ad-related dry-eye syndromes make Ad5-infection a more
etiologically relevant dry-eye model.
Should conjunctival transport abnormalities (e.g., a decrease in Cl' and fluid
secretion, or an increase in mucosal Na+ absorption) occur during the dry eye state,
this Ad5-induced dry eye model would be particularly useful for the development of
methods to overcome such a deficiency.
157
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VII. REFERENCES
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Creator Shiue, Michael Hung-I (author)

Core Title Pharmacological regulation of chloride, fluid, and solute transport in the pigmented rabbit conjunctiva

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

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

Tag biology, cell,biophysics, general,health Sciences, ophthalmology,Health Sciences, Pharmacology,OAI-PMH Harvest

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