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Drug delivery to the posterior region of the eye: Characterization of organic cation transporters

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Drug delivery to the posterior region of the eye: Characterization of organic cation transporters

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Content DRUG DELIVERY TO THE POSTERIOR REGION OF THE EYE:
CHARACTERIZATION OF ORGANIC CATION TRANSPORTERS
by
Ning Zhang
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)
December 2005
Copyright 2005 Ning Zhang
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UMI Number: 3220173
Copyright 2005 by
Zhang, Ning
All rights reserved.
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TABLE OF CONTENTS
Acknowledgements ii
List of Tables iii
List of Figures iv
Abstract vii
Chapter I Introduction 1
Chapter II Characterization of Organic Cation Transport 25
System in Rabbit Conjunctival Epithelial Cells
Chapter III Establishment of in vitro Models of RPE and 50
Identification of Organic Cation Transporters
in RPE
Chapter IV Characterization of Brimonidine Transport in 86
Retinal Pigment Epithelium
Chapter V Conclusions and Future Directions 115
Bibliography 123
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ACKNOWLEDGEMENTS
I wish to express my tremendous appreciation to my mentors Dr. Vincent Lee
and Dr. David Hinton for their scientific guidance and encouragement in my
graduate study. I started my training with very limited knowledge in pharmaceutical
sciences. As accomplished scientists, Dr. Lee and Dr. Hinton showed their patience
and understanding towards me and led me through the transition from a college
student to an independent graduate researcher.
Gratitude is owed to my other dissertation committee members: Dr. Curtis
Okamoto, Dr. Ram Kannan, Dr. Austin Yang and Dr. Robert Farley for their
invaluable guidance and insightful comments on my Ph.D. work. Special thanks to
Dr. Okamoto and Dr. Kannan who have been instrumental in giving me important
directions during the course of my study.
All the people from Dr. Lee and Dr. Hinton’s lab are thanked for their
assistance and the share of all the happy memories.
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LIST OF TABLES
Table 1.1 Substrates and inhibitors of human OCTs (hOCTl, hOCT2
and hOCT3)
Table 1.2 Cloned organic cation transporters
Table 3.1 Electrical Parameters of bovine RPE-choroid explants in
modified Ussing chamber
Table 4.1 Energy-dependency brimonidine uptake from the apical
side or the basolateral side of ARPE-19 cell monolayers.
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LIST OF FIGURES
Figure 1.1 Schematic diagrams of the conjunctiva (A) and the RPE 8
(B)
Figure 1.2 Ocular penetration routes for topically applied drugs 9
Figure 1.3 Schematic illustration of transport pathways across 12
polarized epithelial cell layers.
Figure 1.4 The human organic cation transporters: amino acid 15
sequence and current model of membrane topology.
Figure 1.5 Schematic model of the transport processes associated 23
with the secretion of organic cations (OCs) by renal proximal tubule
cells
Figure 2.1 Directionality uptake of [1 4 C]TEA in primarily cultured 35
RCEC cells
Figure 2.2 Apical and basolateral uptake of [3 H]MPP+ in primarily 37
cultured RCEC cells
Figure 2.3 Effect of L-camitine on apical and basolateral uptake of 39
TEA, or MPP+ in primarily cultured RCEC.
Figure 2.4 Kinetics of apical uptake of MPP+ in primarily cultured 40
RCEC cells
Figure 2.5 RT-PCR analysis of organic cation transporters in ocular 42
tissues
Figure 2.6 Apical uptake of [3 H]MPP+ in primarily cultured RCEC 44
cells was inhibited by various OC drugs
Figure 3.1 Time course of [3 H]mannitol (lOnM) transport across 65
bovine-RPE choroid explants mounted in Ussing Chamber.
iv
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Figure 3.2 Transepithelial electrical resistances (TEER) of ARPE-19 68
cells cultured on transwell filters.
Figure 3.3 Immunofluorecent staining of ZO-1 of ARPE-19 cells 69
cultured transwell filters coated with various extracellular matrix
proteins.
Figure 3.4 Immunofluorecent staining of ZO-1 (3.4-1) and Na+-, K+ - 70
ATPase (3.4-2) on cross sections of ARPE-19 cells.
Figure 3.5 Fluorescent Confocal Z-stack staining of Na+ -, K+- 71
ATPase of ARPE-19 cells.
Figure 3.6 Secretion of VEGF-A in polarized and Non-polarized 72
ARPE-19 cells.
Figure 3.7 Secretion of VEGF-C in polarized and Non-polarized 73
ARPE-19 cells.
Figure 3.8 Polarized secretion of BMP4 protein from the ARPE-19 74
monolayer.
Figure 3.9 RT-PCR of OCT 1, OCT2 and OCT3 in the human retinal 75
pigment epithelial cells.
Figure 3.10 Immunofluorecent staining of OCT3 on cross sections of 76
ARPE-19 cells.
Figure 3.11 Temperature and directionality MPP+ uptake in 78
polarized ARPE-19 cell monolayers.
Figure 3.12 Differential inhibition profiles of apical and basolateral 79
MPP+ uptake in ARPE-19 cells.
Figure 3.13 Concentration dependent uptake of MPP+ from the 80
apical side of ARPE-19 cells.
Figure 4.1 Chemical structure of brimonidine 90
Figure 4.2 The apparent permeability (Pa p p ) of brimonidine in 96
pigmented or non-pigmented bovine-RPE-choroid explants in the
direction of retinal-to-choroidal (R-to-C) or choroidal-to-retinal (C-
to-R).
v
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Figure 4.3 Retinal-to-choroidal (R-to-C) (A) and choroidal-to-retinal 98
(C-to-R) brimonidine transport (B) showed temperature dependency.
Figure 4.4 Total choroidal-to-retinal (C-to-R) brimonidine flux as a 99
function of brimonidine concentration.
Figure 4.5 Temperature-dependent and directional uptake of 20nM 101
[3 H]brimonidine (1.92pCi/ml) in ARPE-19 cell monolayers.
Figure 4.6 Time-dependent uptake of 20nM [3 H]brimonidine in 102
ARPE-19 cell monolayers.
Figure 4.7 Inhibition profile of apical uptake of brimonidine in 106
ARPE-19 cell monolayers.
Figure 4.8 Effect of increasing concentration of unlabeled substrate 107
on basolateral brimonidine uptake at 37°C and 4°C.
Figure 4.9 Dose-dependent inhibition of apical brimonidine 108
(1.62pCi/ml, 20nM) uptake in ARPE-19 cells.
Figure 5.1 Schematic diagram of organic cation transport system in 117
the conjunctival epithelial cells (A) and the retinal pigment
epithelium (B).
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ABSTRACT
Most cases of irreversible blindness result from diseases affecting the
posterior region of the eye. However, drug delivery to the back of the eye remains a
big challenge. Organic cation transporters (OCTs) play a crucial role in the uptake
and active secretion of drugs in organs controlling drug absorption and elimination.
However, OCT-mediated ocular drug transport was not well studied.
We found that a carrier-mediated mechanism contribute to the transport of
organic cations across rabbit conjunctiva mounted in Ussing Chamber. This organic
cation transporter was further localized to the apical side of rabbit conjunctival
epithelial cells and its molecular identity was suggested to be OCT3 using RT-PCR.
Interestingly, OCT3 was also identified in the retinal pigment epithelium (RPE),
which constitutes the outer blood retinal barrier. To investigate if the involvement of
organic cation transport systems provides a valid explanation of the good absorption
of brimonidine, a cationic anti-glaucoma drug, in the vitreous and retina after topical
application, a tissue level model and a cell culture level model of the RPE were
established. Functional studies suggested that more than half of the transport of
brimonidine across RPE was carrier-mediated. Instead of the characterized organic
cation transporters, brimonidine is suggested to be transported by a hitherto
unidentified organic cation transporter which merit further investigation.
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CHAPTER I
INTRODUCTION
A. Diseases associated with the posterior ocular tissues and drug
delivery
Most cases of irreversible blindness result from severe vision loss from
diseases affecting the posterior region of the eye. Such diseases include age-related
macular degeneration (AMD) (Ambati et ah, 2003), diabetic retinopathy (Jawa et al.,
2004), glaucoma-associated retinopathy (David, 1998), and retinitis pigmentosa
(Kalloniatis and Fletcher, 2004). Research performed by Prevent Blindness
America® and National Eye Institute revealed that millions of people in the United
States of America are suffering from these diseases.
While many novel therapeutic agents have been proposed, the treatment of
posterior segment diseases is to a significant extent limited by the difficulty in
delivering therapeutic dose of drugs to posterior eye tissues (Geroski and Edelhauser,
2000). Several approaches have been explored: systemic administration,
intravitreal/periocular injection and topical application. Although systemic
administration can deliver therapeutic levels of drugs to the posterior segments of the
eye, the large dose required can lead to significant side effects. Intravitreal injections
provide the most direct approach and allow the attainment of therapeutic amounts of
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drugs at retina and vitreous. However, potential side effects such as endophthalmitis,
cataract, hemorrhage, and retina detachment were reported. Periocular injections
(including subconjunctival, sub-Tenon’s, and retrobulbar) provide a less invasive
alternative means but are not always as effective as intravitreal injections. Overall,
these injections are not well-tolerated by patients. Drugs injected are rapidly
eliminated, and repeat injections may be frequently required.
Recently, ocular sustained-release injectables or implants have been reported
as exciting new modalities of drug delivery. Bausch and Lomb’s Retisert® and
Allergan’s Posurdex® as well as other sustained’ release systems, including other
implant devices, microspheres, and liposomes, may be alternatives to repeat
injections. Insertion of these devices, however, is invasive and potential
complications associated with such procedures remain inherent. Thus, it becomes
clear that noninvasive drug delivery to the eye’s posterior chamber is an unmet need.
Topical administration is not considered as an effective means to deliver
drugs to posterior eye due to the difficulty in achieving therapeutic amounts of drugs.
However, this mode of administration is still the preferred route because it is non
invasive, the easiest to perform, the most acceptable to patients, and is associated
with minimal side effects. In the past ten to twenty years, there were several reports
of drugs being delivered to the posterior tissues of the eye at the microgram per
milliliter (or gram of tissue) level. Although this level might not be high enough for
many antibiotics (Barza, 1989), it is in the effective range of metabolic enzyme
inhibitors (e.g. carbonic anhydrase inhibitor) or receptor ligands (e.g., P-blockers) for
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the agonist or antagonist function. The compounds that have been studied vary from
small hydrophilic compounds such as L-camitine (Pessotto et al., 1994), to large
hydrophilic compounds as inulin (Ahmed and Patton, 1985), to large lipophilic
compounds such as brimonidine (Acheampong et al., 2002), verapamil (Ettl et al.,
1998), timolol (Acheampong et al., 1995; Sponsel et al., 1999) levobunolol
(Acheampong et al., 1995), and brinzolamide (DeSantis, 2000). Some prodrugs (e.g.
nepafenac (Takahashi et al., 2003)), and peptide drugs (e.g. insulin (Koevary et al.,
2002)) were reported to reach posterior tissues of the eye with therapeutic function as
well.
As potent drugs are being discovered and developed at an accelerated speed
nowadays, topical administration is expected to be adopted more widely. An
improvement in drug bioavailability at the posterior region of the eye can be
achieved by minimizing drug loss due to ocular barriers. An understanding of the
major routes by which drugs reach the posterior eye tissues, and the identification of
facilitative transporters on ocular barriers would help to increase drug bioavailability
via the rational drug design of effective delivery strategies.
B. Anatomy and physiology of conjunctiva and retinal pigment
epithelium
The conjunctiva is a thin, transparent mucous membrane covering the inner
surface of the eyelid (palpebral conjunctiva) and the exterior part of the sclera
(bulbar conjunctiva) and is continuous with the cornea (Figure 1.1). The palpebral
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and bulbar conjunctiva is connected by the fornix conjunctiva. The conjunctiva is
composed of three layers, namely the epithelium, the substantia propria and the
submucosa. The epithelium consists of several layers of nonkeratinized and stratified
surface cells. The surface cells of the conjunctival epithelium are connected by tight
junctions, which render the epithelium a relatively impermeable barrier (Hogan,
1971). The epithelial cells are further attached to each other by means of
desmosomes and to the basal lamina through hemidesmosomes. The substantia
propria contains structural and cellular elements, nerves, lympatics and blood
vessels, while the submucosa providing a loose attachment to the underlying sclera.
While it has been assumed that topically applied drugs penetrated into the
intraocular environment through the cornea, this is currently being reassessed
(Koevary, 2003). Many investigations have shown that the conjunctival route of
entry plays an important role in the penetration of drugs into the anterior segment
(Wang et al., 1991; Ahmed and Patton, 1985; Burstein and Anderson, 1985).
Furthermore, topically applied drugs have been shown to have access to the sclera
from the conjunctiva (Lee and Robinson, 1986; Chien et al., 1990; Koevary, 2002).
As a result, such drugs could find their way to the posterior segment (Figure. 1.2).
Compared to the corneal pathway (Doane et al., 1978), the
conjunctiva/sclera(C/S pathway) pathway (Kompella, 2000) is a more effective
means to deliver drugs to the posterior ocular region for the following reasons: (1)
The conjunctiva provides larger surface area for drug absorption. The conjunctival
surface area is nine times larger than that of cornea in rabbit, and 17 times larger in
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human (Watsky et al., 1988). (2) The conjunctiva is between 2 to 30 times more
permeable to drugs than the cornea (Davies, 2000). The molecular weight cut off for
corneal paracellular transport is below 5 kDa, whereas that for conjunctival
paracellular transport is 20 to 40 kDa (Horibe et al., 1997), which make the delivery
of nucleotide drug or peptide drug feasible. (3) The C/S pathway successfully
bypasses the lens-iris barrier, which is unavoidable by direct entrance via the comeal
pathway. The lens-iris apparatus, including the capsule and suspensory ligaments of
the lens, comprises a barrier to the passage of dmgs between the anterior and the
posterior chambers. In addition, the continuous flow of aqueous humor from the
posterior chamber to the outflow channels in the anterior chamber inhibits the dmgs
to move posteriorly. (4) Anatomically, the proximity of the bulbar conjunctiva and
the sclera to the posterior ocular tissues makes the C/S pathway a more favorable
route.
Same as all the epithelia, the conjunctival epithelium is replete with
intercellular tight junctions; thereby comprising the first barrier to topically applied
hydrophilic dmgs. The sclera and the adjacent layer, the choroid, are permeable to a
wide range of solutes. Dmgs with molecular weight below 150KD traverse the sclera
easily(Maurice and Polgar, 1977; Edelhauser and Maren, 1988).
The vectorial movement of nutrients, metabolites and xenobiotics from
choroids to the retina or vitreous is controlled by the retinal pigment epithelium
(RPE). The RPE cells form a monolayer located between the neural retina and the
vascular choroid (Figure 1.1). The RPE cells are bound together by junctional
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complexes at the lateral domains (Thumann G, 2001; Kniesel and Wolburg, 2000),
among which the intercellular tight junctions as a result of interaction between
extracellular domains of adjacent occludin proteins, lead to the high transepithelial
resistance of the RPE monolayer and rendering the RPE the outer blood-retina
barrier (BRB) by preventing diffusion of large molecules through the space between
adjacent RPE cells (Cunha-Vaz, 2004; Duvvuri et al., 2003). Due to the existence of
the RPE, antibiotics injected by the periocular route reach high concentrations in the
sclera but fail to produce high concentrations in the retina and vitreous humor. The
tight intercellular junctions of this tissue inhibit the passage of drugs into the retina
and vitreous humor (Barza, 1989).
In addition to the barrier function that allows the tight control of the
movement of water and metabolites between retina and fenestrated choriocapillaris,
RPE cells mediate various critical functions of the retina. The RPE maintains retinal
attachment by actively keeping the subretinal space dehydrated by moving water
from the subretinal space with ionic transport systems including Na+ -, K+ -ATPase
pump and HCO3 'transport system (Marmor, 1990; Tsuboi, 1987). RPE cells also
participate in several activities that are critical in photoreceptor function and
viability. These activities include phagocytosis of shed photoreceptor rod outer
segments (ROS), metabolism of retinol, and interaction with light by its melanin
granules. In addition, RPE cells secrete a wide spectrum of cytokines and growth
factors. Secreted cytokines and growth factors in the monolayer may act in an
autocrine or paracrine way on the cell of origin or adjacent RPE, or on adjacent
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photoreceptors or choroidal cell. Due to its critical anatomic location and its ability
to interact with surrounding tissue elements, the RPE cell is often involved in retinal
pathophysiology, especially in age-related diseases (Harman et al., 1997).
Figure 1.1 Schematic diagrams of the conjunctiva (A) and the RPE (B). The RPE
separates the neural retina from the fenestrated capillaries in the choroid, constituting
the outer blood retinal barrier. RPE is polarized. The apical membrane of RPE
interacts with the photoreceptors; the basal membrane interacts with the fenestrated
choriocapillaris. OS: photoreceptor outer segment; AJ: adherent junction; BI:
basolateral infolding; GAP: gap junction M; melanin; MV: microvilli; TJ: Tight
junction. Modified from (Rizzolo, 1997).
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(A)
Lacrimal gland
Fornix
conjunctiva
Sclera
Bulbar _ _
conjunctiva
Cornea
Palpebral__
conjunctiva
(B)
Neural Retina
i-ii
* y
Itruchs
M embrane
Fenestrated Capillary Bed
-R E T IN A L CAPILLARIES BLOOO-RETlNAL
RETINAL PIGMENT E P IT H E L IU M ) BARRIERS
CHOROID
SCLERA
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Precorneal ocular surface
Cornea
iZ
Aqueous
Lens-iris
apparatus
Vitreous
Conjunctiva
^ ....
Sclera
Ciliary body
Choroid
O ' "
RPE
Retina
Figure 1.2 Ocular penetration routes for topically applied drugs, illustrating that the
conjunctiva/sclera pathway is an important route that may facilitate drug transport to
the posterior regions of the eye.
C. Carrier-mediated solute transport and its implication in drug
delivery
Living organisms have evolved selective transport mechanisms for
compounds that do not move freely through the cell membrane due to their size or
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hydrophilicity. Many of these compounds such as glucose, peptides, amino acids,
nucleotides are vital nutrients essential to maintain normal physiological functions.
On the other hand, some of the compounds can be toxic metabolites or xenobiotics.
As a result, cell membranes are equipped with multiple carrier-mediated transport
systems. As illustrated in Figure 1.3, solute transport across polarized epithelial cell
layers occurs through multiple pathways. They include transcellular and paracellular
diffusion, which are both passive processes determined by physicochemical
properties of the solute, and carrier-mediated processes comprising receptor-
mediated endocytosis/transcytosis and transport via3membrane transporter proteins,
which are both processes directly or indirectly utilizing cellular energy and
recognizing substrates with specific chemical structures (Lee, 2001).
For the past decade, scientists in the fields of molecular biology, cell biology,
genetics, bioinformatics, and drug delivery have developed tremendous interest in
membrane transporter biology. While the transport mechanisms lining the epithelium
of major absorption organs in the GI track, the excretion organ such as the kidney
and the metabolism organ such as the liver significantly affect the absorption,
disposition and elimination of oral drugs, it has long been acknowledged that the
availability of poorly absorbed drugs can be significantly improved by changing their
physicochemical properties via modifying their chemical structure or by rational
drug design mocking substrates of the endogenously existed transport systems.
Successful examples include synthesis of amino acid ester prodrugs of antiviral
nucleosides which increased the oral absorption of nucleosides via intestinal di-
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peptide transporter (PepTl) (Inui et al., 2000), synthesis of peptidomimetics such as
beta-lactam antibiotics and angiotensin-converting enzyme inhibitors as substrate of
the di-peptide transporter (Bai and Amidon, 1992), modification of dicarboxylic acid
(carbenicillin) to a monocarboxylic acid (carindacillin) which improved the affinity
of the drug to the intestinal monocarboxylic acid transporter (Li et al., 1999), and
construction of transferring conjugated peptide drugs which improved oral
absorption via stimulation transferring receptor mediated trancytosis (Bai et al.,
2005).
Since the sequence of the entire human genome is available, even more
transporter proteins will be genetically identified. In addition to study transporter as
families, the application of techniques such as molecular cloning, knock down/in and
transgenic allow in depth characterization of the physiological function, structure and
mechanism of an individual transporter. The explosion of information and the
advance in technology will broaden our knowledge of the physiological role of
membrane transporters and their diversity in distribution and function, which will
undoubtedly expand the possibility of using transporters as drug delivery target to
improve the availability of drugs with diverse array of chemical structures.
11
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Receptor-mediated
Endo/transcytosis Membrane
Transcellular Paracellular
Transporter-mediated
^ | i y | u r f M y y y i ( i a Apical
B aso lateral
Passive Transport
Carrier-mediated
Transport
Figure 1.3 Schematic illustration of transport pathways across polarized epithelial
cell layers.
D. Organic cation compounds and the organic cation transporters
(OCT)
Organic cations (OCs) is a diverse array of primary, secondary, tertiary, or
quaternary amines that have a net positive charge on the amine nitrogen at
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physiological pH, which includes many endogenous compounds of high
physiological importance such as monoamine neurotransmitters, choline, and
coenzymes, as well as numerous xenobiotics (Koepsell et al., 2003; Wright, 2005;
Zhang et al., 1998). By a rough estimation, at least 50% of presently available drugs
are organic cations (Groothuis and Meijer, 1996) including the anticholinergic,
antineoplastic, anesthetic, sympathomimetic, antidiabetes and antihistaminic drugs
(Meijer et al., 1990).
In the past decade, several organic cation transporters have been cloned from
various mammalian species. OCT1 was cloned from a rat renal cDNA library
(Grundemann et al., 1994) and proved to be the initial member of a large family of
transport proteins with in the Major Facilitator Superfamily (MFS), which comprises
uniporters, symporters, and antiporters from bacteria, lower eukaryotes, plants, and
mammals including drug resistance proteins, sugar facilitators, facilitators of Krebs
cycle intermediates, organophosphate-phosphate antiporters, and oligosaccharide-H+
symporters in 18 families (Marger and Saier, 1993; Pao et al., 1998). Within the
Human Genome Organization (HUGO) Nomenclature Committee Database, the
OCTs and their homologues have been organized into the SLC22A family of solute
carriers (Hediger et al., 2004). The SLC22A family now includes at least 12 distinct
homologous transport proteins that can be organized into several evolutionarily
distinct subfamilies including the OCTs (organic cation transporters; OCT 1-3) and
OCTNs (organic cation transporters-novel; OCTN1-2), based on phylogenetic
relationship.
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The OCTs and OCTNs are approximately 550-560 amino acids in length
and, as many members in the MFS, the OCTs and OCTNs share a predicted topology
of 12 a-helical transmembrane domains (TMDs) with the N- and C- terminals
pointing to the cytoplasmic side (Figure 1.4) (Meyer-Wentrup et al., 1998; Koepsell
et al., 2003). There are two hydrophilic loops predicted, the latter of which (between
TMD6 and TMD7) is routinely found in proteins of the MFS, whereas the former of
which (between TMD1 and TMD2) is relatively distinctive for the SLC22A family.
There is marked homology between the N- and C-terminal halves of the SLC22A
family. Studies using chemeric protein construct suggested that major elements of
substrate selectivity reside in the C-terminal half of OCT transporters (Wright, 2005;
Feng et al., 2001).
Cation transport by OCTs has been investigated in several heterologous
expression systems including Xenopus oocytes and mammalian cell lines including
human embryonic kidney (HEK) cells, MDCK cells, Chinese hamster ovary (CHO-
K l) cells, and human retinal pigmented epithelium (HRPE) cells. Transport was
determined as uptake of radioactively labeled or fluorescent compounds, and/or as
electrical current oocyte plasma membrane. Both OCTs and OCTNs are
multispecific with overlapped substrate profile covering organic cations of various
structures, some zwitterions and organic anions (Table 1.1). In addition to function
as proton gradient-driven organic cation transporter, OCTNs are also recognized as
Na+ -dependent L-camitine transporter. Deficiency of OCTN2 (SLC22A5) leads to
primary systemic L-camitine deficiency (Wang et al., 1999; Nezu et al., 1999).
14
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Different in the driving force from OCTNs, OCTs translocate organic cations in an
electrogenic manner, independent of Na+ , Cl', or H+ gradient. OCTs transport
substrates across the plasma membrane in either direction. In addition to cation
influx, cation efflux has been demonstrated for rOCTl (Busch et al., 1996; Nagel et
al., 1997), rOCT2 (Kekuda et al., 1998), hOCT2 (Busch et al., 1998), and rOCT3
(Kekuda et al., 1998).
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Figure 1.4 The human organic cation transporters: amino acid sequence and current
model of membrane topology. Amino acids (a.a.) that are conserved in particular
subfamilies of the SLC22 transporter family are color-coded as follows: black, a.a.
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conserved in all members of the SLC22 family; gray, a.a. conserved only in the
mammalian organic cation transporters OCT1/OCT2/OCT3 and/or OCTN1/OCTN2;
gray dots lining the main sequence, consensus sequences for N-glycosylation or
phosphorylation that are conserved in all functional members of the SLC22 family,
or in all functional SLC22 family members except hCT2; black dots lining the main
sequence, consensus sequences for protein kinase C dependent phosphorylation sites
in hOCTl; asterisks, N-glycosylation sites in hOCTl. (adapted from (Koepsell et al.,
2003))
Tissue distribution of the organic cation transporters was found to be
subtype-dependent and species-dependent (Table 1.2). In humans, OCT1 (SLC22A1)
is mainly expressed in the liver, while OCT2 (SLC22A2) is mainly found in the
kidney. The tissue expression pattern of OCT3 (SLC22A3) mRNA is relatively
broad. It was found in the placenta, kidney, heart, skeletal muscle, and liver. OCT2
and OCT3 were also identified in the central nervous system (CNS). Reports on
subcellular localization of the OCTs are limited. Immunofluorescent studies
suggested that rat OCT1 and OCT2 are localized on basolateral membrane of the
renal proximal tubules (Karbach et al., 2000). Rat OCT1 was also found on the
sinusoidal membrane of hepatocytes (Meyer-Wentrup et al., 1998). The subcellular
localization of OCT3 remains to be investigated, while several functional studies
suggested the apical localization on enterocytes (Jonker and Schinkel, 2004; Martel
et al., 2001). OCTN1 (SLC22A4) and OCTN2 (SLC22A5) are expressed in a wide
16
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range of tissues. OCTN2 has been shown to be expressed in the luminal membrane
of the proximal tubules in kidney (Tamai et al., 2001). In contrast, there is no
experimental evidence concerning the subcellular location of OCTN1.
The organic cation transporters play an important role in the intestinal
absorption, renal excretion and hepatic disposition of cationic drugs (Jonker and
Schinkel, 2004). Modulation in the activity of the organic cation transporters directly
affects the pharmacokinetic profile of cationic drugs. Inui and coworkers showed
that the reduced excretion of the cationic drug cimetidine (H2 histamine receptor
blocker) in nephrectomized rats was associated with the downregulation of OCT2 (Ji
et ah, 2002). Another good example in this regard is from the distribution study of
metformin, a biguanide drug widely used in the treatment of type II diabetes, which
is reported to be a substrate of OCT1 (Wang et al., 2002) in the liver and OCT2
(Dresser et ah, 2002) in the kidney. The elimination of OCT1 activity by knocking
out OCT1 gene resulted in a 30 times decrease in liver metformin concentration. In
humans, lower renal expression of OCT2, mutations reducing the activity of hOCT2,
or administration of metformin simultaneously with a drug inhibiting OCT2 in
kidney may cause lactic acidosis due to increased metformin plasma levels as a result
of decreased renal excretion. Furthermore, the regulation of OCTs by hormones such
as testosterone and estradiol, and insulin is believed to be one reason for the gender-
and diabetic-dependent distribution of cationic drugs (Urakami et ah, 1999; Grover
et ah, 2002).
17
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00
Compound Class Charge Km or (IC5 0 ) [pM]
hOCTl hOCT2 hOCT3
Endogenous Compounds
Choline Metabolite +
210
Corticosterone Hormone 0 (7, 22) (34) (0.12, 0.29)
Dopamine Neurotransmitter
(+)
390, 520
/3-Estradiol Hormone 0 (5.7) (>30) (2.9)
Guanidine Metabolite +
(13,000) (6,201)
Histamine Biogenic amine +
1,300 180(140)
Norepinephrine N eurotransmitter 1,900 510
Progesterone Hormone 0 (3.1) (27) (4.3)
Prostaglandin E2 Hormone - 0.66 0.03
Prostaglandin F2 « Hormone - 0.48 0.33
Serotonin N eurotransmitter 80
Drugs
Acebutolol /3-Blocker +
(96)
Acyclovir Antiviral drug
(+)
151
Amantadine Anti-Parkinson + 27 (23)
Cephaloridine Antibiotic +/-
Cefepime Antibiotic +/-
Cefoselis Antibiotic +/-
Cimetidine H2 rec. antagonist
(+)
(166) 8.6
Clonidine c t2-antagonist 4-
(0.55) (373)
Cocaine Stimulant
+ (277)
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Debrisoquine Anti-hypertensive +
7.3
Desipramine Antidepressant + (5.4) (16) (14)
s(+)Disopyramide Na+ ch. blocker +
(30)
r(-)Disopyramide Na+ ch. blocker + (15)
Emetine Anti emetic
(+)
Ganciclovir Antiviral agent
(+)
516
Imipramine Antidepressant +
(42)
Indinavir HIV prot. inhib.
(+)
(62)
Memantine Muscle relaxant +
34
Mepiperphenidol Anticholinergic +
(4.8)
Metformin Antidiabetic +
(2,010) (1,700)
Midazolam Anesthetic + (3.7)
Nelfmavir HIV prot. inhib.
(+)
(22)
o-Mcthyl-isoprenalinc /3-Agonist + (>100) (570) (4.4)
Phenoxybenzamine a-B locker + (2.7) (4.9) (6.1)
Phenformin Antidiabetic + (10) (65)
Procainamide Na+ ch. blocker + (74), (107) (50) (738)
Quinine Antimalaria drug
(+)
(23) (3.4)
Quinidine Na+ ch. blocker
(+)
(18)
Ritonavir HIV prot. inhib.
(+)
(5.2)
Vecuronium Muscle relaxant + (127), (232), (237)
Verapamil Ca2 + ch. blocker + (2.9) (206)
Xenobiotics and model compounds
Cyanine 863 + (0.21)
Decynium-22 +
(1.(M.7) (0.1), (1.1) (0.09)
MPP + 15 (12) 19 (2.4) 47 (54)
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NMN + (7,700) 340 (270)
SKF550 + (>0.3) (0.1) (0.05)
T etrabutylammonium +
(30) (52), (120)
T etraethylammonium + 229 (158-260) 76(156) (1,372)
T etramethylammonium + (12,400) (180), (150)
T etrapentylammonium + (7.5), (8.6) (1.5)
T etrapropylammonium + (90), (102) (128)
Table 1.1 Substrates and inhibitors of human OCTs (hOCTl, hOCT2 and hOCT3). Data are derived from isotope uptake and
uptake-inhibition experiments at physiological pH values using different expression systems (numbers in parentheses, IC5 o
value for inhibition of transport of different substrate; charge symbols are: 0, uncharged and not ionized; +, permanent positive
charge; permanent negative charge; +/-, zwitterion; (+), weak base; (-), weak acid)
to
o
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Sub-family Name Species
Cloned From
Tissue Distribution References
Membrane potential
dependent multi
specific organic
cation transporters
OCT1 rat, mouse,
human, rabbit
liver, intestine, kidney (rat,
mouse and rabbit, but not
detectable in human
kidney), and lung
(Grundemann et al., 1994), (Zhang
et al., 1997), (Green et al., 1999),
(Shen et al., 1999)
OCT2 rat, mouse,
human, Pig
kidney and brain (Okuda et al., 1996), (Gorboulev et
al., 1997), (Grundemann et al.,
1997), (Sweet et al., 2001),
(Kusuhara and Sugiyama, 2004)
OCT3 rat, mouse,
human
placenta, intestine, heart,
brain, kidney, lung, and
RPE
(Kusuhara and Sugiyama, 2004),
(Kekuda et al., 1998), (Wu et al.,
1998), (Verhaagh et al., 1999),
(Rajan et al., 2000)
pH-dependent organic
cation transporter and
Na+-dependent
camtine transporter
OCTN1 rat, human,
mouse
kidney, fetal liver,
intestine, bone marrow,
brain
(Tamai et al., 1997), (Wu et al.,
2000)
OCTN2 rat, human,
mouse
skeletal muscle, kidney,
heart, placenta, brain
(Tamai et al., 1998), (Wu et al.,
1999)
Table 1.2 Cloned organic cation transporters
In addition to the organic cation transporters, there are several transporters
known to be able to transport certain types of organic cations. One example is the
organic anion transporter OAT1, OAT3, and URAT1 (Enomoto et al., 2002), which
also belongs to the SLC22A family and may operate as anion/anion exchangers in
parallel with the organic cation transporters in the renal excretion cimetidine (Cha et
al., 2001). So far, other transporters such as transporters from the OATP (organic
anion-transporting polypeptide) family, the MRPs (the multi-drug resistance
proteins) and the ABC transporter P-glycoprotein (Pg-P) can all recognize some
cation compounds as their substrate. Substrates of OATPs are mainly type II organic
cations, that are usually bulkier (generally >500 MW; e.g., daunomycin) and
frequently polyvalent (Meijer et al., 1990; Hagenbuch and Meier, 2003). P-
glycoprotein and MRPs are active transporters/pumps driven by ATP hydrolysis.
Exogenous and endogenous compounds of various structure and hydrophobicity can
be translocated. A number of cationic drugs that are substrates of OCT1 and OCT2
are substrates of P-gp or MRP (Kruh and Belinsky, 2003). Examples for common
substrates of Pg-p and human OCTs are quinidine, verapamil, cimetidine,
debrisoquine, acyclovir, and ganciclovir (Sakaeda et al., 2002).
Until comparatively recently, models of organic cation transport typically
depicted the process as involving a single apical entry/exit step and/or a single
basolateral exit/entry step, a view that effectively explained existing physiological
data. This scheme, however, is now known to be oversimplified and the process of
transport across epithelial/endothelial barriers involves the concerted activity of a
22
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suite of cellular events operating both in parallel and in series (e.g. organic cation
transport across kidney proximal tubes. Figure 1.5) (Wright, 2005). The delineation
of the transport process requires the investigation of transport characteristics of
multiple reprehensive compounds in models of both intact tissue and isolated apical
or basolateral membranes.
lum en
ocv
oc;
oc
ATP'
,Na’
OC
o c
oc;
'OCT I
Na*
'Oc'vu.tf-inw
Na"
-7 0 m V
choline
Figure 1.5 Schematic model of the transport processes associated with the secretion
of organic cations (OCs) by renal proximal tubule cells. Circles depict carrier-
mediated transport processes. Arrows indicate the direction of net substrate transport.
23
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Solid lines depict what are believed to be principal pathways of OC transport; dotted
lines indicate pathways that are believed to be of secondary importance; the dashed
line indicates diffusive movement. Modified from (Wright, 2005).
24
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CHAPTER II
CHARACTERIZATION OF ORGANIC CATION
TRANSPORT SYSTEM IN RABBIT CONJUNCTIVAL
EPITHELIAL CELLS
A. Summary
Purpose: To investigate the membrane localization and the molecular
identity of the organic cation transport system in rabbit conjunctival epithelial cells.
Methods: Rabbit conjunctival epithelial cells (RCEC) were grown on
transwell filters to exhibit tight barrier properties. Apical and basolateral uptake of
[methyl-3 H]l-methyl-4-phenyl-pyridinium (MPP+ ) and [ethyl-1-
1 4 C]tetraethylammonium (TEA), two prototypic organic cations, were investigated.
The expression of gene transcript of organic cation transporters (OCT1, OCT2 and
OCT3) was examined by RT-PCR using member specific primers. The PCR
products were further confirmed by DNA sequencing.
Results: The apical uptake of [1 4 C]TEA and [3 H]MPP+ were inhibited by
excess unlabeled compounds. The apical uptake of MPP+ was inhibited by the
following organic cations at 2.5mM (histamine (86%), dopamine (88%), and TEA
(88%)), or by steroids at 10/xM (beta-estradiol (22%), and progesterone (15%)), but
not by 2.5mM p-aminohippuric acid (PHA), an organic anion. Basolateral uptake of
25
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[1 4 C]TEA, was not inhibited by excess unlabeled TEA. Although basolateral MPP+
uptake was inhibited by dopamine and excess unlabeled MPP+ , the uptake was
unaffected by exposure to other organic cations (TEA and histamine) at, or steroids
(/3-estradiol and progesterone). L-camitine, a recognized substrate of organic
cation/L-camitine transporter, did not inhibit either the apical or the basolateral
uptake of TEA or MPP+. Apical MPP+ uptake showed saturability with an apparent
Michealis-Menten constant and a maximal uptake rate of 4.2 ± 1.1 pM and 466.7
± 19.4 pmol/mg protein /15min, respectively. While no apparent amplification of
OCT1 or OCT2 was observed by RT-PCR in freshly isolated RCEC, a distinct 357bp
amplicon for OCT3 was identified. The sequence of this fragment showed 99%
identity with the 256bp rabbit OCT3 cDNA fragment, and 85% to 88% identity with
the corresponding sequences of human, rat and mouse OCT3 cDNA. Apical MPP+
uptake was significantly inhibited by various cationic drugs at 2.5mM, including
brimonidine, clonidine, acebutolol, and betaxolol, as well as by verapamil atlmM.
Conclusions: An organic cation transporter was functionally identified on the
apical side of rabbit conjunctival epithelial cells. It may play a significant role in
scavenging OC compounds in the tear fluid and may serve as a conduit for the entry
of cationic drugs into the uveal tract. Among the cloned organic cation transporters,
OCT3 appears to be the major organic cation transporter expressed in the rabbit
conjunctival epithelium.
26
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B. Introduction
The conjunctiva, a thin transparent mucous membrane covering the inner
surface of the eyelids and exterior part of the sclera, plays an important role both in
ocular (Ahmed and Patton, 1985; Koevary, 2003) and systemic absorption (Lee et
al., 1993) of topically administered drugs. Topically applied drugs could find their
way to the posterior ocular region via the conjunctiva and the underlying sclera,
leading to therapeutic or toxic effect at the photoreceptors of neuroretina
(Acheampong et al., 2002; Koevary, 2003). In comparison to the cornea, which
provides direct access to the anterior ocular region, the conjunctiva serves as a route
more direct to the posterior ocular tissues. The increasing requirement in developing
non-invasive approaches to delivery therapeutic compounds for the treatment of
posterior ocular diseases demands an in depth investigation of transport mechanisms
in the conjunctiva.
Organic cations (OCs), including endogenous amines (e.g. catecholamines,
dopamine, and guanidine) as well as drugs, toxins and other xenobiotics, are a broad
class of substances with at least one positive charge at physiological pH. Many
topically applied ophthalmic drugs, such as carbachol, pilocarpine, apraclonidine,
brimonidine and all the /3-blockers belong to this category. Similar to many other
solutes, these drugs or drug metabolites may share naturally existing organic cation
transport mechanisms to penetrate the ocular epithelia.
27
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The movement of OCs across bilipid membrane is controlled by a family of
transporters, the organic cation transporters (OCT). The OCT, including OCT1, 2
and 3 (SLC22A1-3) facilitates the transport of a variety of structurally diverse
organic cations in a membrane potential-dependent and Na+ -independent manner.
Rat OCT1 was the first cloned organic cation transporter, which was detected in
kidney, liver and intestine (Grundemann et al., 1994). In humans, OCT1 is primarily
expressed in the liver (Zhang et al., 1997). OCT2 mRNA was detected
predominantly in the kidney in rats (Okuda et al., 1996) and humans (Gorboulev et
al., 1997) by RT-PCR and was also detected in the dopamine-rich areas of the brain
(Grundemann et al., 1997). In contrast to OCT1 and OCT2, OCT3, first cloned from
rat placenta, has much more widespread tissue distribution. In humans, high
expression of OCT3 mRNA was detected in aorta, skeletal muscle, prostate, adrenal
gland, salivary gland, liver, term placenta, and fetal lung using Northern blot
(Verhaagh et al., 1999). Despite their distinct distribution, functionally, OCT1, 2,
and 3 share an overlapping set of substrates, including the prototypic organic cation
tetraethylammonium (TEA), the parkinsonian neurotoxin l-methyl-4-
phenylpyridinium (MPP+ ), and several biogenic amines (dopamine, histamine and
norepinephrine). Structurally, OCT1, OCT2 and OCT3 bear a typical structure of
Major Facilitator Superfamily (Pao et al., 1998), containing proposed 12
transmembrane domains (Grundemann et al., 1994; Jonker and Schinkel, 2004;
Meyer-Wentrup et al., 1998). In primary amino acid sequences, OCT1 and OCT2 are
more identical, which is exemplified by 67% identity between rat OCT1 and OCT2,
28
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as compared to 48% identity between rat OCT1 and rat OCT3 (Jonker and Schinkel,
2004).
The OCTs are of great physiological and pharmacological importance. The
three OCTs, especially OCT3, are responsible for the extraneuronal clearance of
catecholamines and are predominant components of the uptake-2 system
(Eisenhofer, 2001; Jonker and Schinkel, 2004). In the major secretory organs namely
as the liver, the kidney and the intestine, they not only play a major role in
elimination of, and protection against noxious compounds, but also have a significant
impact on the pharmacokinetic/pharmacodynamic profile of various substrate
organic cations (Jonker and Schinkel, 2004).
In addition to the membrane potential-driven organic cation transporters, the
proton-gradient driven organic cation transporters, known as the OCTNs (novel
proton/organic cation antiporters), also facilitate the translocation of many organic
cations(Tamai et al., 1997; Wu et al., 1998b). The OCTNs, especially OCTN2, was
later identified as a Na+ -dependent, high affinity L-camitine transporter (Tamai et al.,
1998), deficiency of which leads to primary carnitine deficiency (Nezu et al., 1999).
While the importance of OCT and OCTNs are increasingly recognized, they
remain poorly characterized in the ocular surface, which controls the reabsorption of
beneficial compounds from tear fluid, as well as the deposition of topically applied
drugs. Our previous studies suggested a carrier-mediated OC transport process in the
pigmented rabbit conjunctival tissue (Ueda et al., 2000). In the present study, we
provide evidence for the existence and apical localization of organic cation
29
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transporters in a primary rabbit conjunctival epithelial cell culture. We also present
our results from our attempts in elucidating the molecular identity of the OC
transport system in rabbit conjunctival epithelium and other ocular tissues using RT-
PCR.
C. Materials and methods
1. Animals and reagents
Male Dutch-belted pigmented rabbits were purchased from Irish Farms (Los
Angeles, CA). DMEM/F12 cell culture medium was obtained from Mediatech
(Herndon, VA). Tissue culture-treated polyester transwells (6.5mm or 12mm in
outer diameter and 0.4 pm in pore size) were obtained from Costar (Coming, NY).
Cell culture reagents and supplies were obtained from Life Technologies (Grand
Island, NY).
[Methyl-3 H]l-methyl-4-phenyl-pyridinium (85Ci/mmol) and [ethyl-1-
1 4 C]tetraethylammonium bromide (55mCi/mol) were purchased from American
Radiolabeled Chemicals (St. Louis, MO). All the nonlabeled compounds were from
Sigma (St. Louis, MO).
The Trizol reagent for RNA isolation, Superscript II Reverse Transcriptase,
Taq DNA Polymerase and TOPO TA Cloning® kit were purchased from Invitrogen
(Carlsbad, CA). The DNA sequencing was done by Genemed Synthesis (South San
Francisco, CA).
30
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2. Air interfaced primary culture of rabbit conjunctival epithelial
cells
The protocol developed in our laboratory by Yang et al (Yang et al., 2000)
was used. Briefly, the conjunctiva of pigmented rabbit was excised and incubated in
0.2% protease XIV at 37°C for 60 minutes. Epithelial cells were then scraped off,
suspended in a minimum essential medium (S-MEM) containing 10% fetal bovine
serum (FBS) and 0.75mg/ml deoxyribonuclease I, and centrifuged at lOOg for 10
minutes at room temperature. The cells were washed twice with S-MEM containing
10% FBS and filtered through a 40pM cell strainer after the second wash, and
pelleted at lOOg for 10 minutes at room temperature. The cells were then
resuspended in DMEM/F-12 medium supplemented with lOOU/ml penicillin-
streptomycin, 0.5% gentamycin, 0.4% fungizone, 2mM L-glutamine, 1% ITS+,
30pg/ml bovine pituitary extract (BPE), lpM hydrocortisone and lng/ml epidermal
growth factor (EGF). The isolated cells were seeded on transwells coated with 2%
rattail type II collagen, at a seeding density of 1.2x106 cells/cm2 (day 0), and cultured
at a humidified atmosphere of 5%C02 and 95% air at 37°C. On day 4 and thereafter,
culture condition was switched to an air interface condition, in which medium was
only added to basolateral side to mimic the in vivo situation. The cell layers were
used for uptake studies after reaching peak bioelectric parameters
(TEER>1.0kQ*cm2 ) on day 6. Similar criteria for validation of the polarity of RCEC
was followed in all experiments described here (Yang et al., 2000; Saha et al., 1996).
31
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3. Uptake stu d ies by RCEC
Unless otherwise specified, all uptake experiments were performed in a
humidified atmosphere of 5%C02 and 95% air at 37°C in the bicarbonated Ringer’s
solution (BRS) maintained at pH7.4 and an osmolarity of 300mOsm/kg. The BRS
buffer contained 116.4mM NaCl, 5.4mM KC1, 0.78mM NaH2 P C > 4, 0.81mM MgS04,
1.8mM CaCl2, 25mM NaHC03 , 15mM HEPES, and 5.55mM D-glucose. After the
cell layers were incubated in BRS for 30 minutes, the uptake experiments were
initiated by spiking the apical or basolateral fluid with radiolabeled and a
predetermined amount of unlabeled or competitive compounds. After a specified
period of time, uptake was terminated by aspiration of the dosing solution followed
by three washes of the cell layer in ice-cold BRS buffer. The cell layers were then
solubilized in 0.5ml 0.5% Triton X-100 solution. Twenty micro liters of the solution
was taken for protein assay using Bio-Rad Dc Protein assay kit (Hercules, CA),
while the rest of the solution was mixed with 5 ml scintillation cocktail for
quantification of the radioactivity in a liquid scintillation counter (Beckman,
Fullerton, CA).
4. R everse transcription and polym erase chain reaction (RT-PCR)
The conjunctiva, cornea, lens, iris-ciliary body and the retina-choroid were
carefully isolated from male Dutch-belted pigmented rabbits and washed in ice-cold
Hank’s Balanced Salt Buffer. The conjunctival and corneal epithelial cells were
scraped off. A slice of kidney containing both the medulla and the cortex was
excised and used as positive control. All the tissues or cells collected were lysed in
32
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TRIzof reagent (GIBCO/BRL Carlsbad, CA). Total RNA was isolated following
protocol provided by manufacturer.
cDNA was synthesized from total rabbit conjunctival epithelial RNA using
200 units of Superscript II (Invitrogen, Carlsbad, CA). The 20pl reaction mixture
contained 0.5jag oligo(dT) 1 2 -1 8 , lug total RNA, IX First Strand Buffer, 0.01M DTT,
and 0.5mM each deoxyribonucleotide. Reverse transcription was carried out at 42°C
for 50 minutes in a Perkin-Elmer thermal cycler, followed by heating at 70°C for 15
minutes to inactivate the reaction and 99°C for 5 minutes to remove RNA
complementary from the synthesized cDNA. PCR was performed in 50pl mixture
containing 1.5mM MgC^, IX PCR buffer, 0.2mM each deoxyribonucleotides,
0.1 uM each of forward and reverse PCR primers, 5 units of Taq DNA polymerase,
and lul first strand transcript. 35 cycles were carried on in a Perkin-Elmer thermal
cycler. The PCR primers were synthesized by Life Technologies (Rockville, MD).
The primers for OCT1 are s: 5’-CCG CTG ACC TCA AGA TG-3’ and as : 5’-CTG
CCA GAC CTC CAT CA-3’, which would amplify the region between 996 bp and
1531bp of rbOCTl (gi:3236339). The primers for OCT2 are s: 5’-GGA AGC ACA
CCT GCA TCT TG-3’ and as: 5’-GAG ATT CCT GAT GAA CGT GG-3’, which
would amplify the region between 1072bp and 1434bp of rbOCT2 (gi:21956191).
The primers for OCT3 are s:5’-CTG GGT GGT CCC TGA GTC TCC CCG-3’ and
as: 5’-GCG TCG TCC AAG GCG CTC AAT GGT-3’, which would amplify the
region between 891bp and 1247bp of hOCT3. Since the sequence of rabbit OCT3
33
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has not been published, the OCT3 primers were designed based on conserved
sequence of human, mouse and rat OGT3. The PCR products were analyzed in
agarose gel stained with ethidium bromide and were subcloned into pGEM-T vector
for sequencing.
5. Data analysis
To estimate the maximum uptake rate ( V m a x ) and Michealis-Menten constant
(Km ), the uptake rate over various dosing concentrations (S) was fitted based on the
following equation by the means of nonlinear least squares regression analysis using
KaleidaGraph 3.5 (Synergy Software, Reading, PA). All data were expressed as
means ± S.E.M. and statistical analysis was performed with Student-Newman-
Keuls multiple comparison test. The criterion of significant difference was defined as
p < 0.05.
V = V m a x X S / (Km + S)
D. Results
1. Directional uptake of TEA in primarily cultured RCEC
The directionality of TEA uptake was explored in primary RCEC cultures on
permeable transwell filters. Time points over 20-minute period (data not shown)
showed no apparent saturation in TEA uptake, and hence “sink condition” was
assumed during the 5-minute uptake experiments. The initial apical uptake rate of
2/xCi/ml (36/xM) [1 4 C]TEA was 7 times higher than that of the basolateral side
34
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(Figure 2.1). And the difference was abolished by the presence of ImM unlabeled
TEA in the apical dosing medium. The presence of excess unlabeled TEA did not
significantly decrease basolateral TEA uptake rate (p-value = 0.13). The
directionality in TEA uptake suggested that the transport mechanism of the
basolateral uptake of TEA in the RCEC is passive diffusion through cellular
membrane following the concentration gradient, while the rapid apical uptake is
mainly due to a carrier-mediated system.
c
E
jn
120
c
'j£
o
100 -
a
o >
£
80 -
o
E
a
<
60 -
L U
1-
o
*
r
40 -
o
0 )
20 -
*2
a
3
0 -
Unlabeled
TEA:
Apical Basolateral
Figure 2.1 Directionality uptake of [1 4 C]TEA in primarily cultured RCEC. The
uptake of 2pCi/ml (36pM) [1 4 C]TEA was measured for 5 minutes in the presence or
absence of ImM unlabeled TEA. The bars represent means ± SEM, n = 4. The
35
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asterisks (*) represents statistically significant difference from the apical uptake with
no unlabeled TEA in the dosing medium (p < 0.05 by student t-test).
2. Directional uptake of MPP+ in primarily cultured RCEC
The directional uptake of organic cations in RCEC was more extensively
studied using another prototypic OC, MPP+, as the model substrate (Figure 2.2).
Approximately two times higher initial uptake rate of 4.25/iCi/ml (50nM) [3 H]MPP+
was observed for the apical side as compared to the basolateral side. Apical MPP+
uptake was substantially inhibited by excess unlabeled MPP+, and 2.5mM
concentrations of cations, such as TEA, dopamine, and histamine, while low but
statistically significant inhibition by beta-estrodiol and progesterone, the steroid
hormones known to be substrates of organic cation transporters (Grundemann et al.,
1999; Wu et al., 1998a), could be seen at lOuM. The apical MPP+ uptake was not
sensitive to PHA, an organic anion, at a concentration as high as 2.5mM. Basolateral
uptake of 50nM MPP+ was inhibited by excess unlabeled MPP+ and dopamine, but
neither by other tested organic cations, nor by steroid hormones. The difference in
apical and basolateral MPP+ uptake profile is consistent with that of TEA, indicating
that an organic cation transport system existed on the apical side of RCEC.
36
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Apical
B asolateral
Figure 2.2 Apical and basolateral uptake of [3 H]MPP+ in primarily cultured RCEC.
The uptake of 4.25pCi/ml (50nM) [3 H]MPP+ was measured in the presence or
absence of various inhibitors. Beta-estradiol and progesterone were dosed at a final
concentration of 10pM. The concentration used for the other inhibitors was 2.5mM.
The bars represent means ± SEM, n = 3. The asterisks represent statistically
significant difference (p < 0.05 by student t-test) from the apical or basolateral
control where no inhibitors were present.
37
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3. Inhibition of TEA or MPP+ uptake by L-carnitine
To investigate if the organic cation/ L-camitine transporters, exemplified by
OCTN1 and OCTN2, contribute to the absorption of organic cations in RCEC, L-
camitine was used to inhibit the apical or basolateral MPP+ or TEA uptake. As was
shown in Figure 2.3, at the concentration high enough to saturate the organic
cation/L-camitine transporter, L-camitine could not inhibit the absorption of TEA or
MPP+ from either apical or basolateral side, which suggested that the organic
cation/L-camitine transporter did not make a significant contribution in the
absorption of OCs in RCEC.
4. Concentration d e p en d e n cy of apical MPP+ uptake
To estimate the uptake due to passive absorption, excess unlabeled MPP+
(5mM) was added to saturate the carrier-mediated transport. The net transport, which
was contributed by the active transport system, was calculated by subtracting the
passive diffusion from the total uptake. Apical MPP+ uptake showed saturability
over the range of 5 to lOOpM (Figure 2.4). The corresponding Km , and V m ax of apical
MPP+uptake were estimated to be 4.2 ±1.1 jjM and 466.7 ± 19.4 pmol/mg protein
/15mins, respectively. The Eadie-Hofstee plot is compatible with a single uptake
mechanism (Figure 2.3).
38
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(A)
Basolateral
L-camitine:
(B)
© 120
□) 100
Basolateral
L-carnitine:
Figure 2.3 Effect of L-camitine on apical and basolateral uptake of TEA, or MPP+ in
primarily cultured RCEC. (A) The apical or basolateral uptake of 2 pCi/ml (36pM)
[1 4 C]TEA. The uptake was measured for 5 minutes in the presence or absence of
ImM L-camitine; n = 4. (B) The apical or basolateral uptake of 4.25pCi/ml (50nM)
[3 H]MPP+ . The uptake was measured in the presence or absence of 2.5mM L-
camitine; n = 3. The bars represent means ± SEM.
39
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600
♦ Total uptake
■ non-saturable component
“ “ saturable component
c
'£ 500
in
400 -
Q .
O) 300
50 -
40 -
30 -
20 -
200 -
Q .
450 250 350
100 -
Q .
0 20 40 60 80 100
MPP+ (uM)
Figure 2.4 Kinetics of apical uptake of MPP+ in primarily cultured RCEC. All
* 3 i
experiments were conducted in the presence of 4.25pCi/ml (50nM) [ H]MPP and 5
to 1 OOuM unlabeled MPP+. The saturable uptake was measured in the presence of
excessive (5mM) unlabeled MPP+. Inset is the Eadie-Hofstee Plot. Data points
represent means ± SEM; n = 3.
40
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5. Molecular identification of organic cation tran sp o rters in the
pigm ented RCEC
To provide molecular evidence for the existence of members of the organic
cation transporter family in the rabbit conjunctival epithelium, RT-PCR analysis of
mRNA expression of OCT1, OCT2 and OCT3 was performed.
Figure 2.5A showed no apparent amplification for the gene transcript of OCT1 or
OCT2 in the freshly isolated RCEC or in the negative control, where total RNA from
rabbit conjunctival epithelium was used as template in the PCR. In contrast, the
kidney, as a positive control, expressed both OCT1 and OCT2. Thus, our data
indicated lack of expression of OCT1 or OCT2 in the rabbit conjunctival epithelium,
and therefore are unlikely to contribute to the active absorption of the organic cations
in RCEC.
Figure 2.5B showed that a PCR product at the expected 357bp was amplified
using primers designed for OCT3. The sequencing result showed that this 357bp
fragment had 85% to 88% identity with the corresponding fragments of rat, human
and mouse OCT3 cDNA. It had 100% identity with the overlapped region of a 256bp
fragment of rabbit OCT3 reported to NCBI Genbank (gi:9945017). This sequencing
result verified the validity of the RT-PCR experiment, confirming the existence of
OCT3 gene transcript in the rabbit conjunctival epithelium. Interestingly, in addition
to the freshly isolated RCEC and the kidney, OCT3 was also found by RT-PCR in
mRNA samples prepared from the primarily cultured RCEC, the lens and the retina-
choroid suggesting that OCT3 was the major organic cation transporter in the eye.
41
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(A)
GAPDH
^ r
OCT
_ _ A _ _
Kid Cj Neg Kid Cj Ld
OCT1
OCT2
>600bp
-600bp
(B) Neg Kid Cr Len RC IC RCEC Cj
OCT3
► 357bp
Figure 2.5 RT-PCR analysis of organic cation transporters in ocular tissues. (A) RT-
PCR analysis of OCT1 and OCT2 in the freshly isolated RCEC (Cj). The expected
size of the PCR product of OCT1 and OCT2 was 535bp and 363bp, respectively.
Rabbit kidney (Kid) was used as the positive control. GAPDH, a house keeping
gene, was used as a loading control. (B) RT-PCR analysis of OCT3 in the freshly
isolated rabbit conjunctival epithelial cells (Cj), cultured RCEC (RCEC), Iris-ciliary
body (IC), retina-choroid (RC), lens(Len), and the Cornea (Cr). The expected size of
PCR product of OCT3 was 357bp. Rabbit kidney was used as the positive control.
No reverse transcriptase was added in the negative controls. The specificity of the
RT-PCR was verified by DNA sequencing.
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6. Drug interaction with the apical organic cation tran sp o rt sy stem
in primary RCEC culture
To screen the potential drug substrates of the apical organic cation
transporter, the potential for certain drugs to inhibit 50p,M [3 H]MPP+ absorption in
RCEC was evaluated. The drugs selected included (3-blockers (acebutolol and
betaxolol), anticholinergic drugs (procainamide), a2-agonists (brimonidine and
clonidine), and a Ca2 + -channel blocker (verapamil). All the above drugs have
positively charged group(s) at physiological pH fitting the general criteria to act as
potential substrate of organic cation transporters. Interestingly, all the drugs tested
showed statistically significant inhibition on apical MPP+ uptake at a concentration
of 1 or 2.5mM (Figure 2.6), indicating an interaction with the apical organic cation
transporter.
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Figure 2.6 Apical uptake of [3 H]MPP+ in primarily cultured RCEC cells was
inhibited by various OC drugs. The uptake of 4.25pCi/ml (50nM) [3 H]MPP+ was
measured in the presence or absence of various inhibitors dosed at a final
concentration of 2.5mM or l.OmM for verapamil. The bars represent means ± SEM;
n = 3. The asterisks (*) represent statistically significant difference from the control
(p < 0.05 by student t-test).
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E. Discussion
We have obtained evidence on the existence of an organic cation transporter
on the apical side of rabbit conjunctival epithelial cells by showing: a) the uptake of
two model OCs, TEA and MPP+, has directionality; 2) the apical uptake of TEA and
MPP+ exhibits concentration dependency; 3) The apical, but not basolateral, uptake
of MPP+ was inhibited by various OCs and steroid hormones.
More than 30 compounds, including biogenic amines and drugs, has been
identified so far to be substrates of organic cation transporters using cRNA injected
Xenopus laevis oocytes and transfected mammalian cell lines. Among them, MPP+ ,
TEA and guanidine were commonly selected as model substrates both in over
expression systems (Grundemann et al., 1999; Wu et al., 1998a), and in cells
primarily cultured such as renal proximal tubule cells (Grover et al., 2004) and
alveolar epithelial cells (Shen et ah, 1999). They are small hydrophilic molecules
(type I organic cation) permanently charged at physiological pH, with minimal
absorption from passive diffusion across the plasma membrane.
The apical TEA and MPP+ uptake in RCEC are 7 and 2 times higher than that
from the basolateral side, respectively. Given the fact that the difference between
apical and basolateral uptake of 36/dM [I4C]TEA was abolished in the presence of
ImM unlabeled TEA, basolateral uptake of TEA in RCEC is due to the passive
diffusion, and that from the apical side is via a saturable organic cation transporter in
concert with the passive diffusion. This observation is consistent with our previous
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findings that the flux of TEA through rabbit conjunctiva tissue in the mucosal-to-
serosal direction was 49.6 times higher than that in the serosal-to-mucosal direction
(Ueda et al., 2000). The apical uptake of 50nM [3 H]MPP+, but not that for the
basolateral uptake was inhibited by various OCs as well as steroid hormones that are
known to be transported by the OCTs. Different from TEA, basolateral MPP+ uptake
was from passive and carrier-mediated mechanisms, manifested by the substantial
difference between basolateral uptake and that when the active uptake system was
saturated by excess unlabeled MPP+. In addition, the basolateral uptake was inhibited
by dopamine. Another family of transporters well-known to transport MPP+ and
dopamine are the neurotransmitter transporters exemplified by the dopamine
transporter (DAT) (Pifl et al., 1993), the norepinephrine transporter (NET) (Wall et
al., 1995; Roubert et al., 2001) and the serotonin transporter (SERT)(Sitte et al.,
2000) mainly expressed at the synapses. When expressed in epithelial cells, DAT
was detected predominantly in the apical plasma membrane, whereas NET and
SERT were found in the basolateral membrane (Gu et al., 1996). The
neurotransmitter transporters may contribute to the active MPP+ uptake from the
basolateral side of RCEC, but this postulation remains to be confirmed by functional
as well as molecular approaches.
Kinetic studies revealed that the apical MPP+ uptake has a saturable
component with a Michealis-Menten constant (Km ) of 4.2 pM and a maximum
uptake of 466.7 ± 19.4 pmol/mg protein/15mins. The Km of 4.2 pM indicates a
transporter with high affinity to MPP+. It is comparable to the MPP+ uptake mediated
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by organic cation transporters, for example, human OCT1 injected in Xenopus
oocyte (Km = 14.6 ± 4.39 /xM) (Zhang et al., 1997), human OCT2 overexpressed in
mouse inner medullary collecting duct cells (22.2 ±1.8 /xM) (Dudley et al., 2000),
and endogenous rat OCT3 in cerebellar granule neurons (5.3 ±1.2 /xM) (Shang et al.,
2003).
To explore whether the organic cation/L-camitine transporters contribute to
the organic cation uptake in RCEC, L-camitine, at concentrations about 50 times
above the Km of the L-camitine transporter, was added to the uptake medium. L-
camitine failed to inhibit either apical or basolateral uptake of TEA or MPP+. These
results conclusively eliminated the involvement of organic cation/L-camitine
transporters. Furthermore, these findings are consistent with our previous
observation that the OC transport through the rabbit conjunctival tissue was sensitive
to membrane potential modulations instead of extracellular pH (Ueda et al., 2000).
Our finding of the gene transcript for OCT3, but not OCT1 or OCT2,
indicates that OCT3 is most likely to be the prominent organic cation transporter in
the rabbit conjunctival epithelium. In addition to the conjunctival epithelium, OCT3
mRNA was detected in other ocular tissues including the lens, the iris-ciliary body,
and the retina-choroid, which is consistent with observations by Raj an et. al. (Raj an
et al., 2000), that OCT3 is expressed in the retinal pigment epithelium(RPE) and
mouse neural retina.
Although there is no conclusive evidence on the membrane localization of
OCT3 in polarized epithelial cells so far, functional studies suggested that OCT3 are
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putatively responsible for the apical MPP+ absorption in intestine (Jonker and
Schinkel, 2004; Martel et al., 2001). The conjunctiva, together with the cornea,
meibomian glands, lacrimal glands, and the interconnecting neural reflex loops
comprise a functional unit controlling tear production. It is known that alterations of
the quantity or composition of tear output resulted in ocular surface diseases,
exemplified by the dry-eye syndrome (Stem et al., 1998). Recently, Vialou et. al
.(Vialou et al., 2004) demonstrated in the central nervous system that OCT3 is
critical for the neural and behavioral responses to environmentally induced variations
in osmolarity. Located on the mucosal surface of the conjunctiva, OCT3 might play a
major role in maintaining the homeostasis of tear fluid as it does in the regions of the
brain that sense blood osmolarity and regulate salt and water ingestion (Vialou et al.,
2004).
Our identification of the apical organic cation transporter in the conjunctival
epithelium has great pharmacological significance. With recent rapid advances in
drug discovery for the treatment of diseases associated with the back of the eye,
strategies for delivering therapeutic compounds to the posterior ocular region
become increasingly important. Topically applied drugs are believed to reach the
posterior region more efficiently via the conjunctiva and the underlying sclera
compared to the cornea (Ahmed and Patton, 1985; Geroski and Edelhauser, 2000).
The existence of OCT on the apical side of the conjunctival epithelium may facilitate
the entrance of topically applied cationic drugs into the conjunctiva, leading to
increased intra-ocular drug absorption and decreased drug loss due to the tear wash.
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By conducting competitive inhibition experiments in RCEC, we found that many
cationic drugs might serve as substrates of the apical organic cation transporter.
These studies using inhibitors including (3-blockers, a2-agonists, Na+-, and Ca2 + -
channel blockers, indicated the wide applicability of the organic cation transport
system in transport of a variety of drugs in the conjunctiva. In addition, this result
concurs with the observation that OCT3 exists in the rabbit conjunctival epithelium
in that OCT3 is multi-specific. However, the competitive inhibitory mechanism
would require further confirmation in overexpression systems to establish firmly that
OCT3 translocates the above drugs. In fact, the drugs selected fit the criteria to be
OCT substrates in that they are small compounds ranging in size from 229 to 455 Da,
with at least one positively charged amine moiety at physiological pH. Verapamil
(Shu et al., 2001) and the (3-blockers, propranolol and metoprolol (Dudley et al.,
2000) are known to be transported by OCT2, which shares substantial substrate
profile with OCT3.
In conclusion, we identified an organic cation transport system localized on
the apical side of the rabbit conjunctival epithelial cells. The molecular identity of
the transporter strongly corresponds to OCT3. The potential and versatility of this
transporter to mediate transport of a variety of cationic compound categories suggest
its importance in topical drug delivery, as well as in ocular surface-related diseases.
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CHAPTER III
ESTABLISHMENT OF IN VITRO MODELS OF RPE AND
IDENTIFICATION OF ORGANIC CATION
TRANSPORTERS IN RPE
A. Summary
In vitro models of polarized retinal pigment epithelium (RPE) were first
established in the lab. A tissue-level model, bovine RPE-choroid explants mounted
in Ussing Chamber was set up and evaluated by electrophysiology properties. The
tissue showed transepithelial electrical resistance (TEER) and potential difference
(PD) comparable to reported values in the literature. The tissue lasted up to 3 hours
as demonstrated by the flux of mannitol, a paracellular transport marker.
A polarized cell culture model of prolonged culture of ARPE-19 cells was set
up on laminin coated transwell filters in DMEM/F12 medium. ARPE-19 cells
formed monolayers on transwell filters. The cell polarization was supported by
elevated transepithelial electrical resistance (TEER), the expression of tight junctinal
protein, ZO-1 and the polarized secretion of vascular endothelial growth factor
(VEGF) and bone morphogenic protein 4 (BMP4). However, the lack of polarized
localization of Na+ -, K+ -ATPase and the low transepithelial potential difference (PD)
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suggested a potential for improvement of this culture model, and a necessity for new
cell lines or primary culture models.
RT-PCR experiments suggested that organic cation transporter 3 (OCT3) is
the most abundantly expressed organic cation transporters in the RPE. Functional
studies in ARPE-19 cells using prototypic OCT3 substrate, l-methyl-4-
phenylpyridinium (MPP+ ) revealed the apical localization of OCT3 in the RPE.
B. Introduction
The retinal pigment epithelium (RPE) plays a key role in maintaining retinal
function by serving as the metabolic “gatekeeper” between photoreceptors and the
choriocapillaris (Marmor, 1998). RPE dysfunction likely contributes to the
pathogenesis of multiple sight-threatening diseases. In addition, RPE constitutes the
posterior part of the blood retinal barrier, controlling the movement of solute
molecules between choroidal blood circulation and the photoreceptors. Because of
the importance of this structure, the unique properties of RPE cells have attracted
considerable attention. Although much has been learned from in vivo studies, the
limitations imposed by whole animal experimentation have led to the development of
in vitro models including tissue explants (Edelman and Miller, 1991; Frambach et al.,
1989; Frambach et al., 1988b; Steinberg et al., 1978; Koyano et al., 1993) and RPE
cell cultures (Mannagh et al., 1973; Edwards, 1977; Del Priore et al., 1988) from
various species.
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Both the tissue explant models and the cell culture models have their pros and
cons. While tissue explants resemble RPE in its in vivo milieu compared with
cultured cells, the mixed effect from multiple cell types in the explants and the
complicated dissection procedure limit their application. The RPE-choroid explants
or RPE-choroid-sclera explains mounted in two-compartment diffusion chambers or
the Ussing Chamber represent good models for electrophysiology studies and small
scale permeability studies. In Ussing Chamber, the wellness of the RPE as indicated
by the maintenance of RPE polarity can be constantly monitored by measuring the
transepithelial electrical resistance (TEER) and potential difference (PD) throughout
the experiments.
Although RPE culture models are more widely used, mammalian RPE culture
models generally fail to preserve many of the phenotypic characteristics which are
exhibited by RPE in vivo. Such properties include the ability of RPE cells to produce
intercellular tight junctions, to form apical and basolateral membrane domains
through cell polarization, and to phagocytose rod outer segments effectively.
Currently, the most promising human primary RPE culture models are either based
on the long-term growth of RPE in specialized media (Hu and Bok, 2001;
Maminishkis, 2005), or the use of biogenic membranes as matrix support for RPE
cells (Campochiaro and Hackett, 1993; Capeans et al., 2003; Turowski et al., 2004).
The precise mechanism by which extended culture, soluble media supplements, and
extracellular matrix components lead to a specialized RPE phenotype is unclear.
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Cell lines have the advantage over primary cultures in the culture set up, cell
homogeneity, and reproducibility. Several RPE cell lines have been developed
(Dunn et al., 1996; Nabi et al., 1993; Davis et al., 1995), among which ARPE-19 is
the most widely used human RPE cell line. ARPE-19 is a spontaneously arising
human RPE cell line derived from the globes of a 19-year old male donor (Dunn et
al., 1996). The cells maintained normal karyology which formed polarized epithelial
monolayers on porous filter supports, and hence been selected in many studies of
polarized transport (Bridges et al., 2002), polarized protein expression/secretion
(Philp et al., 2003; Holtkamp et al., 1998; Narayan et al., 2003) and barrier
breakdown (Abe et al., 2003).
In this study, for the first time in our lab, ARPE-19 cells were cultured on
transwell filters. The polarity of the cell monolayer was explored via examining the
intercellular tight junctions, the membrane localization of Na+-, K+-ATPase, the
TEER and the differential secretion of vascular endothelial growth factors (VEGF-A
and VEGF-C) and bone morphogenic protein 4 (BMP4). In addition, a tissue level
RPE model, bovine RPE-choroid explants mounted in Ussing Chamber was
established. Properties such as TEER, PD and permeability to mannitol were
evaluated.
The RPE constitutes the major barrier in the posterior region of the eye,
affecting the disposition of ophthalmic drugs administered topically, periocularly or
systemically. The existence of intercellular tight junctions in the RPE significantly
limits the vectorial movement of hydrophilic compounds that mainly penetrate
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paracellularly. The transport of such compounds relies on endogenous transport
systems in the RPE. Several transporters have been identified in the RPE in the past
several years. Among them, the Pg-P (Kennedy and Mangini, 2002), MRP1
(Aukunuru et al., 2001), MCTs (Chidlow et al., 2005; Philp et al., 2003), ATB0 > +
(Naggar et al., 2003), OATPs (Ito et al., 2002; Ito et al., 2003) and OCT3 (Rajan et
al., 2000) may play a significant role in affecting the absorption or elimination of
therapeutic compounds because of their broad range of substrate profile covering
many drugs or drug metabolites.
Organic cation transport systems in the RPE may play a critical role in
modulating the vectorial movement of drugs and endogenous compounds that are
positively charged at physiological pH. In addition to OCT3 (Kekuda et al., 1998),
several other organic cation transporters have been cloned and were shown to control
the absorption and secretion of many organic cations in the kidney, the liver, the
intestine as well as the blood brain barrier (Cucullo et al., 2005), which closely
resembles the blood retinal barrier in anatomy and physiology (Schlosshauer, 2002;
Steuer et al., 2005). These organic cation transporters are OCT1, OCT2, OCTN1 and
OCTN2 (Ohashi et al., 1999). Together with OCT3, they can be subcategorized into
two families, the membrane potential-dependent multispecific organic cation
transporters (OCT1, OCT2 and OCT3) (Urakami et al., 1998; Wu et al., 1999), and
the proton concentration-dependent organic cation transporters (OCTN1 and
OCTN2). The latter was also reported to be L-camitine transporters (Tamai et al.,
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1998; Tamai et al., 2000; Ohashi et al., 2001). The expression of transporters from
the organic cation transporter family remains to be investigated in the RPE.
In this study, the expression of organic cation transporters was studied in
primary human fetal RPE cells and the human adult cell line, ARPE-19 cells. The
function and localization of organic cation transporters were studied in ARPE-19
cells cultured on permeable support that was newly established in the lab.
C. Materials and methods
1. Materials
Human fetal eyes were obtained from the Advanced Bioscience Resources, Inc.,
(Alameda, CA). Bovine and porcine eyes were obtained from Manning Beef, a local
slaughter house, shortly after the animals were killed for food processing.ARPE-19
cells (passage 20) were obtained from American Type Culture Collection (Manassas,
VA) and used between passages 22 to 25.
Tissue culture-treated polyester transwells (6.5mm or 12mm in outer diameter
and 0.4 pm in pore size) were purchased from Costar (Coming, NY). Dulbecco’s
modified Eagles’s medium (DMEM) and Ham’s F-12 medium were purchased from
Mediatech (Hemdon, VA).Laminin, collagen IV and Matrigel was from BD
Biosciences (San Diego, CA). Other cell culture reagents and supplies were obtained
from Life Technologies (Grand Island, NY).
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Polyclonal rabbit anti-rat organic cation transporter 3 antibody was purchase
from Alpha Diagnostic International (San Antonio, TX).The monoclonal antibody
against ZO-1 was from Zymed Laboratories, Inc. (South San Francisco, CA). The
monoclonal antibody against Na+-, K+-ATPase a-\ was from Upstate
Biotechnology (Lake Placid, NY). The FITC-conjugated goat anti-rabbit IgG was
from Jackson frnmunoResearch Laboratories, Inc. (West Grove, PA). Other
secondary antibodies were purchased from Chemicon (Temecula, CA).
ELISA kits for human VEGF-A and BMP4 were ordered from R & D Systems
(Minneapolis, MN), while that for human VEGF-C is from Immuno-Biological
Laboratories Co., Ltd. (Gunma, Japan).
[3 H]Mannitol (17-20Ci/mmol) was purchased from PerkinElmer Life Sciences
(Boston, MA). [Methyl-3 H]l-methyl-4-phenyl-pyridinium (85Ci/mmol) was
purchased from American Radiolabeled Chemicals (St. Louis, MO).The other
chemicals of analytical purity were from Sigma.
2. Bovine RPE-Choroid explants m ounted in U ssing C ham ber
Bovine eyes were obtained form a local slaughter house shortly after the
animals were killed for food processing. The eyes were kept in an ice bucket.
Typically, 3 to 4 hours elapsed before the eyes were studied in the laboratory.
After a brief cleaning of the connective tissues and the muscles, the eye cup
was opened by a circular cut just posterior to the limbus. The antierior segment and
the vitreous were removed. The posterior eye cup with the sclera, choroid, RPE and
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the retina was cut into approximately 1 inch squares. To be consistent, only the
pieces with pigmented RPE were selected. The sclera was removed by a combination
of sharp and blunt dissection under a binocular microscope using microscissors and
delicate forceps. The retina-RPE-choroid preparations were then placed on a nylon
mesh (pore size of 70/«n, Becton Dickinson Labware, NJ), with the retinal side up.
The retina was carefully peeled off, and the remaining RPE-choroid was mounted in
■ y
a custom designed Ussing chamber with an exposed area (A) of 1.0 cm .
The RPE-choroid preparations mounted in a clamping chip were placed
between two chamber halves. The chamber held 6ml bicarbonated Ringer Solution
(BRS) (Frambach et al., 1988a) on each side of the tissue, and was water-jacketed to
keep the temperature within the chamber 37°C. Electronics on either side of the
tissue enabled to measure the voltage produced by the tissues (PD) and the
transepithelial electronic resistance (TEER) of the tissues.
All experiments were performed under short-circuit condition with the use of
an automatic voltage-clamp device (558C-5; Bioengineering Department, University
of Iowa, IA), as describe before (Ueda et al., 2000). Potential difference (PD) was
measured with two matched calomel electrodes. Two polyethylene (PE90) bridges
(containing 3% agar in 3M KC1), whose tips were located near the center of tissue
surfaces, were used to electrically connect the reservoir fluid to electrode wells. The
electrical output of calomel electrodes was amplified by the voltage-clamp unit.
Direct current flowing across the tissue was sent with a pair of matched Ag/AgCl
electrodes with conducting agar bridges, whose tips were positioned away from
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tissue surfaces at the far ends of two reservoirs. The short-circuit current (Isc)
flowing in the bath-tissue-bath circuit was monitored and recorded on a strip chart
recorder (Kipp and Zonen, Delft, The Netherlands). At 60-second intervals, a lmV
pulse (AV) was imposed for 3 seconds across the tissues to estimate the TEER, as a
surface area normalized ratio of applied voltage pulse to the observed deflection in
resultant current (Al) flowing on top of Isc ( TEER= (AV/AI)- A). Before each
experiment, the solution resistance was compensated by the voltage-clamp device.
3. Mannitol tran sp o rt a c ro ss bovine RPE-choroid explants
m ounted in U ssing C ham ber
Basolateral to apical mannitol flux measurement was initiated by adding
[3 H]mannitol (2.0/xCi/ml, Moravek Biochemicals, CA) to the basolateral (choroidal)
donor fluid. At predetermined time periods up to 3 hours, a 0.6ml aliquot was
collected from the receiver (apical) fluid for assay of radioactivity in a liquid
scintillation counter (Beckman, Fullerton, CA). The aliquot removed was
immediately replaced with an equal volume of fresh BRS buffer.
4. Isolation of neutrophil enriched population
A neutrophil enriched population was isolated from murine peritoneal
exudates after glycogen challenge. Briefly, oyster glycogen (Sigma) was dissolved in
sterile 0.9% NaCl at a concentration of 1 mg/ml. A total of 1 ml was injected IP into
male C57BL/6 mice, and the peritoneal exudates were harvested 4 h later. The
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exudate cells were approximately 90% neutrophils determined by characteristic
morphology. The neutrophil enriched population was centrifuged onto superffost
plus micro slides (VWR Scientific, West Chester, PA).
5. Preparation of porcine lens c ap su le m em brane tissu e culture
inserts
The protocol of Greenwood et al. was followed (Turowski et al., 2004). Briefly,
porcine lens capsules (PLCs) were obtained from fresh porcine eyes. After surgical
exposure of the anterior surface of the lens, a vectis loop was used to disinsert the
zonules. The anterior capsule was cut circumferentially along the lens equator.
Transwell polyester tissue culture filters (6 mm diameter; Costar, Cambridge, MA)
were pre-prepared by removing the original membrane and coating the bottom edge
with glue. The support was then gently glued onto the central anterior lens capsule.
Once a complete and tight seal was established between lens capsule and Transwell
support, the remainder of the lens was gently teased away from the capsule. The
prepared PLC membrane inserts were then washed thoroughly with Ca/Mg-free
Hanks Balanced Salt Solution (HBSS) in the presence of 0.5 mg/mL streptomycin
and 500 U/mL penicillin (Sigma, San Diego, CA) and left for 24 hours in a sterile
environment. PLC membranes were then washed and incubated in Dulbecco’s
Modified Eagle’s Medium (DMEM)/ Ham’s F-12 (F-12) growth medium for 48
hours to check sterility before seeding of ARPE-19 cells.
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6. ARPE-19 cell m onolayers on perm eable transw ell filters or PLC
m em branes
Transwell filters (0.4/xm pore size, 6.5mm or 12mm diameter, Coming
Incorporated Life Sciences, Acton, MA) were either coated with one of the following
extracellular matrix protein: 5/xg/cm2 rat laminin (BD Biosciences, San Diego, CA),
thick layers of Matrigel (BD Biosciences, San Diego, CA) and 10 /xg/cm2 mouse
collagen IV (BD Biosciences, San Diego, CA), or replaced by PLC membrane.
ARPE-19 cells (American Type Culture Collection, Manassas, VA) was seeded at a
seeding density of 2.0 x 105 cells/cm2 in DMEM/F-12 culture medium (Mediatech,
Hemdon, VA), supplemented with 100 U/ml penicillin-streptomycin, 2 mM L-
glutamine, and 1% FBS (10% FBS, when PLC membrane was used as support). A
corresponding amount of culture medium was added to the basolateral compartment,
leveling the height of the liquid to prevent hydrostatic pressure. The medium was
changed twice a week. The cell monolayers were cultured for at least a month before
use.
7. Transepithelial resistan c e m easu rem ent
ARPE-19 cells were grown on PLC membrane inserts and Transwell
polyester filters. Subsequently changes in TEER across the monolayers were
monitored weekly at 37°C using STX-2 chopstick electrodes connected to an EVOM
epithelial voltohmmeter (World Precision Instruments, Sarasota, FL). Net TEER
values were calculated by subtracting the mean resistance determined for PLC
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culture cups or plastic filters in the absence of ARPE-19 cells, from the value
recorded for each monolayer grown on PLC membrane inserts or plastic filter,
respectively. Final resistance-area products (in Q -cm2 ) were obtained by
multiplication with the effective growth area.
8. Human fetal RPE culture
RPE cells were isolated from human fetal eyes obtained from the Advanced
Bioscience Resources, Inc. (Alameda, CA), and cultured in DMEM culture medium
(Fisher Scientific, Pittsburgh, PA) with 2 mM L-glutamine, 100 U/ml penicillin, 100
pg/ml streptomycin (Sigma, St. Louis, MO), and 10% heat inactivated FBS (Irvine
Scientific, Santa Ana, CA) as previously described (He et al., 1994).
9. Secretion of grow th factors and cytokines from ARPE-19 cells
Experiments were performed with ARPE-19 cells cultured on transwell filters
of 12mm in diameter. Experiments were started by replacing culture medium with
fresh medium. After incubation in normal growth conditions for predetermined time
period, medium from the upper and lower compartments was collected, snap-frozen,
and stored at -80°C until further analysis.
Medium was concentrated through centrifugal filter devices (Millipore, Bedford,
MA). Each sample of medium was tested in triplicate. Levels of VEGF-A, VEGF-C
and BMP4 production of the ARPE cells toward the basal or the apical side were
assayed by enzyme-linked immunoabsorbent assay (ELISA; R&D Systems,
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Minneapolis, MN, for BMP4 and VEGF-A; IBL Co., Gunma, Japan for VEGF-C),
according to the manufacturer’s instructions.
10. Im m unocytochem istry and Confocal M icroscopy
ARPE-19 cell monolayers, grown for 2-3 months on either PLC membrane
inserts or on Transwell polyester filters, were fixed in 3.7% paraformaldeyde in Tris-
buffered saline (TBS) and permeablized for 30 minutes with 0.1% Triton X-100.
Alternatively cells were fixed and using ice-cold methanol. After washing three
times by TBS for 10 minutes and blocking by 5% rabbit or goat serum in TBS, cells
were reacted with primary antibodies (1:100 dilution) against ZO-1 (from Zymed,
San Francisco, CA), Na/K-ATPase a-1 (Upstate, Waltham, VA) or organic cation
transporter-3 (Alpha Diagnostic International, San Antonio, TX) at 4°C overnight.
The cells were washed and blocked with matching Cy3-, or FITC-conjugated rabbit
or goat secondary antibodies (Jackson Immuno-Research Laboratories, West Grove,
PA) in dark for 1 hour. Finally, immunostained preparations were mounted using
Vectashield with/without DAPI (Vector Laboratories, Burlingame, CA) and
examined by confocal laser-scanning microscopy LSM 510 (Carl Zeiss, Inc.,
Thomwood, NY).
11. RT-PCR of organic cation tran sp o rte rs in RPE cells
Total RNA was isolated from human fetal RPE cells and ARPE-19 cells
using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and the
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contaminating genomic DNA was removed with DNase (DNA-free; Arnbion, Austin,
TX). Reverse transcription was performed using 1 pg total RNA, oligo(dT)i5 primer
and AMV reverse transcriptase (Promega, Madison, WI). PCR were performed for
30 cycles at an annealing temperature of 60°C after the initial denaturation at 95°C
for 5 minutes. The oligonucleotide primers for OCT1, OCT2, and OCT3 were sense:
5’-GAT TTC CTT TAC TCC GCT CTG GTC-3’ and anti-sense: 5’-TTT CTT TGG
GCT TTG CTT TTC TCC-3’ for human OCT1; sense: 5’-GAT TTC TTC TAC TCT
GCC CTG GTT-3’ and anti-sense: 5’-GGA TTT CTA CTT TTG GTC TTG CTG-3’
for human OCT2; and sense: 5’-GAC AAG AGA AGC CCC CAA CCT GAT-3’
and anti-sense: 5’-CAC TAA AGG AGA GCC AAA AAT GTC-3’ for human OCT3.
The PCR products were analyzed in agarose gel stained with ethidium bromide.
12. Uptake stu d ies by ARPE-19 cell m onolayers
Unless otherwise specified, all uptake experiments were performed in a
humidified atmosphere of 5% CO2 and 95% air at 37°C in buffered BRS solution at
pH 7.4. After the cell layers were preincubated in BRS for 40 minutes, the uptake
experiments were initiated by spiking the apical or basolateral fluid with a
predetermined amount of MPP+ (l-methyl-4-phenyl-pyridinium) or competitive
compounds traced with [3 H]MPP+ (85Ci/mmol, American Radiolabeled Chemicals,
St. Louis, MO). After a period of incubation of 5 minutes, uptake was terminated by
aspiration of the dosing solution followed by three quick washes of the cell layers in
ice-cold BRS buffer. The cell layers were then solubilized in 0.5ml 1% SDS solution.
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Five milliliters of scintillation cocktail was added for quantification of the
radioactivity in a liquid scintillation counter (Beckman, Fullerton, CA).
D. Results
1. Bovine R PE -choroid expiants m ounted in Ussing C ham ber
Bovine RPE-choroid explants mounted in Ussing Chamber showed the
baseline PD of 6.5 ± 0.6 mV (retinal side positive) and TEER of 234 ± 34 Q -cm2
after 30 minutes of equilibration in BRS (Table 3.1), which were comparable to
previously reported values (Frambach et al., 1989; Joseph and Miller, 1991).
Laboratory PD (mV)
TEER (Q'cm2)
Hinton (present study) 6.5 ± 0.6 (22) 234 ±34 (15)
Miller (Joseph and Miller, 1991) 6.0 ± 1.0(49) 138.7 ± 7 (49)
Frambach (Frambach et al., 1989) 12 300
Table 3.1 Electrical Parameters of bovine RPE-choroid explants in modified Ussing
chamber. Number in parentheses represents the number of tissues measured. Data are
presented as mean ± SEM.
The mannitol flux across the bovine RPE-choroid explants was measured for
up to 3 hours and showed typical pseudo-steady-state transport properties with a lag
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time of 26 minutes (Figure 3.1). The flux rate increased significantly when mouse
neutrophils were added to the dosing solution to open the juctional barriers of RPE.
6.0
5.0
^ 4.0
ip 3.0
5
c
§ 2.0
1.0
0.0
♦ C o n tro l
■ N e u tro p h il
I
/
V '\
nr
-i
50 100
Time (minute)
150 200
Figure 3.1 Time course of [3 H]mannitol (lOnM) transport across bovine-RPE
choroid explants mounted in Ussing Chamber. The flux of mannitol in the direction
of choroid-to-retina was monitored in the presence (n = 6) or absence (n = 4) of
mouse neutrophils (2 x 105 cells /ml). Data represents mean ± SEM.
65
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2. Cell cultures of the polarized RPE
The effect of extracellular matrix on the tightness of ARPE-19 cell
monolayer was evaluated (Figure 3.2). The TEER increased and peaked in two
weeks post seeding. After 4-week culture, the highest TEER was observed when
anterior porcine lens capsule was used as support. ARPE-19 cells seeded on
transwell filters coated with collagen IV or laminin also showed good electrical
resistance, around 30 0 .cm2. The lowest TEER was observed when a thick layer of
matrigel was used to coat the filters.
To demonstrate the ability of the ARPE-19 cells to polarize and to form
junctional complexes typical for an epithelial phenotype, immunostaining of ZO-1, a
component of the tight junction complex expressed by the ARPE-19 was performed.
As shown in Figure 3.3 and Figure 3.4-1, the intercellular junction outlining each
ARPE cell was stained for ZO-1. ARPE-19 cells seeded on PLC showed the
strongest staining, followed by laminin-coated transwells. On all supports ARPE-19
displayed a cobblestone morphology typically found for epithelial cells in culture.
However, those seeded on PLC were significantly more compact compared with
cells seeded on other support (Figure 3.4).
To evaluate the polarity of the ARPE-19 cells, the localization of Na+ -, K+ -
ATPase was studied using antibody against the a-l unit of Na+-, K+ -ATPase. The
staining revealed the membrane-association of Na+ -, K+ -ATPase (Figure 3.5) with no
apparent apical or basolateral polarity (Figure 3.4-2 and Figure 3.5).
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VEGF-A and -C secretion in polarized ARPE-19 cells was markedly
stimulated as compared to non-polarized cells that reached confluence (Figure 3.6B
and Figure 3.7B). ARPE-19 cells secreted VEGF-A and VEGF-C at both apical and
basolateral surfaces, with more secretion from apical side. The secretion of VEGF-A
increased with 150pM of tertbutyl hydrogen peroxide (TBH) treatment as a function
of time (l-5hrs) with maximal increases at 5 hours (from 402 to 1921 pg/106 cells on
the apical and 269 to 1507 pg/106 cells for basolateral; Figure 3.6A). A similar trend
was observed for VEGF-C secretion (Figure 3.7A). VEGF-A secretion was dose-
dependent for the TBH range of 50-200pM with apical secretion greater than
basolateral secretion (Figure 3.6B). However, VEGF-C did not exhibit a
concentration-dependent increase in apical or basolateral secretion.
In contrast to the secretion pattern of VEGF, BMP4 secretion increased over
time and a preferential BMP4 secretion from the basal surface of the monolayers was
observed (Figure 3.8)
67
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40J CM
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o
£
.c 30.0
o
O '
IXJ
LU
H
20.0
10.0
0.0
- 10.0
T im e (d a y )
Figure 3.2 Transepithelial electrical resistance (TEER) of ARPE-19 cells cultured on
transwell filters. Transwell filters (6mm in diameter, 0.4/xM in pore size) were coated
with various extracellular matrix proteins (n = 5-12). Data points are means ± SEM.
68
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Figure 3.3 Immunofluorecent staining of ZO-1 of ARPE-19 cells cultured transwell
filters coated with various extracellular matrix proteins. A: ARPE-19 cultured on
non-coated transwells; B: ARPE-19 cultured on Collagen IV-coated transwells; C:
ARPE-19 cultured on laminin-coated transwells; D: ARPE-19 cultured PLC (porcine
lens capsule).
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A
\
B
\
C D
\
\
A
\
B
C D
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2)
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Figure 3.4 Immunofluorecent staining of ZO-1 (3.4-1) and Na+ -, K+-ATPase (3.4-2)
on cross sections of ARPE-19 cells. ARPE-19 cells were cultured on laminin-coated
transwell filters for more than 3 month. A: ARPE-19 cells stained with anti-ZO-1
antibody (4.1) or anti- Na+ -, K+ -ATPase antibody (3.4-2); B: transmissive
microscopy of cell morphology; C: merged view of fluorescent staining and
trasmissive image; D: negative control in the absence of secondary antibody. Arrows
indicate the apical side of ARPE-19 cells.
Figure 3.5 Fluorescent Confocal Z-stack staining of Na+-, K+ -ATPase of ARPE-19
cells. ARPE-19 cells were cultured on laminin coated transwell filters for 3 months.
71
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(A)
(B)
« 2500
0 )
0
C O
1
1 1 1
o
a >
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a )
o
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in
O
L X J
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2000
1500
1000
500
-A pical (150uM TBH)
- B asolateral(150uM TBH)
-A- - A pical n o n -tre a te d
-Q - B aso lateral n o n -treated
Tim e (hr)
l/T 1600
" a )
H 1400
co
I
LL1
o 1200
Apical
Basolateral
Non-polarized
g 1000
IS 800
o
o 600
0
i n
< 400
1
u.
g 200
>
0 50 100 150 200
TBH (uM )
Figure 3.6 Secretion of VEGF-A in polarized and Non-polarized ARPE-19 cells. (A)
Time dependent apical and basolateral VEGF-A secretion in ARPE-19 cells treated
with 150uM TBH. (B) Concentration dependent apical and basolateral VEGF-A
secretion in ARPE-19 cells treated with TBH.
72
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(A)
(B)
(0 1000
800 -
7 0 0 -
6 0 0 -
4 0 0 -
3 0 0 -
co
I
ill
o
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3
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0 )
0
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0
0
M
O
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0
ill
>
C O 1400
-A -A p ic a l(1 5 0 u M T B H )
B a s o la te ra l(1 5 0 u M TB H )
S O
T im e (hr)
tra n sw e ll-a p ic a l
- tra n sw e ll-b a so la te ra l
P late
(O 1 2 0 0 -
800 -
A , 600 -
co 400 -
200 -
100 150
TBH (uM)
200 250
Figure 3.7 Secretion of VEGF-C in polarized and Non-polarized ARPE-19 cells. (A)
Time dependent apical and basolateral VEGF-C secretion in ARPE-19 cells treated
with 150uM TBH. (B) Concentration dependent apical and basolateral VEGF-C
secretion in ARPE-19 cells treated with TBH.
73
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48 hr Ohr 24hr
o
S 3
o
• (■4
a >
I.
u
4 >
C/5
Apical
Basal
Figure 3.8 Polarized secretion of BMP4 protein from the ARPE-19 monolayer. Both
apical and basal medium were collected at 0, 24 and 48 hours. After concentration,
the final BMP4 levels were determined using ELISA.
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3. Identification of OCT3 in th e RPE
The RT-PCR experiment was performed using primers specific for human
OCT1, OCT2 and OCT3. Gene transcript of OCT3 was identified both in primary
culture of human fetal RPE cells and ARPE-19 cells (Figure 3.9). The majority of
staining of OCT3 was associated with cell membrane and only sporadic and punctate
staining was observed in the cytoplasm (Figure 3.10).
Negative OCT3 OCT2 OCT1
Primary culture of
human fetal RPE cells
ARPE-19 cells
Figure 3.9 RT-PCR of OCT1, OCT2 and OCT3 in the human retinal pigment
epithelial cells.
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Figure 3.10 Immunofluorecent staining of OCT3 on cross sections of ARPE-19 cells.
ARPE-19 cells were cultured on laminin-coated transwell filters for more than 3
months. A: ARPE-19 cells stained with anti-OCT3 antibody; B: transmissive
microscopy of cell morphology; C: merged view of fluorencent staining and
transmissive image; D: negative control in the absence of secondary antibody.
Arrows indicate the apical side of ARPE-19 cells
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The apical uptake of MPP+, a model substrate of OCT3, is significantly higher
than the basolateral uptake. Apical uptake in ARPE-19 cells showed temperature
dependency (Figure 3.11). The apical uptake of MPP+ was considerably inhibited by
typical organic cations including TEA, MPP+, and dopamine. The specific inhibitor
of OCT3, decynium-22 inhibited OCT3 by 80% at 5/iM (Figure 3.12). The
basolateral uptake of MPP+ was not significantly inhibited by TEA or decynium-22.
However, excess unlabeled MPP+ dopamine and L-alanine inhibited basolateral
MPP+ considerably. No obvious inhibition of apical or basolateral uptake was shown
by L-camitine (substrate of OCTNs) or PHA (an organic anion).
Apical uptake of MPP+ showed a saturable function over the concentration
range of IjJiM to 150uM (Figure 3.13). The Michealis-Menten constant and the
maximum uptake was estimated to be 24 ± 12 uM and 352 ± 51 pmol/5min. The
linear relationship in Eadie-Hofstee plot is consistent with a one transporter-
mediated transport mechanism.
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0.6 n
Apical B asolateral
Figure 3.11 Temperature and directionality MPP+ uptake in polarized ARPE-19 cell
monolayers. Apical uptake of MPP+ also showed temperature dependency, but not
basolateral uptake. (*, p < 0.05)
78
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Decynium 22
■ Basolateral
MPP+
Dopamine
Carnitine
L-Aianine
0 20 40 60 80 100 120 140
Uptake (% of control)
Figure 3.12 Differential inhibition profiles of apical and basolateral MPP+ uptake in
ARPE-19 cells. Except decynium 22 which was dosed at 5pM, all compounds were
dosed at 2.5mM. (*, p < 0.05, n = 3)
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400
Km = 24 ± 12 pM
Vm a x = 352 ± 51 pmol/5min
350
•E 300
E
S 250
O
£ 200
£ 150
re
100
10 -
a
3 200 400
50
150 100 125 25 50 75 0
MPP+ (UM)
Figure 3.13 Concentration dependent uptake of MPP+ from the apical side of ARPE-
19 cells. Apical uptake of MPP+ in ARPE-19 cell monolayers showed concentration
dependency with kinetic parameters comparable to those of OCT3 expressed in
various cell systems. The carrier-mediated uptake was derived by subtracting passive
diffusion and surface binding (determined from uptake at 4°C) from the overall
uptake at 37°C. Inset is the Eadie-Hofstee plot, suggesting a one-component
transport mechanism.
8 0
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E. Discussion
A tissue level model of the retinal pigment epithelium, bovine RPE-choroid
explants mounted in Ussing Chamber, has been established in the lab successfully.
After 30-minute equilibration, the tissue showed trans-tissue potential difference (PD)
and resistance (TEER) comparable to reported values in the literature (Table 3.1).
The tissue lasted up to 3 hour in the chamber for permeability experiments. The flux
rate (represented by the slope in the flux over time plot) of mannitol, a paracellular
marker, remained relatively stable (Figure 3.1), suggesting that the intercellular tight
junctions were unchanged. Neutrophil recruitment caused the breakdown of the
blood retinal barrier (Smith et al., 1994) and the blood brain barrier (Bolton et al.,
1998) under inflammation or ischemia conditions. Pretreatment with antineutrophil
attenuated the vascular permeability increase (Dillon and Duran, 1988). Consistent
with these observations, the presence of neutrophil in the bathing medium increased
the permeability of RPE-choroid explants to mannitol.
It is highly probable that RPE cells require a specific combination of integrin
signaling along with soluble signals to remain fully differentiated. Molecular
integrity of the basement membrane is necessary for RPE function in vivo (Mameros
et al., 2004). ARPE-19 cells prolonged cultured on permeable transwell filters coated
with laminin in DMEM/F12 medium supplemented with 1% FBS formed
monolayers with polarized characteristics, including the increase in TEER over
culture time (Figure 3.2), the expression of tight junction protein ZO-l(Figure 3.3
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and Figure 3.4-1), and the polarized secretion of VEGF (Figure 3.6 and Figure 3.7)
and BMP4 (Figure 3.8). It is interesting that the thick coating of Matrigel did not
induce the polarization of ARPE-19 cells. One possible reason is that the RPE cells
tended to grow into the 3-D matrix rather than to hold hands of the neighbor cells in
the richly formulated coating matrix.
It has long been recognized that VEGF-A played a crucial role in the
physiological function and pathogenesis of the retina and the RPE (Adamis and
Shima, 2005). The polarized secretion of VEGF-A from the RPE cells had been
investigated by several labs. Blaauwgeers and coworkers demonstrated, using
primary human RPE culture, that VEGF-A secretion is primarily basolateral
(Blaauwgeers et al., 1999), which was contradicted by results obtained by
Rosenzweig et al. (Slomiany and Rosenzweig, 2004) and Boulan et al. (Marmorstein
et al., 2000) using human D407 RPE cell line and rat RPE-J cells, respectively. Our
results demonstrated that VEGF-A was secreted to both sides of ARPE-19 cells and
preferentially from the apical side (Figure 3.6). The VEGF-A secretion increased at
elevated oxidative stress induced by TBH. It has been shown recently that VEGF-C
and BMP4 may play a role in the pathogenesis of AMD (unpublished data from our
laboratory). Preferential basal secretion of BMP4 and apical secretion of VEGF-C
were detected in the ARPE-19 monolayers. The polarized BMP4 secretion is
consistent with the observation using immunogold EM that on dry form AMD
posterior eye sections embedded in plastic, BMP4 protein was predominantly
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localized to the Bruch’s membrane and the basal infoldings of RPE (unpublished
data from our laborotary).
It has been shown that culture of ARPE-19 cells on PLC membranes resulted
in a more differentiated phenotype and in expression of a specific set of transcripts
encoding protein products that may affect epithelial differentiation, polarity and
survival (Turowski et al., 2004). The rationale behind the use of PLC membrane as
extracellular support was that PLC constituted a readily available physiological
basement membrane, and it resembled Bruch’s membrane in its high portion of
collagen IV. Although ARPE-19 cells cultured on PLC membrane showed higher
TEER and the stronger staining of ZO-1 compared with those cultured on other
matrixes, the differences were not as prominent as expected. It is also noticed that in
small regions sporadically distributed in the monolayers of ARPE-19 cells cultured
on filters, the ARPE-19 cells showed irregular morphology with breaks in ZO-1
staining (Figure 3.3). Similar observations are from Dr. Rizzolo et al. (Luo, 2005). It
is possible that the ARPE-19 cells currently available from ATCC® are less easily
differentiated in response to matrix signals due to their high passage number (20 -
30).
The Na+ -,K+ -ATPase is a key enzyme that regulates the intracellular Na+ and
K+ homeostasis in animal cells (Delamere and Tamiya, 2004; Matsui and Teraoka,
2004; Schneider, 1992). It catalyses an ATP-dependent transport of three sodium
ions out and two potassium ions into the cell per pump cycle, generating a
transmembrane sodium gradient that is crucial for efficient function of other Na+ -
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coupled transporters and provides the primary energy for the uptake or excretion of a
wide variety of solutes by epithelial cells (Katz, 1988; Lingrel and Kuntzweiler,
1994). It has also been demonstrated that the intracellular Na+ and K+ homeostasis
regulated by Na+ -,K+ -ATPase played an important role in the regulation of tight
junction structure and function (Rajasekaran et al., 2001; Rajasekaran et al., 2003).
An interesting feature of RPE cells is the predominant apical localization of Na+ -,
K+ -ATPase (Miller and Edelman, 1990; Quinn and Miller, 1992), which is found in
most other transporting epithelia at the basolateral plasma membrane (Kyte, 1976).
In the prolonged culture of ARPE-19 cells, a light staining of Na+-, K+-ATPase was
identified. The staining, however, did not restrict to the apical membrane. Some
staining was found on basolateral side of the cells as well. The lack of differentiated
distribution of Na+ -, K+ -ATPase was directly reflected by the low PD value (0.6 ±
0.1 mV) across the cell monolayers compared with intact RPE tissues (~ 6 -13 mV).
RT-PCR experiments indicated that OCT3 was the most abundantly expressed
organic cation transporter in the RPE (Figure 3.9). The physiological significance of
OCT3 in RPE might be associated with the catecholamine homeostasis and the
clearance of dopamine from the subretinal space (Rajan et al., 2000). In addition to
mRNA level of evidence, we provided, for the first time, protein level evidence on
the existence of OCT3 in the RPE. hnmunostaining revealed membrane localization
of OCT3 in ARPE-19 cells (Figure 3.10). Functional studies suggested the apical
localization of OCT3 in ARPE-19 cells. The apical MPP+ uptake in ARPE-19 cells
was significantly inhibited by prototypic organic cations, such as TEA, guanidine,
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and dopamine. Decynium-22, the specific inhibitor of OCT3 at micro molar
concentration, also dramatically inhibited apical MPP+ uptake. The organic anion
PAH, the zwitterion L-camitine, or the amino acid L-alanine did not show significant
inhibition. The basolateral MPP+ uptake was not significantly inhibited by TEA or
guanidine at a concentration up to 2.5mM. Together with the observations of
temperature-dependency and directionality (Figure 3.11), the organic cation
transporter is functionally active on the apical side of ARPE-19 cells. The Km value
of 24/dVI is comparable with OCT3-mediated MPP+ transport in various systems
(Martel et al., 2001; Grundemann et al., 1999; Zwart et al., 2001; Wu et al., 1998).
In summary, a tissue level and a culture level RPE model were established in
the lab for the first time. Although no ideal RPE cell culture model has been reported,
prolonged culture of ARPE-19 cells on laminin coated transwell filters represent one
convenient model with good data reproducibility, which is important for studies that
a lot of cells are required. Among the reported organic cation transporters, OCT3 is
the most abundant organic cation transporter in the RPE, and it is functionally active
on the apical side of RPE.
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CHAPTER IV
CHARACTERIZATION OF BRIMONIDINE TRANSPORT
IN RETINAL PIGMENT EPITHELIUM
A. Summary
Purpose. To investigate the involvement of carrier-mediated transport
mechanisms in brimonidine transport in retinal pigment epithelium (RPE).
Methods. The transport of [3 H]brimonidine in bovine RPE-choroid explants
was evaluated in a modified Ussing chamber. The uptake of [3 H]brimonidine was
evaluated in differentiated ARPE-19 cells cultured on permeable Transwell filters.
Results. The transport of brimonidine into (choroidal-to-retinal, inward) and
out of (retinal-to-choroidal, outward) the eye in bovine RPE-choroid explants was
temperature dependent. Both inward and outward brimonidine transport decreased at
5pM as compared to lOnM. The melanin pigmentation of RPE did not significantly
affect tissue permeability at either brimonidine doses. A saturable component was
identified for the inward transport with the apparent Michaelis-Menten constant and
a maximum transport rate of 51 pM and 148 pmol/(cm2-h), respectively. Both apical
and basolateral brimonidine uptake in ARPE-19 cells showed temperature-
dependency. Apical uptake was higher than basolateral uptake at 37°C, and was
decreased to 70% in the presence of sodium azide, or in the absence of extracellular
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Na+. Besides o2-agonists, apical brimonidine uptake was inhibited by verapamil,
desipramine and quinidine, but not by MPP+ (l-methyl-4-phenylpyridinium), TEA
(tetraethylammonium), decynium-22, carnitine, PHA (p-aminohippurate), alanine or
inosine. Basolateral brimonidine uptake increased by 35% at extracellular pH of 6,
and decreased by 50% under cell depolarized condition of high medium K+ and lpM
valinomycin. Temperature-dependent component of basolateral brimonidine uptake
was not saturated at doses up to 2mM.
Conclusions. The transport of brimonidine into the eye in the bovine RPE-
choroid explant model was carrier-mediated. Evidence for the carrier mediated
transport of brimonidine out of the eye was obtained from the apical uptake in
ARPE-19 cells and the outward transport in bovine RPE-choroid explants. These
transport systems in the RPE may play an important role in the modulation of
brimonidine into and out of the eye.
B. Introduction
The retinal pigment epithelium (RPE), constituting the outer blood-retinal
barrier, is a monolayer of cells resting on Bruch’s membrane, and separating the
neural retina from the choroidal blood supply. Like all epithelia, RPE cells exhibit
polarity. The apical side of RPE faces the subretinal space, and exhibits intercellular
tight junctions that retard paracellular diffusion. The ability of RPE to regulate the
vectorial transport of endogenous and exogenous compounds, metabolites, ions and
87
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fluid relies on an asymmetric distribution of proteins. Studies in the past several
years support RPE as a regulatory barrier by showing the expression of several solute
transporters in the RPE, including glucose transporter-1 (GLUT 1) (Kumagai et al.,
1994), P-glycoprotein (P-gp) (Kennedy and Mangini, 2002), multidrug resistance-
associated protein-1 (MRP1) (Aukunuru et al., 2001), organic cation transporter-3
(OCT3) (Rajan et al., 2000), monocarboxylate transporters (MCTs) (Philp et al.,
2003), and organic anion transporter-2(OATP2) (Ito et al., 2002).
Most cases of irreversible blindness result from diseases affecting the
posterior region of the eye. Such diseases include age-related macular degeneration
(AMD), diabetic retinopathy, glaucoma-associated retinopathy, and retinitis
pigmentosa (Geroski and Edelhauser, 2000). Development of non-surgical local
administration methods to deliver drugs to the posterior region of the eye to avoid
systemic side effects or complications due to surgery is thus of great value.
Knowledge about routes taken by drugs to the get to the retina and vitreous after
local delivery and how to improve drug absorption across ocular barriers is crucial.
Brimonidine is an o2-adrenergic agonist approved for the treatment of open
angle glaucoma (Figure 4.1). In addition to the effect of intraocular pressure
reduction, brimonidine was shown to have a neuroprotective function by promoting
retinal ganglion cell survival (Wheeler et al., 2003). In a recent study, it was reported
that topical brimonidine reduces collateral damage caused by laser photocoagulation
for choroidal neovascularization (Ferencz et al., 2005). Due to the existence of
structural and metabolic barriers, topically applied drugs have difficulty in achieving
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therapeutic levels at the back of the eye, especially in the retina and the vitreous.
However, brimonidine demonstrated good ocular distribution after topical
application in pharmacokinetic studies using monkeys and rabbits. Significant
concentrations of brimonidine were found in the posterior tissues of the eye
(Acheampong et al., 2002). Interestingly, after topical application of a single eye
•5
drop to rats H-bnmonidine was found in optic nerves and tracts as well as in corpus
callosum of the brain within 5 minutes but in extremely low levels in blood
(Abdulrazik M, et al. IOVS 2003;44:ARVO E-Abstract 4271). The mechanism of
the rapid absorption remains to be explored.
Drug absorption can be greatly enhanced by targeting endogenously
expressed transport systems (Steffansen et al., 2004). It was shown that brimonidine
was able to inhibit the passage of guanidine, a model substrate of the organic cation
transporter (OCT), through rabbit conjunctival tissue by 70% at a concentration of
0.1 mM (Ueda et al., 2000), indicating a possible role played by the OCT in ocular
brimonidine absorption. In the present study, we investigated whether a carrier-
mediated transport mechanism contributes to the transport of brimonidine in RPE
using bovine RPE-choroid explants mounted in Ussing Chamber. The brimonidine
transport was further characterized in polarized monolayers of ARPE-19 cells, a
human RPE cell line, cultured on transwell filters.
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Figure 4.1 Chemical structure of brimonidine
C. Materials and Methods
1. M aterials
Bovine eyes were obtained from Manning Beef, a local slaughter house,
shortly after the animals were killed for food processing.
ARPE-19 cells (passage 20) were obtained from American Type Culture
Collection (Manassas, VA) and used between passages 22 to 25. Tissue culture-
treated polyester transwells (6.5mm or 12mm in outer diameter and 0.4 pm in pore
size) were purchased from Costar (Coming, NY). Dulbecco’s modified Eagles’s
medium (DMEM) and Ham’s F-12 medium were purchased from Mediatech
(Hemdon, VA). Mouse laminin was from BD Biosciences (Bedford, MA). Other cell
culture reagents and supplies were obtained from Life Technologies (Grand Island,
NY).
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Brimonidine tartrate was a generous gift from Allergan, Inc. (Irvine, CA).
[3 H]Brimonidine (UK-14,304, [imidazolyl-4,5-3 H]-) (81.2Ci/mmol) was purchased
from PerkinElmer Life Sciences (Boston, MA). The other chemicals of analytical
purity were from Sigma.
2. P rep aratio n of bovine-R P E -choroid e x p la n ts
Bovine eyes were kept in an ice bucket during transfer. Typically, 3 to 4
hours elapsed before the eyes were studied in the laboratory. After a brief cleaning of
the connective tissues and the muscles, the eye cup was opened by a circular cut just
posterior to the limbus. The anterior segment and the vitreous were removed. The
posterior eye cup with the sclera, choroid, RPE and the retina was cut into
approximately 1 inch squares. Unlike human eye, bovine RPE contains non-
pigmented areas over the tapetum and tapetum-ffee melanotic areas. Except in the
studies to compare drug transport in pigmented RPE versus non-pigmented RPE,
only the pieces with pigmented RPE, where the choroid layer was thinner, were
selected. The sclera was removed by a combination of sharp and blunt dissection
under a binocular microscope using microscissors and delicate forceps; only a very
small amount of choroid was included. The retina-RPE-choroid preparation was then
placed on filter paper (Whatman #1, particle retention 11 pM), with the retinal side
up. The retina was then carefully peeled off, and the remaining RPE-choroid was
mounted in a clamping chip, and sealed between two chamber halves of a custom
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designed Ussing Chamber with an exposed area (A) of 1.0 cm2 (Kompella et al.,
1993).
The potential difference (PD) and the transepithelial electrical resistance
(TEER) of the tissues were measured using a voltage-clamp device (558C-5;
Bioengineering Department, University of Iowa, IA). In addition to visual
examination, the integrity of the RPE was examined by PD and TEER
measurements. After 30-minute equilibration at 37°C, the tissues maintained the PD
of 6.5 ± 0.6 mV (retinal side positive) and TEER of 234 ± 34 Q -cm2 (n=22), which
were comparable to reported values (Frambach et al., 1989; Joseph and Miller,
1991). The PD and TEER were monitored for the entire duration of the transport
experiment.
3. T ran sp o rt s tu d y in b o vine R P E -choroid e x p la n ts
The Ussing chamber held 6ml bicarbonated Ringer Solution (BRS) (119 mM
NaCl, 3.6 mM KC1, 10 mM glucose, 0.5 mM Na2 H PC>4, 2.5 mM MgS04, 1.2 mM
CaCb, and 23 mM NaHCCh). The solution was gassed with a 5%C02-95% air gas
mixture to yield a pH of 7.4 on both sides of the tissue, and was water-jacketed to
maintain the temperature within the chamber at 4°C or 37°C.
Choroidal-to-retinal (transport in to the eye, inward) or retinal-to-choroidal
(transport out of the eye, outward) flux measurements were initiated by adding a
specified amount of brimonidine traced with [3 H]brimonidine to the choroidal or the
retinal fluid. At predetermined time periods up to 3 hours, a 0.6ml aliquot was
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collected from the receiver fluid for assay of radioactivity in a liquid scintillation
counter (Beckman, Fullerton, CA). Each aliquot removed was immediately replaced
with an equal volume of fresh BRS buffer.
4. ARPE-19 cell m o n o lay ers on p erm eab le tran sw ell filters
Transwell filters (0.4/un pore size, 6.5mm or 12mm diameter, Costar, MA)
were coated with laminin. ARPE-19 cells were seeded at a seeding density of 1.66 x
105 cells/cm2 in DMEM/F-12 culture medium, supplemented with 100 U/ml
penicillin-streptomycin, 2 mM L-glutamine, and 1% FBS. A corresponding amount
of culture medium was added to the basolateral compartment, leveling the height of
the liquid to prevent hydrostatic pressure. The medium was changed twice a week.
The cells were cultured for at least a month to form differentiated monolayers (Dunn
et al., 1996; Holtkamp et al., 1998) Transepithelial electrical resistance (TEER) was
measured once a week using an EVOM™ Epithelial Voltohmmeter (World Precision
Instruments, FL). The differentiation of ARPE-19 cells was also pre-characterized by
staining for Na+ -,K+ -ATPase and the tight junction protein, Zonules Occludin-1.
5. U ptake s tu d ie s by ARPE-19 cell m o n o la y ers
Unless otherwise specified, all uptake experiments were performed in a
humidified atmosphere of 5% CO2 and 95% air at 37°C in buffered BRS solution at
pFI 7.4. After the cell layers were preincubated in BRS for 40 minutes, the uptake
experiments were initiated by spiking the apical or basolateral fluid with a
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predetermined amount of brimonidine or competitive compounds traced with
■ 5
[ HJbrimonidine. After a specified period of time, uptake was terminated by
aspiration of the dosing solution followed by three quick washes of the cell layers in
ice-cold BRS buffer. The cell layers were then solubilized in 0.5ml 1% SDS
solution. Five milliliters of scintillation cocktail was added for quantification of the
radioactivity in a liquid scintillation counter (Beckman, Fullerton, CA).
6. E stim ation of kinetic p a ra m e te rs
Unidirectional flux (J) for brimonidine was estimated from the steady-state
slope of the cumulative amount appearing in the receiver fluid over time. The
apparent permeability coefficient (Pa p p ) was calculated by normalizing the flux
against nominal surface area (1.0cm2 ) and the initial solute concentration. The Pa p p
for the tissue explants (Pap p ,tissu e ) was calculated from the Pa p p for the tissue explants
mounted on filter paper (Pa p p ) and the Pa p p of the filter paper only (Pap p ,filte r) using the
following equation (1). The activation energy (Ea ) of transport was estimated from
the Pa p p at 37°C (Pa p p ,3 7 ) and 4°C (Pa p p ,4 ) according to equation (2), where R is the
universal gas constant.
1/Papp, tis su e — 1/ Papp — 1 / Papp, filter..........(1)
P app,3 7 /Pap p ,4 = exp {-Ea /R[l/(273 + 37) - 1/(273 +4)]}.........(2)
V=VmaxxS/(K m + S ) (3)
To estimate the maximum uptake rate (Vm ax) and Michealis-Menten constant
(Km ), the uptake rate over various dosing concentrations (S) was fitted based on
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equation (3) by the means of nonlinear least squares regression analysis using
KaleidaGraph 3.5 (Synergy Software, Reading, PA). All data were plotted as means
± S.D. and statistical analysis was performed with analysis of variance (ANOVA)
and Student-Newman-Keuls multiple comparison test. The criterion of significant
difference was defined as p<0.05.
D. Results
1. C haracterization o f brim onidine tra n s p o rt in bovine RPE-
choroid e x p la n ts
Directionality and Pigment Effect
The effect of RPE pigment on brimonidine transport was studied at two
substrate concentrations, lOnM and 5pM, which represented the approximate
concentration of brimonidine in vitreous and choroid of cynomolgus monkeys,
respectively, after a single 0.5% topical application (Acheampong et al., 2002). At
lOnM, the Pa p p of brimonidine in the retinal-to-choroidal direction (R-to-C, outward)
in pigmented and non-pigmented RPE-choroid explants was higher, although
statistically insignificant, than those in the choroidal-to-retinal (C-to-R, inward)
direction. Both R-to-C and C-to-R brimonidine transport decreased to a similar
extent at 5uM. RPE pigmentation did not change the tissue permeability for
brimonidine significantly at either concentration (Figure 4.2).
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Pigmented Non-pigmented
1.4E-06
lOnM
H 5uM
1.2E -06
1.0E -06
o 8.0E -07
q. 6.0E -07
4.0 E -0 7
2.0E -07
0.0E + 00 -i
R -to-C C -to-R R -to-C C -to-R
Figure 4.2 The apparent permeability (Pap p ) of brimonidine in pigmented or non-
pigmented bovine-RPE-choroid explants in the direction of retinal-to-choroidal (R-
to-C) or choroidal-to-retinal (C-to-R). All experiments were conducted in the
presence of tracer alone (0.81 pCi/ml (lOnM) [3 H]brimonidine) (A) or with 5pM
unlabeled brimonidine(B). Data represent mean ± S.D.; n = 3.
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Temperature Dependency
The transport of brimonidine across bovine RPE-choroid explants mounted in
Ussing chamber in the C-to-R direction and the R-to-C direction has a profile of
pseudo steady-state characteristics with no evident lag time. The transport of lOnM
brimonidine showed temperature dependency (Figure 4.3). When the experimental
temperature was lowered to 4°C, the Pa p p for lOnM brimonidine decreased to 20.6%
(Figure 4.3A) and 58.4% (Figure 4.3B) of the Pa p p at 37°C in the R-to-C and C-to-R
direction, respectively. The activation energy (Ea ) of brimonidine transport was
calculated to be 8.2 kcal/mole and 2.8 kcal/mole in the R-to-C direction and C-to-R
direction, respectively.
Concentration Dependency
As shown in Figure 4.4, the initial rate of C-to-R brimonidine flux across
bovine RPE-choroid explants followed a saturable function of substrate
concentration over the range from 1 to 100 pM, with the apparent Michaelis-Menten
constant and the maximal flux rate estimated to be 51 ± 17 pM and 148 ± 22
pmole/(cm -h), respectively.
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(A )
£ 0.06
♦ 37C
S 0.04
o
(B )
20 40 60
tim e (min)
80 100 120
_ 1.0E-06
o 0.05
* 0.04
S
o 0.03
60
Time (min)
120
Figure 4.3 Retinal-to-choroidal (R-to-C) (A) and choroidal-to-retinal (C-to-R)
brimonidine transport (B) showed temperature dependency. [3 H]brimonidine at
0.81uCi/ml (lOnM) was dosed from the retinal or choroidal side of bovine RPE-
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choroid explants. Insets show the Pa p p of R-to-C and C-to-R transport of lOnM
brimonidine at 37°C and 4°C. n = 3-4. Data represent mean ± S.D.
■ total flux
350
— - passive diffusion
■ Saturable component (total flux -
passive diffusion
300
CM
E 250
q
s i
200
O
E
Q .
150
X
3
100
50
80 90 100 10 20 30 40 50 60 70 0
brimonidine (uM)
Figure 4.4 Total choroidal-to-retinal (C-to-R) brimonidine flux as a function of
brimonidine concentration. All experiments were conducted in the presence of
0.81pCi/ml (lOnM) [3 H]brimonidine and 1 to 100pM unlabeled brimonidine. The
linear component of brimonidine flux contributed by passive diffusion was measured
at 4°C. The apparent Michaelis-Menten constant and the maximal flux of the satiable
component contributed by carrier-mediated transport was estimated to be 51 ± 17
pM and 148 ± 22 pmole/(cm2.h), respectively. Data represent mean ± S.D.; n = 3-4.
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2. Characterization of brim onidine transport in ARPE-19 cells
cultured on transwell filters
Directionality and Temperature Dependency
Apical uptake of 20nM [3 H]brimonidine (1.92 pCi/ml) in ARPE-19 cells was
50% higher than basolateral uptake in 5 minutes (Figure 4.5). The difference was
abolished when uptake temperature was lowered to 4°C. About 30% and 50%
brimonidine uptake from the apical or basolateral side, respectively, was contributed
by passive diffusion or surface binding (4°C).
Time Course
Apical and basolateral uptake of 20nM [3 H]brimonidine (1.92 pCi/ml) was
determined in ARPE-19 cells cultured on permeable supports. As shown in Figure
4.6, brimonidine uptake by ARPE-19 cells was rapid over the initial 5 minutes with
faster uptake from the apical side than the basolateral side, and reached equilibration
within 15 minutes.
Energy Dependency
To determine the possible driving force of the carrier-mediated transport
mechanism, the apical and basolateral uptake of 20nM brimonidine was investigated
under conditions of 1) low metabolic energy; 2) without extracellular Na+; 3)
variation in membrane potential; and 4) variation in extracellular proton
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concentration. As shown in Table 1, apical and basolateral brimonidine uptake
exhibited different profiles in response to the above modulations.
p = 0.0002
0.09
0.08
0.07
1 0.06
in
| 0.05
f 0.04
J X .
(5 _ _ _
■ K 0.03
0.02
0.01
4 degree
37 degree
p = 0.007
Apical Basolateral
2 •
Figure 4.5 Temperature-dependent and directional uptake of 20nM [ H]bnmonidine
(1.92|iCi/ml) in ARPE-19 cell monolayers. ARPE-19 cells were cultured on
transwell filters (12mm in diameter and 0.4 um in pore size). Data represent mean ±
S.E.M.; n = 3.
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0.035 n
0.030 -
0.025 -
o
E 0.020 -
CL
O
* 0.015 -
0.010 - ♦ Apical
■ B asolateral
0.005 -
0.000
0 2 4 6 8 10 12 14 16
T im e (m in)
Figure 4.6 Time-dependent uptake of 20nM [3 H]brimonidine in ARPE-19 cell
monolayers. ARPE-19 cells were cultured on transwell filters (6.5mm in diameter,
0.4 um in pore size).
The apical uptake of brimonidine decreased by 30% when the metabolic
energy of ARPE-19 cells was lowered by 30min-pretreatment with lOmM NaN3, an
inhibitor of oxidative phosphorylation. A decrease of equal magnitude was observed
if extracellular Na+ was replaced by N-methyl-D-glucamine (NMDG) or by choline
on the basis of iso-osmolarity. The apical uptake of brimonidine was sensitive to the
changes in extracellular pH with slower uptake in an acidic environment. However,
it was not affected by the elimination of transmembrane proton gradient by 0.5pM
FCCP, a proton ionophore. Changes in membrane potential did not show a
significant effect on apical brimonidine uptake in ARPE-19 cell monolayers.
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The basolateral uptake of brimonidine was not significantly decreased by
decreasing metabolic energy state via NaN3 pretreatment (p = 0.23) (Table 1). The
basolateral uptake decreased moderately (16%) when the extracellular Na+ was
replaced by choline, but no decrease was observed when Na+ was substituted by
NMDG. Basolateral brimonidine uptake decreased significantly (by 50%) when
ARPE-19 cells were depolarized in high extracellular potassium (143mM) and luM
valinomycin. Lowering extracellular pH to 6 increased basolateral brimonidine
uptake by 35%. Similar to the apical uptake, basolateral uptake was not affected by
the elimination of transmembrane proton gradient using 0.5pM FCCP.
Parameter Conditions Uptake (% of control)
Apical Basolateral
Metabolic Energy lOmMNaNs
3 Omin-pretreatment
70.7 ± 4.9'
86.7 ±9.8
Extracellular Na+ replaced by NMDG
replaced by choline
64.9 ± 4.5*
72.3 ± 6.4*
102.0 ±6.2
84.2 ± 5.9*
Membrane Potential + 1 [iM valinomycin
[K+ ] = 143mM
+ l/zM valinomycin
([K+ ] = 143mM)
111.8 ± 8.0
98.2 ±6.0
88.2 ± 12.9
77.8 ± 9.4
76.5 ± 9.4
49.1 ±8.4*
Proton gradient pH = 6
pH = 8.5
+ 0.5 /jlM FCCP (pH = 7.4)
67.1 ±5.0*
109.3 ±3.4*
97.5 ± 5.4
134.6 ± 10.5*
104.4 ±9.7
98.7 ±4.3
NMDG: N-methyl-D-glucamine
Table 4.1 Energy-dependency brimonidine uptake from the apical side or the
basolateral side of ARPE-19 cell monolayers. The experiments were carried out as
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described in the Materials & Methods. Apical or basolateral uptake was measured at
37°C for 3 minutes. The amount of [3 H]brimonidine was 20nM (1.39/iCi/ml). In the
control experiments, BRS at pH 7.4 was used as uptake buffer. The carrier mediated
uptake was calculated by subtracting from uptake at 37°C by that at 4°C. Data
represent mean ± S.E.M.; n = 6. The asterisks (*) denotes significant differences
from control values (p < 0.05).
Substrate Specificity
The effect of various compounds added to the apical dosing solution on
brimonidine uptake is shown in Figure 4.7. The uptake was carried for 3 minutes to
maintain the “sink” condition. The uptake of the carrier mediated process was
estimated by subtracting the uptake at 4°C in the presence of inhibitors from that at
37°C to eliminate surface binding. In addition to other o2-receptor agonists, the
compounds tested comprised cationic or anionic compounds that are substrates or
inhibitors of various known transport systems: the membrane potential-dependent
organic cation transporters (TEA, MPP, verapamil and decynium-22), the pH-
dependent carnitine/organic cation transporters (carnitine), the ATP-dependent
organic anion transporters (PHA), the Na+ -dependent amino acid transporters
(alanine), the Na+ -dependent nucleoside transporter (inosine), the Na+-dependent
neurotransmitter transporter (MPP+ and desipramine). Because -50% of brimonidine
exists as cation at physiological pH, more cation inhibitors were tested in addition to
anions and zwitterions.
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As shown in Figure 4.7, apical brimonidine uptake was inhibited by 02-
receptor agonists, including brimonidine (51% at 0.5mM), clonidine (34% at
0.5mM), and oxymetazoline (55% at ImM). It was not inhibited by TEA, MPP+ or
decynium-22, substrates or inhibitors of the organic cation transporters (OCT), but
was significantly inhibited by other cationic compounds such as verapamil (64% at
500(iM), quinidine (61% at 250pM) and desipramine (85% at ImM). No significant
inhibition of uptake was observed for other compounds tested. Basolateral uptake of
brimonidine decreased by a similar magnitude at 37°C and 4°C as substrate
concentration increased from 0.02qM up to 2mM (Figure 4.8).
To further study the potency of various inhibitors especially the o2-agonists,
the inhibition pattern of apical brimonidine uptake over a range of concentrations
(20nM to 5mM) of verapamil, clonidine, oxymetazoline and unlabeled brimonidine
was investigated and the concentrations for 50% inhibition (K;) was estimated to be
976pM, 1427pM, and 808pM, 351pM, respectively (Figure 4.9).
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120 - i
Figure 4.7. Inhibition profile of apical uptake of brimonidine in ARPE-19 cell
monolayers. The uptake of 1.62pCi/ml (20nM) [3 H]brimonidine was measured in the
presence or absence of various inhibitors. The inhibitors of characterized transport
systems were dosed at concentrations commonly used to achieve sufficient inhibition
in heterologous systems. 1, dosed at 500pM; 2, dosed at ImM; 3, dosed at 2.5mM; 4,
dosed at lOpM; 5, dosed at 250pM. The bars represent mean ± S.D., n = 3. The
asterisks (*) denote statistically significant differences (p < 0.05) from control value,
without inhibitors.
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37 degree
4 degree
0.020
= 5 0.015
0.010
o
® 0.005
ns
0.2 2 20 200
Unlabeled brimonidine concentration (uM)
2000
Figure 4.8. Effect of increasing concentration of unlabeled substrate on basolateral
brimonidine uptake at 37°C and 4°C. The experiment was carried out as described in
Materials & Methods. The bars represent mean ± S.D.; n = 3.
107
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140 i
♦ brim onidine
• clonidine
A oxymetazoline
■ verapamil
120
100
a >
m
a
3
< D
C
j u
'c
o
£
n
■ 3 1 -6 -4 ■ 2 ■ 8 ■ 7 ■ 5
Log [inhibitor concentration (M )]
Figure 4.9. Dose-dependent inhibition of apical brimonidine (1.62pCi/ml, 20nM)
uptake in ARPE-19 cells by verapamil(a), unlabeled brimonidine(4), clonidine(«)
and oxymetazoline(A). The concentrations for 50% inhibition (K j) was estimated to
be 351pM, 808pM, 976pM, and 1427pM for verapamil, brimonidine, clonidine and
oxymetazolin, respectively. The bars represent mean ± S.D., n = 4.
E. Discussion
The blood retinal barrier (BRB) consists of two major components, the
endothelium of retinal blood vessels (inner BRB) and the retinal pigment epithelium
(outer BRB) (Cunha-Vaz, 2004). The retinal blood vessels closely resembling the
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blood-brain-barrier constitute the local blood circulation system for the inner retina,
while the RPE controls penetration of nutrients, metabolic wastes and xenobiotics
from choroid blood supply to the photoreceptor layer of retina that is completely
avascular (Duvvuri et al., 2003). Although the inner and outer BRB cooperate to
create a “privileged” environment in the retina and vitreous, it is indicated that the
outer BRB constitutes the major absorption barrier for trans-scleral (Barza, 1989) or
topically (Maurice, 2002) administrated drugs, especially when low concentration of
drug enters the blood stream.
In the present study, we have presented evidence for the existence of a
carrier-mediated system facilitating the inward (C-to-R) transport of brimonidine
across the outer blood retinal barrier using bovine RPE-choroid-explants. RPE-
choroid explants mounted in two-compartment chambers are commonly used as a
tissue-level model to study the permeability of the outer blood-retina barrier
(Hillenkamp et al., 2004; Kimura et al., 1996; Steuer et al., 2004; To and Hodson,
1998). In this model, most resistance to solute is from the RPE. The influence from
choroid blood flow is excluded, and a contribution from the porous choroid in the
explants is minimal. The integrity of the tissue was monitored by measuring the
TEER and PD throughout the transport experiments. Brimonidine was found to
penetrate through the bovine-RPE-choroid explants rapidly with no obvious lag time.
About a 2-fold difference was observed between C-to-R brimonidine transport at
37°C and 4°C (inset, Figure 4.3), indicating the existence of a carrier-mediated
mechanism, which was further confirmed by the identification of a saturable
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transport component with high affinity (Km of 51 ± 17 pM) (Figure 4.4).
Pharmacokinetic studies in monkeys (Acheampong et al., 2002) showed that after a
single eye drop brimonidine concentration in the sclera (e.g. Cm a x = 6.67-8.19 pg-
Eq/g) was several times more than that in the choroid/retina (e.g. C m aX = 1.84 pg-
Eq/g) and hundreds of times higher than that in the vitreous (e.g. C m aX = 0.011 pg-
Eq/g), indicating a net inward transport of brimonidine at the back of the eye across
the RPE barrier to the therapeutic site under a concentration gradient. If an
intermediate concentration was used to roughly represent drug concentration in
choroid, it can be estimated from our results (Figure 4.4) that, at 5-10pM, more than
50% of the inward brimonidine transport across the RPE barrier might be contributed
by the carrier-mediated transport mechanism.
Brimonidine is known to bind reversibly to ocular melanin with high affinity
(Acheampong et al., 1995). In the present study we studied brimonidine transport in
pigmented and non-pigmented bovine RPE-choroid explants at two concentrations,
lOnM (close to drug concentration in vitreous) and 5pM (close to drug concentration
in choroid). No difference in Pa p p was observed. Although binding with melanin
affects the disposition and retention of brimonidine in RPE, our data indicated an
insignificant effect of melanin on in vitro brimonidine transport through the RPE.
Appropriate RPE function relies highly on the maintainance of its polarity. In
the past decade, the expression, distribution and activity of membrane solute
transporters or ion-channels on the RPE have drawn increased attention. In addition
to establishing the physiologic function of the endogenous transport systems in RPE,
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it is important to also study how they affect the absorption, disposition and
elimination of drugs that are recognized as their substrates. In the present study, we
also investigated whether the active brimonidine transport is mediated by established
nutrient transporters in polarized ARPE-19 cells. ARPE-19, a commercially
available human RPE cell line, forms polarized monolayers after prolonged culture (
> 4 weeks) on permeable support (Dunn et al., 1996), and has been selected as a
model of polarized RPE in studies of polarized transport (Bridges et al., 2002),
polarized protein expression/secretion (Philp et al., 2003; Holtkamp et al., 1998;
Narayan et al., 2003) and barrier breakdown (Abe et al., 2003). Consistent with
tissue-level observations in bovine RPE-choroid explants, uptake studies in ARPE-
19 cells also confirmed the existence of a carrier-mediated process for brimonidine
transport in RPE. Furthermore, similar to the finding of a higher outward
brimonidine transport as compared to the inward transport in RPE-choroid explants,
a carrier-mediated uptake process was primarily located on the apical side of ARPE-
19 cells. Support for this finding came from results on temperature-dependency
(Figure 4.4), directionality of uptake (Figure 4.5 and 4.6), energy-dependency (Table
4.1), substrate specificity (Figure 4.7) and concentration-dependency (Figure 4.9).
Energy dependency studies indicated that the carrier-mediated brimonidine
transport system might be composed of two processes, an active transport process
that was driven by Na+ gradient and a facilitative transport process that was driven
by substrate concentration gradient. Even though acidic bathing medium lowered
apical brimonidine uptake, this decrease was abolished by FCCP, a proton
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ionophore. This suggested that rather than decreasing transmembrane proton
gradient, the decrease in uptake was more likely due to increasing the amount of
ionized brimonidine with a pKa value of 7.4, because the non-protonated
brimonidine can penetrate the lipophilic cell membrane more readily via passive
diffusion, and the amount of non-protonated brimonidine significantly decreased in
acidic pH.
The existence of Na+ -dependent as well as facilitative brimonidine transport
processes on the apical side of RPE may provide a valid explanation for the
observation that the outward brimonidine transport was more efficient than the
inward transport, although brimonidine transport in both directions showed carried-
mediated characteristics. One feature of facilitative transporters, namely bidirectional
movement, may apply for brimonidine. In the in vitro model of bovine RPE-choroid
explants, transport in either direction can be enhanced based on the concentration
gradient created after dosing. Under in vivo conditions, this concentration gradient
across the RPE will depend on various factors including the modality of
administration, the time period post-administration, and the efficacy of drug
elimination by choroidal blood flow.
Basolateral brimonidine uptake in ARPE-19 cells was about 30% higher
when extracelluar pH was lowered from 7.4 to 6. As a consequence, the
protonated/non-protonated brimonidine was estimated to change from 1:1 to 25:1 by
Henderson-Hasselbalch equation. In addition, at pH 7.4, brimonidine uptake
decreased to about 50% when ARPE-19 cells were depolarized with high K+ medium
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containing valinomycin (Table 1). These results showed that protonated brimonidine
might be more readily transported than the non-charged form from the basolateral
side of ARPE-19 cells. Basolateral brimonidine uptake showed temperature
dependency (Figure 4.5). However, the temperature dependent uptake (37°C - 4°C)
of brimonidine was not saturable at concentrations up to 2mM (Figure 4.8).
Basolateral brimonidine uptake, taken together, might be carrier-mediated but via a
low affinity process, which merits further investigation.
The existence of o2-adrenergic receptors on the apical side of RPE cells was
indicated by receptor stimulation studies (Dearry and Burnside, 1988). However, the
apical carrier-mediated transport was more likely via a membrane transporter than
via receptor-mediated endocytosis. This can be supported by several lines of
evidence: 1) brimonidine was introduced at a concentration 10 times higher than its
EC5 0 (2nM) to activate the o2-receptors, indicating the saturation of exposed binding
site; 2) besides o2-adrenergic agonists, brimonidine transport was inhibited by
verapamil with even higher potency (Figure 4.9); 3) Brimonidine uptake is sensitive
to the existence of extracellular Na+ ; 4) although clonidine was much more potent to
induce the o2-adrenergic endocytosis (Olli-Lahdesmaki et al., 2004), it was a less
potent inhibitor of brimonidine uptake (Figure 4.9).
Brimonidine inhibited the inward active transport of guanidine, an model
substrate of the organic cation transporters in rabbit conjunctiva (Ueda et al., 2000).
Organic cation transporter-3 (OCT3), also recognized as the extraneuronal
monoamine transporter, was found in the RPE (Rajan et al., 2000), and was further
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identified primarily on the apical side by immunofluorescent staining and monitoring
MPP+ uptake (Zhang N, et al, AAPS 2004; abstract R6240). Hence, we specifically
explored the involvement of organic cation transporters in brimonidine uptake.
Neither MPP+, TEA and decynium-22, inhibitors of the organic cation transporters,
nor carnitine, an endogenous substrate of the pH-dependent organic cation/carnitine
transporter, inhibited apical brimonidine uptake at a concentration as high as 2.5mM.
These results indicated that brimonidine uptake in the RPE was not via the cloned
organic cation transporters. Other tested cationic compounds including verapamil,
desipramine and quinidine inhibited brimonidine uptake significantly. Interestingly,
unlike the organic cation transporters and the neurotransmitter transporters (Olivier
et al., 2000) which transport many primary and the permanently charged quaternary
amines, the brimonidine transport system in ARPE-19 showed specificity to tertiary
or heterocyclic amines with sp2-hybridized nitrogen. This inhibition profile
resembles that of the “novel cationic drug transporter” functionally characterized in
RPE cells (Han et al., 2001), which recognized verapamil, quinidine and several (3 -
blockers, but not TEA, MPP+ or carnitine. Substrates of several other transporters
(organic anion transporter, amino acid transporter and nucleoside transporter)
showed no inhibition of brimonidine uptake.
In conclusion, based on our observations in both bovine RPE-choroid
explants and polarized ARPE-19 cells, we demonstrated a carrier-mediated transport
process for brimonidine in RPE. This transport system may play a significant role in
modulating the movement of brimonidine into and out of the eye.
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CHAPTER V
CONCLUSIONS AND FUTURE DIRECTIONS
A. Conclusions
The conjunctival epithelium and the retinal pigment epithelium (RPE)
constitute two major barriers in the conjunctival/scleral pathway for topically
administered ophthalmic drug to reach the posterior segment of the eye. Organic
cation transport system was identified and localized on the apical side of the rabbit
conjunctival epithelial cells. The molecular identity of the transporter might be
OCT3. The potential and versatility of this transporter to mediate transport of a
variety of cationic drug categories suggest its importance in topical drug delivery, as
well as ocular surface-related diseases. Together with other membrane transporters
or efflux pumps, OCT3 constitute the organic cation transport system in the
conjunctival epithelium, as illustrated in Figure 5.1 A.
To study the permeability of the RPE, also known as the outer blood retinal
barrier, a tissue level and a culture level RPE model were successfully established in
the lab for the first time. Bovine RPE-choroid explants mounted in Ussing Chamber
showed electrophysiology parameters comparable to similar systems established by
several other labs. The tissue lasted in the chamber for up to 3 hours as indicated by
the mannitol flux. Although no ideal RPE cell culture model has been reported,
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prolonged culture of ARPE-19 cells on laminin coated transwell filters represent one
convenient model with polarized properties. It is valuable for studies in which a large
number of RPE cells are needed.
Among the reported organic cation transporters, OCT3 is the most abundant
organic cation transporter in the RPE, and it is functionally active on the apical side
of RPE (Figure IB). OCT3, on both major epithelium barriers in the
conjunctiva/sclera pathway may serve as a conduct for topically applied cationic
drugs to reach the posterior region of the eye. The availability of topically applied
drugs might be greatly improved by targeting OCT3 via rational drug design or
prodrug approaches.
It has been reported that a significant amount of brimonidine reached the
retina and the vitreous after ocular topical application. Based on our observations in
both bovine RPE-choroid explants and polarized ARPE-19 cells, we demonstrated a
carrier-mediated transport process for brimonidine in RPE. This transport system
may play a significant role in modulating the movement of brimonidine into and out
of the eye. Other than OCT3, functional characteristics of brimonidine transport
suggested the involvement of a novel organic cation transporter that merit further
investigation.
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(A)
Basolateral
Apical
M em brane
M em brane
ATP
ATP
ADP
ADP
Substantia Propria
(loose vascular
supporting tissue)
T ear Fluid
N a 4 "
oc-
2CI
-70 mV
Carnitine
Na+
N a4
pH = 7.4
pH = 5.2 to 8.6
Conjunctival epithelial cells
(B)
Apical
Membrane
Basolateral
Membrane
ATP
ATP
ADP
O C
ADP
Fenestrated
Choriocapillaris
Neural Retina
2CI
Cl'
Retinal pigment epithelial cells
Figure 5.1 Schematic diagram of organic cation transport system in the conjunctival
epithelial cells (A) and the retinal pigment epithelium (B).
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B. Future directions
1. Regulation of organic cation tran sp o rt in the conjunctiva and
the RPE
Same as many transport systems, the organic cation transporters are tightly
regulated by multiple mechanisms. Short-term of OCTs regulation mainly via protein
phosphorylation/dephosphorylation has been studied using isolated proximal tubules
from different species and cell culture systems expressing different OCT isoforms
(Ciarimboli and Schlatter, 2005). OCT3 has multiple potential phosphorylation sites
existed intracellularly. The regulation of the human OCT3 (hOCT3) has been studied
by measuring [3 H]MPP+ uptake in HEK293 cells stably transfected with hOCT3
(Martel et al., 2001). Activation of PKA PKG or PKC does not affect [3 H]MPP
uptake specifically, implying that PKA, PKC or PKG do not play an significant role
in the regulation of hOCT3. Incubation with inhibitors of protein tyrosine
phosphatase or alkaline phosphatase, but not protein serine/threonine phosphatase
reduces [ HJMPP uptake, while inhibitors of exogenous alkaline phosphatase
increases it. This suggests that hOCT3 is active in the dephosphorylated state.
Inhibition of mitogen-activated protein kinase (MAPK) by PD98059 and by
SB203580 significantly reduces [3 H]MPP uptake. Incubation with calmidazolium,
trifluoperazine, (inhibitors of calmodulin (CaM)), and KN62 (an inhibitor of
Calmodulin-Dependent Protein Kinase II (CaMKII)) leads to a concentration-
• 5
dependent inhibition of [ HJMPP uptake. The role of phosphodiesterase (PDE) in the
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-3
regulation of hOCT3-mediated transport of [ HJMPP has also been investigated.
Incubation of the cells with PDE inhibitors causes significant inhibition of [ HJMPP
uptake. Pretreatment with IBMX, a PDE inhibitor, significantly reduces Vm S L X , but not
the Km , for [3 H]MPP uptake, suggesting that the regulatory effects of IBMX are
mediated through a decrease in the number and/or activity of the hOCT3 carriers but
not through changes in their affinity.
Only few studies have been performed on the long-term regulation of OCT3.
Forbes et al. demonstrated that the expression of OCT1, OCT2 and OCT3 decreased
in diabetic rats induced with streptozotocin (32 weeks) (Thomas et al., 2004), which
may be associated with impaired renal clearance in diabetic nephropathy. It is also
shown that OCT3 mRNA level was decreased in kidney, brain and lung of
metamphetamine sensitised rats, while that of OCT1, OCT2 and OCTN2 remained
unchanged (Kitaichi et al., 2003). This down-regulation of OCT3 in sensitised rats
might be crucial for changes in the renal excretion, brain penetration and tissue
distribution of metamphetamine.
To completely understand regulation of the organic cation transport system in
the ocular barriers, it will be necessary to identify both endogenous and exogenous
factors that regulate protein function and expression. Regulation of organic cation
transport system can be investigated in vitro. Such information is valuable for drug
absorption or extrusion in vivo. If we could identify means to up-regulate apical and
basolateral organic cation transporters in the ocular barriers, we could eventually be
able to enhance in vivo absorption of topical applied ocular drugs.
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2. In vitro / in vivo ocular drug delivery targeting OCT3
The presence of the intestinal dipeptide transporter is responsible for
improvement in vivo bioavailability of various peptidomimetics drugs such as the /3 -
lactam antibiotics (Sai et al., 1996), angiotensin-converting enzyme inhibitors (Zhu
et al., 2000) and the antitumor drug bestatin (Saito et al., 1996). Moreover, this
transporter has been specifically targeted for improved bioavailability of drugs such
as zidovudine and acyclovir through prodrug derivatization (Han et al., 1998).
The expression of organic cation transporters in the conjunctival epithelium
and the retinal pigment epithelium is expected to improve the permeability of
cationic drugs across the epithelia as the drugs have limited access of the paracellular
route for transport. If the expression level and functional capacity of the transporters
allow desired level of bioavailability for topically applied ophthalmic drugs, topical
drug delivery to the posterior region of the eye could be a reality. Topical ocular
delivery would be non-invasive with limited systemic side effects. It would be
interesting to conduct in vivo studies in order to determine if the presence of the
organic cation transporter proteins contributes significantly to the improved
bioavailability in posterior ocular region or the elimination of cationic drugs from
ocular tissue. Radio-labeled MPP+ or TEA can be formulated as eye drops and
administer in the conjunctiva cal-de-sac of animals. Drug concentration in the retina
and the vitreous should be monitored to estimate drug absorption rate and extent.
Ocular drug absorption for compounds with similar physicochemical properties such
as LogP and molecular size with MPP+ and TEA but do not belong to the organic
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cation compound family should be measured in the same manner for comparison.
To determine ocular drug absorption from circulation, radio-labeled MPP+ or TEA
can be administered to animals through intravenous injection and the retina and
vitreous should be isolated at different time periods to measure drug absorption.
3. The presence of novel organic cation transporters
So far, six organic cation transporters have been cloned (OCT 1-3 and
OCTN1-3). In addition to the organic cation transporters, several transporters have
been reported recently to be able to transport certain types of organic cations. These
transporters include OAT1, OAT3, and URAT1 (Enomoto et al., 2002), which also
belongs to the SLC22A family and may operate as anion/anion exchangers in
parallel with the organic cation transporters in the renal excretion cimetidine (Cha et
al., 2001), the transporters from the OATP (organic anion-transporting polypeptide)
family; the MRPs (the multi-drug resistance proteins) and the ABC transporter P-
glycoprotein (Pg-P). Substrates of OATPs are mainly type II organic cations, that are
usually bulkier (generally >500 MW; e.g., daunomycin) and frequently polyvalent
(Meijer et al., 1990; Hagenbuch and Meier, 2003). A number of cationic drugs that
are substrates of OCT1 and OCT2 are substrates of P-gp or MRP (Kruh and
Belinsky, 2003), Such as quinidine, verapamil, cimetidine, debrisoquine, acyclovir,
and ganciclovir (Sakaeda et al., 2002). Organic cation transport via uncharacterized
transporters was reported in various systems (Han et al., 2001; Wright, 2005; Fukada
et al., 2002; Shen et al., 1999). The transport properties of brimonidine in RPE also
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indicated the involvement of a novel transporter that may take bulky (type II) organic
cations as substrates. It is interesting to investigate its physiological importance and
it is conceivable that this novel transporter may play a role in the deposition of other
cationic drugs that share same structure motifs as brimonidine.
Following the expression cloning of the first organic cation transporter
(OCT1) in 1994 (Grundemann et al., 1994), and the subtraction cloning of OCTN1
in 1997 (Tamai et al., 1997), additional organic cation transporters were cloning via
homology cloning. For transporters with distinct functional characterizations, they
might not share sufficient sequence homology with the cloned organic cation
transporters for homology cloning. Cloning of such transporters therefore require the
expression cloning approach to screen for organic cation transport activities.
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Creator Zhang, Ning (author)

Core Title Drug delivery to the posterior region of the eye: Characterization of organic cation transporters

School Graduate School

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Degree Program Pharmaceutical Sciences

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Advisor Hinton, David (committee chair), Lee, Vincent H.L. (committee chair), Farley, Robert (committee member), Kannan, Ram (committee member), Okamoto, Curtis T. (committee member), Yang, Austin (committee member)

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