Hypoxia Alters Ocular Drug Transporter Expression and Activity in Rat

Apr 22, 2013 - Department of Medicine, National Jewish Health, Denver, Colorado 80206, United States. Mol. ... *Department of Pharmaceutical Sciences,...
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Hypoxia Alters Ocular Drug Transporter Expression and Activity in Rat and Calf Models: Implications for Drug Delivery Rajendra S. Kadam,† Preveen Ramamoorthy,§ Daniel J. LaFlamme,§ Timothy A. McKinsey,‡ and Uday B. Kompella*,† †

Pharmaceutical Sciences and Ophthalmology and ‡Division of Cardiology and Medicine, University of Colorado, Anschutz Medical Campus, Aurora, Colorado 80045, United States § Department of Medicine, National Jewish Health, Denver, Colorado 80206, United States ABSTRACT: Chronic hypoxia, a key stimulus for neovascularization, has been implicated in the pathology of proliferative diabetic retinopathy, retinopathy of prematurity, and wet age related macular degeneration. The aim of the present study was to determine the effect of chronic hypoxia on drug transporter mRNA expression and activity in ocular barriers. Sprague−Dawley rats were exposed to hypobaric hypoxia (PB = 380 mmHg) for 6 weeks, and neonatal calves were maintained under hypobaric hypoxia (PB = 445 mmHg) for 2 weeks. Age matched controls for rats, and calves were maintained at ambient altitude and normoxia. The effect of hypoxia on transporter expression was analyzed by qRT-PCR analysis of transporter mRNA expression in hypoxic and control rat choroid-retina. The effect of hypoxia on the activity of PEPT, OCT, ATB0+, and MCT transporters was evaluated using in vitro transport studies of model transporter substrates across calf cornea and sclera-choroid-RPE (SCRPE). Quantitative gene expression analysis of 84 transporters in rat choroid-retina showed that 29 transporter genes were up regulated or down regulated by ≥1.5-fold in hypoxia. Nine ATP binding cassette (ABC) families of efflux transporters including MRP3, MRP4, MRP5, MRP6, MRP7, Abca17, Abc2, Abc3, and RGD1562128 were up-regulated. For solute carrier family transporters, 11 transporters including SLC10a1, SLC16a3, SLC22a7, SLC22a8, SLC29a1, SLC29a2, SLC2a1, SLC3a2, SLC5a4, SLC7a11, and SLC7a4 were up regulated, while 4 transporters including SLC22a2, SLC22a9, SLC28a1, and SLC7a9 were down-regulated in hypoxia. Of the three aquaporin (Aqp) water channels, Aqp-9 was down-regulated, and Aqp-1 was up-regulated during hypoxia. Gene expression analysis showed down regulation of OCT-1, OCT-2, and ATB0+ and up regulation of MCT-3 in hypoxic rat choroid-retina, without any effect on the expression of PEPT-1 and PEPT-2. Functional activity assays of PEPT, OCT, ATB0+, and MCT transporters in calf ocular tissues showed that PEPT, OCT, and ATB0+ functional activity was down-regulated, whereas MCT functional activity was up-regulated in hypoxic cornea and SCRPE. Gene expression analysis of these transporters in rat tissues was consistent with the functional transport assays except for PEPT transporters. Chronic hypoxia results in significant alterations in the mRNA expression and functional activity of solute transporters in ocular tissues. KEYWORDS: hypoxia, drug transporters, ocular, blood-retinal barrier

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INTRODUCTION The retina is a metabolically active tissue and needs large amounts of nutrients to produce metabolic energy for phototransduction and neuro-transduction.1 As an extension of brain, the retina is protected by inner and outer blood retinal barriers (BRB) to maintain its controlled environment. The BRB, comprising retinal capillary endothelial cells (inner BRB) and retinal pigmented epithelial cells (RPE; outer BRB), restricts nonspecific transport of solutes from the blood to the retina.2 Metabolic substrates such as glucose and amino acids are hydrophilic, and their passive permeability is restricted by BRB. BRB expresses various nutrient and neurotransmitter transporters to allow their selective entry into the retina.3 Expressions of these transporters in BRB may be altered during chronic hypoxia, which is known to contribute to the neovascular events during age related macular degeneration (AMD), diabetic retinopathy, and retinopathy of prematurity (ROP).4,5 © XXXX American Chemical Society

Hypoxia can influence the expression and functional activity of solute carrier transporters in biological tissues, thereby contributing to the disease pathology. Hypoxia elevates retinal levels of glucose, a casual factor for the development of diabetic retinopathy.6 Hypoxia results in increased expression of glucose transporters that are responsible for increased glucose uptake.7 In pregnant women, placental hypoxia is considered as an underlying cause for fetal growth restriction, preeclampsia, and diabetes.8 Hypoxia results in reduced expression and functional activity of amino acid and glucose transporters in placental barriers.9−11 Hypoxia also alters the expression and functional activity of transporters in kidney, liver, intestines, and cancerous Received: December 17, 2012 Revised: March 29, 2013 Accepted: April 22, 2013

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dx.doi.org/10.1021/mp3007133 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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tissues.12−15 Hypoxia reduces the expression and functional activity of amino acid transporters in the lungs and intestines.14,16 Although tissue hypoxia is a cause of choroid/retinal disorders such as age related macular degeneration17 and diabetic retinopathy,18 there is a dearth of knowledge on the effect of hypoxia on expression and activity of solute and nutrient transporters in the retina. Previous studies from Payet et al. and Takagi et al. characterized the effect of hypoxia on expression of glutamate and glucose transporters in whole retina and retinal capillary endothelial cells, respectively.6,19 Takagi et al. showed up regulation of expression and functional activity of glucose transporter (GLUT1) in retinal capillary endothelial cells under hypoxia and speculated its involvement in the pathology of diabetic retinopathy.6 Monitoring of hypoxia related changes in the expression of transporters will be helpful in elucidating the disease mechanism, while potentially allowing targeted drug delivery to the affected tissue. Due to the difficulty in obtaining human ocular tissues, excised ocular tissues from various animal models (rabbit, bovine, and pig) are commonly used for ocular permeability studies. Rats are widely used disease models for ocular diseases such as diabetic retinopathy, age-related macular degeneration (ARMD), and retinopathy of prematurity (ROP); however, due to their small eye size, excised ocular tissues from rats cannot be used for in vitro permeability studies. In this study, we employed excised eye tissues from calf and rat models exposed to chronic hypoxia. In order to address the paucity of data on hypoxia related changes in expression of transporters in choroid-retina, this study for the first time has characterized the expression of 84 transporters in hypoxic and normoxic rat choroidretina. The functional activity of four solute carrier transporters (SLC), including peptide transporters (PEPT), amino acid transporters (ATB0+), organic cation transporters (OCT), and monocarboxylate transporters (MCT), which are useful for transporter guided drug delivery were compared between hypoxic and normoxic conditions in calf sclera-choroid-RPE (SCRPE). PEPT and ATB0+ were chosen for functional characterization because (1) these transporters have a broad substrate specificity and (2) a high transport capacity.20,21 OCT and MCT were chosen because most of the ocular drugs are either cationic or anionic molecules. These ionic drug molecules may be transported across ocular barriers either through OCT or MCT transporters. The functional activity of PEPT, ATB0+, OCT, and MCT transporter was compared by measuring the transport of specific substrates across hypoxic and normoxic calf sclera-choroid-RPE (SCRPE) and cornea.

eyes were obtained from the Department of Physiology, School of Veterinary Medicine, Colorado State University (Fort Collins, CO). Briefly, 1 day old male Holstein calves (n = 4) were kept in hypobaric hypoxic chambers (PB = 445 mmHg) for 2 weeks. For control experiment, age-matched calves (n = 4) were kept at ambient altitude (PB = 650 mmHg) and normoxia for two weeks. Hypoxic and normoxic rat eyes were obtained from the Department of Medicine, University of Colorado Anschutz Medical campus (Aurora, CO). Male Sprague−Dawley rats weighing 150− 200 g (6 weeks old) were obtained from Charles River Laboratories (Wilmington, DE, USA). A randomly selected test group of four rats was kept in hypobaric hypoxic chambers (PB = 380 mmHg) for 6 weeks, and age-matched four control rats were kept at ambient pressure and normoxia. RNA Extraction and Quality Control Analysis. Isolation of RNA from rat ocular tissues was carried out using QIAzol and RNeasy mini kit as per manufacturer’s protocol (Qiagen, Valencia, CA). Rat eyes were enucleated immediately after euthanasia, snap frozen in liquid nitrogen, and stored at −80 °C until further processing. Eyes were dissected in a frozen condition on an ice-cold ceramic tile placed on a dry ice isopentane bath. Whole choroid-retina was isolated and transferred into RNase free microcentrifuge tube containing 300 μL of RNAlater solution (Cat. No. 76104, Qiagen Inc.) and stored at −80 °C until further processing. At the time of RNA isolation, tissues were removed from RNAlater solution and transferred into a tube containing QIAzol regent (10 times the volume of tissue weight) and homogenized. The isolated total RNA was then further purified using RNeasy mini purification kit (Cat. No. 74104, Qiagen Inc.). On column DNase digestion was carried out during RNA purification to eliminate genomic DNA contamination using a DNA elimination kit (Qiagen, Valencia, CA). Quality control analysis of isolated RNA samples for quantity, purity, and integrity was analyzed using Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) before proceeding to the next step. First Strand cDNA Synthesis. Synthesis of first strand cDNA from isolated RNA samples was carried out using SABiosciences’s RT2 First Strand Kit as per manufacturer’s protocol (Qiagen, Valencia, CA). Briefly, all reagents were centrifuged for 15 s before use. Genomic DNA contamination from the RNA sample (2.5 μg f RNA) was removed by heating the samples at 42 °C for 5 min in genomic DNA elimination buffer. For first strand cDNA synthesis, 10 μL of reverse transcriptase cocktail mixture was incubated with 10 μL of RNA sample treated with genomic DNA elimination mixture. Subsequently, the mixture was incubated at 42 °C for 15 min and then heated at 95 °C for 5 min. Synthesized cDNAs were diluted with water (92 μL) and stored at −80 °C until further use. qPCR. qPCR was performed using 96-well rat drug transporter PCR assay plates (N = 4) and ABI 7900HT FAST block as per manufacturer’s protocol (Qiagen, Valencia, CA). PCR reaction mixture was prepared by mixing 1350 μL of SABiosciences RT2 qPCR master mix, 102 μL of cDNA synthesized and diluted in the above step, and 1248 μL of water. PCR reaction was run in a total incubation volume of 25 μL. The PCR was run at 95 °C for 10 min (initialization step), followed by 40 cycles of 95 °C for 15 s each (denaturation step), and then 60 °C for 1 min each (annealing step), followed by final elongation at 72 °C for 15 min after the last PCR cycle (final elongation step). The setting of threshold (Ct = 35) and baseline was automatically performed by the instrument.

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MATERIALS AND METHODS Materials. Materials required for RNA isolation and q-RTPCR were purchased from Qiagen (Qiagen, Valencia, CA). MPP+ iodide, α-methyl-DL-tryptophan, phenyl acetic acid, valacyclovir, Gly-Sar, metformin, nicotinic acid sodium salt, mannitol, nadolol, and formic acid were purchased from SigmaAldrich (St. Louis, MO). H-Pro-Phe-OH was purchased from Bachem (Torrance, CA). HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). Ammonium formate was purchased from Fluka BioChemika (USA). All other chemicals and reagents used in this study were of analytical reagent grade. Methods. Calf and Rat Ocular Tissues. Animals used in this study were those that were sacrificed as part of other experiments approved by the Institutional Animal Care Committee of the Colorado State University (Fort Collins) and University of Colorado Anschutz Medical campus. Hypoxic and normoxic calf B

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Relative Gene Expression Analysis. Relative gene expression analysis was performed by normalization of gene expression using five rat reference genes, including ribosomal protein P1 (RPLP1), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13A (RPL13A), lactate dehydrogenase-A (LDHA), and β-actin. The geometric mean of five rat reference genes was used as a normalization factor for relative quantification of each gene. The difference in Ct (ΔCt) for each gene in the plate was calculated as the difference between Ct values for the gene of interest and the geometric mean of Ct for reference genes. The fold change in relative gene expression between hypoxic and normoxic choroid-retina was calculated using a web based PCR data analysis software RT2 Profiler PCR Array Data Analysis (Version 3.5) software.22 This integrated web-based software package calculates ΔΔCt based fold-change from raw threshold cycle data and performs pairwise comparison between groups. In Vitro Transport across Calf Cornea and Sclera-ChoroidRPE. In vitro transport studies across hypoxic and control calf cornea and sclera-choroid-RPE (SCRPE) were carried out according to a previously published method23 using the cassette dosing approach. A cassette of drug transporter substrates, GlySar (PEPT), valacyclovir (ATB0+), MPP+ (OCT), and phenylacetic acid (MCT) at a concentration of 100 μM each in assay buffer was prepared. Briefly, the calf eyes were harvested immediately after euthanasia, washed with assay buffer, and cleaned from muscle and unwanted tissues. Anterior and posterior parts were separated by circumferential cut at the limbus. The vitreous was removed, and the neural retina was separated from the choroid-RPE. The eye cup was divided into two pieces (∼1.5 × 1.5 cm) of sclera-choroid-RPE. Isolated tissues were mounted in modified Ussing chambers (Navicyte, Sparks, NV) such that the episcleral side of SCRPE or epithelial side of cornea was facing the donor chamber. Due to the limited availability of calf eyes, and the ability to mount only one chamber with each cornea, the effect of inhibitors on transport across cornea was not evaluated. The chambers were filled with 1.5 mL of assay buffer at 37 °C with (donor side) or without (receiver side) the cocktail of drug transporter substrates. For the study of effect of transporter inhibitors, a cocktail mixture (500 μM each) of transporter inhibitors was added on both donor and acceptor sides. A summary of specific transporter substrates and inhibitors used for transport study is provided in Table 1. During the transport study, the bathing

Phenyl acetic acid was analyzed separately with a negative ionization method and a normal phase separation method. An API-3000 triple quadrupole mass spectrometry (Applied Biosystems, Foster City, CA, USA) coupled with a PerkinElmer series-200 liquid chromatography (Perkin-Elmer, Waltham, Massachusetts, USA) system was used for analysis. Gly-Sar, valacyclovir, and MPP+ were separated on Supelco C-5 column (2.1 × 10 mm, 3 μm) using water containing 0.1% formic acid (A) and acetonitrile:methanol (50:50 v/v) containing 0.1% formic acid (B) as mobile phase. A linear gradient elution at a flow rate of 0.3 mL/min with a total run time of 9 min was employed [90% A (0−1.5 min), 10% A (5.0−6.5 min), 90% A (7.5−9.0 min)]. Phenyl acetic acid was separated in normal phase separation mode on Obelisc-N silica column (2.1 × 10 mm, 3 μM) using 5 mM ammonium formate at pH 3.5 (A) and acetonitrile (B) as mobile phase. A linear gradient mode at a flow rate of 0.3 mL with a total run time of 6 min was used [20% A (0−1.0 min), 80% A (2.5−4.0 min), 20% A (5.0−6.0 min)]. Gly-Sar, valacyclovir, and MPP+ were analyzed in positive ionization mode with the following multiple reaction monitoring (MRM) transitions: 147 → 90 (Gly-Sar); 325 → 152 (valacyclovir); and 170 →128 (MPP+). Phenyl acetic acid was analyzed in negative ionization mode with the following multiple reaction monitoring (MRM) transition: 135 → 91 (penyl acetic acid). Data Analysis. All values in this study are expressed as mean ± SD. Statistical comparisons between two groups were determined using independent sample Student’s t-test. Differences were considered statistically significant at p < 0.05.

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RESULTS Quality Control Analysis of RNA Extracted from Rat Choroid-Retina. Quality control analysis of isolated RNA samples were conducted as per the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines.24 The integrity and purity of isolated RNA samples were analyzed by an Agilent bioanalyzer. Only samples with an RNA integrity number (RIN) above 7 and a rRNA ratio (28s/18s) above 1.5 were used in the qRT-PCR analysis. The RNA concentration, RIN, and rRNA ratio for samples used in the current study are summarized in Table 2. All samples used in the current study had RIN numbers above 8.6 and rRNA ratios above 1.6. Genomic DNA contamination in each RNA samples was analyzed by inclusion of the genomic DNA control well in the RT-PCR plate. In each sample tested, genomic DNA contamination was absent. Further, the effect of impurities present in the RNA samples on reverse transcription and PCR amplification reaction was monitored by inclusion of three wells for reverse transcription control (RTC) and three wells for positive PCR control (PPC) in the qRT-PCR reaction. As per manufacturer’s protocol, the average Ct for PPC should be 20 ± 2 and should not vary by more than two cycles between PCR arrays being compared. For our samples, the average Ct of PPC ranged from 18.1 to 20.9, which were within the acceptable limit (20 ± 2). As per manufacture’s protocol, ΔCt values (ΔCt = Average Ct for RTC − Average Ct for PPC) should be less than 5 to confirm that the isolated RNA samples were free from impurities. In our study we observed that ΔCt values ranged from 3.6 to 4.6, which were below the limit of 5. Transporters mRNA Expression in Rat Choroid-Retina. A summary of the transporter expression patterns in normal rat choroid-retina is shown in Table 3. Transporters with a Ct value above 35 during RT-PCR were considered absent. Out of 84 transporters tested, 9 transporters were absent in rat

Table 1. List of Transporter, Specific Substrates, and Inhibitors for Each Transporter and Inhibition Mechanism transporter specific substrate PEPT OCT ATB0+ MCT

Gly-Sar MPP+ valacyclovir phenyl acetic acid

specific inhibitor

inhibition mechanism

H-Pro-Phe-OH metformin α-methyl tryptophan nicotinic acid

competitive inhibition competitive inhibition specific inhibition competitive inhibition

fluids were maintained at 37 °C, and the fluids were maintained at pH 7.4 using 95% air−5% CO2 aeration. Samples were collected (200 μL) from the receiver side every hour for 6 h, and the removed volume was replaced with fresh assay buffer. Drug levels were analyzed using a LC-MS/MS assay. LC-MS/MS Analysis. Analyte concentrations in transport study samples were measured using an LC-MS/MS method after 5-fold dilution with acetonitrile to reduce the salt concentrations. A cassette analysis method was developed for simultaneous analysis of Gly-Sar, valacyclovir, and MPP+. C

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Table 2. Summary of RNA Quality Control Analysis: RNA Concentrations, RNA Integrity Number (RIN), rRNA Ratios, Positive PCR Control (PPC), and Reverse Transcription Control (RTC) Used during qRT-PCR of Transporter Gene Expression Analysis for mRNA Isolated from Hypoxic and Normoxic Rat Choroid-Retina (CR) sample name

RNA concentrations (ng/μL)

RIN no.

rRNA ratio (28 s/18 s)

hypoxic-CR1 hypoxic-CR2 hypoxic-CR3 normoxic-CR1 normoxic-CR2 normoxic-CR3

603 898 820 637 523 565

9.0 8.9 8.6 9.0 9.1 9.3

1.6 1.6 1.8 1.7 1.7 1.7

positive PCR control (Ct PPC)

ΔCt (Avg Ct RTC − Avg Ct PPC)

± ± ± ± ± ±

3.58 3.87 4.65 3.86 3.82 4.10

18.1 20.6 19.0 20.1 19.1 18.1

0.17 0.12 0.29 0.21 0.23 0.19

TAP-1 were also up regulated by 1.5-fold in hypoxic choroidretina. Effect of Hypoxia on Transport of Transporter Substrate Cassette across Calf SCRPE. Transport of transporter substrate cassette across normoxic and hypoxic calf SCRPE was carried out to evaluate the effect of hypoxia on the functional activity of PEPT, ATB0+, OCT, and MCT transporters in SCRPE. As shown in Figures 4 and 5, transport of Gly-Sar (PEPT substrate), valacyclovir (ATB0+ substrate), and MPP+ (OCT substrate) was significantly decreased in hypoxic calf SCRPE when compared to age matched normoxic calf SCRPE. However, the cumulative % transport and apparent permeability constant (Papp) of phenyl acetic acid (MCT substrate) was increased by several fold in hypoxic condition (Figure 4D and 5D). Transport of all four transporter substrates was significantly inhibited in the presence of transporter specific inhibitors in both normoxic and hypoxic conditions (Figure 4 and 5). Effect of Hypoxia on Transport of Transporter Substrate Cassette across Calf Cornea. Transport of transporters substrate cassette across normoxic and hypoxic calf cornea was carried out to evaluate the effect of hypoxia on functional activity of PEPT, ATB0+, OCT, and MCT in cornea. Similar to SCRPE, the functional activity of PEPT, ATB0+, and OCT transporters was significantly reduced in hypoxic cornea when compared to normoxic cornea. In case of MCT, the functional activity of MCT was significantly increased in hypoxic cornea (Figures 6 and 7). Due to limited availability of hypoxic and normoxic calf eyes, transport studies across cornea were not carried out in the presence of inhibitors.

choroid-retina. Transporters present in the choroid-retina were divided into three categories based on their Ct values. Transporters with Ct values between 30 to 35 were considered as very low expression, Ct values between 25 to 30 were considered as low to medium expression, and transporters with Ct values below 25 were considered as high expression. Out of 75 transporters, 14 transporters exhibited very low expression, 40 transporters showed low to medium expression, and only 18 showed high expressions. Transporters which showed high expression in choroid-retina were glucose transporters, monocarboxylate transporters, nucleoside transporters, organic anion transporting polypeptides, voltage-dependent ion channels, aquaporin 1 transporter, folate and thiamine transporters, and efflux transporters including MRP1, ABCR, and Abc50. Effect of Hypoxia on ATP-Binding Cassette Transporters’ mRNA Expression. Relative gene expression analysis between hypoxic and control rat choroid-retina showed that out of 26 ABC transporters, 9 transporters were up regulated by 1.5fold in hypoxic choroid-retina (Figure 1). Transporters which were up regulated in hypoxia were MRP3, MRP4, MRP5, MRP (member 10), MDR6, Abca17, Abc2, Abc3, and RGD1562128. Effect of Hypoxia on Solute Carrier Transporters’ mRNA Expression. Relative gene expression analysis of solute carrier transporter (SLC) between hypoxic and control rat choroid-retina showed that out of 46 SLC transporters, 11 transporters were up-regulated and 4 transporters were downregulated by ≥1.5-fold in hypoxic choroid-retina (Figure 2). Transporters that were up-regulated in hypoxia are SBACT (sodium/bile acid cotransporter family; SLC10a1), MCT-3/ MCT-4 (monocarboxylate transporter-3; SLC16a3), OAT-2 (organic anion transporter-2; SLC22a7), OAT-3 (organic anion transporter-3; SLC22a8), ENT-1 (equilibrative nucleoside transporter; SLC29a1), ENT-2 (equilibrative nucleoside transporter; SLC29a2), GLUT-1 (facilitated glucose transporter; SLC2a1), MDU-1 (activators of dibasic and neutral amino acid transporter; SLC3a2), SGLT2 (low affinity sodium-glucose cotransporter; SLC5a4), SLC7a11 (cationic amino acid transporter, y+ system; cystine/glutamate transporter), SLC7a4 (cationic amino acid transporter, y+ system; CAT4). Transporters that were downregulated in hypoxic choroid-retina were OCT-2 (organic cation transporter 2; SLC22a2), OAT-5 (organic anion transporter-5; SLC22a9), CNT-1 (sodium coupled concentrative nucleoside transporter; SLC28a1), and ATB0+ (B (0,+)-type amino acid transporter; SLC7a9). Effect of Hypoxia on Miscellaneous Transporters’ mRNA Expression. Relative gene expression analysis of miscellaneous transporters including aquaporin (Aqp), ATPase, voltage-dependent ion channel, and TAP transporters between hypoxic and control rat choroid-retina are shown in Figure 3. Aqp-1 was up-regulated and Aqp-9 and Aqp-7 were downregulated in hypoxic choroid-retina. Further, ATPase-7b and

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DISCUSSION This is the first study to characterize the expression of 84 transporters in rat choroid-retina under normoxic and hypoxic conditions using RT2 Profiler PCR array. Out of the 84 transporters tested, 9 transporters were absent in normal rat choroid-retina, and only 18 showed abundant expression. Induction of hypoxia resulted in significant changes in the expression of transporters; out of 75 transporters present, 23 transporters were up-regulated, and 6 transporters were downregulated by greater than 1.5-fold when compared to agematched normoxic controls. Both mRNA expression and functional activity of OCT and ATB0+ were down-regulated in hypoxia. For PEPT, although functional activity was significantly down-regulated in hypoxic SCRPE and cornea, mRNA analysis showed no change in the expression of PEPT under hypoxia. For MCT, gene expression and functional activity was up-regulated in hypoxia. Due to the highly dynamic nature of mRNA transcription and potential of variability depending on sample handling and processing, quality control analysis is of utmost importance to D

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Table 3. Summary of Expression of 84 Transporter Genes in Normoxic Rat Choroid-Retina: Gene Accession ID, Common Gene Symbols, Gene Name, Mean Ct Value Obtained from Three Assays, and the Expression Levela accession ID

symbol

NM_178095 NM_001106020 NM_001031637 NM_024396 XM_220219 NM_001107721 XM_221101 NM_031760 NM_012623 NM_012690 XM_234725 NM_080582 NM_022281

Abca1 Abca13 Abca17 Abca2 Abca3 Abca4 Abca9 Abcb11 Abcb1b Abcb4 Abcb5 Abcb6 Abcc1

NM_001108201 NM_199377 NM_012833 NM_080581 NM_133411 NM_053924

Abcc10 Abcc12 Abcc2 Abcc3 Abcc4 Abcc5

NM_031013 NM_001108821 NM_012804 NM_001013100 NM_001109883 NM_181381 NM_130414 NM_012778 NM_019157 NM_022960 NM_130823 NM_052803 NM_012511 NM_022715 NM_017047 NM_017222 NM_057121 NM_031672 NM_012716

Abcc6 Abcd1 Abcd3 Abcd4 Abcf1 Abcg2 Abcg8 Aqp1 Aqp7 Aqp9 Atp6v0c Atp7a Atp7b Mvp Slc10a1 Slc10a2 Slc15a1 Slc15a2 Slc16a1

NM_147216 NM_030834 NM_017299 NM_001030024 NM_001108228

Slc16a2 Slc16a3 Slc19a1 Slc19a2 Slc19a3

NM_012697

Slc22a1

NM_031584

Slc22a2

a

gene name Abca1

Abc2 ABCR Bsep/Spgp Abcb1/Mdr1/Pgy1 Mdr2/Pgy3 RGD1566342 MGC93242 Abcc1a/Avcc1a/Mrp/ Mrp1 MRP7 MRP9 Cmoat/Mrp2 Mlp2/Mrp3 Mrp4 Abcc5a/ MGC156604/Mrp5 Mrp6 RGD1562128 PMP70/Pxmp1 MGC105956/Pxmp1l Abc50 BCRP1 CHIP28 MGC93419 Atp6c/Atp6l Mnk Hts/PINA/Wd major vault protein Ntcp/Ntcp1/SBACT ISBAT Pept1 MGC91625 MCT1/RATMCT1/ RNMCT1 MCt8 MCt3 MGC93506/MTX1 MGC124887 ThTr-2/thiamine transporter 2 MGC93570/OCt1/ OrCt1/RoCt1 OCT2/OCT2r/ rOCT2

Ct

expression level

25.306 27.543 34.605 26.064 26.076 20.166 26.740 35.000 29.213 28.583 35.000 28.936 22.511

L to L to VL L to L to H L to A L to L to A L to H

30.151 33.427 27.545 29.486 27.113 25.199

VL VL L to L to L to L to

32.995 27.467 23.421 27.968 22.129 26.968 36.144 24.506 36.575 32.229 20.538 25.274 26.844 27.037 26.505 33.598 29.454 26.724 21.038

VL L to H L to H L to A H A VL H L to L to L to L to VL L to L to H

25.785 26.788 24.709 24.562 29.822

accession ID

gene name

Ct

OCT3/EMT MGC124962/Oat1/ OrCtl1/Paht/Roat1 Oat2 MGC93369/OCT3/ Oat3/RoCt Oat5/Slc22a19 RGD1565889 Cnt1 Cnt2 Cnt3 rENT1 rENT2 GLUTB/GTG1/ Glut1/Gtg3/ RATGTG1 GTT2/Glut2 GLUT3 Ctr1/LRRGT00200 Ata2/Atrc2/Sat2/ Snat2 SN2 D2/NAA-TR/Nbat/ rBAT Mdu1 MGC93553/SGLT1 Slc5a4 cystine/glutamate transporter CAT4 E16/TA1 LAT3 y+LAT1 Lat2/Lat4 ATB0+ OATP-3/Oatp3/ Slc21a7/Slco1a2 Oatp5/Slc21a13 OATP-4/Oatp4/ Slc21a10/Slco1b2/ rlst-1 Matr1/Slc21a2 Slc21a9/moat1 Slc21a11 OATP-E/Slc21a12 Abcb2/Cim/ MGC124549 Abcb3/Cim/ MGC108646 voltage-dependent anion channel 1 voltage-dependent anion channel 2

33.420 34.916

VL A

32.139 26.089

VL L to M

34.306 27.579 35.825 25.951 29.543 22.930 28.633 24.761

VL L to A L to L to H L to H

30.276 26.216 23.622 24.334

VL L to M H H

29.522 20.857

L to M H

22.919 27.548 32.581 24.996

H L to M VL L to M

30.213 24.981 25.441 27.216 23.010 30.503 24.564

VL L to M L to M L to M H VL H

35.963 35.327

A A

26.616 27.831 25.933 22.944 30.635

L to M L to M L to M H VL

26.189

L to M

20.313

H

21.156

H

M M

NM_019230 NM_017224

Slc22a3 Slc22a6

M M

NM_053537 NM_031332

Slc22a7 Slc22a8

NM_173302 XM_342640 NM_053863 NM_031664 NM_080908 NM_031684 NM_031738 NM_138827

Slc22a9 Slc25a13 Slc28a1 Slc28a2 Slc28a3 Slc29a1 Slc29a2 Slc2a1

NM_012879 NM_017102 NM_133600 NM_181090

Slc2a2 Slc2a3 Slc31a1 Slc38a2

NM_138854 NM_017216

Slc38a5 Slc3a1

NM_019283 NM_013033 NM_001106383 NM_001107673

Slc3a2 Slc5a1 Slc5a4a Slc7a11

NM_001107078 NM_017353 NM_001107424 NM_031341 NM_053442 NM_053929 NM_030838

Slc7a4 Slc7a5 Slc7a6 Slc7a7 Slc7a8 Slc7a9 Slco1a5

NM_130736 NM_031650

Slco1a6 Slco1b3

L to M L to M H H L to M

NM_022667 NM_080786 NM_177481 NM_133608 NM_032055

Slco2a1 Slco2b1 Slco3a1 Slco4a1 Tap1

NM_032056

Tap2

35.589

A

NM_031353

Vdac1

33.178

VL

NM_031354

Vdac2

M M M M

M M M M

M M M

M M M M M M

expression level

symbol

M M M M

Gene expression level was assigned based on mean Ct values obtained from qRT-PCR reactions. Ct values above 35 were considered as absent (A); Ct values in the range 30−35 were considered as very low expression (VL); Ct values in the range 25−30 were considered as low to medium expression (L to M); and Ct values less than or equal to 25 were considered as high expression (H). Data are expressed as mean for three biological replicates.

get the reproducible and reliable results during RT2 profiler PCR array analysis. Therefore, we conducted qRT-PCR experiments as per MIQE guidelines to avoid assay-to-assay variability and to obtain reproducible and reliable results.24,25 As shown in Table 2, quality control analysis of RNA samples passed all quality control tests with RIN above 7, a rRNA ratio

(28 s/18 s) above 1.5, and samples were free from genomic DNA contamination. Errors in the quantification of mRNA transcripts are easily compounded with any variation in the amount of starting material between the samples (e.g., errors caused by sample-to-sample variation, variation in RNA integrity, RT efficiency differences, and cDNA sample loading E

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Figure 3. Fold change in miscellaneous transporter expression in hypoxic rat choroid-retina when compared to normoxic rat choroidretina. Values above +1 indicate the up regulation, and values below −1 indicates the down regulation of transporters in hypoxic condition. Thick black lines at ±1.5 are cutoff lines for 50% up regulation and down regulation. Data are expressed as the mean for three biological replicates.

Figure 1. Fold change in ATP-binding cassette (ABC) transporters expression in hypoxic rat choroid-retina when compared to normoxic rat choroid-retina. Values above +1 indicate the up regulation, and values below −1 indicate the down regulation of transporters in hypoxic conditions. Thick black lines at ±1.5 are cutoff lines for 50% up regulation and down regulation. Data are expressed as the mean for three biological replicates.

transporter 1 (MCT1), Atp6v0c, Aquaporin 1, ATP-binding cassette 50, ABCD3, MRP1, and ABCA4 (ABCR) (Table 3). ABCA4 is a retina-specific ABC transporter located in the outer segment of photoreceptor cells and is associated with autosomal retinal degenerative disorders.29 Most of the transporters that showed abundant expression in choroid-retina are nutrient transporters. Retina is a metabolically highly active tissue and needs a large amount of nutrient supply to maintain its metabolic needs. Amino acid transporters such as LAT, rBAT, and Ata2 showed abundant expression because the retina needs a large amount of amino acids for synthesis of various neurotransmitters.30,31 Previous reports showed abundant expression of OATP-E and OATP-1 in rat ocular tissues, most specifically in retinal pigmented epithelium and retina, for the transport of thyroid hormones and organic anions.32,33 Zhang et al. characterized the mRNA expression of drug transporters in human ocular tissues, but their study was limited to 21 transporters, including 5 ABC and 16 SLC transporters.34 While the animal models of our studies are indicative of retinal changes associated with pan hypoxia, they may not truly represent retinal neovascular events associated with focal hypoxia in human subjects. Further, our studies may not be representative of neovascular events in animal models that are subjected to hyperoxia and normoxia cycles. However, a recent study showed that hypobaric hypoxia results in changes in retinal vessel tortuosity similar to retinal angiogenesis.35 Further, both normobaric as well as hypobaric hypoxia result in activation of hypoxia-inducible transcription factor (HIF), which regulates the activation of pathophysiological changes associated with hypoxia.36 Normobaric hypoxia has been previously reported to induce intraretinal angiogenesis.37 Hypoxia results in significant alterations in the expression of transporter genes in rat choroid-retina, with ≥1.5 fold up-regulation of 23 transporters and ≥1.5 fold down regulation of 6 transporters. In the ABC transporter family, 9 transporters including MRP3, MRP4, MRP5, MRP (member 10), MDR6, Abca17, Abc2, Abc3, and RGD1562128 were up-regulated in hypoxia (Figure 1). Although it is not clear whether a hypoxia responsive element is present in the promoter region of ABC transporters,38 few studies have shown up regulation of MRP and MDR transporters during hypoxia.39,40 ABCG2 or BCRP1 transporter was significantly

Figure 2. Fold change in solute carrier transporters (SLC) expression in hypoxic rat choroid-retina when compared to normoxic rat choroid-retina. Values above +1 indicate the up regulation, and values below −1 indicate the down regulation of transporters in hypoxic condition. Thick black lines at ±1.5 are cutoff lines for 50% up regulation and down regulation. Data are expressed as the mean for three biological replicates.

variation). To control for sample-to-sample variations, relative gene expression analysis was performed by normalization of gene expression using five rat reference genes, including RPLP1, HPRT1, RPL13A, LDHA, and β-actin. Results from our study showed hypoxia had little or no effect on the expression of these reference genes. Literature reports also showed that RPLP1, RPL13A, HPRT1, and β-actin are the most stable genes, and the expression of these genes is not altered by hypoxia.26−28 In this study for the first time we have characterized the expression of 84 transporter genes in rat choroid-retina and showed that out of 84 transporters, 9 transporters were absent and only 18 transporters showed abundant expression. Eighteen transporters, which showed abundant expression in rat choroid-retina were voltage-dependent anion channels (Vdac), OATP-E, OATP-1, LAT-2, LAT-1(Slc3a2), B(0,+)-type amino acid transport protein (rBAT), amino acid transporter A2 (Ata2), copper transporter1 (Ctr1), glucose transporter 1 (Glut1), equilibrative nucleoside transporter 1 (ENT1), thiamine transporter (Thtr1), folate transporter (FLOT 1), monocarboxylate F

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Figure 4. Transport of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic calf SCRPE than hypoxic calf SCRPE. On the other hand, transport of phenylacetic acid is significantly higher across hypoxic SCRPE than normoxic SCRPE. Transport of all four transporter substrates was significantly inhibited in the presence of inhibitor cocktail. (A) Gly-Sar; (B) MPP+; (C) valacyclovir; and (d) phenylacetic acid. Data are expressed as mean ± SD for n = 4.

Figure 5. Apparent permeability (Papp) of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic SCRPE than hypoxic SCRPE. For phenylacetic acid, Papp is significantly higher across hypoxic SCRPE than normoxic SCRPE. Apparent permeability of all four transporter substrates was significantly inhibited in the presence of inhibitor cocktail. Effect of hypoxia and transporter inhibitors on apparent permeability of (A) Gly-Sar, (B) MPP +, (C) valacyclovir, and (d) phenylacetic acid across normoxic and hypoxic calf SCRPE. Data are expressed as the mean ± SD for n = 4. * Significantly different from normoxic at P ≤ 0.05. + Significantly different from hypoxic at P ≤ 0.05.

might be due to the down regulation of ABCG2 activity in choroid-retina as a result of hypoxia. In the SLC transporter family, out of 46 SLC transporters, 11 transporters were up-regulated, and 4 transporters were down-regulated by at least 1.5-fold in hypoxic choroid-retina (Figure 2). Transporters which showed greater than 2-fold up

down-regulated in hypoxic choroid-retina. A previous study showed that the ABCG2 is up-regulated in hypoxic stem cells and acts as a cell survival factor by reducing cellular accumulation heme or porphyrin.41 A recent study showed the accumulation of porphyrin and heme in Bruch’s membrane with age.42 Accumulation of porphyrin and heme in Bruch’s membrane G

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Figure 6. Transport of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic calf cornea than hypoxic calf cornea. For phenylacetic acid, transport across hypoxic cornea is significantly higher than normoxic cornea. (A) Gly-Sar; (B) MPP+; (C) valacyclovir; and (d) phenylacetic acid. Data are expressed as the mean ± SD for n = 4.

Figure 7. Apparent permeability (Papp) of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic cornea than hypoxic cornea. For phenylacetic acid, Papp is significantly higher across hypoxic cornea than normoxic cornea. The effect of hypoxia on the apparent permeability of (A) Gly-Sar, (B) MPP +, (C) valacyclovir, and (d) phenylacetic acid across calf cornea. Data are expressed as the mean ± SD for n = 4. *Significantly different from normoxic at P ≤ 0.05.

regulation include MCT-3, GLUT-1, and ENT-1. MCT transporters mediate the diffusion of lactic acid and several other monocarboxylate compounds across plasma membrane.43 MCT-3 expression is largely restricted to the retinal pigmented epithelium in the eye44 and involved in the export of lactic acid produced by the retina to blood. Hypoxia stimulates the expression of various glycolytic enzymes including GLUT1 by transcriptional mechanisms involving hypoxia inducible factor.45

Increased GLUT1 levels in hypoxic retina stimulate lactate production. Nyengaard et al. showed that the retinal lactate levels were 1.7-fold higher in hypoxic rat retina than age matched control rat retina.46 As MCT-3 is the predominant transporter involved in lactic acid export from the retina to choroid, MCT-3 expression is also increased during hypoxia. Ullah et al. showed that only MCT3/4 but not MCT-1 is up-regulated by hypoxia in HeLa and COS cells.47 We also observed that only MCT-3 and H

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not MCT-1 was up-regulated in hypoxic choroid-retina. Previous literature reports reported down regulation of ENT transporters in hypoxia;48,49 however in our study the expression of both ENT1 and ENT2 were up-regulated in hypoxic choroid-retina. The expression of GLUT1 as well as low affinity glucose transporter (Slc5a4a) was up-regulated by 1.6-fold in hypoxic choroid-retina. The stimulation of expression of Slc5a4a in hypoxic conditions might be regulated by the transcriptional mechanisms involving hypoxia inducible factor similar to GLUT1;45 however, very little information is available on Slc5a4a. Other transporters up-regulated in hypoxic choroidretina are activators of dibasic and neutral amino acid transport (SLC3a2), cystine/glutamate transporter (SLC7a11), and CAT4. Cystine/glutamate transporter provides intracellular cystine for the production of glutathione, a major cellular antioxidant.50 Induction of hypoxia results in the development of hypoxia related oxidative stress in choroid-retina. Increased expression of cystine/glutamate transporter in hypoxic choroidretina provides protection from oxidative stress. Overexpression of cystine/glutamate transporter in hypoxic conditions increases the supply of intracellular cystine for production of glutathione in neuronal cells, thereby protecting them from oxidative stress.50 Cationic amino acid transporters (CAT) are involved in the transport of arginine, which is a main precursor for nitric oxide synthesis.51 Hypoxia induces the synthesis of nitric oxide, depending on the supply of precursor L-arginine. L-arginine is a cationic amino acid, and its intracellular transport is mediated by CAT.51 Increased nitric oxide production in hypoxic conditions up regulates CAT mRNA expression as a secondary mechanism to increase the supply of L-arginine.52 SLC transporters down-regulated in hypoxic choroid-retina include OCT-2, OAT5, CNT1, and ATB0+ (Figure 2). Although no direct reports are available on the effect of hypoxia on OCT-1 and OCT-2 expression, literature reports suggest that hypoxia results in down regulation of expression of OCTN-2 in placenta and BeWo cells.53,54 OAT-5 expression in kidney was shown to be down-regulated during ischemia.55 ATB0+ showed 1.6-fold down regulation during hypoxia which is consistent with previous literature reports, which showed down regulation of expression and activity of ATB0+ transporter during hypoxic and ischemic conditions.10,14 Out of 11 miscellaneous transporters, 3 transporters including AQP-1, TAP-1, and ATP7b were up-regulated, and AQP-9 was down-regulated by at least 1.5-fold (Figure 3). In the aquaporin transporter family, AQP-1 was up-regulated, and AQP-9 was down-regulated during hypoxia. AQP-1 is a water channel protein which shows abundant expression in red blood cells and tissues with rapid O2 transport.56 It is known to be upregulated during hypoxia through the hypoxia-inducible factor, and it is associated with inflammatory edema and tumor growth.56 Kaneko et al. showed that AQP1 is required for hypoxia-induced angiogenesis of human retinal vascular endothelial cells and inhibition of AQP1 inhibits angiogenesis.57 Dibas et al. showed the expression of AQP9 in retinal pigment epithelial cells (ARPE-19) and its involvement in the transport of various uncharged molecules such as lactate, glycerol, purines, pyrimidines, urea, and mannitol.58 Hypoxia results in significant down regulation of AQP-9 in rat astrocytes, and subsequent reoxygenation results in restoration of expression of AQP-9 to the basal level.59 Other transporters up-regulated by hypoxia in our study were TAP1 and ATP7b. ATP7a and ATP7b are copper-transporting ATPases that transport copper across cellular membranes,60 and hypoxia is known to up-regulate the activity of ATP7a and ATP7b.61

The effect of hypoxia on the functional activity of four solute carrier transporters including PEPT, ATB0+, OCT, and MCT was evaluated using hypoxic and normoxic calf ocular tissues. Our recent study in human ocular tissues showed the expression and activity of PEPT, OCT, ATB0,+, and MCT transporters in cornea as well as retinal pigmented epithelium (RPE).62 Although the mRNA expression is responsible for protein expression and activity, there are many instances in which mRNA levels show poor correlation with protein levels.63 This is because many complicated post-transcriptional mechanisms are involved in turning mRNA into proteins, and second, different proteins have different biological half-lives in vivo.63 The evaluation of functional activity of selected proteins gives a more realistic picture of disease status and helps to rule out uncertainty. Due to the small dimensions of rat eyes, in vitro transport studies across isolated rat ocular tissues are difficult to perform. Gene expression analysis was not performed in calf ocular tissues due to difficulty in obtaining PCR probes for bovine transporters. Although hypoxia related diseases are most relevant in the retina, we determined transporter activity in the cornea as well, since it is a key barrier for topical ocular drug delivery. It is noteworthy that corneal hypoxic conditions have been reported following corneal transplantation, use of contact lenses, diabetes, ocular infections, and environmental changes.64 Due to limited availability of hypoxic calf ocular tissues, a cassette-dosing approach was used to increase throughput. In cassette-dosing method, we cannot rule out interactions between drug molecules for metabolic enzymes and transporters.65 Transporter substrates and inhibitors in cassette were carefully selected based on literature reports to avoid the cross reactivity with other transporters.62 Valacyclovir is a substrate for both ATB0,+ and PEPT, and it is transported across epithelial barriers via both transporters. Although we have used valacyclovir as a substrate for evaluating functional activity of ATB0,+ in calf SCRPE and cornea, PEPT may also contribute to the carrier-mediated transport of valacyclovir. Valacyclovir is an L-valyl ester of acyclovir and rapidly gets converted to parent acyclovir by esterases and amidases. Ocular tissues including cornea and choroid-RPE are rich in metabolic enzymes, esterases, and amidases.66 Valacyclovir gets converted to the parent acyclovir in ocular tissues during transport, and one needs to measure both valacyclovir and acyclovir to determine the total transport of valacyclovir. A limitation of the current study is that we measured only valacyclovir in receiver chamber, which may have underestimated the cumulative % transport of valacyclovir. Drug molecules may permeate across ocular barriers by passive or carrier mediated transport. To determine the contribution of carrier-mediated transport to drug permeability, in vitro permeability studies were conducted in the presence of transporter inhibitors. As depicted in Figures 4 and 6, cumulative % transport was significantly decreased in the presence of inhibitor cocktail in both normoxic and hypoxic conditions, indicating that the molecules were primarily transported by the respective transporters. For Gly-Sar and MPP+, cumulative % transport across normoxic calf SCRPE was decreased by 85 and 68%, respectively, in the presence of an inhibitor cocktail (Figure 4). The induction of hypoxia resulted in a significant reduction in functional activity of PEPT, ATB0+, and OCT and an increase in the activity of MCT transporters in hypoxic calf SCRPE and cornea, when compared with normoxic controls (Figures 5 and 7). As shown in Figure 8, hypoxia resulted in a reduction of mRNA expression of OCT-1 and OCT-2 by 30% I

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up-regulated, while 4 transporters including SLC22a2, SLC22a9, SLC28a1, and SLC7a9 were down-regulated in hypoxic rat choroid-retina. Functional activity assays in hypoxic calf cornea and SCRPE showed down regulation of PEPT, ATB0+, and OCT activity, whereas up regulation was observed for MCT activity. Hypoxia-induced changes in expression/activity of transporters can potentially result in changes in transporter-mediated solute/ drug entry or removal in eye tissues.

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AUTHOR INFORMATION

Corresponding Author

*Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus 12850 E. 19th Avenue, Aurora, Colorado 80045, United States. Phone: 303-724-4028. Fax: 303-724-4666. E-mail: [email protected].

Figure 8. Relative gene expression of PEPT, ATB0+, OCT, and MCT transporters in hypoxic rat choroid-retina, normalized to normoxic rat choroid-retina. Data are expressed as mean for n = 3. Gene expression in normoxic animal was set to 100%, and the relative change in hypoxic animal was expressed in % up regulation.

Notes

The authors declare the following competing financial interest(s): A patent application is pending (UBK and RSK through University of Colorado).

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and 41%, respectively, in rat choroid-retina. The cumulative % transport of MPP+ (OCT substrate) across hypoxic SCRPE and cornea was decreased by 61% and 49%, respectively (Figures 3 and 5). MPP+ used as a substrate for OCT transporter has broad specificity and interacts with both OCT as well as OCTN transporters.67,68 Hypoxia is known to down-regulate both OCT as well as OCTN transporter activity.54 We observed that hypoxia reduces rat choroid-retina mRNA expression of ATB0,+ by 37% and the cumulative % transport of valacyclovir (ATB0,+ substrate) across SCRPE by 61%. Expression of MCT-1 and MCT-3 mRNA levels were up-regulated by 116% and 253%, respectively, in rat choroid-retina, and cumulative % transport of phenyl acetic acid (MCT substrate) across SCRPE was increased by 387%. In case of PEPT transporters, a functional assay showed a significant reduction in the transport of Gly-Sar (PEPT substrate) across hypoxic calf SCRPE and cornea (Figures 5 and 7). Cumulative % transport of Gly-Sar across hypoxic calf SCRPE was decreased by 85% (Figure 4A). However, there was no change in the expression of both PEPT1 and PEPT2 genes with hypoxia in rat choroid-retina (Figure 8). One reason for this observation might be that hypoxia does not alter PEPT gene expression but affects the protein stability and/or functional activity of PEPT transporters. Another possible reason is interspecies differences in the regulation of transporters by hypoxia. No prior scientific information is available on the expression of transporters in calf ocular tissues, limiting our comparison of expression pattern of transporters between rat and calf models.

ACKNOWLEDGMENTS This work was supported by NIH grants EY018940 and EY017533. The authors are thankful to Drs. Kurt Stenmark and Adil Anwar of University of Colorado Anschutz Medical Campus for providing hypoxic calf eyes.

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REFERENCES

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CONCLUSIONS In summary, under normoxic and hypoxic conditions, this study assessed the expression for 84 transporters in rat ocular tissues and functional activity of 4 SLC transporters in calf ocular tissues. Out of 84 transporters tested, 9 transporters were absent, and only 18 transporters showed abundant expression in rat choroid-retina. Hypoxia results in significant alteration (≥50% up regulation or down regulation) in the expression of drug transporters in rat choroid-retina. Nine out of 29 ATP binding cassette (ABC) families of efflux transporters including MRP3, MRP4, MRP5, MRP6, MRP7, Abca17, Abc2, Abc3, and RGD1562128 were up-regulated. For solute carrier family transporters, 11 transporters including SLC10a1, SLC16a3, SLC22a7, SLC22a8, SLC29a1, SLC29a2, SLC2a1, SLC3a2, SLC5a4, SLC7a11, and SLC7a4 were J

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dx.doi.org/10.1021/mp3007133 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX