Incubation Time Influences Organic Anion Transporter 1 Kinetics and Renal Clearance Predictions.

Accurate predictions of drug uptake transporter involvement in renal excretion of xenobiotics require determination of in vitro transport kinetic parameters under initial-rate conditions. The purpose of the present study was to determine how changing the incubation time from initial rate to steady state influences ligand interactions with the renal organic anion transporter 1 (OAT1), and the impact of the different experimental conditions on pharmacokinetic predictions. Transport studies were performed with Chinese hamster ovary cells expressing OAT1 (CHO-OAT1) and the Simcyp Simulator was used for physiological-based pharmacokinetic predictions. Maximal transport rate and intrinsic uptake clearance (CLint) for PAH decreased with increasing incubation time. The CLint values ranged 11-fold with incubation times spanning from 15 s (CLint,15s, initial rate) to 45 min (CLint,45min, steady state). The Michaelis constant (Km) was also influenced by the incubation time with an apparent increase in the Km value at longer incubation times. Inhibition potency of five drugs against PAH transport was tested using incubation times of either 15 s or 10 min. There was no effect of time on inhibition potency for omeprazole or furosemide, whereas indomethacin was less potent, and probenecid (~2-fold) and telmisartan (~7-fold) more potent with the longer incubation time. Notably, the inhibitory effect of telmisartan was reversible, albeit slowly. A pharmacokinetic model was developed for PAH using the CLint,15s value. The simulated plasma concentration-time profile, renal clearance, and cumulative urinary excretion-time profile of PAH agreed well with reported clinical data, and the PK parameters were sensitive to the time-associated CLint value used in the model.

MATE1 Deficiency Exacerbates Dofetilide-Induced Proarrhythmia.

Dofetilide is a rapid delayed rectifier potassium current inhibitor widely used to prevent the recurrence of atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which predispose patients to ventricular cardiac arrhythmias. The mechanisms involved in the disposition of dofetilide, including its movement in and out of cardiomyocytes, remain unknown. Using a xenobiotic transporter screen, we identified MATE1 (SLC47A1) as a transporter of dofetilide and found that genetic knockout or pharmacological inhibition of MATE1 in mice was associated with enhanced retention of dofetilide in cardiomyocytes and increased QTc prolongation. The urinary excretion of dofetilide was also dependent on the MATE1 genotype, and we found that this transport mechanism provides a mechanistic basis for previously recorded drug-drug interactions of dofetilide with various contraindicated drugs, including bictegravir, cimetidine, ketoconazole, and verapamil. The translational significance of these observations was examined with a physiologically-based pharmacokinetic model that adequately predicted the drug-drug interaction liabilities in humans. These findings support the thesis that MATE1 serves a conserved cardioprotective role by restricting excessive cellular accumulation and warrant caution against the concurrent administration of potent MATE1 inhibitors and cardiotoxic substrates with a narrow therapeutic window.

Quantitative Proteomics and Mechanistic Modeling of Transporter-Mediated Disposition in Nonalcoholic Fatty Liver Disease.

Understanding transporter-mediated drug disposition and pharmacokinetics (PK) in patients with nonalcoholic fatty liver disease (NAFLD) is critical in developing treatment options. Here, we quantified the expression levels of major drug transporters in healthy, steatosis, and nonalcoholic steatohepatitis (NASH) liver samples, via liquid-chromatography tandem mass spectrometry-based proteomics, and used the data to predict the PK of substrate drugs in the disease state. Expression of organic anion transporting polypeptides (OATPs) and multidrug resistance-associated protein (MRP)2 is significantly lower in NASH livers; whereas MRP3 is induced while no change was observed for organic cation transporter (OCT)1. Physiologically-based pharmacokinetic models verified with PK data from healthy subjects well recovered the PK in NASH subjects for morphine (involving OCT1) and its glucuronide metabolites (MRP2/MRP3/OATP1B), 99m TC-mebrofenen (OATP1B/MRP2/MRP3), and rosuvastatin (OATP1B/breast cancer resistance protein). Overall, considerations to altered protein expression can enable quantitative prediction of PK changes in subjects with NAFLD.

ClC Channels and Transporters: Structure, Physiological Functions, and Implications in Human Chloride Channelopathies.

The discovery of ClC proteins at the beginning of the 1990s was important for the development of the Cl- transport research field. ClCs form a large family of proteins that mediate voltage-dependent transport of Cl- ions across cell membranes. They are expressed in both plasma and intracellular membranes of cells from almost all living organisms. ClC proteins form transmembrane dimers, in which each monomer displays independent ion conductance. Eukaryotic members also possess a large cytoplasmic domain containing two CBS domains, which are involved in transport modulation. ClC proteins function as either Cl- channels or Cl-/H+ exchangers, although all ClC proteins share the same basic architecture. ClC channels have two gating mechanisms: a relatively well-studied fast gating mechanism, and a slow gating mechanism, which is poorly defined. ClCs are involved in a wide range of physiological processes, including regulation of resting membrane potential in skeletal muscle, facilitation of transepithelial Cl- reabsorption in kidneys, and control of pH and Cl- concentration in intracellular compartments through coupled Cl-/H+ exchange mechanisms. Several inherited diseases result from C1C gene mutations, including myotonia congenita, Bartter's syndrome (types 3 and 4), Dent's disease, osteopetrosis, retinal degeneration, and lysosomal storage diseases. This review summarizes general features, known or suspected, of ClC structure, gating and physiological functions. We also discuss biophysical properties of mammalian ClCs that are directly involved in the pathophysiology of several human inherited disorders, or that induce interesting phenotypes in animal models.

Influence of age on intestinal bile acid transport in C57BL/6 mice.

Intestinal and hepatic bile acid transporters are important for enterohepatic bile acid circulation and pharmacokinetics. Based on previous literature, we hypothesized that the expression of bile acid transporters and intestinal bile acid absorption are lower in older individuals. Here, we measured active taurocholate absorption across the ileum of male C57BL/6 mice in two different age cohorts - young (age range of 89-224 days) and old (age range of 613-953 days). Also examined in these mice were mRNA expression of the major bile acid transporters - Asbt and Ostα/ß in the ileum, and Ntcp, Oatp1b2 and Bsep in the liver. Mean intestinal taurocholate absorption was significantly lower (~50%) in mice in the older cohort compared to those in the younger cohort. In the ileum, the expression of Asbt was significantly lower in the older cohort, but expression of Ostα/ß was not affected by age. The lower capacity for intestinal bile acid absorption in the older animals is consistent with their lower expression level of Asbt. Of the hepatic bile acid transporters examined, expression of Ntcp and Oatp1b2 were significantly lower in the older mice. This is the first study to directly measure intestinal bile acid absorption as a function of age. The data suggest a lower capacity for intestinal bile acid absorption in older animals. Also, lower expression of Asbt, Ntcp, and Oatp1b2 in older individuals could influence pharmacokinetics of drug substrates.

Drug Transport by the Blood-Aqueous Humor Barrier of the Eye.

The ocular barriers (cornea, blood-retinal barrier, and blood-aqueous humor barrier) make treating eye diseases with therapeutic drugs challenging. The tight capillary endothelium of the iris and the ciliary body epithelium form the blood-aqueous humor barrier. The iris and ciliary body (iris-ciliary body) express a variety of drug transporters in the ATP-binding cassette and solute carrier (SLC) families. ATP-binding cassette family drug transporters that are present in the iris-ciliary body include P-glycoprotein, breast cancer resistance protein, and several multidrug resistance-associated proteins. SLC family drug transporters that are present in the iris-ciliary body include organic anion transporters, organic anion transporting polypeptides, bile acid transporters (apical sodium-dependent bile salt transporter and sodium taurocholate cotransporter), organic cation transporters (novel organic cation transporter and multidrug and toxin extrusion transporter) and peptide transporters. Freshly dissected iris-ciliary body preparations actively accumulate a variety of substrates of SLC drug transporters that are expressed in the tissue. The ciliary body in vitro supports active transport in the aqueous humor-to-blood direction of several substrates of organic anion transporters and multidrug resistance-associated proteins, consistent with the subcellular localization of these transporters in the ciliary body epithelium. In vivo data suggest that drug transporters in the iris-ciliary body reduce the permeation of drugs in the direction of blood-to-aqueous humor, thereby reducing ocular drug bioavailability, and are also involved in active drug elimination from the aqueous humor. An understanding of the influence on pharmacokinetics of drug transporters in the blood-aqueous humor barrier should help improve drug delivery and efficacy in the eye.

Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines.

Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described - OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2), and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby canine kidney (MDCK), or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate - previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1 are in good agreement with previous studies using this variant, but inconsistent with studies using OAT2 with an unidentified sequence. This study shows that organic molecules with diverse physicochemical properties interact with OAT2-tv1, making it a likely site of drug interactions. Many previously identified substrates of OAT2 are not transported by OAT2-tv1, suggesting that variant and/or expression system may contribute. Future work should establish the expression pattern and ligand selectivity of OAT2-tv3.

Organic Anion Transporter 1 Is Inhibited by Multiple Mechanisms and Shows a Transport Mode Independent of Exchange.

The mechanism by which drugs inhibit organic anion transporter 1 (OAT1) was examined. OAT1 was stably expressed in Chinese hamster ovary (CHO) cells, and para-aminohippurate (PAH) and 6-carboxyfluorescein were the substrates. Most compounds (10 of 14) inhibited competitively, increasing the Michaelis constant (Km) without affecting the maximal transport rate (Jmax). Others were mixed-type (lowering Jmax and increasing Km) or noncompetitive (lowering Jmax only) inhibitors. The interaction of a noncompetitive inhibitor (telmisartan) with OAT1 was examined further. Binding of telmisartan to OAT1 was observed, but translocation was not. Telmisartan did not alter the plasma membrane expression of OAT1, indicating that it lowers Jmax by reducing the turnover number. PAH transport after telmisartan treatment and its washout recovered faster in the presence of 10% fetal bovine serum in the washout buffer, indicating that binding of telmisartan to OAT1 and its inhibitory effect are reversible. Together, these data suggest that telmisartan binds reversibly to a site distinct from substrate and stabilizes the transporter in a conformation unfavorable for translocation. In the absence of an exchangeable extracellular substrate, PAH efflux from CHO-OAT1 cells was relatively rapid. Telmisartan slowed PAH efflux, suggesting that some transporter-mediated efflux occurs independent of exchange. Although drug-drug interaction predictions at OAT1 assume competitive inhibition, these data show that OAT1 can be inhibited by other mechanisms, which could influence the accuracy of drug-drug interaction predictions at the transporter. Telmisartan was useful for examining how a noncompetitive inhibitor can alter OAT1 transport activity and for uncovering a transport mode independent of exchange.

A renal-like organic anion transport system in the ciliary epithelium of the bovine and human eye.

The purpose of this study was to determine the direction of organic anion (OA) transport across the ciliary body and the transport proteins that may contribute. Transport of several OAs across the bovine ciliary body was examined using ciliary body sections mounted in Ussing chambers and a perfused eye preparation. Microarray, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to examine OA transporter expression in human ocular tissues. Microarray analysis showed that many OA transporters common to other barrier epithelia are expressed in ocular tissues. mRNA (RT-PCR) and protein (immunoblotting) for OAT1, OAT3, NaDC3, and MRP4 were detected in extracts of the human ciliary body from several donors. OAT1 and OAT3 localized to basolateral membranes of nonpigmented epithelial cells and MRP4 to basolateral membranes of pigmented cells in the human eye. Para-aminohippurate (PAH) and estrone-3-sulfate transport across the bovine ciliary body in the Ussing chambers was greater in the aqueous humor-to-blood direction than in the blood-to-aqueous humor direction, and active. There was little net directional movement of cidofovir. Probenecid (0.1 mM) or novobiocin (0.1 mM) added to the aqueous humor side of the tissue, or MK571 (5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid; 0.1 mM) added to the blood side significantly reduced net active PAH transport. The rate of 6-carboxyfluorescein elimination from the aqueous humor of the perfused eye was reduced 80% when novobiocin (0.1 mM) was present in the aqueous humor. These data indicate that the ciliary body expresses a variety of OA transporters, including those common to the kidney. They are likely involved in clearing potentially harmful endobiotic and xenobiotic OAs from the eye.

Cystic fibrosis transmembrane conductance regulator dysfunction in VIP knockout mice.

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.

A plasma concentration of α-ketoglutarate influences the kinetic interaction of ligands with organic anion transporter 1.

The purpose of the present study was to determine whether a physiologic plasma concentration of α-ketoglutarate (αKG) influences the kinetic interaction of ligands with organic anion transporter 1 (OAT1). The effect of extracellular αKG on the kinetics of para-aminohippurate (PAH) and cidofovir transport was examined along with its effect on the potency of 10 drugs in five different classes (uricosuric, nonsteroidal anti-inflammatories, loop diuretics, angiotensin II receptor antagonists, and ß-lactam antibiotics) to inhibit OAT1 expressed in Chinese hamster ovary cells. Extracellular αKG competitively inhibited PAH and cidofovir transport with Ki values (∼5 µM) approximating its unbound plasma concentration (determined by equilibrium dialysis). When PAH was the substrate, extracellular αKG (5 µM) significantly increased IC50 values for some inhibitors (up to 4-fold), such as probenecid, but not for others (an inhibitor-dependent effect). For some inhibitors, a significant increase in IC50 value was observed when cidofovir was the substrate, but not PAH (a substrate-dependent effect). A significant increase in IC50 value was also observed for inhibition of PAH transport by probenecid in renal basolateral membrane vesicles (5.2-fold). The substrate- and inhibitor-dependent effect of extracellular αKG on ligand interactions with OAT1 highlights the complexity of the OAT1 ligand-binding surface. The effect of extracellular αKG on the potency of OAT1 inhibition should be considered when assessing drug-drug interaction potential at the transporter.

SLC22, SLC44, and SLC47 transporters--organic anion and cation transporters: molecular and cellular properties.

Transporters within the SLC22, SLC44, and SLC47 families of solute carriers mediate transport of a structurally diverse array of organic electrolytes, that is, molecules that are generally charged (cationic, anionic, or zwitterionic) at physiological pH. Transporters in the SLC22 family--all of which are members of the major facilitator superfamily (MFS) of transporters--represent a mechanistically diverse set of processes, including the organic anion transporters (OATs and URAT1) that physiologically operate as organic anion (OA) exchangers, the organic cation transporters (OCTs) that operate as electrogenic uniporters of organic cations (OCs), and the so-called "novel" organic cation transporters (OCTNs) that support Na-cotransport of selected zwitterions. Whereas the OCTNs display a high degree of substrate selectivity, the physiological hallmark of the OATs and OCTs is their multiselectivity--consistent with a principal role in renal and hepatic clearance of a wide array of both endogenous and xenobiotic compounds. SLC47 consists of members of the multidrug and toxin extruder (MATE) family, which are carriers that are obligatory exchangers and that physiologically support electroneutral H⁺ exchange. The MATEs also display a characteristic multiselectivity and are frequently paired with OCTs to mediate transepithelial OC secretion, with the OCTs typically supporting basolateral OC entry and the MATEs supporting apical OC efflux. The SLC44 family contains the choline transporter-like (CTL) transporters. Largely restricted to choline and a limited set of structural congeners, the CTLs appear to support the Na-independent, electrogenic uniport of choline, thereby providing choline for membrane biogenesis. The solution of X-ray crystal structures of representative prokaryotic MFS and MATE transporters has led to the development of homology models of mammalian OAT, OCT, and MATE transporters that, in turn, have supplemented studies of the molecular basis of the complex interactions of ligands with these multiselective proteins.

Functional significance of conserved cysteines in the human organic cation transporter 2.

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.

Expression of organic anion transporter 2 in the human kidney and its potential role in the tubular secretion of guanine-containing antiviral drugs.

The organic anion transporters 1 and 3 (OAT1 and OAT3) and organic cation transporter 2 (OCT2) are important for renal tubular drug secretion. In contrast, evidence for OAT2 expression in the human kidney is limited, and its role in renal drug transport is unknown. Both mRNA (real-time polymerase chain reaction) and protein (Western blotting) for OAT2 were detected in renal cortex from eight donors, and interindividual variability in protein levels was 3-fold. OAT2 protein in the renal cortex was localized (by immunohistochemistry) to the basolateral domain of tubules, as were OAT1 and OAT3. The absolute abundance of OAT2 mRNA was similar to that of OAT1 mRNA and 3-fold higher than that of OCT2 mRNA but 10-fold lower than that of OAT3 mRNA. A previous observation that OAT2 transports cGMP led us to examine whether acyclovir, ganciclovir, and penciclovir are OAT2 substrates; they are guanine-containing antivirals that undergo active tubular secretion. Transport of the antivirals into human embryonic kidney cells was stimulated 10- to 20-fold by expression of OAT2, but there was little to no transport of the antivirals by OAT1, OAT3, or OCT2. The K(m) values for acyclovir, ganciclovir, and penciclovir transport were 94, 264, and 277 µM, respectively, and transport efficiencies were relatively high (6-24 µl · min(-1) · mg protein(-1)). This study provides definitive evidence for the expression of OAT2 in the human kidney and is the first to demonstrate that OAT2, compared with OAT1, OAT3, or OCT2, has a preference for antiviral drugs mainly eliminated in the urine via active secretion.

Differences in the substrate binding regions of renal organic anion transporters 1 (OAT1) and 3 (OAT3).

This study examined the selectivity of organic anion transporters OAT1 and OAT3 for structural congeners of the heavy metal chelator 2,3-dimercapto-1-propanesulfonic acid (DMPS). Thiol-reactive reagents were also used to test structural predictions based on a homology model of OAT1 structure. DMPS was near equipotent in its ability to inhibit OAT1 (IC(50) = 83 µM) and OAT3 (IC(50) = 40 µM) expressed in Chinese hamster ovary cells. However, removal of a thiol group (3-mercapto-1-propanesulfonic acid) resulted in a 2.5-fold increase in IC(50) toward OAT1 vs. a ∼55-fold increase in IC(50) toward OAT3. The data suggested that compound volume/size is important for binding to OAT1/OAT3. The sensitivity to HgCl(2) of OAT1 and OAT3 was also dramatically different, with IC(50) values of 104 and 659 µM, respectively. Consistent with cysteines of OAT1 being more accessible from the external medium than those of OAT3, thiol-reactive reagents reacted preferentially with OAT1 in cell surface biotinylation assays. OAT1 was less sensitive to HgCl(2) inhibition and less reactive toward membrane-impermeant thiol reactive reagents following mutation of cysteine 440 (C440) to an alanine. These data indicate that C440 in transmembrane helix 10 of OAT1 is accessible from the extracellular space. Indeed, C440 was exposed to the aqueous phase of the presumptive substrate translocation pathway in a homology model of OAT1 structure. The limited thiol reactivity in OAT3 suggests that the homologous cysteine residue (C428) is less accessible. Consistent with their homolog-specific selectivities, these data highlight structural differences in the substrate binding regions of OAT1 and OAT3.

Renal transport of organic anions and cations.

Organic anions and cations (OAs and OCs, respectively) comprise an extraordinarily diverse array of compounds of physiological, pharmacological, and toxicological importance. The kidney, primarily the renal proximal tubule, plays a critical role in regulating the plasma concentrations of these organic electrolytes and in clearing the body of potentially toxic xenobiotics agents, a process that involves active, transepithelial secretion. This transepithelial transport involves separate entry and exit steps at the basolateral and luminal aspects of renal tubular cells. Basolateral and luminal OA and OC transport reflects the concerted activity of a suite of separate proteins arranged in parallel in each pole of proximal tubule cells. The cloning of multiple members of several distinct transport families, the subsequent characterization of their activity, and their subcellular localization within distinct regions of the kidney, now allows the development of models describing the molecular basis of the renal secretion of OAs and OCs. New information on naturally occurring genetic variation of many of these processes provides insight into the basis of observed variability of drug efficacy and unwanted drug-drug interactions in human populations. The present review examines recent work on these issues.

Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium of the eye.

The nonpigmented epithelium (NPE) of the ciliary body represents an important component of the blood-aqueous barrier of the eye. Many therapeutic drugs penetrate poorly across the NPE into the aqueous humor of the eye interior. Several of these therapeutic drugs, such as methotrexate, vincristine, and etoposide, are substrates of the multidrug resistance-associated protein 2 (MRP2). Abundant MRP2 protein was detected by Western blot in homogenates of human ciliary body and freshly dissected porcine NPE. In cultured porcine NPE, the intracellular accumulation of the MRP2 substrates calcein (1.8-fold), 5-(and-6)-carboxy-2',7'-dichlorofluorescein (22.1-fold), and doxorubicin (1.9-fold) was significantly increased in the presence of 50 microM MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid), an MRP inhibitor. In addition, the intracellular accumulation of the MRP2 substrate glutathione methylfluorescein was increased by 50 microM MK571 (4.3-fold), 500 microM indomethacin (2.6-fold), and 50 microM cyclosporin A (2.1-fold) but not by 500 microM sulfinpyrazone. These data are consistent with MRP2-mediated transport activity in cultured NPE, and MRP2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) were detected in the cultured cells. Immunolocalization studies in native human and porcine eyes showed MRP2 protein at the apical interface of the NPE and pigmented cell layers. Close examination of MRP2 immunoreactivity supported the conclusion that MRP2 is localized in the apical membrane of the NPE. MRP2 at the apical membrane of NPE cells may be involved in protecting intraocular tissues from exposure to potentially harmful toxins.

Studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium.

PURPOSE: Bicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE). METHODS: Cytoplasmic pH (pH(i)) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. RESULTS: Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO(2)/HCO(3)(-)-free HEPES buffer, exposure to a CO(2)/HCO(3)(-)-containing buffer caused rapid acidification followed by a gradual increase in pH(i). Subsequent removal of CO(2)/HCO(3)(-) with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pH(i). The rate of gradual alkalinization after the addition of HCO(3)(-)/CO(2) was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO(3)(-)/CO(2) was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pH(i). In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively. CONCLUSIONS: The results suggest the NPE uses a Na(+)-HCO(3)(-) cotransporter to import bicarbonate and a Cl(-)/HCO(3)(-) exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.

Influence of estrogen and xenoestrogens on basolateral uptake of tetraethylammonium by opossum kidney cells in culture.

The sex steroid hormone estrogen down-regulates renal organic cation (OC) transport in animals, and it may contribute to sex-related differences in xenobiotic accumulation and excretion. Also, the presence of various endocrine-disrupting chemicals, i.e., environmental chemicals that possess estrogenic activity (e.g., xenoestrogens) may down-regulate various transporters involved in renal accumulation and excretion of xenobiotics. The present study characterizes the mechanism by which long-term (6-day) incubation with physiological concentrations of 17beta-estradiol (E(2)) or the xenoestrogens diethylstilbestrol (DES) and bisphenol A (BPA) regulates the basolateral membrane transport of the OC tetraethylammonium (TEA) in opossum kidney (OK) cell renal cultures. Both 17beta-E(2) and the xenoestrogen DES produced a dose- and time-dependent inhibition of basolateral TEA uptake in OK cell cultures, whereas the weakly estrogenic BPA had no effect on TEA uptake. Treatment for 6 days with either 1 nM 17beta-E(2) or DES reduced TEA uptake by approximately 30 and 40%, respectively. These effects were blocked completely by the estrogen receptor antagonist ICI 182780 (Faslodex, fulvestrant), suggesting that these estrogens regulate OC transport through the estrogen receptor, which was detected (estrogen receptor alpha) in OK cell cultures by reverse transcription-polymerase chain reaction. The J(max) value for TEA uptake in 17beta-E(2)- and DES-treated OK cell cultures was approximately 40 to 50% lower than for ethanol-treated cultures, whereas K(t) was unaffected. This reduction in transport capacity was correlated with a reduction in OC transporter OCT1 protein expression following treatment with both agents.

Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity.

Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO(2)-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272-35280, 2006). Thus it was hypothesized that the environmental contaminant Hg(2+) (as HgCl(2)) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [(3)H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl(2), with an IC(50) of 3.9 +/- 0.11 microM. Treatment with 10 microM HgCl(2) caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg(2+), a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl(2) than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl(2) effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg(2+) with these residues causes reduced TEA transport activity.