Transport mechanism and substrate specificity of human organic anion transporter 2 (hOat2 [SLC22A7])

Human organic anion transporter 2 (hOat2[SLC22A7]) is highly expressed in the human liver. Although localization, gene expression, substrate specificity and transport mechanisms of other human Oat isoforms such as human Oat1 (hOat1), human Oat3 (hOat3) and human Oat4 (hOat4) have been elucidated, information concerning human Oat2 (hOat2) is less defined. The objective of this study was to provide further information on the transport mechanism and substrate specificity of hOat2. When expressed in Xenopus laevis oocytes, the transport of organic compounds mediated by hOat2 was not affected by the replacement of extracellular sodium with lithium, choline and mannitol. The uptake of estrone sulfate (ES) in hOat2-expressing oocytes was significantly trans-stimulated by preloading the oocytes with fumarate and succinate, but not glutarate. Moreover, we observed that hOat2 mediates the transport of bumetanide, ES, glutarate, dehydroepiandrosterone sulfate, allopurinol, prostaglandin E2, 5-fluorouracil, paclitaxel and L-ascorbic acid. These compounds are identified for the first time as hOat2 substrates. A wide range of structurally unrelated organic compounds inhibited the hOat2-mediated uptake of tetracycline, except for sulfobromophthalein. All of these findings indicate that hOat2 is a sodium-independent multi-specific organic anion/dimethyldicarboxylate exchanger. Our present findings thus provide further insights into the role of hOat2 in hepatic drug transport.     

Identification of a mechanism by which the methylmercury antidotes N-acetylcysteine and dimercaptopropanesulfonate enhance urinary metal excretion: transport by the renal organic anion transporter-1

N-Acetylcysteine (NAC) and dimercaptopropanesulfonate (DMPS) are sulfhydryl-containing compounds that produce a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned animals, but the molecular mechanism for this effect is unknown. NAC and DMPS are themselves excreted in urine in high concentrations. The present study tested the hypothesis that the complexes formed between MeHg and these anionic chelating agents are transported from blood into proximal tubule cells by the basolateral membrane organic anion transporters (Oat) 1 and Oat3. Xenopus laevis oocytes expressing rat Oat1 showed increased uptake of [(14)C]MeHg when complexed with either NAC or DMPS but not when complexed with L-cysteine, glutathione, dimercaptosuccinate, penicillamine, or gamma-glutamylcysteine. In contrast, none of these MeHg complexes were transported by Oat3-expressing oocytes. The apparent K(m) values for Oat1-mediated transport were 31 +/- 2 microM for MeHg-NAC and 9 +/- 2 microM for MeHg-DMPS, indicating that these are relatively high-affinity substrates. Oat1-mediated uptake of [(14)C]MeHg-NAC and [(14)C]MeHg-DMPS was inhibited by prototypical substrates for Oat1, including p-aminohippurate (PAH), and was trans-stimulated when oocytes were preloaded with 2 mM glutarate but not glutamate. Conversely, efflux of [(3)H]PAH from Oat1-expressing oocytes was trans-stimulated by glutarate, PAH, NAC, DMPS, MeHg-NAC, MeHg-DMPS, and a mercapturic acid, indicating that these are transported solutes. [(3)H]PAH uptake was competitively inhibited by NAC (K(i) of 2.0 +/- 0.3 mM) and DMPS (K(i) of 0.10 +/- 0.02 mM), providing further evidence that these chelating agents are substrates for Oat1. These results indicate that the MeHg antidotes NAC and DMPS and their mercaptide complexes are transported by Oat1 but are comparatively poor substrates for Oat3. This is the first molecular identification of a transport mechanism by which these antidotes may enhance urinary excretion of toxic metals.     

Mercapturic acids (N-acetylcysteine S-conjugates) as endogenous substrates for the renal organic anion transporter-1

Mercapturic acids are N-acetyl-L-cysteine S-conjugates that are formed from a range of endogenous and exogenous chemicals. Although the kidney is a major site for elimination of mercapturic acids, the transport mechanisms involved have not been identified. The present study examined whether mercapturic acids are substrates for the renal basolateral organic anion transporter-1 (Oat1) from rat kidney. This carrier mediates uptake of organic anions from the bloodstream in exchange for intracellular alpha-ketoglutarate. Uptake of [(3)H]p-aminohippuric acid (PAH) in Oat1-expressing Xenopus laevis oocytes was strongly inhibited by S-(2,4-dinitrophenyl)-N-acetyl-L-cysteine (DNP-NAC) and by all other mercapturic acids tested, including the endogenous mercapturic acid N-acetyl-leukotriene E(4). Inhibition by the mercapturic acids was competitive, which is consistent with the hypothesis that these compounds are substrates for Oat1. This conclusion was supported by the direct demonstration of saturable [(35)S]DNP-NAC uptake in Oat1-expressing oocytes. [(35)S]DNP-NAC uptake was inhibited by PAH and other mercapturic acids and was stimulated in oocytes preloaded with glutarate. The apparent K(m) value for DNP-NAC uptake was only 2 microM, indicating that this mercapturic acid is a high affinity substrate for Oat1. Together, these data indicate that clearance of endogenous mercapturic acids is an important function of the renal organic anion transporter.     

Renal transport of organic compounds mediated by mouse organic anion transporter 3 (mOat3): further substrate specificity of mOat3.

Organic anion transporter 3 [Oat3(Slc22a8)] plays an important role in the renal handling of organic compounds. The substrate specificity of rat Oat3 and human Oat3 has been elucidated; information on mouse Oat3 (mOat3) is less defined. The aim of this study was to extend the substrate selectivity of mOat3. When expressed in Xenopus laevis oocytes, mOat3 mediated the uptake of p-aminohippuric acid and estron sulfate (ES). In addition to these substrates, we found that several organic compounds such as prostaglandin E(2), prostaglandin F(2alpha), allopurinol, 6-mercaptopurine (6-MP), 5-fluorouracil (5-FU), and l-carnitine are substrates of mOat3, compounds identified for the first time. The apparent K(m) values for the uptake of mOat3 that mediated the transport of 6-MP, 5-FU, and l-carnitine were 4.01 +/- 0.7 microM, 53.9 +/- 8.9 nM, and 61.9 +/- 1.1 nM, respectively. Northern blot analysis revealed that gene coding for mOat3 is predominant in the kidney and, to a lesser extent, in the brain and the eye. Our findings thus provide further insights into the role of Oat3 in renal drug transport.     

Interactions of amoxicillin and cefaclor with human renal organic anion and peptide transporters.

Amoxicillin and cefaclor are two of the widely used beta-lactam antibiotics in the treatment of urinary tract infections. Both drugs are eliminated mainly by the kidney and rely on renal excretion to exert their antibacterial activities in the urinary tract. Previous studies have suggested the involvement of organic anion and oligopeptide transporters in membrane transport of beta-lactams. The objective of the current study was to examine the kinetics of amoxicillin and cefaclor interactions with human renal transporters human organic anion transporter 1 (hOAT1), human peptide transporter 1 (hPepT1), and human peptide transporter 2 (hPepT2) in detail, both as substrates and as inhibitors. Using fluorescence protein tagging and cell sorting, we established Madin-Darby canine kidney cell lines stably expressing highly functional hOAT1, hPepT1, and hPepT2. Amoxicillin and cefaclor inhibited hOAT1-mediated [(3)H]para-aminohippuric acid uptake (K(i) = 11.0 and 1.15 mM, respectively). However, our uptake study revealed that neither drug was transported by hOAT1. Amoxicillin and cefaclor competitively inhibited hPepT2-mediated [(3)H]glycylsarcosine uptake (K(i) = 733 and 65 muM, respectively), whereas much lower affinity for hPepT1 was observed with both antibiotics. Direct uptake studies demonstrated that amoxicillin and cefaclor were transported by hPepT1 and hPepT2. Kinetic analysis showed that hPepT2-mediated uptake of both drugs was saturable with K(m) of 1.04 mM for amoxicillin and 70.2 muM for cefaclor. hPepT2, and to a lesser extent hPepT1, may play an important role in apical transport of amoxicillin and cefaclor in the renal tubule. hOAT1, in contrast, is not involved in basolateral uptake of these antibiotics.     

Transport of prostaglandin E1 across the blood-brain barrier in rats

The transport of prostaglandin E(1) (PGE(1)) across the blood-brain barrier (BBB) was characterized using an in-situ rat brain perfusion technique. The uptake of [(3)H]PGE(1) was not affected by shortchain monocarboxylic acids (butyric acid and valeric acid). On the other hand, uptake of [(3)H]PGE(1) was significantly inhibited by medium-chain monocarboxylic acids such as hexanoic acid, enanthic acid and octanoic acid. These medium-chain monocarboxylic acids showed a more potent inhibitory effect on [(3)H]PGE(1) uptake with increasing number of carbon atoms. In contrast, there was no decrease in [(3)H]PGE(1) transport by any dicarboxylic acids with 5-8 carbon atoms. Valproic acid decreased [(3)H]PGE(1) uptake, whereas p-aminohippuric acid, a substrate for the organic anion transporter family, did not inhibit [(3)H]PGE(1) transport. Bromocresol green, an inhibitor of prostaglandin transporter (PGT), strongly decreased [(3)H]PGE(1) transport across the BBB. In addition, digoxin and taurocholate, substrates for organic anion transporting polypeptide subtype 2 (Oatp2), significantly inhibited [(3)H]PGE(1) uptake. RT-PCR analysis revealed that PGT mRNA and Oatp2 mRNA are expressed in a capillary-rich fraction from rat brain. Thus, it is suggested that PGE(1) transport across the BBB is mediated by some specific transport systems, possibly by the members of the Oatp family.     

Mouse organic anion transporter 2 (mOat2) mediates the transport of short chain fatty acid propionate

In this study, we have elucidated that propionate, one of the short chain fatty acids (SCFAs), is the transport substrate for murine organic anion transporter 2 (mOat2), which is expressed in the kidneys and the liver. When expressed in Xenopus oocytes, mOat2-mediated [(3)H]PGE(2) transport was inhibited by three- to five-carbon SCFAs (C3 to C5). Among the SCFAs tested, propionate (3-carbon SCFA) was transported by mOat2 in a time-dependent manner. Since propionate is a potent glucogenic compound, Oat2 may be involved in the regulation of cellular metabolism through the transport of these metabolites in the kidneys and the liver.     

Molecular cloning and functional characterization of a novel gene encoding human prostaglandin carrier, hPrC

In the present study, we isolated and determined the pharmacological characteristics of a novel gene encoding the human prostaglandin carrier (hPrC). The isolated cDNA consisted of 1431 base pairs that encoded a 477-amino acid protein, and we found that isolated hPrC does not belong to any drug transporter families. RT-PCR analysis revealed that the hPrC mRNA is expressed in various human tissues ubiquitously. When expressed in Xenopus laevis oocytes, hPrC mediated the transport of [(3)H]prostaglandin E(2) (PGE(2)) in a sodium-independent manner. The uptake of [(3)H] PGE(2) was not trans-stimulated by PG analogous. Although there are several PG transporters such as multidrug resistance-associated protein 4 (MRP4), organic cation transporter 1 (OCT1) [solute carrier (SLC) 22A1], organic anion transporter 1-3 (OAT1-3) [SLC22A6-8], OAT4 [SLC11], OATP-1 (LST-1) [SLCO1B1], OATP2B1 [SLCO2B1], OATP2A1 (PGT) [SLCO2A1], OATP4A1 (OATP-E) [SLCO4A1] have been isolated and well characterized, our findings suggest that hPrC functions as a novel transport peptide responsible for PG uptake. Our results should provide insight into the novel mechanism of the PG transport in the human body.     

The dietary polyphenol ellagic acid is a potent inhibitor of hOAT1.

Ellagic acid (EA), a polyphenol present in berries, has been demonstrated to be preventive of esophageal and colon cancer in animals. Here, we have studied the ability of organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs) to transport EA. The accumulation of radiolabeled (14)C]EA, [(3)H]p-aminohippuric acid (PAH), [(14)C]glutarate, [(3)H]estrone sulfate, [(3)H]ochratoxin A, and [(3)H]taurocholic acid +/- inhibitor(s) was tested in OAT- and OATP-expressing oocytes. Oocytes expressing human (h)OAT1, rat (r)Oat1, and hOAT4 accumulated 6.5-, 7.1-, and 8.9-fold more EA, respectively, than did water-injected oocytes. This accumulation was prevented by the prototype OAT inhibitors bromosulfophthalein and probenecid. rOatp1, mouse (m)Oat2, hOAT3, and mOat5 showed no EA transport. The uptake of the prototype OAT substrate PAH in hOAT1-expressing oocytes was dose dependently and potently inhibited by EA with an IC(50)of 207 nM. In conclusion, we have demonstrated that the OAT family members hOAT1, rOat1, and hOAT4 mediate transport of EA, with a very high affinity for hOAT1.     

Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake

The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [(14)C]ascorbic acid ([(14)C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di-substituted benzenes. Transepithelial transport of [(14)C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase-mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a K(m) of 83.2muM and V(max) of 94.2pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy-dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca(2+)/CaM mediated pathways. [(14)C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group, i.e., halogen- and nitro-substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A-B) as well as basal to apical side (B-A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile-substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs.     

Ribosomal protein L3 mediated the transport of digoxin in Xenopus laevis oocyte

Ribosomal protein L3 (RPL3) is known to be an indispensable and essential component for the peptidyltransferase center. In the present study, we found a novel function of RPL3 using a Xenopus laevis oocyte expression system. When expressed in X. oocytes, RPL3 mediated the high affinity transport of [(3)H]digoxin (K(m) = 213.3 ± 46.8 nM) in a time-, concentration-, and sodium-dependent manners. The maximum velocity of the transport of [(3)H]digoxin via RPL3 produced at physiological pH. However, we did not observe RPL3-mediated transport of several organic solutes such as [(14)C]androstenedione, [(3)H]dexamethasone, [(3)H]dehydroepiandrosterone sulfate, [(3)H]L-tryptophan, [(14)C]L-ascorbic acid, [(14)C]α-ketoglutarate, [(14)C]glutarate, [(3)H]methotrexate, [(3)H]bumetanide, [(3)H]probenecid, [(14)C]salicylic acid, [(14)C]theophylline and [(3)H]valproate. Our results suggest that RPL3 functions as a drug carrier protein and may be involved in the digoxin toxicity in the human body.     

Oatp2 mediates bidirectional organic solute transport: a role for intracellular glutathione

One member of the OATP family of transporters, rat Oatp1, functions as an anion exchanger that is driven in part by the glutathione (GSH) electrochemical gradient, indicating that other OATP-related transporters may also be energized by this mechanism. The present study examined whether rat Oatp2 is also an anion exchanger, and, if so, whether it is energized by the GSH electrochemical gradient. As with Oatp1, uptake of 10 microM [(3)H]taurocholate in Oatp2-expressing Xenopus laevis oocytes was trans-stimulated by intracellular 0.2 mM unlabeled taurocholate, indicating bidirectional transport. Interestingly, [(3)H]taurocholate uptake in Oatp2-expressing oocytes was also trans-stimulated when oocytes were preloaded with GSH, S-methylglutathione, S-sulfobromophthalein-glutathione, S-dinitrophenyl glutathione, or ophthalmic acid (a GSH analog) but not by glutarate or N-acetylcysteine, suggesting that GSH derivatives and conjugates may function as intracellular substrates for Oatp2. Support for this hypothesis was provided by the demonstration of enhanced [(3)H]GSH and [(3)H]S-(2,4-dinitrophenyl)-glutathione efflux in Oatp2-expressing oocytes. However, in contrast to Oatp1, extracellular GSH failed to cis-inhibit uptake of [(3)H]taurocholate or [(3)H]digoxin in Oatp2-expressing oocytes, indicating that the stimulatory effect of high intracellular GSH concentrations is not due to a coupled exchange mechanism. Taken together, the results indicate that Oatp2 mediates bidirectional transport of organic anions by a GSH-sensitive facilitative diffusion mechanism and suggest that this transporter may play a role in cellular export of specific organic molecules.     

Contribution of organic anion transporting polypeptide OATP-C to hepatic elimination of the opioid pentapeptide analogue [D-Ala2, D-Leu5]-enkephalin

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [(3)H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats. The uptake of [(3)H]DADLE by the isolated hepatocytes was inhibited by several organic anions and pentapeptides, but not by tetra- or tripeptides. When OATP-C was expressed in Xenopus laevis oocytes, a significant increase in uptake of [(3)H]DADLE was observed. Moreover, the inhibitory effects of various compounds, including some peptides, on [(3)H]estrone-3-sulfate uptake by OATP-C were similar to those observed in [(3)H]DADLE uptake by rat isolated hepatocytes. In conclusion, it was demonstrated that OATP-C contributes to the rapid hepatic excretion of peptides and peptide-mimetic drugs.     

Transport and uptake of nateglinide in Caco-2 cells and its inhibitory effect on human monocarboxylate transporter MCT1

1 Nateglinide, a novel oral hypoglycemic agent, rapidly reaches the maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the gastrointestinal tract. The aim of this work is to clarify the intestinal absorption mechanism of nateglinide by means of in vitro studies. 2 We examined the transcellular transport and the apical uptake of [(14)C]nateglinide in a human colon carcinoma cell line (Caco-2). We also examined whether nateglinide is transported via monocarboxylate transport-1 (MCT1) by means of an uptake study using MCT1-expressing Xenopus laevis oocytes. 3 In Caco-2 cells, the transcellular transport of [(14)C]nateglinide from the apical to basolateral side was greater than that in the opposite direction. The uptake of [(14)C]nateglinide from the apical side was concentration-dependent, H(+)-dependent, and Na(+)-independent. Kinetic analysis revealed that the Kt and Jmax values of the initial uptake rate of [(14)C]nateglinide were 448 micro M and 43.2 nmol mg protein(-1) 5 min(-1), respectively. Various monocarboxylates, including salicylic acid and valproic acid, and glibenclamide significantly inhibited the uptake of [(14)C]nateglinide. 4 The uptake study using MCT1-expressing oocytes showed that nateglinide inhibits the MCT1-mediated uptake of [(14)C]L-lactic acid, though nateglinide itself is not transported by MCT1. 5 Taken together, these results suggest that the uptake of nateglinide from the apical membranes of Caco-2 cells is, at least in part, mediated by a proton-dependent transport system(s) distinct from MCT1.     

Characterization of prostaglandin E1 transport in rat renal brush-border membrane

Transport of prostaglandin E(1) (PGE(1)) was investigated in rat renal brush-border membrane vesicles. The uptake of [(3)H]PGE(1) was sensitive to osmosis and temperature. This uptake was saturable and mediated by high-affinity (K(m)=2.1 microM)/low-capacity (V(max)=17.4 pmol/mg protein/30 sec) and low-affinity (K(m)=526.5 microM)/high-capacity (V(max)=1,032.5 pmol/mg protein/30 sec) transport systems. [(3)H]PGE(1) uptake was Na(+)-independent and inhibited by various eicosanoids including PGE(2) and PGF(2alpha). Bromcresol green and sulfobromophthalein, potent inhibitors of prostaglandin transporter (PGT), significantly decreased [(3)H]PGE(1) uptake. Uptake was also inhibited by indomethacin and probenecid, which reportedly have little effect on PGT. Benzylpenicillin and taurocholate decreased the uptake of [(3)H]PGE(1). Like p-[(14)C]aminohippurate (PAH) uptake by vesicles, the uptake of [(3)H]PGE(1) was stimulated by an inside-positive membrane potential, created by applying an inward K(+) gradient and valinomycin. However, the uptake of [(3)H]PGE(1) was not inhibited by PAH, suggesting that PAH and PGE(1) are transported by separate transport systems. [(3)H]PGE(1) uptake was not stimulated by outwardly directed gradients of Cl(-) nor unlabeled PGE(1), indicating that an anion exchanger may not be involved in PGE(1) transport. These findings suggest that the transport of PGE(1) in rat renal brush-border membrane is mediated by specific transport system(s), at least in part, by a potential-sensitive transport system.     

Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2

Vectorial transport of endogenous substances, drugs, and toxins is an important function of polarized cells. We have constructed a double-transfected Madin-Darby canine kidney (MDCK) cell line permanently expressing a recombinant uptake transporter for organic anions in the basolateral membrane and an ATP-dependent export pump for anionic conjugates in the apical membrane. Basolateral uptake was mediated by the human organic anion transporter 8 (OATP8; symbol SLC21A8) and subsequent apical export by the multidrug resistance protein 2 (MRP2; symbol ABCC2). Under physiological conditions, both transport proteins are strongly expressed in hepatocytes and contribute to the hepatobiliary elimination of organic anions. Expression and localization of OATP8 and MRP2 in MDCK cells growing on Transwell membrane inserts was demonstrated by immunoblotting and confocal laser scanning microscopy. (3)H-Labeled sulfobromophthalein (BSP) was a substrate for both transport proteins and was transferred from the basolateral to the apical compartment at a rate at least six times faster by double-transfected MDCK-MRP2/OATP8 cells than by single-transfected MDCK-OATP8 or MDCK-MRP2 cells. Vectorial transport at a much higher rate by double-transfected than by single-transfected cells was also observed for the (3)H-labeled substrates leukotriene C(4), 17 beta-glucuronosyl estradiol, and dehydroepiandrosterone sulfate, for the fluorescent anionic substrate fluo-3, and for the antibiotic rifampicin. Inhibition studies indicated that intracellular formation of S-(2,4-dinitrophenyl)-glutathione from 2,4-chlorodinitrobenzene selectively inhibits the transcellular transport of [(3)H]BSP at the site of MRP2-mediated export. The double-transfected cells provide a useful system for the identification of transport substrates and transport inhibitors including drug candidates.     

Characterization of renal tubular apical efflux of zonampanel, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, in humans

1. Zonampanel, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist, is mainly excreted unchanged via renal tubular secretion. The renal apical transport transport of zonampanel was examined in this study using HEK293 cells expressing human organic anion transporter 4 (OAT4/SLC22A11), and membrane vesicles prepared from Sf-9 insect cells expressing human multidrug resistance-associated protein 2 (MRP2/ABCC2), MRP4 (ABCC4), and breast cancer resistance protein (BCRP/ABCG2). 2. Glutaric acid, a model dicarboxylate, trans-stimulated the uptake of [(14)C]zonampanel by OAT4, suggesting that zonampanel was transported by OAT4 via an exchange with dicarboxylate. Considering the endogenous dicarboxylate gradient, OAT4 seems to transport zonampanel in the direction of reabsorption rather than secretion. For MRP2, MRP4, and BCRP, zonampanel selectively inhibited the activity of MRP4 (K(i) = 41.3 microM). Marked transport of [(14)C]zonampanel was observed only for MRP4 (K(m) = 33.7 microM). 3. In conclusion, the data indicate that MRP4 was the apical efflux transporter that contributed to the active renal tubular secretion of zonampanel in humans, in concert with the apical reabsorption transporter OAT4 and basolateral uptake transporters.     

The organic anion transport polypeptide 1d1 (Oatp1d1) mediates hepatocellular uptake of phalloidin and microcystin into skate liver

Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [(3)H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km approximately 0.4 microM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki approximately 150 microM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km approximately 2.2 microM) and microcystin-LR (Km approximately 27 microM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostalpha/beta, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin.     

High-affinity interaction of sartans with H+/peptide transporters

Sartans are very effective drugs for treatment of hypertension, heart failure, and other cardiovascular disorders. They antagonize the effects of angiotensin II at the AT(1) receptor and display p.o. bioavailability rates of 13 to 80%. Because some sartans sterically resemble dipeptide derivatives, we investigated whether they are transported by peptide transporters. We first assessed the effects of sartans on [(14)C]glycylsarcosine uptake into Caco-2 cells expressing H(+)/peptide transporter (PEPT) 1 and into SKPT cells expressing PEPT2. Losartan, irbesartan, valsartan, and eprosartan inhibited [glycine-1-(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells in a competitive manner with K(i) values of 24, 230, 390, and >1000 microM. Losartan and valsartan also strongly inhibited the total transepithelial flux of [(14)C]Gly-Sar across Caco-2 cell monolayers. In SKPT cells, [(14)C]Gly-Sar uptake was inhibited with K(i) values of 2.2 microM (losartan), 65 microM (irbesartan), 260 microM (valsartan), and 490 microM (eprosartan). We determined by the two-electrode voltage-clamp technique whether the compounds elicited transport currents by PEPT1 or PEPT2 when expressed in Xenopus laevis oocytes. No currents were observed for any of the sartans, but the compounds strongly and reversibly inhibited peptide-induced currents. Uptake of valsartan, losartan, and cefadroxil was quantified in HeLa cells after heterologous expression of human PEPT1 (hPEPT1). In contrast to cefadroxil, no PEPT1-specific uptake of valsartan and losartan was found. We conclude that the sartans tested in this study display high-affinity interaction with PEPTs but are not transported themselves. However, they strongly inhibit hPEPT1-mediated uptake of dipeptides and cefadroxil.     

Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition

The L-type amino acid transporter 1 (LAT1) is an Na(+)-independent neutral amino acid transporter subserving the amino acid transport system L. Because of its broad substrate selectivity, system L has been proposed to be responsible for the permeation of amino acid-related drugs through the plasma membrane. To understand the mechanisms of substrate recognition, we have examined the LAT1-mediated transport using a Xenopus laevis oocyte expression system. LAT1-mediated [(14)C]phenylalanine uptake was strongly inhibited in a competitive manner by aromatic-amino acid derivatives including L-dopa, alpha-methyldopa, melphalan, triiodothyronine, and thyroxine, whereas phenylalanine methyl ester, N-methyl phenylalanine, dopamine, tyramine, carbidopa, and droxidopa did not inhibit [(14)C]phenylalanine uptake. Gabapentin, a gamma-amino acid, also exerted a competitive inhibition on LAT1-mediated [(14)C]phenylalanine uptake. Although most of the compounds that inhibited LAT1-mediated uptake were able to induce the efflux of [(14)C]phenylalanine preloaded to the oocytes expressing LAT1 through the obligatory exchange mechanism, melphalan, triiodothyronine, and thyroxine did not induce the significant efflux. Based on the experimental and semiempirical computational analyses, it is proposed that, for an aromatic amino acid to be a LAT1 substrate, it must have a free carboxyl and an amino group. The carbonyl oxygen closer to the amino group needs a computed charge of -0.55 approximately -0.56 and must not participate in hydrogen bonding. In addition, the hydrophobic interaction between the substrate side chain and the substrate binding site of LAT1 seems to be crucial for the substrate binding. A substrate, however, becomes a blocker once Connolly accessible areas become large and/or the molecule has a high calculated logP value, such as those for melphalan, triiodothyronine, and thyroxine.