The biological monitoring of prenatal exposure to methylmercury.

Several biological media have been used as indicators of the fetal body burden of methylmercury and the levels in the primary target tissue, the developing brain. These media include maternal hair and blood. The relative merits of these media will be considered both with regard to current knowledge of the physiology of mercury disposition in the body and also the practicality of field application with respect to sample, collection, transport, storage and processing.

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.

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.

Intracellular glutathione regulates taurocholate transport in HepG2 cells.

The hepatic organic anion transporter 1, Oatp1, was recently demonstrated to function as a GSH exchanger, indicating that hepatic uptake of drugs and xenobiotics may be sensitive to intracellular GSH levels. The present study characterized taurocholate uptake and efflux mechanisms in HepG2 cells and the effects of intracellular GSH on these transport processes. Taurocholate uptake into HepG2 cells was Na(+)-independent, saturable ( K(m) = 82 +/- 16 microM), and was cis-inhibited by bromosulfophthalein and some bile acids. Intracellular GSH depletion inhibited 3H-taurocholate uptake, and, conversely, the release of GSH from HepG2 cells was stimulated in the presence of extracellular taurocholate and other bile acids, consistent with a role for intracellular GSH in stimulating organic anion uptake. Interestingly, efflux of 3H-taurocholate from HepG2 cells was also sensitive to intracellular GSH concentration: efflux was inhibited in cells with lower intracellular GSH and stimulated in cells with higher GSH. RT-PCR analysis revealed that OATP-A, OATP-D, OATP-E, OATP-8, MRP1, MRP2, and MRP3 are expressed in HepG2 cells but that their expression is not altered by the maneuvers used to lower or raise intracellular GSH. These results provide direct evidence that intracellular GSH levels modulate both uptake and efflux of taurocholate and suggest that GSH plays a regulatory role in the hepatobiliary transport of potentially toxic organic compounds.

Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations.

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 microM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.

Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate.

Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter alpha and beta (Ostalpha, Ostbeta). Ostalpha cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ostbeta contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ostalpha and Ostbeta, and only low identity with sequences in the databases; however, Ostalpha bears a resemblance to some G protein-coupled receptors, and Ostbeta exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-gamma, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ostalpha and Ostbeta, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E(2), but not p-aminohippurate or S-dinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide.

gamma-Glutamyl transpeptidase and l-cysteine regulate methylmercury uptake by HepG2 cells, a human hepatoma cell line.

Mechanisms of methylmercury (MeHg) and inorganic mercury (Hg) uptake were examined in HepG2 cells, a human hepatoma-derived cell line. MeHg uptake was faster when it was present as the l-cysteine complex, as compared to the glutathione (GSH), CysGly, gamma-GluCys, d-cysteine, N-acetylcysteine, l-penicillamine, or albumin complexes. Uptake of MeHg-l-cysteine was independent of Na(+), stereoselective, and was inhibited by the amino acid transport system l substrates l-leucine, l-valine, and l-phenylalanine (5 mM). Moreover, [(3)H]l-leucine uptake was inhibited by MeHg-l-cysteine, suggesting that MeHg-l-cysteine is transported into HepG2 cells by an l-type amino acid carrier. Uptake of MeHg as the GSH complex (MeHg-SG) was dependent on the extracellular GSH concentration, and was diminished when cellular gamma-glutamyl transpeptidase activity was inhibited. Inorganic mercury uptake was slower than that of MeHg, but was also sensitive to the type of thiol ligand present. These findings demonstrate that mercury uptake by HepG2 cells is dependent on the chemical structure of the mercury compound, the thiol ligand, and the activity of gamma-glutamyl transpeptidase. gamma-Glutamyl transpeptidase appears to play a key role in the disposition of MeHg-SG by facilitating the formation of MeHg-l-cysteine, which is readily transported into the cells on an amino acid-type carrier.

ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue.

Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using (3)H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant (K(m)) = 32 +/- 9 microM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of (3)H-labeled S-ethylglutathione and (3)H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (K(m) = 12 +/- 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.

A primitive ATP receptor from the little skate Raja erinacea.

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skate Raja erinacea express a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61-64% similarity to P2Y(1) receptors and 21-37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopus oocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y(1) receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.

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.

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump.

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.

Hepatic toxicity and persistence of ser/thr protein phosphatase inhibition by microcystin in the little skate Raja erinacea.

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 microg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 microg/kg microcystin. Mortality occurred at doses of 500 microg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 microg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitro microcystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.

Carrier-mediated uptake of lucifer yellow in skate and rat hepatocytes: a fluid-phase marker revisited.

Uptake of lucifer yellow (LY), a fluorescent disulfonic acid anionic dye, was studied in isolated skate (Raja erinacea) perfused livers and primary hepatocytes to evaluate its utility as a fluid-phase marker in these cells. However, our findings demonstrated that LY is transported across the plasma membrane of skate hepatocytes largely via carrier-mediated mechanisms. Isolated perfused skate livers cleared 50% of the LY from the recirculating perfusate within 1 h of addition of either 22 or 220 microM LY, with only 4.5 and 9% of the LY remaining in the perfusate after 7 h, respectively. Most of the LY was excreted into bile, resulting in high biliary LY concentrations (1 and 10 mM at the two doses, respectively), indicating concentrative transport into bile canalicular lumen. LY uptake by freshly isolated skate hepatocytes was temperature sensitive, exhibited saturation kinetics, and was inhibited by other organic anions. Uptake was mediated by both sodium-dependent [Michaelis-Menten constant (K(m)), 125 +/- 57 microM; maximal velocity (V(max)), 1.5 +/- 0.2 pmol. min(-1). mg cells(-1)] and sodium-independent (K(m), 207 +/- 55 microM; V(max), 1.7 +/- 0.2 pmol. min(-1). mg cells(-1)) mechanisms. Both of these uptake mechanisms were inhibited by various organic anions and transport inhibitors, including furosemide, bumetanide, sulfobromophthalein, rose bengal, probenecid, N-ethylmaleimide, taurocholate, and p-aminohippuric acid. Fluorescent imaging techniques showed intracellular vesicular compartmentation of LY in skate hepatocyte clusters. Studies in perfused rat livers also indicated that LY is taken up against a concentration gradient and concentrated in bile. LY uptake in isolated rat hepatocytes was saturable, but only at high concentrations, and demonstrated a K(m) of 3.7 +/- 1.0 mM and a V(max) of 1.75 +/- 0.16 nmol. min(-1). mg wet wt(-1). These results indicate that LY is transported into skate and rat hepatocytes and bile largely by carrier-mediated mechanisms, rather than by fluid-phase endocytosis.

ATP-dependent transport of reduced glutathione on YCF1, the yeast orthologue of mammalian multidrug resistance associated proteins.

The transport systems involved in the export of cellular reduced glutathione (GSH) have not been identified, although recent studies implicate a role for some of the multidrug resistance associated proteins (MRP), including MRP1 and MRP2. The present study examined the hypothesis that the yeast orthologue of MRP, Ycf1p, mediates ATP-dependent GSH transport. [3H]GSH transport was measured in vacuolar membrane vesicles isolated from a control strain of Saccharomyces cerevisiae (DTY165), the isogenic DTY167 strain that lacks a functional Ycf1p, and in DTY167 transformed with a 2-micrometer plasmid vector containing YCF1. GSH transport in control vacuolar membrane vesicles was mediated largely by an ATP-dependent, low affinity pathway (Km = 15 +/- 4 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast Ycf1p transporter and inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, probenecid, and sulfinpyrazone, inhibitors of MRP1 and MRP2, but was minimally affected by membrane potential or pH gradient uncouplers. In contrast, ATP-dependent GSH transport was not seen in vacuolar membrane vesicles isolated from the DTY167 yeast strain without a functional Ycf1p but was restored to near wild-type levels in the DTY167 strain transformed with YCF1 and expressing the vacuolar Ycf1p transporter. On the other hand, expression and functional activity of a bile acid transporter, Bat1p, and of the V-type ATPase were similar in all three yeast strains. These results provide direct evidence for ATP-dependent low affinity transport of GSH by the yeast Ycf1p transporter. Because of the structural and functional homology between Ycf1p and MRP1 and MRP2, these data support the hypothesis that GSH efflux from mammalian cells is mediated by these membrane proteins.

Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells.

Apolipoprotein B (apoB) mRNA editing involves a site-specific cytidine to uridine transition catalyzed by the cytidine deaminase, APOBEC-1, in the context of and regulated by a multi-protein-containing editosome. ApoB mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the amount of edited apoB mRNA in rat primary hepatocytes is markedly increased subsequent to transient treatment with ethanol in vitro. The apparent change in editing efficiency was dose-dependent (from 0.1%-2.4% initial ethanol dose) and occurred with rapid onset. The proportion of edited apoB mRNA was also markedly enhanced in a rat hepatoma cell line, McArdle RH7777 cells and in a stable McArdle cell line over-expressing APOBEC-1 by transient treatment with 2.5 % ethanol. In contrast, the apoB mRNA editing in a human hepatoma cell line, HepG2 cells and a stable HepG2 cell line over-expressing APOBEC-1 did not respond to ethanol treatment. The data support the possibility that editing activity is ethanol-responsive but suggest that this change is cell type-specific.

Efficient hepatic uptake and concentrative biliary excretion of a mercapturic acid.

The role of the liver in the disposition of circulating mercapturic acids was examined in anesthetized rats and in the isolated perfused rat liver using S-2,4-dinitrophenyl-N-acetylcysteine (DNP-NAC) as the model compound. When DNP-NAC was infused into the jugular vein (150 or 600 nmol over 60 min) it was rapidly and nearly quantitatively excreted as DNP-NAC into bile (42-36% of the dose) and urine (48-62% of dose). Some minor metabolites were detected in bile (<4%), with the major metabolite coeluting on HPLC with the DNP conjugate of glutathione (DNP-SG). Isolated rat livers perfused single pass with 3 microM DNP-NAC removed 72 +/- 9% of this mercapturic acid from perfusate. This rapid DNP-NAC uptake was unaffected by sodium omission, or by L-cysteine, L-glutamate, L-cystine, or N-acetylated amino acids, but was decreased by inhibitors of hepatic sinusoidal organic anion transporters (oatp), indicating that DNP-NAC is a substrate for these transporters. The DNP-NAC removed from perfusate was promptly excreted into bile, eliciting a dose-dependent choleresis. DNP-NAC itself constituted approximately 75% of the total dose recovered in bile, reaching a concentration of 9 mM when livers were perfused in a recirculating mode with an initial DNP-NAC concentration of 250 microM. Other biliary metabolites included DNP-SG, DNP-cysteinylglycine, and DNP-cysteine. DNP-SG was likely formed by a spontaneous retro-Michael reaction between glutathione and DNP-NAC. Subsequent degradation of DNP-SG by biliary gamma-glutamyltranspeptidase and dipeptidase activities accounts for the cysteinylglycine and cysteine conjugates, respectively. These findings indicate the presence of efficient hepatic mechanisms for sinusoidal uptake and biliary excretion of circulating mercapturic acids in rat liver and demonstrate that the liver plays a role in their whole body elimination.

ATP-dependent transport of reduced glutathione in yeast secretory vesicles.

Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized. The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae. In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19+/-5 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2, 4-dinitrophenyl)glutathione (DNP-SG). Similarly, GSH competitively inhibited ATP-dependent [3H]DNP-SG transport, with a Ki of 18+/-2 mM, but had no effect on ATP-dependent [3H]taurocholate transport. ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter. Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p. These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter.

Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter.

oatp1 is an hepatic sinusoidal organic anion transporter that mediates uptake of various structurally unrelated organic compounds from blood. The driving force for uptake on oatp1 has not been identified, although a role for bicarbonate has recently been proposed. The present study examined whether oatp1-mediated uptake is energized by efflux (countertransport) of intracellular reduced glutathione (GSH), and whether hydrophobic glutathione S-conjugates such as leukotriene C4 (LTC4) and S-dinitrophenyl glutathione (DNP-SG) form a novel class of substrates for oatp1. Xenopus laevis oocytes injected with the complementary RNA for oapt1 demonstrated higher uptake of 10 nM [3H]LTC4 and 50 microM [3H]DNP-SG, and higher efflux of [3H]GSH (2.5 mM endogenous intracellular GSH concentration). The oatp1-stimulated LTC4 and DNP-SG uptake was independent of the Na+ gradient, cis-inhibited by known substrates of this transport protein and by 1 mM GSH, and was saturable, with apparent Km values of 0.27 +/- 0.06 and 408 +/- 95 microM, respectively. Uptake of [3H]taurocholate, an endogenous substrate of oatp1, was competitively inhibited by DNP-SG. Of significance, oatp1-mediated taurocholate and LTC4 uptake was cis-inhibited and trans-stimulated by GSH, and [3H]GSH efflux was enhanced in the presence of extracellular taurocholate or sulfobromophthalein, indicating that GSH efflux down its large electrochemical gradient provides the driving force for uptake via oatp1. The stoichiometry of GSH/taurocholate exchange was 1:1. These findings identify a new class of substrates for oatp1 and provide evidence for GSH-dependent oatp1-mediated substrate transport.