OATP8/1B3-Mediated Cotransport of Bile Acids and Glutathione (original) (raw)
Related papers
Journal of Biological Chemistry, 2006
In cholestasis, the accumulation of organic anions in hepatocytes is reduced by transporters (multidrug resistance-associated proteins and OST␣-OST) able to extrude them across the basolateral membrane. Here we investigated whether organic anion-transporting polypeptides (OATPs) may contribute to this function. Xenopus laevis oocytes expressing human carboxylesterase-1 efficiently loaded cholic acid (CA) methyl ester, which was cleaved to CA and exported. Expression of OATP8/ 1B3 enhanced CA efflux, which was trans-activated by taurocholate but trans-inhibited by reduced (GSH) and oxidized (GSSG) glutathione. Moreover, taurocholate and estradiol 17-D-glucuronide, but not bicarbonate and glutamate, cis-inhibited OATP8/1B3-mediated bile acid transport, whereas glutathione cis-stimulated this process, which involved the transport of glutathione itself with a stoichiometry of 2:1 (GSH/bile acid). No cis-activation by glutathione of OATP-C/1B1 was found. Using real time quantitative reverse transcription-PCR, the absolute abundance of OATP-A/1A2, OATP-C/1B1, and OATP8/1B3 mRNA in human liver biopsies was measured. In healthy liver, expression levels of OATP-C/1B1 were ϳ5-fold those of OATP8/1B3 and >100-fold those of OATP-A/1A2. This situation was not substantially modified in several cholestatic liver diseases studied here. In conclusion, although both OATP-C/ 1B1 and OATP8/1B3 are highly expressed, and able to transport bile acids, their mechanisms of action are different. OATP-C/ 1B1 may be involved in uptake processes, whereas OATP8/1B3 may mediate the extrusion of organic anions by symporting with glutathione as a normal route of exporting metabolites produced by hepatocytes or preventing their intracellular accumulation when their vectorial traffic toward the bile is impaired.
Organic Anion Transporting Polypeptide 1a1 Null Mice Are Sensitive to Cholestatic Liver Injury
Toxicological Sciences, 2012
Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in livers of mice and is thought to transport bile acids (BAs) from blood into liver. Because Oatp1a1 expression is markedly decreased in mice after bile duct ligation (BDL). We hypothesized that Oatp1a1-null mice would be protected against liver injury during BDL-induced cholestasis due largely to reduced hepatic uptake of BAs. To evaluate this hypothesis, BDL surgeries were performed in both male wild-type (WT) and Oatp1a1-null mice. At 24 h after BDL, Oatp1a1-null mice showed higher serum alanine aminotransferase levels and more severe liver injury than WT mice, and all Oatp1a1-null mice died within 4 days after BDL, whereas all WT mice survived. At 24 h after BDL, surprisingly Oatp1a1-null mice had higher total BA concentrations in livers than WT mice, suggesting that loss of Oatp1a1 did not prevent BA accumulation in the liver. In addition, secondary BAs dramatically increased in serum of Oatp1a1-null BDL mice but not in WT BDL mice. Oatp1a1-null BDL mice had similar basolateral BA uptake (Na 1 -taurocholate cotransporting polypeptide and Oatp1b2) and BA-efflux (multidrug resistance-associated protein [Mrp]-3, Mrp4, and organic solute transporter a/b) transporters, as well as BA-synthetic enzyme (Cyp7a1) in livers as WT BDL mice. Hepatic expression of small heterodimer partner Cyp3a11, Cyp4a14, and Nqo1, which are target genes of farnesoid X receptor, pregnane X receptor, peroxisome proliferator-activated receptor alpha, and NF-E2-related factor 2, respectively, were increased in WT BDL mice but not in Oatp1a1-null BDL mice. These results demonstrate that loss of Oatp1a1 function exacerbates cholestatic liver injury in mice and suggest that Oatp1a1 plays a unique role in liver adaptive responses to obstructive cholestasis.
Localization and function of the organic anion–transporting polypeptide Oatp2 in rat liver
Gastroenterology, 1999
Background & Aims: Multispecific organic anion-transporting polypeptides (Oatps) are involved in the transcellular movement of amphipathic compounds in many tissues including the liver, kidney, and blood-brain barrier. Recently, a high-affinity digoxin transporter (Oatp2) was cloned from rat brain and shown to be also expressed in the liver. Methods: We investigated the cellular and subcellular distribution of Oatp2 in rat liver by in situ hybridization technology and immunofluorescence microscopy and compared its substrate specificity with that of Oatp1 in complementary RNA-injected Xenopus laevis oocytes. Results: The results show a selective basolateral (sinusoidal) expression of Oatp2 in midzonal to perivenous hepatocytes, but not in periportal or the innermost layer of perivenous hepatocytes. Common substrates of both Oatp1 and Oatp2 include bile salts, steroid conjugates, thyroid hormones (T3, T4), ouabain, and the endothelin receptor antagonist BQ-123 (Michaelis constants: Oatp1, D600 mol/L; Oatp2, D30 mol/L). Other organic anions including sulfolithotaurocholate, bilirubin monoglucuronide, and sulfobromophthalein were transported only by Oatp1. Conclusions: These results provide definite evidence for the partially overlapping and partially selective substrate specificities of Oatp1 and Oatp2. The unique acinar distribution of Oatp2 might indicate that it represents a high-affinity ''backup'' system for complete hepatocellular removal of certain cholephilic substances from portal blood plasma.
AJP: Gastrointestinal and Liver Physiology, 2007
Organic anion transporting polypeptides (OATP/ SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 ( Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 ( SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C ( SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected...
Localization and Genomic Organization of a New Hepatocellular Organic Anion Transporting Polypeptide
Journal of Biological Chemistry, 2000
Based on sequence homology to the human organic anion transporting polypeptide 2 (OATP2; SLC21A6), we cloned a new member of the SLC21A superfamily of solute carriers, termed OATP8 (SLC21A8). The protein of 702 amino acids showed an amino acid identity of 80% with human OATP2. Based on Northern blotting, the expression of OATP8 was restricted to human liver. Cosmid clones containing the genes encoding human OATP1 (SLC21A3), OATP2 (SLC21A6), and OATP8 (SLC21A8) served to establish their genomic organization. All three genes contained 14 exons with 13 identical splice sites when transferred to the amino acid sequence. An antibody raised against the carboxyl terminus localized OATP8 to the basolateral membrane of human hepatocytes and the recombinant glycoprotein, expressed in MDCKII cells, to the lateral membrane. Transport properties of OATP8 were studied in stably transfected MDCKII and HEK293 cells. Organic anions transported by human OATP8 included sulfobromophthalein, with a K m of 3.3 M, and 17-glucuronosyl estradiol, with a K m of 5.4 M. Several bile salts were not substrates. Thus, human OATP8 is a new uptake transporter in the basolateral hepatocyte membrane with an overlapping but distinct substrate specificity as compared with OATP2, which is localized to the same membrane domain. . The abbreviations used are: SLC, solute carrier superfamily; BSP, sulfobromophthalein; E 2 17G, 17-glucuronosyl estradiol; OATP, organic anion transporting polypeptide; PBS, phosphate-buffered saline; bp, base pair(s); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
Journal of Pharmacology and Experimental Therapeutics, 2005
The organic anion transporting polypeptides OATPs are key membrane transporters for which crystal structures are not currently available. They transport a diverse array of xenobiotics and are expressed at the interface of hepatocytes, renal tubular cells, enterocytes and the choroid plexus. To aid the understanding of the key molecular features for substratetransporter interactions, pharmacophore models were produced for the two OATPs that have been most extensively studied, namely rat Oatp1a1 and human OATP1B1. Literature data from CHO, HeLa, Hek-293 cells and X. laevis oocytes were used to construct pharmacophores for each individual transporter which were later merged to show similarities across cell lines for the same transporter. Additionally, meta-pharmacophores were generated from the combined datasets of each cell system used with the same transporter. The pharmacophores for each transporter consisted of hydrogen bond acceptor and hydrophobic features. There was good agreement between the merged and meta-pharmacophores containing 2 hydrogen bond acceptors and 2 or 3 hydrophobic features for Oatp1a1 and OATP1B1. External test sets were used to validate the individual pharmacophores. The metapharmacophores were also used to make predictions for molecules not included in the models and provided new molecular insight into the key features for these OATP transporters. This approach can be extended to other transporters for which limited data are available. Downloaded from JPET #82370 23 Kanai N, Lu R, Bao Y, Wolkoff AW, Vore M and Schuster VL (1996) Estradiol 17 beta-Dglucuronide is a high-affinity substrate for oatp organic anion transporter. Am J Physiol 270:F326-331. Kim RB (2003) Organic anion-transporting polypeptide (OATP) transporter family and drug disposition. Eur J Clin Invest 33:1-5. Konig J, Cui Y, Nies AT and Keppler D (2000) A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol 278:G156-164. Kontaxi M, Echkardt U, Hagenbuch B, Stieger B, Meier PJ and Petzinger E (1996) Uptake of the mycotoxin ochratoxin A in liver cells occurs via the cloned organic anion transporting polypeptide. J Pharmacol Exp Ther 279:1507-1513. Kouzuki H, Suzuki H, Ito K, Ohashi R and Sugiyama Y (1999) Contribution of organic anion transporting polypeptide to uptake of its possible substrates into rat hepatocytes. J Pharmacol Exp Ther 288:627-634. Kullack-Ublick G-A, Hagenbuch B, Stieger B, Wolkoff AW and Meier PJ (1994) Functional characterization of the basolateral rat liver organic anion transporting polypeptide. Hepatology 20:411-416. Li L, Lee TK, Meier PJ and Ballatori N (1998) Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter. J Biol Chem 273:16184-16191.
Adaptive responses of renal organic anion transporter 3 (OAT3) during cholestasis
AJP: Renal Physiology, 2008
Chen J, Terada T, Ogasawara K, Katsura T, Inui K. Adaptive responses of renal organic anion transporter 3 (OAT3) during cholestasis. During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of -lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis.
Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells
Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane. Double-transfected MDCKII cells stably expressing ABCC2 together with OATP1B1, OATP1B3, or OATP2B1 served as control cells. The quadruple-transfected cells exhibited high rates of vectorial transport of organic anions, including bromosulfophthalein, cholecystokinin peptide (CCK-8), and estrone 3-sulfate. The quadruple-transfected cells enabled the identifi-cation of substrates for uptake or vectorial transport that may be missed in studies with a double-transfected cell line, as exemplified by CCK-8, which is a substrate for OATP1B3 but not for OATP1B1 or OATP2B1. The broad substrate spectrum covered by the three hepatocellular OATP transporters enables representative analyses of the uptake of many organic anions into human hepatocytes. The broad spectrum of organic anions transported vectorially by the quadruple-transfected cells also provides valuable information on the substrate selectivity of ABCC2, without the need for studies in inside-out membrane vesicles containing the ABCC2 protein. The quadruple-transfected MDCKII-ABCC2/OATP1B1/1B3/2B1 cells may thus be useful for the identification of substrates and inhibitors, including drug candidates, undergoing uptake and secretion by human hepatocytes, under conditions that may be better defined than in primary cultures of human hepatocytes.
Functional characterization of the basolateral rat liver organic anion transporting polypeptide
Hepatology, 1994
To characterize the transport functions of a recently cloned basolateral organic anion transporting polypeptide of rat hepatocytes we performed further kinetic transport and substrate cis-inhibition studies in organic anion-transporting polypeptide—cRNA injected Xenopus laevis oocytes. The studies demonstrate saturable Na+-independent sulfobromophthalein (Michaelis-Menten constant, 1.5 μmol/L) and taurocholate (Michaelis-Menten constant, 50 μmol/L) uptake by organic anion-transporting polypeptide. Sulfobromophthalein uptake was inhibited by the following organic anions: 0.01 mmol/L bilirubin (43%), 0.1 mmol/L indocyanine green (81%), 0.1 mmol/L 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 52%) and 1 mmol/L probenecid (74%). Competitive inhibition was shown for indocyanine green (inhibition constant about 1.3 μmol/L). Sulfobromophthalein and taurocholate uptakes were also inhibited by cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate, as well as their glycine and taurine conjugates. Organic anion-transporting polypeptide also mediated uptake of glycocholate, tauroursodeoxycholate and taurochenodeoxycholate. No cis-inhibition of sulfobromophthalein uptake was seen in the presence of ATP, para-aminohippuric acid, bumetanide, digitoxin, reduced glutathione, leukotriene C4, nicotinic acid, ouabain, oxalate, rifampicin, succinate or sulfate. Furthermore, radioactively labeled paraaminohippuric acid, α-ketoglutarate and reduced glutathione were not taken up by organic aniontransporting polypeptide in cRNA-injected frog oocytes. These data confirm that organic aniontransporting polypeptide represents a novel hepatocellular organic anion uptake system that can mediate Na+-independent transport of monovalent (e.g., bile acids) and divalent (e.g., sulfobromophthalein and indocyanine green) cholephilic organic anions. A variety of substrates previously shown to inhibit uptake of sulfobromophthalein and bile acids in perfused rat livers, isolated hepatocytes and basolateral membrane vesicles had no cis-inhibitory effects on organic aniontransporting polypeptide—mediated organic anion transport in X. laevis oocytes. Thus additional Na+-independent organic anion carriers must be present in the basolateral membranes of rat hepatocytes. (Hepatology 1994;20:411-416.)