Taro Tokui

Identification of a Novel Gene Family Encoding Human Liver-specific Organic Anion Transporter LST-1

We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat, and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transports taurocholate (K m = 13.6 μm) in a sodium-independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17β-glucuronide, and estrone-3-sulfate), eicosanoids (prostaglandin E2, thromboxane B2, leukotriene C4, leukotriene E4), and thyroid hormones (thyroxine,K m = 3.0 μm and triiodothyronine,K m = 2.7 μm), reflecting hepatic multispecificity. LST-1 is probably the most important transporter in human liver for clearance of bile acids and organic anions because hepatic levels of another organic anion transporter, OATP, is very low. This is also the first report of the human molecule that transports thyroid hormones.

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3′,5-triiodo-l-thyronine (Km, 0.9μ m), thyronine, and rT3 in a Na+-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The ...

Molecular characterization and functional regulation of a novel rat liver-specific organic anion transporter rlst-1

BACKGROUND & AIMS Recently, we isolated a new complementary DNA (cDNA) encoding human liver-specific organic anion transporter (LST-1), representing the multispecificity of human liver. The aim of this study was to isolate a rat counterpart of human LST-1 and examine the expression regulation of its messenger RNA (mRNA) to clarify the molecular basis of cholestasis. METHODS A rat liver cDNA library was screened with human LST-1 cDNA as a probe. Xenopus oocyte expression system was used for functional analysis. Northern blot analyses were performed using the isolated cDNA (termed rlst-1). The bile duct ligation model and the cecum ligation and puncture model were used for expression analyses. RESULTS rlst-1 encodes 652 amino acids, predicting at least 11 transmembrane regions. The overall homology with human LST-1 was 60.2%, which is the highest among all known organic anion transporters. rlst-1 also belongs to the same new gene family as human LST-1, located between the organic anion transporter family and the prostaglandin transporter. rlst-1 preferably transports taurocholate (K(m), 9.45 micromol/L) in an Na(+)-independent manner. The rlst-1 mRNA is exclusively expressed in the liver. In both the bile duct ligation model and the cecum ligation and puncture model, mRNA expression levels of rlst-1 were down-regulated. CONCLUSIONS rlst-1 is a counterpart of human LST-1 and is one of the important transporters in rat liver for the clearance of bile acid. The expression of rlst-1 may be under feedback regulation of cholestasis by biliary obstruction and/or sepsis.

Thyroid hormone transporters: recent advances

Thyroid hormones, being hydrophobic, were thought to enter target cell membranes by passive diffusion. However, recent studies have documented the existence of numerous organic anion transport systems, about half of which also transport thyroid hormones into (and possibly out of) a variety of target cells. Several of the genes encoding thyroid hormone transporters have been characterized by means of molecular approaches. Here, we discuss the classification of thyroid hormone transporters, with emphasis on how they are influenced by their ionic milieu and what their symported organic anions are.

Immunohistochemical distribution and functional characterization of an organic anion transporting polypeptide 2 (oatp2)

The rabbit polyclonal antibody against rat organic anion transporting polypeptide 2 (oatp2) was raised and immunoaffinity‐purified. Western blot analysis for oatp2 detected two bands (∼74 and 76 kDa) in rat brain and a single band (76 kDa) in the liver. By immunohistochemical analysis, the oatp2 immunoreactivity was specifically high at the basolateral membrane of rat hepatocytes. Functionally, the oatp2‐expressing oocytes were found to transport dehydroepiandrosterone sulfate, δ1 opioid receptor agonist [d‐Pen2,d‐Pen5]enkephalin, Leu‐enkephalin, and biotin significantly, as well as the substrates previously reported. These data reveal the exact distribution of the rat oatp2 at the protein level in the liver, and that oatp2 appears to be involved in the multispecificity of the uptaking substrates in the liver and brain.

OATP1B1, OATP1B3, AND MRP2 ARE INVOLVED IN HEPATOBILIARY TRANSPORT OF OLMESARTAN, A NOVEL ANGIOTENSIN II BLOCKER

Hepatic uptake and biliary excretion of olmesartan, a new angiotensin II blocker, were investigated in vitro using human hepatocytes, cells expressing uptake transporters and canalicular membrane vesicles, and in vivo using Eisai hyperbilirubinemic rats (EHBR), inherited multidrug resistance-associated protein (mrp2)-deficient rats. The uptake by human hepatocytes reached saturation with a Michaelis constant (Km) of 29.3 ± 9.9 μM. Both Na+-dependent and Na+-independent uptake of olmesartan by human hepatocytes were observed. The uptake by Na+-independent human liver-specific organic anion transporters OATP1B1 and OATP1B3 expressed in Xenopus laevis oocytes was also saturable, with Km values of 42.6 ± 28.6 and 71.8 ± 21.6 μM, respectively. The Na+-dependent taurocholate-cotransporting polypeptide expressed in HEK 293 cells did not transport olmesartan. The cumulative biliary excretion in EHBR was one-sixth compared with that in Sprague-Dawley rats. ATP-dependent uptake of olmesartan was observed in both human canalicular membrane vesicles (hCMVs) and MRP2-expressing vesicles. An MRP inhibitor, MK-571 ([[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) completely inhibited the uptake of olmesartan by hCMVs. In conclusion, the hepatic uptake and biliary excretion of olmesartan are mediated by transporters in humans. OATP1B1 and OATP1B3 are involved in hepatic uptake, at least in part, and MRP2 plays a dominant role in the biliary excretion.

Pravastatin, an HMG-CoA Reductase Inhibitor, Is Transported by Rat Organic Anion Transporting Polypeptide, oatp2

AbstractPurpose. We previously demonstrated the HMG-CoA reductase inhibitor, pravastatin, is actively taken up into isolated rat hepatocytes through multispecific organic anion transporters. The present study examined whether a newly cloned organic anion transporting polypeptide (oatp2) transports pravastatin. Methods. We investigated functional expression of oatp2 in Xenopus laevis oocytes, to examine [14C] pravastatin uptake. Results. [14C] Pravastatin (30 μM) uptake into oatp2 cRNA-injected oocytes was 40 times higher than that of water-injected control oocytes. The oatp2-mediated pravastatin uptake was Na+-independent and saturable. The Michaelis-Menten constant was 37.5 ± 9.9 μM, a level comparable to that obtained in isolated rat hepatocytes in our previous study. As is the case with rat hepatocytes, the uptake of pravastatin (30 μM) was inhibited by 300 μM concentrations of taurocholate, cholate, bromosulfophthalein, estradiol-17β-glucuronide, and simvastatin acid, but not by para-aminohippurate. On the other hand, [14C] simvastatin acid (30 μM) uptake of oatp2 cRNA-injected oocytes was not significantly different from that of water-injected oocytes. Conclusions. The cloned oatp2 was identified as the transporter responsible for the active hepatocellular pravastatin uptake.

Carrier-mediated uptake of pravastatin by rat hepatocytes in primary culture

The transport mechanism of pravastatin, a new cholesterol-lowering drug, was compared in vitro with rat hepatocyte primary culture and mouse skin fibroblasts (L-cells). The uptake of 14C-labeled pravastatin by cultured hepatocytes was temperature- and dose-dependent. The temperature-dependent uptake as a function of [14C]pravastatin concentration showed saturation kinetics with Km = 32.2 microM and a maximal uptake rate of 68 pmol/mg protein/min. The uptake of pravastatin was inhibited significantly by metabolic inhibitors such as rotenone, oligomycin A, antimycin A, 2,4-dinitrophenol and KCN. Unlabeled pravastatin as well as R-416 and R-195, structural analogues of pravastatin, effectively competed for the hepatic uptake of [14C]pravastatin at 37 degrees. These results indicate that pravastatin is taken up by the liver by an active transport. In contrast, the transport of pravastatin by L-cells was temperature-independent and non-saturable, suggesting that the uptake of pravastatin by L-cells is mediated by passive diffusion. The marked difference in the uptake mechanism of pravastatin between hepatocytes and L-cells may account for a unique feature of this drug in that the uptake and inhibition of cholesterol biosynthesis occur selectively in the liver.

Inhibition of human organic anion transporter 3 mediated pravastatin transport by gemfibrozil and the metabolites in humans

Coadministration of gemfibrozil (600 mg, b.i.d., 3 days) with pravastatin (40 mg/day) decreased the renal clearance of pravastatin by approximately 40% in healthy volunteers. To investigate the mechanism of this drug–drug interaction in the renal excretion process, we undertook an uptake study of pravastatin using human organic anion transporters (hOATs)-expressing S2 cells. hOAT3 and hOAT4 transported pravastatin in a saturatable manner with Michaelis--Menten constants of 27.7 µM and 257 µM respectively. On the other hand, hOAT1 and hOAT2 did not transport pravastatin. Gemfibrozil and its glucuronide and carboxylic metabolite forms inhibited the uptake of pravastatin by hOAT3 with IC50 values of 6.8 µM, 19.7 µM and 5.4 µM, respectively. Considering the plasma concentrations of gemfibrozil and its metabolites in humans, the inhibition of hOAT3-mediated pravastatin transport by gemfibrozil and its metabolites would lead to a decrease in the renal clearance of pravastatin in clinical settings.

Gemfibrozil and its glucuronide inhibit the hepatic uptake of pravastatin mediated by OATP1B1

When pravastatin (40 mg/day) was co-administered with gemfibrozil (600 mg, b.i.d., 3 days) to man, the AUC of pravastatin increased approximately 2-fold. We have clarified that OATP1B1 is a key determinant of the hepatic uptake of pravastatin in humans. Thus, we hypothesized that gemfibrozil and the main plasma metabolites, a glucuronide (gem-glu) and a carboxylic acid metabolite (gem-M3), might inhibit the hepatic uptake of pravastatin and lead to the elevation of the plasma concentration of pravastatin. Gemfibrozil and gem-glu inhibited the uptake of 14C-pravastatin by human hepatocytes with Ki values of 31.7 µM and 15.7 µM, respectively and also inhibited pravastatin uptake by OATP1B1-expressing Xenopus laevis oocytes with Ki values of 15.1 µM and 7.6 µM. Additionally, we examined the biliary transport of pravastatin and demonstrated that pravastatin was transported by MRP2 using both human canalicular membrane vesicles (hCMVs) and human MRP2-expressing vesicles. However, gemfibrozil, gem-glu and gem-M3 did not affect the biliary transport of pravastatin by MRP2. Considering the plasma concentrations of gemfibrozil and gem-glu in humans, the inhibition of OATP1B1-mediated hepatic uptake of pravastatin by gem-glu would contribute, at least in part, to the elevation of plasma concentration of pravastatin by the concomitant use of gemfibrozil.