Enhanced Expression of the Human Multidrug Resistance Protein 3 by Bile Salt in Human Enterocytes (original) (raw)
Related papers
…, 1998
Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly downregulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine-induced oxidative damage. Therefore, we examined the expression of ABC-transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin-treated rats and performed reversetranscriptase polymerase chain reaction (RT-PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition, mdr1b mRNA was strongly increased, whereas mdr1a and mdr2 mRNA did not change significantly. Both the mRNA and protein levels of the sister of P-glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin-induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up-regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine-induced metabolic stress. (HEPATOLOGY 1998;28:1637-1644.)
European Journal of Gastroenterology & Hepatology, 1998
Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly downregulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine-induced oxidative damage. Therefore, we examined the expression of ABC-transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin-treated rats and performed reversetranscriptase polymerase chain reaction (RT-PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition, mdr1b mRNA was strongly increased, whereas mdr1a and mdr2 mRNA did not change significantly. Both the mRNA and protein levels of the sister of P-glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin-induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up-regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine-induced metabolic stress. (HEPATOLOGY 1998;28:1637-1644.) Excretion of a large variety of endogenous and exogenous compounds from hepatocytes into bile is an adenosine triphosphate (ATP)-dependent process, predominantly performed by members of the P-glycoprotein (Pgp) subfamily and the multidrug-resistance protein (MRP) subfamily of the ATP-binding cassette (ABC) protein superfamily. 1,2 At least four members of the Pgp subfamily are located at the canalicular membrane of rodent liver: mdr1a, mdr1b, mdr2, and spgp. In normal rodent liver, mdr1a and mdr1b are present at low levels. Overexpression of mouse mdr1a/mdr1b confers multidrug resistance against a broad variety of natural product drugs. 3,4 The main physiological function of these multidrug-resistance proteins is presumably the transport of bulky amphiphilic compounds, such as cationic drugs, hydrophobic peptides, steroids, and atypical glycolipids, across the canalicular membrane. 1,4,5 The expression of mdr2 in normal rodent liver is high. This transporter functions as a flippase that translocates phosphatidylcholine across the membrane. 6 From pig liver, Childs et al. cloned part of another member of the Pgp subfamily: the sister gene of Pgp (spgp). 7 Most recently, Gerloff et al. cloned the full-length cDNA of spgp from rat liver. 8 They provided evidence that spgp, which is exclusively present in the liver, most likely is the major canalicular bile salt export pump of mammalian liver. So far, the presence of four members of the mrp subfamily has been demonstrated in liver. 9,10 MRP1/mrp1 is present at very low levels at the lateral membrane of the hepatocyte. 11,12 The human MRP1 and its murine homologue, mrp1, have been shown to confer multidrug resistance to natural product drugs. 13,14 Moreover, MRP1 is able to transport glutathione disulfide 15 and glutathione (GSH) S-conjugates including the lipid peroxidation products, leukotriene C 4 16,17 and 4-hydroxynonenal S-GSH. 18 The recently cloned mrp1 homologue, mrp2, 19,20
Expression and regulation of hepatic drug and bile acid transporters
Toxicology, 2000
Transport across hepatocyte plasma membranes is a key parameter in hepatic clearance and usually occurs through different carrier-mediated systems. Sinusoidal uptake of compounds is thus mediated by distinct transporters, such as Na + -dependent or Na + -independent anionic transporters and by some cationic transporters. Similarly, several membrane proteins located at the apical pole of hepatocytes have been incriminated in the excretion of compounds into the bile. Indeed, biliary elimination of anionic compounds, including glutathione S-conjugates, is mediated by MRP2, whereas bile salts are excreted by a bile salt export pump (BSEP) and Class I-P-glycoprotein (P-gp) is involved in the secretion of amphiphilic cationic drugs, whereas class II-P-gp is a phospholipid transporter. The expression of hepatic transporters and their activity are regulated in various situations, such as ontogenesis, carcinogenesis, cholestasis, cellular stress and after treatment by hormones and xenobiotics. Moreover, a direct correlation between a defect and the absence of transporter with hepatic disease has been demonstrated for BSEP, MDR3-P-gp and MRP2.
Cholesterol modulates human intestinal sodium-dependent bile acid transporter
AJP: Gastrointestinal and Liver Physiology, 2005
Bile acids are efficiently absorbed from the intestinal lumen via the ileal apical sodium-dependent bile acid transporter (ASBT). ASBT function is essential for maintenance of cholesterol homeostasis in the body. The molecular mechanisms of the direct effect of cholesterol on human ASBT function and expression are not entirely understood. The present studies were undertaken to establish a suitable in vitro experimental model to study human ASBT function and its regulation by cholesterol. Luminal membrane bile acid transport was evaluated by the measurement of sodium-dependent 3H-labeled taurocholic acid (3H-TC) uptake in human intestinal Caco-2 cell monolayers. The relative abundance of human ASBT (hASBT) mRNA was determined by real-time PCR. Transient transfection and luciferase assay techniques were employed to assess hASBT promoter activity. Caco-2 cell line was found to represent a suitable model to study hASBT function and regulation. 25-Hydroxycholesterol (25-HCH; 2.5 μg/ml fo...
Journal of Biological Chemistry, 2003
The multidrug resistance proteins MRP2 (ABCC2) and MRP3 (ABCC3) are key primary active transporters involved in anionic conjugate and drug extrusion from the human liver. The major physiological role of MRP2 is to transport conjugated metabolites into the bile canaliculus, whereas MRP3 is localized in the basolateral membrane of the hepatocytes and transports similar metabolites back to the bloodstream. Both proteins were shown to interact with a large variety of transported substrates, and earlier studies suggested that MRPs may work as co-transporters for different molecules. In the present study we expressed the human MRP2 and MRP3 proteins in insect cells and examined their transport and ATPase characteristics in isolated, inside-out membrane vesicles. We found that the primary active transport of estradiol-17--D-glucuronide (E 2 17G), a major product of human steroid metabolism, was differently modulated by bile acids and organic anions in the case of human MRP2 and MRP3. Active E 2 17G transport by MRP2 was significantly stimulated by the organic anions indomethacin, furosemide, and probenecid and by several conjugated bile acids. In contrast, all of these agents inhibited E 2 17G transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E 2 17G, and the results indicated an allosteric cross-stimulation, probably a cotransport of bile acids and glucuronate conjugates through this protein. There was no such stimulation of bile acid transport by MRP3. In conclusion, the different transport modulation of MRPs by bile acids and anionic drugs could play a major role in regulating physiological and pathological metabolite fluxes in the human liver.