3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) induce hepatic expression of the phospholipid translocase mdr2 in rats.

BACKGROUND & AIMS: Biliary cholesterol secretion is coupled to that of phospholipids in a process controlled by mdr2 P-glycoprotein activity and bile salt secretion. Statins, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been shown to affect hepatobiliary lipid secretion in rats. The aim of this study was to relate the effects of statins on bile formation to the expression of mdr2 and other hepatic adenosine triphosphate-dependent transport proteins involved in bile formation in rats. METHODS: Rats received simvastatin- or pravastatin-containing chow continuously for 5 days. In one group of rats, simvastatin treatment was withdrawn 9-12 hours before the end of the experiment to induce biliary cholesterol hypersecretion (rebound). Bile and liver tissue were collected for lipid analysis, and hepatic messenger RNA (mRNA) and protein levels were studied by reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULTS: Simvastatin feeding did not alter biliary bile salt secretion. Secretion of phospholipids and cholesterol was stimulated by 74% and 90%, respectively, in the simvastatin-continuous group and by 72% and 235%, respectively, in the rebound group compared with controls. mdr2 mRNA levels increased only in the continuous group. mdr2 protein levels increased in both simvastatin-fed groups. Induction was most pronounced in periportal hepatocytes. mdr1b mRNA levels were moderately increased in both simvastatin-fed groups. Levels of other hepatic transport proteins did not change. Similar results were obtained in pravastatin-fed rats. CONCLUSIONS: Statins increase expression of mdr2 and mdr1b in rats, revealing a novel effect of these commonly used drugs.

Regulation of hepatic transport systems involved in bile secretion during liver regeneration in rats.

We investigated the expression of hepatic transport systems involved in bile secretion during liver regeneration after partial hepatectomy (PH) in rats. Initial studies showed maximal BrdU incorporation 24 hours after PH. Therefore, transporter expression and bile secretion were analyzed in detail at this time. The mRNA levels of the multidrug resistance genes mdr1a and mrp1 slightly increased, whereas mdr1b mRNA levels showed an extensive increase after PH. The mRNA levels of the conjugate transporter, mrp2, decreased slightly, whereas mrp2 protein levels did not change. Bilirubin secretion did not change, but the biliary glutathione secretion markedly decreased and the hepatic GSH content increased. The messenger RNA levels of the bile salt uptake transporters ntcp, oatp1, and oatp2 and the bile salt exporter, bsep/spgp, all decreased with ntcp showing the most prominent decrease. Protein levels of ntcp dramatically decreased whereas oatp2 only slightly decreased. Oatp1 protein expression slightly increased and bsep/spgp protein levels did not change. Decreased levels of bile salt uptake systems were associated with a 10-fold increase in the plasma bile salt concentration, yet, bile flow and bile salt secretion were increased when expressed per gram liver and unaffected when expressed on the basis of body weight. In conclusion, during the initial phase of rat liver regeneration ntcp is down-regulated whereas other transporter proteins involved in bile secretion are only slightly affected. Despite increased serum bile salt levels the remnant liver is not cholestatic: bile flow is maintained by uptake of bile salts probably via oatp isoforms and their secretion via bsep/spgp.

Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status.

The inducible nitric oxide synthase (iNOS) promoter contains nuclear factor kappaB (NF-kappaB) binding sites. NF-kappaB activation is determined, in part, by the intracellular redox status. The aim of this study was to determine the importance of the cellular glutathione status in relation to NF-kappaB activation and iNOS expression in hepatocytes in vivo and in vitro. For in vivo experiments, rats were injected with endotoxin and sacrificed 6 hours later. Glutathione was depleted by diethylmaleate. For in vitro experiments, cultured hepatocytes from untreated rats were exposed to a cytokine mixture. Glutathione levels were depleted by diethylmaleate and restored by N-acetylcysteine. iNOS expression was assessed by Western blot, reverse transcription polymerase chain reaction, nitric oxide (NO) metabolites, and immunohistochemistry. NF-kappaB binding was assessed by electrophoretic mobility shift assay. Endotoxin-induced iNOS expression in rat liver was prominent in hepatocytes, Kupffer cells, and inflammatory cells, in particular neutrophils. Glutathione depletion prevented iNOS induction in hepatocytes, but not in inflammatory cells. iNOS protein levels were in accordance with iNOS messenger RNA and NO metabolites in plasma. Glutathione depletion did not affect neutrophil infiltration. Cytokines strongly induced iNOS in cultured hepatocytes. Induction was prevented by glutathione depletion and could be restored by addition of N-acetylcysteine. NF-kappaB binding correlated with iNOS induction. In conclusion, in this study we show that iNOS induction in hepatocytes in vivo and in vitro is dependent on the intracellular glutathione status and correlates with NF-kappaB binding. Glutathione-depletion has no effect on the expression of iNOS in inflammatory cells, nor on neutrophil infiltration.

Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver.

Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly down-regulated 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 reverse-transcriptase 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.

Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats.

BACKGROUND & AIMS: During endotoxemia, expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in the liver is increased. NO has been suggested to have a hepatoprotective function. The aim of this study was to investigate the distribution of iNOS and the effect of different NO synthase inhibitors on liver damage and hemodynamics during endotoxemia. METHODS: Rats were injected with lipopolysaccharide (LPS) and received the NOS-inhibitor S-methylisothiourea (SMT) or NG-nitro-L-arginine methyl ester (L-NAME). iNOS induction was assessed by Western blot, immunohistochemistry, and measurement of NO metabolites in plasma and bile. Liver damage was determined by aspartate aminotransferase and alanine aminotransferase and by histology. The effects of both inhibitors on systemic and portal pressure were measured in normal and LPS-treated rats. RESULTS: LPS treatment strongly induced iNOS in inflammatory cells, macrophages, bile duct epithelium, and hepatocytes, especially at the canalicular membrane. LPS-induced liver damage strongly increased after L-NAME. SMT caused a similar reduction of NO production without enhancing liver damage. In LPS-treated rats, SMT increased the systemic and portal pressure significantly more than L-NAME. CONCLUSIONS: During endotoxemia, administration of the NOS-inhibitor L-NAME aggravates liver damage. This liver damage does not seem to be caused by hemodynamic changes. In contrast, SMT caused significant hemodynamic changes but did not increase LPS-induced liver damage.

Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells.

BACKGROUND & AIMS: The multidrug resistance protein (MRP) functions as an organic anion efflux carrier. Recent studies suggest that hepatocytes contain two mrp homologues, named mrp1 and mrp2, localized on the lateral and canalicular membrane, respectively. The aim of this study was to evaluate the role of MRP1. Protein levels and localization of MRP1 in human hepatocytes, HepG2 hepatoma cells, and SV40 large T antigen-immortalized human hepatocytes were studied. METHODS: Using specific antibodies, MRP1 protein levels and cellular localization were examined by Western blotting and fluorescence confocal microscopy, respectively. In addition, a fluorescent substrate, glutathione-methylfluorescein, was used to measure plasma membrane transport activity and to observe intracellular transport activity. RESULTS: The level of MRP1 in normal hepatocytes is very low. In contrast, MRP1 is highly increased in both HepG2 and immortalized hepatocytes. In these cells MRP1 is localized in lateral membranes of adjacent cells. Plasma membrane staining is absent in separate cells. Glutathione-methylfluorescein is transported in the medium and intracellular vesicles. CONCLUSIONS: MRP1 protein level is greatly increased in the lateral membrane of proliferating hepatocyte-derived cells. The presence of a lateral domain seems necessary for plasma membrane localization. These results suggest that MRP1-mediated organic anion transport is important in proliferating hepatocytes, but not in quiescent cells.