CHANGES OF BILIARY EXCRETORY FUNCTION AND CANALICULAR CARRIES IN INTRAHEPATIC CHOLESTASIS

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Transport from the hepatocytes to the canalicular membrane is a rate-limiting step of hepatic transport of bile acids and organic anions, which is mediated by bile salt export pump (Bsep) and multidrug resistance protein 2 (Mrp2), respectively. In intrahepatic cholestasis, biliary excretion of bile acids and organic anions is impaired, and similar results were obtained in experimental cholestasis. Therefore, the changes of these transporters in representative cholestasis induced by phalloidin and lipopolysaccharide was studied. Antibodies against Bsep and Mrp2 were raised by using C-terminal peptides of these transporters coupled to KLH. The specificity of anti-Bsep antibody was confined by plasma membrane of Sf9 cells transfected with Bsep, and that of anti-Mrp2 antibody was confined by bile canalicular membrane of Sprague-Dawley rats and their hyperbilirubinemic mutant, EHBR. Ninety minutes after intravenous phalloidin injection (0.5 mg/kg) or 18 hours after intravenous lipopolysaccharide injection (2.5 mg/kg), the liver was removed, homogenated, and then, the membrane fraction obtained by a sucrose gradient centrifugation was subjected to Western blotting. Both the amount of Bsep and Mrp2 were markedly decreased by phalloidin treatment, suggesting an impaired vesicular targeting of these transporters to the canalicular membrane. By lipopolysaccharide treatment, the amount of Mrp2 was markedly decreased to <5%, whereas the decrease of Bsep was moderate (60%). These results may explain the selective impairment of organic anion excretion by lipopolysaccharide treatment, as well as a marked hyperbilirubinemia only with mild cholestasis in sepsis.