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Intrahepatic cholestasis in the setting of extrahepatic bacterial infection has been attributed to the effects of endotoxin and cytokines such as tumor necrosis factor-alpha (TNF-alpha) on bile acid transport. To define the mechanism of sepsis-associated cholestasis, taurocholate transport was examined in basolateral (bLPM) and canalicular (cLPM) rat liver plasma membrane vesicles derived from control and endotoxin [lipopolysaccharide (LPS)]-treated animals and in plasma membrane vesicles prepared after TNF-alpha treatment. Na(+)-dependent [3H]taurocholate uptake and both membrane-potential-dependent and ATP-dependent [3H]taurocholate transport were reduced in bLPM and cLPM vesicles, respectively, after LPS treatment. In membrane vesicles from TNF-alpha-treated animals, Na(+)-dependent [3H]taurocholate uptake was also reduced. Northern blot hybridization, using cDNA probes for the putative sinusoidal bile acid transporter (Ntcp) and canalicular ecto-adenosinetriphosphatase, demonstrated decreased mRNA levels after LPS and TNF-alpha treatment. Immunoblot analysis of membrane extracts from LPS-treated animals revealed decreased levels of these putative bile acid transporters. Impaired bile acid transport at the sinusoidal and canalicular membrane domains by these and other mediators of the inflammatory response may account for sepsis-associated cholestasis.

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Cholesterol 7 ␣ -hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1 , encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile. Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.

A cDNA encoding a rat liver Na(+)-bile acid cotransporter (Ntcp) has recently been cloned (Hagenbuch, B., B. Steiger, M. Fouget, H. Lubbert, and P. J. Meier. Proc. Natl. Acad. Sci. USA 88: 10629, 1991) using expression cloning in Xenopus laevis oocytes. Although the open reading frame coded for a protein of 39 kDa, in vitro translation experiments produced a 35-kDa protein which increased to a product of 41 kDa after glycosylation by pancreatic microsomes. To more clearly characterize the native protein in rat liver, we have raised antipeptide and anti-fusion protein antibodies to the COOH-terminal part of the cloned transporter. On Western blot analysis both antisera but not preimmune serum specifically detected a protein of approximately 50 kDa in isolated rat liver basolateral plasma membranes (BLPM). The reactivity was abolished when the antiserum was preincubated with the synthetic alpha-337 peptide. Deglycosylation of BLPM with N-glycanase followed by antibody probing led to decrease of the molecular mass to 34.5 kDa, suggesting that the protein is N-glycosylated in vivo. Two-dimensional immunoblotting indicated that the Ntcp protein had an isoelectric point of approximately 6.0. The antibody did not react with any proteins in rat ileal and kidney cortex brush-border membranes, human liver basolateral plasma membranes, or rat hepatoma tissue culture cell homogenates. Immunofluorescence localization studies with both antibodies revealed specific staining of the sinusoidal membrane domain but not of intracellular or bile canalicular membranes. Moreover, there was no acinar gradient in the pattern of staining.(ABSTRACT TRUNCATED AT 250 WORDS)