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Background & Aims: Progressive familial intrahepatic cholestasis 1 (PFIC1) results from mutations in ATP8B1, a putative aminophospholipid flippase. Conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1 deficiency produces cholestasis by altering the activity of the farnesoid X receptor (FXR) or by impairing the structure of the canalicular membrane., Methods: ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes and Caco2 cells using adenoviral and oligonucleotide small interfering RNAs., Results: ATP8B1 messenger RNA and protein expression was greatly reduced in human and rat cells. In contrast, FXR expression and several FXR-dependent membrane transporters (bile salt export pump [BSEP], multidrug resistance-associated protein [MRP] 2) were unchanged at messenger RNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells, as evidenced by 6alpha-ethyl chenodeoxycholic acid-mediated induction of small heterodimer partner, BSEP, and multidrug-resistant protein (MDR) 3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes, although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine, consistent with the function of Atp8b1 as an aminophospholipid flippase., Conclusions: ATP8B1 deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated with cholestasis.

Background/aims: The biochemical mechanism of bile canalicular contraction is similar to that of smooth muscle contraction. Contraction follows inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release, which activates actin-myosin interactions. Nitric oxide is a myorelaxant through the actions of 5'-cyclic guanosine monophosphate (cGMP) and is produced in hepatocytes exposed to endotoxin and cytokines. The aim of this study was to investigate the effect of nitric oxide on canalicular contraction and to determine the mechanism by which cGMP interferes with the contractile signal., Methods: The canalicular motility in rat hepatocyte doublets was measured by microscopic image analysis, and intracellular Ca2+ was measured by fluorescence microscopy. cGMP and InsP3 were determined by radio-immunoassay and high-pressure liquid chromatography. Ca2+ release from liver homogenate was measured by filtration and superfusion assays., Results: Compounds that release nitric oxide stimulated hepatocellular production of cGMP and prevented agonist-induced contraction by inhibiting the increase in intracellular Ca2+. The cGMP analogue bromo-cGMP prevented contraction and the increase in Ca2+. Bromo-cGMP marginally decreased InsP3 production. cGMP blocked InsP3-dependent Ca2+ release from internal stores., Conclusions: These findings suggest that nitric oxide interferes with Ca2+ signals by cGMP-mediated inhibition of the InsP3 receptor/Ca2+ channel and that hepatocellular production of nitric oxide may be cholestatic by impairing canalicular motility.

The influence of sodium taurocholate on bile canalicular contractions was examined by time-lapse cinephotomicrography of isolated rat hepatocytes in primary monolayer cultures. Frame-by-frame analyses indicates that as the dose of taurocholate increased, the number of contractions increased in a dose-dependent manner up to 5 microM. Because bile canaliculi are the smallest biliary passages across which bile secretion occurs, and because it is known that taurocholic acid within the dose range used in these studies causes a linear increase in bile secretion, the results suggest that canalicular contractions may be a function of canalicular bile secretion.

The effect of intravenously administered vasoactive intestinal polypeptide on bile secretion was studied in 11 patients with complete biliary drainage. After infusion of vasoactive intestinal polypeptide, bile volume increased by 65%. In the 2 patients investigated, the output of bicarbonate increased by approximately 250% and the concentration by 50%-70%. Vasoactive intestinal polypeptide thus caused a bicarbonate-rich choleresis. The output of biliary lipids was not affected by infusion of vasoactive intestinal polypeptide, whereas the concentration decreased by approximately 40%. The canalicular bile flow, measured by the clearance of [14C]erythritol, was not affected by infusion of vasoactive intestinal polypeptide. The choleretic effect of vasoactive intestinal polypeptide thus seems to occur only at the ductular level. The ductular bile flow was calculated to be stimulated threefold to fourfold.

We observed the motility of bile canaliculi in isolated rat hepatocytes obtained from animals that were pretreated with phalloidin (500 micrograms/kg body wt) for 1 and 3 days. Time-lapse cinephotomicrographs were taken in each experiment and in controls for 16.25 h. As we previously reported in normal hepatocytes, active contractions of bile canaliculi were observed. The number of contractions was 127.6 in controls, compared with 58.0 in 1-day phalloidin pretreated and 16.2 in 3-day phalloidin pretreated groups. The contraction process itself was not normal in the experimental groups, the contraction phase being slow and sustained. The altered canalicular motility depended on the dose of phalloidin administered. These results suggest that the integrity of actin filaments is necessary for normal bile canalicular motility. Further, dysfunction of actin microfilaments with altered canalicular motility may be involved in the pathogenesis of canalicular cholestasis.

The roles of microfilaments and microtubules in the hepatocellular uptake, translocation, and biliary excretion of horseradish peroxidase and [14C]sodium taurocholate were investigated using the microfilament inhibitor cytochalasin D and the microtubule inhibitor colchicine. In separate studies, horseradish peroxidase and [14C]taurocholate were injected separately as a bolus into rat portal veins after treatment with cytochalasin D or colchicine, and bile was collected and analyzed for the presence of horseradish peroxidase and [14C]taurocholate. Cytochalasin D treatment depressed bile flow by approximately 50% and decreased the biliary secretion of [14C]taurocholate in direct proportion to bile flow. Horseradish peroxidase secretion into bile was unaffected, and total biliary protein secretion was decreased only slightly. Because of the depression of bile secretion, concentrations in bile of horseradish peroxidase and total biliary protein increased significantly. Consistent with reported observations of cytochalasin D, decreases in microfilaments and dilated bile canaliculi were observed by electron microscopy; however, the vesicular transport of horseradish peroxidase as observed using electron microscopy cytochemistry appeared to be normal and unaffected by cytochalasin D treatment. Colchicine, in contrast, had minimal effect on bile flow and did not diminish the biliary secretion of [14C]taurocholate. Colchicine inhibited both the total amount of horseradish peroxidase secreted into bile as well as the rate of its secretion in comparison with control and cytochalasin D-treated animals. Cellular morphology was consistent with published observations for colchicine, which included a marked decrease in microtubules. In addition, after electron microscopy cytochemistry there was a paucity of horseradish peroxidase-containing vesicles within the hepatocytes, suggesting that colchicine interfered with the vesicular transport of horseradish peroxidase. Collectively, the data suggest that (a) the mechanism used by hepatocytes for the secretion of bile acids is independent of the vesicular transport of biliary proteins and is dependent upon intact microfilaments and (b) such vesicular transport of protein into bile requires an intact and functioning microtubular network.