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Background/aims: Progressive familial intrahepatic cholestasis type 2 (PFIC-2) patients have a defect in the hepatocanalicular bile salt secretion. The disease is caused by mutations in the bile salt export pump (BSEP). Ten different missense mutations have been described. In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function., Methods: Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. The effect on expression and subcellular sorting was studied in HepG2 cells, stably expressing enhanced green fluorescent protein (EGFP)-tagged mBsep proteins., Results: The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Protein expression and canalicular sorting were strongly affected by the D482G mutation. Mutant EGFP-mBsep protein was only partly glycosylated and detected in both the canalicular membrane and the cytoplasm. At 30 degrees C, the mutant mRNA and protein levels were strongly increased, and the protein was predominantly glycosylated and efficiently targeted to the canalicular membrane., Conclusions: These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump.

Background: Experimental studies have shown decreased bile acid (BA) uptake and reduced excretion of cholephilic compounds in pregnant rodents., Aim: To assess the expression and function of the main BA importer, the Na(+)/taurocholate cotransporting polypeptide (Ntcp) in pregnant rats., Methods: BA uptake and Ntcp expression were studied in control and timed-pregnant rats in late gestation. Ntcp protein, messenger RNA (mRNA) expression, and Ntcp tissue localization were determined by Northern blotting, Western analysis, and tissue immunofluorescence. The activity of three transactivators of the Ntcp promoter: hepatocyte nuclear factor 1-alpha (HNF1-alpha), nuclear receptor heterodimer retinoid X receptor:retinoid acid receptor (RXR:RAR) and signal transducer and activator of transcription 5 (Stat5) was assessed using gel electrophoretic mobility shift assays., Results: A significantly reduced BA uptake and decreased Ntcp mRNA levels (-40%) and protein mass (-60%) was observed in pregnant rats. Nuclear extracts from pregnant rats showed a marked decrease of HNF1-alpha and RXR:RAR binding activities by -80 and -40% of basal activity, respectively. In contrast, binding activity of Stat-5 was increased by 50% in nuclear extracts from pregnant rats., Conclusions: Pregnancy is associated with reduced Ntcp expression and function in the rat. Our findings suggest that Ntcp down-regulation during pregnancy occurs primarily at the transcriptional level.

Background/aims: Ntcp-mediated uptake of bile salts at the basolateral membrane of hepatocytes is required for maintenance of their enterohepatic circulation. Expression of Ntcp is reduced in various experimental models of cholestasis associated with increased plasma bile salt concentrations. Mdr2 P-glycoprotein-deficient mice lack biliary phospholipids and cholesterol but show unchanged biliary bile salt secretion and increased bile flow. These mice are evidently not cholestatic, but plasma bile salt concentrations are markedly increased. The aim of this study was to investigate the role of Ntcp in the elevated bile salt levels in mdr2 P-glycoprotein-deficient (-/-) mice., Methods: Plasma membranes were isolated from male wild-type (+/+) and mdr2 (-/-) mice for measurement of Na+-dependent taurocholate transport and assessment of Ntcp protein levels by Western blotting. Northern blot analysis and competitive reverse transcription-polymerase chain reaction were used to determine hepatic Ntcp mRNA levels., Results: Kinetic analysis showed a 2-fold decrease in the Vmax of Na+-dependent taurocholate transport, with an unaffected Km in (-/-) mice compared with (+/+) controls. Ntcp protein levels were 4-6-fold reduced in plasma membranes of (-/-) mice relative to sex-matched controls. Surprisingly, hepatic Ntcp mRNA levels were not significantly affected in the (-/-) mice., Conclusions: Elevated plasma bile salt levels in mdr2 P-glycoprotein-deficient mice in the absence of overt cholestasis are associated with reduced Ntcp expression and transport activity. This is due to posttranscriptional down-regulation of Ntcp.

The usefulness of ATP-depleted rat hepatocytes in transport studies was examined. ATP-depleted hepatocytes were prepared by incubating cell suspensions with 30 microM rotenone. In ATP-depleted hepatocytes, plasma membrane permeability was increased and mitochondrial membrane potential decreased, while both intracellular volume and pH remained normal. Furthermore, in the presence of valinomycin, the initial uptake rates of 3H-tetraphenyl phosphonium (TPP+) with varied medium concentrations of potassium were predicted according to the Goldman-Hodgkin-Katz equation, which demonstrated that a potassium diffusion potential could be produced in this system. Using the thus-characterized ATP-depleted cells, the uptake mechanism of taurocholate was investigated. In the presence of an inwardly directed Na gradient, the taurocholate uptake was markedly stimulated and bile acid was transiently accumulated at a concentration 3-times higher than at equilibrium ('overshoot') in ATP-depleted cells. No overshoot was observed in viable cells, however, which suggests that in ATP-depleted cells the Na gradient, a driving force for taurocholate uptake, decreased with time. In both viable and ATP-depleted cells, the relationship between medium concentrations of Na and the Na-dependent initial uptake rate were sigmoidal, and the Hill coefficients were close to 2. The Na-dependent initial uptake rate of taurocholate was stimulated by a valinomycin-induced inside negative potassium-diffusion potential in ATP-depleted cells, and the movement of a 'one plus' (as a net) charge was revealed by fitting the data to the Goldman-Hodgkin-Katz equation. These results support the hypothesis that sodium-coupled hepatic uptake of taurocholate occuthrough an electrogenic process with the stoichiometry of 2 Na: 1 taurocholate, although this issue is controversial. In the presence of an outwardly directed sodium gradient, efflux of taurocholate from ATP-depleted cells was not stimulated. Consequently, the physiological transport vector of taurocholate from blood to cell is not only due to the direction of the sodium gradient (blood to cell) but also to membraneous orientation of transport carriers. In conclusion, kinetic analysis using ATP-depleted hepatocytes allowed the formulation of a new approach to clarify the as yet unresolved issues concerning transport stoichiometry and the mechanism for vectorial transport of taurocholate.