Intestinal absorption of bile acids depends on a sodium-bile acid cotransport protein in the apical membrane of the ileal epithelial cell. Transport is Na ؉ -dependent, but the Na ؉ -bile acid stoichiometry and electrogenicity of transport are not known. Studies in whole intestine, isolated cells, and ileal membrane vesicles have been unable to resolve this issue because transport currents are small and can be obscured by other ionic conductances and transport proteins present in these membranes. In this study, the human apical sodium-bile acid transporter was expressed in stably transfected Chinese hamster ovary cells that lack other bile acid transporters. The Na ؉ -dependent transport of a fluorescent bile acid analog, chenodeoxycholyl-N⑀-nitrobenzoxadiazol-lysine, was monitored by fluorescence microscopy in single, voltage-clamped cells. Bile acid movement was bidirectional and voltage-dependent with negative intracellular voltage-stimulating influx. A 3-fold reduction in extracellular Na؉ produced a negative 52 mV shift of the flux-voltage relationship, consistent with a 2:1 Na ؉ :bile acid coupling stoichiometry. No Na ؉ -or voltage-dependent uptake was observed in nontransfected Chinese hamster ovary cells. These results indicate that the cotransport of bile acids and Na ؉ by human apical sodium-bile acid transporter is electrogenic and bidirectional and is best explained by a 2:1 Na ؉ :bile acid coupling stoichiometry. These results suggest that membrane potential may regulate bile acid transport rates under physiological and pathophysiological conditions.

The transport properties of three different synthetically prepared fluorescent conjugated bile acid analogs (FBA), all with the fluorophore on the side chain, were determined using isolated rat hepatocytes and hepatocyte couplets. The compounds studied were cholylglycylamidofluorescein (CGamF), cholyl(N epsilon-nitrobenzoxadiazolyl [NBD])-lysine (C-NBD-L), and chenodeoxycholyl-(N epsilon-NBD)-lysine (CDC-NBD-L). When hepatocytes were incubated at 37 degrees C with 0.3 mumol/L of FBA and 0.15 mol/L of Na+, cell fluorescence increased linearly with time at a rate (U/min) of 7.8 +/- 0.5 for CGamF, 7.2 +/- 0.3 for C-NBD-L, and 13.7 +/- 1.0 for CDC-NBD-L (mean, +/- SE; n = 40 to 90). Uptake was concentration dependent for concentrations less than 20 mumol/L and was saturable. The Michaelis constant (Km) value (mumol/L) for CGamF was 10.8, for C-NBD-L was 3.8, and for CDC-NBD-L was 3.0. In the absence of Na+, the uptake rate was decreased by 50% for CGamF and by 38% for C-NBD-L; but uptake of CDC-NBD-L was unchanged and thus Na+ independent. Cellular uptake of all three derivatives was specific to hepatocytes and was absent in several nonhepatocyte cell lines. For CGamF and C-NBD-L, both Na(+)-dependent and Na(+)-independent uptake was inhibited by 200-fold excess concentrations of cholyltaurine, dehydrocholyltaurine, and cholate, but for CDC-NBD-L, these nonfluorescent bile acids did not inhibit initial uptake. The intracellular fluorescence of CGamF was strongly pH dependent at an excitation wavelength of 495 nm, but pH independent at 440 nm excitation. In contrast, intracellular fluorescence of C-NBD-L and CDC-NBD-L was pH independent. All three FBA were secreted into the canalicular space of approximately 50% to 60% of couplets. Cellular adenosine triphosphate (ATP) depletion with either CN- or atractyloside inhibited secretion of all three FBA. The multispecific organic anion transporter (MOAT) inhibitor, chlorodinitrobenzene, blocked secretion of fluorescent MOAT substrates at a concentration of 1 mumol/L. At this concentration it did not affect secretion of the three FBA. At higher concentrations, chlorodinitrobenzene partially inhibited the canalicular secretion of CGamF but not the other two FBA. In conclusion, all three FBA are secreted by canalicular membrane bile acid transporters, but the sinusoidal uptake characteristics of CGamF and C-NBD-L are more similar than those of CDC-NBD-L to the transport properties of cholyltaurine. Therefore, C-NBD-L appears to be the best of the three for studies of conjugated trihydroxy-bile acid transport in hepatocytes.

Endotoxin (LPS) can cause hepatocellular injury under several circumstances, and leukotrienes have been implicated as a contributing factor. Since ion channel activation has been associated with cytotoxicity, the aim of this study was to determine the circumstances under which LPS and/or leukotrienes activate ionic conductances in hepatocytes. LPS treatment of rats increased Cl