TAUROCHOLATE INDUCES PERICANALICULAR LOCALIZATION OF C6-NBD-CERAMIDE IN ISOLATED HEPATOCYTE COUPLETS

The mechanisms and pathways involved in hepatocellular transport of lipid destined for biliary excretion remain poorly understood. Using fluorescence microscopy of rat hepatocyte couplets in primary culture, we examined the effects of taurocholate (TC) on the intracellular distribution of 6-N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] aminocaproylsphingosine (C6-NBD-ceramide), a lipid that accumulates in the Golgi apparatus. Microscopic findings were quantified with morphometric and digital image analysis and were correlated with the metabolism of C6-NBD-ceramide in isolated hepatocyte suspensions and the biliary excretion of fluorescent lipid in the isolated perfused liver. After plasma membrane uptake of C6-NBD-ceramide from albumin at 0-degree-C, the lipid was rapidly internalized at 37-degrees-C, but exhibited only a modest concentration of fluorescence in intracellular organelles. With 17-mu-M TC in the medium, there was enhanced localization of fluorescence to organelles and significant recruitment of fluorescent lipid to the pericanalicular region of the couplets within 30 min. C6-NBD-ceramide was partially metabolized to C6-NBD-sphingomyelin and -glucosylceramide, indicative of transit through the Golgi apparatus. The generation of C6-NBD-sphingomyelin was significantly increased by TC. After a similar loading protocol in the perfused liver, there was little biliary excretion of fluorescent lipid at 37-degrees-C under control conditions. However, infusion of TC markedly enhanced the biliary output of fluorscent lipid over the first 30 min, primarily as C6-NBD-sphingomyelin and -glucosylceramide. We conclude that TC modulates the distribution of C6-NBD-ceramide in hepatocytes by inducing translocation of lipid to a pericanalicular location, most likely the Golgi apparatus, before excretion of its metabolites in bile. Our findings support the concept that bile salt-induced biliary lipid excretion is facilitated by the interaction of bile salts with lipids at the level of intracellular organelles.