LACK OF EVIDENCE FOR VESICLE TRAFFICKING OF FLUORESCENT BILE-SALTS INRAT HEPATOCYTE COUPLETS

The role of intracellular vesicles in the movement of bile salts throu gh hepatocytes from blood to bile has not been resolved. To determine whether bile salts are sequestered during transit, rat hepatocyte coup lets were incubated with the fluorescent bile salts cholyl-lysyl-fluor escein (CLF) and chenodeoxycholyl-lysyl-fluorescein (CDCLF). Cellular and canalicular fluorescence were measured by confocal scanning fluore scence microscopy; inhomogeneity in intracellular fluorescence was use d to evaluate potential sequestering of bile salts. Mean cellular and canalicular fluorescence increased in parallel over 10 min, slightly e xceeding (P < 0.05) the degree of increase in intracellular inhomogene ity. The microtubule inhibitor colchicine had no effect on cellular or canalicular fluorescence patterns. In contrast, the nonfluorescent bi le salt taurocholate enhanced the recovery of microtubules from cold-i nduced depolymerization, measured by confocal immunofluorescence of be ta-tubulin. Thus no evidence was obtained for intracellular sequesteri ng of bile salts or microtubule-dependent trafficking before canalicul ar secretion; cellular uptake and distribution occurred in parallel wi th canalicular secretion. The previously documented dependence of bile salt secretion on intact microtubule function therefore appears to be an indirect rather than a direct consequence of microtubule-dependent events. In particular, enhanced microtubule assembly may play a role in bile salt-induced delivery of bile salt transporters to the canalic ular membrane.