Lack of evidence for vesicle trafficking of fluorescent bile salts in rat hepatocyte couplets

Abstract

The role of intracellular vesicles in the movement of bile salts through hepatocytes from blood to bile has not been resolved. To determine whether bile salts are sequestered during transit, rat hepatocyte couplets were incubated with the fluorescent bile salts cholyl-lysyl-fluorescein (CLF) and chenodeoxycholyl-lysyl-fluorescein (CDCLF). Cellular and canalicular fluorescence were measured by confocal scanning fluorescence microscopy; inhomogeneity in intracellular fluorescence was used to evaluate potential sequestering of bile salts. Mean cellular and canalicular fluorescence increased in parallel over 10 min, slightly exceeding (P < 0.05) the degree of increase in intracellular inhomogeneity. The microtubule inhibitor colchicine had no effect on cellular or canalicular fluorescence patterns. In contrast, the nonfluorescent bile salt taurocholate enhanced the recovery of microtubules from cold-induced depolymerization, measured by confocal immunofluorescence of beta-tubulin. Thus no evidence was obtained for intracellular sequestering of bile salts or microtubule-dependent trafficking before canalicular secretion; cellular uptake and distribution occurred in parallel with 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 canalicular membrane.