SERIAL QUANTITATIVE IMAGE-ANALYSIS AND CONFOCAL MICROSCOPY OF HEPATIC-UPTAKE, INTRACELLULAR-DISTRIBUTION AND BILIARY-SECRETION OF A FLUORESCENT BILE-ACID ANALOG IN RAT HEPATOCYTE DOUBLETS

To characterize the poorly understood mechanisms of intracellular transport of bile acids, fluorescein isothiocyanate-glycocholate was synthesized and its ring-OH-linked structure established by fast atom bombardment, mass spectroscopy and C-13 nuclear magnetic resonance. Biliary secretion of fluorescein isothiocyanate-glycocholate and [C-14]-labeled glycocholate in rats was similar, in contrast to the biliary secretion of sodium fluorescein and methylamine-conjugated fluorescein isothiocyanate, which are nonbile acid organic anions. After incubation of cultured hepatocyte doublets with fluorescein isothiocyanate-glycocholate, serial quantitative image analysis of fluorescence was performed in the cellular cytoplasm, perinuclear zone and bile canaliculus. Uptake of fluorescein isothiocyanate-glycocholate into the cytoplasm was inhibited by removal of sodium from the medium and by addition of glycocholate or taurocholate. After preincubation with colchicine, but not lumicolchicine, the proportion of perinuclear to cytoplasmic fluorescein isothiocyanate-glycocholate increased during incubation with fluorescein isothiocyanate-glycocholate. Neither fluorescein isothiocyanate-glycocholate uptake nor canalicular secretion was affected. by confocal microscopy the perinuclear zone containing fluorescein isothiocyanate-glycocholate was identified as Golgi apparatus by fluorescent colocalization with C6-NBD-ceramide, which specifically identifies the Golgi apparatus. We conclude that colchicine inhibits fluorescein isothiocyanate-glycocholate transfer from the Golgi apparatus to the bile canaliculus. These results suggest that intracellular bile acid transport involves microtubule-dependent vesicular movement from the Golgi apparatus to the bile canaliculus. The role of this process in normal physiology in uncertain.