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

Hepatology : Official Journal of the American Association for the Study of Liver Diseases
T KitamuraI M Arias

Abstract

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 13C nuclear magnetic resonance. Biliary secretion of fluorescein isothiocyanate-glycocholate and [14C]-labeled glycocholate in rats was similar, in contrast to the biliary secretion of sodium fluorescein and methylamine-conjugated fluorescein isothiocyanate, which are non-bile 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...Continue Reading

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