Biliary secretion of sodium fluorescein in primary monolayer cultures of adult rat hepatocytes and its stimulation by nicotinamide

Abstract

Addition of sodium fluorescein to primary cultures of rat hepatocytes resulted in a rapid uptake of the dye by the hepatocytes and a subsequent accumulation in bile canaliculi-like structures. A similar distribution was obtained with fluorescein diacetate. Concentrations of Na-fluorescein accumulating within canaliculi varied over a wide range, often far exceeding that used in the medium. Ouabain strongly blocked cellular uptake, thus also impairing secretion of Na-fluorescein, whereas colchicine affected neither process. Taurolithocholate had virtually no influence on uptake, but markedly reduced the number of fluorescent canaliculi. Furthermore, fluorescent canaliculi could be discharged by addition of I M-sucrose in Hank's buffer, without affecting viability of the cultured cells. The percentage of canicular structures accumulating high amounts of Na-fluorescein markedly increased during cultivation for 7 days, concomitant with the progressive development of originally small and sporadic canaliculi into an anastomosing network of slender channels. This canalicular proliferation was strikingly reinforced by 20 mM-nicotinamide, resulting in an impressive network of canaliculi within 2-3 days. Nicotinamide also supported the secretion of Na-fluorescein, which could be stimulated further by addition of dehydrocholate. These results suggest that cultured hepatocytes are able to re-create a functional biliary polarity at least with respect to the biliary secretion of Na-fluorescein.