Biliary Secretion of Sodium Fluorescein in Primary Monolayer Cultures of Adult Rat Hepatocytes and Its Stimulation by Nicotinamide

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.