Is vesicular exocytosis and membrane recycling a mechanism for secretin-induced choleresis in bile duct epithelium?

Abstract

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated, from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10−12 - 10−7M) increase in acridine orange fluorescence by acridine orangeloaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 × 10−9M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p<0.05) or trypsin (90% inhibition, p<0.001); no inhibition was seen with lumicolchicine and heatinactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p<0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.