Involvement of Rab4 in regulated exocytosis of rat pancreatic acini

Abstract

Background & Aims: Rab4, a Ras-related small guanosine triphosphate (GTP)-binding protein, has been suggested to participate in exocytosis. The function of Rab4 in regulated exocytosis of pancreatic acini was examined in this study. Methods: Subcellular localization of Rab4 was determined by Western blotting and immunohistochemistry. The Rab4 function in regulated exocytosis was examined by introducing Rab4 hypervariable carboxy-terminal domain peptide (Rab4 peptide) and anti-Rab4 antibody into streptolysin O–permeabilized acini. The regulation of Rab4 by cholecystokinin (CCK) and 12- O-tetradecanoyl-phorbol 13-acetate (TPA) was investigated by examining their effects on [ 32P]GTP binding rate into the Rab4 immunoprecipitates. The participation of protein kinase C in the Rab4 regulation by CCK was confirmed by calphostin C pretreatment of acini. Results: Rab4 was localized on zymogen granule membranes. Both Rab4 peptide and anti-Rab4 antibody enhanced calcium-stimulated amylase release from streptolysin O–permeabilized acini, suggesting the inhibitory role of Rab4 in exocytosis. CCK and TPA increased GTP binding to Rab4. Calphostin C attenuated the stimulatory effect of CCK on GTP binding to Rab4. Conclusions: Rab4 negatively modulates regulated exocytosis of pancreatic acini and is controlled by CCK through a protein kinase C pathway. GASTROENTEROLOGY 1999;116:943-952