Involvement of Rab4 in regulated exocytosis of rat pancreatic acini.
OBJECTIVE
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.
Ohnishi
H
H
Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo. hohnishi.tky@umin.u-tokyo.ac.jp
Mine
T
T
Shibata
H
H
Ueda
N
N
Tsuchida
T
T
Fujita
T
T
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002766:Cholecystokinin / Q000494:pharmacology; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000502:physiology*; D051275:Glucose Transporter Type 4; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D009281:Naphthalenes / Q000494:pharmacology; D010179:Pancreas / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020695:rab4 GTP-Binding Proteins
D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009281:Naphthalenes; C495694:Slc2a4 protein, rat; D006160:Guanosine Triphosphate; D002766:Cholecystokinin; D000681:Amylases; D019204:GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium