cholecystokinin and herbimycin

In isolated rat pancreatic acini, Src, RhoA, PI3-K, Vav-2, G(alpha12), and G(alpha13) were detected by immunoblotting. CCK enhanced the levels of these proteins, and the levels of Src and RhoA were reduced by the Src inhibitor herbimycin A and the Rho inhibitor pravastatin. The PI3-K inhibitor wortmannin reduced the level of PI3-K. These inhibitors also decreased amylase secretion in CCK-treated pancreatic acini without altering basal secretion. Immunoprecipitation studies indicated that CCK caused Src to associate with Vav-2, RhoA, and PI3-K and RhoA and Src to associate with Vav-2. Ras, RasGAP, and SOS did not coimmunoprecipitate with Vav-2, and RasGAP and SOS did not coimmunoprecipitate with RhoA. CCK also enhanced Vav-2 and RhoA to coimmunoprecipitate with G(alpha13). We conclude that CCK stimulates the recruitment of the Src-RhoA-PI3-K signaling pathway by Vav-2 downstream of G(alpha13) in pancreatic acini.

Topics: Amylases; Androstadienes; Animals; Benzoquinones; Cholecystokinin; GTP-Binding Protein alpha Subunits, G12-G13; In Vitro Techniques; Lactams, Macrocyclic; Male; Pancreas, Exocrine; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pravastatin; Proto-Oncogene Proteins c-vav; Quinones; Rats; Rats, Sprague-Dawley; rhoA GTP-Binding Protein; Rifabutin; Signal Transduction; src-Family Kinases; Wortmannin

Cholecystokinin (CCK), a known mitogen for the exocrine pancreas, is shown to activate 70-kDa S6 kinase in isolated pancreatic acini. In this study, we examined the kinetics and cellular mechanisms of CCK-induced p70 S6 kinase activation in vivo and in vitro. Fasted mice were intraperitoneally injected with 0.01-10 microg/kg CCK analoge cerulein. Cerulein caused a concentration-dependent activation of p70 S6 kinase, with the maximal effect at 1-10 microg/kg. After 1 microg/kg cerulein administration, the kinase activity was increased at 5 min, peaked at 10 min, and subsequently decreased. Cerulein also caused a rapid and transient activation of Src. Prior administration of the tyrosine kinase inhibitor herbimycin A compeletely inhibited cerulein-induced Src activation, while the inhibition of p70 S6 kinase activity was partial. Similar results were obtained with pancreatic acinar cell line AR42J cells. These results suggest that tyrosine kinases, including Src as a possible candidate, are partly implicated in the signaling pathway of CCK-induced p70 S6 kinase activation in the exocrine pancreas.

Topics: Animals; Benzoquinones; Cells, Cultured; Ceruletide; Cholecystokinin; Enzyme Activation; Enzyme Inhibitors; In Vitro Techniques; Lactams, Macrocyclic; Male; Mice; Mice, Inbred ICR; Mitogens; Pancreas; Quinones; Ribosomal Protein S6 Kinases; Rifabutin

Three sialagogues, isoproterenol (IPR), carbachol, and methoxamine, caused induction of ornithine decarboxylase (ODC) in cultured rat parotid explants. All the protein tyrosine kinase inhibitors tested suppressed this ODC induction but enhanced sialagogue-dependent amylase secretion. Sodium orthovanadate showed the reverse effects as the kinase inhibitors. Immunoblot analysis with anti-phosphotyrosine antibody revealed that herbimycin A depresses IPR-stimulated tyrosine phosphorylation of parotid proteins. Herbimycin A did not affect the IPR- or dibutyryl cAMP-induced surge of the parotid cAMP level but inhibited these agonist-dependent ODC inductions. These results suggest that sialagogue-induced ODC induction and amylase secretion are mediated by different signal transduction pathways and that protein tyrosine kinase participates in IPR-dependent ODC induction and amylase secretion in the process subsequent to the cAMP surge.

Topics: Amylases; Animals; Benzoquinones; Bucladesine; Carbachol; Chamomile; Cholecystokinin; Cinnamates; Cyclic AMP; Enzyme Induction; Enzyme Inhibitors; Flavonoids; Genistein; Isoflavones; Isoproterenol; Lactams, Macrocyclic; Male; Methoxamine; Oils, Volatile; Organ Culture Techniques; Ornithine Decarboxylase; Parotid Gland; Phenols; Plants, Medicinal; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Wistar; Rifabutin; Sialic Acids; Vanadates