title: Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine.
abstract: Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, Mr = 60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50 mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-gamma-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 microM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+-dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 microM and 2.5 microM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.
mesh_terms: D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D006224:Cricetinae; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007700:Kinetics; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D009374:Neoplasms, Experimental / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology*
chemical_list: D007328:Insulin; D009363:Neoplasm Proteins; D010455:Peptides; D014268:Trifluoperazine; D011188:Potassium; D002118:Calcium