Ca²⁺-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine

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, M(r)=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 50mM K⁺ was Ca²⁺-dependent since omission of extracellular Ca²⁺ or addition of the Ca²⁺ channel blocker α-isopropyl-α-[(N-methyl-N-homoveratryl)-γ-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 μM), 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 μM and 2.5 μM 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.