High K+ rapidly stimulates Ca2+-dependent phosphorylation of three proteins concomitant with insulin secretion from HIT cells.
We have previously shown in a glucose-responsive insulin-secreting beta-cell line (HIT cells) that a cell membrane-depolarizing concentration of K+ causes an influx of extracellular Ca2+ that elevates the free cytosolic Ca2+ concentration ([Ca2+]i) and activates insulin secretion. To identify the cellular mechanisms that couple Ca2+ influx to exocytosis, studies of protein phosphorylation were conducted. HIT cells were loaded with 32P and stimulated with either 40 mM K+ or 19.4 mM glucose, both of which trigger the immediate release of insulin. Phosphoproteins were examined with two-dimensional polyacrylamide gel electrophoresis and autoradiography. With addition of 40 mM K+, after the rise in [Ca2+]i, proteins of Mr 17,500, 20,000, and 24,000 were rapidly and specifically phosphorylated. The 17,000-Mr protein showed maximum phosphorylation by 1 min, whereas phosphorylation of the 24,000- and 20,000-Mr proteins continued to increase at 5 min. Despite stimulating the immediate release of insulin, high glucose did not result in a change in [Ca2+]i or the phosphorylation of the three Ca2+-dependent phosphoproteins. Insulin secretion, the increase in [Ca2+]i, and phosphorylation of the proteins by 40 mM K+ was inhibited by 100 microM verapamil, an organic Ca2+-channel-blocking drug. The rapid phosphorylation of three proteins, after a rise in [Ca2+]i and the coordinate inhibition of insulin secretion and phosphorylation, are consistent with the hypothesis that Ca2+ enters the beta-cell through voltage-dependent Ca2+ channels, regulating insulin release by effects on protein phosphorylation. However, glucose activates the immediate release of insulin by another, as yet undefined, mechanism.
D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism*; D011863:Radioimmunoassay; D014700:Verapamil / Q000494:pharmacology
D007328:Insulin; D010750:Phosphoproteins; D011506:Proteins; D014700:Verapamil; D005947:Glucose; D011188:Potassium; D002118:Calcium