We have previously shown in a glucose-responsive insulin-secreting β-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+],) 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+]1 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+], or the phosphorylation of the three Ca2+-dependent phosphoproteins. Insulin secretion, the increase in [Ca2+]1, and phosphorylation of the proteins by 40 mM K+ was inhibited by 100 μM verapamil, an organic Ca2+-channel–blocking drug. The rapid phosphorylation of three proteins, after a rise in [Ca2+]1 and the coordinate inhibition of insulin secretion and phosphorylation, are consistent with the hypothesis that Ca2+ enters the β-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.
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Original Contributions|
July 01 1987
High K+ Rapidly Stimulates Ca2+-Dependent Phosphorylation of Three Proteins Concomitant With Insulin Secretion From HIT Cells
James M Oberwetter;
James M Oberwetter
Departments of Cell Biology and Medicine, Baylor College of Medicine
Houston, Texas
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A E Boyd, III
A E Boyd, III
Departments of Cell Biology and Medicine, Baylor College of Medicine
Houston, Texas
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Address correspondence and reprint requests to A. E. Boyd III, MD, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
Diabetes 1987;36(7):864–871
Article history
Received:
July 07 1986
Revision Received:
January 28 1987
Accepted:
January 28 1987
PubMed:
3556284
Citation
James M Oberwetter, A E Boyd; High K+ Rapidly Stimulates Ca2+-Dependent Phosphorylation of Three Proteins Concomitant With Insulin Secretion From HIT Cells. Diabetes 1 July 1987; 36 (7): 864–871. https://doi.org/10.2337/diab.36.7.864
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