Protein kinase C modulates the insulin secretory process by maintaining a proper function of the β-cell voltage-activated Ca2+ channels

P. Arkhammar, L. Juntti-Berggren, O. Larsson, M. Welsh, E. Nanberg, A. Sjoholm, M. Kohler, P. O. Berggren

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Abstract

In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the β-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse β-cells and in similar β-cells deprived of PKC activity. [Ca2+]i was measured with the intracellular fluorescent Ca2+ indicator fura-2 and the Ca2+ channel activity was estimated by the whole cell configuration of the patch-clamp technique. To reveal the various isoenzymes of PKC present in the mouse β-cell, proteins were separated by one-dimensional gel electrophoresis and Western blotting was performed. The production of inositol phosphates was measured by ion-exchange chromatography and insulin release was measured radioimmunologically. Acute stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate resulted in suppression of both the carbamylcholine-induced increase in [Ca2+]i and production of inositol 1,4,5-trisphosphate. Under these conditions the increase in [Ca2+]i in response to glucose was similar to that found in control cells. When β-cells were deprived of PKC, by exposure to 200 nM 12-O-tetradecanoylphorbol-13-acetate for 24-48 h, there was an enhanced response to carbamylcholine. This response constituted increases in both the [Ca2+]i signal and production of inositol 1,4,5-trisphosphate. Interestingly, cells with down-regulated PKC activity responded more slowly to glucose stimulation, when comparing the initial increase in [Ca2+]i, than control cells. On the other hand, the maximal increase in [Ca2+]i was similar whether or not PKC was present. Moreover, PKC down-regulated cells exhibited a significant reduction of maximal whole cell Ca2+ currents, a finding that may explain the altered kinetics with regard to the [Ca2+]i increase in response to the sugar. Both the α and β1 forms of the PKC isoenzymes were present in the mouse β-cell and were also subjected to PKC down-regulation. Hence, either of these isoenzymes or both may be involved in the modulation of phospholipase C and Ca2+ channel activity. Since insulin release under physiological conditions is critically dependent on Ca2+-influx through the voltage-gated L-type Ca2+ channels, the kinetics of hormone release was expected to demonstrate a similar delay as that of the [Ca2+]i increase. Although not as pronounced, such a delay was indeed also observed in the onset of insulin release. There was, however, no effect on the total amounts of hormone released. The present study confirms that PKC has multiple roles and thereby interacts at different sites in the complex series of events constituting the β-cell signal-transduction pathway. It is suggested that PKC may be tonically active and effective in the maintenance of the phosphorylation state of the voltage-gated L-type Ca2+ channel, enabling an appropriate function of this channel in the insulin secretory process.

Original languageEnglish (US)
Pages (from-to)2743-2749
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number4
StatePublished - Aug 4 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Arkhammar, P., Juntti-Berggren, L., Larsson, O., Welsh, M., Nanberg, E., Sjoholm, A., Kohler, M., & Berggren, P. O. (1994). Protein kinase C modulates the insulin secretory process by maintaining a proper function of the β-cell voltage-activated Ca2+ channels. Journal of Biological Chemistry, 269(4), 2743-2749.