| 1. |
- Arkhammar, P., et al.
(författare)
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Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage-activated Ca2+ channels
- 1994
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Ingår i: Journal of Biological Chemistry. - : Baishideng Publishers. - 0021-9258 .- 1083-351X. ; 269:4, s. 2743-2749
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Tidskriftsartikel (refereegranskat)abstract
- In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the beta-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse beta-cells and in similar beta-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 beta-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 beta-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 alpha and beta 1 forms of the PKC isoenzymes were present in the mouse beta-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 Ca(2+)-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. There was,h owever, no effect on thet otal amounts of hormone released. The present study con- firms that PKC has multiple roles and thereby interacta at different sites in the complex series of events consti- tuting the #?-cell signal-transduction pathway. It is sug- gested 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 appro- priate function of this channel in the insulin secretory process.
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| 3. |
- Haby, Christelle, et al.
(författare)
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Inhibition of serine/threonine protein phosphatases promotes opening of voltage-activated L-type Ca2+ channels in insulin-secreting cells
- 1994
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 298:Pt 2, s. 341-346
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Tidskriftsartikel (refereegranskat)abstract
- The biological activity of many proteins, including voltage-sensitive ion channels, is controlled by their state of phosphorylation. Ca2+ influx through voltage-activated L-type Ca2+ channels serves as the major stimulatory signal in insulin-secreting cells. We have now investigated the extent to which Ca2+ handling in clonal insulin-secreting RiNm5F cells was affected by okadaic acid, an inhibitor of various serine/threonine protein phosphatases. Whole-cell patch-clamp experiments showed that okadaic acid generated an increase in membrane current, suggesting that it promotes Ca2+ influx through L-type voltage-gated Ca2+ channels probably by modifying their phosphorylation state. Okadaic acid was found to provoke a transient rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i) but had no further effect on the K(+)-induced increase. The Ca2+ transient induced by okadaic acid was dependent on the presence of extracellular Ca2+ and was abolished by D600, a blocker of voltage-activated L-type Ca2+ channels. Concomitant with the rise in [Ca2+]i, okadaic acid induced insulin secretion, a phenomenon that was also dependent on extracellular Ca2+. It is proposed that hyperphosphorylation of voltage-activated L-type Ca2+ channels in insulin-secreting cells lowers the threshold potential for their activation.
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