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. 1990 Dec 15;272(3):637–645. doi: 10.1042/bj2720637

Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells.

G D Li 1, R Regazzi 1, S Ullrich 1, W F Pralong 1, C B Wollheim 1
PMCID: PMC1149756  PMID: 2176469

Abstract

The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.

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Selected References

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