Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells.

Abstract

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.

Urotensin‐II regulates intracellular calcium in dissociated rat spinal cord neurons

“…The lack of a major PKC participation in U-II-effects on spinal cord neurons is further supported by experiments using bisindolylmaleimide I, a specific inhibitor (Toullec et al 1991;Yashpal et al 1995). Some atypical PKC isotypes are PMA-insensitive (Mellor and Parker 1998) and bisindolylmaleimide I fails to block translocation of atypical PKC isozyme zeta in RINm5F cells (Tang and Sharp 1998) Kase et al 1987) are the most specific protein kinase A and protein kinase G inhibitors currently available. Moreover, they are plasma membrane-permeable and have been previously demonstrated to be effective in neurons at the concentrations used in our study (Santschi et al 1999;Hou and Wang 2001).…”

Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.

“…We have previously shown that carbachol effectively elevates inositol phosphates in INS-1 cells, which provides evidence that muscarinic receptor signaling is normal in these cells (21). Perhaps INS-1 cells have low or absent levels of expression of the atypical protein kinase C isozyme ( ) that has been implicated in carbachol-stimulated insulin secretion in RIN cells (30).…”

Protein kinase C isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from mouse pancreatic islets

“…The above parameters were all measured at the steady state in 11 mM glucose or 11 mM glucose + 100 nM TMX (i.e., after 20 min exposures to TMX). Figure 2 depicts the effects of TMX on [Ca 2+ ] i and 5-HT oscillations in the continued presence of 11 mM glucose and 10 μM bisindolylmaleimide I, a specific broad-range PKC inhibitor (Martiny-Baron et al, 1993;Tang and Sharp, 1998), which did not appear to affect by itself the islet oscillatory activity. In this particular experiment, an irregular activity was observed, with slow oscillations underlying a pattern of high-frequency oscillations both in control and in presence of TMX; this is a pattern that is seen occasionally in islets under physiological conditions and, therefore, unrelated to the presence of bisindolylmaleimide.…”

“…Since, at the concentration used, TMX reportedly activates cPKC isoforms only (Ryves et al, 1991), the PKC isoform involved in toxin action is likely to be either PKC-α, PKC-β or both. Indeed, rat pancreatic β-cells and insulinoma cell lines express the cPKC isoforms α and β (Knutson and Hoenig, 1994;Tang and Sharp, 1998;Tian et al, 1996). We are investigating currently whether mouse β-cells express the same cPKC isoforms.…”

“…The nature of the PKC isoforms involved is contradictory. Studies involving β-cell lines show that, while in RINm5F cells the cholinergic response is mediated by an atypical isoform (aPKC, i.e., Ca 2+ -and DAG/phorbol ester-insensitive) (Tang and Sharp, 1998), in MIN6 cells, the contributing isoforms are phorbol estersensitive (i.e., cPKC and/or nPKC) (Pinton et al, 2002;Tian et al, 1996). Studies using whole rat islets indicate the involvement of nPKC isoforms (Harris et al, 1996;Ishikawa et al, 2005).…”