Dissimilar effects of the protein kinase C inhibitors, staurosporine and H‐7, on cholecystokinin‐induced enzyme secretion from rabbit pancreatic acini

Abstract

The effects of two putative inhibitors of protein kinase C activity, staurosporine and H-7, on partially purified protein kinase C and amylase secretion from isolated rabbit pancreatic acini were investigated. Staurosporine dose-dependently inhibited amylase release stimulated by an optimal concentration of cholecystokinin C-terminal octapeptide. At a concentration of 100 nM, the drug inhibited the secretory response to the secretagogue by approximately 50%. At the same concentration, staurosporine inhibited 12-0-tetradecanoylphorbol 13-acetatestimulated enzyme secretion by 90%. Moreover, the potentiating effect of this phorbol ester on cholecystokinininduced amylase release was completely abolished in the presence of staurosporine. Interestingly, amylase release was decreased to the level observed with the combination of cholecystokinin and staurosporine. In contrast, H-7, potentiated rather than inhibited cholecystokinin-stimulated enzyme secretion, whereas the secretory response to 12-0-tetradecanoylphorbol 13-acetate was not affected by the drug. Both staurosporine and H-7, however, inhibited protein kinase C purified from exocrine pancreatic tissue. Kinetic analysis revealed that both compounds inhibited protein kinase C competitively with respect to ATP. The Ki value for staurosporine was 0.55 nM and for H-7 13.5 pM. Our results obtained with staurosporine are in line with a stimulatory role of protein kinase C in cholecystokinin-induced enzyme secretion from the exocrine pancreas. The results obtained with H-7 emphasize that care has to be taken in interpreting the biological effects of this drug.Protein kinase Cis an ubiquitous Ca2+-and phospholipiddependent protein kinase which is thought to play a role in a wide variety of cellular processes involved in hormone-induced cell activation [l]. In pancreatic acini, as in most tissues, the activation of the enzyme appears to be linked to the secretagogue-induced turnover of inositol phospholipids in the plasma membrane [2 -51. Interaction of a hormone, like cholecystokinin, or a neurotransmitter, such as acetylcholine, with its membrane receptor stimulates a phospholipase C activity that cleaves phosphatidylinositol 4,5-bisphosphate leading to the generation of two important second messengers, inositol 1,4,5-trisphosphate [6, 71 and diacylglycerol [8, 91. Inositol 1,4,5-trisphosphate stimulates the release of Ca2 + from intracellular stores [lo]. Diacylglycerol increases the Ca2+-affinity of protein kinase C [l, 111, leading, in conjunction with the increase in intracellular Ca", to activation of protein kinase C.Evidence for the involvement of the diacylglycerol-activated protein kinase C in the mechanism of action of calciummobilizing pancreatic secretagogues has come primarily from studies with phorbol esters. These compounds, such as 12-0-tetradecanoylphorbol 13-acetate (TPA), mimic the stimulatory effect of diacylglycerol on protein kinase C [ll] and stimulate pancreatic enzyme secretion [12 -141. However, phorbol esters can also be inhibitory active. F...

The possible involvement of protein kinase C(s) and inositol phosphate metabolism in the basal but not in the prolactin stimulated casein release by the lactating rabbit mammary epithelial cell

“…The latter authors concluded that protein kinase C could exert a negative feedback role in receptor-mediated pancreatic enzyme secretion. However, as regards pancreatic secretion (Ederveen et al, 1990) and mammary epithelial cell secretion, this hypothesis is not supported by the results obtained with other putative protein kinase C inhibitors.…”

“…It has been shown to dosedependently inhibit the pancreatic secretory response (Ederveen et al, 1990 (Judd et al, 1989 (Caufield and Bolander, 1986).…”

“…At a concentration of 100 11M, it exerted a slight stimulatory effect on the release of newly-synthesized caseins in mammary cells and potentiated the PRL secretagogue effect. It should be noted that this compound, which inhibits acinar protein kinase C activity in rabbit pancreatic acini, also potentiates stimulated pancreatic amylase secretion (Pandol and Schoeffield, 1986;Ederveen et al, 1990). The latter authors concluded that protein kinase C could exert a negative feedback role in receptor-mediated pancreatic enzyme secretion.…”

Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

“…It has been reported that the PKC inhibitor H7 actually potentiates amylase release elicited by CCK and acetylcholine, suggesting that PKC plays an inhibitory role in the control of exocytosis (Pandol & Schoeffield, 1986). Subsequently the more specific inhibitor staurosporine was shown to inhibit secretion partially (Verme et al, 1989;Ederveen et al, 1990), supporting the generally accepted view of PKC as stimulatory in the exocytic pathway. Care should be taken in the evaluation of these data, as staurosporine has been subsequently shown to affect a wide variety of second-messenger systems other than PKC (Ruegg & Burgess, 1989).…”

Cholecystokinin-stimulated enzyme secretion from dispersed rabbit pancreatic acinar cells: phosphorylation-dependent changes in potency and efficacy

“…'Thus, TPA caused both a narrowing of the dose/response curve for CCKs and an increase in the efficacy of the secretagogue. These observations urged us to investi gate the effects of kinase activation and inactivation on that artificial activation of each one of these two routes tho shape of the dose/response curve for the stimulaonly moderately stimulates secretion [2,4 -X, 11, 14, 15, 19 21, 25, 29, 30], whereas simultaneous activation of both routes mimics the stimulatory effect of the Ca~}-mobilizing seeretagogues [5,19,25], Putative inhibitors of PKC' activity, such as 11-7 and staurosporine, have been used in an attempt to ascer tain the relative importance of both routes in the mech anism of action of the Ca-' -mobilizing secretagogues [7,21,25, 29|. Staurosporine was found to completely inhibit the secretory response to the phorbol ester 12-iMetradeeanoylphorbol 13-acetate (TPA) [7,21.…”