Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: Evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state

Abstract

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca2+ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokinin-octapeptide (CCK8) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca2+ concentration ([Ca2+]i,av) in a suspension of acinar cells. However, in the presence of 1.0 microM staurosporine the stimulatory effect of submaximal concentrations of CCK8 was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca2+]i,av in cells prestimulated with a submaximal concentration of CCK8. The data obtained with staurosporine indicate that CCK8-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca2+ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK8-induced Ca2+ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK8-evoked increase in [Ca2+]i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK8 at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca2+]i,av evoked by the CCK8 analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca2+]i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK8 concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca2+]i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca2+ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein kinase C activation inhibits receptor-evoked inositol trisphosphate formation and induction of cytosolic calcium oscillations by decreasing the affinity-state of the cholecystokinin receptor in pancreatic acinar cells

“…We have previously demonstrated that CCKs dose-dependently recruits freshly isolated pancreatic acinar cells in terms of receptor-evoked Ca mobili zation [5]. The present study shows that activation The inhibitory effects of TPA described here are consistent with those originally reported by Honda et aL [20] and support the idea that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state into a low-affinity state [18]. Moreover, they demonstrate that the TPAevoked reduction in peak increase of the average However, it should be noted that this conclusion is not supported by the observation that phorbol esters can effectively block ongoing Ca oscillations evoked by the G-protein activating agent mastoparan [23], Furthermore, the observation that Ca oscillations do occur in TPA-treated cells demon strates that protein kinase C is not directly involved in the mechanism by which higher CCKs concentra tions prevent the occurrence of oscillatory changes in [Ca2+]i.…”

“…In a recent study, we have demonstrated that ac tivation of protein kinase C by means of the phorbol ester 12-0~tetradecanoylphorbol 13-acetate (TPA) causes both a rightward shift of the dose-response curve for the stimulatory effect of CCKs on the peak increase in average [Ca2+]i and complete in hibition of JMV-180-evoked Ca2+ mobilization [18]. In other words, protein kinase C activation leads to inhibition of Ca2+ signaling through the high-affinity receptor, without interfering with Ca signaling through the low-affinity receptor.…”

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

“…This inhibitory action of the phorbol ester was found to be paralleled by inhibition of CCK-induced inositol 1,4,5-trisphosphate production [23] and Ca 2+ mobilization [20,22,23], suggesting an effect upstream of the phospholipase-C-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate. The inhibitory effect of TPA on CCK-induced inositol 1,4,5-trisphosphate production, Ca 2+ mobilization and enzyme secretion appeared to be restricted to submaximally effective CCK concentrations and was overcome by increasing the hormone concentration [20,22,23]. In contrast, TPA-evoked inhibition of the increase in [Ca 2+ ] i in response to the high-affinity receptor agonist JMV-180 was not overcome by increasing the agonist concentration [22].…”

“…The inhibitory effect of TPA on CCK-induced inositol 1,4,5-trisphosphate production, Ca 2+ mobilization and enzyme secretion appeared to be restricted to submaximally effective CCK concentrations and was overcome by increasing the hormone concentration [20,22,23]. In contrast, TPA-evoked inhibition of the increase in [Ca 2+ ] i in response to the high-affinity receptor agonist JMV-180 was not overcome by increasing the agonist concentration [22]. From these observations it was concluded that PKC directly or indirectly inhibits signalling through the high-affinity state of the CCK receptor.…”

“…The Fura-2-loaded cells were transferred to a cuvette placed in a Shimadzu RF-5000 spectrofluorophotometer equipped with a magnetic stirrer and a thermostatic cuvette holder. Fluorescence measurements were carried out at 37°C as described previously [21,22]. The fluorescence emission ratio at 490 nm was monitored as a measure of the average cytosolic free Ca 2+ concentration after excitation at 340 and 380 nm.…”