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The role of extracellular calcium in modulating the actions of cholecystokinin octapeptide (CCK8) on phosphatidylinositol 4,5-bisphosphate hydrolysis was studied in freshly isolated rat pancreatic acini and cultured AR42J cells. In both cell types, CCK8 rapidly induced the formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and 1,3,4, 5-tetrakisphosphate [Ins(1,3,4,5)P4]. The actions of CCK8 were inhibited by lanthanum and manganese, agents that block transmembrane calcium fluxes, and by chelation of extracellular calcium with EGTA. In pancreatic acini, lanthanum and manganese also partially inhibited the effects of carbachol and bombesin on Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. In acini, the CCK8-mediated increases in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels were progressively greater as the extracellular calcium concentration was raised from the micromolar range to 1.28 mM and progressively smaller as the manganese concentration was raised from 10 microM to 1 mM. Furthermore, the CCK8-mediated rise in Ins(1,4,5)P3 levels was partially attenuated by the calcium channel blockers verapamil, diltiazem, and nifedipine. These findings indicate that extracellular calcium enhances the ability of CCK8 and other calcium-mobilizing agonists to generate biologically active inositol phosphates in pancreatic acinar cells.

The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.