Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] may be the initiating event for the increase in [³²P]P(i) incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2 h, [³²P]P(i) incorporation into PtdA, PtdIns(4,5)P₂ and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused ³²P in PtdIns(4,5)P₂ to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [³²P]P(i) incorporation into PtdA and PtdIns. Similar changes in ³²P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in ³²P in chromatographically-purified PtdIns(4,5)P₂ reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P₂ was prelabelled with [2-³H]inositol, and (b) when PtdIns(4,5)P₂ was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P₂ was not inhibited by Ca²⁺ deficiency (severe enough to inhibit amylase secretion and Ca²⁺-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P₂ hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P₂ and the increase in [³²P]P(i) incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P₂ is an early event in the action of pancreatic secretogogues that mobilize Ca²⁺, and it is possible that this hydrolysis may initiate the Ca²⁺-independent labelling of PtdA and PtdIns. Ca²⁺ mobilization may follow these responses, and subsequently cause Ca²⁺-dependent hydrolysis of PtdIns and exocytosis.