Phospholipase C-γ Mediates the Hydrolysis of Phosphatidylinositol, but Not of Phosphatidylinositol 4,5-Bisphoshate, in Carbamylcholine-stimulated Islets of Langerhans

Abstract

In pancreatic islets the activation of phospholipase C (PLC) by the muscarinic receptor agonist carbamyol-choline (carbachol) results in the hydrolysis of both phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol (PtdIns). Here we tested the hypothesis that PtdIns hydrolysis is mediated by PLCγ1, which is known to be regulated by activation of tyrosine kinases and PtdIns 3-kinase. PtdIns breakdown was more sensitive than that of PtdInsP2 to the tyrosine kinase inhibitor, genistein. Conversely, the tyrosine phosphatase inhibitor, vanadate, alone promoted PtdIns hydrolysis and acted non-additively with carbachol. Vanadate did not stimulate PtdInsP2 breakdown. Carbachol also stimulated a rapid (maximal at 1-2 min) tyrosine phosphorylation of several islet proteins, although not of PLCγ1 itself. Two structurally unrelated inhibitors of PtdIns 3-kinase, wortmannin and LY294002, more effectively attenuated the hyrolysis of PtdIns compared with PtdInsP2. Adenovirally mediated overexpression of PLCγ1 significantly increased carbachol-stimulated PtdIns hydrolysis without affecting that of PtdInsP2. Conversely overexpression of PLCβ1 up-regulated the PtdInsP2, but not PtdIns, response. These results indicate that the hydrolysis of PtdIns and PtdInsP2 are independently regulated in pancreatic islets and that PLCγ1 selectively mediates the breakdown of PtdIns. The activation mechanism of PLCγ involves tyrosine phosphorylation (but not of PLCγ directly) and PtdIns 3-kinase. Our findings point to a novel bifurcation of signaling pathways downstream of muscarinic receptors and suggest that hydrolysis of PtdIns and PtdInsP2 might serve different physiological ends.