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 carbamyolcholine (carbachol) results in the hydrolysis of both phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) and phosphatidylinositol (PtdIns). Here we tested the hypothesis that PtdIns hydrolysis is mediated by PLC␥1, which is known to be regulated by activation of tyrosine kinases and PtdIns 3-kinase. PtdIns breakdown was more sensitive than that of PtdInsP 2 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 PtdInsP 2 breakdown. Carbachol also stimulated a rapid (maximal at 1-2 min) tyrosine phosphorylation of several islet proteins, although not of PLC␥1 itself. Two structurally unrelated inhibitors of PtdIns 3-kinase, wortmannin and LY294002, more effectively attenuated the hyrolysis of PtdIns compared with PtdInsP 2 . Adenovirally mediated overexpression of PLC␥1 significantly increased carbachol-stimulated PtdIns hydrolysis without affecting that of PtdInsP 2 . Conversely overexpression of PLC␤1 up-regulated the PtdInsP 2 , but not PtdIns, response. These results indicate that the hydrolysis of PtdIns and PtdInsP 2 are independently regulated in pancreatic islets and that PLC␥1 selectively mediates the breakdown of PtdIns. The activation mechanism of PLC␥ involves tyrosine phosphorylation (but not of PLC␥ 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 PtdInsP 2 might serve different physiological ends.Many receptor tyrosine kinases and GPCRs 1 couple to PLC that catalyzes the cleavage of PtdInsP 2 and resultant generation of the two second messengers Ins(1,4,5)P 3 , which mobilizes Ca 2ϩ from intracellular stores, and DAG, which activates protein kinase C (1-4). These pathways are well described in the regulation of insulin secretion following occupation of m3 and m1 muscarinic receptors on the surface of pancreatic ␤Ϫcells (5-8). However we have shown that in addition to the classical pathway of PtdInsP 2 hydrolysis, exposure of pancreatic islets to the muscarinic receptor agonist carbachol also promotes the hydrolysis of PtdIns (9, 10). The latter predominates quantitatively over, and is a precursor of, PtdInsP 2 . Although hydrolysis of either phosphoinositide species results in generation of DAG, only PtdInsP 2 gives rise to a Ca 2ϩ signal via Ins(1,4,5)P 3 production. Therefore the two hydrolytic events may have different functional consequences. The inositol phosphate, corresponding to Ins(1,4,5)P 3 , that is derived from PtdIns, is Ins(1)P 1 . This has no biological function but can be used to quantify PtdIns hydrolysis, at least under the conditions verified in our previous studies (9).The PLC family is comprised of four major groups (PLCs ␤, ␥, ␦, and ⑀). Members of the PLC␤ family are activated by Gproteins in response to o...

Evidence for Selective Coupling of α1-Adrenergic Receptors to Phospholipase C-β1 in Rat Neonatal Cardiomyocytes

“…It is possible that the large excess of PLC-␤ 3 relative to PLC-␤ 1 in Ad-PLC-␤ 3 NCM competes for access to endogenous G␣ q (13). Overexpression of PLC-␥ 1 (27) did not alter [ 3 H]InsP responses to either NE or ATP, and the tyrosine kinase inhibitor genistein (50 M) was not inhibitory (data not shown). Thus, neither receptor appears to be coupled to PLC-␥ under the conditions of our experiments (35).…”

“…Blank virus (Ad-MX) contained the adenoviral backbone but expressed no additional gene product. Rat PLC␥ 1 was subcloned into pShuttle-CMV and recombined with pAdEasy-1 (27).…”

Oscillations of Phospholipase C Activity Triggered by Depolarization and Ca2+ Influx in Insulin-secreting Cells

“…In contrast, depolarization was reported to inhibit PLC-mediated hydrolysis of phosphatidylinositol without effect on PIP 2 in rat islets (29). The different regulation of phosphatidylinositol and PIP 2 hydrolysis may reflect the involvement of different PLC isoforms with distinct substrate specificities (30 . Although the phenomenon still remains to be shown in primary ␤-cells, we have observed oscillations in PLC activity also in murine insulin-secreting MIN6 cells, which exhibit a glucose sensitivity similar to that of primary ␤-cells.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…The eukaryotic PI-PLCs can use PtdIns, PtdIns4P, and PtdIns(4,5)P 2 but not 3-phosphorylated inositides as substrates in vitro, but they are believed to act primarily on PtdIns(4,5)P 2 in their native cellular environment. Only a few studies addressed this question rigorously, and a few reports suggested that PLCs act on PtdIns (1066) and that they are present in intracellular locations (149,354), where they could act on PtdIns or PtdIns4P.…”