AGONIST-MEDIATED ACTIVATION OF PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D IN INTESTINAL SMOOTH-MUSCLE

The contributions of phosphoinositide (PI)- and phosphatidylcholine (PC)-specific phospholipases [PI-specific phospholipase C (PI-PLC), PC-specific phospholipase C (PC-PLC), and phospholipase D (PLD)] to diacylglycerol (DAG) formation and regulation of the enzymes by G proteins, Ca2+, and protein kinase C (PKC) were examined in dispersed intestinal circular and longitudinal muscle cells. DAG formation induced by cholecystokinin was biphasic and paralleled by PKC activity. The initial phase (similar to 1 min) was mediated by PI-PLC in circular muscle cells and by both PI- and PC-PLC in longitudinal muscle cells, whereas the sustained phase was mediated by PC-PLC and PLD in both cell types. PC-PLC activity during the initial phase was identified by rapid formation of the initial products [H-3]phosphocholine (5 sec) and [H-3]myristate-labeled DAG (similar to 15 sec). PLD activity did not contribute to DAG formation during the initial phase, and PI hydrolysis had no effect on PC-PLC or PLD activity during the initial or sustained phases. PLD activity during the sustained phase was evident by the formation of [H-3]phosphatidylethanol, a PLD-specific transphosphatidylation product. Dephosphorylation of phosphatidic acid (PA) by phosphatidate phosphohydrolase (PPH) accounted for about 50% of DAG formation; inhibition of PPH activity by propranolol or suppression of PA formation by ethanol inhibited DAG formation by 59-69% and 57-62%, respectively. Residual DAG in the presence of ethanol was augmented 55-57% by DAG kinase inhibitor, whereas residual PA was inhibited by 60-67%, implying that PA was derived from DAG, and DAG from PLC-mediated PC hydrolysis, In the presence of ethanol, calphostin C inhibited phosphatidylethanol formation but had no effect on PA or DAG levels, implying that only PLD activity was modulated by PKC. Maintenance of resting intracellular Ca2+ concentrations, rather than an agonist-induced increase in the intracellular Ca2+ concentration, was required for optimal PC-PLC and PLD activity. Guanosine-5'-O-(beta-thio)diphosphate abolished DAG and PA formation in reversibly permeabilized muscle cells. We conclude that DAG formation in intestinal muscle is mediated by time-dependent activation of three phospholipases (PI-PLC, PC-PLC, and PLD) and two converting enzymes (DAG kinase and PPH). PC-PLC and PLD are Ca2+ dependent and appear to be G protein coupled; only PLD is PKC sensitive.