INVOLVEMENT OF PHOSPHATIDYLCHOLINE HYDROLYSIS BY PHOSPHOLIPASE-D IN EXTRACELLULAR ATP-INDUCED ARACHIDONIC-ACID RELEASE IN AORTIC SMOOTH-MUSCLE CELLS

We investigated the effect of extracellular ATP on phosphatidylcholine -hydrolyzing phospholipase D activity and the role of phospholipase D activation in extracellular ATP-induced arachidonic acid release in cu ltured rat aortic smooth muscle cells. ATP significantly stimulated th e formation of choline in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. However, ATP had no effect on the formation of p hosphocholine. Staurosporine, an inhibitor of protein kinases, did not affect the ATP-induced formation of choline. ATP significantly stimul ated arachidonic acid release in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. DL-Propranolol hydrochloride (propranolol ), an inhibitor of phosphatidic acid phosphohydrolase, significantly i nhibited the ATP-induced release of arachidonic acid. 1,6-Bis(cyclohex yloximinocarbonylamino)-hexane (RHC-80267), a potent and selective inh ibitor of diacylglycerol lipase, reduced ATP-induced arachidonic acid release. Quinacrine, a phospholipase A(2) inhibitor, suppressed ATP-in duced arachidonic acid release. Both propranolol and RHC 80267 markedl y inhibited the ATP-induced synthesis of 6-keto-prostaglandin F-1 alph a, a stable metabolite of prostacyclin. These results strongly suggest that extracellular ATP activates phosphatidylcholine-hydrolyzing phos pholipase D independently of protein kinase C in aortic smooth muscle cells and that the arachidonic acid release induced by extracellular A TP is mediated, at least in part, through phosphatidylcholine hydrolys is by phospholipase D activation.