ARACHIDONIC-ACID RELEASE FROM NIH 3T3 CELLS BY GROUP-I PHOSPHOLIPASE A(2) - INVOLVEMENT OF A RECEPTOR-MEDIATED MECHANISM

Group I pancreatic phospholipase A(2) (PLA(2) I) is primarily a digest ive enzyme. Recently, however, in addition to its catalytic activity a receptor-mediated function has been described for this enzyme. PLAL(2 ) I binding to its receptor induces cellular chemokinesis, proliferati on, and smooth muscle contraction. This enzyme also induces the produc tion of prostaglandin E(2) in certain cells and may have a proinflamma tory role. However, despite its ability to hydrolyze phospholipids in in vitro assays, PLA(2)-I does not efficiently catalyze release of AA from intact cells. Here, we demonstrate that while short-term exposure of NIH 3T3 cells to PLA(2) is ineffective, exposure of 6 h or longer significantly increases the basal release of AA. Dose-response curve o f PLA(2)-l-induced AA release was saturable with an EC(50) of 14.01 +/ - 1.36 nM (n = 3). [H-3]-AA was preferentially released over [H-3]olei c acid by PLA(2)-I. PLA(2)-I, inactivated with 4-bromophenacyl bromide , was fully capable of mediating AA release. These data suggest that a non-catalytic, receptor-mediated mechanism is involved in PLA(2)-l-in duced AA release in NIH-3T3 cells. This release of AA is not dependent on protein kinase C or Ca2+ concentration. Comparison of the effect o f PLA(2)-I with those of ATP and platelet-derived growth factor indica tes that each of these agonists regulates AA release via independent p athways. Neither the basal enzymatic activity of the 85-kDa cytosolic PLA(2) nor the protein level of this enzyme was affected by treatment of cells with PLA(2)-I. However, the increase in basal enzymatic activ ity of 85 kDa PLA(2) due to protein kinase C activation was further en hanced by pretreatment of cells with PLA(2)-I. We conclude that: (1) s hort-term exposure oi cells to PLA(2) I does not cause measurable AA r elease; (2) release of AA from intact cells by this enzyme requires lo ng-term exposure; (3) AA release is not mediated by a direct catalytic effect of PLA(2) I; and (4) AA release by PLA(2) I is accomplished vi a a receptor-mediated process. Taken together, these results raise the possibility that PLA(2) I, in addition to its digestive function, may also contribute to aggravate preexisting inflammatory processes and/o r to initiate new ones when chronic exposure of cells to this enzyme o ccurs. (C) 1995 Wiley-Liss, Inc.