Role of an aprotinin-sensitive protease in protein kinase Cα-mediated activation of cytosolic phospholipase A 2 by calcium ionophore (A23187) in pulmonary endothelium

Abstract

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A 2 (PLA 2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF 3), a cPLA 2 inhibitor, but not bromoenollactone (BEL), a iPLA 2 inhibitor, prevents A23187 stimulated PLA 2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA 2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 μM)-treated cells with polyclonal PKCα antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA 2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA 2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 μM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA 2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 μM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCα, which subsequently stimulates cPLA 2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA 2 activity and AA release caused by both low and high doses of A23187.