title: Role of an aprotinin-sensitive protease in protein kinase Calpha-mediated activation of cytosolic phospholipase A2 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 A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 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 cPLA2 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 microM)-treated cells with polyclonal PKCalpha 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 cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 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 microM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 microM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA2 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 cPLA2 activity and AA release caused by both low and high doses of A23187.
authors.affiliation: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India. saj_chakra@rediffmail.com
authors.forename: Sajal
authors.initials: S
authors.lastname: Chakraborti
authors.forename: John R
authors.initials: JR
authors.lastname: Michael
authors.forename: Tapati
authors.initials: T
authors.lastname: Chakraborti
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism; D001095:Arachidonic Acids / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011651:Pulmonary Artery / Q000201:enzymology*
chemical_list: D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; C081565:arachidonyltrifluoromethane; D016718:Arachidonic Acid; D000001:Calcimycin; D007611:Aprotinin; D037342:Pertussis Toxin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010741:Phospholipases A; D054467:Phospholipases A2