Proteolytic activation of protein kinase Calpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein.
We sought to determine the roles of PKCalpha and G(i)alpha in regulating cPLA(2) activity in bovine pulmonary artery endothelial cell membrane under peroxynitrite (ONOO(-)) stimulation. Treatment of bovine pulmonary artery endothelial cells with ONOO(-) markedly stimulates the cell membrane associated protease activity, protein kinase C (PKC) activity, phospholipase A(2) (PLA(2)) activity, and arachidonic acid (AA) release from the cells. ONOO(-) significantly increases (Ca(2+))(i) in the cells, and pretreatment with the intracellular Ca(2+) chelator BAPTA-AM prevents the increase in (Ca(2+))(i), protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment of the cells with arachidonyl trifluoromethyl ketone (AACOCF(3)) (a cPLA(2) inhibitor) prevents ONOO(-)-stimulated cPLA(2) activity and AA release without producing a significant alteration of the protease activity. Pretreatment with vitamin E and aprotinin prevents ONOO(-)-induced increase in the protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment with the PKC inhibitor calphostin C prevents ONOO(-)-caused increase in PKC activity and cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot study of the cell membrane isolated from the ONOO(-)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. An immunoblot study with anti-nitrotyrosine antibody revealed that ONOO(-) induces nitration of tyrosine residues in PKCalpha. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. An immunoblot study of the endothelial cell membrane with polyclonal cPLA(2) antibody revealed that treatment of the cells with ONOO(-) markedly increases the cPLA(2) immunoreactive protein profile in the membrane. Pretreatment of the endothelial cells with Go6976, a PKCalpha inhibitor, prevents the increase in PKC activity and cPLA(2) activity in the cell membrane under ONOO(-)-triggered condition. It, therefore, appears from the present study that treatment of the cells with ONOO(-) causes an increase in the protease activity, and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot assay with polyclonal G(i)alpha antibody elicited an immunoreactive band having a molecular mass of 41 kDa. Pretreatment of the cells with pertussis toxin markedly inhibits ONOO(-)-induced increase in cPLA(2) activity and AA release without significantly altering (Ca(2+))(i), protease activity, and PKC activity in the cell membrane. Treatment of the cells with ONOO(-) causes phosphorylation of G(i)alpha in the cell membrane, and pretreatment with Go6976 prevents its phosphorylation. We suggest the existence of a pertusssis toxin sensitive G protein-mediated mechanism for activation of cPLA(2) by ONOO(-) in bovine pulmonary artery endothelial cell membrane, which is regulated by PKCalpha-dependent phosphorylation and sensitive to aprotinin for its inhibition.
Chakraborti
Tapati
T
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Das
Sudip
S
Chakraborti
Sajal
S
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D020013:Calcium Signaling; D002417:Cattle; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D008954:Models, Biological; D010447:Peptide Hydrolases / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011506:Proteins / Q000378:metabolism; D011651:Pulmonary Artery / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism
D011506:Proteins; D030421:Peroxynitrous Acid; D016718:Arachidonic Acid; D037342:Pertussis Toxin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010741:Phospholipases A; D054467:Phospholipases A2; D010447:Peptide Hydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go