Oxidant-mediated activation of cytosolic phospholipase a(2) in pulmonary endothelium: role of protein kinase C alpha and a pertussis toxin-sensitive protein.
The authors have previously demonstrated that the oxidant t-buOOH stimulates phospholipase A(2) (PLA(2)) activity in bovine pulmonary artery endothelial cells (S. Chakraborti et al. American Journal of Physiology, 257, L430-L437, 1989). Herein, the authors sought to investigate the mechanism by which t-buOOH stimulates PLA(2) activity and the role of protein kinase C (PKC) in this scenario. Treatment of bovine pulmonary artery endothelial cells with t-buOOH stimulated an aprotinin-sensitive protease activity, PKC activity, and PLA(2) activity in the cell membrane. Pretreatment with intracellular Ca(2+) chelator (BAPTA-AM), PKCalpha inhibitor (Go6976), cPLA(2) inhibitor (AACOCF(3)), and pertussis toxin prevented t-buOOH-stimulated PLA(2) activity. Immunoblot studies with aprotinin, cPLA(2), PKCalpha, and Gialpha antibodies revealed their presence in the endothelial membrane. Immunoblot studies of the cell membrane isolated from t-buOOH-stimulated cells with cPLA(2) and PKCalpha antibodies elicited an apparent increase in their immunoreactive protein profiles along with an additional 47-kDa immunoreactive fragment in the membrane. t-buOOH caused Gialpha phosphorylation in the membrane and pretreatment with Go6976 prevented the phosphorylation. Overall, these results suggest that t-buOOH stimulates an aprotinin-sensitive protease activity that proteolytically activates PKCalpha and that subsequently phosphorylates a pertussis toxin-sensitive protein, resulting in the stimulation of cPLA(2) activity in the cell membrane.
Chakraborti
Sajal
S
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India. saj_chakra@rediffmail.com
Das
Sudip
S
Chakraborti
Tapati
T
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D010447:Peptide Hydrolases / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D016877:Oxidants; D020122:tert-Butylhydroperoxide; D037342:Pertussis Toxin; D051571:Protein Kinase C-alpha; D010741:Phospholipases A; D010447:Peptide Hydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go