title: Phorbol ester mediated activation of inducible nitric oxide synthase results in platelet profilin nitration.
abstract: Nitric oxide is an important precursor for peroxynitrite production under in vivo conditions leading to cell injury and cell death. In platelets, a number of cytosolic and actin binding proteins were shown to be nitrated [K.M. Naseem, S.Y. Low, M. Sabetkar, N.J. Bradley, J. Khan, M. Jacobs, K.R. Bruckdorfer, The nitration of platelet cytosolic proteins during agonist-induced activation of platelets. FEBS Lett. 473 (1) (2000) 199-122 and M. Sabetkar, S.Y. Low, K.M. Naseem, K.R. Bruckdorfer, The nitration of proteins in platelets: significance in platelet function, Free Radic. Biol. Med. 33 (6) (2002) 728-736]. We investigated the possible mechanism that regulates profilin (an actin binding protein) nitration in platelets. Activation of bovine platelets with arachidonic acid, thrombin, and phorbol 12,13-dibutyrate resulted in nitration of profilin on tyrosine residue. In vivo profilin nitration showed a four- and eight-fold increase in the presence of thrombin and phorbol 12,13-dibutyrate, respectively. Analysis of nitroprofilin levels in the presence of NOS inhibitors (1400W and EGTA), indicated that profilin nitration in phorbol 12,13-dibutyrate treated platelets is mediated by inducible nitric oxide synthase. Phorbol ester treated platelets exhibited higher levels by inducible nitric oxide synthase (491% over control), while total nitric oxide synthase activity increased by 5% over control. Higher levels of peroxynitrite in platelets treated with phorbol 12,13-dibutyrate indicated that profilin nitration is mediated by peroxynitrite. Increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity in platelets treated with thrombin and phorbol 12,13-dibutyrate indicates that nitration of platelet profilin could be mediated by PI 3-kinase. A decrease in the level of nitroprofilin in PDBu treated platelets in the presence of inducible nitric oxide synthase inhibitor, 1400W, was observed suggesting that profilin nitration is mediated by PI 3-kinase dependent activation of inducible nitric oxide synthase.
authors.affiliation: Department of Biochemistry, Osmania University, Hyderabad, India [corrected]
authors.forename: S
authors.initials: S
authors.lastname: Kasina
authors.forename: R
authors.initials: R
authors.lastname: Wasia
authors.forename: Aneesa
authors.initials: A
authors.lastname: Fasim
authors.forename: K V N
authors.initials: KV
authors.lastname: Radhika
authors.forename: Surya S
authors.initials: SS
authors.lastname: Singh
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002118:Calcium / Q000097:blood; D002417:Cattle; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000097:blood; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood; D051304:Profilins / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D009566:Nitrates; D051304:Profilins; D009569:Nitric Oxide; D015240:Phorbol 12,13-Dibutyrate; D052247:Nitric Oxide Synthase Type II; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium