Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes' peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Znsuperoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid) 2 , and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inf lammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.Prostaglandins and thromboxanes are important mediators of inflammation, hyperalgesia, cell growth, and hemostasis inter alia. The committed step in prostaglandin biosynthesis is the oxygenation of arachidonic acid (AA) by prostaglandin endoperoxide (PGH) synthase, a bifunctional, membrane-bound hemeprotein (1-4). Inhibition of the cyclooxygenase activity of PGH synthase is the basis for the pharmacological action of nonsteroidal antiinflammatory drugs (5, 6). Two different PGH synthases exist in vertebrates-PGH synthase-1, which is expressed constitutively and occurs in many tissues, and PGH synthase-2, which is inducible and expressed transiently (7-10). PGH synthase-2 is present at high levels in monocytes͞ macrophages, where it appears to play a major role in the production of inflammatory prostaglandins (7,11).Several recent reports demonstrate that NO stimulates prostaglandin biosynthesis in vivo, in perfused organs, and in macrophages (12-17). The stimulatory effect of NO is rapid and appears to be the result of direct activation of cyclooxygenase activity (16). However, conflicting reports exist regarding the ability of NO to stimulate purified PGH synthase, and it is possible that a derivative of NO is responsible for the activation in inflammatory cells (16,(18)(19)(20).The principal mechanism described for direct activation of the cyclooxygenase activity of PGH synthase is reaction of fatty acid hydroperoxides with the heme prosthetic group to generate a protein radical. This protein radical (probably Tyr-385) serves as the catalytic oxidant of AA (21-23). The identity of the hydroperoxides that activate PGH synthase in different cell types is uncertain because fatty acid hydroperoxides are excellent substrates for glutathione peroxidase (GSH-Px)-catalyzed reduction by glutathione (GSH) (24). The potential for control of prostaglandin biosynthesis by GSH-Px͞GSH reduction of hydroperoxide activators exhibits s...

The modification of reduced cysteines of proteins by nitric oxide alters protein function, structure, and potentially, interactions with downstream signaling targets. We assessed the effect of the S-nitroso compounds S-nitrosoglutathione and S-nitroso-N-acetyl-penicillamine, the NO donor 2-(N,N-diethylamino)-diazenolate 2-oxide, and the nitroxyl donor Angeli's salt on the cysteines of the abundant cytoskeletal protein, tubulin. Total cysteine modification by each compound was quantitated and compared to peroxynitrite anion, an oxidant that we have studied previously. Angeli's salt was most effective at modifying the cysteines of tubulin and at inducing the formation of tubulin interchain disulfide bonds followed by peroxynitrite anion, S-nitrosoglutathione, S-nitroso-N-acetyl-penicillamine, and 2-(N,N-diethylamino)-diazenolate 2-oxide. S-nitrosation of tubulin by S-nitrosoglutathione and S-nitroso-N-acetyl-penicillamine was detected by the Saville assay. Our data show that tubulin interchain disulfide bond formation by these molecules correlated with inhibition of tubulin polymerization. Closer examination of the reaction of tubulin with S-nitrosoglutathione showed a concentration-dependent shift in the type of cysteine modification detected. More tubulin disulfides were detected at lower concentrations of S-nitrosoglutathione than at higher concentrations, suggesting that reduced glutathione, generated by the reaction of S-nitrosoglutathione with tubulin cysteines, reduced disulfides initially formed by S-nitrosoglutathione.

Prostaglandins and NO. are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO. biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO. and PGHS, but this is not universally observed. Clarification of the role of NO. in PGHS catalysis has been attempted by examining NO. interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO. has not yet emerged. Available studies suggest that NO. may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2.-+NO.) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2.- from RAW 267.4 cells that produce NO. and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.