15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and nafazatrom

Phenylbutazone (PB), a nonsteroidal anti-inflammatory drug, is an efficient reducing cofactor for the peroxidase activity of prostaglandin H synthase (PHS). Most reducing cofactors for the peroxidase protect PHS and prostacyclin synthase from inactivation by hydroperoxides. PB, however, does not protect these enzymes, but rather augments their hydroperoxide-dependent inactivation. Using ram seminal vesicle microsomes as a source of PHS and prostacyclin synthase, we have examined the interaction of PB and exogenous hydroperoxides. Chromatographic analysis of the metabolism of 14C-labeled arachidonic acid in this system revealed that PB-dependent inactivation of PHS is markedly increased in the presence of 100 microM H2O2. This inactivation is a linear function of PB concentration between 10 and 250 microM, with a half-maximal effect in this range at about 100 microM PB. Prostacyclin synthase is even more sensitive to inactivation by the combined PB and H2O2 treatment, with a corresponding half-maximal effect at PB concentrations near 25 microM. This PB- and H2O2-dependent inactivation is demonstrable whether PGH2 is generated in situ from arachidonic acid or is added exogenously, supporting a direct effect of the treatment on prostacyclin synthase. As PB undergoes peroxide-dependent co-oxygenation catalyzed by PHS, we propose that it is an oxygenated derivative of PB, rather than the parent compound, which is responsible for the inactivation of PHS and prostacyclin synthase. Nafazatrom, a competitive inhibitor of PB co-oxygenation, blocks the effects of the PB and H2O2 treatment, supporting our proposal.

Topics: Animals; Arachidonic Acids; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Epoprostenol; Fibrinolytic Agents; Hydrogen Peroxide; Intramolecular Oxidoreductases; Kinetics; Leukotrienes; Lipid Peroxides; Male; Microsomes; Peroxides; Phenylbutazone; Prostaglandins; Pyrazoles; Pyrazolones; Seminal Vesicles; Sheep

Nafazatrom, an antithrombotic and antimetastatic agent containing a pyrazolone functionality, is a reducing substrate for the peroxidase activity of prostaglandin H (PGH) synthase. Nafazatrom inhibits the hydroperoxide-dependent oxidation of phenylbutazone, stimulates the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, and is oxidized by microsomal or purified enzyme preparations from ram seminal vesicles. Consonant with the effects of other peroxidase-reducing substrates, nafazatrom stimulates the oxygenation of arachidonic acid to prostaglandin endoperoxides by the cyclooxygenase component of PGH synthase. In addition, nafazatrom causes an elevation in the levels of 6-keto-prostaglandin F1 alpha, the non-enzymatic hydrolysis product of prostacyclin (PGI2) biosynthesized from arachidonic acid by ram seminal vesicle microsomes. Elevation of PGI2 biosynthetic capacity by nafazatrom occurs under conditions in which prostaglandin endoperoxide biosynthesis is maximal, suggesting that nafazatrom has a stimulatory effect on the conversion of prostaglandin endoperoxides to PGI2. Nafazatrom has no effect on the ability of ram seminal vesicle microsomes to convert PGH2 to PGI2 but protects microsomal PGI2 synthase from inactivation by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. Nafazatrom stimulates PGI2 biosynthesis in ram seminal vesicle microsomes by acting as a substrate for the peroxidase-catalyzed reduction of hydroperoxy fatty acids that are irreversible inactivators of PGI2 synthase. Several other compounds, including dipyridamole and triiodothyronine, exert similar effects. This may contribute to the reported ability of nafazatrom and related compounds to elevate the levels of bioassayable PGI2 in vivo and to the antithrombotic and antimetastatic activities of nafazatrom.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Epoprostenol; Fibrinolytic Agents; Hydrogen Peroxide; Kinetics; Leukotrienes; Lipid Peroxides; Male; Oxidation-Reduction; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins H; Pyrazoles; Pyrazolones; Seminal Vesicles; Sheep

Topics: Animals; Antineoplastic Agents; Arachidonic Acids; Cell Line; DNA Replication; Epoprostenol; Fibrinolytic Agents; Leukotrienes; Lipid Peroxides; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms, Experimental; Oxidoreductases; Peroxides; Pyrazoles; Pyrazolones; Thromboxane-A Synthase