ABSTRACT
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.
Subject(s)
Fibrinolytic Agents/pharmacology , Leukotrienes , Prostaglandin-Endoperoxide Synthases/metabolism , Pyrazoles/pharmacology , Pyrazolones , Seminal Vesicles/enzymology , Animals , Arachidonic Acid , Arachidonic Acids/metabolism , Arachidonic Acids/pharmacology , Epoprostenol/biosynthesis , Hydrogen Peroxide/metabolism , Kinetics , Lipid Peroxides/pharmacology , Male , Oxidation-Reduction , Prostaglandin Endoperoxides, Synthetic/metabolism , Prostaglandin H2 , Prostaglandins H/metabolism , SheepABSTRACT
The ESR spin-trapping technique has been used to identify a free radical involved in the oxygenation of arachidonic acid by ram seminal vesicle microsomes. The ESR spectrum of the radical adduct indicates that a carbon-centered arachidonic acid free radical has been observed. The formation of this species is inhibited by indomethacin, but not by phenol, and it is probably the first intermediate formed during the prostaglandin synthetase-catalyzed oxidation of arachidonic acid. The chemical identity of the trapped radical was substantiated with an independent synthesis of a closely related radical adduct.
Subject(s)
Arachidonic Acids , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Electron Spin Resonance Spectroscopy , Free Radicals , Male , Microsomes/enzymology , Oxidation-Reduction , Oxygen Consumption , Seminal Vesicles/enzymology , SheepABSTRACT
We have investigated the time course of formation of thromboxane A2, thromboxane B2, and the C-17 hydroxy fatty acid, HHT, from arachidonic acid in a washed human platelet suspension. Our results indicate that HHT is not a breakdown product of thromboxane A2, but rather thromboxane A2 decomposes exclusively into thromboxane B2. The kinetics of formation of thromboxane B2 from the endoperoxide prostaglandin H2 in human platelet microsomes was examined. Our data suggest that a bimolecular reaction is involved in the formation of thromboxane A2 from prostaglandin H2 and that thromboxane synthase is not an isomerase, but may be acting via a dismutase-type reaction. One possibility is that thromboxane and HHT are produced simultaneously from two molecules of prostaglandin H2.
