Studies on the reduction of endogenously generated prostaglandin G₂ by prostaglandin H synthase

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G₂ (PGG₂) via its cyclooxygenase activity and reduces PGG₂ to prostaglandin H₂ by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG₂ is the preferred substrate for the peroxidase compared with exogenous PGG₂. Arachidonic acid and varying concentrations of exogenous PGG₂ were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP^.+) served as an index of peroxide reduction. The simultaneous addition of PGG₂ with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP^.+. This suggests that the formation of AP^.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG₂. In addition to the AP^.+ assay, the reduction of exogenous versus endogenous PGG₂ was studied by using [5,6,8,9,11,12,14,15-²H]arachidonic acid and unlabeled PGG₂ as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG₂. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG₂ was observed even under conditions in which all of the endogenous PGG₂ was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG₂ using similar experimental conditions. Our findings indicate that PGG₂ formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.