The thromboxane antagonist, 13-azaprostanoic acid, inhibits arachidonic acid-induced Ca 2+ release from isolated platelet membrane vesicles

Abstract

In the present study we investigated the ability of the arachidonic acid metabolites, prostaglandin H 2 and thromboxane A 2, to release Ca 2+ from isolated platelet vesicles. The vesicles were prepared through modification of previously described procedures. 45Ca uptake and release were determined by Millipore filtration and isotope counting of the filter paper. Incubation of the vesicles (25°C) with 50 μM CaCl 2 (plus 45Ca) resulted in the accumulation of 13 nmol Ca 2+ per mg of protein under steady-state conditions. Addition of arachidonic acid (25 μM) resulted in a 42% release of the accumulated Ca 2+ and the production of 150 ng thromboxane B 2/mg protein. Pretreatment of the vesicles with indomethacin (4 μM) completely inhibited arachidonic acid-induced Ca 2+ release and reduced thromboxane B 2 synthesis by 82%. Pretreatment of the vesicles with the specific thromboxane A 2/prostaglandin H 2 antagonist, 13-azaprostanoic acid (20 μM), also resulted in complete inhibition of Ca 2+ release but no inhibition of thromboxane B 2 production. Addition of prostaglandin H 2 (0.3 μM) to the platelet vesicles produced a significant release of Ca 2+ only in the presence of the adenylate cyclase inhibitor, 2′,5′-dideoxyadenosine (100 μM). This Ca 2+ release was totally blocked by 13-azaprostanoic acid (20 μM). The thromboxane synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I 3.6 μM), in the presence of 2′,5′-dideoxyadenosine, only slightly inhibited Ca 2+ release in response to added prostaglandin H 2, even though thromboxane B 2 production was blocked by 95%.