The thromboxane antagonist, 13-azaprostanoic acid, inhibits arachidonic acid-induced Ca2+ release from isolated platelet membrane vesicles.

ABSTRACT

In the present study we investigated the ability of the arachidonic acid metabolites, prostaglandin H2 and thromboxane A2, to release Ca2+ 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 degrees C) with 50 microM CaCl2 (plus 45Ca) resulted in the accumulation of 13 nmol Ca2+ per mg of protein under steady-state conditions. Addition of arachidonic acid (25 microM) resulted in a 42% release of the accumulated Ca2+ and the production of 150 ng thromboxane B2/mg protein. Pretreatment of the vesicles with indomethacin (4 microM) completely inhibited arachidonic acid-induced Ca2+ release and reduced thromboxane B2 synthesis by 82%. Pretreatment of the vesicles with the specific thromboxane A2/prostaglandin H2 antagonist, 13-azaprostanoic acid (20 microM), also resulted in complete inhibition of Ca2+ release but no inhibition of thromboxane B2 production. Addition of prostaglandin H2 (0.3 microM) to the platelet vesicles produced a significant release of Ca2+ only in the presence of the adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (100 microM). This Ca2+ release was totally blocked by 13-azaprostanoic acid (20 microM). The thromboxane synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I, 3.6 microM), in the presence of 2',5'-dideoxyadenosine, only slightly inhibited Ca2+ release in response to added prostaglandin H2, even though thromboxane B2 production was blocked by 95%.