KINETIC EVIDENCE THAT ARACHIDONATE-INDUCED CALCIUM EFFLUX FROM PLATELET MICROSOMES INVOLVES A CARRIER-TYPE IONOPHORIC MECHANISM

Arachidonate, at concentrations up to 50 μM, induced dose-dependent calcium efflux from preloaded microsomes prepared from human platelets, but not from unilamellar egg phosphatidylcholine vesicles. Arachidonate-induced efflux from microsomes was not inhibited by indomethacin, 13-azaprostanoic acid, or catalase and superoxide dismutase, indicating that the release was due to arachidonate and not a metabolite. Linolenate (18:3, cis) and linoleate (18:2, cis) induced calcium efflux in a manner similar to arachidonate (20:4, cis), while arachidate (20:0), linolelaidate (18:2, trans, elaidate (18:1, trans), oleate (18:1, cis), stearate (18:0) and palmitate (16:0) had no effect. An experimental method was developed for distinguishing between carrier ionophore, small aqueous pore (i.e., calcium channel), or large aqueous pore (i.e., detergent effect) mechanisms in vesicular efflux systems in which calcium efflux occurs over a period of minutes. This development predicted that with a carrier ionophore mechanism, an increase in either internal or external calcium should competitively inhibit 45Ca efflux. In contrast, 45Ca efflux by diffusion through a small aqueous pore or a large aqueous pore should be measurably insensitive to variations in internal or external calcium. These predictions were experimentally verified in the platelet microsomal system using efflux agents with known mechanisms. Efflux of 45Ca by A23187, a calcium ion carrier ionophore, was sensitive to internal or external calcium competition, while alamethicin, a small aqueous pore channel model, and Triton X-100, a detergent which forms large aqueous pores, mediated 45Ca efflux which was measurably insensitive to variations in internal or external calcium concentration. Arachidonate-induced 45Ca efflux was inhibited by increasing either internal and external calcium concentration, suggesting that the fatty acid functions as a carrier ionophore. Arachidonate-induced 45Ca efflux was also inhibited with extravesicular Sr2+, but not Mn2+ or Ba2+. The dependence of the initial arachidonate efflux rate on arachidonate concentration showed that at least two aracidonates were contained in the calcium-carrier complex. These results are consistent with a model in which arachidonate (A) and an endogenous microsomal component (B) translocate calcium across the membrane through a carrier ionophore mechanism as part of a complex with a stoichiometry of A2B·Ca. © 1990.