Kinetic evidence that arachidonate-induced calcium efflux from platelet microsomes involves a carrier-type ionophoric mechanism.

Arachidonate, at concentrations up to 50 microM, 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 arachidonates 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.

Cyclic AMP-dependent protein kinase does not increase calcium transport in platelet microsomes.

Cyclic AMP inhibits platelet activation, at least in part, by reducing intracellular levels of ionic calcium. Previous studies using platelet microsomal fractions have suggested that one mechanism for this effect is stimulation by cyclic AMP and its protein kinase of calcium uptake into microsomal storage sites. In the present study, the effect of cyclic AMP and its protein kinase on calcium uptake by microsomal membranes has been re-examined using the active catalytic subunit of cyclic AMP-dependent protein kinase. The catalytic subunit increased calcium uptake two-fold, but this effect was not inhibited by boiling the catalytic subunit or by recombination with the regulatory subunit of cyclic AMP-dependent protein kinase, conditions that inhibited catalytic subunit activity. Conversely, dialysis of the catalytic subunit preparation against low phosphate buffer, which did not inhibit catalytic subunit activity, inhibited the stimulation of calcium uptake by the catalytic subunit preparation. Finally, the addition of high phosphate buffer, similar in phosphate concentration to that of the catalytic subunit preparation, stimulated calcium uptake. We conclude that the catalytic subunit does not directly stimulate calcium uptake by platelet microsomes.

The identification of sarcoplasmic reticulum terminal cisternae proteins in platelets.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.