Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Abstract

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca 2+ + Mg 2+)-ATPase activity of about 10 nmol (min·mg) −1 and an ATP-dependent Ca 2+ uptake of about 10 nmol (min·mg) −1. When incubated in the presence of Mg[γ- 32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [ 32P]phosphate-labeled Ca 2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca 2+ or Ca 2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ( M r = 94 000, 87 000, 60 000 and 43 000 ). Some minor calmodulin-binding proteins were enriched in the membrane fractions ( M r = 69 000, 57 000, 39 000 and 37 000 ). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca 2+ uptake is essentially unaltered, while the Ca 2+ capacity is diminished as a consequence of a doubling in the rate of Ca 2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca 2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.