AGONIST-INDUCED CA2+ INFLUX INTO HUMAN PLATELETS IS SECONDARY TO THE EMPTYING OF INTRACELLULAR CA2+ STORES

We have studied the relation between the filling state of the intracellular Ca2+ stores and the plasma-membrane permeability to Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Emptying of the intracellular Ca2+ stores either by incubation in Ca2+-free medium or by treatment with low concentrations of the Ca2+ ionophore ionomycin accelerated the influx of Mn2+. Refilling of the Ca2+ stores by incubation in Ca2+-containing medium restored low Mn2+ permeability. This Ca2+-store-regulated permeability was inhibited by Ni2+ and by cytochrome P-450 inhibitors. Stimulation of platelets with thrombin produced Ca2+ release from the intracellular stores, which was followed, after a temperature-dependent lag (2 s at 37-degrees-C; 5 s at 18-degrees-C), by an acceleration of Mn2+ influx. Cytochrome P-450 inhibitors prevented the thrombin-induced Mn2+ influx, with little effect on the Ca2+ mobilization from the intracellular stores. K(i) values were similar to those estimated for inhibition of the store-regulated permeability in non-stimulated platelets. Similar results were found in platelets stimulated by platelet-activating factor or by ADP. We propose that agonist-induced Ca2+ (Mn2+) influx in platelets is secondary to the emptying of the intracellular Ca2+ stores. The activation of the plasma-membrane Ca2+ (Mn2+) pathway may take place by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197] and neutrophils [Montero, Alvarez & Garcia-Sancho (1991) Biochem. J. 277, 73-79].