Indirect regulation of Ca2+ entry by cAMP‐dependent and cGMP‐dependent protein kinases and phospholipase C in rat platelets

Abstract

The Ca2+ responses of rat platelets are dominated by the influx of extracellular Ca2+ across the plasma membrane [Heemskerk, J. W. M., Feijge, M. A. H., Rietman, E. & Hornstra, G. (1991) FEBS Lett. 284, 223], which allows the study of Ca2+ entry into these cells by measuring increases in cytosolic Ca2+ concentration, [Ca2+]i. Several pieces of evidence indicated that, as in human platelets [Sage, S. O., Reast, R., & Rink, T. J. (1990) Biochem. J. 265, 675–680; Alonso, M., Alvarez, J., Montero, M., Sanchez, A. & García‐Sancho, J. (1991) Biochem. J. 280, 783–789], agonist‐stimulated Ca2+ entry was linked to the mobilisation of Ca2+ from intracellular stores: there was good correlation between the potency of receptor agonists in elevating [Ca2+]i in the presence or absence of external CaCl2; agonist‐induced Ca2+ entry was inhibited to a similar degree as internal mobilisation by activators of cAMP‐dependent or cGMP‐dependent protein kinase or by the phospholipase C inhibitor, U73122; thapsigargin (an inhibitor of endomembrane Ca2+ ‐ATPases) evoked store depletion and Ca2+ entry, which were both reduced by prior activation of cAMP‐dependent or cGMP‐dependent protein kinase but were not affected by U73122. In platelets with depleted Ca2+ stores, the addition of CaCl2 resulted in a considerable entry of Ca2+ which was insensitive to cAMP‐dependent and cGMP‐dependent protein kinase activation. In control platelets with full Ca2+ stores, CaCl2 potentiated the thrombin‐induced generation of myo‐inositol phosphates, suggesting that Ca2+ entry potentiated phospholipase C activity. Taken together, these results indicate that Ca2+ entry in rat platelets, (a) is mostly secondary to store depletion, (b) is not directly downregulated by cAMP‐dependent and cGMP‐dependent protein kinase, but indirectly by inhibition of store depletion, (c) can proceed in the absence of phospholipase C activation, but is stimulated by this activity probably by increased mobilisation of Ca2+ from the stores. These results lead to the concept that a major part of receptor‐mediated Ca2+ entry in rat platelets is regulated in an indirect way by factors that stimulate or inhibit the degree of Ca2+ mobilisation from the internal stores. Copyright © 1994, Wiley Blackwell. All rights reserved