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.