INDIRECT REGULATION OF CA2-DEPENDENT AND CGMP-DEPENDENT PROTEIN-KINASES AND PHOSPHOLIPASE-C IN RAT PLATELETS( ENTRY BY CAMP)

The Ca2+ responses of rat platelets are dominated by the influx of ext racellular Ca2+ across the plasma membrane [Heemskerk, J. W. M., Feijg e, M. A. H., Rietman, E. & Hornstra, G. (1991) FEBS Lett. 284, 223], w hich allows the study of Ca2+ entry into these cells by measuring incr eases in cytosolic Ca2+ concentration, [Ca2+](i). Several pieces of ev idence 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, & Garcia-Sancho, J. (1991) Biochem. J. 280, 783-789], agonist-stimulated Ca2+ entry was linked to the mobilisatio n of Ca2+ from intracellular stores: there was good correlation betwee n the potency of receptor agonists in elevating [Ca2+](i) in the prese nce or absence of external CaCl2; agonist-induced Ca2+ entry was inhib ited to a similar degree as internal mobilisation by activators of cAM P-dependent or cGMP-dependent protein kinase or by the phospholipase C inhibitor, U73122; thapsigargin (an inhibitor of endomembrane Ca2+-AT Pases) 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+ stor es, the addition of CaCl2 resulted in a considerable entry of Ca2+ whi ch was insensitive to cAMP-dependent and cGMP-dependent protein kinase activation. In control platelets with full Ca2+ stores, CaCl2 potenti ated the thrombin-induced generation of myo-inositol phosphates, sugge sting that Ca2+ entry potentiated phospholipase C activity. Taken toge ther, 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 b y inhibition of store depletion, (c) can proceed in the absence of pho spholipase C activation, but is stimulated by this activity probably b y increased mobilisation of Ca2+ from the stores. These results lead t o the concept that a major part of receptor-mediated Ca2+ entry in rat platelets is regulated in an indirect way by factors that stimulate o r inhibit the degree of Ca2+ mobilisation from the internal stores.