Open AccessIndirect regulation of Ca 2+ entry by cAMP‐dependent and cGMP‐dependent protein kinases and phospholipase C in rat platelets
Author(s) -
HEEMSKERK Johan W. M.,
FEIJGE Marion A. H.,
SAGE Stewart O.,
WALTER Ulrich
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb19023.x
Subject(s) - thapsigargin , phospholipase c , extracellular , protein kinase c , protein kinase a , platelet , intracellular , chemistry , cytosol , microbiology and biotechnology , kinase , receptor , biology , biochemistry , medicine , enzyme , immunology
The Ca 2+ responses of rat platelets are dominated by the influx of extracellular Ca 2+ 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 Ca 2+ entry into these cells by measuring increases in cytosolic Ca 2+ concentration, [Ca 2+ ] 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 Ca 2+ entry was linked to the mobilisation of Ca 2+ from intracellular stores: there was good correlation between the potency of receptor agonists in elevating [Ca 2+ ] i in the presence or absence of external CaCl 2 ; agonist‐induced Ca 2+ 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 Ca 2+ ‐ATPases) evoked store depletion and Ca 2+ 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 Ca 2+ stores, the addition of CaCl 2 resulted in a considerable entry of Ca 2+ which was insensitive to cAMP‐dependent and cGMP‐dependent protein kinase activation. In control platelets with full Ca 2+ stores, CaCl 2 potentiated the thrombin‐induced generation of myo ‐inositol phosphates, suggesting that Ca 2+ entry potentiated phospholipase C activity. Taken together, these results indicate that Ca 2+ 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 Ca 2+ from the stores. These results lead to the concept that a major part of receptor‐mediated Ca 2+ entry in rat platelets is regulated in an indirect way by factors that stimulate or inhibit the degree of Ca 2+ mobilisation from the internal stores.