Indirect regulation of Ca2+ entry by cAMP-dependent and cGMP-dependent protein kinases and phospholipase C in rat platelets.
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 Ca(2+)-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.
Heemskerk
J W
JW
Department of Human Biology, University of Limburg, Masstricht, The Netherlands.
Feijge
M A
MA
Sage
S O
SO
Walter
U
U
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases / Q000097:blood*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000097:blood*; D004357:Drug Synergism; D004533:Egtazic Acid / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D008297:Male; D015539:Platelet Activation / Q000187:drug effects; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000097:blood*
D004963:Estrenes; D011450:Prostaglandin Endoperoxides, Synthetic; D011760:Pyrrolidinones; D013729:Terpenes; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D004533:Egtazic Acid; D013928:Thromboxane A2; D019284:Thapsigargin; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D010738:Type C Phospholipases; D013917:Thrombin; D000252:Calcium-Transporting ATPases; D002122:Calcium Chloride; D002118:Calcium