Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and the tyrphostin ST271 inhibit phospholipase C in human platelets by preventing Ca2+ entry.
In the present study, the roles of Ca2+ and fibrinogen receptor occupancy in the regulation of phospholipase C by G protein-coupled and tyrosine kinase-linked receptor pathways in human platelets have been investigated. Agonist stimulation of phospholipase C was not altered significantly in the absence of stirring or in the presence of the fibrinogen receptor antagonist arginine-glycine-aspartate-serine, conditions that prevent platelet aggregation. Similarly, elevation of intracellular Ca2+ levels by the ionophores A23187 or ionomycin did not induce formation of inositol phosphates. In contrast, chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) reduced formation of inositol phosphates by G protein receptor (thrombin)- and tyrosine kinase (Fc receptor and peroxovanadate)-regulated pathways. Similarly, short term exposure to Ni2+ ions, which also prevent Ca2+ entry, inhibited thrombin-stimulated formation of inositol phosphates. Loading of platelets with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) markedly suppressed elevation of intracellular Ca2+ and formation of inositol phosphates in platelets stimulated by G protein receptor- and tyrosine kinase-regulated pathways. The greater inhibition of phospholipase C by BAPTA, relative to that induced by EGTA, is consistent with the more pronounced inhibition of intracellular Ca2+ elevation. The tyrphostin tyrosine kinase inhibitor ST271 also reduced intracellular Ca2+ levels and inhibited activation of phospholipase C. The degree of inhibition of phospholipase C by ST271 was slightly greater than that induced by EGTA but was not additive with the effect of EGTA, suggesting a common mode of action. It is concluded that elevation of intracellular Ca2+ regulates agonist-induced activation of phospholipase C and that this contributes to the inhibition of thrombin-induced formation of inositol phosphates by the tyrphostin ST271.
Watson
S P
SP
Department of Pharmacology, University of Oxford, UK.
Poole
A
A
Asselin
J
J
D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D013343:Styrenes / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D007295:Inositol Phosphates; D010980:Platelet Membrane Glycoproteins; D013343:Styrenes; C086531:ST 271; D000001:Calcimycin; D004533:Egtazic Acid; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium