Intracellular calcium fluxes in human platelets

Abstract

Fluorescence changes and secretory response were measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membrane-associated Ca2+. When extracellular [Ca2+] is decreased to less than 0.1 .mu.M by addition of EGTA [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid] a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilization of membrane-associated Ca2+ on stimulation by maximal doses of 5 excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve. Similar ED50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence. The data provide further evidence supporting the proposal that chlortetracycline can be used as a probe to monitor mobilization of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilization and Ca2+ removal can be used under some conditions involve sites which are not monitored by this probe.