Agonist-induced Ca2+ influx into human platelets is secondary to the emptying of intracellular Ca2+ stores.
We have studied the relation between the filling state of the intracellular Ca2+ stores and the plasma-membrane permeability to Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Emptying of the intracellular Ca2+ stores either by incubation in Ca(2+)-free medium or by treatment with low concentrations of the Ca2+ ionophore ionomycin accelerated the influx of Mn2+. Refilling of the Ca2+ stores by incubation in Ca(2+)-containing medium restores low Mn2+ permeability. This Ca(2+)-store-regulated permeability was inhibited by Ni2+ and by cytochrome P-450 inhibitors. Stimulation of platelets with thrombin produced Ca2+ release from the intracellular stores, which was followed, after a temperature-dependent lag (2 s at 37 degrees C; 5 s at 18 degrees C), by an acceleration of Mn2+ influx. Cytochrome P-450 inhibitors prevented the thrombin-induced Mn2+ influx, with little effect on the Ca2+ mobilization from the intracellular stores. Ki values were similar to those estimated for inhibition of the store-regulated permeability in non-stimulated platelets. Similar results were found in platelets stimulated by platelet-activating factor or by ADP. We propose that agonist-induced Ca2+ (Mn2+) influx in platelets is secondary to the emptying of the intracellular Ca2+ stores. The activation of the plasma-membrane Ca2+ (Mn2+) pathway may take place by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & García-Sancho (1991) Biochem. J. 274, 193-197] and neutrophils [Montero, Alvarez & García-Sancho (1991) Biochem. J. 277, 73-79].
Alonso
M T
MT
Departamento de Bioquímica, Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Spain.
Alvarez
J
J
Montero
M
M
Sanchez
A
A
García-Sancho
J
J
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism*; D065607:Cytochrome P-450 Enzyme Inhibitors; D016257:Fura-2; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009532:Nickel / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000494:pharmacology
D002120:Calcium Channel Agonists; D065607:Cytochrome P-450 Enzyme Inhibitors; D010972:Platelet Activating Factor; D008345:Manganese; D015759:Ionomycin; D000244:Adenosine Diphosphate; D009532:Nickel; D013917:Thrombin; D002118:Calcium; D016257:Fura-2