title: The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets.
abstract: We have investigated the sub-second kinetics of changes in cytosolic free calcium, [Ca2+]i, in fura-2-loaded human platelets by stopped-flow fluorimetry. Thrombin, vasopressin, platelet-activating factor, and the thromboxane A2 analogue U46619 all evoked a rise in [Ca2+]i which was delayed in onset by 200-400 ms in the presence of 1 mM external Ca2+. The responses to these agonists in media containing 1 mM EGTA or 1 mM Ni2+, to prevent Ca2+ influx, were delayed by an additional 60-100 ms. These results indicate that agonist-evoked Ca2+ influx precedes the release of Ca2+ from internal stores. The delays in onset of both responses are sufficient for one or more biochemical steps to lie between ligand-receptor binding and Ca2+ flux generation. ADP responses in media containing EGTA or Ni2+ were similar to those evoked by other agonists, but the response in the presence of external Ca2+ was markedly shorter, occurring without measurable delay at optimal ligand concentration. Analysis of this response showed some delay in ADP-evoked influx at lower concentrations, but this delay was markedly less than that observed with thrombin at doses giving the same elevation in [Ca2+]i. These results suggest that ADP evokes influx using a different transduction system, more closely coupled to the Ca2+ entry system than that used by other agonists. Differences between thrombin- and ADP-evoked influx were further demonstrated by the inhibitory actions of cAMP, which reduced and substantially increased the delay in onset of thrombin-evoked influx but did not measurably delay the influx evoked by an optimal concentration of ADP.
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D001572:Benzofurans; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D005576:Colforsin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D005470:Fluorometry; D016257:Fura-2; D006801:Humans; D007700:Kinetics; D009532:Nickel / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000494:pharmacology; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D001572:Benzofurans; D010972:Platelet Activating Factor; D011450:Prostaglandin Endoperoxides, Synthetic; D014667:Vasopressins; D005576:Colforsin; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D009532:Nickel; D013917:Thrombin; D002118:Calcium; D016257:Fura-2