title: ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.
abstract: Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.
authors.affiliation: Physiological Laboratory, University of Cambridge, U.K.
authors.forename: S O
authors.initials: SO
authors.lastname: Sage
authors.forename: R
authors.initials: R
authors.lastname: Reast
authors.forename: T J
authors.initials: TJ
authors.lastname: Rink
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001572:Benzofurans; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D013696:Temperature
chemical_list: D001572:Benzofurans; D005456:Fluorescent Dyes; D007093:Imidazoles; D005576:Colforsin; D008345:Manganese; D000244:Adenosine Diphosphate; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; D016257:Fura-2