title: Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry.
abstract: We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.
authors.affiliation: Physiological Laboratory, University of Cambridge, UK.
authors.forename: P
authors.initials: P
authors.lastname: Sargeant
authors.forename: W D
authors.initials: WD
authors.lastname: Clarkson
authors.forename: S O
authors.initials: SO
authors.lastname: Sage
authors.forename: J W
authors.initials: JW
authors.lastname: Heemskerk
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002451:Cell Compartmentation; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004464:Econazole / Q000494:pharmacology; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D007424:Intracellular Fluid / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects*; D008345:Manganese / Q000378:metabolism; D008825:Miconazole / Q000494:pharmacology; D009994:Osmolar Concentration; D010972:Platelet Activating Factor / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D011950:Receptors, Cholinergic / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D065607:Cytochrome P-450 Enzyme Inhibitors; D006873:Hydroquinones; D010972:Platelet Activating Factor; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D013729:Terpenes; D014667:Vasopressins; C019359:2,5-di-tert-butylhydroquinone; D008345:Manganese; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D004464:Econazole; D008825:Miconazole; D015544:Inositol 1,4,5-Trisphosphate; D003577:Cytochrome P-450 Enzyme System; D013917:Thrombin; D000252:Calcium-Transporting ATPases; D002118:Calcium