Calcium influx evoked by Ca 2+ 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 Ca 2+ entry in human platelets appears to be promoted following the release of Ca 2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca 2+ stores and plasma membrane Ca 2+ permeability in Fura-2-loaded human platelets. Ca 2+ 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 Mn 2+. It has been suggested that cytochrome P-450 may couple Ca 2+ store depletion to an increased plasma membrane Ca 2+ permeability. In apparent agreement with this, Mn 2+ 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 Mn 2+ influx was only partially inhibited by these compounds at the same concentration (3 μM). Econazole (3 μM) reduced the Mn 2+ 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 Mn 2+ entry or rises in [Ca 2+] i. These data confirm the existence of a Ca 2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca 2+ 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 Ca 2+ content, rather than in promoting Ca 2+ influx in the initial stages of platelet Ca 2+ signal generation.