The effects of the Ca2+-ATPase inhibitors thapsigargin (Tg) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) were examined by using Ca2+-regulatory systems of platelet mixed membranes, saponin-permeabilized and intact platelets. Both agents inhibit Ca2+-ATPase activities of platelet mixed membranes, without any effect on the basal Mg2+-ATPase activity. Tg is more effective (EC50 = 35 nM) than tBuBHQ (EC50 = 580 nM). The effect of the two inhibitors on Ca-45(2+) release from saponin-permeabilized platelets has also been characterized. Ca-45(2+) uptake into non-mitochondrial intracellular stores occurs via an ATP-dependent mechanism, and if added at equilibrium the second messenger Ins(1,4,5)P3 releases 50% of the accumulated Ca-45(2+). Maximally effective concentrations of Tg (1 muM) and tBuBHQ (50 muM) release 77% and 68% of the accumulated Ca-45(2+). Addition of Ins(1,4,5)P3 together with either Tg or tBuBHQ resulted in a non-additive release which was the same as with either Tg or tBuBHQ alone, indicating that the Ins(1,4,5)P3-sensitive Ca2+ pool was a subset of the pool that is sensitive to the Ca2+-ATPase inhibitors. Release of Ca-45(2+) by either Tg or tBuBHQ was not affected by heparin, which totally blocked Ins(1,4,5)P3-induced Ca2+ release, and Tg was found not to affect [P-32]Ins(1,4,5)P3 binding to its receptor on mixed membranes. Thus both Tg and tBuBHQ release Ca2+ from a pool that totally overlaps the Ins(1,4,5)P3-sensitive pool without affecting Ins(1,4,5)P3 function. In intact indomethacin-treated Fura 2-loaded platelets, Tg and tBuBHQ cause Ca2+ elevation, arising from release from intracellular stores and influx from the outside. Both Tg and tBuBHQ elevated Ca2+ to similar levels, which were less and slower than those observed with thrombin. Addition of thrombin to cells already treated with Tg or tBuBHQ produced further elevation of Ca2+, indicating agonist utilization of a Ca2+-ATPase inhibitor-insensitive pool. In aggregation experiments Tg and tBuBHQ showed different functional effects. In indomethacin-treated cells Tg induces slow aggregation and secretion responses, whereas tBuBHQ only induces shape change. Both agents show synergistic secretory responses with the protein kinase C activator dioctanoylglycerol (DiC8). Tg also showed greater ability than tBuBHQ to release [H-3]arachidonic acid (AA) from [H-3]AA-labelled platelets. Additionally, in [P-32]P1-labelled platelets both Tg and tBuBHQ induced phosphorylation of myosin light chain, a 27 kDa protein and the 45 kDa protein pleckstrin, but Tg showed a greater ability than tBuBHQ to cause phosphorylation of pleckstrin. These studies indicate that Tg and tBuBHQ are effective in releasing the Ins(1,4,5)P3-sensitive Ca2+ pool in platelets. The differences obtained in Tg- and tBuBHQ-induced functional responses may reflect additional effects of Tg on protein phosphorylation.
