DIFFERENT EFFECTS OF ENDOTHELIN-3 ON THE CA2+ DISCHARGE INDUCED BY AGONISTS AND CA2+-ATPASE INHIBITORS IN HUMAN PLATELETS

1 The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca2+-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca2+-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2 Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic, However, ET-3 did not affect Ca2+ movements induced by 1 mu M ADP. Addition of Tg (0.5 to 5 mu M) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3 ET-3 did not modify the Ca2+ response to another Ca2+-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A(2) receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 mu M Tg, which became similar to that induced by 15 mu M tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4 ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A(2) receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.