Nitric Oxide Inhibits Capacitative Cation Influx in Human Platelets by Promoting Sarcoplasmic/Endoplasmic Reticulum Ca-ATPase–Dependent Refilling of Ca Stores

Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca/Mn influx in intact platelets and to monitor Ca uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca and Mn influx developed rapidly (within 30 s) after fast release of Ca from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) with 30 mmol/L 2,5-di-(tert-butyl)-1,4benzohydroquinone (BHQ). NO (1 mmol/L) inhibited capacitative Ca and Mn influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca and Mn influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca-free medium, NO reduced the peak Ca rise caused by thrombin and significantly promoted Ca back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 mmol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca concentration. Inhibition of Ca uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca into the stores after inositol-1,4,5-trisphosphate– induced Ca release or after addition of Ca (1 mmol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca stores. (Circ Res. 1999;84:201-209.)