Nitric Oxide Inhibits Capacitative Cation Influx in Human Platelets by Promoting Sarcoplasmic/Endoplasmic Reticulum Ca 2+ -ATPase–Dependent Refilling of Ca 2+ Stores

Abstract

Abstract —Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca 2+ concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca 2+ /Mn 2+ influx in intact platelets and to monitor Ca 2+ uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca 2+ and Mn 2+ influx developed rapidly (within 30 s) after fast release of Ca 2+ from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca 2+ leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) with 30 μmol/L 2,5-di-( tert -butyl)-1,4-benzohydroquinone (BHQ). NO (1 μmol/L) inhibited capacitative Ca 2+ and Mn 2+ influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca 2+ and Mn 2+ influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca 2+ -free medium, NO reduced the peak Ca 2+ rise caused by thrombin and significantly promoted Ca 2+ 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 μmol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca 2+ concentration. Inhibition of Ca 2+ uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca 2+ into the stores after inositol-1,4,5-trisphosphate–induced Ca 2+ release or after addition of Ca 2+ (1 μmol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca 2+ 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 2+ stores.