Mechanism of Nitric Oxide–Induced Vasodilatation: Refilling of Intracellular Stores by Sarcoplasmic Reticulum Ca2+ATPase and Inhibition of Store-Operated Ca2+Influx

Abstract

Abstract—The precise mechanisms by which nitric oxide (NO) decreases free [Ca 2+]i, inhibits Ca 2+influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca 2+]i were resistant to nifedipine, suggesting Ca 2+entry through non–L-type Ca 2+channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca 2+]i and the rate of Mn 2+influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca 2+from the cytoplasm, decreased [Ca 2+]i, and inhibited Ca 2+and Mn 2+influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca 2+exchanger or the plasma membrane Ca 2+ATPase had no influence on the ability of NO to decrease [Ca 2+]i. NO maximally decreased [Ca 2+]i within 5 s, whereas significant inhibition of AII-induced Ca 2+and Mn 2+influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca 2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca 2+influx, the influx of Ca 2+and the uptake into intracellular stores are required. In the absence of [Ca 2+]o, NO diminished the AII-induced [Ca 2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca 2+in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca 2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca 2+by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca 2+influx through non–L-type Ca 2+conducting ion channels and that this maintains the decrease in [Ca 2+]i and NO-induced relaxation.