Mechanism of Nitric Oxide–Induced Vasodilatation

Abstract

—The precise mechanisms by which nitric oxide (NO) decreases free [Ca²⁺]_i, inhibits Ca²⁺ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca²⁺]_i were resistant to nifedipine, suggesting Ca²⁺ entry through non–L-type Ca²⁺ 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 [Ca²⁺]_i and the rate of Mn²⁺ 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 Ca²⁺ from the cytoplasm, decreased [Ca²⁺]_i, and inhibited Ca²⁺ and Mn²⁺ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na⁺/Ca²⁺exchanger or the plasma membrane Ca²⁺ ATPase had no influence on the ability of NO to decrease [Ca²⁺]_i. NO maximally decreased [Ca²⁺]_i within 5 s, whereas significant inhibition of AII-induced Ca²⁺ and Mn²⁺ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca²⁺]_o and functional SERCA, suggesting that during the delay before NO inhibits Ca²⁺ influx, the influx of Ca²⁺ and the uptake into intracellular stores are required. In the absence of [Ca²⁺]_o, NO diminished the AII-induced [Ca²⁺]_i transient by a SERCA-dependent mechanism and increased the amount of Ca²⁺ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca²⁺]_i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca²⁺ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca²⁺ influx through non–L-type Ca²⁺ conducting ion channels and that this maintains the decrease in [Ca²⁺]_i and NO-induced relaxation.