Antioxidant Improves Smooth Muscle Sarco/Endoplasmic Reticulum Ca ²⁺ -ATPase Function and Lowers Tyrosine Nitration in Hypercholesterolemia and Improves Nitric Oxide–Induced Relaxation

Abstract

Abstract-Antioxidants improve endothelial function in hypercholesterolemia (HC); however, whether this includes improvement of the vascular smooth muscle response to NO is unknown. NO relaxes arteries, in part, by stimulating Ca 2ϩ uptake via sarco/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) in aortic smooth muscle, and HC impairs SERCA function and the response to NO. HC induces oxidative stress, which could impair SERCA function. To study the effect of antioxidants, which are known to improve endothelium-dependent relaxation in HC, smooth muscle SERCA activity and NO-induced relaxation were studied in rabbits fed normal chow or a 0.5% cholesterol diet for 13 weeks. The antioxidant t-butylhydroxytoluene (BHT, 1%) was mixed with the HC diet in the last 3 weeks. HC impaired acetylcholine-and NO-induced relaxation, and these were restored by BHT. After inhibiting SERCA with thapsigargin, no difference existed in NO-induced relaxation among the three groups. Reduced aortic SERCA activity in HC was restored by BHT without changing SERCA protein expression. 3-Nitrotyrosine was notably increased in the media of the HC aorta, where it colocalized with SERCA. Tyrosine-nitrated SERCA protein was immunoprecipitated in the aortas of HC rabbits, where it was decreased by BHT, and it was also detected in the aortas of atherosclerotic humans. Thus, the antioxidant reverses impaired smooth muscle SERCA function in HC, and this is correlated with the improved relaxation to NO. These beneficial effects may depend on reducing the direct effects on SERCA of reactive oxygen species that are augmented in HC. Key Words: t-butylhydroxytoluene Ⅲ nitric oxide Ⅲ sarco/endoplasmic reticulum Ca 2ϩ -ATPase Ⅲ atherosclerosis Ⅲ nitrotyrosine H ypercholesterolemia (HC) and other vascular diseases, including hypertension, 1-3 diabetes, 4,5 and hyperhomocysteinemia, 6,7 may increase oxidative stress and decrease NO bioactivity, which may be associated with promoting atherosclerosis. 8 Antioxidants, including ␤-carotine, 9 vitamin E, 9,10 vitamin C, 11 and probucol, 12,13 improve endotheliumdependent relaxation in HC. The effects of these antioxidants do not depend on lowering plasma cholesterol levels, 12,13 suggesting that oxidative stress in HC is related to vascular dysfunction. 14 However, the protective mechanism of antioxidants on NO bioactivity has not yet been elucidated.Oxidative stress may impair NO release 15 or endothelial NO synthase function, 16 although increased NO release in HC has also been reported. 17 Increased scavenging of NO by superoxide may decrease NO bioactivity; however, a few hours of incubation with antioxidants 18,19 or adenovirusmediated gene transfer of superoxide dismutase 20 fails to normalize endothelium-dependent relaxation, suggesting that a decrease in superoxide generation is not enough to immediately restore NO bioactivity in HC.Another possible factor is that oxidative stress impairs the function of target proteins of NO in aortic smooth muscle. NO activates guanylyl cyclase and relaxes arteries, and its...

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