Reduced Sarco/Endoplasmic Reticulum Ca ²⁺ Uptake Activity Can Account for the Reduced Response to NO, but Not Sodium Nitroprusside, in Hypercholesterolemic Rabbit Aorta

Abstract

Background-Hypercholesterolemia (HC) impairs acetylcholine-induced relaxation but has little effect on that caused by the NO donor sodium nitroprusside (SNP), suggesting that acetylcholine releases less NO from the endothelium in HC. The relaxation to authentic NO gas, however, is also impaired in HC aortic smooth muscle, indicating an abnormal smooth muscle response. NO relaxes arteries by both cGMP-dependent and -independent mechanisms, and the response involves calcium (Ca 2ϩ ) store refilling via the sarco/endoplasmic reticulum calcium ATPase (SERCA). We studied the involvement of cGMP and SERCA in the smooth muscle response to NO and SNP in HC rabbit aorta. Methods and Results-A selective guanylyl cyclase inhibitor, 1H-[1,2,4]-oxadiazole-[4,3-a]quinoxalin-1-one, eliminated SNP-induced relaxation but only partially blocked NO-induced relaxation in both normal and HC aorta. The residual relaxation to NO was still less in HC and, in both normal and HC aorta, was abolished by concomitant administration of the SERCA inhibitor cyclopiazonic acid (CPA). In contrast, CPA did not affect SNP-induced relaxation in either normal or HC aorta. SERCA activity measured by 45 Ca 2ϩ uptake was markedly decreased in HC, although SERCA2 protein expression did not change significantly. Conclusions-These data suggest that NO-induced relaxation but not that to SNP is partially mediated by cGMPindependent Ca 2ϩ uptake into sarco/endoplasmic reticulum and that reduced sarco/endoplasmic reticulum Ca 2ϩ pump function can account for the impaired response to NO in HC. Key Words: hypercholesterolemia Ⅲ nitric oxide Ⅲ sarcoplasmic reticulum Ⅲ muscle, smooth Ⅲ calcium H ypercholesterolemia (HC) impairs endotheliumdependent relaxation but has little effect on sodium nitroprusside (SNP)-induced relaxation. 1,2 Therefore, it is widely accepted that HC impairs endothelial release of NO and that the smooth muscle response to nitric oxide (NO) is preserved. Previously, we used authentic NO gas solution and showed that the decreased relaxation to acetylcholine in rabbit 1 and mouse 3 aortic rings was mirrored by decreased relaxation to NO. The decrease in intracellular free calcium caused by NO in smooth muscle from HC aorta in primary culture is also decreased in magnitude. 1 It is not clear why the relaxation to NO and SNP is affected differently in HC aorta, but the question of whether the release and/or the response to NO is impaired is an important one from both an experimental and a therapeutic viewpoint. NO gas requires diffusion, which can be interfered with by oxygen, superoxide anion, and metals. 4 SNP can release NO by 1 electron reduction at the plasma membrane of smooth muscle cells, 5 where it might not be as susceptible as NO to scavenging. HC increases superoxide production, 6 and therefore increased scavenging of NO might account for the discrepancy between NO and SNP. Although long-term treatment with antioxidants improves vascular function in HC rabbit aorta, antioxidants like glutathione ester 7 or other scavengers of super...

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

“…24,25 This process is, at least partially, cGMP independent. 19,25 Moreover, we have shown that HC impairs SERCA function and decreases the cGMPindependent SERCA-dependent smooth muscle response to NO. 19 The reduced SERCA activity in HC is not due to decreased SERCA protein levels.…”

“…NO activates guanylyl cyclase and relaxes arteries, and its activity is preserved at least during the early stages of HC. 19,21,22 Thus, sodium nitroprusside-induced relaxations that depend on the activation of guanylyl cyclase and those caused by 8-bromo-cGMP are preserved in HC. 19 K ϩ channels are directly activated by NO 23 ; however, K ϩ channelmediated relaxation by NO is preserved in HC carotid arteries.…”

“…19,21,22 Thus, sodium nitroprusside-induced relaxations that depend on the activation of guanylyl cyclase and those caused by 8-bromo-cGMP are preserved in HC. 19 K ϩ channels are directly activated by NO 23 ; however, K ϩ channelmediated relaxation by NO is preserved in HC carotid arteries. 22 We have provided evidence that NO induces relaxation, in part, by stimulating the refilling of intracellular Ca 2ϩ stores via smooth muscle sarco/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA), which thereby decreases intracellular Ca 2ϩ levels and inhibits store-dependent Ca 2ϩ influx via plasma membrane ion channels.…”

“…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.…”

Hypoxic regulation of nitric oxide signaling in vascular smooth muscle

“…Additional evidence supports a role for cGMP-independent activation of delayed rectifier K ϩ channels in pulmonary arterial smooth muscle cells (32). Other direct effects of NO include stimulation of SERCA in rabbit aortic smooth muscle, leading to increased Ca 2ϩ reuptake by intracellular stores, a fall in [Ca 2ϩ ] i , and consequent inhibition of store-operated Ca 2ϩ influx (1,2,9). This latter mechanism of cGMP-independent activation of SERCA is consistent with the findings of Mingone et al (16), which identify regulatory influences of hypoxia on this pathway.…”