Abstract
Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) and the enzyme responsible for the conversion of GTP into cGMP. As such, the NO-receptor sGC is crucially involved in the physiology of the cardiovascular system by modulating vessel tone. Indeed, mice lacking this receptor are hypertensive (5). Oxidative stress is associated with cardiovascular diseases (CVDs) such as hypertension, atherosclerosis, and diabetes. Most oxidative CVDs are accompanied by endothelial dysfunction and impaired vascular reactivity with decreased NO bioavailability. Nonetheless, it should be pointed out that oxidative stress affects as well the smooth muscle cell (SMC) layers where sGC is expressed. It has been reported that reactive oxygen species (ROS) alter sGC expression and activity (19, 25, 29). We (21) have previously shown that in vitro and in vivo S-nitrosation of sGC impairs its ability to be activated by NO. In particular, we established that infusion of low therapeutic doses of nitroglycerin in rats for 3 days induces S-nitrosation of sGC and correlates with desensitization of sGC to NO stimulation. Importantly, the nitroglycerin treatment was associated with reduced relaxation of arterioles to exogenous NO donors, thus implicating directly sGC activity (22). Association between protein S-nitrosation and diseases such as diabetes (2, 30) has been reported but association with other CVDs such as hypertension has not been directly addressed. Angiotensin II (ANG II) is a peptide involved in blood pressure regulation and renovascular hypertension (11, 14, 18) and elevated levels of ANG II induce hypertension. ANG II infusion or treatment, which is clearly established as an inducer of oxidative stress via activation of NADPH oxidases in vivo (17) and in vascular smooth muscle cells such as A7r5 (31), was recently reported to generate global S-nitrosation (3).Most studies have focused on endothelial dysfunction in ANG II-linked hypertension, yet a decreased NO responsiveness, e.g., NO resistance, has been observed and could be due to scavenging of NO by reactive oxygen species, to decreased sGC activity, or to a decreased sGC expression, as reported in overactive renin-angiotensin system of hypertensive TGR rats (7). Based on our finding that sGC is desensitized to NO by S-nitrosation leading to decreased relaxation in vivo and that ANG II generates nitrosative stress, we hypothesized that sGC activity could be impaired via S-nitrosation in ANG II-induced hypertension thereby contributing to decreased vascular reactivity. Thus we assayed in ANG II-induced hypertension rat model, the level of S-nitrosation of the sGC, whether sGC was desensitized to NO, and measured vascular NO resistance in vivo (so far studies by others on NO responsiveness and vascular reactivity have been done on isolated vessels in organ chamber). In parallel, we explored the molecular mechanism of sGC desensitization by ANG II in A7r5 SMC, which are depleted for endogenous sGC, by infecting the SMC with adenoviruses expressing wild-type (WT) sGC and Cys mutants of sGC and measuring NO-stimulated sGC activity in the infected cells treated or not with ANG II.