Nitric oxide (NO; which plays a critical role in vascular health) is produced by eNOS in endothelial cells (EC) and activates soluble guanylate cyclase (sGC) to induce vasorelaxation. NO can also exist as the vasorelaxant nitroxyl (HNO) although the precise role for this form is not fully understood. Given that gender-specific variations exist in vascular activity we investigated if HNO generation underlies any differences between the sexes. Using isolated rings of aorta from female (n=16) and male (n=20) Sprague Dawley rats, and following sub-maximal contraction with phenylephrine (PE), responses to ACh (1nM - 3µM) were measured isometrically in EC-containing or -denuded rings with and without a range of pharmacological inhibitors. In separate experiments, the depression of PE (1nM - 3µM)-induced contraction was used as a measure of the tonic depressor influence of the EC in the absence of ACh. Data are given as means ± SEM, compared by ANOVA, n≥6 for all observations. In aortic rings from both females and males, ACh-induced EC-dependent relaxation was completely abolished following incubation with either L-NAME (300µM; P<0.001), hydroxocobalamin (100µM; P<0.001) or ODQ (30nM; P<0.001) suggesting that the relaxation was dependent entirely on eNOS activity, generation of NO and sGC activation, respectively. As determined by the depression of PE-induced contraction, the tonic vasodepressor influence of the EC in aortic rings from females was shown to be completely reliant on eNOS, the NO radical and sGC activation (i.e. treatment with L-NAME, hydroxocobalamin and ODQ, respectively, altered PE-induced contraction so that it was indistinguishable from EC-denuded vessels, P>0.05). In aorta from males, EC-dependent depression of PE (max contraction in EC-containing rings was 1.0±0.3g vs. 2.0±0.2g in EC-denuded preparations; P<0.001) was also shown to be eNOS and sGC dependent (contraction in L-NAME and ODQ-treated tissue was 1.9±0.1g and 2.1±0.2g, respectively; neither statistically different from EC-denuded rings, P>0.05). However, in powerful contrast to females, inactivation of NO with hydroxocobalamin only partly impaired the influence of EC (1.6±0.2g; P<0.01 compared to EC-denuded rings) suggesting the presence of a non-NO mediator. Use of the established HNO scavenger L-cysteine (3mM) produced a partial inhibition of the vasodepressor influence of EC (1.5±0.1g; P<0.05 compared to EC-containing rings) but in combination with hydroxocobalamin produced an effect greater than when either was used independently (1.9±0.2g; indistinguishable from EC-denuded rings, P>0.05). In conclusion, tonically active eNOS in aortic EC from male, but not female, rats generates both NO and HNO but the HNO contribution is lost when the EC are stimulated by ACh. These data identify a gender-specific role for HNO.
|Publication status||Published - 2012|