Novel Nitric Oxide Synthase-Dependent Mechanism of Vasorelaxation in Small Arteries from Hypertensive Rats

Abstract

Endothelial dysfunction in hypertension is associated with impaired endothelium-dependent vasorelaxation, which is consistently observed in conduit vessels. However, the controversial observation of either impaired or intact vasorelaxation of small resistance arteries from hypertensive animals suggests that the mechanism(s) of endothelium-dependent vasorelaxation in small resistance arteries may be different from that observed in conduit vessels under hypertensive condition. Vasorelaxation in small resistance arteries is mediated via multiple pathways including nitric oxide synthase (NOS)-, cyclooxygenase (COX)-, and endothelium-derived hyperpolarizing factor (EDHF)-mediated pathway. Therefore, the overall goal of these studies was to determine the mechanism(s) involving vasorelaxation of small arteries from hypertensive rats. For these studies, normotensive (NORM), angiotensin II-infused (ANG), high salt (HS), ANG high salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt (DOCA) rats were studied. The studies with pharmacological blockade of each pathway demonstrated that the NOS-dependent component was increased to maintain acetylcholine (ACh)-induced vasorelaxation in small mesenteric arteries from hypertensive rats. Furthermore, increased NOS-dependent pathway appears to compensate for the dysfunctional Ca2+-activated K+ channel-sensitive EDHF pathway in small mesenteric arteries from ANG compared to NORM. These results led us to design further experiments to test the hypothesis that both NO and H2O2 serve as NOS-dependent mediators to maintain vasorelaxation in small mesenteric arteries from hypertensive rats. In small arteries from ANG, ACh increased NOS-dependent cGMP production. ACh also increased NOS3 phosphorylation at Ser 633 and decreased phosphorylation at Thr 495. While, NOS3 phosphorylation at Ser 1177 was impaired in response to ACh in ANG, which was accompanied by reduced basal and a less extended ACh-stimulated cGMP production in ANG compared to NORM. To investigate the alteration of signal transduction pathways related to impaired NOS3 phosphorylation at Ser 1177 in response to ACh, Akt phosphorylation at Ser 473 and VASP phosphorylation at Ser 239 were tested. These pathways were not changed by ACh in the small mesenteric arteries from ANG. Our results indicate that the NO/cGMP signaling is present in response to ACh in small mesenteric arteries from ANG, however this signaling pathway-mediating vasorelaxation may be facilitated via neither Akt nor PKG. On the other hand, ACh stimulated L-NAME-sensitive H2O2 production in small mesenteric arteries from ANG, but not NORM. H2O2 induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation in small mesenteric arteries from ANG. Reduced BH4/BH2 ratio was observed in small mesenteric arteries from ANG compared to NORM, which might be one of the mechanisms of NOS-mediated H2O2 production. Antioxidant enzyme capacity was also determined in small mesenteric arteries from ANG and NORM. Total superoxide dismutase (SOD) activity and protein expression of CuZn SOD and ecSOD were reduced in ANG compared to NORM, while Mn SOD expression was comparable between groups. Interestingly, both activity and expression of catalase were reduced in ANG compared to NORM, whereas GPx activity and expression were not changed. These results indicate that reduced catalase activity and expression may contribute to the augmentation of H2O2 in small mesenteric arteries from ANG, whereas reduced SOD does not greatly influence the H2O2 production in both basal and ACh-stimulated condition. In conclusion, the NOS pathway appears to be the primary endothelium-derived relaxing factor (EDRF) pathway in small mesenteric arteries from experimental animal models of hypertension. The increased dependence on the NOS pathway in ACh-induced vasorelaxation is mediated by both NOS-derived NO/cGMP signaling and NOS-mediated H2O2.