Abstract 15259: Genetically Engineered eNOS Dimer Destabilization Impairs Blood Pressure Reducing Activity of eNOS in Mice

Abstract

Endothelial dysfunction and oxidative stress are associated with hypertension but whether endothelial superoxide plays a role in the early development of essential hypertension remains uncertain. In this study we investigated whether eNOS-derived endothelial oxidative stress is involved in the regulation of blood pressure (BP). Normal bovine eNOS (eNOS-Tg) or a novel dimer-destabilized eNOS-mutant harboring a partially disrupted zinc-finger (C101A-eNOS-Tg) was introduced in C57BL/6 mice using the endothelium-specific tie-2 promoter. Mice were monitored for aortic endothelium-dependent relaxation, systolic BP, levels of superoxide and several post-translational protein modifications indicating activity and/or increased vascular oxidative stress. Some groups of mice underwent voluntary exercise training or treatment with SOD mimetic Tempol (1mM). C101A-eNOS-Tg (aorta, skeletal muscle, left ventricular myocardium and lung) showed significantly increased superoxide generation, protein- and eNOS-tyrosine-nitration, eNOS-S-glutathionylation, eNOS1176/79 phosphorylation and Thr172-AMP kinase (AMPKα) phosphorylation as compared to eNOS-Tg and wildtype (WT)-controls. The localization of eNOS-Tg was restricted to endothelium as evidenced by immunohistochemical staining for eNOS and an endothelial-specific marker CD31. Exercise training increased phosphorylation of eNOS and AMPKα in WT while these physiologic adaptations were absent in C101A-eNOS-Tg. Aortic endothelium-dependent relaxation was similar in all strains. In striking contrast, C101A-eNOS-Tg displayed normal BP despite higher levels of eNOS, while eNOS-Tg showed significant hypotension. Tempol completely reversed the occurring protein modifications and significantly reduced BP in C101A-eNOS-Tg but not in WT. We observed that oxidative stress generated by endothelial-specific expression of genetically destabilized C101A- eNOS selectively prevents BP reducing activity of vascular eNOS, while having no effect on aortic endothelial dependent relaxation. Our data suggest that oxidative stress generated by eNOS dimer destabilization contributes to the regulation of blood pressure.