Abstract-Recently, we demonstrated that the heptapeptide angiotensin-(1-7) (Ang- [1][2][3][4][5][6][7]) exhibits a favorable kinetic of nitric oxide (NO) release accompanied by extremely low superoxide (O 2 Ϫ ) production. In this report we describe AVE 0991, a novel nonpeptide compound that evoked effects similar to Ang-(1-7) on the endothelium. AVE 0991 and unlabeled Ang-(1-7) competed for high-affinity binding of [125 I]-Ang-(1-7) to bovine aortic endothelial cell membranes with IC 50 values of 21Ϯ35 and 220Ϯ280 nmol/L, respectively. Stimulated NO and O 2 Ϫ release from bovine aortic endothelial cells was directly and simultaneously measured on the cell surface by selective electrochemical nanosensors. Peak concentrations of NO and O 2 Ϫ release by AVE 0991 and Ang-(1-7) (both 10 mol/L) were not significantly different (NO: 295Ϯ20 and 270Ϯ25 nmol/L; O 2 Ϫ : 18Ϯ2 and 20Ϯ4 nmol/L). However, the released amount of bioactive NO was Ϸ5 times higher for AVE 0991 in comparison to Ang-(1-7). The selective Ang-(1-7) antagonist [D-Ala 7 ]-Ang-(1-7) inhibited the AVE 0991-induced NO and O 2 Ϫ production by Ϸ50%. A similar inhibition level was observed for the Ang II AT 1 receptor antagonist EXP 3174. In contrast, the Ang II AT 2 receptor antagonist PD 123,177 inhibited the AVE 0991-stimulated NO production by Ϸ90% but without any inhibitory effect on O 2 Ϫ production. Both NO and O 2 Ϫ production were inhibited by NO synthase inhibition (Ϸ70%) and by bradykinin B 2 receptor blockade (Ϸ80%). AVE 0991 efficiently mimics the effects of Ang-(1-7) on the endothelium, most probably through stimulation of a specific, endothelial Ang-(1-7)-sensitive binding site causing kinin-mediated activation of endothelial NO synthase.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Angiotensin II receptor blockers improve endothelial cell-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide synthase (eNOS) function. WHAT THIS STUDY ADDS• The key finding from this study is that angiotensin II receptor blockers (ARBs) differentially enhanced nitric oxide (NO) release in a manner influenced by certain genetic variants of eNOS. This finding provides new insights into the effects of ARBs on endothelial cell-dependent vasodilation and eNOS function that are of high importance in vascular medicine and clinical pharmacology. AIMAngiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk. METHODSThe effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 mM, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 mM) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism. RESULTSAll of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n = 4-5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n = 4-5 in all eNOS variants) than increases observed with other ARBs. CONCLUSIONSThe ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.

Vascular and renal NO was significantly reduced in diabetic hypertensive rats and correlated with metabolic changes. Nebivolol reversed these effects in a manner consistent with enhanced endothelial function.

The endothelium plays a critical role in maintaining vascular tone by releasing nitric oxide (NO). Endothelium derived NO diffuses to smooth muscles, triggering their relaxation. The dynamic of NO production is a determining factor in signal transduction. The present studies were designed to elucidate dynamics of NO release from normal and dysfunctional endothelium. The nanosensors (diameter 100-300 nm) exhibiting a response time better than 100 ms and detection limit of 1.0´10 -9 mol L -1 were used for in vitro monitoring of NO release from single endothelial cells from the iliac artery of normotensive (WKY) rats, hypertensive (SHR) rats, and normal and cholesterolemic rabbits. Also, the dynamics and distribution of NO in left ventricular wall of rabbit heart were measured. The rate of NO release was much higher (1200 ± 50 nmol L -1 s -1 ) for WKY than for SHR (460 ± 10 nmol L -1 s -1 ). Also, the peak NO concentration was about three times higher for WKY than SHR. Similar decrease in the dynamics of NO release was observed for cholesterolemic rabbits. The dynamics of NO release changed dramatically along the wall of rabbit aorta, being highest (0.86 ± 0.12 mmol L -1 ) for the ascending aorta, and lowest for the iliac aorta (0.48 ± 0.15 mmol L -1 ). The distribution of NO in the left ventricular wall of rabbit heart was not uniform and varied from 1.23 ± 0.20 mmol L -1 (center) to 0.90 ± 0.15 mmol L -1 (apex). Both, the maximal concentration and the dynamics of NO release can be useful diagnostic tools in estimating the level of endothelial dysfunction and cardiovascular system efficiency.Nitric oxide generated by endothelium is a unique, ubiquitous messenger of cellular signals (Miyamoto et al., 1998;Palmer et al., 1987;Quyyumi et al., 1995;Zeiher et al., 1991). NO is not only involved in the regulation of blood pressure but also has been characterized as a neurotransmitter and plays an important role in the immune system. Its