Pharmacological characterization of 1-nitrosocyclohexyl acetate, a long-acting nitroxyl donor that shows vasorelaxant and antiaggregatory effects.

Nitroxyl (HNO) donors have potential benefit in the treatment of heart failure and other cardiovascular diseases. 1-Nitrosocyclohexyl acetate (NCA), a new HNO donor, in contrast to the classic HNO donors Angeli's salt and isopropylamine NONOate, predominantly releases HNO and has a longer half-life. This study investigated the vasodilatative properties of NCA in isolated aortic rings and human platelets and its mechanism of action. NCA was applied on aortic rings isolated from wild-type mice and apolipoprotein E-deficient mice and in endothelial-denuded aortae. The mechanism of action of HNO was examined by applying NCA in the absence and presence of the HNO scavenger glutathione (GSH) and inhibitors of soluble guanylyl cyclase (sGC), adenylyl cyclase (AC), calcitonin gene-related peptide receptor (CGRP), and K(+) channels. NCA induced a concentration-dependent relaxation (EC(50), 4.4 µM). This response did not differ between all groups, indicating an endothelium-independent relaxation effect. The concentration-response was markedly decreased in the presence of excess GSH; the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide had no effect. Inhibitors of sGC, CGRP, and voltage-dependent K(+) channels each significantly impaired the vasodilator response to NCA. In contrast, inhibitors of AC, ATP-sensitive K(+) channels, or high-conductance Ca(2+)-activated K(+) channels did not change the effects of NCA. NCA significantly reduced contractile response and platelet aggregation mediated by the thromboxane A(2) mimetic 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F(2)(α) in a cGMP-dependent manner. In summary, NCA shows vasoprotective effects and may have a promising profile as a therapeutic agent in vascular dysfunction, warranting further evaluation.

Dimethylarginine dimethylaminohydrolase1 is an organ-specific mediator of end organ damage in a murine model of hypertension.

BACKGROUND: The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is an independent predictor of cardiovascular and overall mortality. Moreover, elevated ADMA plasma concentrations are associated with the extent of hypertension. However, data from small-sized clinical trials and experimental approaches using murine transgenic models have revealed conflicting results regarding the impact of ADMA and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) in the pathogenesis of hypertension. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we investigated the role of ADMA and DDAH1 in hypertension-induced end organ damage using the uninephrectomized, deoxycorticosterone actetate salt, and angiotensin II-induced hypertension model in human DDAH1 (hDDAH1) overexpressing and wild-type (WT) mice. ADMA plasma concentrations differed significantly between hDDAH1 and WT mice at baseline, but did not significantly change during the induction of hypertension. hDDAH1 overexpression did not protect against hypertension-induced cardiac fibrosis and hypertrophy. In addition, the hypertension-induced impairment of the endothelium-dependent vasorelaxation of aortic segments ex vivo was not significantly attenuated by hDDAH1 overexpression. However, hDDAH1 mice displayed an attenuated hypertensive inflammatory response in renal tissue, resulting in less hypertensive renal injury. CONCLUSION/SIGNIFICANCE: Our data reveal that hDDAH1 organ-specifically modulates the inflammatory response in this murine model of hypertension. The lack of protection in cardiac and aortic tissues may be due to DDAH1 tissue selectivity and/or the extent of hypertension by the used combined model. However, our study underlines the potency of hDDAH1 overexpression in modulating inflammatory processes as a crucial step in the pathogenesis of hypertension, which needs further experimental and clinical investigation.

Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012.

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10-100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.