cyclic-gmp and oxyhyponitrite

Pre-clinical studies have identified nitroxyl (HNO), the reduced congener of nitric oxide (NO•), as a potent vasodilator which is resistant to tolerance development. The present study explores the efficacy of HNO in human blood vessels and describes, for the first time, a vasodilator for humans that is not susceptible to tolerance. Human radial arteries and saphenous veins were obtained from patients undergoing coronary artery graft surgery and mounted in organ baths. Repeated vasodilator responses to the HNO donor Angeli's salt (AS) and NO• donor glyceryl trinitrate (GTN) were determined. AS- and GTN-induced concentration-dependent vasorelaxation of both human radial arteries (AS pEC50: 6.5 ± 0.2; -log M) and saphenous veins (pEC50: 6.7 ± 0.1) with similar potency. In human radial arteries, GTN-induced relaxation was reduced by the NO• scavenger hydroxocobalamin (HXC; P<0.05) but was unaffected by the HNO scavenger L-cysteine. Alternately, AS was unaffected by HXC but was reduced by L-cysteine (5-fold shift, P<0.05). The sGC (soluble guanylate cyclase) inhibitor ODQ abolished responses to both AS and GTN in arteries and veins (P<0.05). Inhibition of voltage-dependent potassium channels (Kv channels) with 4-AP also significantly reduced responses to AS (pEC50: 5.5) and GTN, suggesting that the relaxation to both redox congeners is cGMP- and Kv channel-dependent. Critically, a concentration-dependent development of tolerance to GTN (1 and 10 μM; P<0.05), but not to AS, was observed in both saphenous veins and radial arteries. Like GTN, the HNO donor AS causes vasorelaxation of human blood vessels via activation of a cGMP-dependent pathway. Unlike GTN, however, it does not develop tolerance in human blood vessels.

Topics: Cyclic GMP; Dose-Response Relationship, Drug; Drug Tolerance; Enzyme Inhibitors; Guanylate Cyclase; Humans; In Vitro Techniques; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Nitroglycerin; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Radial Artery; Receptors, Cytoplasmic and Nuclear; Saphenous Vein; Soluble Guanylyl Cyclase; Vasodilation; Vasodilator Agents

New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated.. Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli's salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.. We now demonstrate that Angeli's salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli's salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli's salt were mimicked by BNP. We also demonstrate that the effects of Angeli's salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP).. Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.

Topics: Angiotensin II; Animals; Cardiomegaly; Cell Adhesion Molecules; Cyclic GMP; Endothelin-1; Guanylate Cyclase; Microfilament Proteins; Myocytes, Cardiac; NADPH Oxidases; Natriuretic Peptide, Brain; Nitrites; Nitrogen Oxides; p38 Mitogen-Activated Protein Kinases; Phosphoproteins; Phosphorylation; Rats; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Superoxides

Nitroxyl (HNO) displays distinct pharmacology to its redox congener nitric oxide (NO(•)) with therapeutic potential in the treatment of heart failure. It remains unknown if HNO donors are resistant to tolerance development following chronic in vivo administration. Wistar-Kyoto rats received a 3-day subcutaneous infusion of one of the NO(•) donors, glyceryl trinitrate (GTN) or diethylamine/NONOate (DEA/NO), or the HNO donor Angeli's salt (AS). GTN infusion (10 μg/kg/min) resulted in significantly blunted depressor responses to intravenous bolus doses of GTN, demonstrating tolerance development. By contrast, infusion with AS (20 μg/kg/min) or DEA/NO (2 μg/kg/min) did not alter their subsequent depressor responses. Similarly, ex vivo vasorelaxation responses in isolated aortae revealed that GTN infusion elicited a significant 6-fold decrease in the sensitivity to GTN and reduction in the maximum response to acetylcholine (ACh). Chronic infusion of AS or DEA/NO had no effect on subsequent vasorelaxation responses to themselves or to ACh. No functional cross-tolerance between nitrovasodilators was evident, either in vivo or ex vivo, although an impaired ability of a nitrovasodilator to increase tissue cGMP content was not necessarily indicative of a reduced functional response. In conclusion, HNO donors may represent novel therapies for cardiovascular disease with therapeutic potential over clinically used organic nitrates.

Topics: Acetylcholine; Animals; Aorta; Cyclic GMP; Diethylamines; In Vitro Techniques; Male; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Nitroglycerin; Rats

The free radical form of nitric oxide (NO(.)) is a well-known mediator of vascular tone. What is not so well recognized is that NO(.) exists in several different redox forms. There is considerable evidence that NO(.) and its one-electron reduction product, nitroxyl (HNO), have pharmacologically distinct actions that extend into the regulation of the vasculature. The aim of this study was to compare the vasorelaxation mechanisms of HNO and NO(.), including an examination of the ability of these redox variants to hyperpolarize and repolarize vascular smooth muscle cells from rat mesenteric arteries. The HNO donor Angeli's salt (0.1 nM-10 microM) caused a concentration-dependent hyperpolarization of vessels at resting tone and a simultaneous, concentration-dependent vasorelaxation and repolarization of vessels precontracted and depolarized with methoxamine. Both vasorelaxation and repolarization responses to Angeli's salt were significantly attenuated by both the HNO scavenger l-cysteine (3 mM) and the voltage-dependent K(+) (K(v)) channel inhibitor 4-aminopyridine (4-AP; 1 mM) and virtually abolished by the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM) or 30 mM K(+). In contrast, NO(.) (0.01-1 microM) repolarized arteries to a lesser extent than HNO, and these responses were resistant to inhibition by ODQ (10 microM) and 4-AP (1 mM). Blockade of K(v) channels (1 mM 4-AP) also significantly inhibited the repolarization response to YC-1 (0.1-10 microM), confirming a role for sGC/cGMP in the activation of K(v) channels in this preparation. We conclude that HNO causes vasorelaxation via a cGMP-dependent activation of K(v) channels and that there are different profiles of vasorelaxant activity for the redox siblings HNO and NO(.).

Topics: 4-Aminopyridine; Animals; Cyclic GMP; Cysteine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Guanylate Cyclase; In Vitro Techniques; Male; Membrane Potentials; Mesenteric Artery, Superior; Muscle, Smooth, Vascular; Nitric Oxide; Nitrites; Nitrogen Oxides; Oxadiazoles; Oxidation-Reduction; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vascular Resistance; Vasodilation; Vasodilator Agents

Topics: Animals; Biological Factors; Cyclic GMP; Guanylate Cyclase; Humans; Membrane Potentials; Muscle, Smooth, Vascular; Nitric Oxide; Nitrites; Nitrogen Oxides; Oxidation-Reduction; Potassium Channels, Voltage-Gated; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vascular Resistance; Vasodilation; Vasodilator Agents

There is a growing body of evidence on the role of nitric oxide (NO) in human platelet physiology regulation. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO-), because it is formed by a number of diverse biochemical reactions. The aim of the present study was to comparatively analyze the effect of HNO and NO on several functional parameters of human platelets. For this purpose, sodium trioxodinitrate (Angeli's salt,AS) and sodium nitroprusside (SNP) were used as HNO and NO releasers, respectively. BothAS and SNP significantly inhibited platelet aggregation and ATP release induced by different agonists and adrenaline. AS or SNP did not modify the expression of platelet glycoproteins (Ib, IIb-IIIa, la-IIa, IV), whereas they substantially decreased the levels of CD62P, CD63 and of PAC-1 (a platelet activated glycoprotein IIb/IIIa epitope) after the stimulation with ADP. AS and SNP significantly increased cGMP accumulation in a 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-1-one (ODQ)-sensitive manner. However, while L-cysteine reduced the effect of AS, it increased the effect of SNP on this parameter. Accordingly, a differential effect of L-cysteine was observed on the antiaggregatory effect of both compounds. In summary, these results indicate that HNO is an effective inhibitor of human platelet aggregation.

Topics: Adenosine Triphosphate; Antigens, CD; Blood Platelets; Cyclic GMP; Cysteine; Dose-Response Relationship, Drug; Drug Interactions; Humans; In Vitro Techniques; Nitric Oxide; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Nitroprusside; P-Selectin; Platelet Aggregation; Platelet Membrane Glycoproteins; Tetraspanin 30; Time Factors

Diazeniumdiolates, more commonly referred to as NONOates, have been extremely useful in the investigation of the biological effects of nitric oxide (NO) and related nitrogen oxides. The NONOate Angeli's salt (Na(2)N(2)O(3)) releases nitroxyl (HNO) under physiological conditions and exhibits unique cardiovascular features (i.e., positive inotropy/lusitropy) that may have relevance for pharmacological treatment of heart failure. In the search for new, organic-based compounds that release HNO, we examined isopropylamine NONOate (IPA/NO; Na[(CH(3))(2)CHNH(N(O)NO]), which is an adduct of NO and a primary amine. The chemical and pharmacological properties of IPA/NO were compared to those of Angeli's salt and a NO-producing NONOate, DEA/NO (Na[Et(2)NN(O)NO]), which is a secondary amine adduct. Under physiological conditions IPA/NO exhibited all the markers of HNO production (e.g., reductive nitrosylation, thiol reactivity, positive inotropy). These data suggest that primary amine NONOates may be useful as HNO donors in complement to the existing series of secondary amine NONOates, which are well-characterized NO donors.

Topics: Animals; Azo Compounds; Calcitonin Gene-Related Peptide; Cardiovascular System; Cell Survival; Cells, Cultured; Cricetinae; Cricetulus; Cyclic GMP; Dogs; Glutathione; Hemodynamics; Hydrazines; Lethal Dose 50; Male; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Uric Acid

Nitroxyl anion (NO(-)) is the one-electron reduction product of nitric oxide (NO( small middle dot)) and is enzymatically generated by NO synthase in vitro. The physiologic activity and mechanism of action of NO(-) in vivo remains unknown. The NO(-) generator Angeli's salt (AS, Na(2)N(2)O(3)) was administered to conscious chronically instrumented dogs, and pressure-dimension analysis was used to discriminate contractile from peripheral vascular responses. AS rapidly enhanced left ventricular contractility and concomitantly lowered cardiac preload volume and diastolic pressure (venodilation) without a change in arterial resistance. There were no associated changes in arterial or venous plasma cGMP. The inotropic response was similar despite reflex blockade with hexamethonium or volume reexpansion, indicating its independence from baroreflex stimulation. However, reflex activation did play a major role in the selective venodilation observed under basal conditions. These data contrasted with the pure NO donor diethylamine/NO, which induced a negligible inotropic response and a more balanced veno/arterial dilation. AS-induced positive inotropy, but not systemic vasodilatation, was highly redox-sensitive, being virtually inhibited by coinfusion of N-acetyl-l-cysteine. Cardiac inotropic signaling by NO(-) was mediated by calcitonin gene-related peptide (CGRP), as treatment with the selective CGRP-receptor antagonist CGRP(8-37) prevented this effect but not systemic vasodilation. Thus, NO(-) is a redox-sensitive positive inotrope with selective venodilator action, whose cardiac effects are mediated by CGRP-receptor stimulation. This fact is evidence linking NO(-) to redox-sensitive cardiac contractile modulation by nonadrenergic/noncholinergic peptide signaling. Given its cardiac and vascular properties, NO(-) may prove useful for the treatment of cardiovascular diseases characterized by cardiac depression and elevated venous filling pressures.

Topics: Animals; Anions; Baroreflex; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Cyclic GMP; Dogs; Male; Myocardial Contraction; Nitrates; Nitric Oxide; Nitrites; Nitrogen Oxides; Oxidation-Reduction; Peptide Fragments; Signal Transduction

Considerable controversy exists in the literature with regard to the nature of the agent mediating the biological effects of nitroxyl (NO-) donors. Here it is demonstrated that Angeli's salt (AS), a generator of NO-, enhanced human neutrophil migration. Under aerobic conditions, AS was converted to peroxynitrite to a small extent. However, using methionine, a scavenger of peroxynitrite, it was shown that peroxynitrite was not involved in AS-induced migration. AS equally enhanced human neutrophil migration under aerobic and anaerobic conditions, which strongly suggests that extracellular conversion of NO- to .NO by oxygen was not required. Furthermore, metHb and L-cysteine, which react more readily with NO- than with .NO, inhibited AS-induced migration, whereas the response towards gaseous .NO remained unaffected. AS induced an increase in the intracellular level of cGMP, although the curves for migration and cGMP level appeared to be slightly different in their concentration dependence. An inhibitor of soluble guanylate cyclase and antagonists of cGMP-dependent protein kinase had a more pronounced inhibitory effect on .NO-induced migration than on AS-induced migration. This suggests that the cGMP signalling cascade is partially, but not solely, responsible for AS-induced migration. As it has been demonstrated that soluble guanylate cyclase can only be activated by .NO, and not by NO-, these data indicate that NO- is at least partly converted intracellularly to .NO.

Topics: Chemotaxis, Leukocyte; Cyclic GMP; Free Radicals; Humans; In Vitro Techniques; Methionine; Neutrophils; Nitrates; Nitric Oxide; Nitrites; Nitrogen Oxides; Oxidants