HNO Donor Research Articles

Water soluble acyloxy nitroso compounds: HNO release and reactions with heme and thiol containing proteins

Nitroxyl (HNO) has gained interest as a potential treatment of congestive heart failure through the ability of the HNO donor, Angeli's salt (AS), to evoke positive inotropic effects in canine cardiac muscle. The release of nitrite during decomposition limits the use of AS requiring other HNO sources. Acyloxy nitroso compounds liberate HNO and small amounts of nitrite upon hydrolysis and the synthesis of the water-soluble 4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate allows for pig liver esterase (PLE)-catalysis increasing the rate of decomposition and HNO release. The pivalate derivative does not release HNO, but the addition of PLE catalyzes hydrolysis (t1/2=39min) and HNO formation (65% after 30min). In the presence of PLE, this compound converts metmyoglobin (MetMb) to iron nitrosyl Mb and oxyMb to metMb indicating that these compounds only react with heme proteins as HNO donors. The pivalate in the presence and the absence of PLE inhibits aldehyde dehydrogenase (ALDH) with IC50 values of 3.5 and 3.3μM, respectively, in a time-dependent manner. Reversibility assays reveal reversible inhibition of ALDH in the absence of PLE and partially irreversible inhibition with PLE. Liquid chromatography–mass spectrometry (LC–MS) reveals formation of a disulfide upon incubation of an ALDH peptide without PLE and a mixture of disulfide and sulfinamide in the presence of PLE. A dehydroalanine residue forms upon incubation of this peptide with excess AS. These results identify acyloxy nitroso compounds as unique HNO donors capable of thiol modification through direct electrophilic reaction or HNO release.

89 NOVEL VASOPROTECTIVE ACTIONS OF NITROXYL (HNO) IN THE VASCULATURE

Background: Nitric oxide (NO•) plays a fundamental role as a vascular protectant with its well reported ability to induce vasorelaxation in human vessels and reduce leukocyte to endothelium adhesion, an initiating event of vascular inflammation. Nitroxyl (HNO), the one-electron reduced redox sibling of NO•, has distinct pharmacology to NO• and the effects of HNO in this context are not known. Objective: We aimed to determine the vasodilatory and anti-inflammatory actions of HNO using the HNO donor, Angeli's Salt (AS) compared to the NO• donor, GTN. Methods and Results: Cumulative concentration-response curves (CRCs) to AS or GTN were constructed in radial arteries (RA) and saphenous veins (SV) obtained from patients undergoing coronary artery graft surgery mounted in organ baths. AS induced vasorelaxation of RA (EC50 = 6.5 ± 0.1 M; n = 8) and SV (EC50 = 6.6 ± 0.1 M; n = 9) with similar potency to GTN, and AS-induced vasorelaxation was inhibited by the HNO scavenger L-cysteine (3 mM) but not the NO• scavenger hydroxocobalamin (100 μM). Adhesion of monocytic THP-1 cells to activated HUVECs (with tumour necrosis factor α (TNFα)) was decreased in a concentration-dependent manner by AS and GTN (P<0.05). Furthermore, experiments which allow the visualization of leukocyte-endothelium adhesion in isolated mouse (C57BL/6) aorta, using whole blood, showed that AS (10 μM) and GTN (10 μM) significantly attenuated (5 ± 2 and 3 ± 1 cells/field, respectively, n = 4-5;P<0.05) the TNFα-stimulated increase (16 ± 3 cells/field, n = 5-11) in leukocyte adhesion. Conclusions: This study shows for the first time that HNO can relax human blood vessels and reduce leukocyte-endothelium adhesion which will aid in determining the therapeutic potential of HNO donors.

Abstract 261: Nitroxyl Anion Induced Vasorelaxation in Pudendal Arteries is Partially Mediated via the Large Conductance Calcium-activated Potassuim Channel

Nitroxyl anion (HNO) - a redox congener of nitric oxide (NO) - has been demonstrated to be a potent vasodilator of both conduit and resistance vessels. Previous data from our laboratory have demonstrated that the mechanism of HNO-mediated vasorelaxation varied according to the vascular bed. However, no studies address the role of HNO in erectile function. We have demonstrated that the corpus cavernosum does not exhibit a relaxation response to HNO. However, we have shown that the pudendal artery (PA), which supplies blood flow to the penis, does exhibit a relaxation response to HNO and that ACh-mediated relaxation responses may be partially attributed to HNO as compared to NO. We hypothesized that HNO mediates vasorelaxation in pudendal arteries via activation of potassium (K + ) channels. PAs were isolated from Sprague-Dawley rats and concentration-response curves (CRC) to an HNO donor: Angeli’s Salt (AS) were performed in phenylephrine contracted vessels (1μM) in the absence and the presence of the calcium (Ca 2+ ) -activated K + channel inhibitors: TRAM-34 (intermediate Ca 2+ -activated K + -channel inhibitor, IK Ca , 10 μM) , UCL-1689 (Small conductance calcium-activated potassium channel, SK Ca ,1 μM), iberiotoxin (large conductance Ca 2+ -activated K + -channel inhibitor, BK Ca ,0.1μM). There were no observed differences in relaxation responses to AS with IK Ca and SK Ca channel inhibition. In contrast, inhibition of BK Ca channels resulted in a rightward shift in the CRC to AS (logEC 50 -4.74±0.11 vs . -5.26±0.11, p&lt;0.01) with no differences in R max . These data suggest that AS-dependent vasorelaxation in PA is partially mediated via BK Ca channel activation.

Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli’s salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.

Endothelium-dependent nitroxyl-mediated relaxation is resistant to superoxide anion scavenging and preserved in diabetic rat aorta

The aim of the study was to investigate whether diabetes-induced oxidant stress affects the contribution of nitroxyl (HNO) to endothelium-dependent relaxation in the rat aorta. Organ bath techniques were employed to determine vascular function of rat aorta. Pharmacological tools (3mM l-cysteine, 5mM 4-aminopyridine (4-AP), 200μM carboxy-PTIO and 100μM hydroxocobalamin, HXC) were used to distinguish between NO and HNO-mediated relaxation. Superoxide anion levels were determined by lucigenin-enhanced chemiluminescence. In the diabetic aorta, where there is increased superoxide anion production, responses to the endothelium-dependent relaxant ACh were not affected when the contribution of NO to relaxation was abolished by either HXC or carboxy-PTIO, indicating a preserved HNO-mediated relaxation. Conversely, when the contribution of HNO was inhibited with l-cysteine or 4-AP, the sensitivity and maximum relaxation to ACh was significantly decreased, suggesting that the contribution of NO was impaired by diabetes. Furthermore, whereas HNO appears to be derived from eNOS in normal aorta, in the diabetic aorta it may also arise from an eNOS-independent source, perhaps derived from nitrosothiol stores. Similarly, exposure to the superoxide anion generator, pyrogallol (100μM) significantly reduced the sensitivity to the NO donor, DEANONOate and ACh-induced NO-mediated relaxation but had no effect on responses to the HNO donor, Angeli's salt and ACh-induced HNO-mediated relaxation in the rat aorta. These findings demonstrate that NO-mediated relaxation is impaired during oxidative stress but the HNO component of relaxation is preserved under those conditions.

Nitrite- and nitroxyl-induced relaxation in porcine coronary (micro-) arteries: Underlying mechanisms and role as endothelium-derived hyperpolarizing factor(s)

To investigate the vasorelaxant efficacy of nitrite and nitroxyl (HNO) in porcine coronary (micro)arteries (PC(M)As), evaluating their role as endothelium-derived hyperpolarizing factors (EDHFs), preconstricted PCAs and PCMAs were exposed to UV light (a well-known inductor of nitrite; wave-length: 350–370nm), nitrite, the HNO donor Angeli's salt, or bradykinin. UV light-induced relaxation of PCAs increased identically after endothelium removal and endothelial nitric oxide (NO) synthase (eNOS) blockade. UV light-induced relaxation diminished during Na+-K+-ATPase inhibition and S-nitrosothiol-depletion, and disappeared during NO scavenging with hydroxocobalamin or soluble guanylyl cyclase (sGC) inhibition with ODQ. Nitrite-induced relaxation of PCAs required millimolar levels, i.e., >1000 times endogenous vascular nitrite. Angeli's salt relaxed PCMAs more potently than PCAs, and this was due to the fact that HNO directly activated sGC in PCMAs, whereas in PCAs this occurred following its conversion to NO only. sGC activation by NO/HNO resulted in Na+-K+-ATPase stimulation and Kv channel activation. The HNO scavenger l-cysteine blocked bradykinin-induced relaxation in PCAs, and potentiated it in PCMAs. The latter did not occur in the presence of hydroxocobalamin, suggesting that it depended on l-cysteine-induced generation of vasorelaxant S-nitrosothiols. In all experimental setups, incubation with red wine extract mimicked the effects of ODQ. In conclusion, nitrite, via its conversion to NO and S-nitrosothiols, and HNO, either directly, or via its conversion to NO, mediate relaxant effects involving the sGC-cGMP pathway, Na+-K+-ATPase and/or Kv channels. Red wine extract counteracts these beneficial effects. NO blocks nitrite activation, and HNO, but not nitrite, may act as EDHF in the coronary vascular bed.

Abstract 156: The Nitroxyl Donor Angeli's Salt Elicits Concomitant Concentration-Dependent Coronary Vasodilatory and Inotropic Actions in the Intact Rat Heart

Nitroxyl (HNO) is a novel redox sibling of NO• that elicits vasodilator, antihypertrophic and superoxide-suppressing actions. HNO is however resistant to scavenging by superoxide, and has high reactivity with thiols (enabling potent enhancement of LV function). In the present study, hearts from adult male Sprague-Dawley rats were Langendorff-perfused at constant pressure. Dose-response curves to the HNO donor Angeli’s salt were performed following preconstriction with U46619 to ∼50% of baseline coronary flow (CF). Angeli’s salt (10pmol-10µmol, n=6) elicited significant dose-dependent increases in left ventricular systolic pressure (LVSP), LV developed pressure (LVDP), LV end-diastolic pressure (LVEDP), LV±dP/dt, and CF. The effects of the highest dose of Angeli's salt studied on LV function are shown in the Table; these were significantly inhibited by co-administration of the HNO scavenger L-cysteine (4mM, n=6) or the soluble guanylyl cyclase inhibitor ODQ (10µM, n=5). The EC 50 for Angeli's salt-induced vasodilatation was increased from -8.6±0.3mol to -7.5±0.2mol (p&lt;0.01) and -7.6±0.3mol (p&lt;0.05) by L-cysteine and ODQ, respectively. Although CGRP is a putative mediator of HNO actions, both the inotropic and vasodilator actions of Angeli's salt were insensitive to the CGRP antagonist CGRP 8-37 (0.1µM, n=5). The vehicle for Angeli’s salt, 0.01M NaOH, had no effect on any of these parameters (n=3, results not shown). Our results demonstrate that the cardiac effects of Angeli’s salt are thiol-sensitive, and mediated by HNO and cGMP, but not CGRP. HNO donors represent potential therapy for the failing heart, alone or in addition to standard care.

Nitroxyl-mediated disulfide bond formation between cardiac myofilament cysteines enhances contractile function.

In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery. To determine the effects of HNO modification in cardiac myofilament proteins. The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (F(max)) and reducing the concentration of Ca(2+) for 50% activation (Ca(50)) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase F(max) without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin-tropomyosin and myosin heavy chain-myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin-tropomyosin and myosin heavy chain-myosin light chain 1 interactions independently correlated with increased Ca(2+) sensitivity and force generation, respectively. HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca(2+) responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.

Effective methods for preparation and characterization of HNO‐adducts of heme proteins

Driven by the documented effectiveness of cryoradiolysis coupled with resonance Raman spectroscopy for the preparation and structural characterization of unstable peroxo and hydroperoxo intermediates of catalytic and oxidative enzymes, similar strategies have now been applied to prepare and study the still relatively unexplored chemistry of nitrosyl hydride (HNO) adducts of heme proteins and their derivatives. Previously, such HNO derivatives of heme proteins were prepared by chemical addition of reducing agents or by direct addition of an HNO donor. Here, for the first time, we report effective cryoradiolytic preparation of Fe–N(H)O species of myoglobin and their structural characterization by resonance Raman spectroscopy. Our results are in excellent agreement with those previously reported for chemically prepared derivatives and with computational results. Furthermore, the present study provides new data for the deuterated analogue; i.e. bearing a nitrosyl deuteride (DNO) fragment. Copyright © 2012 John Wiley &amp; Sons, Ltd.

The mechanism underlying nitroxyl and nitric oxide formation from hydroxamic acids

BackgroundThe pharmacological effects of hydroxamic acids (RC(O)NHOH, HX) are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Given the development and use of HXs as therapeutic agents, elucidation of the oxidation mechanism is needed for more educated selection of HX-based drugs. MethodsAcetohydroxamic and glycine-hydroxamic acids were oxidized at pH 7.0 by a continuous flux of radiolytically generated ·OH or by metmyoglobin and H2O2 reactions system. Gas chromatography and spectroscopic methods were used to monitor the accumulation of N2O, N2, nitrite and hydroxylamine. ResultsOxidation of HXs by ·OH under anoxia yields N2O, but not nitrite, N2 or hydroxylamine. Upon the addition of H2O2 to solutions containing HX and metmyoglobin, which is instantaneously and continuously converted into compound II, nitrite and, to a lesser extent, N2O are accumulated under both anoxia and normoxia. ConclusionsOxidation of HXs under anoxia by a continuous flux of ·OH, which solely oxidizes the hydroxamate moiety to RC(O)NHO·, forms HNO. This observation implies that bimolecular decomposition of RC(O)NHO· competes efficiently with unimolecular decomposition processes such as internal disproportionation, hydrolysis or homolysis. Oxidation by metmyoglobin/H2O2 involves relatively mild oxidants (compounds I and II). Compound I reacts with HX forming RC(O)NHO· and compound II, which oxidizes HX, RC(O)NHO·, HNO and NO. The latter reaction is the main source of nitrite. General significanceHXs under oxidative stress release HNO, but can be considered as NO-donors provided that HNO oxidation is more efficient than its reaction with other biological targets.

Abstract 144: Endothelium-Derived Nitroxyl-Mediated Relaxation Is Resistant to Superoxide Scavenging and Preserved in Diabetic Rat Aorta

Nitric oxide (NO)-mediated relaxation is impaired in diabetes due to oxidative stress. Nitroxyl (HNO) also contributes to endothelium-dependent relaxation however its role in diabetes-induced oxidative stress is not known. The aim of this study is to investigate whether acute exposure to oxidative stress or diabetes affects the contribution of HNO to endothelium-dependent relaxation in the rat aorta. Vascular responses to the endothelium-dependent relaxant, ACh, the HNO donor, Angeli’s salt and the NO donor, DEANONOate, were determined using standard organ bath techniques. Pharmacological tools (3 mM L-cysteine), a selective HNO scavenger and 100 μM hydroxocobalamin (HXC, a selective NO scavenger), were used to distinguish between NO and HNO-mediated relaxation. Exposure to the superoxide generator pyrogallol (100 μM) significantly reduced the sensitivity to DEANONOate (pEC 50 : control, 8.03±0.10 vs. pyrogallol, 7.48±0.09, n=6, p&lt;0.01) but had no effect on responses to Angeli’s salt in the rat aorta (pEC 50 : control, 7.30±0.15 vs. pyrogallol, 7.31±0.08, n=5-6, p&gt;0.05). In the presence of L-cysteine, pyrogallol caused a further inhibition to ACh-induced NO-mediated relaxation. In contrast, the addition of pyrogallol to HXC did not cause any further inhibition of endothelium-dependent relaxation (R max : HXC, 80±2% vs. pyrogallol+HXC, 67±7%, n=7, p&gt;0.05), indicating that HNO-mediated relaxation was not affected by superoxide. Similarly, in the diabetic aorta, responses to ACh were not affected when the contribution of NO to relaxation was abolished by HXC (R max : normal, 55±7% vs. diabetic, 60±8%, n=8-9, p&gt;0.05), indicating a preserved HNO-mediated relaxation. Conversely, when the contribution of HNO was inhibited with L-cysteine, the responses to ACh (pEC 50 : normal, 7.01±0.04 vs. diabetic, 6.72±0.1, n=7-9, p&lt;0.05), were significantly decreased in diabetic compared to normal aorta, suggesting that the contribution of NO was impaired by diabetes. These findings demonstrate that NO-mediated relaxation is impaired in the presence of oxidative stress but the HNO component of relaxation is preserved under those conditions.

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation.

BackgroundNew 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.MethodsNeonatal 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.ResultsWe 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).ConclusionsOur 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.

Endothelium‐Derived Nitroxyl‐Mediated Relaxation Is Resistant To Superoxide Scavenging And Preserved In Diabetic Rat Aorta

The aim of this study is to investigate whether acute exposure to oxidative stress or diabetes affects the contribution of HNO to endothelium‐dependent relaxation in the rat aorta. Vascular responses to the endothelium‐dependent relaxant, ACh, the HNO donor, Angeli's salt and the NO donor, DEANONOate, were determined using organ bath techniques. Exposure to the superoxide generator pyrogallol (100 μM) significantly reduced the sensitivity to DEANONOate (pEC50: control, 8.03±0.10 vs. pyrogallol, 7.48±0.09, n=6, p&lt;0.01) but had no effect on responses to Angeli's salt. In the presence of L‐cysteine (a selective HNO scavenger), pyrogallol caused a further inhibition to ACh‐induced NO‐mediated relaxation. In contrast, the addition of pyrogallol to hydroxocobalamin (HXC, a selective NO scavenger) did not cause any further inhibition of ACh‐induced relaxation, indicating that HNO‐mediated relaxation was resistant to superoxide. Similarly, in the diabetic aorta, responses to ACh were not affected when NO was abolished by HXC, indicating a preserved HNO‐mediated relaxation. Conversely, when HNO was inhibited with L‐cysteine, the responses to ACh (pEC50: normal, 7.01±0.04 vs. diabetic, 6.72±0.1, n=7–9, p&lt;0.05), were significantly decreased in diabetic aorta, suggesting that NO was impaired by diabetes. These findings demonstrate that HNO component of relaxation is unaffected by oxidative stress.

Photoinduced Upregulation of Calcitonin Gene-Related Peptide in A549 Cells through HNO Release from a Hydrophilic Photocontrollable HNO Donor

Nitroxyl (HNO), a one-electron-reduced form of nitric oxide, has various biological activities, including a cardioprotective effect. Here, we first synthesized another, more hydrophilic photocontrollable HNO donor (3), which can release HNO in a spatially and temporally controlled manner, and then examined the properties of our series of compounds as practical HNO donors in a cellular system under photocontrol. We selected compound 2 as the preferred donor, and used it to show that calcitonin gene-related peptide (CGRP) can be upregulated in A549 cells via photocontrolled HNO release. This result demonstrates the suitability of this photocontrollable HNO donor for biological investigations.

Nitroxyl (HNO) acutely activates the glucose uptake activity of GLUT1

Nitroxyl (HNO) is a molecule of significant interest due to its unique pharmacological properties, particularly within the cardiovascular system. A large portion of HNO biological effects can be attributed to its reactivity with protein thiols, where it can generate disulfide bonds. Evidence from studies in erythrocytes suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond. However, there are no reports that document the effects of HNO on glucose uptake. Therefore, we examined the acute effects of Angeli’s salt (AS), a HNO donor, on glucose uptake activity of GLUT1 in L929 fibroblast cells. We report that AS stimulates glucose uptake with a maximum effective concentration of 5.0 mM. An initial 7.2-fold increase occurs within 2 min, which decreases and plateaus to a 4.0-fold activation after 10 min. About 60% of the 4.0-fold activation recovers within 10 min, and 40% remains after an hour. The activation is blocked by the pretreatment of cells with thiol-reactive compounds, iodoacetamide (0.75 mM), cinnamaldehyde (2.0 mM), and phenylarsine oxide (10 μM). The effects of AS are not additive to the stimulatory effects of other acute activators of glucose uptake in L929 cells, such as azide (5 mM), berberine (50 μM), or glucose deprivation. These data suggest that GLUT1 is acutely activated in L929 cells by the formation of a disulfide bond, likely within GLUT1 itself.

The effect of nitro substitution on the photochemistry of benzyl benozhydroxamate: Photoinduced release of benzohydroxamic acid

Abstract The redox congener of the important signaling agent nitric oxide (NO), nitroxyl or nitrosyl hydride (HNO) has also been demonstrated to induce distinct physiological effects. The aim of this study was to determine if benzohydroxamic acid, which was selected as a stable model compound of HNO donors, could be released by the o -nitrobenzyl photolabile protecting group (PPG) in a wavelength-dependent manner. It was expected that selective irradiation of the o -nitrobenzyl chromophore would favor the release of benzohydroxamic acid over undesired products associated with N–O bond cleavage. Quantum yields for the release of benzohydroxamic acid protected by the o -nitrobenzyl PPG increased at longer wavelengths, with a concomitant decrease in the yield of minor products. Through the use of triplet photosensitizers, triplet quenchers, computational methods, and the position of the nitro substituent, insights into the nature of the mechanism were suggested.

ChemInform Abstract: Novel Protection—Deprotection Strategies in Diazeniumdiolate Chemistry: Synthesis of V‐IPA/NO.

AbstractA five‐step synthesis of potentially liver selective HNO donor V‐IPA/NO (VIII) is elaborated using a protection—deprotection strategy.

Vasorelaxant and antiaggregatory actions of the nitroxyl donor isopropylamine NONOate are maintained in hypercholesterolemia

Nitroxyl (HNO) displays pharmacological and therapeutic actions distinct from those of its redox sibling nitric oxide (NO(•)). It remains unclear, however, whether the vasoprotective actions of HNO are preserved in disease. The ability of the HNO donor isopropylamine NONOate (IPA/NO) to induce vasorelaxation, its susceptibility to tolerance development, and antiaggregatory actions were compared with those of a clinically used NO(•) donor, glyceryl trinitrate (GTN), in hypercholesterolemic mice. The vasorelaxant and antiaggregatory properties of IPA/NO and GTN were examined in isolated carotid arteries and washed platelets, respectively, from male C57BL/6J mice [wild-type (WT)] maintained on either a normal diet (WT-ND) or high fat diet (WT-HFD; 7 wk) as well as apolipoprotein E-deficient mice maintained on a HFD (ApoE(-/-)-HFD; 7 wk). In WT-ND mice, IPA/NO (0.1-30 μmol/l) induced concentration-dependent vasorelaxation and inhibition of collagen (30 μg/ml)-stimulated platelet aggregation, which was predominantly soluble guanylyl cyclase/cGMP dependent. Compared with WT-HFD mice, ApoE(-/-)-HFD mice displayed an increase in total plasma cholesterol levels (P < 0.001), vascular (P < 0.05) and platelet (P < 0.05) superoxide (O(2)(·-)) production, and reduced endogenous NO(•) bioavailability (P < 0.001). Vasorelaxant responses to both IPA/NO and GTN were preserved in hypercholesterolemia, whereas vascular tolerance developed to GTN (P < 0.001) but not to IPA/NO. The ability of IPA/NO (3 μmol/l) to inhibit platelet aggregation was preserved in hypercholesterolemia, whereas the actions of GTN (100 μmol/l) were abolished. In conclusion, the vasoprotective effects of IPA/NO were maintained in hypercholesterolemia and, thus, HNO donors may represent future novel treatments for vascular diseases.

Aorta from angiotensin II hypertensive mice exhibit preserved nitroxyl anion mediated relaxation responses

Hypertension is a disorder affecting millions worldwide, and is a leading cause of death and debilitation in the United States. It is widely accepted that during hypertension and other cardiovascular diseases the vasculature exhibits endothelial dysfunction; a deficit in the relaxatory ability of the vessel, attributed to a lack of nitric oxide (NO) bioavailability. Recently, the one electron redox variant of NO, nitroxyl anion (NO −) has emerged as an endothelium-derived relaxing factor (EDRF) and a candidate for endothelium-derived hyperpolarizing factor (EDRF). NO − is thought to exist protonated (HNO) in vivo, which would make this species more resistant to scavenging. However, no studies have investigated the role of this redox species during hypertension, and whether the vasculature loses the ability to relax to HNO. Thus, we hypothesize that aorta from angiotensin II (AngII)-hypertensive mice will exhibit a preserved relaxation response to Angeli's Salt, an HNO donor. Male C57Bl6 mice, aged 12–14 weeks were implanted with mini-osmotic pumps containing AngII (90 ng/min, 14 days plus high salt chow) or sham surgery. Aorta were excised, cleaned and used to perform functional studies in a myograph. We found that aorta from AngII-hypertensive mice exhibited a significant endothelial dysfunction as demonstrated by a decrease in acetylcholine (ACh)-mediated relaxation. However, vessels from hypertensive mice exhibited a preserved response to Angeli's Salt (AS), the HNO donor. To confirm that relaxation responses to HNO were maintained, concentration response curves (CRCs) to ACh were performed in the presence of scavengers to both NO and HNO (carboxy-PTIO and L-cys, resp .). We found that ACh-mediated relaxation responses were significantly decreased in aorta from sham and almost completely abolished in aorta from AngII-treated mice. Vessels incubated with l-cys exhibited a modest decrease in ACh-mediated relaxations responses. These data demonstrate that aorta from AngII-treated hypertensive mice exhibit a preserved relaxation response to AS, an HNO donor, regardless of a significant endothelial dysfunction.

A protective protein matrix improves the discrimination of nitroxyl from nitric oxide by Mn III protoporphyrinate IX in aerobic media

The selectivity of Mn III/II porphyrinates toward nitroxyl or nitric oxide donors provides a convenient starting point for the development of new materials for the speciation of these nitrogen-containing redox relatives. In the present report, we describe the insertion of Mn III protoporphyrinate IX in apomyoglobin and its chemical behavior toward HNO or NO donors, either under anaerobic or aerobic conditions. For comparison and discussion, the Mn III porphyrinate, devoid of the protein matrix, was studied in parallel. The Mn III reconstituted globin successfully reacted with the nitroxyl donor trioxodinitrate, while it was unreactive toward NO or NO donors, in good agreement with previously reported data on water soluble Mn III porphyrinates. The estimated association rate constant for the reaction with the nitroxyl donor was of the same order of magnitude for the reconstituted globin and the free porphyrinate, suggesting that the protein environment is not involved in the reaction mechanism. In contrast, the reaction product exhibited enhanced stability in the presence of dioxygen only when the porphyrinate was included in the protein matrix; this feature is ascribed to the role of the distal residues on the metal centered reactivity. This behavior is required for spectroscopic detection under biologically relevant conditions.