No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The nitric oxide redox sibling nitroxyl partially circumvents impairment of platelet nitric oxide responsiveness
... NO• resistance affects multiple sites in the cardiovascular system, including the myocardium, vasculature and platelets (Qin et al., 2020). As such, patients with diabetes fail to respond to the anti-aggregatory and vasodilator effects of NO•-based pharmacotherapies during cardiovascular emergencies, such as acute myocardial infarction, transient myocardial ischemia and acute decompensated heart failure (Dautov et al., 2013). Several pharmacotherapies including statins, some angiotensin-converting enzyme (ACE) inhibitors, perhexiline, and insulin (in the presence of severe hyperglycemia) ameliorate NO• resistance (Chirkov and Horowitz, 2007), while sGC activators primarily circumvent the problem (Costell et al., 2012). ...
... NO• resistance occurs largely due to oxidative stress, where ROS scavenge NO• and reversibly inactivate sGC, resulting in impaired tissue responsiveness to endogenous or exogenous NO• (Dautov et al., 2013;Tare et al., 2017). Oxidative stress refers to an imbalance between the generation of ROS and their clearance by endogenous antioxidants, such as superoxide dismutase, catalase and glutathione peroxidase (Wink et al., 2001). ...
... •Anti-platelet effect diminished in patients with IHD •Does not reduce infarct size post-acute myocardial infarction (Dautov et al., 2013;Siddiqi et al., 2014;Jones et al., 2015) Nitroxyl donors •Vasodilator, anti-aggregatory, positive cardiac inotropic/lusitropic actions •sGC-dependent & -independent signaling •Resistant to oxidative stress ...
Diabetes is associated with an increased mortality risk due to cardiovascular complications. Hyperglycemia-induced oxidative stress underlies these complications, leading to an impairment in endogenous nitric oxide (NO•) generation, together with reductions in NO• bioavailability and NO• responsiveness in the vasculature, platelets and myocardium. The latter impairment of responsiveness to NO•, termed NO• resistance, compromises the ability of traditional NO•-based therapeutics to improve hemodynamic status during diabetes-associated cardiovascular emergencies, such as acute myocardial infarction. Whilst a number of agents can ameliorate (e.g. angiotensin converting enzyme [ACE] inhibitors, perhexiline, statins and insulin) or circumvent (e.g. nitrite and sGC activators) NO• resistance, nitroxyl (HNO) donors offer a novel opportunity to circumvent NO• resistance in diabetes. With a suite of vasoprotective properties and an ability to enhance cardiac inotropic and lusitropic responses, coupled with preserved efficacy in the setting of oxidative stress, HNO donors have intact therapeutic potential in the face of diminished NO• signaling. This review explores the major mechanisms by which hyperglycemia-induced oxidative stress drives NO• resistance, and the therapeutic potential of HNO donors to circumvent this to treat cardiovascular complications in type 2 diabetes mellitus.
... Much of the evidence regarding NO-resistance in the cardiovascular system has been derived from the studies of Horowitz and Chirkov, particularly in human platelets [17,39,151,[153][154][155]. Impaired vascular NO-responsiveness has also been closely linked to poorer prognosis in patients with coronary artery disease, with increased incidence of major cardiovascular events (across a broad composite of endpoints including death, cardiac surgery, stroke, etc), independent of other cardiac risk factors [18]. ...
... In the vasculature, we, and others, have shown that HNO is a potent vasorelaxant [49,150,[176][177][178][179] and anti-aggregatory agent [150,155,180], prevents adherence of leukocytes to the endothelium [181] and targets Nox2 oxidase to limit vascular oxidative stress [182] (Fig. 3). Whilst the vasoprotective actions of HNO are mediated predominantly via activation of sGC and a subsequent increase in cGMP [48,179,183], we have provided the first evidence for a sGC/cGMP-independent action of HNO in the vasculature [182]. ...
... Moreover, the vasorelaxant and anti-aggregatory actions of HNO are preserved in pre-clinical models of hypercholesterolemia [150], hypertension [171,172] and diabetes [169] where impaired NO signalling is evident. Importantly, Horowitz and colleagues have shown that HNO donors circumvent, in part, the impaired platelet responsiveness to NO (NO resistance) observed in patients with coronary artery disease [155]. Specifically in patients in whom the anti-aggregatory actions of the NO donor, SNP were impaired, the HNO donor, IPA-NO was found to exert substantially greater sGC stimulation and anti-aggregatory Fig. 3. Overview of the vaso-and cardio-protective actions of nitroxyl (HNO). ...
Nitric oxide (NO) plays a pivotal role in the maintenance of cardiovascular homeostasis. A reduction in the bioavailability of endogenous NO, manifest as a decrease in the production and/or impaired signaling, is associated with many cardiovascular diseases including hypertension, atherosclerosis, stroke and heart failure. There is substantial evidence that reactive oxygen species (ROS), generated predominantly from NADPH oxidases (Nox), are responsible for the reduced NO bioavailability in vascular and cardiac pathologies. ROS can compromise NO function via a direct inactivation of NO, together with a reduction in NO synthesis and oxidation of its receptor, soluble guanylyl cyclase. Whilst nitrovasodilators are administered to compensate for the ROS-mediated loss in NO bioactivity, their clinical utility is limited due to the development of tolerance and resistance and systemic hypotension. Moreover, efforts to directly scavenge ROS with antioxidants has had limited clinical efficacy. This review outlines the therapeutic utility of NO-based therapeutics in cardiovascular diseases and describes the source and impact of ROS in these pathologies, with particular focus on the interaction with NO. Future therapeutic approaches in the treatment of cardiovascular diseases are highlighted with a focus on nitroxyl (HNO) donors as an alternative to traditional NO donors and the development of novel Nox inhibitors.
... Nitroxyl (HNO) is the one-electron reduced product of NO• (Miranda et al., 2003). Similar to its redox sibling, HNO also has antiaggregatory (Dautov et al., 2013) and vasodilator (Tare et al., 2017) effects. However, unlike NO•, HNO lacks reactivity with the superoxide anion and thus remains effective during oxidative stress (Leo et al., 2012). ...
... The contribution of sGC to inotropic, lusitropic, and coronary vasodilator effects of DEA/NO and Angeli's salt was examined using ODQ, which irreversibly inhibits sGC by oxidising its ferrous (Fe 2+ ) heme group to its ferric (Fe 3+ ) state (Zhao et al., 2000). Such findings may indicate that HNO is able to activate sGC in its oxidised form; however, this remains a matter of contention due to conflicting evidence that either supports this hypothesis (Dautov et al., 2013;Miranda et al., 2003;Qin et al., 2020) or refutes it (Miller et al., 2009;Zeller et al., 2009). We also demonstrated that ODQ attenuated coronary vasodilation in response to Angeli's salt in non-diabetic and diabetic hearts. ...
Background and Purpose
The risk of fatal cardiovascular events is increased in patients with type 2 diabetes mellitus (T2DM). A major contributor to poor prognosis is impaired nitric oxide (NO•) signalling at the level of tissue responsiveness, termed NO• resistance. This study aimed to determine if T2DM promotes NO• resistance in the heart and vasculature and whether tissue responsiveness to nitroxyl (HNO) is affected.
Experimental Approach
At 8 weeks of age, male Sprague–Dawley rats commenced a high‐fat diet. After 2 weeks, the rats received low‐dose streptozotocin (two intraperitoneal injections, 35 mg·kg⁻¹, over two consecutive days) and continued on the same diet. Twelve weeks later, isolated hearts were Langendorff‐perfused to assess responses to the NO• donor diethylamine NONOate (DEA/NO) and the HNO donor Angeli's salt. Isolated mesenteric arteries were utilised to measure vascular responsiveness to the NO• donors sodium nitroprusside (SNP) and DEA/NO, and the HNO donor Angeli's salt.
Key Results
Inotropic, lusitropic and coronary vasodilator responses to DEA/NO were impaired in T2DM hearts, whereas responses to Angeli's salt were preserved or enhanced. Vasorelaxation to Angeli's salt was augmented in T2DM mesenteric arteries, which were hyporesponsive to the relaxant effects of SNP and DEA/NO.
Conclusion and Implications
This is the first evidence that inotropic and lusitropic responses are preserved, and NO• resistance in the coronary and mesenteric vasculature is circumvented, by the HNO donor Angeli's salt in T2DM. These findings highlight the cardiovascular therapeutic potential of HNO donors, especially in emergencies such as acute ischaemia or heart failure.
... In other words, NO resistance refers to the decreased response to any available NO in targeted cells or tissues, even if pure NO is provided [86][87][88]. The accumulating evidence has been derived from human platelets studies [89][90][91]. Absence of NO-responsiveness is linked with poor prognosis in subjects suffering with coronary artery disease, independent of other cardiovascular risk factors [88]. It is supposed that oxidative stress is a crucial player in the process of NO resistance [92,93]. ...
... These studies highlight the emerging roles of HNO in VSMC proliferation and migration, and HNO may be taken as a novel candidate for the therapy of various cardiovascular diseases. Importantly, HNO donor circumvents the impaired platelet responsiveness to NO in subjects with coronary artery disease [91]. In addition, the pure HNO donors including CXL-1020 and CXL-1427 can undergo translational research in the near future [164]. ...
Cardiovascular diseases are recognized to be a major cause of people morbidity and mortality. A host of stress signals contribute to the pathogenesis of cardiovascular disorders. Deficiency of hydrogen sulfide (H2S) or nitric oxide (NO) coordinately plays essential roles in the development of cardiovascular diseases. Recent studies have shown that interaction between the two gaseostransmitters, H2S and NO, may give rise to nitroxyl (HNO), one-electron-reduced product of NO. HNO is found to exhibit a variety of biological and pharmacological properties including positive inotropy and cardiovascular protective effects, etc. In this review, recent progresses regarding HNO generation, detection, biochemical and pharmacological functions are discussed.
... In addition, HNO sensitizes myofilaments to Ca 2? , leading to increased myocardial contractility [32]. Further distinguishing features of HNO, as compared with NO, are its resistance to scavenging by superoxide [33,34], the lack of tolerance development with continued use [21,35,36], and preserved anti-aggregatory actions in the setting of coronary artery disease [37]. As such, the efficacy of HNO may be preserved under conditions of oxidative stress in which NO signaling is compromised. ...
... However, the beneficial effects of HNO donors may go beyond their acute inotropic and vasodilatory capacity. Indeed, with their anti-thrombotic [28,37,85] and antiremodeling properties [22,23,86] and ability to limit oxidative stress [22,23,87] and inflammation [88], together with their lack of tolerance development [21,35,36], there is potential for the use of HNO donors in the long-term treatment of HF and other cardiovascular pathologies such as angina and atherothrombotic syndromes [89]. We await the development of orally active, long-lasting HNO donors to fully ascertain the therapeutic potential of this class of drug in the chronic treatment of HF and the associated myocardial remodeling. ...
Heart failure (HF) is a major cause of hospital admission in the Western world, yet there remains a paucity of effective pharmacological management options. With the recent development of synthetic, next-generation nitroxyl (HNO) donors and their progress into clinical trials, it is timely to now provide an update on the therapeutic potential of HNO donors in the management of acute decompensated heart failure. In this article, we summarize current understanding of the pharmacology of HNO (in comparison with its redox sibling, nitric oxide), its spectrum of cardioprotective actions, and efforts to translate these into the clinic. Future research directions for this exciting new class of HF drugs are also considered.
... It has been previously demonstrated that in diabetes, and other causes of oxidative stress, there is impairment of vascular and cardiac responses to NO• donors (Van Etten et al., 2002;Okon et al., 2005;Shemyakin et al., 2012;Qin et al., 2020) referred to as NO• resistance. Further, while tolerance to the chronic use of NO• donors is a well-established phenomenon, HNO donors maintain their efficacy with continued use (Irvine et al., 2007;Kemp-Harper, 2011;Dautov et al., 2013;Tare et al., 2017). Efforts are underway to develop new generation HNO donors with optimal half-lives to enhance their therapeutic utility (Cowart et al., 2019). ...
Introduction
Endothelial dysfunction is a major risk factor for several of the vascular complications of diabetes, including ischemic stroke. Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO•), is resistant to scavenging by superoxide, but the role of HNO in diabetes mellitus associated endothelial dysfunction in the carotid artery remains unknown.
Aim: To assess how diabetes affects the role of endogenous NO• and HNO in endothelium-dependent relaxation in rat isolated carotid arteries.
Methods: Male Sprague Dawley rats were fed a high-fat-diet (HFD) for 2 weeks prior to administration of low dose streptozotocin (STZ; 35 mg/kg i. p./day) for 2 days. The HFD was continued for a further 12 weeks. Sham rats were fed standard chow and administered with citrate vehicle. After 14 weeks total, rats were anesthetized and carotid arteries collected to assess responses to the endothelium-dependent vasodilator, acetylcholine (ACh) by myography. The combination of calcium-activated potassium channel blockers, TRAM-34 (1 μmol/L) and apamin (1 μmol/L) was used to assess the contribution of endothelium-dependent hyperpolarization to relaxation. The corresponding contribution of NOS-derived nitrogen oxide species to relaxation was assessed using the combination of the NO• synthase inhibitor, L-NAME (200 μmol/L) and the soluble guanylate cyclase inhibitor ODQ (10 μmol/L). Lastly, L-cysteine (3 mmol/L), a selective HNO scavenger, and hydroxocobalamin (HXC; 100 μmol/L), a NO• scavenger, were used to distinguish between NO• and HNO-mediated relaxation.
Results: At study end, diabetic rats exhibited significantly retarded body weight gain and elevated blood glucose levels compared to sham rats. The sensitivity and the maximal relaxation response to ACh was significantly impaired in carotid arteries from diabetic rats, indicating endothelial dysfunction. The vasorelaxation evoked by ACh was abolished by L-NAME plus ODQ, but not affected by the apamin plus TRAM-34 combination, indicating that NOS-derived nitrogen oxide species are the predominant endothelium-derived vasodilators in sham and diabetic rat carotid arteries. The maximum relaxation to ACh was significantly decreased by L-cysteine in both sham and diabetic rats, whereas HXC attenuated ACh-induced relaxation only in sham rats, suggesting that diabetes impaired the contribution of NO•, whereas HNO-mediated vasorelaxation remained intact.
Conclusion: Both NO• and HNO contribute to endothelium-dependent relaxation in carotid arteries. In diabetes, NO•-mediated relaxation is impaired, whereas HNO-mediated relaxation was preserved. The potential for preserved HNO activity under pathological conditions that are associated with oxidative stress indicates that HNO donors may represent a viable therapeutic approach to the treatment of vascular dysfunction.
... Like NO, HNO also significantly expands the capacity of blood vessels and resistance vessels [4,5], but HNO does not have unpaired electrons. Therefore, HNO should not be scavenged by reactive oxygen species (ROS) [6], which can be stably present in depleted hearts and exert special biological effects. This has sparked interest in the treatment of HF with HNO. ...
Heart failure is the end stage of various heart diseases such as ischemic heart disease, dilated cardiomyopathy, valvular heart disease, congenital heart disease, and hypertensive myocardial damage. It is characterized by a decrease in myocardial contractility, but there is currently no ideal treatment. Nitroxyl hydrogen (HNO) is considered to be a protonated form of NO. It has special chemical properties compared to other nitrogen oxides. In the body of organisms, HNO can participate in all aspects of the occurrence and development of heart failure (HF) and react with some proteins closely related to cardiac activity, changing its spatial structure and exerting cardioprotective effects. In recent years, studies have shown that HNO can inhibit cardiomyocyte hypertrophy, reduce inflammation, enhance myocardial contractility, dilate coronary arteries as well as peripheral blood vessels in early heart failure, and protect the heart against heart failure. This paper, combined with the latest research results at home and abroad, clarifies that nitrosyl hydrogen exerts cardioprotective effects through various processes that occur in the development of heart failure.
... It has been demonstrated that HNO donors act as potent vasodilators and induce positive inotropic/lusitropic effects. It has also been shown for different HNO donors that they possess antiaggregatory effect on platelets [26,[31][32][33][34][35]. Due to those effects, the applicability of HNO donors as therapeutics in the treatment of heart failure has been proposed [36][37][38]. ...
The recent interest in the clinical applications of Piloty's acid derivatives as HNO donors for the treatment of cardiovascular system dysfunction has led us to the examination of factors controlling HNO release from selected ortho-substituted N-hydroxysulfonamides. Here we present the kinetic and quantum mechanical studies on the mechanism of HNO release from selected ortho-substituted N-hydroxysulfonamides and in vivo examination of the antiaggregatory properties of N-hydroxy-(2-bromobenzene)sulfonamide complex with sodium salt of β-cyclodextrin sulfobutyl ethers-ethyl ethers as compared with Angeli's salt.
... HNO is a potent dilator of the conduit (Andrews et al. 2015;Bullen et al. 2011a;Irvine et al. 2007) and resistance vasculature (Andrews et al. 2009), and it inhibits platelet aggregation (Bermejo et al. 2005;Bullen et al. 2011a) by signalling predominantly via the soluble guanylate cyclase (sGC)/cGMP pathway. In addition, HNO is not directly scavenged by ·O 2 ─ (Leo et al. 2012;Miranda et al. 2002), limits vascular ROS generation (Miller et al. 2013), circumvents NO resistance (Dautov et al. 2013) and does not develop tolerance (Andrews et al. 2015;Irvine et al. 2007Irvine et al. , 2011. Importantly, like NO·, HNO can be generated by the endothelium and serve as an endothelium-derived relaxing factor (Bullen et al. 2011a). ...
Endothelial dysfunction is a major risk factor for the vascular complications of diabetes. Increased reactive oxygen species (ROS) generation, a hallmark of diabetes, reduces the bioavailability of endothelial vasodilators, including nitric oxide (NO·). The vascular endothelium also produces the one electron reduced and protonated form of NO·, nitroxyl (HNO). Unlike NO·, HNO is resistant to scavenging by superoxide anions (·O2(─)). The fate of HNO in resistance arteries in diabetes is unknown. We tested the hypothesis that the vasodilator actions of endogenous and exogenous HNO are preserved in resistance arteries in diabetes. We investigated the actions of HNO in small arteries from the mesenteric and femoral beds as they exhibit marked differences in endothelial vasodilator function following 8 weeks of streptozotocin (STZ)-induced diabetes mellitus. Vascular reactivity was assessed using wire myography and ·O2(─) generation using lucigenin-enhanced chemiluminescence. The HNO donor, Angeli's salt, and the NO· donor, DEA/NO, evoked relaxations in both arteries of control rats, and these responses were unaffected by diabetes. Nox2 oxidase expression and ·O2(─) generation were upregulated in mesenteric, but unchanged, in femoral arteries of diabetic rats. Acetylcholine-induced endothelium-dependent relaxation was impaired in mesenteric but not femoral arteries in diabetes. The HNO scavenger, L-cysteine, reduced this endothelium-dependent relaxation to a similar extent in femoral and mesenteric arteries from control and diabetic animals. In conclusion, HNO and NO· contribute to the NO synthase (NOS)-sensitive component of endothelium-dependent relaxation in mesenteric and femoral arteries. The role of HNO is sustained in diabetes, serving to maintain endothelium-dependent relaxation.
... 94 More recently, Dautov and colleagues tested whether HNO can circumvent NO d resistance in platelets isolated from human subjects with or without ischemic heart disease. 95 These authors reported that IPA/NO was more effective than SNP in inhibiting platelet aggregation in normal subjects. Importantly, in NO-resistant individuals the IPA/NO: SNP response ratio was markedly increased, consistent with partial circumvention of NO d resistance by HNO. ...
Reactions based on the transfer of one or more electrons from a donor (reductant) to an acceptor (oxidant) are at the basis of several physiologically relevant cellular processes. Both reactive oxygen and nitrogen species (ROS and RNS, respectively) can signal through these reduction/oxidation (redox) reactions, particularly via reversible interaction with highly reactive thiols. Nitroxyl (HNO), the one-electron reduction product of nitric oxide (NO), is an RNS that in the last few lusters has garnered a lot of attention owing to its pharmacological properties that are quite dissimilar from those exhibited by its sibling NO or other RNS such as nitrite/nitrate. HNO uniqueness becomes particularly evident in the cardiovascular system. One aim of this chapter is to put the following three fundamental chemical properties of HNO in a cardiovascular physiology and therapeutic perspective: (1) HNO elective, and likely selective thiophilic nature; (2) inertness toward ROS; and (3) modest reactivity with molecular O2. With this conceptual framework in mind, here we will first review routes accounting for possible HNO endogenous formation as they may be relevant to govern basal and stress-stimulated cardiac and vascular function. Then, we will provide an updated account of the pharmacological properties of HNO donors in the heart and in vessels, both in vivo and in vitro, under normal and disease conditions, flanking this evidence with additional HNO pharmacological properties that may nicely complement its main cardiovascular actions. We will conclude discussing the perspective of HNO donors as a treatment for heart failure, more specifically for acute decompensated heart failure (ADHF), comparing HNO therapeutic portfolio to current mainstay ADHF therapies.
Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO•), has emerged as a nitrogen oxide with a suite of vasoprotective properties and therapeutic advantages over its redox sibling. Whilst HNO has garnered much attention due to its cardioprotective actions in heart failure, its ability to modulate vascular function, without the limitations of tolerance development and NO• resistance is desirable in the treatment of vascular disease. HNO serves as a potent vasodilator and anti-aggregatory agent and has an ability to limit vascular inflammation and reactive oxygen species generation. In addition, its resistance to scavenging by reactive oxygen species and ability to target distinct vascular signalling pathways (Kv, KATP, CGRP), contributes to its preserved efficacy in hypertension, diabetes and hypercholesterolemia. In this review, the vasoprotective actions of HNO will be compared with those of NO• and the therapeutic utility HNO donors in the treatment of angina, acute cardiovascular emergencies and chronic vascular disease discussed.
Open image in new window
Both nitric oxide (NO) and nitroxyl (HNO) donors signal through soluble guanylyl cyclase (sGC). NO binds to the Fe(II) form of sGC and nitroxyl possibly to both sGC heme and thiol groups. In the vasculature, nitroxyl can also signal through voltage-dependent (Kv) and ATP-sensitive (KATP) K⁺ channels as well as calcitonin gene-related peptide (CGRP). In the heart, HNO directly targets critical thiols to increase myocardial contractility, an effect not seen with NO. The qualitative effects via elevation of cGMP are similar, i.e. lusitropic in the heart and inhibitory on vasoconstriction, inflammation, aggregation and vascular remodelling. Of pathophysiological significance is the fact the efficacy of NO donors is impaired by ROS, e.g. through chemical scavenging of NO, to generate reactive nitrogen oxide species (RNOS), whilst nitroxyl is apparently not.
Aim:
Impairment of tissue responsiveness to exogenous and endogenous nitric oxide (NO•), known as NO• resistance, occurs in many cardiovascular disease states, prominently in diabetes and especially in the presence of marked hyperglycemia. In the current study, we sought to determine in moderate and severe diabetes 1) whether NO• resistance also occurs in myocardium, 2) whether NO• redox sibling nitroxyl (HNO) circumvents this.
Results:
The spectrum of acute NO• effects (induced by diethylamine-NONOate) including vasodilation, and enhanced myocardial contraction and relaxation, were impaired by moderately diabetic rats ([blood glucose]~20 mM). In contrast, acute HNO effects (induced by isopropylamine-NONOate) were preserved even in more severe diabetes ([blood glucose]>28 mM). Intriguingly, the positive inotropic effects of HNO were significantly enhanced in diabetic rat hearts. Further, progressive attenuation of soluble guanylyl cyclase (sGC) contribution to myocardial NO• responses occurred with increasing severity of diabetes. Nevertheless, activation of sGC by HNO remained intact in the myocardium.
Discussion:
These results provide the first evidence that NO• resistance occurs in diabetic hearts, and HNO largely circumvents this problem. Further, positive inotropic and lusitropic effects of HNO are enhanced in severely diabetic myocardium, a finding which warrants further mechanistic interrogation. The results support a potential role for therapeutic HNO administration in acute treatment of ischemia and/or heart failure in diabetics.
Innovation:
Diabetes is associated with marked attenuation of vascular and myocardial effects of NO and NO donors, and this NO• resistance is circumvented by HNO, suggesting potential therapeutic utility for HNO donors in cardiovascular emergencies in diabetics.
We have introduced herein, a novel copper complex based fluorescent probe [CuII(DQ468)Cl]+ exhibiting a significant fluorescence turn-on response towards nitroxyl (HNO) with high selectivity over other biological reactive oxygen, nitrogen and sulfer species including nitric oxide (NO). A smart strategy, involving HNO induced reduction of paramagnetic [CuII(DQ468)Cl]+ to diamagnetic [CuI(DQ468)]+ with concomitant fluorescence enhancement via PET mechanism is focused here. This reduction based strategy was also supported by X-band EPR response and mass spectroscopy. The metal free probe (DQ468) shows high affinity towards Cu2+ to form [CuII(DQ468)Cl]+ with 0.091 μM detection limit, which subsequently detects HNO in organo-aqueous medium at biologicalin pH (7.4) in the green region (λem= 543 nm) with LOD 0.41 μM. The ground state geometries of DQ468, [CuII(DQ468)Cl]+ and [CuI(DQ468)]+ are optimized by DFT calculation which displayed that the central metal ion in [CuII(DQ468)Cl]+ is in distorted tetrahedral geometry with C1 point group. Negligible cytotoxicity and bio-compatibility make our probe useful for in vitro detection of HNO.
In this work, we developed a novel fluorescent probe 1 for quantitative detection of nitroxyl (HNO). Probe 1 consisted of a naphthalene backbone and 2-(diphenylphosphino)benzoate which was used as a nitroxyl recognition unit. The probe exhibited an intense fluorescence turn-on response to nitroxyl via the aza-ylide formation and its subsequent Stadinger ligation to release compound 2. The probe can be applied to the quantification of nitroxyl with a linear range covering from 5.0×10-8 to 9.0×10-6 mol•L-1. The detecting limit of probe 1 toward nitroxyl was estimated to be 43 nM. Furthermore, probe 1 displayed a much higher selectivity for nitroxyl than other biologically relevant species. Importantly, owing to the high cell permeability and low cytotoxicity of probe 1, it was successfully applied to cell imaging of nitroxyl in living cells. The probe was expected to be a useful chemical tool for investigating the detailed functions and mechanisms of HNO in living systems.
Nitroxyl plays crucial roles in many biological pathways and can serve as a potent therapeutic agent for the treatment of heart failure. Recent studies suggest that HNO may be produced in mitochondria and the HNO formed might have functional consequences for mitochondrial activity. However, in order to study the function of HNO in mitochondria, a suitable research method is needed. Herein, through rational design, we synthesized a new mitochondria-targeted fluorescent nitroxyl probe (Mito-HNO). The developed probe was highly selective toward HNO over other reactive nitrogen species and reducing species. In addition, the probe Mito-HNO was rapidly responsive and suitable for visualization of HNO in mitochondria in living cells. The probe is expected to be employed in further revealing the biological function of HNO in subcellular mitochondria.
A excited-state intramolecular proton transfer (ESIPT) fluorescent turn-on probe (FLA-1) for the detection of nitroxyl (HNO) is designed and synthesized. It consists of a flavone derivative moiety as the fluorophore, linked via an ester moiety to a diphenylphosphinobenzoyl group as a recognition receptor, which forms an aza-ylide upon reaction with HNO. Intramolecular attack of the aza-ylide on the ester carbonyl group releases a fluorescent flavone derivative. In addition, the results showed that FLA-1 exhibited a selective response to HNO over other biological reductants and can quantitatively detect HNO in the range of 0-20 μM with the detection limit of 1.28 × 10-7 M in aqueous solution. Importantly, FLA-1 was successfully applied to detect HNO in the range of 2-20 μM with the detection limit of 3.96 × 10-7 M in complex biological samples (serum) demonstrating its value in practical application.
Key message
β-Cyclodextrin–hemin complex-induced tomato lateral root formation was associated with nitric oxide and heme oxygenase 1 by modulating cell cycle regulatory genes.
Abstract
β-Cyclodextrin–hemin complex (β-CDH), a complex by combining β-cyclodextrin (β-CD) with hemin, a heme oxygenase 1 (HO1) inducer, was a trigger of cucumber adventitious root formation by enhancing HO1 gene expression. In this report, our results identified the previously unknown function of β-CDH in plants: the inducer of tomato lateral root (LR) formation. β-CDH-triggered LR formation is hemin-specific, since β-CD failed to induce LR development. Because nitric oxide (NO) is involved in LR formation, the correlation of β-CDH with NO and HO1 was investigated. Our analysis suggested that β-CDH induced an increase in endogenous NO production, followed by up-regulation of tomato HO1 gene and LR formation, all of which were mimicked by hemin and two NO-releasing compounds (SNP and GSNO). The induction of HO1 gene expression and LR formation triggered by β-CDH or hemin were significantly blocked by an inhibitor of HO1. Further results revealed that both β-CDH- and SNP-stimulated HO1 gene expression and thereafter LR formation were sensitive to the removal of NO with a potent NO scavenger, and the responses of SNP were significantly blocked by an inhibitor of HO1. Molecular evidence illustrated that representative cell cycle regulatory genes, including SlCDKA1, SlCYCA3;1, SlCYCA2;1, and SlCYCD3;1, were significantly up-regulated by β-CDH and SNP, but obviously blocked when seedlings were co-treated with the scavenger of NO or the inhibitor of HO1. In summary, our physiological and molecular evidence demonstrated that both NO and HO1 were involved in the β-CDH-induced LR formation with, at least partially, HO1 acting downstream of NO signaling.
The nitric oxide (NO)/soluble guanylate cyclase (sGC) system is fundamental to endothelial control of vascular tone, but also plays a major role in the negative modulation of platelet aggregation. The phenomenon of platelet NO resistance, or decreased antiaggregatory response to NO, occurs increasingly with advanced age, as well as in the context of cardiovascular disease states such as heart failure, ischemic heart disease, and aortic valve disease. The central causes of NO resistance are "scavenging" of NO and dysfunction of sGC. In the current review, we discuss the roles of several modulators of NO synthesis and of the NO/sGC cascade on changes in platelet physiology with aging, together with potential therapeutic options to reduce associated thrombotic risk.
Previous studies in non-human blood vessels and in platelets have demonstrated that under hypoxic conditions release of NO from nitrite (NO2−) is potentiated by deoxyhaemoglobin. In the current study, we characterized hypoxic potentiation of NO2− effects in human vasculature and platelets in vitro, addressing underlying mechanisms.
The vasodilator efficacy of NO2−, in comparison with glyceryl trinitrate (GTN), was evaluated in vitro, using segments of human saphenous vein. Under hypoxic conditions, there was a leftward shift of the NO2− concentration–response curve (EC50: 22 μM in hyperoxia vs 3.5 μM in hypoxia; p < 0.01), but no significant potentiation of GTN effect. In the presence of red blood cells, hypoxic potentiation of NO2− vasodilator effect was accentuated.
In whole blood samples and platelet-rich plasma (PRP) we assessed inhibition of platelet aggregation by NO2− (1 mM), in comparison with that of sodium nitroprusside (SNP, 10 μM). In individual subjects (n = 37), there was a strong correlation (r = 0.75, p < 0.0001) between anti-aggregatory effects of NO2− and SNP in whole blood, signifying that resultant sGC activation underlies biological effect and responses to NO2− are diminished in the presence of NO resistance. In PRP, the effects of NO2− were less pronounced than in whole blood (p = 0.0001), suggesting an important role of Hb (within RBCs) in the bioconversion of NO2− to NO. Inhibition of platelet aggregation by NO2− was almost 3-fold greater in venous than in arterial blood (p < 0.0001), and deoxyHb concentration directly correlated (r = 0.69, p = 0.013) with anti-aggregatory response. Incremental hypoxia applied to venous blood samples (in hypoxic chamber) caused a progressive increase in both deoxyHb level and anti-aggregatory effect of NO2−. When subjects inhaled a 12% O2 mixture for 20 min, there was a 3-fold rise in blood deoxyHb fraction (p < 0.01). In PRP, response to NO2− also increased under hypoxia, and was further enhanced (p < 0.01) by deoxyHb. Furthermore, deoxyHb exerted significant anti-aggregatory effects even in the absence of added NO2−, suggesting a role for endogenous NO2−.
The results of this work provide further mechanistic insights into hypoxic potentiation of vasodilator and anti-aggregatory actions of NO2−. In human saphenous veins and blood, the balance of evidence suggests differential rates of NO release from NO2− (largely modulated by deoxyHb) as the fundamental mechanism.
The first near-infrared fluorescent turn-on sensor for the detection of nitroxyl (HNO), the one-electron reduced form of nitric oxide (NO), is reported. The new copper-based probe, CuDHX1, contains a dihydroxanthene (DHX) fluorophore and a cyclam-derivative as a Cu(II) binding site. Upon reaction with HNO, CuDHX1 displays a 5-fold fluorescence turn-on in cuvettes and is selective for HNO over thiols and reactive nitrogen and oxygen species. CuDHX1 can detect exogenously applied HNO in live mammalian cells and in conjunction with the zinc-specific, green-fluorescent sensor ZP1, can perform multicolor/multianalyte microscopic imaging. These studies reveal that HNO treatment elicits an increase in the concentration of intracellular mobile zinc.
Nitric oxide (NO) signaling regulates key processes in cardiovascular physiology, specifically vasodilation, platelet aggregation, and leukocyte rolling. Soluble guanylate cyclase (sGC), the mammalian NO sensor, transduces an NO signal into the classical second messenger cyclic GMP (cGMP). NO binds to the ferrous (Fe2+) oxidation state of the sGC heme cofactor and stimulates formation of cGMP several hundred fold. Oxidation of the sGC heme to the ferric (Fe3+) state desensitizes the enzyme to NO. The heme-oxidized state of sGC has emerged as a potential therapeutic target in the treatment of cardiovascular disease. Here, we investigate the molecular mechanism of NO-desensitization and find that sGC undergoes a reductive nitrosylation reaction that is coupled to the S-nitrosation of sGC cysteines. We further characterize the kinetics of NO-desensitization and find that heme-assisted nitrosothiol formation of β1Cys78 and β1Cys122 causes the NO-desensitization of ferric sGC. Finally, we provide evidence that the mechanism of reductive nitrosylation is gated by a conformational change of the protein. These results bear insight into function and dysfunction of sGC in cardiovascular disease.
The effects of L-cysteine were tested in rat aortic rings on responses to nitric oxide free radical (NO•), nitroxyl (NO−) derived from Angeli's salt and endothelium-derived relaxing factor (EDRF) activated by acetylcholine, ATP and the calcium ionophore A23187. Concentrations of 300 μM or less of L-cysteine had no effect on responses.
Relaxations produced by exogenous NO• (0.25–2.5 μM) were markedly prolonged and relaxations produced by sodium nitroprusside (0.001–0.3 μM) were enhanced by 1 and 3 mML-cysteine. The enhancements by L-cysteine of responses to NO• and sodium nitroprusside may be attributed to the formation of S-nitrosocysteine.
Relaxations mediated by the nitroxyl anion (0.3 μM) donated from Angeli's salt were more prolonged than those produced by NO•, and nitroxyl-induced relaxations were reduced by L-cysteine (1 and 3 mM).
EDRF-mediated relaxations produced by acetylcholine (0.01–10 μM), ATP (3–100 μM) and the calcium ionophore A23187 (0.1 μM) were significantly reduced by 3 mML-cysteine.
The similarity between the inhibitory effects of L-cysteine on responses to EDRF and on those to nitroxyl suggests that a component of the response to EDRF may be mediated by nitroxyl anion.
British Journal of Pharmacology (2000) 129, 315–322; doi:10.1038/sj.bjp.0703058
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.
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 (90ng/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.
The nitric oxide (NO(•)) sibling, nitroxyl or nitrosyl hydride (HNO), is emerging as a molecule whose pharmacological properties include providing functional support to failing hearts. HNO also preconditions myocardial tissue, protecting it against ischemia-reperfusion injury while exerting vascular antiproliferative actions. In this review, HNO's peculiar cardiovascular assets are discussed in light of its unique chemistry that distinguish HNO from NO(•) as well as from reactive oxygen and nitrogen species such as the hydroxyl radical and peroxynitrite. Included here is a discussion of the possible routes of HNO formation in the myocardium and its chemical targets in the heart. HNO has been shown to have positive inotropic/lusitropic effects under normal and congestive heart failure conditions in animal models. The mechanistic intricacies of the beneficial cardiac effects of HNO are examined in cellular models. In contrast to β-receptor/cyclic adenosine monophosphate/protein kinase A-dependent enhancers of myocardial performance, HNO uses its "thiophylic" nature as a vehicle to interact with redox switches such as cysteines, which are located in key components of the cardiac electromechanical machinery ruling myocardial function. Here, we will briefly review new features of HNO's cardiovascular effects that when combined with its positive inotropic/lusitropic action may render HNO donors an attractive addition to the current therapeutic armamentarium for treating patients with acutely decompensated congestive heart failure.
It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating
the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein,
we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating
this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly,
HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect
enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory
heme and cysteine thiols.
In brain and other tissues, nitric oxide (NO) operates as a diffusible second messenger that stimulates the soluble form of the guanylyl cylase enzyme and so elicits an accumulation of cGMP in target cells. Inhibitors of NO synthesis have been used to implicate NO in a wide spectrum of physiological and pathophysiological mechanisms in the nervous system and elsewhere. The function of cGMP in most tissues, however, has remained obscure. We have now identified a compound, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), that potently and selectively inhibits NO-stimulated guanylyl cyclase activity. In incubated slices of cerebellum, ODQ reversibly inhibited the NO-dependent cGMP response to glutamate receptor agonists (IC50 approximately nM) but did not affect NO synthase activity. The compound did not affect synaptic glutamate receptor function, as assessed in hippocampal slices, nor did it chemically inactivate NO. ODQ did, however, potentially inhibit cGMP generation in response to NO-donating compounds. An action on NO-stimulated soluble guanylyl cyclase was confirmed in studies with the purified enzyme. ODQ failed to inhibit NO-mediated macrophage toxicity, a phenomenon that is unrelated to cGMP, nor did it affect the activity of particulate guanylyl cyclase or adenylyl cyclase. ODQ is the first inhibitor that acts selectively at the level of a physiological NO "receptor" and, as such, it is likely to prove useful for investigating the function of the cGMP pathway in NO signal transduction.
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.
Nitroxyl anion (HNONO(-)), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/NO(-) augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts beta-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO(-) generated by Angelis' salt (AS) was infused (10 microg/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed beta-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted beta-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO(-) cardiotropic action. Thus, HNO/NO(-) has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to beta-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/NO(-) donors for the treatment of heart failure.
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.
Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl (HNO/NO−) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO.
A labile inorganic free radical, nitric oxide (.NO), is produced by nitric oxide synthase from the substrate L-arginine in various cells and tissues. It acts as an endothelium-derived relaxing factor (EDRF) or as a neurotransmitter in vivo. We investigated the reactivity of stable radical compounds, imidazolineoxyl N-oxides such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), carboxy-PTIO, and carboxymethoxy-PTIO against .NO/EDRF in both chemical and biological systems. By using electron spin resonance (ESR) spectroscopy, imidazolineoxyl N-oxides were found to react with .NO in a stoichiometric manner (PTIO/.NO = 1.0) in a neutral solution (sodium phosphate buffer, pH 7.4) with rate constants of approximately 10(4) M-1 s-1, resulting in the generation of NO2-/NO3- and imidazolineoxyls such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl (PTI), carboxy-PTI, or carboxymethoxy-PTI. Furthermore, the effects of imidazolineoxyl N-oxides on acetylcholine- or ATP-induced relaxation of the smooth muscle of rabbit aorta were tested. The vasorelaxations were inhibited by all three imidazolineoxyl N-oxides markedly. The inhibitory effects of carboxy-PTIO was almost 2-fold stronger than those of .NO synthesis inhibitors, N(omega)-nitro-L-arginine and N(omega)-monomethyl-L-arginine. Generation of EDRF/.NO was identified by reacting the PTIO in aortic strips and quantitating the reaction product with ESR spectroscopy. Thus, it was clarified that imidazolineoxyl N-oxide antagonize EDRF/.NO via a unique radical-radical reaction with .NO.
Background and purpose:
Nitrite (NO₂⁻) has recently been shown to represent a potential source of NO, in particular under hypoxic conditions. The aim of the current study was to compare the haemodynamic effects of NO₂⁻ in healthy volunteers and patients with stable congestive heart failure (CHF).
Experimental approach:
The acute haemodynamic effects of brachial artery infusion of NO₂⁻ (0.31 to 7.8 μmol·min⁻¹) was assessed in normal subjects (n = 20) and CHF patients (n = 21).
Key results:
NO₂⁻ infusion was well tolerated in all subjects. Forearm blood flow (FBF) increased markedly in CHF patients at NO₂⁻ infusion rates which induced no changes in normal subjects (ANOVA: F = 5.5; P = 0.02). Unstressed venous volume (UVV) increased even with the lowest NO₂⁻ infusion rate in all subjects (indicating venodilation), with CHF patients being relatively hyporesponsive compared with normal subjects (ANOVA: F = 6.2; P = 0.01). There were no differences in venous blood pH or oxygen concentration between groups or during NO₂⁻ infusion. Venous plasma NO₂⁻ concentrations were lower in CHF patients at baseline, and rose substantially less with NO₂⁻ infusion, without incremental oxidative generation of nitrate, consistent with accelerated clearance in these patients. Plasma protein-bound NO concentrations were lower in CHF patients than normal subjects at baseline. This difference was attenuated during NO₂⁻ infusion. Prolonged NO₂⁻ exposure in vivo did not induce oxidative stress, nor did it induce tolerance in vitro.
Conclusions and implications:
The findings of arterial hyper-responsiveness to infused NO₂⁻ in CHF patients, with evidence of accelerated transvascular NO₂⁻ clearance (presumably with concomitant NO release) suggests that NO₂⁻ effects may be accentuated in such patients. These findings provide a stimulus for the clinical exploration of NO₂⁻ as a therapeutic modality in CHF.
Nox2 oxidase activity underlies the oxidative stress and vascular dysfunction associated with several vascular-related diseases. We have reported that nitric oxide (NO.) decreases reactive oxygen species production by endothelial Nox2 oxidase. This study tested the hypothesis that nitroxyl (HNO), the redox sibling of NO., also suppresses vascular Nox2 oxidase activity. Specifically, we examined the influence of two well-characterized HNO donors, Angeli's salt and isopropylamine NONOate (IPA/NO), on Nox2 oxidase-dependent responses to angiotensin II (reactive oxygen species production and vasoconstriction) in mouse cerebral arteries. Angiotensin II (0.1μmol/L)-stimulated superoxide (measured by lucigenin-enhanced chemiluminescence) and hydrogen peroxide (Amplex Red fluorescence) levels in cerebral arteries (pooled basilar and middle cerebral [MCA]) from wild-type (WT) mice were ~60% lower (P<0.05) in the presence of either Angeli's salt (1μmol/L) or IPA/NO (1μmol/L). Similarly, phorbyl 12,13-dibutyrate (10μmol/L; Nox2 activator)-stimulated hydrogen peroxide levels were ~40% lower in the presence of IPA/NO (1μmol/L; P<0.05). The ability of IPA/NO to decrease superoxide levels was reversible and abolished by the HNO scavenger L-cysteine (3mmol/L; P<0.05), but was unaffected by hydroxocobalamin (100μmol/L; NO. scavenger), ODQ (10μmol/L; soluble guanylyl cyclase [sGC] inhibitor), or Rp-8-pCPT-cGMPs (10μmol/L; cyclic guanosine monophosphate [cGMP]-dependent protein kinase inhibitor). Angiotensin II-stimulated superoxide was substantially less in arteries from Nox2-deficient (Nox2-/y) versus WT mice (P<0.05). In contrast to WT, IPA/NO (1μmol/L) had no effect on superoxide levels in arteries from Nox2-/y mice. Finally, angiotensin II (1-1000μmol/L)-induced constriction of WT MCA was virtually abolished by IPA/NO (1μmol/L), whereas constrictor responses to either the thromboxane A2 mimetic U46619 (1-100nmol/L) or high potassium (122.7mmol/L) were unaffected. In conclusion, HNO suppresses vascular Nox2 oxidase activity via a sGC-cGMP-independent pathway. Thus, HNO donors might be useful therapeutic agents to limit and/or prevent Nox2 oxidase-dependent vascular dysfunction.
Nitric oxide (NO) and nitroxyl (HNO) are reported to have numerous biological activities with significant therapeutic potential. Many of these activities are overlapping. The chemistry by which these two species act is likely to be distinct in spite of their apparent close structural similarities. Discussed in this review is the chemistry of NO and HNO with their likely biological targets - thiolproteins, metalloproteins (more specifically iron heme proteins) and free radical processes. Based on the chemistry discussed, it can be concluded that the biological actions of NO are likely due primarily to its interactions with metal centers and reaction with radical species. The likely biological targets for HNO are, similarly, metal centers and radical species (albeit with different chemistry compared to NO). HNO is also particularly good at directly modifying thiols while NO-mediated thiol modification requires other reactants to be present and is not as facile. Thus, a fundamental difference between NO and HNO that likely distinguishes them with regards to their biological activity is the greater propensity for HNO to react with thiols compared to NO.
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 (t(1/2)=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 IC(50) 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.
Using 2 sequential studies in HOPE (Heart Outcomes Prevention Evaluation) study-type patients, the aims of this study were: 1) to test the hypothesis that ramipril improves platelet nitric oxide (NO) responsiveness: and 2) to explore biochemical and physiological effects of ramipril in a cohort selected on the basis of platelet NO resistance.
Ramipril prevents cardiovascular events, but the bases for these effects remain uncertain. NO resistance at both the platelet and vascular levels is present in a substantial proportion of patients with diabetes or ischemic heart disease and is an independent risk factor for cardiovascular events.
Study 1 was a double-blind, randomized comparison of ramipril (10 mg) with placebo in a cohort of patients (n = 119) with ischemic heart disease or diabetes plus additional coronary risk factor(s), in which effects on platelet responsiveness to NO were compared. Study 2 was a subsequent short-term evaluation of the effects of ramipril in a cohort of subjects (n = 19) with impaired platelet NO responsiveness in whom additional mechanistic data were sought.
In study 1, ramipril therapy increased platelet responsiveness to NO relative to the extent of aggregation (p < 0.001), but this effect occurred primarily in patients with severely impaired baseline NO responsiveness (n = 41). In study 2, ramipril also improved platelet NO responsiveness (p < 0.01), and this improvement was correlated directly with increased NO-stimulated platelet generation of cyclic guanosine monophosphate (p < 0.02) but not with changes in plasma thrombospondin-1 levels.
Ramipril ameliorates platelet NO resistance in HOPE study-type patients, with associated increases in soluble guanylate cyclase responsiveness to NO. This effect is likely to contribute to treatment benefit and define patients in whom ramipril therapy is particularly effective.
Left ventricular hypertrophy (LVH), an increased left ventricular (LV) mass, is common to many cardiovascular disorders, initially developing as an adaptive response to maintain myocardial function. In the longer term, this LV remodelling becomes maladaptive, with progressive decline in LV contractility and diastolic function. Indeed LVH is recognised as an important blood-pressure independent predictor of cardiovascular morbidity and mortality. The clinical efficacy of current treatments for LVH is reduced, however, by their tendency to slow disease progression rather than induce its reversal, and thus the development of new therapies for LVH is paramount. The signalling molecule cyclic guanosine-3′,5′-monophosphate (cGMP), well-recognised for its role in regulating vascular tone, is now being increasingly identified as an important anti-hypertrophic mediator. This review is focused on the various means by which cGMP can be stimulated in the heart, such as via the natriuretic peptides, to exert anti-hypertrophic actions. In particular we address the limitations of traditional nitric oxide (NO•) donors in the face of the potential therapeutic advantages offered by novel alternatives; NO• siblings, ligands of the cGMP-generating enzymes, soluble (sGC) and particulate guanylyl cyclases (pGC), and phosphodiesterase inhibitors. Further impact of cGMP within the cardiovascular system is also discussed with a view to representing cGMP-based therapies as innovative pharmacotherapy, alone or concurrent with standard care, for the management of LVH.
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.
The growing evidence that nitroxyl (HNO) has a rich pharmacological potential that differs from that of nitric oxide (NO) has intensified interest in HNO donors. Recently, the diazeniumdiolate (NONOate) based on isopropylamine (IPA/NO; Na[(CH(3))(2)CHNH(N(O)NO)]) was demonstrated to function under physiological conditions as an organic analogue to the commonly used HNO donor Angeli's salt (Na(2)N(2)O(3)). The decomposition mechanism of Angeli's salt is dependent on pH, with transition from an HNO to an NO donor occurring abruptly near pH 3. Here, pH is shown to also affect product formation from IPA/NO. Chemical analysis of HNO and NO production led to refinement of an earlier, quantum mechanically based prediction of the pH-dependent decomposition mechanisms of primary amine NONOates such as IPA/NO. Under basic conditions, the amine proton of IPA/NO is able to initiate decomposition to HNO by tautomerization to the nitroso nitrogen (N(2)). At lower pH, protonation activates a competing pathway to NO production. At pH 8, the donor properties of IPA/NO and Angeli's salt are demonstrated to be comparable, suggesting that at or above this pH, IPA/NO is primarily an HNO donor. Below pH 5, NO is the major product, while IPA/NO functions as a dual donor of HNO and NO at intermediate pH. This pH-dependent variability in product formation may prove useful in examination of the chemistry of NO and HNO. Furthermore, primary amine NONOates may serve as a tunable class of nitrogen oxide donor.
Acyloxy nitroso compounds hydrolyze to nitroxyl (HNO), a nitrogen monoxide with distinct chemistry and biology. Ultraviolet-visible spectroscopy and mass spectrometry show hydrolysis rate depends on pH and ester group structure with the observed rate being trifluoroacetate (3) > acetate (1) > pivalate (2). Under all conditions, 3 rapidly hydrolyzes to HNO. A combination of spectroscopic, kinetic, and product studies show that addition of thiols increases the decomposition rate of 1 and 2, leading to hydrolysis and HNO. Under conditions that favor thiolates, the thiolate directly reacts with the nitroso group, yielding oximes without HNO formation. Biologically, 3 behaves like Angeli's salt, demonstrating thiol-sensitive nitric oxide-mediated soluble guanylate cyclase-dependent vasorelaxation, suggesting HNO-mediated vasorelaxation. The slow HNO-donor 1 demonstrates weak thiol-insensitive vasorelaxation, indicating HNO release kinetics determine HNO bioavailability and activity. These results show that acyloxy nitroso compounds represent new HNO donors capable of vasorelaxation depending on HNO release kinetics.
Due to recent discoveries of important and novel biological activity, nitroxyl (HNO) has become a molecule of significant interest. Although it has been used in the past as a treatment for alcoholism, it is currently being touted as a treatment for heart failure. It is becoming increasingly clear that many of the biological actions of HNO can be attributed to its ability to react with specific thiol- and, possibly, heme-proteins. Herein is discussed the chemistry of HNO with likely biological targets. A particular focus is given to targets associated with the pharmacological utility of HNO as a cardiovascular agent and for the treatment of alcoholism.
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.
Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO(•)), is rapidly emerging as a novel nitrogen oxide with distinct pharmacology and therapeutic advantages over its redox sibling. Whilst the cardioprotective effects of HNO in heart failure have been established, it is apparent that HNO may also confer a number of vasoprotective properties. Like NO(•), HNO induces vasodilatation, inhibits platelet aggregation, and limits vascular smooth muscle cell proliferation. In addition, HNO can be putatively generated within the vasculature, and recent evidence suggests it also serves as an endothelium-derived relaxing factor (EDRF). Significantly, HNO targets signaling pathways distinct from NO(•) with an ability to activate K(V) and K(ATP) channels in resistance arteries, cause coronary vasodilatation in part via release of calcitonin-gene related peptide (CGRP), and exhibits resistance to scavenging by superoxide and vascular tolerance development. As such, HNO synthesis and bioavailability may be preserved and/or enhanced during disease states, in particular those associated with oxidative stress. Moreover, it may compensate, in part, for a loss of NO(•) signaling. Here we explore the vasoprotective actions of HNO and discuss the therapeutic potential of HNO donors in the treatment of vascular dysfunction.
Nitric oxide (NO) regulates a number of essential physiological processes by activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. The mechanism of NO sensing was previously thought to result exclusively from NO binding to the sGC heme; however, recent studies indicate that heme-bound NO only partially activates sGC and additional NO is involved in the mechanism of maximal NO activation. Furthermore, thiol oxidation of sGC cysteines results in the loss of enzyme activity. Herein the role of cysteines in NO-stimulated sGC activity investigated. We find that the thiol modifying reagent methyl methanethiosulfonate specifically inhibits NO activation of sGC by blocking a non-heme site, which defines a role for sGC cysteine(s) in mediating NO binding. The nature of the NO/cysteine interaction was probed by examining the effects of redox active reagents on NO-stimulated activity. These results show that NO binding to, and dissociation from, the critical cysteine(s) does not involve a change in the thiol redox state. Evidence is provided for non-heme NO in the physiological activation of sGC in context of a primary cell culture of human umbilical vein endothelial cells. These findings have relevance to diseases involving the NO/cGMP signaling pathway.
Nitroxyl (HNO) may be formed endogenously by uncoupled nitric-oxide (NO) synthases, enzymatic reduction of NO or as product of vascular nitroglycerin bioactivation. The established HNO donor Angeli's salt (trioxodinitrate, AS) causes cGMP-dependent vasodilation through activation of soluble guanylate cyclase (sGC). We investigated the mechanisms underlying this effect using purified sGC and cultured endothelial cells. AS (up to 0.1 mM) had no significant effect on sGC activity in the absence of superoxide dismutase (SOD) or dithiothreitol (DTT). In the presence of SOD, AS caused biphasic sGC activation (apparent EC(50) approximately 10 nM, maximum at 1 microM) that was accompanied by the formation of NO. DTT (2 mM) inhibited the effects of <10 microM AS but led to sGC activation and NO release at 0.1 mM AS even without SOD. AS had no effect on ferric sGC, excluding activation of the oxidized enzyme by HNO. The NO scavenger carboxy-PTIO inhibited endothelial cGMP accumulation induced by AS in the presence but not in the absence of SOD (EC(50) approximately 50 nM and approximately 16 microM, respectively). Carboxy-PTIO (0.1 mM) inhibited the effect of <or=10 microM AS in the presence of SOD but caused NO release from 0.1 mM AS in the absence of SOD. These data indicate that AS activates sGC exclusively via NO, formed either via SOD-catalyzed oxidation of HNO or through a minor AS decomposition pathway that is unmasked in the presence of HNO scavenging thiols.
Oxidative stress, a risk factor for several cardiovascular disorders, interferes with the NO/sGC/cGMP signalling pathway through scavenging of NO and formation of the strong intermediate oxidant, peroxynitrite. Under these conditions, endothelial and vascular dysfunction develops, culminating in different cardio-renal and pulmonary-vascular diseases. Substituting NO with organic nitrates that release NO (NO donors) has been an important principle in cardiovascular therapy for more than a century. However, the development of nitrate tolerance limits their continuous clinical application and, under oxidative stress and increased formation of peroxynitrite foils the desired therapeutic effect. To overcome these obstacles of nitrate therapy, direct NO- and haem-independent sGC activators have been developed, such as BAY 58-2667 (cinaciguat) and HMR1766 (ataciguat), showing unique biochemical and pharmacological properties. Both compounds are capable of selectively activating the oxidized/haem-free enzyme via binding to the enzyme's haem pocket, causing pronounced vasodilatation. The potential importance of these new drugs resides in the fact that they selectively target a modified state of sGC that is prevalent under disease conditions as shown in several animal models and human disease. Activators of sGC may be beneficial in the treatment of a range of diseases including systemic and pulmonary hypertension (PH), heart failure, atherosclerosis, peripheral arterial occlusive disease (PAOD), thrombosis and renal fibrosis. The sGC activator HMR1766 is currently in clinical development as an oral therapy for patients with PAOD. The sGC activator BAY 58-2667 has demonstrated efficacy in a proof-of-concept study in patients with acute decompensated heart failure (ADHF), reducing pre- and afterload and increasing cardiac output from baseline. A phase IIb clinical study for the indication of ADHF is currently underway.
Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O(2)(*)(-)) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O(2)(*)(-) and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague-Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 micromol/kg bw, orally), and TNF+ Tempol (Temp; 300 micromol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O(2)(*)(-) production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.
Selected nucleophile/nitric oxide adducts [compounds which contain the anionic moiety, XN(O-)N = O] were studied for their ability to release nitric oxide spontaneously in aqueous solution and for possible vasoactivity. The diversity of structures chosen included those in which the nucleophile residue, X, was that of a secondary amine [Et2N, as in [Et2NN(N = O)O]Na, 1], a primary amine [iPrHN, as in [iPrHNN(N = O)O]Na, 2], a polyamine, spermine [as in the zwitterion H2N(CH2)3NH2+(CH2)4N[N(N = O)O-](CH2)3NH2, 3], oxide [as in Na[ON(N = O)O]Na, 4], and sulfite [as in NH4[O3SN(N = O)O]NH4, 5]. The rate constants (k) for decomposition in pH 7.4 phosphate buffer at 37 degrees C, as measured by following loss of chromophore at 230-260 nm, were as follows: 1, 5.4 x 10(-3) s-1; 2, 5.1 x 10(-3) s-1; 3, 0.30 x 10(-3) s-1; 4, 5.0 x 10(-3) s-1; and 5, 1.7 x 10(-3) s-1. The corresponding extents of nitric oxide release (ENO) were 1.5, 0.73, 1.9, 0.54, and 0.001 mol/mol of starting material consumed, respectively, as determined from the integrated chemiluminescence response. Vasodilatory activities expressed as the concentrations required to induce 50% relaxation in norepinephrine-constricted aortic rings bathed in pH 7.4 buffer at 37 degrees C (EC50) were as follows: 1, 0.19 microM; 2, 0.45 microM; 3, 6.2 microM; 4, 0.59 microM; and 5, 62 microM. Vasorelaxant potency (expressed as 1/EC50) was strongly correlated with the quantity of .NO calculated from the physicochemical data to be released in the interval required to achieve maximum relaxation at the EC50 doses (r = 0.995). This suggests that such nucleophile/.NO adducts might generally be useful as vehicles for the nonenzymatic generation of nitric oxide, in predictable amounts and at predictable rates, for biological purposes. The particular significance for possible drug design is underscored in the very favorable potency comparison between several of these agents and the established nitrovasodilators sodium nitroprusside and glyceryl trinitrate (EC50 values of 2.0 and greater than 10 microM, respectively) in parallel aortic ring tests.
The expanding role of intravenous nitroglycerin (GTN) in the management of critically ill hospitalized patients demands a clear knowledge of its pharmacodynamics and kinetics in both normal and diseased states. Accordingly, we studied 16 patients with congestive cardiac failure to establish the relationship between blood levels of GTN and its physiologic effects during and after an i.v. infusion. The end point of this study was either a greater than 25% fall in pulmonary capillary wedge pressure or more than a tenfold increment over the initial GTN infusion rate. Infusion rate of GTN and blood concentration correlated well (r=0.75, p<0.001). Patients were divided into two groups based on their blood GTN concentration. Group 1 patients (n=8) achieved blood GTN concentrations of 1.2-11.1 ng/ml and all reached the hemodynamic end point. The minimum effective blood GTN concentration was 1.2 ng/ml at an infusion rate of 15 μg/min. Group 2 patients (n=8) had blood levels greater than 11.1 ng/ml and only three achieved the hemodynamic end point. Group 2 had greater systemic venous congestion than group 1 (right atrial pressure 19 ± 4 mm Hg (SD) vs 10 ± 4 mm Hg [p<0.001]). In addition, group 2 had lower total body clearance of GTN (3.6 ± 1.8 l/min) than group 1 (13.8 ± 5.8 l/min) (p<0.005). The low clearance of GTN in group 2 patients may be explained in part by impaired hepatic metabolism secondary to severe systemic venous congestion. Complete blood GTN data were available on five patients after cessation of the GTN infusion and revealed a short half-life of 1.9 minutes. Some patients failed to reach the hemodynamic end point with high infusion rates of GTN (220-440 μg/min), and blood levels of 42.2-481.3 ng/ml. There was no evidence of toxicity despite these high GTN blood levels.
Nitroxyl (NO-) is the one-electron reduction product of nitric oxide (NO.). Recently, NO- generating compounds were shown to possess potent vasorelaxant activity and this was attributed to the ready conversion of NO- to NO.. Because of its metastable character, direct chemical detection of NO- or its conjugated acid, HNO, has not been accomplished yet. In order to gain further insight into the cellular mode of action of NO- generating compounds we aimed at finding a means to discriminate NO- from NO. by bioassay. Using isolated rat aortic rings in organ baths, we here show that high concentrations of L-cysteine cause complete inhibition of the vasorelaxant response to NO- (generated from Angeli's salt and sodium nitroxyl) whereas responses to authentic NO. and S-nitrosocysteine are largely enhanced. Preliminary results indicate that the inhibition by L-cysteine of NO- activity may be mediated in part by enzymatic and non-enzymatic mechanisms. Whether or not NO- generating compounds will have promising therapeutic potential as a new class of NO.- donors will not least depend on their interference with enzymatic routes susceptible to inhibition by NO-.
Platelet hyperactivity plays an important role in the pathogenesis of cardio-vascular diseases. In patients with stable angina pectoris, we have recently demonstrated that nitroglycerin suppressed the increased platelet aggregability. The anti-aggregating effect of NTG and other nitrovasodilators is mediated by platelet guanylate cyclase, which generates cyclic GMP (cGMP) in response to nitric oxide (NO) liberated from the nitrovasodilator molecule. In the current study we utilised a more “direct” NO donor, sodium nitroprusside (SNP), to examine reversal of ADP-induced platelet aggregation in comparison with intraplatelet cGMP elevation in platelets from normal subjects (n = 22) and patients with stable angina pectoris (n = 23).
Concentrations of SNP associated with 50% reversal of aggregation were 2.7 ± 0.4 × 10−7 mol/L with normal subjects and 4.5 ± 0.5 × 10−6 mol/L with patients (P < 0.01). SNP produced a concentration-dependent elevation of intraplatelet cGMP content: with 10−4 mol/L SNP this was 17-fold for normals and 5-fold for patients (P < 0.01). An increase in cAMP content was seen only with 10−4 mol/L SNP, and was 157 ± 11% of baseline in platelets from normal subjects and 138 ± 14% in patients. There was a strong interrelationship between cGMP-stimulating and anti-aggregating effects of SNP. The decrease in cGMP responsiveness to SNP was not related to a dysfunction of platelet guanylate cyclase; neither basal nor SNP-stimulated activity of the enzyme varied significantly between normal subjects and patients. Lipophilic derivatives of cGMP (db-cGMP) and cAMP (db-cAMP) caused reversal of aggregation; there was a nonsignificant trend towards decreased responsiveness of platelets from patients to both db-cGMP and db-cAMP.
The observed decrease in responsiveness of platelets from angina patients to anti-aggregating effects of the exogenous NO donor, SNP, can therefore be attributed to suppressed cGMP accumulation. These results imply reduced platelet sensitivity to endogenous NO (endothelium-derived relaxing factor); this might contribute to platelet hyperaggregability observed in angina pectoris.
Hemodynamic resistance to nitrates has been previously documented in congestive heart failure. In patients with stable angina pectoris (SAP), we have observed a similar phenomenon: decreased platelet response to disaggregating effects of nitroglycerin (NTG) and sodium nitroprusside (SNP).
In blood samples from normal subjects (n=32) and patients with SAP (n=56), we studied effects of NO donors (NTG and SNP) on ADP-induced platelet aggregation and on intraplatelet cGMP. NTG and SNP inhibited platelet aggregation in patients to lesser extents than in normal subjects (P<0.01). The cGMP-elevating efficacy of NTG and SNP was diminished in platelets from patients in comparison with those from normals (P<0.001). Inhibition of the anti-aggregatory effects of NTG and SNP by ODQ, a selective inhibitor of NO-stimulated guanylate cyclase, was significantly less pronounced in patients than in normal subjects. Content of O2- was higher in blood samples from patients than in those from normal subjects (P<0. 01). In blood samples from patients with SAP, but not in normal subjects, the O2- scavenger superoxide dismutase (combined with catalase) suppressed platelet aggregation (P<0.01) and increased the extent of anti-aggregatory effect of SNP (P<0.01).
In patients with SAP, platelets are less responsive to the anti-aggregating and cGMP-stimulating effects of NO donors; this may reflect both reduction in guanylate cyclase sensitivity to NO and inactivation of the released NO by O2-. The implied impairment of anti-platelet efficacy of endogenous NO (in the form of EDRF) may contribute to platelet hyperaggregability associated with angina pectoris.
Endothelial vasodilator dysfunction is a characteristic feature of patients at risk for coronary atherosclerosis. Therefore, we prospectively investigated whether coronary endothelial dysfunction predicts disease progression and cardiovascular event rates.
Coronary vasoreactivity was assessed in 147 patients using the endothelium-dependent dilator acetylcholine, sympathetic activation by cold pressor testing, dilator responses to increased blood flow, and dilation in response to nitroglycerin. Cardiovascular events (cardiovascular death, unstable angina, myocardial infarction, percutaneous transluminal coronary angioplasty, coronary bypass grafting, ischemic stroke, or peripheral artery revascularization) served as outcome variables over a median follow-up period of 7.7 years. Patients suffering from cardiovascular events during follow-up (n=16) had significantly increased vasoconstrictor responses to acetylcholine infusion (P=0. 009) and cold pressor testing (P=0.002), as well as significantly blunted vasodilator responses to increased blood flow (P<0.001) and the intracoronary injection of nitroglycerin (P=0.001). Impaired endothelial and endothelium-independent coronary vasoreactivity were associated with a significantly higher incidence of cardiovascular events by Kaplan-Meier analysis. By multivariate analysis, all tests of coronary vasoreactivity were significant, independent predictors of a poor prognosis, even after adjustment for traditional cardiovascular risk factors or the presence of atherosclerosis itself.
Coronary endothelial vasodilator dysfunction predicts long-term atherosclerotic disease progression and cardiovascular event rates. Thus, the assessment of coronary endothelial vasoreactivity can provide pivotal information as both a diagnostic and prognostic tool in patients at risk for coronary heart disease.
Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.
The study examined possible clinical determinants of platelet resistance to nitric oxide (NO) donors in patients with stable angina pectoris (SAP) and acute coronary syndromes (ACS), relative to nonischemic patients and normal subjects.
We have shown previously that platelets from patients with SAP are resistant to the antiaggregating effects of nitroglycerin (NTG) and sodium nitroprusside (SNP).
Extent of adenosine diphosphate (1 micromol/liter)-induced platelet aggregation (impedance aggregometry in whole blood) and inhibition of aggregation by NTG (100 micromol/liter) and SNP (10 micromol/liter) were compared in normal subjects (n = 43), nonischemic patients (those with chest pain but no fixed coronary disease, (n = 35) and patients with SAP (n = 82) or ACS (n = 153). Association of NO resistance with coronary risk factors, coronary artery disease (CAD), intensity of angina and current medication was examined by univariate and multivariate analyses.
In patients with SAP and ACS as distinct from nonischemic patients and normal subjects, platelet aggregability was increased (both p < 0.01), and inhibition of aggregation by NTG and SNP was decreased (both p < 0.01). Multivariate analysis revealed that NO resistance occurred significantly more frequently with ACS than with SAP (odds ratio [OR] 2.3:1), and was less common among patients treated with perhexiline (OR 0.3:1) or statins (OR 0.45:1). Therapy with other antianginal drugs, extent of CAD, intensity of angina and coronary risk factors were not associated with variability in platelet responsiveness to NO donor.
Patients with symptomatic ischemic heart disease, especially ACS, exhibit increased platelet aggregability and decreased platelet responsiveness to the antiaggregatory effects of NO donors. The extent of NO resistance in platelets is not correlated with coronary risk factors. Pharmacotherapy with perhexiline and/or statins may improve platelet responsiveness to NO.
Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO·; NO gas solution) and nitroxyl ion (NO−; from Angeli's salt).
The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 μM), concentration-dependently inhibited responses to all agents. 10 μM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate.
The NO· scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; 100 μM) and hydroxocobalamin (100 μM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine.
The NO− inhibitor, L-cysteine (3 mM), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution.
The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO· and NO−. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.
British Journal of Pharmacology (2001) 134, 463–472; doi:10.1038/sj.bjp.0704269
To examine whether the prophylactic antianginal agent perhexiline potentiates platelet responsiveness to nitric oxide (NO) in patients with stable angina pectoris (SAP) and acute coronary syndromes (ACS: unstable angina pectoris or non-Q-wave myocardial infarction).
Blood samples were obtained from patients before and after initiation of treatment with perhexiline. ADP-induced platelet aggregation and its inhibition by the NO donor sodium nitroprusside (SNP) were determined via impedance aggregometry in whole blood (WB) and platelet-rich plasma (PRP). Intraplatelet cGMP content was assayed by RIA, and superoxide (O(2)(-)) level by lucigenin-derived chemiluminescence. In patients with ACS not receiving perhexiline (n=12), platelet responsiveness to SNP did not vary significantly over the first 3 days post admission to hospital. Therapy with perhexiline for 3 days was associated with increases in SNP-induced inhibition of aggregation from 29+/-2% to 43+/-4% (n=50,P <0.001) in WB and from 20+/-5% to 42+/-7% (n=12, P<0.01) in PRP. Resolution of symptomatic ischaemia (n=39) was associated with significantly greater (P<0.01) increases than non-resolution (n=11). Similar increases in SNP responsiveness (P<0.001) occurred following institution of perhexiline therapy in patients with SAP (n=30), associated with a 85% decrease in anginal frequency. Treatment with perhexiline potentiated the cGMP-elevating effects of SNP in platelets (n=9,P =0.03). Although perhexiline did not alter whole blood O(2)(-) concentration ex vivo, it inhibited (P<0.01) O(2)(-) release from neutrophils in vitro.
Perhexiline potentiates platelet responsiveness to NO both in SAP and ACS patients; in the latter group this improvement was predictive of resolution of ischaemic symptoms. The predominant mechanism of perhexiline effect is an increase in platelet cGMP responsiveness. Perhexiline also may reduce the potential for NO clearance by neutrophil-derived O(2)(-).
Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl (HNO/NO(-)) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO.
Nitric oxide (NO) plays an important role in the control of vascular tone. Traditionally, its vasorelaxant activity has been attributed to the free radical form of NO (NO*), yet the reduced form of NO (NO-) is also produced endogenously and is a potent vasodilator of large conduit arteries. The effects of NO- in the resistance vasculature remain unknown. This study examines the activity of NO- in rat small isolated mesenteric resistance-like arteries and characterizes its mechanism(s) of action. With the use of standard myographic techniques, the vasorelaxant properties of NO* (NO gas solution), NO- (Angeli's salt), and the NO donor sodium nitroprusside were compared. Relaxation responses to Angeli's salt (pEC50=7.51+/-0.13, Rmax=95.5+/-1.5%) were unchanged in the presence of carboxy-PTIO (NO* scavenger) but those to NO* and sodium nitroprusside were inhibited. l-Cysteine (NO- scavenger) decreased the sensitivity to Angeli's salt (P<0.01) and sodium nitroprusside (P<0.01) but not to NO*. The soluble guanylate cyclase inhibitor ODQ (3 and 10 micromol/L) concentration-dependently inhibited relaxation responses to Angeli's salt (41.0+/-6.0% versus control 93.4+/-1.9% at 10 micromol/L). The voltage-dependent K+ channel inhibitor 4-aminopyridine (1 mmol/L) caused a 9-fold (P<0.01) decrease in sensitivity to Angeli's salt, whereas glibenclamide, iberiotoxin, charybdotoxin, and apamin were without effect. In combination, ODQ and 4-aminopyridine abolished the response to Angeli's salt. In conclusion, NO- functions as a potent vasodilator of resistance arteries, mediating its response independently of NO* and through the activation of soluble guanylate cyclase and voltage-dependent K+ channels. NO- donors may represent a novel class of nitrovasodilator relevant for the treatment of cardiovascular disorders such as angina.
1. Hypercholesterolaemia has been associated with impaired endothelial function. However, there are no available data as to whether hypercholesterolaemia is also associated with platelet dysfunction.
2. In a group of asymptomatic adults with (n = 16) and without (n = 13) mild hypercholesterolaemia, we evaluated inhibition of platelet aggregation in response to the nitric oxide (NO) donor sodium nitroprusside (SNP), as well as the augmentation index (AIx), a parameter of arterial stiffness that is impaired in the presence of endothelial dysfunction.
3. Neither SNP response nor AIx varied significantly between normocholesterolaemic (NC) and hypercholesterolaemic (HC) subjects.
4. Three months treatment with pravastatin (40 mg/day) in HC subjects lowered mean (±SEM) total cholesterol levels from 6.6 ± 0.2 to 5.5 ± 0.2 mmol/L. Platelet response to SNP increased in platelet-rich plasma and tended to increase in whole blood. The AIx did not change significantly. However, falls in low-density lipoprotein (P = 0.03) and total cholesterol (P = 0.08) correlated with reductions in AIx in individual subjects.
5. These data provide evidence that moderate reduction of cholesterol levels may improve platelet responses to NO, whereas improvement in arterial stiffness may be detectable only with more extensive and/or a prolonged reduction in cholesterol levels.
In 15 patients with chronic heart failure of ischemic origin who were not previously treated with angiotensin-converting enzyme inhibitors, platelets exhibited hyperaggregability and impaired responsiveness to the antiaggregatory and cyclic guanosine monophosphate-stimulatory effects of nitric oxide donor sodium nitroprusside compared with normal subjects; this was paralleled by increased blood levels of superoxide radicals. Treatment with perindopril for 4 days significantly improved platelet responses to sodium nitroprusside; there was also a trend toward a decrease in superoxide radical levels.
The options available for distinguishing the effects of cGMP mediated by cGK versus those mediated by other cGMP targets are discussed and evaluated. These include the unnecessary but often sole reliance on synthetic, small-molecule activators and inhibitors of cGK which are increasingly recognized as deficient in specificity. Other important adjunct options include cGK overexpression using adenoviral vectors and transgenic animals, or use of cGK-deficient systems, i.e. cells which have spontaneously lost cGK during repetitive passaging in cell culture, cells treated with siRNA, or genetically-engineered cGK-deficient mice. Finally, cGK-dependent phosphorylation of substrates such as vasodilator stimulated phosphoprotein (VASP) and phosphodiesterase 5 (PDE 5) is described as a useful monitor of cGK presence and activity associated with physiological functions or dysfunctions of signaling pathways.
Nitric oxide (NO) is critically important in the regulation of vascular tone and the inhibition of platelet aggregation. We have shown previously that patients with acute coronary syndromes (ACS) or stable angina pectoris have impaired platelet responses to NO donors when compared with normal subjects. We tested the hypotheses that platelet hyporesponsiveness to NO is a predictor of (1) cardiovascular readmission and/or death and (2) all-cause mortality in patients with ACS (unstable angina pectoris or non-Q-wave myocardial infarction).
Patients (n=51) with ACS had evaluation of platelet aggregation within 24 hours of coronary care unit admission using impedance aggregometry. Patients were categorized as having "normal" (> or =32% inhibition of ADP-induced aggregation with the NO donor sodium nitroprusside; 10 micromol/L; n=18) or "impaired" (<32% inhibition of ADP-induced aggregation; n=33) NO responses. We then compared the incidence of cardiovascular readmission and death during a median of 7 years of follow-up in these 2 groups. Using a Cox proportional hazards model adjusting for age, sex, index event, postdischarge medical treatment, revascularization status, left ventricular systolic dysfunction, concurrent disease states, and cardiac risk factors, impaired NO responsiveness was associated with an increased risk of the combination of cardiovascular readmission and/or death (relative risk, 2.7; 95% CI, 1.03 to 7.10; P=0.041) and all-cause mortality (relative risk, 6.3; 95% CI, 1.09 to 36.7; P=0.033).
Impaired platelet NO responsiveness is a novel, independent predictor of increased mortality and cardiovascular morbidity in patients with high-risk ACS.
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.
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.
Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca(2+). A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca(2+) pump and the ryanodine receptor 2, leading to increased Ca(2+) uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca(2+)-release channels and accelerates Ca(2+) reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca(2+) transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca(2+) cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.