Nitrite in action: a commentary on "Low-dose intravenous nitrite improves hemodynamics in a canine model of acute pulmonary thromboembolism".
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Acute Disease↗
Biomedical subjects
Tienush Rassaf
Publications and source records attributed to Tienush Rassaf.
Positive effects of nitric oxide on left ventricular function in humans.
AIMS: The myocardial effect of tonically released nitric oxide (NO) in humans is still not known. We tested the hypothesis that low-dose NO exerts positive effects on left ventricular (LV) function. METHODS AND RESULTS: Twelve healthy volunteers, 26+/-4 years, were enrolled in this study. Magnetic resonance imaging was used to precisely measure the direct effects of NO on stroke volume index (SVI). The NO pool was monitored by chemiluminescence. We reduced endogenous NO levels with intravenous infusion of the NO synthase-inhibitor N(G)-monomethyl-l-arginine. Replenishment of the NO pool was achieved with the NO donor S-nitrosoglutathione (GSNO) (0.5 micromol iv). To differentiate load-dependent from the direct effects of NO on LV function, changes in SVI in response to GSNO were compared with changes in the NO-independent vasodilator dihydralazine (2.5 mg iv) at matched arterial pressure and heart rate. Inhibition of NO synthesis was followed by reduction in SVI. Subsequent replenishment of the circulating NO with GSNO significantly increased SVI (39+/-8 to 54+/-7 mL m(-2); P=0.001), whereas no significant changes were observed with the NO-independent vasodilator dihydralazine (39+/-8 to 46+/-8 mL m(-2); P=0.0626). CONCLUSION: Inhibition of endogenous NO release reduces, whereas replenishment with exogenous NO increases LV function, pointing towards a positive effect of tonically released NO on LV function in healthy humans.
Adult↗
Plasma nitrite reserve and endothelial function in the human forearm circulation.
Attenuation of endothelium-derived nitric oxide (NO) synthesis is a hallmark of endothelial dysfunction. Early detection of this disorder may have therapeutic and prognostic implications. Plasma nitrite mirrors acute and chronic changes in endothelial NO-synthase activity. We hypothesized that local plasma nitrite concentration increases during reactive hyperemia of the forearm, reflecting endothelial function. In healthy subjects (n = 11) plasma nitrite and nitrate were determined at baseline and during reactive hyperemia of the forearm using reductive gas-phase chemiluminescence and flow-injection analysis, respectively. Endothelium-dependent dilation of the brachial artery was measured as flow-mediated dilation (FMD) using high-resolution ultrasound. Results were compared to patients with endothelial dysfunction as defined by reduced FMD (n = 11). Reactive hyperemia of the forearm increased local plasma nitrite concentration from 68 +/- 5 to 126 +/- 13 nmol/L (p < 0.01), whereas in endothelial dysfunction nitrite remained unaffected (116 +/- 12 to 104 +/- 10 nmol/L; n.s.), corresponding to nitrite reserves of 94 +/- 21 and -8 +/- 4%. This was accompanied by a significantly greater increase in brachial artery diameter (FMD: 8.5 +/- 0.4% vs 2.9 +/- 0.5%, for healthy subjects and endothelial dysfunction, respectively; p < 0.001). This observation suggests that nitrite changes reflect endothelial function. Assessment of local plasma nitrite during reactive hyperemia may open new avenues in the diagnosis of vascular function.
Adult↗
Circulating NO pool in humans.
Nitric oxide (NO) generated from L-arginine by NO synthases in the endothelium and in other cells plays a central role in several aspects of vascular biology and has been linked to many regulatory functions in mammalian cells. Whereas for a long time the signaling actions of NO in the vasculature have been thought to be short-lived as a result of the rapid reaction of NO with hemoglobin, recent studies changed the biochemical thinking of NO. NO is not anymore the paracrine agent with only local effects, but, like a hormone, it disseminates throughout the body. Thus, a circulating pool of NO exists, opening new considerable pharmacological and therapeutical avenues in the diagnosis and therapy of cardiovascular diseases. In this review we briefly discuss the major routes of NO metabolism and transport in the mammalian circulation, considering plasma, red blood cell and tissue compartments separately, with a special focus on the implication of the circulating NO pool in clinical research.
Amines↗
Plasma nitroso compounds are decreased in patients with endothelial dysfunction.
OBJECTIVES: We investigated whether plasma nitros(yl)ated species (RXNOs) that mediate systemic nitric oxide (NO) bioactivity are depleted in individuals with cardiovascular risk factors and endothelial dysfunction. BACKGROUND: Endothelium-derived NO acts not only as a regional messenger but exerts significant systemic effects via formation of circulating RXNOs delivering NO to sites of impaired production. METHODS: Endothelial function was assessed in 68 patients with one to four major cardiovascular risk factors (RF) and 39 healthy control subjects (C) by measurement of flow-mediated dilation (FMD) of the brachial artery using high-resolution ultrasound. In parallel, plasma RXNOs were determined by reductive gas phase chemiluminescence. RESULTS: Increasing numbers of risk factors were accompanied by a progressive decrease in FMD: 6.5 +/- 0.4% (C); 4.7 +/- 0.5% (one RF); 2.8 +/- 0.4% (two RF); 2.2 +/- 0.4% (three RF); and 1.0 +/- 0.3% (four RF). Progressively impaired vascular function was associated with a concomitant decrease in plasma RXNOs (p < 0.01): 39 +/- 2 nmol/l (C); 30 +/- 2 nmol/l (one RF); 24 +/- 3 nmol/l (two RF); 22 +/- 3 nmol/l (three RF); and 15 +/- 2 nmol/l (four RF), with univariate correlation between FMD and RXNO (r = 0.41, p < 0.001). In a multivariate regression model, RXNO was an independent predictor of endothelial function. CONCLUSIONS: Endothelial dysfunction in patients with cardiovascular risk factors is associated with decreased levels of circulating RXNOs. Plasma RXNOs may be diagnostically useful markers of NO bioavailability and a surrogate index of endothelial function. Whether the observed decrease in concentration reflects impaired NO formation, accelerated decomposition, and/or consumption of RXNOs and whether these processes play a causal role in the pathophysiology of arteriosclerosis remain to be investigated.
Adult↗
Flavanols and cardiovascular health: effects on the circulating NO pool in humans.
Atherosclerosis is the major cause for chronic vascular diseases. The key event in the pathogenesis of atherosclerosis is believed to be dysfunction of the endothelium and disruption of endothelial homeostasis, leading to vasoconstriction, inflammation, leukocyte adhesion, thrombosis, and proliferation of vascular smooth muscle cells. Endothelium-derived nitric oxide (NO) plays a major role in vascular homeostasis and a decrease in NO-bioavailability accelerates the development of atherosclerosis. Given that endothelial dysfunction is at least in part reversible, the characterization of endothelial function and therapeutical approaches have gained much attention over the past years. Recent studies demonstrated that especially the consumption of plant-derived foods rich in certain flavonoids can improve endothelial function in both compromised and healthy humans. Furthermore, various physiologic and biochemical measures have been used previously as biomarkers for the assessment of the proposed beneficial effects of flavonoids in this context. More recently, the analysis of plasma nitros(yl)ated species (RXNOs), referred to as the circulating NO pool, has gained recognition, especially as a marker for endothelial function. This review is aimed at evaluating the suitability of quantifying this NO pool as a biomarker for cardiovascular function in humans, in particular during dietary interventions with flavonoid-rich foods.
Endothelium, Vascular↗
Red blood cells express a functional endothelial nitric oxide synthase.
The synthesis of nitric oxide (NO) in the circulation has been attributed exclusively to the vascular endothelium. Red blood cells (RBCs) have been demonstrated to carry a nonfunctional NO synthase (NOS) and, due to their huge hemoglobin content, have been assumed to metabolize large quantities of NO. More recently, however, RBCs have been identified to reversibly bind, transport, and release NO within the cardiovascular system. We now provide evidence that RBCs from humans express an active and functional endothelial-type NOS (eNOS), which is localized in the plasma membrane and the cytoplasm of RBCs. This NOS is regulated by its substrate L-arginine, by calcium, and by phosphorylation via PI3 kinase. RBC-NOS activity regulates deformability of RBC membrane and inhibits activation of platelets. The NOS-dependent conversion of L-arginine in RBCs is comparable to that of cultured human endothelial cells. RBCs in eNOS-/- mice in contrast to wild-type mice lack NOS protein and activity, strengthening the evidence of an eNOS in RBCs. These data show an eNOS-like protein and activity in RBCs serving regulatory functions in RBCs and platelets, which may stimulate new approaches in the treatment of NO deficiency states inherent to several vascular and hematologic diseases.
Base Sequence↗
Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues.
Mammalian tissues produce nitric oxide (NO) to modify proteins at heme and sulfhydryl sites, thereby regulating vital cell functions. The majority of NO produced is widely assumed to be neutralized into supposedly inert oxidation products including nitrite (NO2(-)). Here we show that nitrite, also ubiquitous in dietary sources, is remarkably efficient at modifying the same protein sites, and that physiological nitrite concentrations account for the basal levels of these modifications in vivo. We further find that nitrite readily affects cyclic GMP production, cytochrome P450 activities, and heat shock protein 70 and heme oxygenase-1 expression in a variety of tissues. These cellular activities of nitrite, combined with its stability and abundance in vivo, suggest that this anion has a distinct and important signaling role in mammalian biology, perhaps by serving as an endocrine messenger and synchronizing agent. Thus, nitrite homeostasis may be of great importance to NO biology.
Animals↗
Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo.
There is mounting evidence that the established paradigm of nitric oxide (NO) biochemistry, from formation through NO synthases, over interaction with soluble guanylyl cyclase, to eventual disposal as nitrite/nitrate, represents only part of a richer chemistry through which NO elicits biological signaling. Additional pathways have been suggested that include interaction of NO-derived metabolites with thiols and metals to form S-nitrosothiols (RSNOs) and metal nitrosyls. Despite the overwhelming attention paid in this regard to RSNOs, little is known about the stability of these species, their significance outside the circulation, and whether other nitros(yl)ation products are of equal importance. We here show that N-nitrosation and heme-nitrosylation are indeed as ubiquitous as S-nitrosation in vivo and that the products of these reactions are constitutively present throughout the organ system. Our study further reveals that all NO-derived products are highly dynamic, have fairly short lifetimes, and are linked to tissue oxygenation and redox state. Experimental evidence further suggests that nitroso formation occurs substantially by means of oxidative nitrosylation rather than NO autoxidation, explaining why S-nitrosation can compete effectively with nitrosylation. Moreover, tissue nitrite can serve as a significant extravascular pool of NO during brief periods of hypoxia, and tissue nitrate/nitrite ratios can serve as indicators of the balance between local oxidative and nitrosative stress. These findings vastly expand our understanding of the fate of NO in vivo and provide a framework for further exploration of the significance of nitrosative events in redox sensing and signaling. The findings also raise the intriguing possibility that N-nitrosation is directly involved in the modulation of protein function.
Animals↗
Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues.
The formation of nitric oxide (NO) has been linked to many regulatory functions in mammalian cells. With the appreciation that NO-mediated nitrosation reactions are involved in cell signaling and pathology there is a need to elucidate and better characterize the different biochemical pathways of NO in vivo. Despite significant methodological advances over the years one major obstacle in assessing the significance of nitrosated species and other NO-related metabolites remains: their reliable measurement in complex biological matrices. In this review we briefly discuss the major routes of NO metabolism and transport in the mammalian circulation, considering plasma, red blood cell, and tissue compartments separately. In addition, we attempt to give a recommendation as to the most appropriate analytical technique and sample processing procedures for the reliable quantification of either species.
Biological Transport↗
Bound NO in human red blood cells: fact or artifact?
There has been considerable debate over the nature and chemistry of the interaction between nitric oxide (NO) and red blood cells (RBCs), in particular whether hemoglobin consumes or conserves NO bioactivity. Given the vast range of nitrosation levels reported for human RBCs in the literature, we sought to investigate whether there was a common denominator that could account for such discrepancies across different methodologies and reaction conditions and if such a pathway may exist in physiology. Here, we show that there are marked differences in reactivity toward NO between human and rat hemoglobin, which offers a mechanistic explanation for why basal levels of NO-adducts in primate RBCs are considerably lower than those in rodents. We further demonstrate that the inadvertent introduction of trace amounts of nitrite and incomplete thiol alkylation lead to rapid heme and thiol nitros(yl)ation, with generation of nitrosylhemoglobin (NOHb) and S-nitrosohemoglobin (SNOHb), while neither species is detectable in human RBCs at physiological nitrite concentrations. Thus, caution should be exercised in interpreting experimental results on SNOHb/NOHb levels that were obtained in the absence of knowledge about the degree of nitrite contamination, in particular when a physiological role for such species is implicated.
Adult↗
Thiols enhance NO formation from nitrate photolysis.
Nitrate is generally considered an inert oxidative breakdown product of nitric oxide (NO). Whereas it has been shown that limited amounts of NO are produced during the photolysis of nitrate in aqueous solution, the photochemistry of nitrate in biological matrices such as plasma is unknown. We hypothesized that thiols, which are ubiquitously present in biological systems, may significantly enhance NO-quantum yields from nitrate photolysis. Exposure of fresh human plasma to high-intensity UV-light resulted in NO-formation (19 +/- 3 nmol/l/min) as measured by gas phase chemiluminescence, and this signal was almost completely abolished by the removal of plasma N-oxides (2 +/- 1 nmol/l/min). Reconstitution of NOx-depleted plasma samples with a physiological concentration of nitrate, but not nitrite, restored photolytic NO-generation to values comparable to naïve plasma. Addition of the thiol-reducing agent, dithiothreitol or the sulfhydryl-bearing amino acid, L-cysteine increased NO-formation above control levels. Thiol-blockade by either N-ethylmaleimide (NEM) or mercuric chloride (HgCl2) reduced basal NO formation from 19 +/- 3 to 7 +/- 2 and 4 +/- 1 nmol/l/min, respectively. Exposure of plasma to UV-light increased NO-adduct concentrations from 18 +/- 5 to 1662 +/- 658 nmol/l. Collectively, our results show that thiols facilitate photolytic conversion of nitrate to NO and NO-adducts such as S-nitrosothiols. This may lead to substantial overestimation of the latter when photolysis-based methodologies are used for their determination. Whether this novel reaction channel also has in vivo relevance remains to be investigated.
Nitrates↗
Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals.
Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.
Animals↗
NO adducts in mammalian red blood cells: too much or too little?
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Animals↗
Chemical nature of nitric oxide storage forms in rat vascular tissue.
Endothelial NO production results in local formation of adducts that may act as storage forms of NO. Because little is known about their chemical nature, concentrations, and possible role in vascular biology, we sought to characterize those species basally present in rat aorta, using two independent approaches. In the first approach, tissue homogenates were analyzed by using chemiluminescence- and ion-chromatography-based techniques that allow trace-level quantification of NO-related compounds in complex biological matrices. In the second approach, NO stores were characterized by their ability to release NO when illuminated with light and subsequently relax vascular smooth muscle (photorelaxation). The latter included a careful assessment of action spectra for photorelaxation, taking into account the light-scattering properties of the tissue and the storage depletion rates induced by exposure to controlled levels of light. Biochemical analyses revealed that aortic tissues contained 10 +/- 1 microM nitrite, 42 +/- 7 microM nitrate, 40 +/- 6 nM S-nitroso, and 33 +/- 6 nM N-nitroso compounds (n = 4-8). The functional data obtained suggest that the NO photolytically released in the tissue originated from species with photophysical properties similar to those reported for low-molecular-weight S-nitrosothiols, as well as from nitrite. The relative contribution of these potential NO stores to the extent of photorelaxation was consistent with their concentrations detected biochemically in vascular tissue when their photoactivity was taken into account. We conclude that intravascular nitroso species and nitrite both have the potential to release physiologically relevant quantities of NO independent of enzymatic control by NO synthase.
Animals↗
Concomitant presence of N-nitroso and S-nitroso proteins in human plasma.
Nitric oxide (NO)-mediated nitrosation reactions are involved in cell signaling and pathology. Recent efforts have focused on elucidating the role of S-nitrosothiols (RSNO) in different biological systems, including human plasma, where they are believed to represent a transport and buffer system that controls intercellular NO exchange. Although RSNOs have been implicated in cardiovascular disease processes, it is yet unclear what their true physiological concentration is, whether a change in plasma concentration is causally related to the underlying pathology or purely epiphenomenological, and to what extent other nitrosyl adducts may be formed under the same conditions. Therefore, using gas phase chemiluminescence and liquid chromatography we sought to quantify the basal plasma levels of NO-related metabolites in 18 healthy volunteers. We find that in addition to the oxidative products of NO metabolism, nitrite (0.20 +/- 0.02 micromol/l nitrite) and nitrate (14.4 +/- 1.7 micromol/l), on average human plasma contains an approximately 5-fold higher concentration of N-nitroso species (32.3 +/- 5.0 nmol/l) than RSNOs (7.2 +/- 1.1 nmol/l). Both N- and S-nitroso moieties appear to be associated with the albumin fraction. This is the first report on the constitutive presence of a high-molecular-weight N-nitroso compound in the human circulation, raising the question as to its origin and potential physiological role. Our findings may not only have important implications for the transport of NO in vivo, but also for cardiovascular disease diagnostics and the risk assessment of nitrosamine-related carcinogenesis in man.
Adult↗
Plasma nitrosothiols contribute to the systemic vasodilator effects of intravenously applied NO: experimental and clinical Study on the fate of NO in human blood.
Higher doses of inhaled NO exert effects beyond the pulmonary circulation. How such extrapulmonary effects can be reconciled with the presumed short half-life of NO in the blood is unclear. Whereas erythrocytes have been suggested to participate in NO transport, the exact role of plasma in NO delivery in humans is not clear. Therefore, we investigated potential routes of NO decomposition and transport in human plasma. NO consumption in plasma was accompanied by a concentration-dependent increase in nitrite and S-nitrosothiols (RSNOs), with no apparent saturation limit up to 200 micro mol/L. The presence of red blood cells reduced the formation of plasma RSNOs. Intravenous infusion of 30 micro mol/min NO in healthy volunteers increased plasma levels of RSNOs and induced systemic hemodynamic effects at the level of both conduit and resistance vessels, as reflected by dilator responses in the brachial artery and forearm microvasculature. Intravenous application of S-nitrosoglutathione, a potential carrier of bioactive NO, mimicked the vascular effects of NO, whereas nitrite and nitrate were inactive. Changes in plasma nitrosothiols were correlated with vasodilator effects after intravenous application of S-nitrosoglutathione and NO. These findings demonstrate that in humans the pharmacological delivery of NO solutions results in the transport and delivery of NO as RSNOs along the vascular tree.
Adult↗
Nitric oxide is consumed, rather than conserved, by reaction with oxyhemoglobin under physiological conditions.
Although irreversible reaction of NO with the oxyheme of hemoglobin (producing nitrate and methemoglobin) is extremely rapid, it has been proposed that, under normoxic conditions, NO binds preferentially to the minority deoxyheme to subsequently form S-nitrosohemoglobin (SNOHb). Thus, the primary reaction would be conservation, rather than consumption, of nitrogen oxide. Data supporting this conclusion were generated by using addition of a small volume of a concentrated aqueous solution of NO to a normoxic hemoglobin solution. Under these conditions, however, extremely rapid reactions can occur before mixing. We have thus compared bolus NO addition to NO generated homogeneously throughout solution by using NO donors, a more physiologically relevant condition. With bolus addition, multiple hemoglobin species are formed (as judged by visible spectroscopy) as well as both nitrite and nitrate. With donor, only nitrate and methemoglobin are formed, stoichiometric with the amount of NO liberated from the donor. Studies with increasing hemoglobin concentrations reveal that the nitrite-forming reaction (which may be NO autoxidation under these conditions) competes with reaction with hemoglobin. SNOHb formation is detectable with either bolus or donor; however, the amounts formed are much smaller than the amount of NO added (less than 1%). We conclude that the reaction of NO with hemoglobin under normoxic conditions results in consumption, rather than conservation, of NO.
Hemoglobins↗