No abstract

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent two-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Nitrite has now been proposed to play an important physiological role in signaling, blood flow regulation and hypoxic nitric oxide homeostasis. A recent two-day symposium at the US National Institutes of Health highlighted recent advances in the understanding of nitrite biochemistry, physiology and therapeutics.

The plasma level of NOx, i.e., the sum of NO2 ؊ and NO3 ؊ , is frequently used to assess NO bioavailability in vivo. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NO x concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO 2 ؊ and NO3 ؊ concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO 2 ؊ or NO3 ؊ . Endothelial NO synthase (eNOS) stimulation with acetylcholine (1-10 g͞min) dose-dependently augmented venous NO 2 ؊ levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO 2 ؊ . Intraarterial infusion of NO2 ؊ had no effect on FBF. NOS inhibition (N G -monomethyl-L-arginine; 4 -12 mol͞min) dosedependently reduced basal NO 2؊ and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO3 ؊ and total NOx remained unchanged as did systemic arterial NO 2 ؊ and NO3 ؊ levels during all these interventions. FBF and NO release showed a positive association (r ‫؍‬ 0.85; P < 0.001). These results contradict the current paradigm that plasma NO 3 ؊ and͞or total NOx are generally useful markers of endogenous NO production and demonstrate that only NO2 ؊ reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive.endothelium ͉ blood flow ͉ red blood cells ͉ endothelial dysfunction

Maintenance of vascular homeostasis by EPCs may be attenuated with age based on functional deficits rather than depletion of CD34/KDR or CD133/KDR cells.

The steady-state concentration and thus the biological effects of NO are critically determined not only by its rate of formation, but also by its rate of decomposition. Bioreactivity of NO at physiological concentrations may differ substantially from that suggested by in vitro experiments. The charge neutrality and its high diffusion capacity are hallmarks that characterize NO bioactivity. Reactive oxygen derived species are major determinants of NO breakdown. Biotransformation of NO and its related N-oxides occurs via different metabolic routes within the body. S-Nitrosothiols formed upon reaction of NO with redox-activated thiols represent an active storage pool for NO. The major oxidative metabolites represent nitrite and nitrate, the ratio of both is determined by the microenvironmental redox conditions. In humans, circulating nitrite represents an attractive estimate of regional endothelial NO formation, whereas nitrate, with some caution, appears useful in estimating overall nitrogen/NO turnover. Within the near future, more specific biochemical tools for diagnosis of reduced NO bioactivity will become available. Increasing knowledge on the complex metabolism of NO in vivo will lead to the development of new therapeutic strategies to enhance bioactivity of NO via modulation of its metabolism.

Nitric oxide (NO) induces vasodilatatory, antiaggregatory, and antiproliferative effects in vitro. To delineate potential beneficial effects of NO in preventing vascular disease in vivo, we generated transgenic mice overexpressing human erythropoietin. These animals induce polyglobulia known to be associated with a high incidence of vascular disease. Despite hematocrit levels of 80%, adult transgenic mice did not develop hypertension or thromboembolism. Endothelial NO synthase levels, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels were markedly increased. Administration of the NO synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME) led to vasoconstriction of peripheral resistance vessels, hypertension, and death of transgenic mice, whereas wild-type siblings developed hypertension but did not show increased mortality. L-NAMEtreated polyglobulic mice revealed acute left ventricular dilatation and vascular engorgement associated with pulmonary congestion and hemorrhage. In conclusion, we here unequivocally demonstrate that endothelial NO maintains normotension, prevents cardiovascular dysfunction, and critically determines survival in vivo under conditions of increased hematocrit.

The nitrite anion is reduced to nitric oxide (NO • ) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO • signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type ( +/+ ) and knockout ( −/− ) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO • generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO • generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO • by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin +/+ but not in myoglobin −/− hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin +/+ mice, whereas in myoglobin −/− mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation. myoglobin knockout mice