Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 micromol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extraerythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases. Nitric oxide (NO)3 is an endothelium-derived relaxation factor that is synthesized in endothelial cells (1-4). To elicit its vasodilatory activity, NO must diffuse to the smooth muscle cells and activate soluble guanylate cyclase. In 1994, Lancaster suggested that the proximity of the endothelium to the millimolar concentrations of hemoglobin (Hb), an avid NO scavenger, would severely compromise the efficiency of the NO/soluble guanylate cyclase pathway (5). However, later studies have indicated that the physical compartmentalization of hemoglobin within the red blood cell (RBC) effectively reduces the apparent rate at which NO is consumed by Hb (6 -15). One contributory element to this effect is a RBC-free zone at the blood/endothelium interface that is present during laminar flow (7, 9, 10). In addition, the rate of NO consumption has been reported to occur up to 1000 times more slowly by red blood cells than by an equivalent concentration of cell-free hemoglobin. Two potential mechanisms for this effect involve either the presence of an unstirred layer surrounding the red blood cell that is formed as a result of NO diffusion (6, 13) or a physical barrier to NO diffusion that is integral to the protein-rich RBC submembrane (11). The faster effective reaction of NO with cell-free Hb compared with RBC-encapsulated hemoglobin may ...

Recent evidence suggests that the reaction between nitrite and deoxygenated hemoglobin provides a mechanism by which nitric oxide is synthesized in vivo. This reaction has been previously defined to follow second order kinetics, although variable product stoichiometry has been reported. In this study we have re-examined this reaction and found that under fully deoxygenated conditions the product stoichiometry is 1:1 (methemoglobin:nitrosylhemoglobin), and unexpectedly, the kinetics deviate substantially from a simple second order reaction and exhibit a sigmoidal profile. The kinetics of this reaction are consistent with an increase in reaction rate elicited by heme oxidation and iron-nitrosylation. In addition, conditions that favor the "R" conformation show an increased rate over conditions that favor the "T" conformation. The reactivity of nitrite with heme is clearly more complex than has been previously realized and is dependent upon the conformational state of the hemoglobin tetramer, suggesting that the nitrite reductase activity of hemoglobin is under allosteric control.The reaction between nitrite and deoxyhemoglobin (deoxyHb 1 NO] 2ϩ ) (1) through the intermediacy of nitric oxide (NO) (2). This reaction has recently been invoked to explain nitrite-mediated effects on blood flow at rest and during exercise. At supra-and near-physiologic concentrations, nitrite produces increased blood flow in a dose-dependent fashion, regardless of NO synthase inhibition or metabolic demand (3). Furthermore, HbNO production, formed from nitrite infusion, was shown to be inversely proportional to hemoglobin oxygen saturation (3). An important characteristic of nitrite is that HbNO formation only occurs with deoxyHb and not oxygenated hemoglobin (oxyHb or Hb[Fe II O 2 ] 2ϩ ), suggesting an oxygen-dependence to the NOforming reaction. The reaction of nitrite with oxyHb occurs via a complex autocatalytic mechanism that has not yet been fully elucidated (4). The potential nitrite-reductase activity of deoxyHb has previously been invoked as a potential source of bioactive NO (5). The reaction between nitrite and deoxyHb was extensively examined by Doyle et al. (2). These investigators concluded that the reaction was second order and that the major products of the reaction were metHb and HbNO. Their observations were largely consistent with the reactions shown in Equations 1-4. As reaction 3 is extremely fast (k 3 ϳ 4 ϫ 10 7 ), the kinetics of the reaction are dictated by the second order reaction shown in Equation 2, which was calculated to have a rate constant of 1. In this study we have re-examined this reaction under conditions of molar excess Hb to nitrite and vice versa. In contrast to the earlier study (2), we concluded that although the initial rate of the reaction has a first order dependence on both nitrite and hemoglobin concentrations, the kinetics profile does not fit a second order/reaction but exhibits autocatalytic kinetics. In addition we found that under strictly anaerobic conditions the stoichiometry o...

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin.Here we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4,400 μm/s and 5,100 μm/s while the permeability under deoxygenated conditions is greater than 64,000 μm/s. The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.

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