Red blood cells prevent inhibition of hypoxic pulmonary vasoconstriction by nitrite in isolated, perfused rat lungs

Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 muM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of approximately 35 muM) independent of perfusate Po(2). Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.     

Soluble nitric oxide donor and surfactant improve oxygenation and pulmonary hypertension in porcine lung injury.

Acute lung injury (ALI) is associated with diminished surfactant activity and pulmonary hypertension. NONOates are soluble NO donors which release NO in solution. Intratracheal NONOates reduce pulmonary hypertension and improve oxygenation in ALI. We hypothesized that the pharmacologic properties of NO donors would be unaltered after surfactant admixture in vitro and that aerosolized NONOate activity would be enhanced by surfactant pretreatment in vivo. NO donors were added to saline or surfactant and analyzed for nitrite/nitrate production and aortic ring vasodilation. Surfactant did not alter nitrate/nitrite production or aortic ring vasodilation. A porcine model of ALI with pulmonary hypertension was produced using intravenous oleic acid. Animals were assigned to Surfactant-Saline, Surfactant-NONOate, Saline-Saline, or Saline-NONOate groups. Saline or surfactant was instilled into the trachea, followed by gas exchange, pulmonary function, and hemodynamic measurements. NONOate or saline was then aerosolized, and additional data were collected. Oxygenation was improved in the Surfactant-NONOate group, while pulmonary hypertension was selectively reduced in both NONOate groups. Aerosolized NONOate following surfactant pretreatment improves oxygenation and reduces pulmonary hypertension in ALI.     

Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis

Hemoglobin (Hb) potently inactivates the nitric oxide (NO) radical via a dioxygenation reaction forming nitrate (NO(3)(-)). This inactivation produces endothelial dysfunction during hemolytic conditions and may contribute to the vascular complications of Hb-based blood substitutes. Hb also functions as a nitrite (NO(2)(-)) reductase, converting nitrite into NO as it deoxygenates. We hypothesized that during intravascular hemolysis, nitrite infusions would limit the vasoconstrictive properties of plasma Hb. In a canine model of low- and high-intensity hypotonic intravascular hemolysis, we characterized hemodynamic responses to nitrite infusions. Hemolysis increased systemic and pulmonary arterial pressures and systemic vascular resistance. Hemolysis also inhibited NO-dependent pulmonary and systemic vasodilation by the NO donor sodium nitroprusside. Compared with nitroprusside, nitrite demonstrated unique effects by not only inhibiting hemolysis-associated vasoconstriction but also by potentiating vasodilation at plasma Hb concentrations of <25 muM. We also observed an interaction between plasma Hb levels and nitrite to augment nitroprusside-induced vasodilation of the pulmonary and systemic circulation. This nitrite reductase activity of Hb in vivo was recapitulated in vitro using a mitochondrial NO sensor system. Nitrite infusions may promote NO generation from Hb while maintaining oxygen delivery; this effect could be harnessed to treat hemolytic conditions and to detoxify Hb-based blood substitutes.     

Effects of BAY 41-2272, a soluble guanylate cyclase activator, on pulmonary vascular reactivity in the ovine fetus

Nitric oxide (NO)-cGMP signaling plays a critical role during the transition of the pulmonary circulation at birth. BAY 41-2272 is a novel NO-independent direct stimulator of soluble guanylate cyclase that causes vasodilation in systemic and local circulations. However, the hemodynamic effects of BAY 41-2272 have not been studied in the perinatal pulmonary circulation. We hypothesized that BAY 41-2272 causes potent and sustained fetal pulmonary vasodilation. We performed surgery on 14 fetal lambs (125-130 days gestation; term = 147 days) and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic flow transducer was placed on the left pulmonary artery (LPA) to measure blood flow, and a catheter was placed in the LPA for drug infusion. Pulmonary vascular resistance (PVR) was calculated as pulmonary artery pressure minus left atrial pressure divided by LPA blood flow. BAY 41-2272 caused dose-related increases in pulmonary blood flow up to threefold above baseline and reduced PVR by 75% (P < 0.01). Prolonged infusion of BAY 41-2272 caused sustained pulmonary vasodilation throughout the 120-min infusion period. The pulmonary vasodilator effect of BAY 41-2272 was not attenuated by N(omega)-nitro-l-arginine, a NO synthase inhibitor. In addition, compared with sildenafil, a phosphodiesterase 5 inhibitor, the pulmonary vasodilator response to BAY 41-2272 was more prolonged. We conclude that BAY 41-2272 causes potent and sustained fetal pulmonary vasodilation independent of NO release. We speculate that BAY 41-2272 may have therapeutic potential for pulmonary hypertension associated with failure to circulatory adaptation at birth, especially in the setting of impaired NO production.     

Intermediates detected by visible spectroscopy during the reaction of nitrite with deoxyhemoglobin: the effect of nitrite concentration and diphosphoglycerate

The reaction of nitrite with deoxyhemoglobin (deoxyHb) results in the reduction of nitrite to NO, which binds unreacted deoxyHb forming Fe(II)-nitrosylhemoglobin (Hb(II)NO). The tight binding of NO to deoxyHb is, however, inconsistent with reports implicating this reaction with hypoxic vasodilation. This dilemma is resolved by the demonstration that metastable intermediates are formed in the course of the reaction of nitrite with deoxyHb. The level of intermediates is quantitated by the excess deoxyHb consumed over the concentrations of the final products formed. The dominant intermediate has a spectrum that does not correspond to that of Hb(III)NO formed when NO reacts with methemoglobin (MetHb), but is similar to metHb resulting in the spectroscopic determinations of elevated levels of metHb. It is a delocalized species involving the heme iron, the NO, and perhaps the beta-93 thiol. The putative role for red cell reacted nitrite on vasodilation is associated with reactions involving the intermediate. (1) The intermediate is less stable with a 10-fold excess of nitrite and is not detected with a 100-fold excess of nitrite. This observation is attributed to the reaction of nitrite with the intermediate producing N2O3. (2) The release of NO quantitated by the formation of Hb(II)NO is regulated by changes in the distal heme pocket as shown by the 4.5-fold decrease in the rate constant in the presence of 2,3-diphosphoglycerate. The regulated release of NO or N2O3 as well as the formation of the S-nitroso derivative of hemoglobin, which has also been reported to be formed from the intermediates generated during nitrite reduction, should be associated with any hypoxic vasodilation attributed to the RBC.     

Hemoglobin mediated nitrite activation of soluble guanylyl cyclase

Nitrite has long been known to be vasoactive when present at large concentrations but it was thought to be inactive under physiological conditions. Surprisingly, we have recently shown that supraphysiological and near physiological concentrations of nitrite cause vasodilation in the human circulation. These effects appeared to result from reduction of nitrite by deoxygenated hemoglobin. Thus, nitrite was proposed to play a role in hypoxic vasodilation. We now discuss these results in the context of nitrite reacting with hemoglobin and effecting vasodilation and present new data modeling the nitric oxide (NO) export from the red blood cell and measurements of soluble guanylate cyclase (sGC) activation. We conclude that NO generated within the interior of the red blood cell is not likely to be effectively exported directly as nitric oxide. Thus, an intermediate species must be formed by the nitrite/deoxyhemoglobin reaction that escapes the red cell and effects vasodilation.     

Hemoglobin oxygen fractional saturation regulates nitrite-dependent vasodilation of aortic ring bioassays

Nitrite reacts with deoxyhemoglobin to generate nitric oxide (NO). This reaction has been proposed to contribute to nitrite-dependent vasodilation in vivo and potentially regulate physiological hypoxic vasodilation. Paradoxically, while deoxyhemoglobin can generate NO via nitrite reduction, both oxyhemoglobin and deoxyhemoglobin potently scavenge NO. Furthermore, at the very low O(2) tensions required to deoxygenate cell-free hemoglobin solutions in aortic ring bioassays, surprisingly low doses of nitrite can be reduced to NO directly by the blood vessel, independent of the presence of hemoglobin; this makes assessments of the role of hemoglobin in the bioactivation of nitrite difficult to characterize in these systems. Therefore, to study the O(2) dependence and ability of deoxhemoglobin to generate vasodilatory NO from nitrite, we performed full factorial experiments of oxyhemoglobin, deoxyhemoglobin, and nitrite and found a highly significant interaction between hemoglobin deoxygenation and nitrite-dependent vasodilation (P < or = 0.0002). Furthermore, we compared the effect of hemoglobin oxygenation on authentic NO-dependent vasodilation using a NONOate NO donor and found that there was no such interaction, i.e., both oxyhemoglobin and deoxyhemoglobin inhibited NO-mediated vasodilation. Finally, we showed that another NO scavenger, 2-carboxyphenyl-4,4-5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibits nitrite-dependent vasodilation under normoxia and hypoxia, illustrating the uniqueness of the interaction of nitrite with deoxyhemoglobin. While both oxyhemoglobin and deoxyhemoglobin potently inhibit NO, deoxyhemoglobin exhibits unique functional duality as an NO scavenger and nitrite-dependent NO generator, suggesting a model in which intravascular NO homeostasis is regulated by a balance between NO scavenging and NO generation that is dynamically regulated by hemoglobin's O(2) fractional saturation and allosteric nitrite reductase activity.     

Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation

BACKGROUND: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. METHODOLOGY/FINDING: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 μM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. CONCLUSION: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.     

Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling, cytoprotection, and vasodilation

Accumulating evidence suggests that the simple and ubiquitous anion salt, nitrite (NO(2)(-)), is a physiological signaling molecule with potential roles in intravascular endocrine nitric oxide (NO) transport, hypoxic vasodilation, signaling, and cytoprotection after ischemia-reperfusion. Human and animal studies of nitrite treatment and NO gas inhalation provide evidence that nitrite mediates many of the systemic therapeutic effects of NO gas inhalation, including peripheral vasodilation and prevention of ischemia-reperfusion-mediated tissue infarction. With regard to nitrite-dependent hypoxic signaling, biochemical and physiological studies suggest that hemoglobin possesses an allosterically regulated nitrite reductase activity that reduces nitrite to NO along the physiological oxygen gradient, potentially contributing to hypoxic vasodilation. An expanded consideration of nitrite as a hypoxia-dependent intrinsic signaling molecule has opened up a new field of research and therapeutic opportunities for diseases associated with regional hypoxia and vasoconstriction.     

Pulmonary vascular effects of nitric oxide-cGMP augmentation in a model of chronic pulmonary hypertension in fetal and neonatal sheep

Persistent pulmonary hypertension of the newborn (PPHN) is partly due to impaired nitric oxide (NO)-cGMP signaling. BAY 41-2272 is a novel direct activator of soluble guanylate cyclase, but whether this drug may be an effective therapy for PPHN is unknown. We hypothesized that BAY 41-2272 would cause pulmonary vasodilation in a model of severe PPHN. To test this hypothesis, we compared the hemodynamic response of BAY 41-2272 to acetylcholine, an endothelium-dependent vasodilator, and sildenafil, a selective inhibitor of PDE5 in chronically instrumented fetal lambs at 1 and 5 days after partial ligation of the ductus arteriosus. After 9 days, we delivered the animals by cesarean section to measure their hemodynamic responses to inhaled NO (iNO), sildenafil, and BAY 41-2272 alone or combined with iNO. BAY 41-2272 caused marked pulmonary vasodilation, as characterized by a twofold increase in blood flow and a nearly 60% fall in PVR at day 1. Effectiveness of BAY 41-2272-induced pulmonary vasodilation increased during the development of pulmonary hypertension. Despite a similar effect at day 1, the pulmonary vasodilator response to BAY 41-2272 was greater than sildenafil at day 5. At birth, BAY 41-2272 dramatically reduced PVR and augmented the pulmonary vasodilation induced by iNO. We concluded that BAY 41-2272 causes potent pulmonary vasodilation in fetal and neonatal sheep with severe pulmonary hypertension. We speculate that BAY 41-2272 may provide a novel treatment for severe PPHN, especially in newborns with partial response to iNO therapy.     

Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc(1) complex of the mitochondria are known to reduce nitrite anaerobically in vitro, their relative contribution to the hypoxic vasodilatory response has remained unsolved. Using a wire myograph, we have investigated how the nitrite-dependent vasodilation in rat aortic rings is controlled by oxygen tension, norepinephrine concentration, soluble guanylate cyclase (the target for vasoactive NO), and known nitrite reductase activities under hypoxia. Vasodilation followed overall first-order dependency on nitrite concentration and, at low oxygenation and norepinephrine levels, was induced by low-nitrite concentrations, comparable to those found in vivo. The vasoactive effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc(1) complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction of deoxygenated heme), the effect of nitrite was not different from that observed with inositol hexaphosphate alone, indicating that under the conditions investigated here deoxygenated hemoglobin did not enhance nitrite vasoactivity. Together, our results indicate that the mechanism for nitrite vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S-nitrosothiols within the arterial smooth muscle, are discussed.     

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation

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.     

Assessments of the chemistry and vasodilatory activity of nitrite with hemoglobin under physiologically relevant conditions

Hypoxic vasodilation involves detection of the oxygen content of blood by a sensor, which rapidly transduces this signal into vasodilatory bioactivity. Current perspectives on the molecular mechanism of this function hold that hemoglobin (Hb) operates as both oxygen sensor and a condition-responsive NO reactor that regulates the dispensing of bioactivity through release of the NO group from the beta-cys93 S-nitroso derivative of Hb, SNO-Hb. A common path to the formation of SNO-Hb involves oxidative transfer of the NO-group from heme to thiol. We have previously reported that the reaction of nitrite with deoxy-Hb, which furnishes heme-Fe(II)NO, represents one attractive route for the formation of SNO-Hb. Recent literature, however, posits that the nitrite-reductase reaction of Hb might produce physiological vasodilatory effects through NO that evades trapping on heme-Fe(II) and may be stored before release as Fe(III)NO. In this article, we briefly review current perspectives in NO biology on the nitrite-reductase reaction of Hb. We report in vitro spectroscopic (UV/Vis, EPR) studies that are difficult to reconcile with suggestions that this reaction either generates a heme-Fe(III)NO reservoir or significantly liberates NO. We further show in bioassay experiments that combinations of nitrite and deoxy-Hb--under conditions that suppress SNO-Hb formation--exhibit no direct vasodilatory activity. These results help underscore the differences between physiological, RBC-regulated, hypoxic vasodilation versus pharmacological effects of exogenous nitrite.     

The role of nitrite in nitric oxide homeostasis: A comparative perspective

Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O₂ saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O₂ saturations. The hemoglobin nitrite reductase activity depends on heme O₂ affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia-reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.     

Formation of nitric oxide from nitrous acid in ischemic tissue and skin.

Nitric oxide (NO) can form from nitrous acid under conditions of low pH and formation of the gas N2O3 is the rate-determining step. Published data allow us to calculate the rate at which NO forms from nitrite in a closed system such as circulating blood plasma. Because of the bimolecular reactions involved, and the very low concentration of nitrite, the rate of formation of NO is very slow. It might take at least 12 days, when the pH of nitrite solution is lowered, for the concentration of NO to reach a level sufficiently high to activate guanylyl cyclase and so it seems unlikely that naturally circulating nitrite is involved in vasodilation in ischemic tissue through its conversion into NO. It is more realistic to consider that NO is produced at biologically significant concentrations from nitrite in perspiration on the skin.     

Inhaled carbon monoxide does not cause pulmonary vasodilation in the late-gestation fetal lamb

As observed with nitric oxide (NO), carbon monoxide (CO) binds and may activate soluble guanylate cyclase and increase cGMP levels in smooth muscle cells in vitro. Because inhaled NO (I(NO)) causes potent and sustained pulmonary vasodilation, we hypothesized that inhaled CO (I(CO)) may have similar effects on the perinatal lung. To determine whether I(CO) can lower pulmonary vascular resistance (PVR) during the perinatal period, we studied the effects of I(CO) on late-gestation fetal lambs. Catheters were placed in the main pulmonary artery, left pulmonary artery (LPA), aorta, and left atrium to measure pressure. An ultrasonic flow transducer was placed on the LPA to measure blood flow to the left lung. After baseline measurements, fetal lambs were mechanically ventilated with a hypoxic gas mixture (inspired O(2) fraction < 0.10) to maintain a constant fetal arterial PO(2). After 60 min (baseline), the lambs were treated with I(CO) [5-2,500 parts/million (ppm)]. Comparisons were made with I(NO) (5 and 20 ppm) and combined I(NO) (5 ppm) and I(CO) (100 and 2,500 ppm). We found that I(CO) did not alter left lung blood flow or PVR at any of the study doses. In contrast, low-dose I(NO) decreased PVR by 47% (P < 0.005). The combination of I(NO) and I(CO) did not enhance the vasodilator response to I(NO). To determine whether endogenous CO contributes to vascular tone in the fetal lung, zinc protoporphyrin IX, an inhibitor of heme oxygenase, was infused into the LPA in three lambs. Zinc protoporphyrin IX had no effect on baseline PVR, aortic pressure, or the pressure gradient across the ductus arteriosus. We conclude that I(CO) does not cause vasodilation in the near-term ovine transitional circulation, and endogenous CO does not contribute significantly to baseline pulmonary vascular tone or ductus arteriosus tone in the late-gestation ovine fetus.     

Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by NG-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.     

Endothelial nitric oxide synthase reduces nitrite anions to NO under anoxia

In this work, we demonstrate that endothelial nitric oxide synthase is capable of anoxic reduction of nitrite anions to nitric oxide at physiological pH by absorption and EPR spectroscopy and electrochemical measurements. The nitrite reduction is achieved at the oxygenase domain of the protein and proceeds even in the absence of the tetrahydrobiopterin cofactor. The nitrite pathway increases by sixfold the NO production with respect to the regular arginine pathway under hypoxia, which is largely blocked. Therefore, basal levels of NO release could be sustained by anoxic nitrite reduction. The reaction suggests a new pathway for fast NO delivery under hypoxia, precisely when the vasodilating properties of nitric oxide are most needed.     

Erythrocyte storage increases rates of NO and nitrite scavenging: implications for transfusion-related toxicity

Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to RBCs (red blood cells). It has been suggested that these changes have potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion-related toxicities. However, the mechanisms linking RBC storage and toxicity remain unclear. In the present study we tested the hypothesis that storage of leucodepleted RBCs results in cells that inhibit NO (nitric oxide) signalling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from haemolysis or microparticles, our data indicate that the consumption rates of NO increased ~40-fold and NO-dependent vasodilation was inhibited 2-4-fold comparing 42-day-old with 0-day-old RBCs. These results are probably due to the formation of smaller RBCs with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBCs to affect NO formation via deoxygenated RBC-mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBCs did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole-blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBCs stored for >25?days, but not with younger RBCs. Collectively, these data suggest that increased rates of reactions between intact RBCs and NO and nitrite may contribute to mechanisms that lead to storage-lesion-related transfusion risk.