The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation

Nitric oxide (NO) plays a fundamental role in maintaining normal vasomotor tone. Recent data implicate a critical function for hemoglobin and the erythrocyte in regulating the activity of NO in the vascular compartment. Intravascular hemolysis releases hemoglobin from the red blood cell into plasma (cell-free plasma hemoglobin), which is then able to scavenge endothelium-derived NO 600-fold faster than erythrocytic hemoglobin, thereby disrupting NO homeostasis. This may lead to vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression (ET-1), and end-organ injury, thus suggesting a novel mechanism of disease for hereditary and acquired hemolytic conditions such as sickle cell disease and cardiopulmonary bypass. Furthermore, therapy with NO gas inhalation or infusion of sodium nitrite during hemolysis may attenuate this disruption in vasomotor balance by oxidizing plasma cell-free hemoglobin, thereby preventing the consumption of endogenous NO and the associated pathophysiological changes. In addition to providing an NO scavenging role in the physiological regulation of NO-dependent vasodilation, hemoglobin and the erythrocyte may deliver NO as the hemoglobin deoxygenates. While this process has previously been ascribed to S-nitrosated hemoglobin, recent data from our laboratories suggest that deoxygenated hemoglobin reduces nitrite to NO and vasodilates the human circulation along the physiological oxygen gradient. This newly described role of hemoglobin as a nitrite reductase is discussed in the context of blood flow regulation, oxygen sensing, and nitrite-based therapeutics.     

Nitric oxide scavenging by hemoglobin regulates hypoxic pulmonary vasoconstriction

Although the importance of red blood cells in augmenting hypoxic pulmonary vasoconstriction has been recognized for decades, only recently has it become clear that this occurs primarily because of the inactivation of nitric oxide (NO) by hemoglobin. This interaction between red blood cells, NO, and the pulmonary circulation is critical in understanding the effects of anemia and polycythemia on pulmonary blood flow distribution, gas exchange, and global O2 delivery and in understanding the development of hemoglobin-based oxygen carriers. This review will discuss the proposed mechanisms for initiation of hypoxic pulmonary vasoconstriction and regulation of hypoxic pulmonary vasoconstriction by red blood cells with an emphasis on hemoglobin-NO interactions. In addition, the review will discuss how biologic (S-nitrosation) or pharmacologic (cross-linking) modification of hemoglobin may affect pulmonary circulatory-hemoglobin interactions.     

Rate of NO scavenging alters effects of recombinant hemoglobin solutions on pulmonary vasoreactivity.

Many hemoglobin-based oxygen carriers (HBOCs) produce systemic and pulmonary hypertension and may increase microvascular permeability as a consequence of nitric oxide (NO) scavenging. In this study, we examined the effects of two recombinant human hemoglobin solutions, rHb1.1 and rHb2.0 for injection (rHb2.0), with different rates of NO scavenging on vasoconstrictor reactivity and vascular permeability in isolated, saline-perfused rat lungs. We hypothesized that rHb1.1, a first-generation HBOC with an NO scavenging rate similar to that of native human hemoglobin, would exacerbate pulmonary vasoconstriction and permeability and that rHb2.0, a second-generation HBOC with an NO scavenging rate approximately 20- to 30-fold lower than that of rHb1.1, would minimally influence these responses. Consistent with this hypothesis, rHb1.1 enhanced pulmonary vasoconstrictor reactivity to both hypoxia and thromboxane mimetic U-46619 in a dose-dependent fashion. In contrast, rHb2.0 produced little or no change in reactivity to these stimuli. Furthermore, whereas rHb1.1 abrogated pulmonary vasodilation to the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP), dose-dependent responses to SNAP were preserved, albeit attenuated, in lungs treated with rHb2.0. Finally, the capillary filtration coefficient was unaltered by either rHb1.1 or rHb2.0. We conclude that pulmonary hemodynamic responses to rHb2.0 are greatly reduced compared with those observed with rHb1.1, consistent with rHb2.0 having a diminished capacity to scavenge NO. In addition, neither hemoglobin solution measurably altered microvascular permeability in this preparation.     

NO dioxygenase activity in hemoglobins is ubiquitous in vitro, but limited by reduction in vivo

Genomics has produced hundreds of new hemoglobin sequences with examples in nearly every living organism. Structural and biochemical characterizations of many recombinant proteins reveal reactions, like oxygen binding and NO dioxygenation, that appear general to the hemoglobin superfamily regardless of whether they are related to physiological function. Despite considerable attention to "hexacoordinate" hemoglobins, which are found in nearly every plant and animal, no clear physiological role(s) has been assigned to them in any species. One popular and relevant hypothesis for their function is protection against NO. Here we have tested a comprehensive representation of hexacoordinate hemoglobins from plants (rice hemoglobin), animals (neuroglobin and cytoglobin), and bacteria (Synechocystis hemoglobin) for their abilities to scavenge NO compared to myoglobin. Our experiments include in vitro comparisons of NO dioxygenation, ferric NO binding, NO-induced reduction, NO scavenging with an artificial reduction system, and the ability to substitute for a known NO scavenger (flavohemoglobin) in E. coli. We conclude that none of these tests reveal any distinguishing predisposition toward a role in NO scavenging for the hxHbs, but that any hemoglobin could likely serve this role in the presence of a mechanism for heme iron re-reduction. Hence, future research to test the role of Hbs in NO scavenging would benefit more from the identification of cognate reductases than from in vitro analysis of NO and O(2) binding.     

Reversal of hemoglobin-induced vasoconstriction with sustained release of nitric oxide

Erythrocyte free hemoglobin (Hb) induces vasoconstriction due to nitric oxide (NO) scavenging, limiting the NO available for vascular smooth muscle. The central objective of this study was to restore NO bioavailability using long-lived circulating NO-releasing nanoparticles (NO-np) to reverse the vasoconstriction and hypertension induced by polymerized bovine Hb (PBH) NO scavenging. PBH (13 g/dl) was infused in a volume equal to 10% of the animal blood volume. Intravascular NO supplementation was provided with an infusion of NO-np (10 and 20 mg/kg body wt). This study was performed using the hamster window chamber model to concurrently access systemic and microvascular hemodynamics. Infusion of PBH increased blood pressure and induced vasoconstriction. Treatment with 10 and 20 mg/kg NO-np reduced the blood pressure and vasoconstriction induced by PBH. Moreover, the higher dose of NO-np decreased blood pressure and induced vasodilation compared with baseline, respectively. Treatment with NO-np to decrease PBH-induced vasoconstriction increased methemoglobin levels and plasma nitrite and nitrate. In conclusion, NO-np counteracted both systemic hypertension and decreased the vasoconstrictor effects of PBH infusion, improving systemic and microvascular function. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent to replenish NO in situations where NO production is impaired, insufficient, or consumed, thereby preventing vascular complications.     

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.     

Computation of plasma hemoglobin nitric oxide scavenging in hemolytic anemias

Intravascular hemoglobin limits the amount of endothelial-derived nitric oxide (NO) available for vasodilation. Cell-free hemoglobin scavenges NO more efficiently than red blood cell-encapsulated hemoglobin. Hemolysis has recently been suggested to contribute to endothelial dysfunction based on a mechanism of NO scavenging by cell-free hemoglobin. Although experimental evidence for this phenomenon has been presented, support from a theoretical approach has, until now, been missing. Indeed, due to the low amounts of cell-free hemoglobin present in these pathological conditions, the role of cell-free hemoglobin scavenging of NO in disease has been questioned. In this study, we model the effects of cell-free hemoglobin on NO bioavailability, focusing on conditions that closely mimic those under known pathological conditions. We find that as little as 1 microM cell-free intraluminal hemoglobin (heme concentration) can significantly reduce NO bioavailability. In addition, extravasation of hemoglobin out of the lumen has an even greater effect. We also find that low hematocrit associated with anemia increases NO bioavailability but also leads to increased susceptibility to NO scavenging by cell-free hemoglobin. These results support the paradigm that cell-free hemoglobin released into plasma during intravascular hemolysis in human disease contributes to the experimentally observed reduction in NO bioavailability and endothelial dysfunction.     

Model of nitric oxide diffusion in an arteriole: impact of hemoglobin-based blood substitutes

Administration of hemoglobin-based oxygen carriers (HBOCs) frequently results in vasoconstriction that is primarily attributed to the scavenging of endothelium-derived nitric oxide (NO) by cell-free hemoglobin. The ensuing pressor response could be caused by the high NO reactivity of HBOC in the vascular lumen and/or the extravasation of hemoglobin molecules. There is a need for quantitative understanding of the NO interaction with HBOC in the blood vessels. We developed a detailed mathematical model of NO diffusion and reaction in the presence of an HBOC for an arteriolar-size vessel. The HBOC reactivity with NO and degree of extravasation was studied in the range of 2-58 x 10(6) M(-1) x s(-1) and 0-100%, respectively. The model predictions showed that the addition of HBOC reduced the smooth muscle (SM) NO concentration in the activation range (12-28 nM) for soluble guanylate cyclase, a major determinant of SM contraction. The SM NO concentration was significantly reduced when the extravasation of HBOC molecules was considered. The myoglobin present in the parenchymal cells scavenges NO, which reduces the SM NO concentration.     

Nitroso-redox balance in control of coronary vasomotor tone

Reactive oxygen species (ROS) are essential in vascular homeostasis but may contribute to vascular dysfunction when excessively produced. Superoxide anion (O(2)(·-)) can directly affect vascular tone by reacting with K(+) channels and indirectly by reacting with nitric oxide (NO), thereby scavenging NO and causing nitroso-redox imbalance. After myocardial infarction (MI), oxidative stress increases, favoring the imbalance and resulting in coronary vasoconstriction. Consequently, we hypothesized that ROS scavenging results in coronary vasodilation, particularly after MI, and is enhanced after inhibition of NO production. Chronically instrumented swine were studied at rest and during exercise before and after scavenging of ROS with N-(2-mercaptoproprionyl)-glycine (MPG, 20 mg/kg iv) in the presence or absence of prior inhibition of endothelial NO synthase (eNOS) with N(ω)-nitro-L-arginine (L-NNA, 20 mg/kg iv). In normal swine, MPG resulted in coronary vasodilation as evidenced by an increased coronary venous O(2) tension, and trends toward increased coronary venous O(2) saturation and decreased myocardial O(2) extraction. These effects were not altered by prior inhibition of eNOS. In MI swine, MPG showed a significant vasodilator effect, which surprisingly was abolished by prior inhibition of eNOS. Moreover, eNOS dimer/monomer ratio was decreased after MI, reflecting eNOS uncoupling. In conclusion, ROS exert a small coronary vasoconstrictor influence in normal swine, which does not involve scavenging of NO. This vasoconstrictor influence of ROS is slightly enhanced after MI. Since inhibition of eNOS abolished rather than augmented the vasoconstrictor influence of ROS in swine with MI, while eNOS dimer/monomer ratio was decreased, our data imply that uncoupled eNOS may be a significant source of O(2)(·-) after MI.     

The anoxic plant mitochondrion as a nitrite: NO reductase

Under the conditions of oxygen deprivation, accumulating nitrite can be reduced in the mitochondrial electron transport chain forming free radical nitric oxide (NO). By reducing nitrite to NO, plant mitochondria preserve the capacity to oxidize external NADH and NADPH and retain a limited power for ATP synthesis complementing glycolytic ATP production. NO participates in O(2) balance in mitochondria by competitively inhibiting cytochrome c oxidase which can oxidize it to nitrite when oxygen concentration increases. Some of the NO escapes to the cytosol, where the efficient scavenging system involving non-symbiotic hemoglobin oxygenates NO to nitrate and supports continuous anaerobic turnover of nitrogen species.     

Involvement of nitrogen oxide in the cerebral vasoconstriction during respiration with high pressure oxygen

High pressure oxygen evokes a cerebral vasoconstriction and diminishes cerebral blood flow with the aid of mechanisms which are not yet sufficiently studied. We were checking a hypothesis that the hyperbaric oxygen (HBO2) inactivates cerebral nitrogen oxide (NO), interrupts its basal relaxing effect, and evokes a vasoconstriction. In our experiments, HBO2 decreased cerebral blood flow depending on the pressure. Inhibiting the NO-synthase weakened basal vasorelaxation in breathing with atmosphere air and eliminated the vasoconstriction in exposure to the HBO2. Inactivation of O2 prevented the HBO2-induced vasoconstriction. The data obtained reveal that diminishing of cerebral blood flow in HBO is related to the NO inactivation and weakening of its basal vasorelaxing effect. Possible mechanisms of the NO inactivation may involve its reaction with oxygen and superoxide anion which lead to diminishing of the tissue NO concentration and weakening of its vasorelaxing effect.     

Nitric oxide red blood cell membrane permeability at high and low oxygen tension.

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 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). 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.     

Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin

Cell-free hemoglobin, released from the red cell, may play a major role in regulating the bioavailability of nitric oxide. The abundant serum protein haptoglobin, rapidly binds to free hemoglobin forming a stable complex accelerating its clearance. The haptoglobin gene is polymorphic with two classes of alleles denoted 1 and 2. We have previously demonstrated that the haptoglobin 1 protein–hemoglobin complex is cleared twice as fast as the haptoglobin 2 protein–hemoglobin complex. In this report, we explored whether haptoglobin binding to hemoglobin reduces the rate of nitric oxide scavenging using time-resolved absorption spectroscopy. We found that both the haptoglobin 1 and haptoglobin 2 protein complexes react with nitric oxide at the same rate as unbound cell-free hemoglobin. To confirm these results we developed a novel assay where free hemoglobin and hemoglobin bound to haptoglobin competed in the reaction with NO. The relative rate of the NO reaction was then determined by examining the amount of reacted species using analytical ultracentrifugation. Since complexation of hemoglobin with haptoglobin does not reduce NO scavenging, we propose that the haptoglobin genotype may influence nitric oxide bioavailability by determining the clearance rate of the haptoglobin–hemoglobin complex. We provide computer simulations showing that a twofold difference in the rate of uptake of the haptoglobin–hemoglobin complex by macrophages significantly affects nitric oxide bioavailability thereby providing a plausible explanation for why there is more vasospasm after subarachnoid hemorrhage in individuals and transgenic mice homozygous for the Hp 2 allele.     

The influence of radial RBC distribution, blood velocity profiles, and glycocalyx on coupled NO/O2 transport.

The purpose of this investigation was to study the effect of the presence of red blood cells (RBCs) in the plasma layer near the arteriole wall on nitric oxide (NO) and oxygen (O2) transport. To this end, we extended a coupled NO and O2 diffusion-reaction model in the arteriole, developed by our group, to include the effect of the presence of RBCs in the plasma layer and the effect of convection. Two blood flow velocity profiles (plug and parabolic) were tested. The average hematocrit in the bloodstream was assumed to be constant in the central core and decreasing to zero in the boundary layer next to the endothelial surface layer. The effect of the presence or absence of RBCs near the endothelium was studied while varying the endothelial surface layer and boundary layer thickness. With RBCs present in the boundary layer, the model predicts that 1) NO decreases significantly in the endothelium and vascular wall; 2) there is a very small increase in endothelial and vascular wall Po2; 3) scavenging of NO by hemoglobin decreases with increasing thickness of the boundary layer; 4) the shape of the velocity profile influences both NO and Po2 gradients in the bloodstream; and 5) the presence of RBCs in the boundary layer near the endothelium has a much larger effect on NO than on O2 transport.     

Effects of the RBC membrane and increased perfusate viscosity on hypoxic pulmonary vasoconstriction.

Red blood cells (RBCs) augment hypoxic pulmonary vasoconstriction (HPV) in part by scavenging of nitric oxide (NO) by Hb (Deem S, Swenson ER, Alberts MK, Hedges RG, and Bishop MJ, Am J Respir Crit Care Med 157: 1181-1186, 1998). We studied the contribution of the RBC compartmentalization of Hb to augmentation of HPV and scavenging of NO in isolated perfused rabbit lungs. Lungs were initially perfused with buffer; HPV was provoked by a 5-min challenge with hypoxic gas (inspired O(2) fraction 0.05). Expired NO was measured continuously. Addition of free Hb to the perfusate (0.25 mg/ml) resulted in augmentation of HPV and a fall in expired NO that were similar in magnitude to those associated with a hematocrit of 30% (intracellular Hb of 100 mg/ml). Addition of dextran resulted in a blunting of HPV after free Hb but no change in expired NO. Blunting of HPV by dextran was not prevented by NO synthase inhibition with N(omega)-nitro-L-arginine and/or cyclooxygenase inhibition. RBC ghosts had a mild inhibitory effect on HPV but caused a small reduction in expired NO. In conclusion, the RBC membrane provides a barrier to NO scavenging and augmentation of HPV by Hb. Increased perfusate viscosity inhibits HPV by an undetermined mechanism.     

Role of 20-HETE in the pial arteriolar constrictor response to decreased hematocrit after exchange transfusion of cell-free polymeric hemoglobin.

The cerebrovascular response to decreases in hematocrit and viscosity depends on accompanying changes in arterial O2 content. This study examines whether 1) the arteriolar dilation seen after exchange transfusion with a 5% albumin solution can be reduced by the K(ATP) channel antagonist glibenclamide (known to inhibit hypoxic dilation), and 2) the arteriolar constriction seen after exchange transfusion with a cell-free hemoglobin polymer to improve O2-carrying capacity can be blocked by inhibitors of the synthesis or vasoconstrictor actions of 20-HETE. In anesthetized rats, decreasing hematocrit by one-third with albumin exchange transfusion dilated pial arterioles (14 +/- 2%; SD), whereas superfusion of the surface of the brain with 10 muM glibenclamide blocked this response (-10 +/- 7%). Exchange transfusion with polymeric hemoglobin decreased the diameter of pial arterioles by 20 +/- 3% without altering arterial pressure. This constrictor response was attenuated by superfusing the surface of the brain with a 20-HETE antagonist, WIT-002 (10 microM; -5 +/- 1%), and was blocked by two chemically dissimilar selective inhibitors of the synthesis of 20-HETE, DDMS (50 microM; 0 +/- 4%) and HET-0016 (1 microM; +6 +/- 4%). The constrictor response to hemoglobin transfusion was not blocked by an inhibitor of nitric oxide (NO) synthase, and the inhibition of the constrictor response by DDMS was not altered by coadministration of the NO synthase inhibitor. We conclude 1) that activation of K(ATP) channels contributes to pial arteriolar dilation during anemia, whereas 2) constriction to polymeric hemoglobin transfusion at reduced hematocrit represents a regulatory response that limits increased O2 transport and that is mediated by increased formation of 20-HETE, rather than by NO scavenging.     

Nitric oxide in the human respiratory cycle

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O(2)) by subserving the classical physiological responses of hypoxic vasodilation and hyperoxic vasconstriction in the human respiratory cycle. Here we show that in in vitro and ex vivo systems as well as healthy adults alternately exposed to hypoxia or hyperoxia (to dilate or constrict pulmonary and systemic arteries in vivo), binding of NO to hemes (FeNO) and thiols (SNO) of Hb varies as a function of HbO(2) saturation (FeO(2)). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO(2) over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO(2). Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O(2) uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O(2) and carbon dioxide, may therefore prove useful in the diagnosis and treatment of many human conditions and in the development of new therapies. Our results also help elucidate the link between RBC dyscrasias and cardiovascular morbidity.     

Nitric Oxide in Plants: The Roles of Ascorbate and Hemoglobin

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.     

Modulation of the NO/cGMP pathway reduces the vasoconstriction induced by acellular and PEGylated haemoglobin

Activation of the NO/cGMP pathway modulates smooth muscle cells relaxation and hence vasoconstriction, a major hindrance for the use of cell-free haemoglobin (Hb) as blood substitute, despite conjugation with 5-kDa maleimide poly(ethylene)-glycol (PEG) reduces vasoconstriction in vivo. We aimed at assessing how a recently developed PEGylated-Hb (Deoxy-PEGHb) and manipulation of the NO/cGMP pathway enable modulation of vasoconstriction in isolated rat hearts. Hearts were Langendorff-perfused with oxygenated Krebs-Henseleit (15 ml/min) while monitoring the coronary pressure (CPP) after injection (1 min) of 50 nM norepinephrine followed by a 1 microM Hb or Deoxy-PEGHb bolus, without altering the flow. Deoxy-PEGHb induced less vasoconstriction than Hb. Although the presence of PEG could contribute to vasoconstriction, Deoxy-PEGHb did not contain appreciable amounts of free PEG. Whereas reducing endothelial NO release by 0.2 mM L-NAME increased vasoconstriction, abolishing NO scavenging by Hb using its cyanomet derivative almost completely blunted it. Furthermore, maintaining intracellular cyclic GMP by inhibiting phosphodiesterase-5 with 0.02 mM sildenafil enabled control of Hb-induced vasoconstriction. We conclude that, although PEG-Hb represents a possible approach to limit Hb-induced vasoconstriction, manipulating the NO/cGMP pathway may provide a powerful way to circumvent this problem.