© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0006ED
The Role of Red Blood Cells and Hemoglobin–Nitric Oxide Interactions on Blood Flow
National Institutes of Health
University of Alabama at Birmingham Salazar Vazquez (1) and colleagues have examined the hemodynamic responses to isovolemic infusions of red blood cells that have been oxidized to contain 100% methemoglobin. Because methemoglobin will not directly scavenge nitric oxide (NO) (2), this experimental approach allows for an analysis of the opposing effects of shear stress–mediated endothelial-dependent vasodilation and viscosity effects of red blood cells on blood pressure, while eliminating the NO scavenging effects of ferrous or reduced hemoglobin. The authors find that methemoglobin-containing red blood cells produce a decrease in vascular resistance and mean arterial blood pressure as hematocrit increases by approximately 20% from baseline. The infusion of red cells that cannot scavenge NO reveals an unopposed shear-mediated release of endothelial vasodilators, such as NO. As hematocrit is increased by more than 20% above baseline, the effects of viscocity on increasing blood pressure begin to dominate, resulting in a "U-shaped" effect of hematocrit on blood pressure. By comparing these results with data derived from infusions of oxyhemoglobin-containing red blood cells that scavenge NO, the authors determine that the extent of shear-mediated vasodilation is increased when NO scavenging is minimized. Endothelial NO production by the endothelial isoforms of NO synthases controls approximately 25% of our resting blood flow and is responsible for shear- or flow-mediated vasodilation (3). Because viscosity is a critical determinant of shear stress and the red blood cells have high intrinsic viscosity, the red cell mass provides a major contribution to shear activation of the endothelium. In addition, the red blood cell scavenges NO on one hand, but on the other is proposed to contribute to hypoxic vasodilation via ATP release and a nitrite reductase activity of deoxyhemoglobin (4–7). These complex and often opposing effects of red cells are important to normal physiologic blood flow regulation and will participate in the pathogenesis of human disease.
The concentration of NO available for the activation of soluble guanylate cyclase depends on the rate of production from endothelial NO synthases and the extent of scavenging reactions with intra-erythocytic hemoglobin (8). Nitric oxide reacts with oxy- and deoxy-hemoglobin at the near diffusion limit (107 M–1s–1) to produce methemoglobin and nitrate or iron-nitrosyl-hemoglobin, respectively (Equations 1 and 2) (9).
Since mammals do not possess nitrate reductase enzymes and the off-rate of NO from iron-nitrosyl-hemoglobin is relatively slow, these two reactions represent irreversible scavenging pathways. Indeed, the half-life of NO in a free (i.e., not encapsulated by a red cell membrane) solution of 10 mM oxyhemoglobin (the concentration of hemoglobin in whole blood) is estimated to be 1 microsecond, and this NO can only diffuse 1 micrometer (8). This effect is only slightly diminished by the fact that the smooth muscle cells are on one side of the endothelium and the blood is on the other side. Considering these factors, how can NO be the endothelium-derived relaxing factor if the massive concentrations of intravascular hemoglobin should scavenge it and limit its ability to diffuse from endothelium to smooth muscle? This paradox has been largely solved by the understanding that there are major diffusional barriers for NO between the source of production, endothelial NO synthases, and hemoglobin that is compartmentalized within the red blood cell (recently reviewed in Refs. 4 and 8). These diffusional barriers for NO include the unstirred layer around the erythrocyte, the cell-free zone that forms along the endothelium in laminar flowing blood, and possibly in the red cell submembrane, formed from the components of the inner membrane scaffolding (10–13). These three major diffusional barriers reduce the reaction of NO with intracellular hemoglobin by approximately 1,000-fold and allow sufficient NO diffusion for paracrine signaling from endothelium to smooth muscle. For example, with a cell-free zone of 5 µm, the lifetime of NO would increase from 1 microsecond to about 7.5 milliseconds; thus, the lifetime of NO increases by a factor of almost 10,000 in this situation (8). In addition to these diffusional barriers, the current study illustrates a second important mechanism that limits NO scavenging by intact red blood cells, that is the direct mechanotransduction of shear-forces of flowing red cells into endothelial signals that generate NO from endothelial NO synthase. Within the normal physiologic range of hematocrits, these opposing forces (NO scavenging, shear-mediated NO generation, viscocity, and red cell–derived NO [via ATP and hemoglobin nitrite reductase activity]) are all balanced to maintain a dynamic and adaptive blood flow that synergizes with metabolic oxygen requirements (4–7). Understanding this balance between NO production and scavenging reactions with intracellular hemoglobin helps explain the clear toxicity observed in the clinical development of the stroma-free hemoglobin-based blood substitutes and the vasculopathy of the chronic hemolytic anemias (recently reviewed in Ref. 14). The infusion of cell-free hemoglobin solutions into normal volunteers and patients immediately disrupts the NO diffusion barriers and produces dose-dependent vasconstriction (systemic and pulmonary hypertension), smooth muscle dystonias (gastroparesis, esophageal spasm, and abdominal and chest pain), platelet activation, and death (9, 14). The toxicity of cell-free hemoglobin solutions has resulted in serious morbidity (myocardial infarction) and excess mortality in most clinical trials of blood substitutes in at-risk patients (15). In sickle cell disease, thalassemia, malaria, and other acquired, infectious and hereditary hemolytic conditions, intravascular hemolysis similarly disrupts the NO diffusional barriers created by the red cell membrane that limit NO reactions with hemoglobin, and the cell-free plasma hemoglobin destroys NO at a rate 1,000-fold faster than intra-erythrocytic hemoglobin (2, 16). As a result of hemolysis, hemoglobin is released into plasma, where it reacts with and destroys NO, resulting in abnormally high rates of NO consumption, and produces a state of resistance to NO bioactivity. In support of this NO resistance mechanism, plasma from patients with sickle cell disease contains cell-free ferrous oxyhemoglobin, which stoichiometrically consumes micromolar quantities of NO and abrogates forearm blood flow responses to NO donor infusions (16). Finally, as illustrated by the "U-shaped" relationship between hematocrit and mean arterial blood pressure in the current study by Salzar Vasquez and colleagues, we can now explain the systemic and pulmonary hypertension, and cardiovascular morbidity and mortality, that characterize clinical polycythemia. Erythropoietin therapy, aggressive transfusion, and diseases such as polycythemia vera and Chuvash polycythemia are all associated with increased cardiovascular risk (17–19); these common effects are likely linked to increased red cell mass and the associated increase NO scavenging (20). Even considering the diffusional barriers to NO scavenging described above, these barriers become less effective as the hematocrit increases (21, 22). The current study informs us that NO scavenging by the high intracellular hemoglobin concentrations associated with increasing red cell mass, as well as classically considered increases in intrinsic viscosity, overwhelm homeostatic NO diffusion barriers and shear-mediated NO generation. Future work that carefully dissects the relative contributions of classical viscosity, shear-mediated endothelial NO generation, red cell NO scavenging, and rates of NO generation from the erythrocyte will help us better understand, and potentially therapeutically modulate, the complete blood and vascular hemodynamic system. Footnotes Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. References
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