Nitric Oxide Scavenging by Red Blood Cells as a Function of Hematocrit and Oxygenation

Abstract

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 ...

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

“…Preferential reactivity of AS with cell-free Hb compared to that encapsulated in red blood cells was performed based on the original conception of the Liao group [40] and modified as described in detail previously [45]. Briefly, AS (50 μM to 150 μM) was added to a mixture of Hb (final concentration of 30 μM to 100 μM) and RBCs at a hematocrit (Hct) comparable to that found in normal physiology (45%) or comparable to that of patients during sickle cell crisis (18% [75]).…”

“…That NO is made in a compartment adjacent to the blood where there is about 10 mM Hb (heme concentration * ), led to questioning how it can function without being scavenged by the Hb [34]. In normal physiology, the reason that endothelial-derived NO is not scavenged to the extent predicted, based purely on kinetic calculations, is that red blood cell (RBC) encapsulated Hb in the blood reacts with NO much more slowly than does cell-free Hb [35][36][37][38][39][40][41][42][43][44][45]. Three mechanisms contribute to reduced NO scavenging by RBCs [46].…”

Hemoglobin binding to Aβ and HBG2 SNP association suggest a role in Alzheimer's disease

“…adult vs. fetal) of RBCs plays a role in RBC lysis, RBC bound Aβ can cause oxidative injury to Hb within the RBC [42] and Aβ binding to Hb, in particular Hb A as we have demonstrated here, may effect the stabilization of the heme group mediating its release and potential for endothelial injury. Free or oxyHb may not only directly injure the vasculature, including endothelial cells, but may mediate blood flow through NO scavenging, resulting in hypoperfusion to the tissue [4,35,43,59]. Indeed, hypoperfusion may be an early pathogenic event in AD [38,45,93].…”

Interactions of nitrosylhemoglobin and carboxyhemoglobin with erythrocyte

“…Ordinary differential equations (Eqns. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] describe the mass actions of chemical species. For the interaction of HbFe II CO and RBCs (Box 1), reactions in both the phase inside of RBCs and the extracellular solution were considered.…”

“…It is therefore necessary to preserve NO bioactivity in the proximity of a pool of hemoglobin (∼10 mM) in the lumen. Several solutions to this paradox have been proposed, including i) the diffusion and transport limitation arises from the encapsulation of hemoglobin (Hb) in erythrocyte [8,9,10,11,12,13,14,5,15,16,17,18], ii) the formation of S-nitroso-hemoglobin (SNO-Hb) from the intramolecular transfer of nitrosylhemoglobin (HbFe II NO) [19,20,21,22,23,24,5,25], and iii) the NO bioactivity transduced from nitrite [26,27,28,29]. In the latter two cases, the formation of HbFe II NO can be argued to play a role for preserving NO bioactivity.…”