Modulation of nitric oxide bioavailability by erythrocytes

Abstract

Nitric oxide (NO) activates soluble guanylyl cyclase in smooth muscle cells to induce vasodilation in the vasculature. However, as hemoglobin (Hb) is an effective scavenger of NO and is present in high concentrations inside the red blood cell (RBC), the bioavailability of NO would be too low to elicit soluble guanylyl cyclase activation in the presence of blood. Therefore, NO bioactivity must be preserved. Here we present evidence suggesting that the RBC participates in the preservation of NO bioactivity by reducing NO influx. The NO uptake by RBCs was increased and decreased by altering the degree of band 3 binding to the cytoskeleton. Methemoglobin and denatured hemoglobin binding to the RBC membrane or cytoskeleton also were shown to contribute to reducing the NO uptake rate of the RBC. These alterations in NO uptake by the RBC, hence the NO bioavailability, were determined to correlate with the vasodilation of isolated blood vessels. Our observations suggest that RBC membrane and cytoskeleton associated NO-inert proteins provide a barrier for NO diffusion and thus account for the reduction in the NO uptake rate of RBCs. E xtensive studies have established the bioactivity of nitric oxide (NO) in vasoregulation through activation of soluble guanylyl cyclase (ref. 1 and references therein). On the other hand, NO is rapidly deactivated by oxygenated hemoglobin (HbO 2 ) and myoglobin (MbO 2 ) to form nitrate. As NO reacts rapidly (k ϳ 10 7 M Ϫ1 ⅐s Ϫ1) with HbO 2 and deoxygenated Hb (deoxyHb), infusion of cell-free normoxic Hb (hereafter, Hb denotes both HbO 2 and deoxyHb) at M levels into animal models, human subjects, or isolated blood vessels (2-5) causes significant vessel constriction due to NO scavenging. However, normal blood containing RBC-encapsulated Hb (rbcHb) at an equivalent concentration of about 10 mM shows insignificant NO reactivity under physiological conditions (4,6,7). This discrepancy is difficult to explain in view of the high permeability of NO through lipid bilayers. Thus, NO bioactivity in blood must be preserved by a yet unclear mechanism under physiological conditions. This problem recently was addressed by a NO bioactivity export theory (8). According to this theory, NO enters the RBC and preferentially binds with the free heme on Hb to form heme-nitrosylHb (HbNO) rather than being oxidized by O 2 -conjugated heme (9). HbNO then transfers the conjugated NO to ␤-93Cys to form S-nitrosoHb (9). NO bioactivity is then exported out of RBCs through the anion exchange protein, band 3 (or AE1). Although detailed mechanisms of SNO (Snitrosothiol species) formation and NO bioactivity export from band 3 are still unclear (10, 11), the theory provides an explanation for the preservation of NO bioactivity and highlights the importance of the RBC in preserving NO bioavailability, which has been suggested previously (12).In parallel to the NO bioactivity export theory, an independent, but not mutually exclusive, explanation that NO bioavailability is preserved by reducing interactions between NO and r...

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

“…Several mechanisms act to reduce NO scavenging by red blood cells (RBCs) compared to cell-free hemoglobin, thus enabling NO to act as the endothelium-derived relaxation factor [7][8][9][10][11][12][13][14][15][16]. These mechanisms include (1) a RBC free zone created adjacent to the endothelium due to the velocity gradient in laminar flow [8,10,11], (2) an unstirred layer surrounding the RBCs that results in NO uptake being rate-limited by diffusion of NO to the RBC [7,14], and (3) an intrinsic, physical RBC membrane barrier to NO diffusion [12,13,[15][16][17][18]. The relative importance of each of these factors is the subject of much debate [12,14,16,19].…”

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

“…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].…”

“…Three mechanisms contribute to reduced NO scavenging by RBCs [46]. : (1) the rate of the reaction is largely limited by external diffusion of NO through the plasma to the surface of the RBC [44], especially due to the presence of a red cell free zone adjacent to the vessel walls where NO is made [37][38][39]; (2) NO diffusion is partially blocked by a physical barrier across the RBC membrane [40,43,47]; and (3) RBC-encapsulated Hb is efficiently compartmentalized in the lumen; it does not extravasate into the endothelium and interstitium [44,[48][49][50][51][52].…”

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.…”