Nitric oxide from nitrite reduction by hemoglobin in the plasma and erythrocytes

Abstract

Experimental evidence has shown that nitrite anion plays a key role in one of the proposed mechanisms for hypoxic vasodilation, in which the erythrocyte acts as a NO generator and deoxygenated hemoglobin in pre-capillary arterioles reduces nitrite to NO, which contributes to vascular smooth muscle relaxation. However, because of the complex reactions among nitrite, hemoglobin, and the NO that is formed, the amount of NO delivered by this mechanism under various conditions has not been quantified experimentally. Furthermore, paracrine NO is scavenged by cell-free hemoglobin, as shown by studies of diseases characterized by extensive hemolysis (e.g., sickle cell disease) and the administration of hemoglobin-based oxygen carriers. Taking into consideration the free access of cell-free hemoglobin to the vascular wall and its ability to act as a nitrite reductase, we have now examined the hypothesis that in hypoxia this cell-free hemoglobin could serve as an additional endocrine source of NO. In this study, we constructed a multicellular model to characterize the amount of NO delivered by the reaction of nitrite with both intraerythrocytic and cell-free hemoglobin, while intentionally neglecting all other possible sources of NO in the vasculature. We also examined the roles of hemoglobin molecules in each compartment as nitrite reductases and NO scavengers using the model. Our calculations show that: (1) approximately 0.04pM NO from erythrocytes could reach the smooth muscle if free diffusion were the sole export mechanism; however, this value could rise to approximately 43pM with a membrane-associated mechanism that facilitated NO release from erythrocytes; the results also strongly depend on the erythrocyte membrane permeability to NO; (2) despite the closer proximity of cell-free hemoglobin to the smooth muscle, cell-free hemoglobin reaction with nitrite generates approximately 0.02pM of free NO that can reach the vascular wall, because of a strong self-capture effect. However, it is worth noting that this value is in the same range as erythrocytic hemoglobin-generated NO that is able to diffuse freely out of the cell, despite the tremendous difference in hemoglobin concentration in both cases (microM hemoglobin in plasma vs. mM in erythrocyte); (3) intraerythrocytic hemoglobin encapsulated by a NO-resistant membrane is the major source of NO from nitrite reduction, and cell-free hemoglobin is a significant scavenger of both paracrine and endocrine NO.

Fig. 1

Model schematic. The model consists of five layers: (1) the intraluminal layer containing discrete erythrocytes and plasma; (2) endothelium and the interstitial space; (3) smooth muscle cells; (4) non-perfused tissue containing nerve fibers and parenchymal cells; and (5) tissue perfused by capillaries. NO from nitrite reduction by hemoglobin is the sole source of NO in the model.

Fig. 2

NO concentration profiles when nitrite reduction by intraerythrocytic hemoglobin is the sole source of NO. The contour plots (a, c, and e) represent the cross-sections of an arteriole that contains erythrocytes and its surrounding tissue. The elevation plots (b, d, and f) show the NO concentration from the center of the lumen to the perivascular region along a path. The starting and ending points of the path are represented as 1 and 2, respectively, in the inset. The small circles in the inset represent erythrocytes. NO delivery by nitrite reductions is calculated under three conditions: (1) NO formation by nitrite reduction occurs uniformly inside erythrocytes and the subsequent transport of NO occurs through free diffusion (a and b); (2) formed NO is transported out of the cell membrane through a facilitated mechanism (c and d); (3) the NO bioactivity is preserved as an intermediate species, which does not liberate NO until it reaches the vascular wall (e and f). The erythrocyte membrane permeability was 0.001 μm/s in (e) and (f). All other parameters are listed in Table 1. Hematocrit was 45%. The NO concentration inside each erythrocyte was nearly zero.

Fig. 3

NO concentration profile when nitrite reduction by cell-free hemoglobin is the sole source of NO. The concentration of cell-free hemoglobin was 20 μM. The reduction of nitrite by intracellular hemoglobin was not considered. The erythrocyte distribution and other simulation conditions were the same as in Fig. 2. The contour plot (a) represents the cross-sections of an arteriole that contain erythrocytes and its surrounding tissue. The elevation plot (b) shows the NO concentration from the center of the lumen to the perivascular region along a path. The starting and ending points of the path are presented as 1 and 2, respectively, in the inset. The circles in the inset represent erythrocytes, which are sinks for NO. NO is produced by the reaction between cell-free hemoglobin and extracellular nitrite.

Fig. 4

NO delivered to smooth muscle through nitrite reduction by cell-free hemoglobin, as a function of the (a) cell-free hemoglobin concentration or (b) extracellular nitrite concentration. All other NO production sources, including nitrite reduction by intracellular hemoglobin, were not considered in the simulation. In (a), the concentration of extracellular nitrite was 0.121 μM and that of cell-free hemoglobin was varied from 1 to 100 μM. In (b), the concentration of cell-free hemoglobin was 20 μM and that of nitrite was varied from 0.121 to 30 μM. The erythrocyte distribution and other simulation conditions were the same as shown in Fig. 3.

Fig. 5

The effect of extravasation of cell-free hemoglobin into the interstitial space on the endocrine signaling of NO reduced by nitrite. In addition, the presence of 20 μM cell-free hemoglobin in the lumen was also included in the simulation in the case of extravasation. The concentration of the cell-free hemoglobin in the interstitial space was the same as that in the lumen (20 μM).

Fig. 6

NO concentration profiles when NO production from nitrite reduction by both intracellular and extracellular hemoglobin is considered. (a) NO release from erythrocytes occurs through the facilitated mechanism, as discussed in section Model formulation; (b) NO formation in erythrocytes occurs uniformly inside the cells, and NO transport out of the cells occurs through free diffusion.

Fig. 7

Contribution of nitrite reduction in capillaries to the NO concentration around an arteriole. The homogeneous NO production rate from the capillary-perfused region was 0.11 nM/s. All other simulation conditions were the same as those in Fig. 2a and b. The NO concentration curves, when the nitrite reduction in capillaries was considered or not, nearly overlapped, indicating that NO from this source did not significantly alter the NO concentration around an arteriole.