Mechanisms of Slower Nitric Oxide Uptake by Red Blood Cells and Other Hemoglobin-containing Vesicles

Janet S Lee,Ivan Azarov,Hannah Reynolds,Chen Liu,Daniel B Kim-Shapiro,Zaharo Tsekouras,Mark T Gladwin

doi:10.1074/jbc.m111.228650

Janet S Lee, Ivan Azarov + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m111.228650

Copy DOI

Abstract

Nitric oxide (NO) acts as a smooth muscle relaxation factor and plays a crucial role in maintaining vascular homeostasis. NO is scavenged rapidly by hemoglobin (Hb). However, under normal physiological conditions, the encapsulation of Hb inside red blood cells (RBCs) significantly retards NO scavenging, permitting NO to reach the smooth muscle. The rate-limiting factors (diffusion of NO to the RBC surface, through the RBC membrane or inside of the RBC) responsible for this retardation have been the subject of much debate. Knowing the relative contribution of each of these factors is important for several reasons including optimization of the development of blood substitutes where Hb is contained within phospholipid vesicles. We have thus performed experiments of NO uptake by erythrocytes and microparticles derived from erythrocytes and conducted simulations of these data as well as that of others. We have included extracellular diffusion (that is, diffusion of the NO to the membrane) and membrane permeability, in addition to intracellular diffusion of NO, in our computational models. We find that all these mechanisms may modulate NO uptake by membrane-encapsulated Hb and that extracellular diffusion is the main rate-limiting factor for phospholipid vesicles and erythrocytes. In the case of red cell microparticles, we find a major role for membrane permeability. These results are consistent with prior studies indicating that extracellular diffusion of several gas ligands is also rate-limiting for erythrocytes, with some contribution of a low membrane permeability.

Highlights

The difference in the scavenging rates has been attributed to four possible factors: 1) a red blood cell (RBC)-free zone adjacent to the endothelium due to the velocity gradient in laminar flow (2– 4), 2) Nitric oxide (NO) uptake being rate-limited by diffusion of NO to the red blood cells (RBCs), which contributes to the phenomenon of an unstirred layer around the RBC (1, 6, 14 –16), 3) an intrinsic, physical RBC membrane barrier to NO diffusion (14, 17–20), and 4) NO uptake being rate-limited by diffusion of NO within the RBC (17, 21)
We have simulated the uptake of NO by deoxygenated hemoglobin encapsulated in phospholipid vesicles (as in the recent experiments carried out by another group of researchers (21)) and by oxygenated hemoglobin encapsulated in red blood cell microparticles, and conducted experiments on NO uptake by red blood cells and red cell microparticles
We found that in the experiments of Sakai et al (21) and in our experiments with red cells, extracellular diffusion of NO to the encapsulated Hb is the major rate-limiting factor, whereas, in our experiments with red cell microparticles, membrane permeability of NO appears to be rate-limiting

Summary

EXPERIMENTAL PROCEDURES

Computational Model Development—We have constructed a three-dimensional model to simulate stopped-flow experiments of the anaerobic reaction between phospholipid vesicleencapsulated hemoglobin and NO within the software package COMSOL Multiphysics (Comsol Inc., Burlington, MA, version 3.5), a partial differential equation-based finite element modeling environment. In these experiments deoxygenated hemoglobin within vesicles binds NO to form a ferrous heme-NO complex (FeII-NO). Cex and Cin represent the concentrations of NO on the outside (extracellular region) and inside (intracellular region) of the vesicle, respectively, n is the unit vector normal to the sphere surface pointing in the radial direction and. Pm is the lipid membrane permeability coefficient of NO (Table 1)

Simulation Parameters for NO Uptake by Phospholipid

RESULTS

DISCUSSION