Extracellular diffusion and permeability effects on NO-RBCs interactions using an experimental and theoretical model

Abstract

Nitric oxide (NO) is a potent vasodilator and its homeostasis depends on interaction with RBCs. A key factor in understanding NO-RBC interactions in vascular lumen is a comprehensive analysis of product identification and quantification. In this context, administration of NO during in vitro NO-RBC interactions becomes a crucial variable. In this study, we designed a bioreactor that maintains a precise NO concentration in the headspace that diffuses to RBCs suspension to study the quantitative effect of NO concentration and hematocrit (Hct) on NO-RBC interactions. The products of NO-RBC reaction (nitrite and total nitrogen species (total NOx)) were measured by chemiluminescence assay. A mathematical model simulating NO biotransport to a single RBC was developed to (1) estimate NO-RBC reaction rate constant; (2) predict the NO concentrations in the bulk RBC suspension and at the RBC membrane for RBC membrane NO permeability (P(m)) values of 0.0415-40 cm/s. Measured nitrite and total NOx concentrations increased with increase in headspace NO concentration whereas nitrite concentrations decreased with hematocrit and total NOx concentrations increased with hematocrit. This indicates that the extracellular resistance is a controlling factor for RBC uptake of NO. Modeling results showed that the effective reaction rate constant (k(eff)) for NO-RBC interactions was 2.32 x 10(4)-1.08 x 10(6) M(-1) s(-1). Results also predict that the membrane permeability in the range of 0.0415-0.4 cm/s is required to maintain physiologically relevant levels of NO at the smooth muscle cell layer. The effective reaction rate constant increased with increase in P(m) and magnitude of increase was higher at 45% Hct. For all P(m) values, the k(hb)/k(eff) ratios were lower for 45% Hct as compared to 5% Hct indicating extracellular resistance is important for RBC NO uptake. Our experimental and mathematical analyses of NO-RBC interactions indicate that both unstirred layer and RBC membrane have a significant effect on NO transport to RBCs. In addition, the membrane permeability in the range of 0.0415-0.4 cm/s is required to maintain sufficient NO concentrations at the smooth muscle cell layer.

Figure 1. a) Schematic diagram for bioreactor

Gaseous NO and N₂ mixture is allowed in the bioreactor headspace. NO diffuses from the headspace to the RBCs in suspension and reacts with RBC-Hb. The RBC suspension is kept stirring at low speed by a magnetic stirrer to prevent RBCs from settling. Samples are taken after the reaction through a sample port. b) NO transport to the RBCs (in vivo and in the bioreactor).

Figure 2. Model geometry for simulating NO biotransport to a single RBC

The model geometry consists of two concentric spheres. The outer sphere represents the unstirred layer surrounding the RBC and the inner cylinder represents a single RBC.

Figure 3. Characterization of bioreactor

a) NO–DI water reaction was carried out for 10 min. at equilibrium NO concentration of 1.33 μM. Samples are collected every 2 min and measured for nitrite formation (n = 1); b) NO–PBS reaction was carried out for 30 min and NO equilibrium concentration was kept at 1.33 μM. Samples were collected every 5 min and measured for nitrite formation (n = 3).

Figure 4. Effect of time on NO-RBC interaction

a) Nitrite formation over 10 min for 5% Hct at equilibrium NO concentration of 1.33 μM (n=3); b) Total NOx formation over 10 min for 5% Hct at equilibrium NO concentration of 1.33 μM (n=3).

Figure 5. Effect of NO concentrations on NO-RBC interaction

a) Nitrite formation at the end of 10 min; b) Total NOx formation at the end of 10 min. The hematocrit used was 5% and the equilibrium NO concentrations used were 0.8, 1.33 and 1.9 μM (n=3).

Figure 6. Effect of hematocrit on NO-RBC interaction

a) Nitrite formation at the end of 10 min; b) Total NOx formation at the end of 10 min. The hematocrit used were 5% and 45% and the equilibrium NO concentration was kept at 1.33 μM (n=3).

Figure 7. Estimation of C_NO^m and C_NO^b using mathematical model

a) NO concentrations at the membrane of RBC; and b) NO concentrations in the unstirred layer surrounding the RBC are plotted as a function of NO flux based on total NOx formations at different equilibrium NO concentration. The permeability values used were 0.0415, 0.4, 4.5 and 40 cm/s.

Figure 8. Estimation of C_NO^m and C_NO^b using mathematical model

a) NO concentrations at the membrane of the RBC; and b) NO concentrations in the unstirred layer surrounding the RBC are plotted as a function of NO permeability of the membrane for 5% and 45% Hct.

Figure 9. Effect of permeability

on a) effective reaction rate constant of NO-RBC reaction (k_eff) and b) the ratio of NO-Hb reaction rate constant and NO-RBC reaction rate constant (k_hb/k_eff).