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Nitric oxide (NO) consumption by red blood cell (RBC) hemoglobin (Hb) in vasculature is critical in regulating the vascular tone. The paradox of NO production at endothelium in close proximity of an effective NO scavenger Hb in RBCs is mitigated by lower NO consumption by RBCs compared to that of free Hb due to transport resistances including membrane resistance, extra- and intra-cellular resistances for NO biotransport to the RBC. Relative contribution of each transport resistance on NO-RBC interactions is still not clear. We developed a mathematical model of NO transport to a single RBC to quantify the contributions from individual transport barriers by analyzing the effect of RBC membrane permeability (P(m)), hematocrit (Hct) and NO-Hb reaction rate constants on NO-RBC interactions. Our results indicated that intracellular diffusion of NO was not a rate limiting step for NO-RBC interactions. The extracellular diffusion contributed 70-90% of total transport resistance for P(m)>1 cm s(-1) whereas membrane resistance accounts for 50-75% of total transport resistance for P(m)<0.1 cm s(-1). We propose a narrow P(m) range of 0.21-0.44 cm s(-1) for 10-45% Hct, respectively, below which membrane resistance is more significant and above which extracellular diffusion is a dominating transport resistance for NO-RBC interactions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bioavailability of vasoactive endothelium-derived nitric oxide (NO) in vasculature is a critical factor in regulation of many physiological processes. Consumption of NO by RBC plays a crucial role in maintaining NO bioavailability. Recently, Deonikar and Kavdia (2009b) reported an effective NO-RBC reaction rate constant of 0.2×10(5)M(-1)s(-1) that is ~7 times lower than the commonly used NO-RBC reaction rate constant of 1.4×10(5)M(-1)s(-1). To study the effect of lower NO-RBC reaction rate constant and nitrite and nitrate formation (products of NO metabolism in blood), we developed a 2D mathematical model of NO biotransport in 50 and 200μm ID arterioles to calculate NO concentration in radial and axial directions in the vascular lumen and vascular wall of the arterioles. We also simulated the effect of blood velocity on NO distribution in the arterioles to determine whether NO can be transported to downstream locations in the arteriolar lumen. The results indicate that lowering the NO-RBC reaction rate constant increased the NO concentration in the vascular lumen as well as the vascular wall. Increasing the velocity also led to increase in NO concentration. We predict increased NO concentration gradient along the axial direction with an increase in the velocity. The predicted NO concentration was 281-1163nM in the smooth muscle cell layer for 50μm arteriole over the blood velocity range of 0.5-4cms(-1) for k(NO-RBC) of 0.2×10(5)M(-1)s(-1), which is much higher than the reported values from earlier mathematical modeling studies. The NO concentrations are similar to the experimentally measured vascular wall NO concentration range of 300-1000nM in several different vascular beds. The results are significant from the perspective that the downstream transport of NO is possible under the right circumstances., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In blood vessels, nitric oxide homeostasis is maintained by its formation by endothelial nitric oxide synthase and its consumption in smooth muscle cells and in vascular lumen by red blood cell (RBC) encapsulated hemoglobin (Hb). Free hemoglobin has a very high reaction rate (k(Hb-NO) approximately 10(7) M(-1) s(-1)) with NO as compared to RBC-Hb. Mechanisms of reduced NO uptake by RBC-Hb has been extensively studied in recent years. A critical factor in the investigation of NO-RBC interactions is delivery of NO. Common NO delivery methods include use of NO donors and bolus saturated NO solutions, which delivers NO homogeneously and only in the vicinity of bolus, respectively. In this study, we developed a flow system that uses gaseous delivery of NO through a polymeric semi-permeable membrane to obtain precise and uniform NO concentrations for NO-RBC interactions. We conducted experiments using the flow system to study the effect of NO concentrations, hematocrit and RBC suspension flow rates on NO-RBC interactions. We developed a computational model to simulate NO transport and to estimate the reaction rate constant for NO-RBC interaction in the flow system. Our results showed that NO consumption of RBCs (i) increased linearly with an increase in available NO, and (ii) decreased with increase in RBCs suspension flow rate. We estimated the reaction rate constant for NO-RBC interactions to be 0.2 x 10(5) M(-1) s(-1) which is approximately 1250-fold lower than NO consumption by free hemoglobin and approximately 2.5-20 fold slower than reported NO-RBC reaction rate.

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

A plasma stratum (cell free layer or CFL) generated by flowing blood interposed between the red blood cell (RBC) core and the endothelium affects generation, consumption, and transport of nitric oxide (NO) in the microcirculation. CFL width is a principal factor modulating NO diffusion and vessel wall shears stress development, thus significantly affecting NO bioavailability. Since the CFL is bounded by the surface formed by the chaotically moving RBCs and the stationary but spatially non-uniform endothelial surface, its width fluctuates randomly in time and space. We analyze how these stochastic fluctuations affect NO transport in the CFL and NO bioavailability. We show that effects due to random boundaries do not average to zero and lead to an increase of NO bioavailability. Since endothelial production of NO is significantly enhanced by temporal variability of wall shear stress, we posit that stochastic shear stress stimulation of the endothelium yields the baseline continual production of NO by the endothelium. The proposed stochastic formulation captures the natural continuous and microscopic variability, whose amplitude is measurable and is of the scale of cellular dimensions. It provides a realistic model of NO generation and regulation. [ABSTRACT FROM AUTHOR]

Background: Nitrite acts as a reservoir of nitric oxide (NO) bioactivity in the vasculature. Catalytic reduction of nitrate, diet and in vivo NO production are major sources of nitrite. Nitrite reaction with RBC-encapsulated deoxy-hemoglobin can lead to formation of NO or vasoactive species that can be further exported to cause vasodilation. Quantification of nitrite formation from NO, NO–RBC interactions metabolites and nitrite–RBC interaction metabolites is necessary to understand the nitrite reductase activity of deoxyHb. Methods: We developed an unsteady state model to quantify the formation of NO-RBC and nitrite-RBC reaction metabolites such as methemoglobin (metHb) and nitrosyl hemoglobin (HbNO) under oxygenated and deoxygenated conditions. The model geometry consists of two concentric spheres. The outer sphere represents plasma surrounding the RBC and the inner sphere represents the RBC. We estimated the concentrations of all the NO–RBC and nitrite–RBC reaction metabolites by solving the mass balance equations for each metabolite over a period of 1min. Results and conclusions: Using this model, we studied the effects of hematocrit and fractional oxygen saturation on NO–RBC and nitrite–RBC interactions. Our results showed that metHb, HbNO and nitrite concentrations increased with time. MetHb concentrations increased whereas HbNO and nitrite concentrations decreased with increase in fractional oxygen saturation. For a given oxygen saturation, MetHb, HbNO and nitrite increased with hematocrit. The results obtained from this model will assist in quantifying the amount and the distribution of NO or NO active species resulting from nitrite reductase activity in RBC, which can be exported to smooth muscles for vasodilation. Disclosure: Supported by NIH Grant R01 HL084337. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) bioavailability is implicated in determining fate of many physiological and pathophysiological processes. In vasculature, nitric oxide homeostasis is maintained by its production by endothelial nitric oxide synthase and consumption by red blood cells (RBC) and leads to smooth muscle cells vasorelaxation. The reaction between NO and RBCs is under intense investigation. We studied the quantitative effects of oxygenation and hematocrit on NO -- RBC interactions. For this purpose, we designed a novel stirred-bioreactor with a headspace to maintain controlled NO gas concentration. Gaseous NO was reacted with oxygenated and deoxygenated RBC suspension for a period of 10 min and samples were taken every 2 min. To study the effect of hematocrit on NO -- RBC interactions, three hematocrit suspensions (5%, 20% and 45%) were reacted with NO for 10 min. Nitrite and total nitrogen species (total NOx) contents of all samples were measured by chemiluminescence assay and hemoglobin derivatives were measured using spectrophotometric analysis. Oxygenated NO-RBC interactions resulted in higher nitrite and total NOx concentrations than that of deoxygenated NO-RBC interactions. Nitrite concentrations decreased with hematocrit and total NOx concentrations increased with hematocrit. These results will enhance the present understanding of hematocrit and oxygenation effects on NO-RBC interactions. [ABSTRACT FROM AUTHOR]

Red blood cells (RBCs) modulate nitric oxide (NO) bioavailability in the vasculature. Extracellular free hemoglobin (Hb) in the vascular lumen can cause NO bioavailability related complications seen in pathological conditions such as pancreatitis, sickle cell disease and malaria. In addition, the role of extracellular free Hb has been critical to estimate kinetic and transport properties of NO–RBCs interactions in ‘competition experiments’. We recently reported a strong dependence of NO transport on RBC membrane permeability and hematocrit. NO donors combined with anti-inflammatory drugs are an emergent treatment for diseases like cancer, cardiovascular complications and wound healing. However, the role of RBCs in transport NO from NO donors is not clearly understood. To understand the significance of extracellular free Hb in pathophysiology on NO availability and estimation of the NO-RBC interactions, we developed a computational model to simulate NO biotransport to the RBC in the presence of extracellular free Hb. Using this model, we studied the effect of hematocrit, RBC membrane permeability and NO donors on NO–RBC interactions in the presence and absence of extracellular free Hb. The plasma NO concentration gradients and average plasma NO concentrations changed minimally with increase in extracellular free Hb concentrations at the higher hematocrit as compared to those at the lower hematocrit irrespective of the NO delivery method, indicating that the presence of extracellular free Hb affects the NO transport only at a low hematocrit. We also observed that NO concentrations increased with NO donor concentrations in the absence as well as in the presence of extracellular free Hb. In addition, NO donor supplementation may increase NO availability in the plasma in the event of loss of endothelium-derived NO activity. [ABSTRACT FROM AUTHOR]