Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release

Previously, we reportedthat red blood cells (RBCs) of rabbits and humans release ATP inresponse to mechanical deformation and that this release of ATPrequires the activity of the cystic fibrosis transmembrane conductanceregulator (CFTR). It was reported that cAMP, acting through acAMP-dependent protein kinase, PKA, is an activator of CFTR. Here weinvestigate the hypothesis that cAMP stimulates ATP release from RBCs.Incubation of human and rabbit RBCs with the direct activator ofadenylyl cyclase, forskolin (10 or 100 µM), with IBMX (100 µM),resulted in ATP release and increases in intracellular cAMP. Inaddition, epinephrine (1 µM), a receptor-mediated activator ofadenylyl cyclase, stimulated ATP release from rabbit RBCs. Moreover,incubation of human and rabbit RBCs with an active cAMP analog[adenosine 3'5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP, 100 µM)] resulted in ATP release. In contrast, forskolin and Sp-cAMPwere without effect on dog RBCs, cells known not to release ATP inresponse to deformation. When rabbit RBCs were incubated with theinactive cAMP analog and inhibitor of PKA activity, adenosine3',5'-cyclic monophosphorothioate Rp-isomer (100 µM),deformation-induced ATP release was attenuated. These results areconsistent with the hypothesis that adenylyl cyclase and cAMP arecomponents of a signal-transduction pathway relating RBC deformation toATP release from human and rabbit RBCs.

     

Dependence of red blood cell dynamics in microvessel bifurcations on the endothelial surface layer’s resistance to flow and compression

Red blood cells (RBCs) make up 40–45% of blood and play an important role in oxygen transport. That transport depends on the RBC distribution throughout the body, which is highly heterogeneous. That distribution, in turn, depends on how RBCs are distributed or partitioned at diverging vessel bifurcations where blood flows from one vessel into two. Several studies have used mathematical modeling to consider RBC partitioning at such bifurcations in order to produce useful insights. These studies, however, assume that the vessel wall is a flat impenetrable homogeneous surface. While this is a good first approximation, especially for larger vessels, the vessel wall is typically coated by a flexible, porous endothelial glycocalyx or endothelial surface layer (ESL) that is on the order of 0.5–1 µm thick. To better understand the possible effects of this layer on RBC partitioning, a diverging capillary bifurcation is analyzed using a flexible, two-dimensional model. In addition, the model is also used to investigate RBC deformation and RBC penetration of the ESL region when ESL properties are varied. The RBC is represented using interconnected viscoelastic elements. Stokes flow equations (viscous flow) model the surrounding fluid. The flow in the ESL is modeled using the Brinkman approximation for porous media with a corresponding hydraulic resistivity. The ESL’s resistance to compression is modeled using an osmotic pressure difference. One cell passes through the bifurcation at a time, so there are no cell–cell interactions. A range of physiologically relevant hydraulic resistivities and osmotic pressure differences are explored. Decreasing hydraulic resistivity and/or decreasing osmotic pressure differences (ESL resistance to compression) produced four behaviors: (1) RBC partitioning nonuniformity increased slightly; (2) RBC deformation decreased; (3) RBC velocity decreased relative to blood flow velocity; and (4) RBCs penetrated more deeply into the ESL. Decreasing the ESL’s resistance to flow and/or compression to pathological levels could lead to more frequent cell adhesion and clotting as well as impaired vascular regulation due to weaker ATP and nitric oxide release. Potential mechanisms that can contribute to these behaviors are also discussed.

     

Red blood cell shape transitions and dynamics in time-dependent capillary flows

The dynamics of single red blood cells (RBCs) determine microvascular blood flow by adapting their shape to the flow conditions in the narrow vessels. In this study, we explore the dynamics and shape transitions of RBCs on the cellular scale under confined and unsteady flow conditions using a combination of microfluidic experiments and numerical simulations. Tracking RBCs in a comoving frame in time-dependent flows reveals that the mean transition time from the symmetric croissant to the off-centered, nonsymmetric slipper shape is significantly faster than the opposite shape transition, which exhibits pronounced cell rotations. Complementary simulations indicate that these dynamics depend on the orientation of the RBC membrane in the channel during the time-dependent flow. Moreover, we show how the tank-treading movement of slipper-shaped RBCs in combination with the narrow channel leads to oscillations of the cell's center of mass. The frequency of these oscillations depends on the cell velocity, the viscosity of the surrounding fluid, and the cytosol viscosity. These results provide a potential framework to identify and study pathological changes in RBC properties.     

Conductometric study of shear-dependent processes in red cell suspensions. II. Transient cross-stream hematocrit distribution

A novel experimental approach based on electrical properties of red blood cell (RBC) suspensions was applied to study the effects of the size and morphology of RBC aggregates on the transient cross-stream hematocrit distribution in suspensions flowing through a square cross-section flow channel. The information about the effective size of RBC aggregates and their morphology is extracted from the capacitance (C) and conductance (G) recorded during RBC aggregation, whereas a slower process of particle migration is manifested by delayed long-term changes in the conductance. Migration-induced changes in the conductance measured at low shear rates (< or =3.1 s(-1)) for suspensions of RBCs in a strongly aggregating medium reveal an increase to a maximum followed by a decrease to the stationary level. The ascending branch of G(t) curves reflects the aggregate migration in the direction of decreasing shear rate. A further RBC aggregation in the region of lower shear stresses leads to the formation of RBC networks and results in the transformation of the rheological behavior of suspensions from the thinning to the thickening. It is suggested that the descending branches of the G(t) curves recorded at low shear rates reflect an adjustment of the Hct distribution to a new state caused by a partial dispersion of RBC networks. For suspensions of non-aggregating RBCs it is found that depending on whether the shear rate is higher or lower compared with the prior value, individual RBCs migrate either toward the centerline of the flow or in the opposite direction.     

Impaired release of ATP from red blood cells of humans with primary pulmonary hypertension

Previously, we reported that in the isolated perfused rabbit lung, red blood cells (RBCs) obtained from either rabbits or healthy humans were a required component of the perfusate to unmask evidence of nitric oxide (NO) participation in regulation of the pulmonary circulation. In addition, we found that mechanical deformation of rabbit and healthy human RBCs released ATP, a known agonist for enhanced NO synthesis. In contrast, RBCs obtained from patients with cystic fibrosis (CF) did not release ATP in response to mechanical deformation. The coexistence of airway disease and alveolar hypoxia in patients with CF precluded the drawing of conclusions relating a defect in RBC ATP release with the pulmonary hypertension associated with CF. Airway disease and alveolar hypoxia are not, however, features of primary pulmonary hypertension (PPH), a human condition of unknown etiology. We postulated that a defect in NO generation might contribute to the increased pulmonary vascular resistance in PPH, and as a first step, we hypothesized that RBCs obtained from patients with PPH would not release ATP. In contrast to RBCs of healthy humans, when RBCs of PPH patients were passed through filters (average pore size 12, 8, or 5 microm), ATP was not released and the RBCs exhibited reduced deformability. Moreover, when incubated with the active cAMP analogue, Sp-cAMP (100 microM), an activator of the CF transmembrane conductance regulator, ATP was not released. These results demonstrate that RBCs obtained from patients with PPH fail to release ATP whether the stimulus is mechanical or pharmacological. Thus, failure of RBCs to release ATP in patients with PPH might be a major pathogenetic factor that accounts for the heretofore unknown etiology of their pulmonary hypertension.     

Mechanism of CD47-induced alpha4beta1 integrin activation and adhesion in sickle reticulocytes

We recently reported that CD47 (integrin-associated protein) on sickle red blood cells (SS RBCs) activates G-protein-dependent signaling, which promotes cell adhesion to immobilized thrombospondin (TSP) under relevant shear stress. These data suggested that signal transduction in SS RBCs may contribute to the vaso-occlusive pathology observed in sickle cell disease. However, the CD47-activated SS RBC adhesion receptor(s) that mediated adhesion to immobilized TSP remained unknown. Here we demonstrate that the alpha4beta1 integrin (VLA-4) is the receptor that mediates CD47-stimulated SS RBC adhesion to immobilized TSP. This adhesion requires both the N-terminal heparin-binding domain and the RGD site of TSP. CD47 signaling induces an "inside-out" activation of alpha4beta1 on SS RBCs as indicated by an RGD-dependent interaction of this integrin with soluble, plasma fibronectin. However, CD47 engagement also induces an alpha4beta1-mediated, RGD-independent adhesion of SS RBCs to immobilized vascular cell adhesion molecule-1 (VCAM-1). CD47 signaling in SS RBCs appears to be independent of large scale changes in cAMP formation but nonetheless promotes alpha4beta1-mediated adhesion via a protein kinase A-dependent, serine phosphorylation of the alpha4 cytoplasmic domain. CD47-activated SS RBC adhesion absolutely requires the Src family tyrosine kinases and is also enhanced by treatment of SS RBCs with low concentrations of cytochalasin D, which may release alpha4beta1 from cytoskeletal restraints. In addition, CD47 co-immunoprecipitates with alpha4beta1 in a sickle reticulocyte-enriched fraction of SS RBCs. These studies therefore identify the alpha4beta1 integrin on SS RBCs as a CD47-activated receptor for TSP, VCAM-1, and plasma fibronectin, revealing novel binding characteristics of this integrin.     

Visualization study of motion and deformation of red blood cells in a microchannel with straight,divergent and convergent sections

The size of red blood cells (RBC) is on the same order as the diameter of microvascular vessels. Therefore, blood should be regarded as a two-phase flow system of RBCs suspended in plasma rather than a continuous medium of microcirculation. It is of great physiological and pathological significance to investigate the effects of deformation and aggregation of RBCs on microcirculation. In this study, a visualization experiment was conducted to study the microcirculatory behavior of RBCs in suspension. Motion and deformation of RBCs in a microfluidic chip with straight, divergent, and convergent microchannel sections have been captured by microscope and high-speed camera. Meanwhile, deformation and movement of RBCs were investigated under different viscosity, hematocrit, and flow rate in this system. For low velocity and viscosity, RBCs behaved in their normal biconcave disc shape and their motion was found as a flipping motion: they not only deformed their shapes along the flow direction, but also rolled and rotated themselves. RBCs were also found to aggregate, forming rouleaux at very low flow rate and viscosity. However, for high velocity and viscosity, RBCs deformed obviously under the shear stress. They elongated along the flow direction and performed a tank-treading motion.     

Ticagrelor induces adenosine triphosphate release from human red blood cells

RationaleThe novel P2Y₁₂ antagonist ticagrelor inhibits ADP-induced platelet aggregation more rapidly and more potently than clopidogrel. Clinical trials have revealed dyspnea and asymptomatic ventricular pauses as side effects of ticagrelor. The mechanism behind these side effects is not known, but it is plausible that they are mediated by adenosine.ObjectiveTicagrelor is known to increase adenosine concentrations by inhibiting red blood cell reuptake, but the potency of this effect may be too low to fully explain the adenosine related effects. The purpose of the present study was to determine whether ticagrelor has other effects on red blood cells (RBCs) that could contribute to explain the pleiotropic effects seen with ticagrelor treatment.Methods and resultsUsing a luciferase-based bioluminescence assay, we studied ATP release in human blood. Human RBCs responded to ticagrelor in vitro by releasing substantial amounts of ATP in a dose-dependent manner (IC₅₀ 14 μM). The rapid effect indicates release through membrane channels, which was supported by a depolarizing effect of ticagrelor and inhibition of ATP release by anion channel blockers.ConclusionIn conclusion, our data show that, in vitro, ticagrelor can induce ATP release from human RBCs, which is subsequently degraded to adenosine. Further studies are warranted to determine what role this mechanism may play in the clinical effects of ticagrelor.     

Effect of hemodilution on RBC velocity, supply rate, and hematocrit in the cerebral capillary network.

The effect of isovolemic hemodilution on the circulation of red blood cells (RBCs) in the cerebrocortical capillary network was studied by intravital videomicroscopy with use of a closed-cranial-window technique in the rat. Velocity and supply rate of RBCs were measured by tracking the movement and counting the number of fluorescently labeled cells. Arterial blood was withdrawn in increments of 2 ml and replaced by serum albumin. Arterial blood pressure was maintained constant with an infusion of methoxamine. Both velocity and supply rate of RBCs increased, by approximately equal amounts, as arterial hematocrit was reduced from 44 to 15%. The maximum increase in RBC velocity was 4.6 and in RBC supply rate was 5.2 times the baseline value. Calculated lineal density of RBC, an index of capillary hematocrit, did not change with hemodilution. The results suggest that RBC flow and oxygen supply in the cerebral capillary network are maintained during isovolemic hemodilution. The "optimal hematocrit" is as low as 15%.     

Rheological study on coagulation of blood with special reference to the triggering mechanism of venous thrombus formation

A markedly reduced blood flow, an elevation of hematocrit and an increased aggregability of erythrocytes [red blood cells (RBCs)] are risk factors for venous thrombus formation (intravascular blood coagulation). However, these risk factors alone seem to be insufficient to stimulate the coagulation cascade in the absence of a primary triggering mechanism. In this paper, our rheological and biochemical studies on blood coagulation, especially focusing on procoagulant activity of RBCs, are summarized. It is shown that the intrinsic coagulation pathway is triggered by the activation of factor IX (F-IX) by RBCs. The F-IX-activating enzyme in normal human erythrocyte (RBC) membranes was purified, identified and characterized. The activation of F-IX by RBCs was enhanced by a decrease in flow shear rate and an elevation in hematocrit. The procoagulant ability of RBCs and coagulation of blood obtained from individuals with a relatively high level of hypercoagulability were enhanced compared with those for normals. The studies demonstrated a new triggering mechanism for coagulation or thrombus formation that may occur under stagnant flow conditions.     

Inflow/Outflow Boundary Conditions for Particle-Based Blood Flow Simulations: Application to Arterial Bifurcations and Trees

When blood flows through a bifurcation, red blood cells (RBCs) travel into side branches at different hematocrit levels, and it is even possible that all RBCs enter into one branch only, leading to a complete separation of plasma and RBCs. To quantify this phenomenon via particle-based mesoscopic simulations, we developed a general framework for open boundary conditions in multiphase flows that is effective even for high hematocrit levels. The inflow at the inlet is duplicated from a fully developed flow generated in a pilot simulation with periodic boundary conditions. The outflow is controlled by adaptive forces to maintain the flow rate and velocity gradient at fixed values, while the particles leaving the arteriole at the outlet are removed from the system. Upon validation of this approach, we performed systematic 3D simulations to study plasma skimming in arterioles of diameters 20 to 32 microns. For a flow rate ratio 6:1 at the branches, we observed the “all-or-nothing” phenomenon with plasma only entering the low flow rate branch. We then simulated blood-plasma separation in arteriolar bifurcations with different bifurcation angles and same diameter of the daughter branches. Our simulations predict a significant increase in RBC flux through the main daughter branch as the bifurcation angle is increased. Finally, we demonstrated the effectiveness of the new methodology in simulations of blood flow in vessels with multiple inlets and outlets, constructed using an angiogenesis model.     

Liposome-loaded phenylalanine or tryptophan as sickling inhibitor: a possible therapy for sickle cell disease

Phenylalanine or tryptophan entrapped in small unilamellar liposomes was used to transport Phe or Trp across the red blood cell membrane. The incorporation of Phe or Trp into RBCs via liposomes markedly inhibited and reversed the in vitro sickling of deoxy Hb S. Furthermore, normal and SS RBCs loaded with Phe or Trp did not exhibit significant change in osmotic fragility, mechanical fragility, autohemolysis, and glycolysis when compared to untreated RBCs. In addition, the oxygen affinity measured as the P50 and concentrations of 2,3-DPG and ATP were not affected by the incorporation of Phe or Trp into AA or SS RBCs. These results demonstrate that this liposomal transport system which transferred Phe and Trp into intact RBCs did not have any adverse effect on RBC metabolism and function, and may have therapeutic implications in the treatment of sickle cell disease.     

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

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.     

Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.     

Hematocrit,viscosity and velocity distributions of aggregating and non-aggregating blood in a bifurcating microchannel

Microscale blood flow is characterised by heterogeneous distributions of hematocrit, viscosity and velocity. In microvascular bifurcations, cells are unevenly distributed between the branches, and this effect can be amplified in subsequent branches depending on a number of parameters. We propose an approach to infer hematocrit profiles of human blood flowing through a bifurcating microchannel. The influence of aggregation, induced by the addition of Dextran 2000 to the samples, is also considered. Averaged values indicate plasma skimming, particularly in the presence of red blood cell (RBC) aggregation. Using an empirical model, the hematocrit profiles are used to estimate local relative viscosity distributions. Simulations are used to predict how the non-uniform viscosity influences the velocity profiles. Comparing these data to velocity profiles of RBCs measured using particle image velocimetry provides validation of the model. It is observed that aggregation blunts velocity profiles after a long straight section of channel. Downstream of the bifurcation, skewing of the velocity profiles is detected, which is enhanced by aggregation. The proposed methodology is capable of providing hitherto unreported information on important aspects of microscale blood rheology.     

Sublethal Supraphysiological Shear Stress Alters Erythrocyte Dynamics in Subsequent Low-Shear Flows

Blood is a non-Newtonian, shear-thinning fluid owing to the physical properties and behaviors of red blood cells (RBCs). Under increased shear flow, pre-existing clusters of cells disaggregate, orientate with flow, and deform. These essential processes enhance fluidity of blood, although accumulating evidence suggests that sublethal blood trauma—induced by supraphysiological shear exposure—paradoxically increases the deformability of RBCs when examined under low-shear conditions, despite obvious decrement of cellular deformation at moderate-to-higher shear stresses. Some propose that rather than actual enhancement of cell mechanics, these observations are “pseudoimprovements” and possibly reflect altered flow and/or cell orientation, leading to methodological artifacts, although direct evidence is lacking. This study thus sought to explore RBC mechanical responses in shear flow using purpose-built laser diffractometry in tandem with direct optical visualization to address this problem. Freshly collected RBCs were exposed to a mechanical stimulus known to drastically alter cell deformability (i.e., prior shear exposure (PSE) to 100 Pa × 300 s). Samples were subsequently transferred to a custom-built slit-flow chamber that combined laser diffractometry with direct cell visualization. Cell suspensions were sheared in a stepwise manner (between 0.3 and 5.0 Pa), with each step being maintained for 15 s. Deformability and cell orientation indices were recorded for small-scatter Fraunhofer diffraction patterns and also visualized RBCs. PSE RBCs had significantly decreased visualized and laser-derived deformability at any given shear stress ≥1 Pa. Novel, to our knowledge, observations demonstrated that PSE RBCs had increased heterogeneity of direct visualized orientation with flow vector at any shear, which may be due to greater vorticity and thus instability in 5-Pa flow compared with unsheared control. These findings indicate that shear exposure and stress-strain history can alter subsequent RBC behavior in physiologically relevant low-shear flows. These findings may yield insight into microvascular disorders in recipients of mechanical circulatory support and individuals with hematological diseases that alter physical properties of blood.     

Cross talk between endothelial and red blood cell glycocalyces via near-field flow

Vascular endothelial cells and circulating red blood cell (RBC) surfaces are both covered by a layer of bushy glycocalyx. The interplay between these glycocalyx layers is hardly measurable and insufficiently understood. This study aims to investigate and qualify the possible interactions between the glycocalyces of RBCs and endothelial cells using mathematical modeling and numerical simulation. Dissipative particle dynamics (DPD) simulations are conducted to investigate the response of the endothelial glycocalyx (EG) to varying ambient conditions. A two-compartment model including EG and flow and a three-compartment model comprising EG, RBC glycocalyx, and flow are established. The two-compartment analysis shows that a relatively fast flow is associated with a predominantly bending motion of the EG, whereas oscillatory motions are predominant in a relatively slow flow. Results show that circulating RBCs cause the contactless deformation of EG. Its deformation is dependent on the chain layout, chain length, bending stiffness, RBC-to-EG distance, and RBC velocities. Specifically, shorter EG chains or RBC-to-EG distance leads to greater relative deflections of EG. Deformation of EG is enhanced when the EG chains are rarefied or RBCs move faster. The bending stiffness maintains stretching conformation of EG. Moreover, a compact EG chain layout and shedding EG chains disturb the neighboring flow field, causing disordered flow velocity distributions. In contrast, the movement of EG chains on RBC surfaces exerts a marginal driving force on RBCs. The DPD method is used for the first time, to our knowledge, in the three-compartment system to explore the cross talk between EG and RBC glycocalyx. This study suggests that RBCs drive the EG deformation via the near-field flow, whereas marginal propulsion of RBCs by the EG is observed. These new, to our knowledge, findings provide a new angle to understand the roles of glycocalyx in mechanotransduction and microvascular permeability and their perturbations under idealized pathophysiologic conditions associated with EG degradation.     

An automated cell analysis sensing system based on a microfabricated rheoscope for the study of red blood cells physiology

An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. RBCs suspended in a high viscosity medium were filmed flowing through a microchannel. Under these conditions, RBCs exhibit different orientations and deformations according to their location in the velocity profile. The rheoscope system produces valuable data such as velocity profile of RBCs, spatial distribution within a microchannel and deformation index (DI) curves. The variation of DI across the channel height, due to change in shear stress, was measured carrying implications for diffractometry methods. These curves of DI were taken at a constant flow rate and cover most of the relevant shear stress spectrum. This is an improvement of the existing techniques for deformability measurements and may serve as a diagnostic tool for certain blood disorders. The DI curves were compared to measurements of the flowing RBCs velocity profile. In addition, we found that RBCs flowing in a microchannel are mostly gathered in the center of the flow and maintain a characteristic spatial distribution. The spatial distribution in this region changes slightly with increasing flow rate. Hence, the system described, provides means for examining the behavior of individual RBCs, and may serve as a microfabricated diagnostic device for deformability measurement.     

Tank-treading dynamics of red blood cells in shear flow: On the membrane viscosity rheology

In this article, extensive three-dimensional simulations are conducted for tank-treading (TT) red blood cells (RBCs) in shear flow with different cell viscous properties and flow conditions. Apart from recent numerical studies on TT RBCs, this research considers the uncertainty in cytoplasm viscosity, covers a more complete range of shear flow situations of available experiments, and examines the TT behaviors in more details. Key TT characteristics, including the rotation frequency, deformation index, and inclination angle, are compared with available experimental results of similar shear flow conditions. Fairly good simulation-experiment agreements for these parameters can be obtained by adjusting the membrane viscosity values; however, different rheological relationships between the membrane viscosity and the flow shear rate are noted for these comparisons: shear thinning from the TT frequency, Newtonian from the inclination angle, and shear thickening from the cell deformation. Previous studies claimed a shear-thinning membrane viscosity model based on the TT frequency results; however, such a conclusion seems premature from our results and more carefully designed and better controlled investigations are required for the RBC membrane rheology. In addition, our simulation results reveal complicate RBC TT features and such information could be helpful for a better understanding of in vivo and in vitro RBC dynamics.     

Red blood cells prevent inhibition of hypoxic pulmonary vasoconstriction by nitrite in isolated, perfused rat lungs

Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 muM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of approximately 35 muM) independent of perfusate Po(2). Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.