Oxidation-induced calcium-dependent dehydration of normal human red blood cells

Phenazine-methosulphate (PMS) is a strong oxidant that induces reactive oxygen species (ROS) formation in cells. Though it has been shown that PMS increases the red blood cell (RBC) membrane permeability to K⁺, the hypotheses on the mechanism of PMS-induced effects are contradictory and there are no data on volume changes induced by this oxidant. Therefore, the influence of the PMS + ascorbate oxidative system on the volume of normal human RBCs was studied. In a Ca^2 + -containing medium, PMS + ascorbate caused dehydration (shrinking) of RBCs judged by: (1) changes in the density and osmotic resistance distributions of RBCs, and (2) a decrease in their low-angle scattering assessed by FACS analysis. The dehydration resulted from activation of the Gardos channels, was PMS and ascorbate concentration-dependent, was associated with broadening of the density and osmotic resistance distributions of the RBCs, and decreased in the presence of the taxifolin and rutin antioxidants. These findings contribute to a better understanding of the physiology and pathology of oxidatively-modified RBCs and may be of practical significance in estimating the antioxidant activity of various substances.     

Oxidation-induced calcium-dependent dehydration of normal human red blood cells

Phenazine-methosulphate (PMS) is a strong oxidant that induces reactive oxygen species (ROS) formation in cells. Though it has been shown that PMS increases the red blood cell (RBC) membrane permeability to K⁺, the hypotheses on the mechanism of PMS-induced effects are contradictory and there are no data on volume changes induced by this oxidant. Therefore, the influence of the PMS + ascorbate oxidative system on the volume of normal human RBCs was studied. In a Ca^{2 +} -containing medium, PMS + ascorbate caused dehydration (shrinking) of RBCs judged by: (1) changes in the density and osmotic resistance distributions of RBCs, and (2) a decrease in their low-angle scattering assessed by FACS analysis. The dehydration resulted from activation of the Gardos channels, was PMS and ascorbate concentration-dependent, was associated with broadening of the density and osmotic resistance distributions of the RBCs, and decreased in the presence of the taxifolin and rutin antioxidants. These findings contribute to a better understanding of the physiology and pathology of oxidatively-modified RBCs and may be of practical significance in estimating the antioxidant activity of various substances.     

Effect of inositol hexaphosphate on the transient behavior of red cells following a DMSO-induced osmotic pulse

An osmotic pulse can be used to incorporate inositol hexaphosphate (IHP) into red cells. The pulse is induced by equilibrating a red cell suspension with DMSO and then rapidly diluting with an isotonic IHP solution. Since IHP binds to hemoglobin and lowers the affinity for oxygen, this method may find application in the preparation of low-affinity cells for experimental and clinical use. The experiments reported here examined the dynamic changes of several red cell variables immediately following the osmotic pulse. The effect of IHP, which has been shown to dissociate red cell cytoskeletons, was evaluated by comparison with a matched phosphate-buffered saline (PBS) diluent. Red cell morphology, volume, and hemoglobin permeability were studied by fixing the cells at times ranging from 0.06 to 300 sec after dilution. Mechanical fragility was measured by subjecting the cells to a short period of shear stress at the same times after dilution. With both diluents, the cells underwent a rapid increase in volume followed by a return towards normal volume with a maximum at less than 250 msec. With IHP diluent, the period of hemoglobin permeability immediately followed the size peak and was completed by about 1 sec after dilution. PBS also induced a second leakage at longer times (10-120 sec), which resulted in a morphological dichotomy with ghosts and intact cells. The choice of diluent also affected sensitivity to shear stress. The IHP-treated cells had a mechanical fragility maximum at about 1 sec. The PBS-treated cells exhibited no enhanced mechanical fragility. An unexpected result was the inhibition of the second phase of lysis in PBS-treated cells by a properly timed shear stress.     

Effect of procaine hydrochloride on the aggregation behavior and suspension viscoelasticity of human red blood cells

The present study examined the effects of procaine hydrochloride (PRHCL), a cationic local anesthetic, on the aggregation behavior of human red blood cells (RBC); the effects of PRHCL on RBC suspension viscoelasticity, cell shape, volume and density were also investigated. Four indices of RBC aggregation, induced by autologous plasma or 3 g% dextran T70, were evaluated by a computerized light transmission method, and the viscous and elastic components of the complex viscosity were determined by oscillatory viscometry. Low concentrations of PRHCL (8 x 10(-5) to 8 x 10(-4) M) significantly (p less than 0.05 or better) reduced the extent of aggregation (maximal decrease of 22% at 8 x 10(-4) M), but did not alter the viscoelastic components, cell shape, volume or density. The anti-aggregating effect of PRHCL (8 x 10(-4) M) in plasma significantly (p less than 0.005) decreased with time; this temporal effect was abolished by addition of eserine (1 x 10(-4) M). High concentrations of PRHCL (8 x 10(-2) M) caused: 1) increased extent of aggregation and decreased strength of the aggregates (p less than 0.01 or better); 2) elevation of both viscoelastic components for cells in plasma or buffer; 3) a discocyte-stomatocyte shape change; 4) decreased cell density (p less than 0.001) without alteration of cell volume. Our results at low concentrations of PRHCL suggest a mechanism based on an increase of RBC negative surface potential; at the highest concentration, the effects are most likely due to altered cell shape and deformability, and to decreased RBC negative surface potential.     

Effect of polyamines on the electrokinetic properties of red blood cells.

At the physiological pH 7.4, the zeta potential of the normal red blood cell in 1.5% glycine buffer was found to be ?52 mv, whereas that of sickling erythrocytes is ?45 mv. Addition of spermidine to normal red blood cells reduced the zeta potential by approximately 20 mv. In sickling red blood cells, where the polyamine content is determined to be 5 to 6 times greater than in the normal erythrocyte, addition of spermidine reduced the zeta potential by only 5 mv, indicating that little more polyamine binding occurs. The polyamine content of whole blood taken from 24 patients having sickle cell anemia was found to be more than ten times that of whole blood from normal donors. Binding of polyamines to the normal red blood cell was analyzed from the surface charge potential variation as a function of polyamine concentration and the apparent binding constant determined to be 130 d1/g. The difference in the electrokinetic properties of normal and sickling red blood cells in this system may be attributed, in part, to a variation in the polyamine content of the two types of erythrocytes.     

Temperature dependence of the viscoelastic recovery of red cell membrane.

The time-dependent recovery of an elongated red cell is studied as a function of temperature. Before release, the elongated cell is in static equilibrium where external forces are balanced by surface elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of a viscoelastic solid material undergoing large ("finite") deformation. The recovery process is characterized by a time constant, tc, that decreases from approximately 0.27 s at 6 degrees C to 0.06 s at 37 degrees C. From this measurement of the time constant and an independent measurement of the shear modulus of surface elasticity for red cell membrane, the value for the membrane surface viscosity as a function of temperature can be calculated.     

Effect of noradrenaline on the methaemoglobin concentration of rainbow trout red cells.

We studied the effect of noradrenaline on the methaemoglobin (metHb) concentration in rainbow trout red cells. The erythrocytes were incubated in physiological medium with or without noradrenaline and the percentage of metHb of total Hb content was measured. Noradrenaline lowered the metHb content significantly as compared to controls. To study if the effect of noradrenaline was caused by adrenergic intracellular alkalinization, cells were treated with noradrenaline + carbonic anhydrase or noradrenaline + acetazolamide. Carbonic anhydrase inhibits the adrenergic increase in intracellular pH, but did not reduce the effect of noradrenaline on the metHb concentration. Acetazolamide accentuates the increase in intracellular pH. However, there was no difference in the methaemoglobin content of noradrenaline-incubated and noradrenaline + acetazolamide-incubated cells. These results show that the effect of noradrenaline on the methaemoglobin content is independent from the adrenergic increase in intracellular pH. However, amiloride treatment inhibited the effect of noradrenaline on the methaemoglobin content, suggesting that the protein mediating sodium/proton exchange may also be involved in controlling cellular methaemoglobin levels.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

Molecular basis for red cell membrane viscoelastic properties.

An unusual combination of membrane properties allows the red cell to undergo extensive deformation without cell fragmentation, enabling it to effectively perform its function of oxygen delivery during its long life span in the circulation. These material properties are the consequence of a composite structure in which a plasma membrane envelope made up of amphiphilic surfactant molecules is anchored to a network of skeletal proteins through tethering sites (transmembrane proteins) in the bilayer. Explosive growth in our understanding of the primary structure of the various red cell membrane proteins, definition of specific mutations in various red phenotypes, and detailed biophysical characterization of membrane properties of normal and mutant red cells has enabled development of models of molecular and structural basis for red cell properties.     

Effect of calcium on the membrane potential ofAmphiuma red cells

Summary An increase in extracellular Ca concentration causes the membrane of giant red cells of the salamander,Amphiuma means, to undergo a marked, transient hyperpolarization. This hyperpolarization is caused by an increase in K permeability of the membrane as judged from the K sensitivity of the membrane potential and from the rate of K loss under influence of raised extracellular Ca concentration. At constant external pH, the induction of hyperpolarization by increased extracellular Ca has a relatively well-defined threshold concentration. Furthermore the phenomenon is of an all or none type with most of the cells having membrane potential values either in the normal range (about –15 mV) or in the range –40 to –70 mV. Shortly after suspension in Ringer's with 15mM Ca, most if not all of the individual cells are hyperpolarized. Upon continued exposure (5–20 min) to the higher Ca concentration the membrane potential returns to the normal value in a fashion compatible with an all or none response. The observed Ca effect is sensitive to the pH of the suspending medium. At pH 6.2 the response is absent whereas the hyperpolarization is markedly stronger at pH 8.2 than at pH 7.2. It is argued that a reliable transport number for K under influence of Ca cannot be estimated from the slope of membrane potentialvs. log (extracellular K concentration). This is probably related to the fact that the membrane potentials of the cells in the population do not stay constant in time. The above phenomenon is compared with the Ca-induced K permeability in poisoned human red cells or red cell ghosts. It is important to note that the cells employed in the present study are neither poisoned nor mechanically disrupted. This study emphasizes that the role of Ca in regulating cell membrane permeability to K seems to be a general feature.     

A simulation study of the anomalous osmotic behavior of red cells

The factors responsible for movements of water across cell membranes were described mathematically and incorporated into a model which simulates water balance in the cell. Included in the model are a variable charge and osmotic coefficient of hemoglobin, a Na/K pump whose rate varies with ionic concentrations, and the standard electroneutrality and osmotic equilibrium assumptions. The model was used to investigate the phenomena whereby human red cells placed in media of varying tonicities exhibit steady state volume changes less than those predicted by van't Hoff's Law. The model results showed that this anomalous osmotic behavior was primarily due to changes in the osmotic coefficient of hemoglobin as its concentration in the cell varied. A second factor accounting for a part of this behavior was the alteration in the rate of the Na/K pump due to intracellular ionic concentration changes as cell volume varied. The effect of variable electrical charge on the hemoglobin molecule was found to be in the wrong direction to account for the observed osmotic behavior. Also, this effect was seen to produce relatively large changes in cell membrane potential, a result inconsistent with experimental data. It was concluded from the model results that the anomalous osmotic behavior of human red cells is primarily due to the variation in the osmotic coefficient of hemoglobin as the cell volume changes, and that the variable charge effect on the hemoglobin molecule, if it exists, does not play a role in this response.     

Solvent effects on the viscoelastic behavior of porcine submaxillary mucin

Rheological methods have been used to investigate the intermolecular interactions of porcine submaxillary mucins (PSM) in solution. PSM is a high molecular weight glycoprotein consisting of a linear, semi-flexible protein backbone to which a large number of oligosaccharides (1–5 saccharide units) are attached as side chains. Concentrated aqueous solutions of PSM containing different amounts of guanidine hydrochloride (GdnHCl) were subjected to both controlled stress and controlled strain rheological analyses. In the absence of GdnHCl, PSM solutions exhibit viscoelastic properties characteristic of a gel: the storage modulus, G′, is much larger than the loss modulus, G″, at all deformation frequencies, and the compliance is 100% recoverable at small stresses, indicative of strong intermolecular interactions. In 3.0 M aqueous GdnHCl, PSM forms a viscoelastic solution, with G″ > G′ at all frequencies and a relatively small recoverable compliance, pointing to disruption of the intermolecular interactions by the chaotropic salt. Intermediate behavior is observed in 1.5 M GdnHCl, characteristic of a marginal gel: G′ ≈ G″ and greater than 50% recoverable compliance. In dilute solution, PSM behaves viscoelastically as a typical polyelectrolyte. However, concentrated solutions are turbid, the turbidity decreasing as GdnHCl is added, indicating that extensive intermolecular association accompanies the gelation process. The results show that although PSM is secreted in nature as a viscous solution, it can form gels that are similar to those of tracheobronchial and gastric mucins, and suggest common features to the gelation mechanism, with the strength of the gel correlated with the length of the oligosaccharide side chains.     

Effect of osmolality on the hydraulic permeability coefficient of red cells

The osmotic water permeability coefficient, L_p, for human and dog red cells has been measured as a function of medium osmolality, and found to depend on the osmolality of the bathing medium. In the case of human red cells L_p falls from 1.87 x 10^-11 cm³/dyne sec at 199 mOSM to 0.76 x 10^-11 cm³/dyne sec at 516 mOSM. A similar decrease was observed for dog red cells. Moreover, L_p was independent of the direction of water movement and the nature of the solute used to provide the osmotic pressure gradient; it depended only on the final osmolality of the medium. Furthermore, L_p was not affected by pH in the range of 6 to 8 nor by the presence of drugs such as valinomycin (1 x 10^-6 M) and tetrodotoxin (3.2 x 10^-6 M). The instantaneous nature of the response to changes in external osmolality suggests that the hydraulic conductivity of the membrane is controlled by a thin layer at the outer face of the membrane.