A method for estimating free Ca within human red blood cells,with an application to the study of their Ca-dependent K permeability

Summary Murphy, Coll, Rich and Williamson (J. Biol. Chem. 255:6600–6608, 1980) described a null-point method for estimating intracellular free Ca in liver cells. They used digitonin to lyse the cells in solutions of varying Ca concentration. This method has been adapted for use with human red cells. The values found are about 0.4 m Ca in fresh cells, and from 0.4 to 0.7 m Ca in blood-bank cells, at pH 7.2 and 37°C. These are likely to be overestimates, and the errors and limitations of the method are discussed. Red cells may be loaded with Ca by metabolic depletion in Ca-containing solutions. Such cells have an elevated K permeability, and the relationships between free Ca, total Ca and K permeability were investigated, using⁸⁶Rb as a tracer for K.⁸⁶Rb flux studies show that the affinity of the K channel for Ca is the same in cells as in resealed ghosts where intracellular Ca can be controlled with Ca buffers, but the rate of tracer equilibration is 3–6 times faster in ghosts than in cells.     

Studies on the cation permeability of human red cell ghosts

Summary Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant at pH 7.2 for Ca (K _D1) of 3–5×10^–7 m, for Sr of 7×10^–6 m. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes.K _D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mm. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1–140mm) or K(0.1–6mm) concentrations had little effect and did not changeK _D1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K _D2) varied between 6×10^–7 and 4×10^–6 m at pH 7.2. Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca>Sr>Ba>Mg.K _D2 was independt on the pH value in the range between 6.0 and 8.0. Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill coefficients were affected neither by the ion composition nor by the pH values of the intra- and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific channel has properties similar to the K channel in excitable tissues.     

Improved stop-flow apparatus to measure permeability of human red cells and ghosts

An improved stop-flow apparatus has been designed and constructed to measure the permeability characteristics of human red cells, which can be inferred from the time course of red cell volume changes following a sudden change in cellular environment produced by a rapid mixing device. The improved apparatus is directly coupled to a computer which automates thesubtraction and averaging procedures that have been developed to minimize the noise generated in the system by the cessation of red cell forward motion when the flow is suddenly stopped. Real time data acquisition also makes it possible to increase the number of data points by an order of magnitude, thus improving accuracy significantly. The apparatus has been tested by measurements of the human red cel hydraulic permeability coefficient. Data are presented to validate the subtraction procedure. Experiments have also been carried out on red cell ghosts which indicate that the hydraulic conductivity of the ghost is similar to that of the undisturbed red cell.     

Protonophore anion permeability of the human red cell membrane determined in the presence of valinomycin

Summary A transport model for translocation of the protonophore CCCP across the red cell membrane has been established and cellular CCCP binding parameters have been determined. The time course of the CCCP redistribution across the red cell membrane, following a jump in membrane potential induced by valinomycin addition, has been characterized by fitting values of preequilibrium extracellular pHvs. time to the transport model. It is demonstrated, that even in the presence of valinomycin, the CCCP-anion is well behaved, in that the translocation can be described by simple electrodiffusion. The translocation kinetics conform to an Eyring transport model, with a single activation energy barrier, contrary to translocation across lipid bilayers, that is reported to follow a transport model with a plateau in the activation energy barrier. The CCCP anion permeability across the red cell membrane has been calculated to be close to 2.0×10^–4 cm/sec at 37°C with small variations between donors. Thus the permeability of CCCP in the human red cell membrane deviates from that found in black lipid membranes, in which the permeability is found to be a factor of 10 higher.     

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.     

K-Permeabilized human red cells lose an alkaline,hypertonic fluid containing excess K over diffusible anions

Summary Experiments were performed to test specific predictions of an integrated red cell model developed by Lew and Bookchin [Lew, V.L., Bookchin, R.M.J. Membrane Biol. 92:57–74 (1986)], that K-permeabilized human red cells suspended in low-K media would dehydrate and lose an alkaline, hypertonic fluid with excess K over accompanying anions, and that cell dehydration would precede medium alkalinization. Red cells were suspended at about 30% hematocrit in an initially K-free Na-saline and permeabilized to K by the addition of valinomycin. The results showed that by the time a quasi-steady state had been reached the cells had lost the equivalent of a hypertonic fluid containing about 180 mM KCl (SCN) and 10 mM KOH, and that cell dehydration did precede alkalinization of the medium, in good agreement with the theoretical predictions. Since these experiments critically test the interaction between transport, pH and volume regulatory functions in the human red cell, the observed agreement validates the basic assumptions and structure of the integrated model. The functional implications of these results are discussed.     

Melittin lysis of red cells

Summary This paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3×10^–8 m and maximum binding capacity of 1.8×10⁷ molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2×10⁶ molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.     

Calcium pump phosphoenzyme from young and old human red cells

An increase in intracellular Ca(2+) occurs during ageing of human erythrocytes in vivo. The aged cells show a reduced capacity for active Ca(2+) extrusion. Such a defect may arise from pump proteolysis, due to calpain activation by the raised intracellular Ca(2+). To test this possibility, Ca(2+) pump phosphorylation by [gamma-(32)P]ATP was studied on percoll-separated young and old human erythrocytes. After phosphorylation for 30 s with Ca(2+), the amount of phosphoenzyme produced by the young cell membranes was 50% that of the old cells. With Ca(2+) plus La(3+), in contrast, the phosphoenzyme level was nearly the same in both preparations. After a prolonged phosphorylation period (50-90 s), the phosphoenzyme reached almost identical equilibrium levels in both membrane preparations. On the other hand, a single Ca(2+)-dependent radioactive band of about 150 kDa was apparent in both preparations after acidic electrophoresis. Likewise, Western blotting using 5F10 monoclonal antibody also detected a single band of similar molecular weight. These results demonstrate that there is no alteration in either molecular mass or number of active Ca(2+) pump units during cell ageing, thus indicating that the reduced Ca(2+) pumping activity of aged cells does not arise from pump proteolysis.     

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.     

Inhibition of the phosphate self-exchange flux in human erythrocytes and erythrocyte ghosts

Summary The phosphate self-exchange flux in resealed erythrocyte ghosts and in amphotericin B (5.5 m) permeabilized erythrocytes has been studied. The phosphate self-exchange flux exhibits an S-shaped concentration dependence and a self-inhibition in permeabilized red cells while in erythrocyte ghosts no self-inhibition of the phosphate flux has been observed. The apparent halfsaturation constants and the apparent Hill coefficients were assessed by the double reciprocal Hill plots of versus 1/[P] ⁿ . The phosphate half-saturation constants amount to approx. 125mm in ghosts and to about 75mm in permeabilized cells while the apparent Hill coefficients amount to 1.15 and to 1.65 (pH 7.2, 25°C), respectively. Both chloride and sulfate elicit a mixed-type inhibition of the phosphate self-exchange flux. In permeabilized cells, chloride and sulfate shift the flux optimum towards higher phosphate concentrations and reduce the apparent Hill coefficients. In erythrocyte ghosts, the apparent Hill coefficients are insensitive to these anions. The double reciprocal Hill plots indicate a mixed-type inhibition of the phosphate self-exchange flux by DNDS, salicylate and dipyridamole and a noncompetitive inhibition of the phosphate self-exchange flux by phlorhizin. By contrast, the Hill-Dixon plots for chloride and sulfate indicate a competitive inhibition of the phosphate self-exchange flux in erythrocyte ghosts and a mixed-type inhibition in permeabilized cells and provide Hill coefficients of greater than unity for chloride and sulfate. The Dixon plots for DNDS, salicylate, phlorhizin and dipyridamole show a noncompetitive inhibition of the phosphate flux and provide apparent Hill coefficients of 0.95–1.0 for inhibitor binding. Using the Debye-Hückel theory, the effects of ionic strength upon phosphate transport and inhibitor binding can be eliminated. The results of our studies provide strong evidence for the assumption that electrostatic forces are involved in phosphate transport and in inhibitor binding.     

Conjugation of methoxypolyethylene glycol to the surface of bovine red blood cells

Methoxypolyethylene glycol (mPEG) covalently bound to the surface of human red blood cells (hRBCs) has been shown to decrease immunological recognition of hRBC surface antigens (Bradley et al., 2002). However, there is an increasing shortage of hRBC donations, thus making hRBCs scarce and expensive (Davey, 2004; Riess, 2001). The goal of this study is to similarly PEGylate the surface of bovine RBCs (bRBCs) with the aim of reducing the demand on human blood donations needed for blood transfusions. This study investigates the feasibility of modifying the surface of bRBCs with the succinimidyl ester of methoxypolyethylene glycol propionic acid (SPA-mPEG) for use as a potential blood substitute. The oxygen binding affinity of PEGylated bRBCs was moderately increased with increasing initial SPA-mPEG concentrations up to 4 mM when reacted with bRBCs at a hematocrit of 12%. Oxygen transport simulations verified that SPA-mPEG conjugated bRBCs could still transport oxygen to pancreatic islet tissues even under extreme conditions. PEGylated bRBCs reconstituted to a hematocrit of 40% exhibited viscosities on the order of approximately 3 cp, similar to hRBCs at the same hematocrit. Taken together, the results of this study demonstrate the success of PEGylating bRBCs to yield modified cells with oxygen binding, transport and flow properties similar to that of hRBCs.     

Effect of solution environment on the permeability of red blood cells

The cell membrane permeability governs the rate of solute transport into and out of the cell, significantly affecting the cell's metabolic processes, viability, and potential usefulness in both biotechnological applications and physiological systems. Most previous studies of the cell membrane permeability have neglected the possible effects of suspending medium on membrane transport, even though there is extensive experimental evidence that suspending phase composition can significantly affect other properties related to the cell membrane (e.g., cell deformability, fragility, and aggregation rate). This study examined the effects of suspending phase composition (both proteins and electrolytes) on the permeability of human red blood cells to the metabolites creatinine and uric acid. Data were obtained using a stirred ultrafiltration device with direct cell- and proteinfree sampling through a semipermeable membrane. Both the uric acid and creatinine permeabilities were strongly affected by the suspending phase composition, with the permeabilities in different buffer solutions varying by as much as a factor of three. The predominant factors affecting the permeability were the presence (or absence) of chloride, phosphate/adenine, and proteins, although the magnitude and even the direction of these effects were significantly different for creatinine and uric acid transport. The dramatic differences in behavior for uric acid and creatinine reflect the different transport mechanisms for these solutes, with uric acid transported by a carrier-mediated mechanism and creatinine transported by passive diffusion through the lipid bilayer. These results provide important insights into the effects of solution environment on cell membrane transport and other cell membrane-mediated properties. (c) 1994 John Wiley & Sons, Inc.     

All or none cell responses of Ca2+-dependent K channels elicited by calcium or lead in human red cells can be explained by heterogeneity of agonist distribution

Summary We have studied the all or none cell response of Ca²⁺-dependent K⁺ channels to added Ca in human red cells depleted of ATP by incubation with iodoacetate and inosine. A procedure was used which allows separation and differential analysis of responding and nonresponding cells. Responding (H for heavy) cells incubated in medium containing 5mM K lose KCl and water and increase their density to the point of sinking on diethylphthalate (specific gravity=1.12) on centrifugation. Nonresponding (L for light) cells do not lose KCl at all. There is no intermediate behavior. Increasing the Ca concentration in the medium increases the fraction of cells which become H. No differences in the sensitivity to Ca²⁺ of the individual K⁺ channels were detected in inside-out vesicles prepared either from H or from L cells. The Ca content of H cells was higher than that of L cells. Cells depleted of ATP by incubation with iodoacetate and inosine sustain pump-leak Ca fluxes of about 15 mol/liter cells per hour. ATP seems to be resynthesized in these cells at the expense of cell 2,3-diphosphoglycerate stores at a rate of about 150 mol/liter cells per hour. Inhibition of 2,3-diphosphoglycerate phosphatase by tetrathionate increased 6–8 times the measured rate of uptake of external⁴⁵Ca. This was accompanied by an increase in the fraction of H cells. All or none cell responses of Ca²⁺-dependent K channels have also been evidenced in intact human red cells on addition of Pb. They have the same characteristics as those in responding and nonresponding cells. The detailed study of the kinetics of Pb-induced shrinkage of red cells suspended in medium containing 5mM K showed that changes of Pb concentration changed not only the fraction of H cells but also the rate of shrinkage of responding cells. H cells generated by Pb treatment contained significantly more lead than L cells. The above results suggest that the two all or none cell responses studied here can be explained by heterogeneity of agonist distribution among cells. Since pump-leak fluxes exist in both cases, differences of agonist distribution could be generated by heterogeneity of pumping among cells. This interpretation turns interest from K channels to Ca pumps to explain the heterogeneous behavior of red cells in response to a uniform stimulus.     

天花粉蛋白对红细胞损伤作用的AFM研究

目的：利用原子力显微镜（atomic force microscopy,简称AFM）观察红细胞（red blood cells,简称RBC）与天花粉蛋白（trichosanthin,简称TCS）作用后形态上的变化以及细胞膜的损伤情况。方法：将1.2mg/ml的TCS溶液与红细胞的PBS缓冲溶液（pH7.4)按1：4的比例混合,在35℃温度下作用2h后,用原子力显微镜观察受损的红细胞,与正常红细胞进行对比。结果：（1）与正常红细胞相比,与TCS作用后的红细胞的高度明显降低,凹陷部分更加明显。（2）对红细胞上小范围扫描成像的结果显示,受损后的红细胞膜表面结构发生了变化,膜表面颗粒排列的特征依然存在,但颗粒之间开始产生连接。结论：TCS能损伤红细胞膜,改变细胞膜的微结构,引起红细胞的溶血作用。     

Cold activation of Na influx through the Na-H exchange pathway in guinea pig red cells

Summary Previous work showed that amiloride partially inhibits the net gain of Na in cold-stored red cells of guinea pig and that the proportion of unidirectional Na influx sensitive to amiloride increases dramatically with cooling. This study shows that at 37°C amiloride-sensitive (AS) Na influx in guinea pig red blood cells is activated by cytoplasmic H⁺, hypertonic incubation, phorbol ester in the presence of extracellular Cat²⁺ and is correlated with cation-dependent H⁺ loss from acidified cells. Cytoplasmic acidification increases AS Na efflux into Na-free medium. These properties are consistent with the presence of a Na-H exchanger with a H⁺ regulatory site. Elevation of cytoplasmic free Mg^2– above 3 mm greatly increases AS Na influx: this correlates with a Na-dependent loss of Mg^2–, indicating the presence of a Na-Mg exchanger.At 20°C activators of Na-H exchange have little or no further stimulatory effect on the already elevated AS Na influx. AS Na influx is much larger than either Na-dependent H⁺ loss or AS Na efflux at 20°C. The affinity of the AS Na influx for cytoplasmic H⁺ is greater at 20°C than at 37°C. Depletion of cytoplasmic Mg²⁺ does not abolish the high AS Na influx at 20°C.Thus, elevation of AS Na influx with cooling appears to be due to increased activity of a Na-H exchanger (operating in a slippage mode) caused by greater sensitivity to H⁺ at a regulatory site.     

Thermodynamics of all-or-none water channel closure in red cells

Summary The relation of osmotic to diffusional water permeability of human red blood cells was compared after treating the cells with different concentrations of PCMBS (p-chloromercuribenzene sulfonate). After subtracting the PCMBS-insensitive permeability (presumably the water permeability of the lipid bilayer) from each, the ratio of osmotic to diffusional permeability remains invariant (11) as more and more water channels are inhibited by increasing concentrations of PCMBS. This result implies that the channels close in an all-or-none way and suggests a two-state model. Analysis of the dependence of osmotic water permeability on PCMBS concentration in terms of the model reveals a 11 stoichiometry and a dissociation constant for the PCMBS/membrane receptor complex of about 0.019mm at 37°C. Temperature dependence studies show that the reaction is entropically driven (H ^o25 kcal/mol, S ^o100 cal/moldeg) and suggest the involvement of hydrophobic interactions.     

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H₂O₂) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H₂O₂ in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H₂O₂ is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H₂O₂ between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H₂O₂ permeation. The permeability coefficient of H₂O₂ through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10⁻⁴ and 5 × 10⁻³ cm s⁻¹, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H₂O₂ at 37 °C, on the other hand, was 1.6 × 10⁻³ cm s⁻¹. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H₂O₂. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H₂O₂ as normal human RBCs. Therefore, these results indicate that H₂O₂ does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.     

Demonstration of adenylate cyclase activity in human red blood cell ghosts

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine ′,5′-monophosphatase (cyclic AMP) · 2 h^?1 · mg^?1 protein. This enzymatic activity is strongly stimulated by NaF and 5′-guanylimidodiphosphate, is slightly stimulated by epinephrine, norephrine, soproterenol, and prostaglandin E, and is inhibited by calcium. The hormone stimulation is not potentiated by 5′-guanylylimidodiphosphate.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.