Effects of nystatin on intracellular contents and membrane transport of alkali cations,and cell volume in HeLa cells

Summary Nystatin (50 g/ml) had strong influence on the intracellular contents and membrane transports of monovalent ions and water in HeLa cells. The nystatin-induced changes in the intracellular ion content and cell volume were inhibited by sucrose, and Donnan and osmotic equilibria were attained. Using cells under conditions for these equilibria, the concentrations of intracellular impermeant solutes, their mean valence, the differences of their intra- and extracellular osmotic concentrations, and the circumferential tension of the cell membrane were determined. Stimulation by nystatin of the influx of one cation species, e.g. Rb, was inhibited by another cation species, e.g. Na. The stimulatory effect of nystatin on cation fluxes was reversible within 1 hr after ionophore addition, and after 1-hr treatment the intracellular contents of Na and K became proportional to their extracellular concentrations, provided that the sum of these concentrations was constant (300mm). Similar proportionality was also observed in the presence of choline, provided that the choline concentration was less than those of the alkali cations. The implications of these results in relation to the osmotic properties of cultured cells, and the experimental regulation of alkali cations in the cells, are discussed.     

Proton (or hydroxide) fluxes and the biphasic osmotic response of human red blood cells

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'- dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside- positive transmembrane voltage, followed by a slow DIDS (4,4'- diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs- Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).     

Osmotic effects of protein polymerization: Analysis of volume changes in sickle cell anemia red cells following deoxy-hemoglobin S polymerization

Summary Polymerization-depolymerization of proteins within cells and subcellular organelles may have powerful osmotic effects. As a model to study these we analyzed the predicted volume changes following hemoglobin (Hb) S polymerization in sickle cell anemia (SS) red cells with different initial volumes. The theoretical analysis predicted that dehydrated SS red cells may sustain large polymerization-induced volume shifts whose direction would depend on whether or not small solutes were excluded from polymer-associated water. Experiments with SS cells from promptly fractionated venous blood showed oxygenation-induced swelling, maximal in the densest cells, in support of nonexclusion models. The predicted extent of cell dehydration on polymerization was strongly influenced by factors such as the dilution of residual soluble Hb and the increased osmotic contribution of Hb in cells dehydrated by salt loss, largely overlooked in the past. The osmotic effects of polymer formation may thus play an important part in microcirculatory infarction by dense SS cells, as they become even denser and stiffer during deoxygenation in the capillaries.     

Metabolic dynamics in the human red cell. Part II--Interactions with the environment

The maintenance of human red cell volume under multitude of trying physiological conditions is a self regulated dynamic process. Theoretical and experimental studies on red cell osmotic states have been primarily focussed on three different interdependent areas: the permeative properties of the red cell membrane, the kinetic studies of transmembrane fluxes of various ionic and nonionic chemical constituents of the red cell and plasma, and the ideal and non-ideal thermodynamic formulation of the osmotic states. The primary objective of this work is to provide a general model that converges the above mentioned components of the red cell and its environment under one umbrella. Such a model facilitates the simultaneous interpretation and prediction of quantitative changes in the red cell volume, pH, Donnan ratios, osmotic effects, plasma volume, transmembrane fluxes, and permeable and impermeable solute concentration.     

Control of Cell Volume in Skeletal Muscle

Regulation of cell volume is a fundamental property of all animal cells and is of particular importance in skeletal muscle where exercise is associated with a wide range of cellular changes that would be expected to influence cell volume. These complex electrical, metabolic and osmotic changes, however, make rigorous study of the consequences of individual factors on muscle volume difficult despite their likely importance during exercise. Recent charge-difference modelling of cell volume distinguishes three major aspects to processes underlying cell volume control: (i) determination by intracellular impermeant solute; (ii) maintenance by metabolically dependent processes directly balancing passive solute and water fluxes that would otherwise cause cell swelling under the influence of intracellular membrane-impermeant solutes; and (iii) volume regulation often involving reversible short-term transmembrane solute transport processes correcting cell volumes towards their normal baselines in response to imposed discrete perturbations. This review covers, in turn, the main predictions from such quantitative analysis and the experimental consequences of comparable alterations in extracellular pH, lactate concentration, membrane potential and extracellular tonicity. The effects of such alterations in the extracellular environment in resting amphibian muscles are then used to reproduce the intracellular changes that occur in each case in exercising muscle. The relative contributions of these various factors to the control of cell volume in resting and exercising skeletal muscle are thus described.     

The kinetics of colloid osmotic hemolysis. I. Nystatin-induced lysis

A kinetic model of colloid osmotic hemolysis for cation-permeable cells has been developed. The model consists of three essential components. The first is a set of flux equations, under the assumption that the membrane potential is equal to the chloride equilibrium potential and that cation fluxes are described by the Goldman flux equation. The second is the osmotic equilibrium model of Freedman and Hoffman that takes into account the non-ideal osmotic behavior of erythrocytes. The third is an empirical relation between hemolysis and cell volume, developed from the lysis behavior in hypoosmotic media. Model simulations are compared with lysis experiments using the antibiotic nystatin to raise cation permeability. The form of the kinetics and inhibition of lysis by sucrose are described well by the model. In additional lysis experiments at different external pH the small pH dependence is accounted for by the model.     

The behavior of the human erythrocyte as an imperfect osmometer: a hypothesis

The human erythrocyte does not behave as a perfect osmometer that is its volume does not change as predicted with the change of the tonicity of the medium, as if there was a fraction of the cell water not participating in the osmotic exchange. A mechanism of control of the erythrocyte shape has been previously proposed in which Band 3 (AE1), the protein anion exchanger of Cl(-) and HCO(3)(-), plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation and inward-facing conformation (Band 3(o)/Band 3(i)) contract and relax the membrane skeleton, thus favoring echinocytosis and stomatocytosis, respectively. The equilibrium Band 3(o)/Band 3(i) ratio is determined by the Donnan equilibrium ratio of anions and protons, increasing with it (r=Cl(i)(-)/Cl(o)(-)=HCO 3(i)(-)/HCO 3(o)(-)=H(o)(+)/H(i)(+)). The Donnan ratio is influenced by the erythrocyte transport and metabolic activities. The volume change of the human erythrocyte alters the skeleton conformation as it is accompanied by a change of the membrane curvature. Thus, the mechanism could be a hypothesis for explaining the behavior of the human erythrocyte as an imperfect osmometer since the Donnan ratio controls the Band 3(o)/Band 3(i) ratio which controls the volume by a control of the degree of contraction or relaxation of the skeleton. Predictions made by the hypothesis on the Ponder's coefficient R' values in the presence of sucrose or Band 3 substrates slowly transported as well as on the participation of Band 3 in the osmotic hemolysis appear to be corroborated by previous observations. If the hypothesis was valid, it would follow that there is a pressure gradient across the erythrocyte membrane. The equilibrium volume is antagonistically determined by the Donnan ratio per se and Band 3. Band 3, rather than the ratio of surface-to-volume, primarily controls the osmotic hemolysis.     

Pseudo-streaming potentials in Necturus gallbladder epithelium. II. The mechanism is a junctional diffusion potential

The mechanisms of apparent streaming potentials elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution were studied by assessing the time courses of: (a) the change in transepithelial voltage (Vms). (b) the change in osmolality at the cell surface (estimated with a tetrabutylammonium [TBA+]-selective microelectrode, using TBA+ as a tracer for sucrose), and (c) the change in cell impermeant solute concentration ([TMA+]i, measured with an intracellular double-barrel TMA(+)-selective microelectrode after loading the cells with TMA+ by transient permeabilization with nystatin). For both sucrose addition and removal, the time courses of Vms were the same as the time courses of the voltage signals produced by [TMA+]i, while the time courses of the voltage signals produced by [TBA+]o were much faster. These results suggest that the apparent streaming potentials are caused by changes of [NaCl] in the lateral intercellular spaces, whose time course reflects the changes in cell water volume (and osmolality) elicited by the alterations in apical solution osmolality. Changes in cell osmolality are slow relative to those of the apical solution osmolality, whereas lateral space osmolality follows cell osmolality rapidly, due to the large surface area of lateral membranes and the small volume of the spaces. Analysis of a simple mathematical model of the epithelium yields an apical membrane Lp in good agreement with previous measurements and suggests that elevations of the apical solution osmolality elicit rapid reductions in junctional ionic selectivity, also in good agreement with experimental determinations. Elevations in apical solution [NaCl] cause biphasic transepithelial voltage changes: a rapid negative Vms change of similar time course to that of a Na+/TBA+ bi-ionic potential and a slow positive Vms change of similar time course to that of the sucrose-induced apparent streaming potential. We conclude that the Vms changes elicited by addition of impermeant solute to the apical bathing solution are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow from the cells to the apical bathing solution and from the lateral intercellular spaces to the cells. Our results do not support the notion of junctional solute-solvent coupling during transepithelial osmotic water flow.     

Non linear volume flow dependence on osmotic pressure difference in frog skin.

The volume flow dependence upon the osmotic pressure difference of both impermeant (sucrose) and permeable (NaCl) species has been investigated in leg skin bags of Rana esculenta. It is concluded: 1. The hydration-dehydration error in the flow measurement with leg skin bags is negligible. 2. The flow-force relationship is non-linear. 3. Unstirred layers and solute permeation have little, if any, influence on non linearity. 4. Structural modifications of the skin induced with hypertonic solutions have been observed and may contribute to non linearity, as well as the multiple-barrier effect.     

Cell volume and osmotic properties of erythrocytes after complement lysis measured by flow cytometry

The changes of volume distribution curves of erythrocytes during and after lysis by complement or nystatin or in hypotonic buffers were measured by flow cytometry. Biconcave and spheroidal ghosts were observed after complement lysis and spheroidal ghosts were seen only after nystatin and hypotonic lysis. The spheroidal ghosts derived from red cells lysed by complement or nystatin were permeable to sucrose; those from hypotonic lysis were sucrose-impermeable. Spheroidal ghosts after complement lysis remained permeable for sucrose whereas spheroidal ghosts after nystatin lysis resealed after removal of the drug by washing. Biconcave ghosts produced by complement lysis were almost impermeable to sucrose initially and therefore responded to osmotic changes, but they became sucrose-permeable upon prolonged incubation at 37 degrees C. The rate of sucrose equilibration increased as the stability of the biconcave shape diminished with increasing numbers of C5b-9 complexes. At 850 C5b-9 complexes/ghost, the biconcave shape and impermeability for sucrose were completely lost. The results support the hypothesis that complement C5b-9 complexes, in addition to the interaction with the lipid bilayer, may interact with the cytoskeleton of the erythrocyte membrane.     

Correlation of the internal microviscosity of human erythrocytes to the cell volume and the viscosity of hemoglobin solutions

The microviscosity of the cytoplasm of human erythrocytes as well as of membrane-free hemoglobin solutions was investigated measuring the rotation of the small spin-label molecule, Tempone. The dependence of the intracellular microviscosity on the extracellular pH and osmotic pressure which was varied by NaCl or sucrose was sufficiently explained on the basis of alterations of the red blood cell volume. The intracellular microviscosity depended exclusively on the hemoglobin concentration. It did not differ from that of comparable membrane-free hemoglobin solutions. It was not necessary to take into account long-range interactions between hemoglobin molecules. The conclusion therefore was that the intracellular viscosity is not modified by cytoplasmic structures or the cell membrane. Above a hemoglobin concentration of 6 mM the viscosity of hemoglobin solutions increased much faster than the microviscosity. From measurements obtained with different spin-labels it followed that also the charge of these molecules is of importance.     

An electrophysiological technique to measure change in hepatocyte water volume

We have applied an electrophysiologic technique (Reuss, L. (1985) Proc. Natl. Acad. Sci. USA 82, 6014) to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA+) during transient exposure of cells to nystatin. Intracellular TMA+ activity (alpha 1TMA) was measured with TMA(+)-sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions alpha iTMA remained constant over 4 min in a single impalement. Hyperosmotic solutions (50, 100 and 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 and 100 mM) increased and decreased alpha iTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(alpha iTMA)0/(alpha iTMA)4min] -1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less predicted for a perfect osmometer. Corresponding measurements of Vm showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba2+ (2 mM) was present during hyposmotic stress of 0.66 X 286 mosmol (control), cell water volume increased by a factor of 1.44 +/- 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 +/- 0.02, P less than 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 +/- 0.04 to 1.24 +/- 0.09, P less than 0.01. We conclude that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of Vm in the presence of K(+)-channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.     

A note on osmotic pressure measured with ionophore treated red blood cells

A method is described by which the osmotic pressure of macromolecules or many low molecular weight substances can be measured relative to the known osmotic pressure of a reference substance. Measurements can also be made in the presence of univalent electrolytes. The method involves the use of ionophore treated mammalian red blood cells as osmometers. Details are given for the establishment of the isosmotic identity line for dextran M_w = 9 400, M_n = 5 500 and sucrose using nystatin treated human red blood cells. The sucrose concentrations used were from 20 to 33 mOsm (50–80 kPa).     

Osmotic properties of human red cells

Summary When an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of nonosmotic water which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol. 52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (z_Hb). A number of investigators, particularly Freedman and Hoffman (1979,J. Gen. Physiol. 74:157) have been unable to confirm Gary-Bobo and Solomon's experimental evidence for this concentration dependence of z_Hb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5±0.8% of the total isosmotic cell water (P0.0005,t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.     

Factors affecting solute entrapment in phospholipid vesicles prepared by the freeze-thaw extrusion method: a possible general method for improving the efficiency of entrapment

It is often assumed that the internal solute concentrations of phospholipid vesicles are equal to those in the medium in which they were prepared, particularly when freeze-thaw cycles are employed during the procedure. Conditions are reported here which when used to prepare vesicles by the polycarbonate filter extrusion method, produce approximately 12- and approximately 7-fold higher internal concentrations of Ca2+ and sucrose, respectively, than exist in the external medium. Formation of these large gradients is dependent upon the use of freeze-thaw cycles during preparation, on the presence of tetraethylammonium perchlorate in the medium, and is independent of media pH across the region of pH 5-9. Gradient formation is antagonized by high concentrations of an impermeant solute (NaCl). It is proposed that gradients form because solutes are concentrated by exclusion from ice during freezing but that they are normally dissipated by osmotic lysis during thawing. The presence of a permeant solute such as tetraethylammonium perchlorate provides an alternative mechanism to balance osmotic pressure, thereby preserving the gradients of impermeable species.     

Volume-dependent and NEM-stimulated K+,Cl- transport is elevated in oxygenated SS, SC and CC human red cells

Mechanisms involved in cell volume regulation are important in SS, SC cells as they might be involved in determining the extent of sickling and the generation of dense cells and irreversibly sickled cells. We have studied in these cells the response to cell swelling of the K+,Cl- transporter. We found that Hb SS, SC and CC red cells have higher values of a ouabain-resistant, chloride-dependent and NEM-stimulated K+ efflux than AA red cells. In contrast, the Na+,K+,Cl- cotransport estimated from the bumetanide-sensitive component of K+ efflux was not significantly different in SS, SC and CC red cells. The (ouabain + bumetanide)-resistant K+ efflux from SS, SC and CC red cells was stimulated by cell swelling induced by reduction of the osmotic pressure (300 to 220 mosmol/l) and pH (8 to 7) of the flux media (140 mM NaCl). The Cl--dependent K+ efflux stimulated by osmotic swelling highly correlated with the NEM-stimulated component (r = 0.8, p less than 0.001, n = 22) and the acid-pH-induced swelling (r = 0.969, p less than 0.001, n = 22), indicating that it is driven by the K+,Cl- transporter.     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

Ionic dependence of the extracellular ATP-induced permeabilization of transformed mouse fibroblasts: role of plasma membrane activities that regulate cell volume

Extracellular ATP rendered the plasma membrane of transformed mouse fibroblasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since 1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and 2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+,K+-ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+,K+,Cl- -cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+,K+,Cl- -cotransporter-mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+,K+,Cl- -cotransporter.     

Swelling and de-swelling kinetics of gelatin hydrogels in ethanol-water marginal solvent

Controlled osmotic swelling and de-swelling measurements have been performed on gelatin, a polyampholyte, hydrogels suspended in water-ethanol marginal solvent at room temperature (20 degrees C) where the alcohol concentration was changed from 0 to 100% (v/v). The change in gel mass was monitored as function of time until osmotic equilibrium was established with the surrounding solvent. It was observed that osmotic pressure of polymer-solvent mixing, pi(m)相似文献   

Effects of different saccharose and electrolyte concentrations on preserved erythrocytes

Preservation solutions for buffy coat-free red cell concentrates with sucrose concentrations from 234 decreasing up to 15 mmol per 1 solution were tested. The hemolysis rate increased from 0.5 up to 1.9% by decreasing the sucrose concentration. The red cell volume was unchanged at low sucrose concentrations. No differences were noticed in ATP content and morphological changes. A considerable extracellular pH shift at high sucrose concentration exists only at the beginning of storage. A sucrose concentration of 30-50 mmol/l solution (3-5 mmol per unit red cell concentrate) at an ionic strength of 0.16 proves to be most suitable.