Macromolecular crowding and volume perception in dog red cells

To differentiate whether the primary volume signal in dog red cells arises from a change in cell configuration or the concentration and dilution of cell contents, we prepared resealed ghosts that had the same surface area and hemoglobin concentration as intact cells but less than 1/3 their volume. Shrinkage of both intact cells and resealed ghosts triggered Na/H exchange. Activation of this transporter in the two preparations correlated closely with cytosolic protein concentration but not at all with volume. The Na/H exchanger was more sensitive to shrinkage in albumin-loaded resealed ghosts than in intact cells or ghosts containing only hemoglobin. Similar results were obtained for the swelling-induced [K-Cl] cotransporter. We believe perception of cell volume originates with changes in cytoplasmic protein concentration. We think the kinases and phosphatases that control the activation of membrane transporters in response to cell swelling or shrinkage are regulated by the mechanism of macromolecular crowding.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.     

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.     

Thermohaemolysis of human erythrocytes in sucrose containing isotonic media

1. 1.|The thermohaemolysis of human erythrocytes in NaCl/sucrose isotonic media can be best accounted for in terms of the colloid-osmotic theory of haemolysis.

2. 2.|The thermohaemolysis in NaCl saline was preceded by leakage of K⁺ and cell swelling. If the inner oncotic osmoactivity was balanced with external sucrose the cells progressively shrinked losing K⁺, but the haemolysis was strongly reduced.

3. 3.|Time dependence of the shrinking of cells and one-step resealed ghosts suspended in isotonic 60 mOsm NaCl/sucrose media was studied between 50 and 58°C.

4. 4.|After a lag period for cells only, this shrinking proceeded with apparently constant rate for cells and ghosts.

5. 5.|The rate constant of shrinking for cells and ghosts obeys the Arrhenius relation, giving the value of 250 ± 15 kJ/mol for the activation energy of shrinking in both cases. This is also the case for the activation energy of the membrane ion permeability constant.

6. 6.|These results are consistent with the thermal inactivation of membrane associated protein(s) acting as a trigger for the ion permeability barrier disturbance.

7. 7.|The mid-point temperature for these membrane events was about 61°C.

Volume-regulating behavior of human platelets

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidimetry. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCl via volume-activated conductive permeability pathways for K+ and anions, presumably Cl-. In media containing greater than 50 mM KCl, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K+ plus Cl-. The K+ pathway was specific for Rb+ and K+ compared to Li+ and Na+. The Cl- pathway accepted NO-3 and SCN- but not citrate or SO4(2-). In isotonic medium, the permeability of platelets to Cl- appeared to be low compared to that of K+. After hypotonic swelling both permeabilities were increased, but the Cl- permeability exceeded that of K+. The Cl- conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na+/H+ exchange, was present or unless Na+ was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pHi appeared to activate Na+/H+ exchange, with a resultant uptake of Na+ and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na+/H+ exchange, but regulatory volume increase was not detectable.     

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.     

Increased anion permeability during volume regulation in human lymphocytes

Peripheral blood lymphocytes (p.b.ls) readjust their volumes after swelling in hypotonic media. An essential component of the regulatory response is an increase in K+ and Cl- permeability. No evidence was found for a tightly coupled co-transport of K+ and Cl-. The flux of either ion proceeds normally in the virtual absence of the transported counterion. Furthermore, alterations in membrane potential recorded during the phase of volume readjustment can be qualitatively accounted for by an increase in Cl- conductance. In tonsillar lymphocytes, a failure of the K+-permeability is nevertheless increased upon swelling. This further suggests that K+ and Cl- are transported during volume regulation through independent pathways. Cytoplasmic free Ca2+ appears to be involved in regulatory volume decrease. K+ and Cl-. Moreover, swelling and shrinking can be induced in isotonic K+-rich and K+-free media, respectively, by the Ca2+ ionophore. The ion flux and volume changes produced by either swelling or internal Ca2+ can be inhibited by similar concentrations of quinine and phenothiazines. The inhibitory activity of the latter drugs, which are powerful antagonists of calmodulin, suggests the participation of this Ca2+-regulator protein in volume regulation.     

Are osmotic forces involved in influenza virus-cell fusion?

The kinetics of the fusion process of unsealed and resealed erthyrocyte ghosts with influenza virus (A/PR8/34, A/Chile 1/83), were measured under hypotonic, isotonic and hypertonic conditions using a recently developed fluorescence assay (Hoekstraet al. (1984)Biochemistry 23:5675–5681]. No correlation between the external osmotic pressure and kinetics and extent of fusion was observed. Influenza viruses fuse as effectively with unsealed ghosts as with resealed ghosts. It is concluded that osmotic forces as well as osmotic swelling of cells are not necessary for virus-cell membrane fusion.     

Temperature effects on osmotic fragility, and the erythrocyte membrane

Results are reported on the temperature-dependence of intact-cell surface area, isotonic volume, hemolytic volume, and ghost steady-state surface area and volume, using several techniques of resistive pulse spectroscopy. Temperature was found not to alter the intact cell surface area permanently: the area remains constant at 130 +/- 1 micron 2, at temperatures ranging from 0 to 40 degrees C. Temperature does alter the steady-state volume of the cells, with a colder temperature inducing swelling by about 0.29 micron 3/deg. C. Such a temperature-induced volume change is sufficient to explain only approximately half of the fragility differences which result from temperature changes. The remainder was found to result from higher temperatures enabling a substantial transient increase in surface area of intact cells (up to at least 14% of 40 degrees C), with a corresponding increase in the cell's hemolytic volume (up to 21%). The hemolytic volume apparently increases linearly with temperature, since steady-state ghost volumes are found to increase linearly with the temperature at which the ghosts were produced. In the steady state (at high temperature), the membranes of electrically-impermeable resealed ghosts can remain extended by more than 10%, compared with membranes of the corresponding unhemolyzed, intact red cells.     

Regulation of sheep erythrocyte volume in anisotonic media.

Sheep erythrocytes of high and low potassium types were incubated in non-haemolytic hypotonic and hypertonic media for 4-5 h at 30 degrees. After initial swelling or shrinking, they readjusted their volume toward their initial isotonic volume. The volume regulation was associated with specific changes in cation fluxes. In the swollen cells, efflux of both sodium and potassium was increased and influx of both cations was slightly decreased; the converse was true for the shrunken cells. All four fluxes were changed in a direction that led to return to normal volume. The difference in the response of the two types of sheep erythrocytes to changes of extracellular fluid osmolality resided in the different activity of their cation transport systems. It is concluded that sheep erythrocytes possess some means of regulating their volume in vitro which is linked to cation permeability. The exact nature of the physical mechanisms by which they accomplish this remains to be elucidated.     

Permeability properties of erythrocyte ghosts

1. Erythrocyte ghosts from human blood were produced by gentle water hemolysis. The ghost-containing hemolysate (about 20 mN) was added to media of different composition (KCl, NaCl, glucose, sucrose, etc.) and varying concentration ranging from 8 to 840 mN. The volume changes of the ghost cells were followed by a light absorption method. The potassium and sodium concentrations were also analyzed in some representative cases. 2. The ghosts shrank, or swelled, in two stages. An initial phase with a momentary expulsion, or uptake, of water leading to an osmotic equilibrium, was followed by a second phase in which a slow swelling or shrinking proceeded toward a final constant volume. 3. The ghosts were semipermeable in the sense that water always passed rapidly in either direction so as to maintain isotonicity with the external medium. The relation between ghost cell volumes (V) and the total concentration (C(e)) of the suspension medium can be expressed by a modified van't Hoff-Mariotte law: (C(e) + a)(V - b) = constant. Here a is a term correcting for an internal pressure and b is the non-solvent volume of the ghost cells. This means that the ghosts behave as perfect osmometers. 4. On the other hand appreciable concentration differences of the K and Na ions could be maintained across the intact ghost cell membranes for long periods. Whether this phenomenon is due simply to very low cation permeability or to active transport processes cannot be decided, although the first assumption appears more probable. 5. When the ghosts were treated with small concentrations of a lytic substance like Na oleate, the alkali ion transfer was greatly increased. This seems to be a simple exchange diffusion process with simultaneous, continued maintenance of osmotic equilibrium (= the second phase). A simplified theory is also given for the kinetics of the volume variations and ion exchange during the second phase (cf. the Appendix). 6. Miscellaneous observations on the effects of pH, and of some other substances are discussed. Some shape transformations of the ghost cells are also described.     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape.

Na+ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na+ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the 31P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg2+/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.     

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.     

Investigations on chromatin condensation of hen erythrocyte nuclei in vitro. An ultrastructural and biochemical study

Hen erythrocyte nuclei, isolated in non-buffered sucrose, (3 mM Mg²⁺, at low ionic strength) have a condensed chromatin to which hemoglobin is bound. Incubation of the isolated nuclei in 0.125 M phosphate-buffer solutions of increasing pH induces a release of the bound hemoglobin and a swelling of the nuclei. Between pH 6.4 and 7.0 both processes reach a plateau and above pH 7.0 a second steep increase in nuclear volume is observable, leading to nuclear disruption above pH 7.4. Titration at low ionic strength shifts the process of hemoglobin release and nuclear swelling to higher pH values. Hemoglobin release and nuclear swelling are fully reversible by backtitration to pH 5.8. The nuclear swelling and shrinking is observed electron-microscopically as decondensation and condensation of the chromatin. The results of these investigations suggest that hemoglobin acts like a cation in the maintenance of nuclear condensation under the conditions used for the isolation of hen erythrocyte nuclei, but that this action is unlikely the physiological mechanism causing chromatin condensation during the maturation of erythroid cells.     

Na+/H+ exchange in volume regulation and cytoplasmic pH homeostasis in lymphocytes

Osmotic shrinking activates an amiloride-sensitive Na+/H+ exchange in the membrane of blood and thymic lymphocytes. The exchange, which is virtually quiescent in isotonic conditions, can also be activated by lowering the cytoplasmic pH (pHi). Activation by pHi is largely caused by an allosteric interaction of H+ with a kinetic modifier site, different from the internal substrate site. The set point or threshold pHi for activation of the exchanger is dictated by the protonation of the modifier. Evidence is presented that indicates that cell shrinking alters the pHi sensitivity of the modifier, shifting the set point to more alkaline levels. In the presence of HCO3- and Cl- a volume increase will accompany the change in pHi. Volume changes can also be produced in isotonic solutions if the exchange is activated by acidification of the cytoplasm, e.g., by addition of propionate to the medium. The latter phenomenon provides a simple method for the detection of the Na+/H+ antiport by electronic cell sizing.     

血红蛋白对人红细胞膜脂质分子侧向运动的影响

用荧光漂白恢复法测定了血红蛋白对红细胞膜脂质分子侧向扩散的限制作用.血红蛋白主要是通过和内侧膜脂质的结合而产生影响的,pH6及PH7.7时都显示出效应的存在.和膜结合较强的高铁血红蛋白,表现出对膜脂质侧向扩散亦有较大的限制作用.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 micron)3. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Characterization of PEG-mediated electrofusion of human erythrocytes.

Polyethylene glycol (PEG) and electrofusion were applied together in a simple and highly efficient cell fusion method. PEG (8000 M(r)) was used to bring human erythrocytes into contact, and a single 4.4 kV/cm, 80 microseconds duration pulse was applied to cell suspensions. The fusion yield (FY) is PEG concentration-dependent. A maximum FY (50%) was found at about 10% PEG. Higher PEG concentrations (> 10%) suppressed FY caused by colloid osmotic shrinkage. Morphological changes, such as colloidal osmotic swelling and shrinking, and the expanding and contraction of fusion lumen, when suspension media were changed from PBS to isotonic 15% dextran solutions, was examined by microscopy. FY was found to depend on both simple osmotic and colloidal-osmotic swelling. From the swelling behavior, we propose two types of electropores: the pre-fusion sites between cell pairs, and electropores on each individual cell connecting intracellular and extracellular space. The latter type is responsible for the colloidal osmotic swelling and shrinking of cell which, together with simple osmotic swelling, is responsible for expanding the pre-fusion sites into fusion lumens. Resealing of electropores resulted in reducing FY, but the FY can be restored by simple osmotic shock. Apparently, PEG plays two opposite roles in this fusion method; one is to promote pre-pulse and post-pulse cell-cell contact, protecting pre-fusion sites, and the other suppresses FY by colloid osmotic shrinkage of cells after pulsing, especially when high PEG concentration is used. 10% PEG 8000 represents the optimal combination of these properties.     

Kinetics of acid hemolysis in an isotonic sugar medium]

The kinetics of acid hemolysis of human erythrocytes suspended in isotonic sucrose media was investigated. Three successive changes on the curve of the suspension extinction derivative were discriminated and characterized as changes due to morphological transformation, swelling and lysis of the cells. The change due to the reversible morphological transformation could develop in two opposite directions depending on some conditions. The cell swelling was apparently induced by the protonization of hemoglobin with protons entering through the cell membrane. The phases of cell swelling and lysis partly covered each other by isotonicity, but at higher tonicities they were completely separated. These results demonstrate the increased informability of the method applied under such conditions.     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape

Na⁺ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na⁺ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the ³¹P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg²⁺/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.