Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

Effects of impermeant medium ions on the composition of rabbit renal cortical slices

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)     

The diversity of sulfhydryl groups in the human erythrocyte membrane

Human bank blood erythrocytes were exposed to the mercurials p-chloromercuribenzoate (PCMB), chlormerodrin (CM), p-chloromercuribenzenesulfonate (PCMBS), and 1-bromomercuri-2-hydroxypropane (BMHP) for different time intervals, at different concentrations and in combination with n-ethylmaleimide (NEM) added before, and 2-mercaptoethylguanidine (MEG) and reduced glutathione (GSH) added after the mercurial. Binding patterns of the mercurials to the cells and effects on permeability of the cells were measured. The results indicate that the erythrocyte membrane contains multiple classes of sulfhydryl groups, alteration of which has a variety of effects on cell permeability. PCMB, chlormerodrin and PCMBS react with at least three classes of sulfhydryls, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, sodium accumulation and hemolysis. BMHP reacts with at least two classes of sulfhydryls, one of which is associated with permeability, and, when altered, results in hemolysis in isotonic solutions of choline chloride or lactose. The results provide additional insight into the structure and function of the erythrocyte membrane.     

Osmotic stress as a mechanism of freezing injury

When red cells are suspended in solutions of nonpenetrating solutes of osmolality in excess of 1300 mosm, changes in membrane permeability are seen: there is an influx of extracellular solute, cell volume increases, and the cells will hemolyze on return to isotonic suspension. Furthermore, if cells suspended in such a solution are exposed to a downward temperature change (thermal shock), many will hemolyze.     

Motile activities of Dictyostelium discoideum differ from those in Protista or vertebrate animal cells

Cell movement in the amoebae Dictyostelium discoideum has been examined in media differing in monovalent cation concentration (i.e. Na+ and K+). Under isotonic or even slightly hypertonic conditions, the cells move equally well in solutions in which either potassium or sodium ions dominate. However, in strongly hypertonic solutions the amoebae showed motility in a 2% potassium chloride solution, but remained motionless in a hypertonic 2% sodium chloride solution. This inhibition of D. discoideum amoebae movement in a hypertonic sodium chloride solution was fully reversible. Such behaviour corresponds to that of plant, fungi, and some invertebrate animal cells rather than protozoan or vertebrate cells. These observations suggest that studies using D. discoideum as a model for cell motility in vertebrate animal tissue cells should be considered with caution, and would seem to confirm the classification of cellular slime moulds as related rather to Fungi than to Protista. This also shows that the cell membrane models should consider the asymmetry in sodium/potassium ion concentrations found in vertebrate animal cells as one of various possibilities.     

Evidence for chloride dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost,Opsanus tau

Summary Red blood cells of the marine teleost,Opsanus tau (oyster toadfish), were characterized as to their normal hemoglobin, ion and water contents. Cells were exposed to ouabain containing, hyposmotic salt solutions (osmolarity reduced to 2/3 of normal) in which the cation or anion composition was varied. It was found that the initial cell volume expansion due to water influx was independent of the anion present. However, a secondary volume reduction was dependent on the presence of chloride or bromide anions. During volume reduction, cellular potassium and chloride ion contents fell by about equal amounts. Potassium loss was commensurate to the total amount of potassium ions detected extracellularly about 1.5h after the initial osmotic shock. No major changes were seen in the cellular sodium ion contents. When chloride ions within the cells and in the suspending medium were replaced by nitrate, iodide or thiocyanate, the cells failed to return to volumes close to those of isosmotically suspended controls, and the cellular potassium content also remained constant. In hypotonic potassium chloride the cells failed to extrude potassium chloride and water, and hence retained their expanded volume. Neither potassium loss nor volume decrease occurred in cells swollen in hypotonic sodium chloride media containing furosemide or 4,4 diisothiocyano-2,2-stilbene-disulfonic acid (DIDS). These two compounds are known inhibitors of monovalent cation cotransport and anion self exchange, respectively, in mammalian red cells. Hence toadfish red cells respond to osmotic swelling primarily by activation of an ouabain-insensitive, chloride dependent potassium transport system which is sensitive to inhibition by furosemide and DIDS.     

A study of passive potassium efflux from human red blood cells using ion-specific electrodes

Summary Using ion-specific electrodes, the potassium leakage induced by ouabain in human erythrocytes can be measured continuously and precisely near physiological conditions. Upon small additions of isotonic sucrose solution to a suspension of red cells in physiological saline the passive potassium efflux increases proportionally to the chloride ratio. The same result is obtained upon addition of hypertonic sucrose solution, suggesting that neither osmolarity nor intracellular concentrations have any influence on the passive potassium efflux. The independence of the potassium efflux and osmolarity can be verified by addition of a penetrating substance like glucose to the cell suspension. Adding water or hypertonic sodium chloride solution shows that the potassium efflux increases slightly in more concentrated salt solutions. Inasmuch as it can be interpreted as a pure ionic strength effect, this result supports the hypothesis of independence of potassium efflux and intracellular concentrations. The results of this investigation together with other studies show that the passive permeability of the human red blood cell to potassium depends uniquely on the membrane potential near physiological conditions, while it depends on parameters such as pH or concentrations for large membrane potentials. This suggests that two different mechanisms of transport might be involved: one would control the permeability under normal conditions; the other would represent a leak through the route normally used by anions and become important only under extreme conditions.     

Droplet freezing of antibody-linked indicator red cells of sheep, ox, and human origin

A droplet freezing technique for the cryopreservation of indicator red cells is described. Recovery was crucially dependent on the composition of the solution in which the cells were suspended. Preliminary experiments to determine the relative importance of sucrose, glucose, sodium chloride and hydroxyethyl starch (HES) in determining the survival of trypsin-treated sheep red cells showed that the addition of sucrose or HES or both to isotonic sodium chloride solution increased recovery, whereas the additional inclusion of glucose was detrimental. It was shown that glucose penetrated the cells whereas sucrose did not. The optimum combination of sucrose and sodium chloride concentration, in the presence of 6 g/dl HES, was 7 g/dl sucrose plus 0.3 g/dl sodium chloride. Recovery was increased by increasing the concentration of HES, and maximal recovery was obtained by thawing the frozen droplets in phosphate-buffered saline at 40 °C. Trypsintreated ox and human cells gave much lower recovery than sheep cells when HES was used in the freezing mixture but the substitution of dextran (10 g/dl) for HES gave greater than 80% recovery with all three species. Ten different antibody-coupled reagent cells all gave >83% recovery. The effects of hematocrit, incubation time, and storage temperature are described. The preservation technique described is simple and convenient, and will make it possible to extend the use of immunoassay procedures using antibody-coupled red cells.     

Further studies on the partial double Donnan. Is isosmotic KCl solution isotonic with cells of respiratory trees of the holothurian Isostichopus badionotus Selenka?

As potassium, chloride and water traverse cell membranes, the cells of stenohaline marine invertebrates should swell if exposed to sea water mixed with an isosmotic KCl solution as they do when exposed to sea water diluted with water. To test this hypothesis respiratory tree fragments of the holothurian Isostichopus badionotus were exposed to five isosmotic media prepared by mixing artificial sodium sea water with isosmotic (611 mmol/l) KCl solution to obtain 100, 83, 71, 60 and 50% sea water, with and without 2 mmol/l ouabain. For comparison, respiratory tree fragments were incubated in sea water diluted to the same concentrations with distilled water, with and without ouabain. Cell water contents and potassium and sodium concentrations were unaffected by KCl-dilution or ouabain in isosmotic KCl-sea water mixtures. In tissues exposed to H(2)O-diluted sea water, cell water increased osmometrically and potassium, sodium and chloride concentrations decreased with dilution; ouabain caused a decrease in potasium and an increase in sodium but no effect on chloride concentrations. The isotonicity of the isosmotic KCl solution cannot be adscribed to impermeability of the cell membrane to KCl as both ions easily traverse the cell membrane. Rather, operationally immobilized extracellular sodium ions, which electrostatically hold back anions and consequently water, together with the lack of a cellward electrochemical gradient for potassium, resulting from membrane depolarization caused by high external potassium concentration, would explain the isotonicity of isosmotic KCl solution. The high external potassium concentration also antagonizes the inhibitory effect of ouabain on the Na(+)/K(+) ATPase responsible for sodium and potassium active transport.     

Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium

When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium.     

The specific requirement for sodium chloride for the active uptake of l-glutamate by Halobacterium salinarium

1. Uptake of l-glutamate by Halobacterium salinarium is dependent on high concentrations of sodium chloride in the environment. When the sodium chloride is replaced by isomolar concentrations of potassium chloride, sodium acetate or potassium acetate, only negligible uptake occurs. 2. Most of the glutamate taken up can be shown to be in the cells in the free state and at a concentration of at least 50 times that in the medium. Sodium chloride is therefore required for an active transport of the glutamate into the cells. 3. The question whether sodium chloride is essential for the actual migration of glutamate across the cell envelope or for the mechanism supplying energy for this migration is discussed on the basis of experiments on endogenous respiration and with inhibitors.     

Aluminum transfer as glutamate complex through blood-brain barrier

In vitro distribution of aluminium between plasma and erythrocytes has been studied in the presence of variable amounts of sodium L-glutamate. With a red blood cell suspension in isotonic sodium chloride, aluminium remains confined in erythrocytes even when the sodium L-glutamate concentration increases in the medium. Aluminium initially present in plasma penetrates red blood cells when sodium L-glutamate increases in whole blood, showing that this metal is able in vitro to cross the erythrocyte membrane as glutamate complex. In vivo experiments with male Wistar rats prove that aluminium is also able to pass the blood--brain barrier as glutamate complex and deposit in the brain cortex.     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Sodium, potassium, and chloride concentrations in the Schwann cell and axon of the squid nerve fiber

Sodium, potassium, and chloride concentrations were determined in the sheath cells and axoplasm of the nerve fiber of the squid Sepioteuthis sepioidea. The sheaths were obtained by slitting the nerve fiber, the extracellular electrolytes were washed out in isotonic sucrose solution, and the concentrations in the cells were determined after different soaking times in the sucrose solution. Values for the Schwann cell were calculated by extrapolation to zero time from the plots of the logarithms of the concentrations in the cells as a function of soaking time in sucrose solution. The Schwann cells made up 84 per cent of the sheath''s total cellular volume. The Schwann cell concentrations in millimols per liter, are: 312 (404-241) for sodium, 220 (308-157) for potassium, and 167 (208-138) for chloride. The concentrations in the axoplasm (mean ± SE), in millimols per liter are: 52 ± 10 for sodium, 335 ± 25 for potassium, and 135 ± 14 for chloride. The possibility that some fraction of the Schwann cell electrolytes, especially of sodium, is bound, cannot be discarded.     

The mechanism of mitochondrial swelling. VIII. Permeability of mitochondria to alkali metal acetates

The capacity of beef heart mitochondria to undergo osmotically induced volume changes in decimolar M⁺-acetate or other weak acid anion media is characterized by the following features: (1) mitochondria resist swelling when suspended in potassium or rubidium acetate media in the presence of respiratory inhibitors; (2) mitochondria swell extensively when suspended in ammonium or sodium acetate media in the presence of respiratory inhibitors; and (3) actively respiring mitochondria swell extensively whether suspended in ammonium, sodium, potassium, or rubidium acetate media. These findings have been interpreted to mean that (1) the nonenergized mitochondrial inner membrane is permeable to acetate anions, (2) the nonenergized mitochondrial inner membrane is permeable to ammonium and sodium ions in the presence of acetate or other weak acid anions, (3) the nonenergized mitochondrial inner membrane is relatively impermeable to potassium and rubidium ions in the presence of acetate or other weak acid anions, and (4) energized mitochondria are considerably more permeable to potassium and rubidium (acetate) ions than are non-energized mitochondria. The experiments described in this communication which provide the evidence for these interpretations involve methods which are independent of volume changes. The results confirm the first three of the above interpretations but are inconsistent with the fourth. A general theory for passive ion movements in mitochondria is presented and the results are discussed in terms of the development of an energy dependent ion gradient as the key to energized swelling in potassium or rubidium acetate.     

The effects of osmotic stress on human platelets

The effect of osmotic stress on human platelets was investigated at 0, 25, and 37 degrees C. The osmolality of the suspending plasma was decreased by adding water or increased by adding sodium chloride or sucrose. After 5 min, isotonicity was restored by dilution with an excess of isotonic phosphate-buffered saline. After centrifugation, the platelets were resuspended in autologous plasma and then incubated for 1 hr at 37 degrees C before assaying the active transport of 5-hydroxytryptamine (5-HT) and the hypotonic stress response. Anisosmotic conditions had a greater effect on the extent of volume reversal in the hypotonic stress test than on 5-HT uptake. At 25 degrees C, only moderate degrees of hypotonicity (0.25 osmol/kg) or hypertonicity (0.59 osmol/kg) were sufficient to depress the hypotonic stress response. In general, platelets tolerated departures from isotonic conditions better at 0 degree C than at the higher temperatures. Furthermore, at 0 and 25 degrees C approximately equiosmolal concentrations of sucrose and sodium chloride depressed the hypotonic stress response to similar extents, but at 37 degrees C high osmolalities (greater than 2 osmol/kg) were tolerated better when the additive was sucrose than when it was sodium chloride. Platelets shrank when subjected to hyperosmotic conditions, but their discoid shape and the peripheral band of microtubules were maintained.     

Cell volume regulation in renal cortical cells

Both proximal renal tubule cells and cultured Madin-Darby canine kidney (MDCK) cells are capable of regulating their volume in hypotonic media. Regulatory cell volume decrease in proximal straight tubules is impaired by barium, amiloride and acetazolamide and depends on the presence of bicarbonate and of sodium, whereas it is unaffected by complete removal of extracellular chloride. The observations may point to parallel loss of potassium through potassium channels as well as of bicarbonate and sodium via a bicarbonate-sodium cotransport. Alternatively, potassium/hydrogen ion exchange or potassium bicarbonate cotransport could be involved. In MDCK cells, exposure to hypotonic media apparently leads to the activation of an anion channel, while potassium conductance is rather decreased. In both proximal tubules and MDCK cells, volume regulatory decrease is possibly triggered by leucotrienes, which may be released during cell swelling. Cell volume is altered in a variety of conditions even at isotonic extracellular fluid and cell volume-regulatory mechanisms are likely to participate in regulation of renal transepithelial transport.     

Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium.

Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport. However, when the deenergized cells were treated with valinomycin and diluted into sodium phosphate to create both an artificial membrane potential and a chemical sodium gradient, rates of serine uptake were fivefold greater than in cells having only a sodium gradient. If deenergized cells were preloaded with sodium (no membrane potential or sodium gradient), there was little serine transport. Nigericin and monensin, ionophores capable of reversing sodium gradients across membranes, strongly inhibited sodium-dependent uptake of the three amino acids. Membrane vesicles loaded with potassium and diluted into either lithium or choline chloride were unable to transport serine, but rapid uptake was evident if sodium chloride was added to the assay mixture. Serine transport had an extremely poor affinity for sodium, and more than 30 mM was needed for half-maximal rates of uptake. Serine transport was inhibited by an excess of threonine, but an excess of alanine had little effect. Results indicated that S. bovis had separate sodium symport systems for serine or threonine and alanine, and either the membrane potential or chemical sodium gradient could drive uptake.