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

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

Osmoregulation in red blood cells of Bufo viridis

The effect of hyperosmotic solution of NaCl, urea and mannitol on Bufo viridis red blood cells were studied. The percentage of water content in B. viridis red blood cells decreased significantly in NaCl and mannitol hypertonic solutions compared to urea hypertonic solution. The urea concentration found in red blood cells in a urea hypertonic solution was significantly higher than in red blood cells acclimated to NaCl and mannitol hypertonic solutions. The Na+ concentration was significantly lower in red blood cells immersed in urea hypertonic solution than in red blood cells immersed in hypertonic NaCl and mannitol solutions. However, the K+ concentration increased at a similar rate in three different hypertonic solutions.     

Changes in red blood cell volume on fixation in glutaraldehyde solutions

Summary Light scattering (nephelometry) was used to determine directly the change in volume of red blood cells immersed in a variety of buffer and fixative solutions. Cells immersed in saline or phosphate buffer solutions showed a change in volume that reflected the osmolarity of the solution, shrinkage taking place in hypertonic solutions and swelling and haemolysis occurring in strongly hypotonic solutions. On the other hand, while there was considerable shrinkage in hypertonic glutaraldehyde solutions, swelling was more restricted and haemolysis was prevented in the weaker glutaraldehyde solutions. Thus, while glutaraldehyde exerts a definite osmotic effect on cells in fixative solutions, the magnitude of this effect seems to be limited by its direct action as a fixative.     

The salting-in hypothesis of post-hypertonic lysis

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done.A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are “salted in” to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then “salted out” of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze–thaw injury in human red blood cells.     

The salting-in hypothesis of post-hypertonic lysis

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done.A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are “salted in” to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then “salted out” of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze–thaw injury in human red blood cells.     

Thermal shock and dilution shock as the causes of freezing injury

We suggest that during slow freezing, cellular membranes are altered by the hypertonic solutions produced. This alteration in itself does not cause membrane leakage of normally impermeant solutes but it renders the cells susceptible to solute leakage on the application of a stress, which is provided during freezing by the reduction in temperature (thermal shock) and during thawing by dilution (dilution shock).During slow freezing the effects of cooling rate changes are due to the different times available for the hypertonic solutions to affect the membrane. At a given cooling rate cryoprotective agents reduce the effect on the cells at each temperature during freezing perhaps by reducing the ionic strength. The thermal shock stress during cooling and the dilution shock during thawing thus damages the cells less. With rapid freezing, there is insufficient time for these effects to take place during cooling, which allows the cells to reach low temperatures without thermal shock damage. However, the presence of extracellular ice and the formation of intracellular ice provide hypertonic conditions that render the cells liable to dilution shock on thawing. The slower the rate of thawing of rapidly cooled cells the greater will be the damage from this dilution shock.     

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.     

Autoregulation of the electrogenic sodium pump

The dependence of electrogenic sodium pump activity on changes in the cell volume of Helix pomatia neurons with different levels of intracellular sodium ion concentration was studied. Hypertonic solutions caused hyperpolarization of the membrane and increased membrane resistance in cells with a low sodium content (low-sodium cells; LSC). The activity of the electrogenic sodium pump in hypertonic solutions was increased compared to the activity in hypotonic solutions in LSC and decreased in cells with a high sodium content (high-sodium cells; HSC). The concentration of ouabain which led to maximal inhibition of active 22Na efflux from the neurons was 10(-4) M. Lower concentrations of ouabain (10(-8) M and lower) did not inhibit the sodium pump but stimulated it. The swelling of neurons in hypotonic solutions was accompanied by an increase in the number of binding sites for ouabain, while shrinking in hypertonic solutions led to the opposite effect--a decrease in binding sites. An increase in the number of binding sites also took place in normal isotonic potassium-free solutions compared with normal Ringer's solution. Two saturable components of ouabain binding were detectable in all solutions examined. gamma-Aminobutyric acid (GABA) and acetylcholine (ACh) increased the number of ouabain binding sites on the membrane. The results suggest that there are two opposite mechanisms by which cell volume changes can modulate the pump activity. One of them depends on the intracellular sodium ion concentration and causes pump activation in hypertonic solutions in LSC and saturation in HSC, while a second mechanism mediates the activating effect of cell swelling on the sodium pump in HSC. In addition, there may be a negative feedback between the pump activity and the number of functioning pump units in the membrane.     

Solute transport and epithelial cell volume regulation

1. The regulation of epithelial cell volume is an essential requirement for normal tissue function and the maintenance of cellular integrity. 2. Renal papillary epithelial cells utilize an organic to compensate for the shrinkage associated with exposure to hypertonic solutions. 3. These cells synthesize the polyol, sorbitol, to increase their intracellular solute content. 4. Sorbitol is synthesized from glucose by the enzyme aldose reductase; exposure of the cells to hypertonic media causes aldose reductase synthesis and subsequent sorbitol generation over a two or three day period. 5. The intracellular signal for the formation of aldose reductase is not yet identified.     

Solute-dependent activation of cell motility in strongly hypertonic solutions in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>, human melanoma HTB-140 cells and walker 256 carcinosarcoma cells

Published data concerning the effects of hypertonicity on cell motility have often been controversial. The interpretation of results often rests on the premise that cell responses result from cell dehydration, i.e. osmotic effects. The results of induced hypertonicity on cell movement of Dictyostelium discoideum amoebae and human melanoma HTB-140 cells reported here show that: i) hypertonic solutions of identical osmolarity will either inhibit or stimulate cell movement depending on specific solutes (Na⁺ or K⁺, sorbitol or saccharose); ii) inhibition of cell motility by hypertonic solutions containing Na⁺ ions or carbohydrates can be reversed by the addition of calcium ions; iii) various cell types react differently to the same solutions, and iv) cells can adapt to hypertonic solutions. Various hypertonic solutions are now broadly used in medicine and to study modulation of gene expression. The observations reported suggest the need to examine whether the other responses of cells to hypertonicity can also be based on the solute-dependent cell responses besides cell dehydration due to the osmotic effects.     

Light scattering and cell volumes in osmotically stressed and frozen-thawed cells

Recent reports, indicating that under some conditions the intensity of light scattering from cells is a nonlinear function of cell volume, have led to the widespread generalization that intensity of low-angle light scattering indicates cell size. This study was performed to measure the relationships between light scattering and cell volumes in an-isotonic solutions and after a freeze-thaw stress. Cell volumes in isolated human lymphocytes, human granulocytes, and hamster fibroblasts were deliberately altered by exposure to anisotonic solutions. Boyle-vant Hoff plots of cell volume as a function of inverse osmotic pressure showed that the cells behaved as osmometers. Similar plots of right-angle and low-angle light scattering showed that the intensity of light scattering varied inversely with cell volume. In other experiments where cells were frozen without cryoprotectant at various sub zero temperatures to -25 degrees C and then thawed rapidly, cell viability decreased progressively with decreasing temperature, as did the intensity of both low-angle and right-angle light scattering, although cell volumes remained relatively constant. The intensity of both low- and high-angle light scattering varied inversely with cell volumes in hypertonic and hypotonic solutions, but cell damage induced by freezing and thawing resulted in significant reductions in the intensity of low-angle light scattering with little change in cell volume. These observations show that light scattering and cell volumes can vary independently, and they underline the need for a better understanding of the phenomenon of light scattering from living cells.     

Some combined effects of hypertonic solutions and changes in temperature on posthypertonic hemolysis of human red blood cells

The combined effects of hypertonic solutions and temperature changes on the posthypertonic hemolysis of human red blood cells have been investigated. Cells were exposed to hypertonic solutions of sodium chloride and also to hypertonic solutions of the extracellular cryoprotective additive sucrose, such as would occur during the freezing of cells in an isotonic salt solution to which 15% $\text{w}\text{v}$ sucrose had been added. In both cases the extent of posthypertonic hemolysis was increased by temperature reduction per se when the osmolality of the extracellular solution exceeded about 1400 mOsm/kg water. The posthypertonic hemolysis of cells exposed to a hypertonic solution at 0 °C was reduced with the temperature of the resuspension solution up to 35 °C.     

Role of Cell Adhesion Molecules and Immune-Cell Migration in the Initiation,Onset and Development of Atherosclerosis

Atherosclerosis is currently the leading factor of death in developed countries. It is now recognized as a chronic immune-inflammatory disease, whose initial stages involve the interaction of leukocytes with the endothelial monolayer. The initial stage of atherosclerosis requires the interplay of various cell adhesion molecules and immune cells to trigger leukocyte and lymphocyte migration from the circulating blood into the arterial intima. Studies have unveiled the role of inflammatory mediators in the initiation, onset and progression of the disease. During the last few years we have gained a greater understanding of the mechanism that modulates monocyte, macrophage and T cell infiltration, the role these cells play in the atherosclerotic lesion, in the formation of the fibrous plaque formation with the consequent narrowing of the arteries, and the mechanisms that lead to plaque rupture and the formation of thrombi and emboli. This review talks about the leukocyte recruitment in early atherosclerosis, the formation of the plaque and the mechanisms that lead to thrombosis in advanced atherosclerosis. Finally, we discuss the potential for novel therapies to treat this disease.     

Features of consequences of osmotic modification at different steps of cell heating]

The influence of media with different osmotic pressure (NaCl water solutions) on survival and permeability of Escherichia coli B/r and Escherichia coli Bs-1 cells heated up to 50, 52 and 60 degrees C was investigated. Hypotonic media increased, while hypertonic media, within a certain range of sodium chloride concentrations, decreased the damaging action of heating independently of the temperature. The effectiveness of thermoprotection was seen to increase, and the range of osmolyte concentrations, at which the highest effect of protection takes place, to move markedly towards higher concentrations of NaCl with increase in heating temperature. A certain relationship is suggested between the observed phenomenon and the osmotic homeostasis system of microorganisms under condition of thermogenic and tonic stress.     

Thymidine kinase deficient human cells have increased UV sensitivity in their capacity to support herpes simplex virus but normal UV sensitivity for colony formation

A thymidine kinase deficient (tk-) and two thymidine kinase proficient (tk+) human cell lines were compared for UV sensitivity using colony-forming ability as well as their capacity to support the plaque formation of herpes simplex type 1 (HSV-1). The tk- line (143 cells) was a derivative of one of the tk+ lines (R970-5), whereas the other tk+ line (AC4 cells) was a derivative of the 143 cells obtained by transfection with purified sheared HSV-2 DNA encoding the viral tk gene. 143, R970-5 and AC4 cells showed a similar UV sensitivity for colony-forming ability. In contrast, the capacity to support HSV-1 plaque formation immediately (within 1 h) after UV-irradiation was reduced to a greater extent in the 143 cells compared to the R970-5 and AC4 cells. Capacity curves for plaque formation of the HSV-1: KOS wild-type (tk+) strain were similar to those for the HSV-1: PTK3B mutant (tk-) strain in the 3 cell strains, indicating that the viral tk gene does not influence the ability of HSV-1 to form plaques in UV-irradiated compared to unirradiated human cells. Cellular capacity for HSV-1 plaque formation was found to recover in both tk- and tk+ cells for cultures infected 24 h after UV-irradiation. These results suggest that repair of UV-damaged DNA takes place to a similar extent in both tk- and tk+ human cells, but the kinetics of repair are initially slower in tk- compared to tk+ human cells.     

Changes in osmotic pressure and structure at the initial stages of zygote formation inClosterium ehrenbergii

Summary The behavior of gamete cells ofClosterium ehrenbergii in hypertonic solutions was observed and the significance of changes in osmotic pressure of the protoplasts is discussed in relation to zygote formation. The osmotic pressure of fusing gamete protoplasts was calculated to be 0.063 Osm at the original cell volume. The osmotic pressure of immature gamete protoplasts was 0.24 Osm at incipient plasmolysis. This lowering of cell osmotic pressure may serve to protect the rupture of the plasma membrane during migration of protoplasts in the conjugation tube after dissolution of cell walls. During maturation of gamete cells, chloroplasts and dictyosomes differed greatly in their ultrastructure from those of vegetative cells. These structural changes may be induced by changes of the physiological condition including osmotic pressure in the cells.     

Construction and Preliminary Characterization of a Library of “Lethal” Preterminal Protein Mutant Adenoviruses

Adenoviruses containing lethal in-frame insertion mutant alleles of the preterminal protein (pTP) gene were constructed with cell lines that express pTP. Thirty in-frame insertion mutant alleles, including 26 alleles previously characterized as lethal and 4 newly constructed mutant alleles, were introduced into the viral chromosome in place of the wild-type pTP gene. The viruses were tested for ability to form plaques at 37 degrees C in HeLa-pTP cells and at 32 degrees C and 39.5 degrees C in HeLa cells. Two of the newly constructed viruses exhibited temperature sensitivity for plaque formation, one virus did not form plaques in the absence of complementation, seven additional mutants exhibited a greater than 10-fold reduction in plaque formation in the absence of complementation, and another eight mutants exhibited stronger phenotypes than did previously characterized in-frame insertion mutants in the plaque assay. These mutant viruses offer promise for analysis of pTP functions.     

A plasmolytic cycle: The fate of cytoskeletal elements

Summary In most plant cells, transfer to hypertonic solutions causes osmotic loss of water from the vacuole and detachment of the living protoplast from the cell wall (plasmolysis). This process is reversible and after removal of the plasmolytic solution, protoplasts can re-expand to their original size (deplasmolysis). We have investigated this phenomenon with special reference to cytoskeletal elements in onion inner epidermal cells. The main processes of plasmolysis seem to be membrane dependent because destabilization of cytoskeletal elements had only minor effects on plasmolysis speed and form. In most cells, the array of cortical microtubules is similar to that found in nonplasmolyzed states except that longitudinal patterns seen in some control cells were never observed in plasmolyzed protoplasts of onion inner epidermis. As soon as deplasmolysis starts, cortical microtubules become disrupted and only slowly regenerate to form an oblique array, similar to most nontreated cells. Actin microfilaments responded rapidly to the plasmolysis-induced deformation of the protoplast and adapted to its new form without marked changes in organization and structure. Both actin microfilaments and microtubules can be present in Hechtian strands, which, in plasmolyzed cells, connect the cell wall to the protoplast. Anticytoskeletal drugs did not affect the formation of Hechtian strands.Abbreviations DIC differential interference contrast - DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Effect of salt solutions on the radiosensitivity of mammalian cells as a function of the state of adhesion and the water structure.

The radiation isodose survival curve of attached Chinese hamster (V79) cells, subjected to a wide concentration range of salt or sucrose solutions, is characterized by two maxima separated by a minimum. Cells are radioprotected at the maxima (high and low hypertonic salt concentrations) while they are radiosensitized at the minimum (intermediate hypertonic salt concentrations). Both cations and anions can alter the cellular radiosensitivity above and beyond the (osmotic) effect observed for cells treated with sucrose solutions. However, the basic curve shape, except in the case of sulphate salts, remains the same. When these experiments are repeated with single cells in suspension, the isodose survival curve is quite different in that high salt concentrations (greater than 0.9 M) do not protect cells in suspension unlike the case with attached cells. The curve shape is also altered in that the second maximum is absent with many salt solutions. If multicellular spheroids are used for these experiments, the data resemble those for single cell suspensions rather than for attached cells. The radiation survival data for cells in suspension in salt solutions correlate with water proton spin-lattice relaxation time (T1) and, in hypo- and iso-tonic solutions, with cell volume.     

The effect of low concentrations of adaptogen solutions on the functional activity of murine bone marrow cells in vitro]

Influence of water solutions of chemically pure adaptogens--a synthetic analog of Rhodiola rosea extract phenol combination (SAR) and Dibazol on the functional activity of mice hemopoietic cells in vitro was studied. A clear periodical character of drugs effects manifestation with a tendency to the stimulating activity domination was revealed. A reliable stimulation of clonogenic activity was in correlation with 4 x 10(-11) and 4 x 10(-15) adaptogens molecules in SAR and Dibazol solutions per 1 blood marrow cell. This phenomenon is suggested to be connected with the solvent (water) molecules changes and the formation of structures, keeping the information of adaptogenes with possible translation of the latter during the process of consistent dissolution of the solvent.