The role of the dehydration stage in the post-hypertonic hemolysis of mammalian erythrocytes

The aim of this study was to assess the level of damage to mammalian erythrocytes under post-hypertonic shock depending on the concentration of NaCl in the dehydration medium and to determine the effect of hypertonic NaCl solutions on the condition of mammalian erythrocytes by flow cytometry. To achieve this goal, spectrophotometric and cytometry research methods were used. The data obtained showed that post-hypertonic lysis of mammalian erythrocytes depends on the concentration of NaCl in the dehydration medium. The most sensitive to the effects of post-hypertonic shock are rat erythrocytes, the least sensitive are rabbit cells. Cytometry studies revealed significant changes in the histograms of the distribution of erythrocytes of all mammalian species with increasing salt concentration in the dehydration medium. These changes are species-specific and are probably related to changes in cell volume and morphology. The data revealed a relationship between the level of post-hypertonic hemolysis and the values of such indicators as the median distribution and the coefficient of variation. Thus, an increase in the sensitivity of mammalian erythrocytes to post-hypertonic shock with increasing salt concentration in dehydration medium was usually accompanied by a decrease in the median cell division, and higher values of the coefficient of variation are characteristic of mammalian erythrocytes resistant to post-hypertonic shock.

About the Mechanism of Mammalian Erythrocytes Osmotic Stability

The peculiarities of the effect of hypertonic shock and hypotonic stress on erythrocytes of different species of mammals (human, bull, horse, rabbit, dog, rat) have been investigated. Based on the results of correlation analysis (using the Spearman’s rank correlation coefficient), the relationship between osmotic sensitivity of mammalian erythrocytes and the well-known structural and functional characteristics of these cells was assessed. The paper presents and analyzes the significant relationships. Under hypotonic stress of mammalian erythrocytes, the values of the threshold concentration of NaCl and the one of osmotic fragility were found to correlate with the size of cells (diameter). Under hypertonic shock of mammalian erythrocytes, the values of the threshold concentrations of NaCl and that of hemolysis of cells in a medium containing 4.0 mol/L NaCl correlated with the membrane permeability to water. Mammalian erythrocytes with a high value of the coefficient of diffusion water transport due to the protein channels are more resistant to hypertensive shock.

Transient active osmotic swelling of epithelium upon curvature induction

Generation of tissue curvature is essential to morphogenesis. However, how cells adapt to changing curvature is still unknown because tools to dynamically control curvature in vitro are lacking. Here we developed self-rolling substrates to study how flat epithelial cell monolayers adapt to a rapid, anisotropic change of curvature. We show that the primary response is an active and transient osmotic swelling of cells. This cell volume increase is not observed on inducible wrinkled substrates, where concave and convex regions alternate each other over short distances, identifying swelling as a collective response to changes of curvature with persistent sign over large distances. It is triggered by a drop in membrane tension and actin depolymerization, perceived by cells as a hypertonic shock. Osmotic swelling restores tension while actin reorganizes, probably to comply with curvature. Epithelia are thus unique materials that transiently, actively swell while adapting to large curvature induction.

Impact of amphiphilic compounds on post-hypertonic shock of human erythrocytes

When the cryopreserved erythrocytes are thawed, with the ice melting the extracellular hypertonic medium changes to isotonic one, resulting in post-hypertonic cell lysis development. Under experimental conditions, the post-hypertonic shock of erythrocytes simulates the influence of cryodamage factors, acting at the erythrocyte thawing stage, as well as when the cells, cryopreserved under protection of penetrating cryoprotectant are transferred into bloodstream. Post-hypertonic shock of erythrocytes was carried out by transferring the cells from a hypertonic solution contained 1.65 mol/l NaCl (dehydration medium) into an isotonic one with 0.15 mol/l NaCl (rehydration medium) at 0ºС. The effect of specimens of various classes of amphiphilic compounds (anionic sodium decyl sulfate, non-ionic decyl-β,D-glucopyranoside, and cationic chlorpromazine) on the human erythrocyte sensitivity to post-hypertonic shock, was studied. Amphiphilic substances were supplemented into rehydration medium prior to cell introduction into it. It was shown that under post-hypertonic shock of erythrocytes, all the studied amphiphilic substances, when used in efficient concentrations, manifested a high anti-hemolytic activity (at the level of 70%). A comparative study of the efficiency of amphiphilic substances under post-hypertonic shock of erythrocytes showed differences in size of the plateau (the concentration range of amphiphilic compound, within the limits of which the minimum level of erythrocyte hemolysis was observed). Thus, it was found that for non-ionic decyl-β,D-glucopyranoside the plateau was 3 times more than for anionic sodium decyl sulfate and cationic chlorpromazine. The minimum efficient concentration for sodium decyl sulfate and the maximum one for decyl-β,D-glucopyranoside under post-hypertonic shock of erythrocytes were revealed. It is assumed that the revealed protective effect of amphiphilic compounds under post-hypertonic shock of erythrocytes is associated with their capability to integrate into membrane. This entails an increase in the surface area of the membrane and, therefore, the critical hemolytic volume of cell, which allows it to swell to a larger volume.

Influence of chlorpromazine on the resistance of erythrocytes of rats of different ages to hypertonic conditions

The osmotic stability of native and modified with chlopromazine (CPR) erythrocytes of 1- and 12-month rats to hypertonic conditions in sucrose solutions and hypertonic shock (4.0 M NaCl) has been studied. It has been shown that 2-min incubation of rat erythrocytes of different ages in hypertonic sucrose media does not reveal any differences in the osmotic stability of these cells. In this case, CPR does not affect cell hemolysis. An increase of the incubation time in hypertonic sucrose solutions to 30 minutes allowed detecting a greater osmotic sensitivity of erythrocytes of 1-month animals to the action. In this case, the protective effect of CPR for older age rat erythrocytes (12 months) has been established. It has been found that in 4.0 M NaCl the hemolysis level of animal erythrocytes of both age groups increases with preliminary incubation (2 min) in a sucrose medium with a concentration of 0.7 M and above. With increasing exposure time (30 min) in sucrose hypertonic solutions, the sensibilization of animal erythrocytes of both age groups to the action of hypertonic shock is also intensified. In this study it has been shown that the influence of CPR on the sensitivity of 1-month-old animal erythrocytes to the transfer in 4.0 M NaCl depends on the tonicity and duration of the cell initial incubation in sucrose solutions. Thus, CPR increases the osmotic resistance of erythrocytes, which were preexposed in sucrose solutions at a concentration of 0.6–0.8 M for 2 and 30 min. Some increase of hemolysis level of these animals erythrocytes modified with CPR in 4.0 M NaCl has been observed after 2 min of incubation in sucrose solutions at a concentration of 0.27–0.5 M. The exclusively protective influence of CPR on 12-month-old animal erythrocytes in conditions of hypertonic shock has been revealed. A quantitative estimation of the efficiency of CPR at hypertonic shock (4.0 M NaCl) of different age animal erythrocytes has been carried out by calculation of the antihemolytic activity value (AG). Short-term incubation (2 min) in a sucrose media does not reveal any differences in the values of AG of CPR for erythrocytes of both age groups. For the cells of young rats, increase of AG of CPR is observed at incubation in sucrose medium to 30 min and for erythrocytes of the older group – to 10 min. With an increase in incubation time of up to 60 min the CPR efficiency in hypertonic saline media is reduced for rat cells in both age groups but in varying degrees.