The purpose of the work was to conduct a comparative analysis of the sensitivity of human and equine erythrocytes to hypertonic shock (4.0 mol/L NaCl) in conditions of the combined action of temperature, preliminary partial dehydration of cells and chlorpromazine. Material and methods. Erythrocytes were obtained from donor blood by conventional methods. All media were prepared on 0.01 mol / l phosphate buffer, pH 7.4. Chlorpromazine from Calbiochem was used in the study. Hypertensive shock was performed as follows: using a Gilson piston dispenser, 50 μl of erythrocyte pellet was transferred into 0.5 ml of NaCl solution (0.15 mol / l or 0.4 mol / l) and incubated for 2 min at 37 or 0°C. (pre-incubation step). Then from each sample 50 μl of erythrocyte suspension was transferred into 1 ml of 4.0 mol / l NaCl (stage of hypertensive shock) containing chlorpromazine, at a constant temperature (37 or 0°C). The cells were then pelleted by centrifugation and the amount of hemoglobin in the supernatant was determined spectrophotometrically at a wavelength of 543 nm. Results and discussion. The study results showed that the lowest level of hypertonic hemolysis of control human and equine erythrocytes ( with no chlorpromazine) was observed with the combined use of preliminary incubation of cells in 0.4 mol/L NaCl and low temperature. In all the cases, the level of hypertonic hemolysis of the control equine cells was lower than that of human erythrocytes. This fact suggests that equine erythrocytes were more resistant to hypertonic shock. It is known that, unlike many mammal erythrocytes, the cells of horse are deprived of one of the components of the cytoskeletal complex, i.e. band 4.2 protein. This protein binds to the cytoplasmic domain of the band 3 protein and interacts with ankyrin in human erythrocytes. The compositional feature of the equine erythrocyte cytoskeleton-membrane complex is responsible for their higher resistance to the action of hypertonic shock if compared to human cells. The use of chlorpromazine reduced the level of hypertonic damage to human and equine erythrocytes, initially incubated in a medium containing both 0.15 mol/L and 0.4 mol/L NaCl at 37 and 0°С. Under these conditions, the values of the maximum anti-hemolytic activity of chlorpromazine are quite high and are in the range of 38-83%. Despite the different level of initial damage to erythrocytes (transferred to 4.0 mol/L NaCl from 0.15 mol/L or 0.4 mol/L NaCl) and the values of minimal hemolysis in the chlorpromazine presence, the effectiveness of the substance is similar for human and equine cells at 37°C and differs at 0°C. Under low temperature, the antihemolytic activity of chlorpromazine when using human cells was by 1.2-1.4 times higher. Conclusion. The distribution of chlorpromazine molecules in plasma membrane of erythrocytes is heterogeneous. The plasma membranes of erythrocytes of various mammal species differ in lipid composition. Perhaps at 0°C, the sites of building-in of amphiphilic molecules into the membrane change due to the structural transition of certain lipids at 0°C and differ for human and horse erythrocytes that lead to various manifestations of the antihemolytic activity of the test substance at low temperature.
Keywords: human and equine erythrocytes, hypertonic shock, partial dehydration, temperature, chlorpromazine
Full text: PDF (Ukr) 195K