EFFECT OF ERYTHROPOIETIN ON T-LYMPHOCYTES OF THE THYMUS OF RATS IN VITRO AFTER EXPOSURE TO INHIBITORS OF DIFFERENT CLASSES
Abstract
Introduction. Erythropoietin (EPO) – physiological stimulator of erythropoiesis. It activates the mitosis and maturation of red blood cells from progenitor cells erythroid series. One of the main effects of EPO is the slowdown in the rate of apoptosis of erythroid progenitor cells in the bone marrow. Protective properties of EPO demonstrated in various diseases in clinical and experimental conditions. Previously, it was found that activating EPO-effect on T-lymphocytes (TLC) may result not only to the increase of the number of fluorescent mitochondria in cell (nm/c) with proton potential (Δφm), but also external growth of growth of external membrane potential electric potential of plasma (Δφp). The objective of this work was to investigate the TLC response upon EPO after exposure to specific inhibitors of phosphorylation reactions in the respiratory chain. Material and methods. We studied EPO («Eprex», Cilag) effect on rat TLC in vitro after their deenergization by several inhibitors: dinitrophenol (DNP) - uncoupler of oxidative phosphorylation and ingibitor of respiratory chain), pentachlorphenol (PCP)- uncoupler of oxidative phosphorylation, N,N -dicyclohexylcarbodiimide (DCCD)- of Ca2+- inhibitor of the membranebound part of the mitochondrial membrane ATP-ase with the help of a potential-sensitive vital fluorescent probe-cation 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). Rat TLC were isolated from thymuses according to the standard method. The microfluorimetric studies of DSM-stained TLC were performed by means of fluorescent microscope («Lumam I- 2», «LOMO», Russia) with thermostatic table. Fluorescence of 50–70 single cells was measured in each specimen, mean fluorescence intensity of TLC (F ~ ) was calculated. In addition, n m/c was calculated in each fluorescent cell. Statistical processing of the experimental data was performed by the Spearman’s rank correlation coefficient. Results and discussion. In experiments with TLC from different thymuses, we registered a decrease in F ~ and n m/c after incubation with all used inhibitors. It was found that the difference in decrease velocity of nm/c and of F ~ depended on the type of inhibitor and on the duration of incubation. Maximum reduction of energy of the TLC achieved by incubation with a DNF, after which the EPO does not recover F ~ and m/c. After incubation with PCP, EPO restores ~ 20–23 % m/c and F ~ . The reaction of TLC on the DCCD confirms the important role of the ATP-ase in the maintenance of mitochondrial membrane potential. After deenergization of TLC under the action of DCCD, EPO has the maximum effect: restored ~ 42 % n m/c and ~ 38 % F ~ . Conclusions. EPO is able to partially recover the polarization of the mitochondria membranes in TLC violated as a result of exposure to DCCD.
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