[Ultrastructural and X-ray spectral analysis of cells U-937 during apoptosis process induced by hypertony].

The results of this work concerning ultrastructural changes of U-937 cells in a state of apoptosis are largely in consistent with the same information available in the literature. However, we have got the original data on the ultrastructural changes of cell organelles and immune localization and distribution of proteasomes. It has been demonstrated that Golgi apparatus is located close to the plasma membrane in the case of apoptosis induced by incubating the cells in a hypertonic suchrose solution (200-400 mM). The fact can be considered as an indirect indication of depolymerization of cytoskeletal elements, in particular, MTs maintaining Golgi apparatus in a cell centre. In the later stages of apoptosis, the distances between Golgi cisterna are significantly increased. It can be explained by hydrolysis of golgins binding cisterna between each other. Mitochondria are not significantly changed in these cells. They have regularly disposed crista and sufficiently dense matrix with a few vacuoles. Proteasomes as rod-shaped osmiophilic particles (12 x 30 nm) have been revealed during each apoptosis stage both in nuclei and cytopl;asm of cells studied. The particles form aggregates of different densitities and sizes unlimited by membrane. It has been proposed that the particle aggregates revealed in the work are analogous to "processing bodies" or aggresomes described in the literature. They can be detected in cells under conditions of suppressed nucleus transcriptional processes in the nucleus and participate in storing and degradation of various mRNAs, RNP and proteins. The changes of intracellular contents of Na and K in a single cell during apoptosis induced by osmotic shock have been revealed using method of X-ray microanalysis. It has been demonstrated the increase in the ratio of intracellular contents Na+/K+ in the most of apoptotic cells in comparing with control cells.

[Li/Na exchange and Li active transport in human lymphoid cells U937 cultured in lithium media].

Lithium transport across the cell membrane is interesting in the light of general cell physiology and becau- se of its alteration during numerous human diseases. The mechanism of Li4 transfer has been studied mainly in erythrocytes with a slow kinetics of ion exchange and therefore under the unbalanced ion distribution. Prolife- rating cultured cells with a rapid ion exchange have not been used practically in study of Li4 transport. In pre- sent paper, the kinetics of Li4 uptake and exit as well as its balanced distribution across the plasma membrane of U937 cells were studied at minimal external Li+ concentrations and after the whole replacement of external Na+ for Li+. It has been found that a steady state Li+ distribution is attained at a high rate similar to that for Na+ and Cl- and that Li+/Na+ discrimination under the balanced ion distribution at 1-10 mM external Li+ keeps on 3 and drops to 1 following blocking of the Na,K-ATPase pump by ouabain. About of 80% of the total Li+ flux across the plasma membrane under the balanced Li+ distribution at 5 mM external Li+ accounts for the equiva- lent Li+/Li+ exchange. The most part of the Li+ flux into the cell down the electrochemical gradient is a flux through channels and its small part may account for the NC and NKCC cotransport influxes. The downhill Li+ influxes are balanced by the uphill Li+ efflux involved in Li+/Na+ exchange. The Na+ flux involved in the countertransport with the Li+ accounts for about 0.5% of the total Na+ flux across the plasma membrane. The study of Li+ transport is an important approach to understand the mechanism of the equivalent Li+/Li+/Na+/Na+ exchange because no blockers of this mode of ion transfer are known and it cannot be revealed by electrophysiological methods. Cells treated with the medium where Na+ is replaced for Li+ are recommended as an object for studying cells without the Na,K-ATPase pump and with very low intracellular Na+ and K+ concentration.

[Integration of monovalent ion fluxes across plasma membrane under the balanced state: ion gradients and water balance in animal cells].

Interrelationships between monovalent ion transport, the membrane potential, and intracellular ion and water content of an animal cell are analyzed by computation of the balance of ion fluxes across the plasma membrane when the sodium pump and electroconductive channels operate in parallel with NKCC or Na-Cl (NC) symport. The behavior of the system is described in terms of the integral permeability of Cl- channels, the activities of NKCC or NC symport and two dimensionless parameters characterizing the activity of the Na/K pump relative to that of electroconductive channels. The latter two parameters are shown to largely determine the plasma membrane potential and intracellular concentration of K+ and Na+ whereas NC and NKCC symports and the permeability of Cl- channels regulate cell water balance and only slightly influence the intracellular K+/Na+ ratio and membrane potential.

[Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells placed into hyperosmotic medium].

Time course of changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium with addition of 200 mM sucrose was studied. Ouabain-sensitive and ouabain-resistant Rb+ (K+) influxes were measured during regulatory cell volume increase (RVI) and apoptotic volume decrease (AVD). Microscopy of cells stained by Acrydine orange, Ethydium bromide, APOPercenrage Dye and polycaspase marker FLICA was performed. We found that initial osmotic cell shrinkage induced both RVI and AVD responses. RVI dominated at the early stage whereas AVD prevailed at the later stage. In view of the data obtained in U937 cells the current opinion that RVI "dysfunction" is a prerequisite for apoptosis and AVD (Subramanyam et al., 2010) should be revised. U937 cells are capable to trigger of apoptosis and AVD in spite of the unimpaired RVI response. It is concluded that AVD plays a significant role in preventing osmotic lysis of apoptotic cells rather than in the initiation of apoptosis.

[Changes in K+, Na+ and Cl- contents and K+ and Cl- fluxes during apoptosis of U937 cells by staurosporine. On the mechanism of cell dehydration in apoptosis].

K+, Na+ and Cl- balance and K+ (Rb+) and 36Cl fluxes in during apoptosis of U937 cells caused by 0.2 or 1 microM staurosporine were studied by flame emission and radiotracer techniques. It is found that monovalent ion redistribution accounts for 2/3 of all decrease in the amount of intracellular osmolytes in apoptotic cells while 1/3 is due to the loss of other intracellular osmolytes. Na+ gain in apoptotic cells hampers dehydration is caused by K+ and Cl- loss. It is found that the rate of equilibration of 36Cl, Rb+ (K+) and 22Na+ between cells and the medium exceeds significantly the rate of alteration of cell ion content associated with apoptosis. It is concluded that apoptotic changes should be considered as a drift of the balanced ion distribution. Alteration of the ion balance in apoptosis, caused by 0.2 microM staurosporine, is associated with an increase in the uabain-resistant Rb+ (K+) "channel" influx and insignificant alteration of the uabain-sensitive "pump" influx. Stronger apoptosis, induced by 1 microM staurosporine, is associated with a decrease in the pump fluxes and insignificant changes in the "channel" Rb+ (K+) fluxes. Decreasing of the Cl- level in apoptotic cells by a factor 1.4-1.8 is accompanied with a decrease in the flux, by a factor 1.2-1.6.

[Computation of the K+, Na+ and Cl- fluxes through plasma membrane of animal cell with Na+/K+ pump, NKCC, NC cotransporters, and ionic channels with and without non-Goldman rectification in K+ channels. Norma and apoptosis].

The balance of K+, Na+ and Cl- fluxes through cell membrane with the Na+/K+ pump, ion channels and NKCC and NC cotransporters is considered. It is shown that all unidirectional K+, Na+ and Cl- fluxes through cell membrane, permeability coefficients of ion channels and membrane potential can be computed for balanced ion distribution between cell and the medium if K+, Na+ and Cl- concentration in cell water and three fluxes are known: total Cl- flux, total K+ influx and ouabain-inhibitable "pump" component of the K+ influx. Changes in the mortovalent ion balance in lymphoid cells U937 induced to apoptosis by 1 microM staurosporine are analyzed as an example. It is found that the apoptotic shift in ion and water balance in studied cells is caused by a decrease in the pump activity which is accompanied by a decrease in the integral permeability of Na+ channels without significant increase in K+ and Cl- channel permeabilities. Computation shows that only a small part of the total fluxes of K+, Na+ and Cl- accounts for the fluxes via NKCC and NC cotransporters. Therefore, cotransport fluxes can not be studied using inhibitors.

[Cell shrinkage during apoptosis is not obligatory. Apoptosis of U937 cells induced by staurosporine and etoposide]. / Degidratsionnoe sokrashchenie ob"ema kletok pri apoptoze--fakul'tativnyi priznak. Apoptoz kletok U937, vyzvannyi staurosporinom i étopozidom.

A study was made of apoptotic cell shrinkage, which is generally believed to be a hallmark of apoptosis. The two conventional models of apoptosis were used for examination of changes in cell water balance--one is apoptosis caused in human lymphoma cell line U937 by staurosporine, and the other by etoposide. Intracellular water was determined by measuring buoyant density of cells in continuous Percoll gradient. Apoptosis was recognized by microscopy and flow cytometry. Apoptosis caused by staurosporine (1 microM, 4 h) was found to be associated with a decrease in cell water content by almost 24%. In contrast, no decrease in cell water content was observed in U937 cells incubated with etoposide (50 microM, 4 h), in spite of the number of features suggesting the presence of apoptosis, such as the appearance of apoptotic bodies, chromatin condensation and fragmentation and disappearance of S-phase cells in DNA histogram. It is concluded that definition of apoptosis as "shrinkage-necrosis" (Kerr, 1971) needs correcting: the distinction of apoptotic cells involves the absence of swelling, rather than cell shrinkage.

[Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Ion-osmotic model of cell volume decrease]. / Vodnyi i ionnyi balans timotsitov krysy pri apoptoze, vyzvannom deksametazonom i étopozidom. Iono-osmoticheskaia model' umen'sheniia ob'ema kletki.

Cell ion and water balance was studied with respect to analysis of the osmotic model of apoptotic volume decrease (AVD) in rat thymocytes under dexamethasone (1 microM, 4-6 h) or etoposide (50 microM, 5 h) treatment. Intracellular water content was determined by measurement of cell buoyant density in continuous Percoll gradient, while intracellular potassium and sodium contents were determined by flame emission analysis. Apoptosis was verified by an increase in cell buoyant density, fluorescence of cells stained with Acridine orange and Ethidium bromide (flow cytometry), by changes in the cell cycle and the appearance of sub-diploid peak in the DNA histogram (flow cytometry), and by a decrease in cell size examined with light microscope. A separate fraction of dense cells with reduced size was found to appear after dexamethasone or etoposide treatment. This fraction was considered as apoptotic. An increase in buoyant density of apoptotic cells corresponded to a decrease in cell water content. In apoptotic cells vs. cells with normal buoyant density, the intracellular potassium content was lower, but sodium content was higher. The sum of potassium and sodium contents was lower in apoptotic cells. Taken into account the loss of anions, associated with the loss of cations, the bulk decrease in ions content has been sufficient to be accounted for cell volume decrease on the basis of the ion-osmotic model.

[Study of "group" expression of mRNA of the ion transporters ATP1B1, NHE1 and NKCC1, beta-actin, glycerophosphosphate dehydrogenase, proteins regulating proliferation and apoptosis of p53, Bcl-2, IL-2 and hSGK kinase at the prereplicative stage of human lymphocyte activation]. / Issledovanie "gruppovoi" ékspressii mRNK ionnykh transporterov ATP1B1, NHE1 i NKCC1, beta-aktina, glitserofosfatdegidrogenazy, belkov--reguliatorov proliferatsii i apoptoza p53, Bcl-2, IL-2 i kinazy hSGK na predreplikativnoi stadii aktivatsii limfotsitov cheloveka.

Previously, we found no segregation in F2 obtained from crosses between two Dileptus anser clones differing (under the same culture conditions) in their serotypes, i.e. in their immobilization antigens (i-antigens); indeed, all the F2 clones had mixed, i.e. hybrid serotype, being immobilized simultaneously with both immune sera developed against either parental clone (Uspenskaya, Yudin, 2000). Presently, experiments were carried out to see if this unusual phenotype would be re-expressed after a temporary switching off. To switch off both expressed i-antigens, serotype transformation was induced in the F2 clones by shifting the culture temperature from 25 to 17 degrees C. Two weeks later, when the clones returned to the initial temperature conditions, each of them was seen to re-express both parental i-antigens. This result is discussed with reference to the role of i-antigens in regulation of their own expression as has been suggested by some authors.