[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.

Analysis of the monovalent ion fluxes in U937 cells under the balanced ion distribution: recognition of ion transporters responsible for changes in cell ion and water balance during apoptosis.

Unidirectional (22)Na, Li(+) and Rb(+) fluxes and net fluxes of Na(+) and K(+) were measured in U937 human leukemic cells before and after induction of apoptosis by staurosporine (1 microM, 4 h) to answer the question which ion transporter(s) are responsible for changes in cell ion and water balance at apoptosis. The original version of the mathematical model of cell ion and water balance was used for analysis of the unidirectional ion fluxes under the balanced distribution of major monovalent ions across the cell membrane. The values of all major components of the Na(+) and K(+) efflux and influx, i.e. fluxes via the Na(+),K(+)-ATPase pump, Na(+) channels, K(+) channels, Na/Na exchanger and Na-Cl symport were determined. It is concluded that apoptotic cell shrinkage and changes in Na(+) and K(+) fluxes typical of apoptosis in U937 cells induced by staurosporine are caused by a complex decrease in the pump activity, Na-Cl symport and integral Na(+) channel permeability.

[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.

Differential transcription of ion transporters, NHE1, ATP1B1, NKCC1 in human peripheral blood lymphocytes activated to proliferation.

This work, using RT PCR, studied expression of mRNAs encoding ion transporters, the Na/H antiporter (NHE1), the beta subunit of the Na,K-ATPase pump (ATP1B1), the NaK2Cl symporter (NKCC1), and some proteins unrelated to ion transport: the serum and glucocorticoid dependent kinase (hSGK), beta-actin, a glycolytic enzyme (GAPDH), and regulators of proliferation and apoptosis (p53, Bcl-2) during activation of human lymphocytes with phytohemagglutinin for 4-24 h. Within 24 hours the mRNA levels of NHE1, beta-actin, Bcl-2, and p53 increased by more than 100%, the mRNA levels of ATP1B1, GAPDH, and hSGK, by about 50%, while the mRNA levels of NKCC1 decreased transiently. These results indicate a differential transcriptional control of NHE1, ATP1B1, and NKCC1 following a proliferative stimulus of human lymphocytes.

The role of actin cytoskeleton in the generation of surface oscillations of red blood cell ghosts.

We have studied the effects of three compounds on surface oscillations of human red blood cell ghosts: the P-ATPase inhibitor, suramin; the fluorescent dye of a similar structure, 1,8-anilinonaphthalene sulfonate (ANS); and subfragment 1 of skeletal muscle myosin (S1). It has been found that suramin (10 microM), ANS (100 microM) and S1 (2 mg/ml) suppress the surface oscillations reversibly. The shape of the ghosts remains unchanged. We have also found that suramin and ANS inhibit the ghosts' non-transport (presumably, F-actin-associated) ATPase. The results of the present study suggest the important role of actin ATPase in the generation of cell surface oscillations. The effect of S1, the protein which increases the torsional, but not the bending, rigidity of F-actin upon binding to filaments, favours the possibility that just the torsional dynamics of actin protofilaments leads to the observed oscillations of the ghosts' surface.

Functional expression of the Na/K pump is controlled via a cyclosporin A-sensitive signalling pathway in activated human lymphocytes.

An immunosuppressant cyclosporin A (CsA) inhibits T-cell proliferation by blocking the nuclear factor of activated T-cells (NFAT) required for expression of the interleukin-2 (IL-2) gene. This work has demonstrated for the first time that in human blood lymphocytes (HBLs) activated by phytohemagglutinin (PHA), CsA at anti-proliferative doses inhibits the late sustained increase in ouabain-sensitive Rb(K) influxes, which accompanies the growth phase of G0/G1/S transition. CsA affects neither the initial, transient activation of the pump in response to PHA nor the ouabain-resistant ion fluxes during cell cycle progression. When the HBLs were rendered competent to proliferate by phorbol 12,13-dibutyrate ester and ionomycin in the presence of CsA, the exogenous IL-2 did not bypass the initial inhibitory effect of CsA on the long-term pump enhancement. When applied after the competence induction, CsA produced no effect on the sustained increase in ouabain-sensitive Rb influxes during the IL-2-induced progression phase. These results indicate that in activated HBLs, (1) IL-2 is involved in functional expression of the Na/K pump during cell transition from quiescence to proliferation, (2) the cell cycle-associated upregulation of the pump is related to a CsA-sensitive signalling pathway.

Na, K-ATPase pump in activated human lymphocytes: on the mechanisms of rapid and long-term increase in K influxes during the initiation of phytohemagglutinin-induced proliferation.

Functional expression of Na, K-ATPase pump as determined by ouabain-sensitive Rb influxes has been investigated in human peripheral blood lymphocytes, activated by phytohemagglutinin (PHA) from resting state to proliferation. It is found that a rapid twofold elevation of ouabain-sensitive Rb influx in response to PHA is followed by a long-term increase in pump activity, which precedes the DNA synthesis and is temporally related to the growth phase of mitogenic response. Unlike the early pump activation, the late enhanced pump activity is not the result of elevated cell Na content, it is inhibited by cycloheximide and requires new protein synthesis. Actinomycin D and alpha-amanitin, in doses, which suppress the PHA-induced increase in the RNA synthesis, do not abolish the elevated Rb influx until 20-24h of mitogenic activation and inhibit the late, growth-associated increase in Rb influx. It is concluded that (1) in mitogen-activated cells both short- and long-term control is involved in the enhanced pump activity, and (2) translational and transcriptional mechanisms may contribute to the long-term up-regulation of Na, K-ATPase pump during blast transformation of human lymphocytes.

Cyclosporin A inhibits long-term activation of Na+,K+ pump in phytohemagglutinin-stimulated human lymphocytes.

The effect of the immunosuppressive drug cyclosporin A (CsA) on the K (Rb) influx, intracellular K and Na contents, and on the major parameters of lymphocyte activation have been investigated in human peripheral blood lymphocytes activated by phytohemagglutinin (PHA). CsA suppressed protein, RNA, DNA syntheses and cell proliferation by 49.8 +/- 4.3, 67.6 +/- 10.1, 60.4 +/- 5.3 and 60.0 +/- 5.1%, respectively (n = 10) within 48 h. It also inhibited the late long-term Na+,K+ pump activation, as determined from the ouabain-sensitive Rb uptake, and prevented the increase in the intracellular K content at the late stages of G0/G1/S progression. Cyclosporin A did not affect the early transient pump activation, the dynamics, of ouabain-resistant influxes and the intracellular Na content in PHA-activated lymphocytes. When added 1 h after PHA, CsA neither affected the activation of the pump-mediated Rb influxes nor the increase in the intracellular K content. It is concluded that in activated human lymphocytes, the long-term activation of Na+,K+ pump associated with the mitogen-induced blast transformation, as well as the late increase in K content depend on the T-cell growth factor interleukin-2.

[The effect of NaK2Cl symport and chloride channel permeability on ion flux balance and on transmembrane ion distribution in different types of animal cells]. / Vliianie simporta NaK2Cl i pronitsaemosti khlornykh kanalov na balans ionnykh potokov i transmembrannoe raspredelenie ionov u zhivotnykh kletok raznogo tipa.

The relationships between monovalent ion fluxes via major cell membrane pathways (Na/K pump, NaK2Cl symporter, electroconductive sodium, potassium and chloride channels) and steady state transmembrane ion distribution, membrane potential and cell water content were calculated for the high potassium animal cells with high and low membrane potential. It is found that variation in NaK2Cl symport or chloride electroconductive permeability causing changes in cell water content of high magnitude do not lead to significant changes in the intracellular Na/K ratio or membrane potential, in contrast to the effects caused by variation in the Na/K pump fluxes or permeability of the Na and K channels. It is shown that water content in cells with a high membrane potential, e.g. of about 70 mV, cannot be increased due to an increase in NaK2Cl symport by more than 1.6 times. In cells with a low membrane potential an increase in symport leads to a decrease in water content, which is also limited. In cells with membrane potential of about 10 mV the water content cannot be decreased more than by 1.8 times. When NaK2Cl symporter is operating, the effect of chloride channel permeability on the ion and water balance is quite opposite to the symport and is limited by the same boundaries. It is shown that effects caused by changes in symport and in chloride permeability can be differentiated only by the analysis of kinetic (fluxes, transport rate constants etc.) but not "static" characteristics of ion distribution. It is shown that under some circumstances the influence of NaK2Cl symport and chloride channel permeability on ion and water balance can be strong even at a very small symport share in the overall flux.