[Potassium transport in erythrocytes from patients with gentamicin intolerance]. / Issledovanie transporta kaliia v éritrotsitakh bol'nykh s lekarstvennoi neperenosimost'iu k gentamitsinu.

A study was made of rubidium (potassium analog) influxes via ouabain-sensitive Na/K pump, bumetanide-sensitive cotransport and resistant to ouabain bumetanide membrane ion pathways and of the intracellular potassium and sodium contents in red blood cells from patients with high gentamicin sensitivity (GSP) and healthy patients (HP). It is found that red blood cells from two groups of donors do not differ in both intracellular potassium and sodium contents and pump-mediated rubidium influxes, however, bumetanide-sensitive rubidium influxes were twice as low in GSP as compared to HP (0.28 against 0.46 mumole per gram of hemoglobin). In the presence of gentamicin (10(-6) M) bumetanide-sensitive rubidium influxes were shown to decrease in red blood cells of GSP, being unchanged in erythrocytes of HP. It is suggested that the increased rates of hemolysis in response to hypotonicity in red blood cells of GSP may be due to a decreased activity of bumetanide-sensitive cotransport in plasma membrane of these cells.

[Comparative study of the functional expression of the Na/K-pump in human lymphocytes, activated by phytohemagglutinin, phorbol ester, ionomycin, and interleukin-2]. / Sravnitel'noe issledovanie funktsional'noi ékspressii Na/K-nasosa v limfotsitakh cheloveka, aktivirovannykh fitogemaggliutininom, forbolovym éfirom, ionomitsinom i interleikinom-2.

Rubidium (Rb) influxes via Na/K pump and ouabain-resistant pathways, and protein, RNA and DNA syntheses have been studied in human blood lymphocytes during the cell transit from quiescence to proliferation. In lymphocytes, stimulated either by phytohemagglutinin (PHA), phorbol 12,13-dibutyrate (PDBu) calcium ionophore, ionomycin, or by PDBu and interleukin-2 (IL-2), the late stages of the G0/G1-->S transit are accompanied by sustained 3-fold increased ouabain-sensitive Rb influxes. Using a two-pulse activation by a brief (1 h) PDBu/ionomycin treatment, followed by incubation with PDBu or IL-2 for 48 h, it has been found that both IL-2 and IL-2 receptor (IL-2R) are necessary for a long-term enhancement of Na/K pump. When present at the early stage of PDBu and ionomycin induction, cyclosporin A (CsA, 1 microgram/ml) inhibits the late increase in pump-mediated Rb influxes. However, when applied after the competence induction, CsA produced no effect on the flux increase at the progression stage. It is concluded that in activated human lymphocytes the functional expression of Na/K pump may by associated with IL-2-dependent progression of competent cells in the cell cycle.

Na/K-pump and the cell response to mitogenic signal: regulatory mechanisms and relation to the blast transformation of human blood lymphocytes.

It has been shown for the human peripheral blood lymphocytes activated with phytohemagglutinin (PHA) that the cell transition from the resting stage to proliferation is accompanied by an increase in the ouabain-sensitive influx of rubidium between the 16th and 48 hours of activation, which is confined to the growth stage and precedes DNA synthesis. The long-term activation of the Na/K-pump is not the result of the increased intracellular sodium concentration, it is inhibited by cycloheximide, actinomycin D, and alpha-amanitin at concentrations sufficient to inhibit the increase in PHA-induced RNA and protein syntheses. It has been shown for the lymphocytes activated by PHA, phorbol ester, ionomycin, and/or interleukin-2 in the presence or absence of cyclosporin A that the Na/K-pump activation, accompanying the human lymphocyte blast transformation, is due to the cyclosporin A-dependent initiation of interleukin-2 expression.

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.

Long-term enhancement of Na,K-ATPase pump during blasttransformation of human lymphocytes is controlled first by translational, then by transcriptional mechanisms.

The transition of phytohemagglutinin-activated human lymphocytes from resting state to proliferation is accompanied by a long-term increase in ouabain-sensitive Rb(K) influx which is closely related to a cyclosporin A-sensitive step of G0/G1/S progression. At least two distinct phases of the up-regulation of cation pump has been revealed: the initial stage (5-20 h) which is cycloheximide-inhibitable and actinomycin D (alpha-amanitin)-unaffected, and the later stage (after 20 h) which is cycloheximide- and actinomycin D (alpha-amanitin)-inhibitable. Thus, the enhanced Na,K-ATPase pump during the cell progression from quiescence to proliferation is controlled both at translational and transcriptional levels.

[The Na,K-ATPase pump and blast transformation of human peripheral blood lymphocytes]. / Na,K-ATFaznyi nasos i blasttransformatsiia limfotsitov perifericheskoi krovi cheloveka.

A transition of phytohemagglutinin (PHA)-activated human lymphocytes from resting state to proliferation is accompanied by an early and a long-term, delayed increase in ouabain-sensitive Rb influx. The long-term (between 16 and 48 h of PHA action) activation of the Na,K-ATPase pump is concomitant with the enlargement of lymphocytes and precedes the onset of DNA synthesis. When inhibiting the protein synthesis in activated lymphocytes, cycloheximide fails to suppress the early rise in Rb influx; however, it abolishes completely the increase in ouabain-sensitive Rb fluxes after 2 h of PHA stimulation. The inhibitors of transcription, actinomycin D and alpha-amantin, in doses suppressing the increase in the PHA-induced RNA synthesis, do not abolish the elevated Rb influx within 20-24 h of PHA action, and inhibit a growth-related increase in Rb influx during the 2nd day of activation. It is concluded that in mitogen-stimulated human lymphocytes the protein synthesis dependent enhancement of Na,K-ATPase pump activity is different in its nature at different phases of G0/G1/S progression. Unlike the early pump stimulation (0.5-2 h), the late elevation of K influxes associated with the growth of lymphocytes, is due to the regulation of the Na,K-ATPase pump at translational (2-20 h) and transcriptional (after 20 h) levels.

Expression of mRNAs encoding the alpha 1 and the beta 1 subunits of Na+, K(+)-ATPase in human lymphocytes activated with phytohaemagglutinine.

Increase in Na+, K(+)-ATPase mRNAs was detected in activated lymphocytes by the RT-PCR method. alpha 1 subunit mRNA gradually increased with time and by 36 h was 2.4 times higher than at the start. Increase in the beta 1 mRNA was transient reaching a maximum in the 8 h probe and declining to the initial level in the 24 and 36 h probes. The elevation of Na+, K(+)-ATPase mRNAs does not underlie a cycloheximide-inhibited increase in cation pumping peculiar to the prereplicative period as can be judged from the fact that Act D fails to eliminate PHA-induced enhancement of pump fluxes.

[The transport and distribution of monovalent cations during the blast transformation of human peripheral blood lymphocytes activated by phytohemagglutinin]. / Transport i raspredeleneie monovalentnykh kationov pri blasttransformatsii limfotsitov perifericheskoi krovi cheloveka, aktivirovannykh fitogemaggliutininom.

Potassium (rubidium) influx, sodium and potassium contents, as well as size distribution, DNA and protein contents and synthesis have been examined in PHA-activated human lymphocytes within 0.5-72 h. A complex set of ionic events was found to include at least two stages of the increase in potassium and sodium contents per g cell protein and in ouabain-sensitive potassium influx which are preceded by a decrease in potassium content by almost 17% within the first 2-5 h. The kinetics of potassium and sodium changes has own pattern for each of cations, thus indicating definite changes in the ouabain-resistant transport of potassium and sodium during the G0----G1----S progression. The late increase in potassium content per g cell protein was found to correlate with the growth in cell size. This finding confirms the rule which was stated earlier for other animal cells, i. e. cells that prepare to proliferate are to raise their potassium per g cell protein up to the level of 0.8-1.0 mmole (Vereninov. Marakhova, 1986).

[The relationship between the cation and protein content, cationic transport and the rate of protein synthesis detected in cells in the presence and in the absence of ouabain]. / Sviaz' mezhdu soderzhaniem kationov, belka, kationnym transportom i skorost'iu sinteza belka, obnaruzhennaia na kletkakh v prisutstvii i v otsutstvii uabaina.

The measurements were made of monovalent cation (K+ + Rb+, Na+) and protein contents, of leucin incorporation into the protein (protein synthesis), and of ouabain-sensitive K+ influx during the growth of the Jurkat culture cells in the presence or in the absence of ouabain. All the data were calculated per cell. The time dependences of these parameters are nonlinear [correction of unline] curves. The results of three independent measurements are not reproduced. The problem was to find out an intracellular "main" parameter, so that the dependences between this and the rest of parameters be of line character. It has been found that it is the protein synthesis rate that may be taken as the main parameter, because between the protein synthesis rate and all the other parameters there is the line connection. Comparative results obtained with or without ouabain showed that K+ and Na+ concentrations in the cell were different, although the constants of the rate of protein synthesis were the same. This points to the same type of change of the protein synthesis process in the cells growing with or without ouabain.

[Heavy water inhibition of alkali cation transport across the muscle membrane. II. A comparison of the action of D20 and ouabain on the sodium efflux and rubidium influx in magnesium media]. / Tormozhenie tiazheloi vody transporta shchelochnykh kationov cherez myshechnuiu membranu. II. Sravnenie deistviia D20 i uabaina na vykhod natriia i vkhod rubidiia v magnievykh sredakh.

The action of heavy water and ouabain on sodium effluxes and rubidium influxes has been measured and compared in frog muscles (m. sartorius, R. temporaria). Approximately half of muscle sodium was substituted by lithium by preliminary incubation in mixed sodium-lithium media. The ratio of the ouabain-sensitive parts of rubidium influx and sodium efflux is 7.3:10.5, and that of D2O-sensitive parts of corresponding parameters is 7.5:11.3. A conclusion is made that D2O-effect on the Na, K-ATPase system of muscles under investigation resembles ouabain-effect on sodium effluxes as well as on rubidium influxes.

[Heavy water inhibition of the transport of alkaline cations across the muscle membrane. I. A comparison of different types of sodium transfer]. / Tormozhenie tiazheloi vodoi transporta shchelochnykh kationov cherez myshechnuiu membranu. I. Sravnenie raznykh vidov perenosa natriia.

The sodium efflux from the frog sartorius muscle into the media of different ion composition, prepared with ordinary and heavy water, was measured by radiotracer and flame-emission techniques. About the half of the sodium in muscles was substituted for lithium. The ouabain-sensitive, as well as external potassium- and external sodium-dependent components of the efflux were found to be totally inhibited in D2O, whereas the residual efflux observed in sodium- and potassium-free magnesium medium was diminished in D2O only by one half. A conclusion is made that the decrease in sodium efflux in D2O is due to the inhibition of sodium transfer through the Na, K-ATPase transport system.

Effect of gramicidin A and thallium ions on cation effluxes in frog sartorius muscle.

The effluxes of potassium, rubidium, sodium and lithium from the sartorius muscle of Rana temporaria in magnesium-Ringer solution free of sodium and potassium have been studied with the flame-emission technique. The channel-forming antibiotic gramicidin A (2.5 X X10(-7)-1 X 10(-6) mol/l) enhanced the efflux of potassium and rubidium and increased the rate constants of these effluxes. Gramicidin had small if any effect on sodium and lithium effluxes and rate constants. After 60-100 min in a gramicidin-containing medium, the potassium efflux and the corresponding rate constant reached a steady-state level. This steady-state value depended on gramicidin concentration. Effect of gramicidin on both the potassium efflux and the rate constant was partially reversible. Thallium ions (2.5 X 10(-3) and 5 X 10(-3) mol/l) in sodium- and potassium- free magnesium Ringer solution caused a large increase in effluxes of all the cations examined (K+, Rb+ and Na+) both in presence and absence of gramicidin. Possible mechanisms of gramicidin and thallium effects are discussed.

[Membrane potential, permeability coefficients and the ratio of the influx/efflux rates for alkaline cations across the muscle fiber membrane of the frog in a bi-ionic system]. / Membrannyi potentsial, koéffitsienty pronitsaemosti i otnoshenie skorostei priamogo i obratnogo perenosa shchelochnykh kationov cherez membranu myshechnykh volokon liagushki v biionnoi sisteme.

Membrane potential and 42K, 22Na, 86Rb fluxes were measured in frog muscles immersed in sucrose--sulfate media with cation of one type. For elimination of the internal sodium and chloride ions muscles were preincubated in sucrose solution. It was found that dependence of membrane potential on the cation concentration deviated from the Goldman--Hodgkin--Katz relation. The variation in the permeability coefficients which should be assumed to correlate to experimental dependence with the Goldman--Hodgkin--Katz formula, were estimated. The required values of the permeability, coefficients were shown to differ from those found by fluxes. The influx/efflux rate constants ratio for all of the cations tested did not obey the Ussing criterion for independent passive movement of ion across membrane. It is concluded that the Goldman--Hodgkin--Katz formula cannot be used for a description of passive ion movement in the systems similar to those studied in this work.

[Use of flame emission analysis for measuring the alkaline cation flux across a cell membrane]. / Primenenie plamenno-émissionnogo analiza dlia izmereniia potokov shchelochnykh kationov cherez kletochnuiu membranu.

Concentration-emission relations are presented for assayed alkaline cations within 1-100 micro M and interfering alkaline cations at concentrations 1-150 mM. All the data were obtained with the Perkin-Elmer AA spectrophotometer (AA 306) and with an air-propane flame. The advantages of flame-emission technique for measurements of cation fluxes across cell membrane are demonstrated on frog muscle and neuroblastoma cells.

[Sodium flux across a muscle fiber membrane in saline media lacking sodium and potassium]. / Issledovanie perenosa natriia cherez membranu myshecknykh volokon v solevykh sredakh, defitsitnykh po natriiu i kaliiu.

Unidirectional sodium fluxes were measured in frog sartorius muscle with radioisotopes and emission techniques. A comparison was made of sodium rate constants, evaluated from the efflux of sodium preexisting in muscle, and from the efflux of 22Na. The rate constants for sodium influx and efflux were determined in solutions in which sodium and potassium were substituted for magnesium, choline and tris. Ouabain inhibits about 40% of sodium efflux investigated. In sodium- and potassium -free media, the rate constant for sodium efflux does not depend on the internal sodium concentration both for ouabaine-resistant and ouabain-sensitive components. The ouabain-sensitive and ouabain-resistant sodium fluxes and operation pump in cation deficient medium have been discussed.

[The "anomalous" relationship between the concentration of potassium in the medium and the membrane potential of muscle fibers with a decreased intracellular potassium concentration. III. Change in the membrane potential during prolonged muscle incubation in saccharose-sulfate media containing 2.5 or 75 mM of potassium]. / Issledovanie "anomal'noi" zavisimosti membrannogo potensiala myshechnykh volokon s ponizhennoi vnutrikletochnoi kontsentratsiei kaliia ot kontsentratsii kaliia v srede. III. Izmenenie membrannogo potentsiala pri dlitel'noi inkubatsii myshts v sakharozno-sul'fatnykh sredakh, soderzhashchikh 2.5 ili 75 mM kaliia

At the external potassium concentration 2.5 mM, The value Em--Ek diminishes, but at 75 mM it increases, although the potassium fluxes are nearly balanced and nosignificant changes in internal potassium occur. The contribution of other ions to the electrogenesis is examined. An attempt is made to describe the movement of these ions by the Goldman equations. The permeability coeficients should have been much higher than potassium coefficient, and besides it should be admitted that the coefficients and the internal activity of ions discussed may vary with time.

[The "anomalous" relationship between the concentration of potassium in the medium and the membrane potential of muscle fibers with a decreased intracellular potassium concentration. I. Movement of potassium, accompanying ions and water]. / Issledovanie "anomal'noi" zavisimosti membrannogo potentsiala myshechnykh volokon s ponizhennoi kontsentratsiei kaliia ot kontsentratsii kaliia v srede. I. Dvizhenie kaliia, soputstvuiushchikh ionov i vody

A study was made of the electrogenesis on (Na+C1)-free muscles in potassiumsulfate media, with the membrane potential not corresponding to Ek. It was found that the potassium content in these muscles may change: at 2.5 mM external potassium, the internal potassium content decreases, whereas at 75 mM it increases. Undoubtedly, some other ions (phosphates?) apart from K+ do penetrate the muscle membrane. The evidence obtained disprove a previous idea that under the condition described it is potassium alone which is involved in potential genesis.