[The STAT5 signaling in the expression of alpha-subunit of interleukin-2 receptor in human blood lymphocytes].

The comparative study of the STAT3 and STAT5 activity (as assessed by tyrosine phosphorylation level) and the expression of a α-subunit of interleukin-2 receptor (as examined by cytophotometric evaluation of the number of CD25+ cells) during the phytohemagglutinin (PHA)-induced proliferation of human blood lymphocytes (HBL) have been made. It has been revealed that the level of STAT3 phosphorylation is high in both res ting and competent HBL and remains unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen in both resting and competent HBL. We observed phosphorylation of STAT5 no earlier than 5 h after PHA stimulation and the maximum phosphorylation was detected following 24 h. Exogenous IL-2 induced high level of STAT5 phosphorylation in the competent HBL as early as at 30 min and this level of STAT5 phosphorylation kept in the next 24-48 h. The correlation between alterations in tyrosine phosphorylation level of STAT5 and the expression of CD25 has been established. WHI-P131, an inhibitor of JAK3 kinase, prevents STAT5 activation, cell surface expression of CD25 and lymphocyte proliferation. It has been concluded that JAK3/STAT5 signaling via IL-2 receptor is necessary to maintain the long-term expression of the high-affinity αßγ(c)-receptor of IL-2 and optimal proliferation of HBL.

[The functional characteristics oF TRPV5 and TRPV6 channels in normal and transformed human blood lymphocytes].

Calcium signaling and Ca(2+)-conducting channels are involved in the development of immune response, cell proliferation, growth and differentiation of lymphocytes. In this paper the calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) were studied in the plasma membrane of T cell line Jurkat and normal human blood lymphocytes. The channels were activated by removing Ca2+ and Mg2+ from surrounding solution, characterized by inward rectification, and were inactivated by the effective blocker of TRPV5 and TRPV6, ruthenium red. Channel activity was significantly higher in Jurkat cells, than in normal human lymphocytes. Quantitative PCR analysis revealed higher levels of mRNA genes encoding channels TRPV5 and TRPV6 in the proliferation cells compared with resting lymphocytes. In general these data showed that TRPV5/TRPV6 in human lymphocytes are functionally active, and their activity is associated with proliferative status of blood cells.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. I. The role of JAK and Src tyrosine kinases as revealed by inhibitory analysis].

The long-lasting expression of an alpha-subunit of interleukin-3 receptor (IL-2Ralpha) was found to accompany the PHA-induced proliferation of human blood lymphocytes (HBL), so that to the end of the second day of mitogenic stimulation only, the large blasts may express the high affinity alphabetagamma(c) receptor for IL-2. With the selective pharmacological drugs to JAK (WHI-P131) and Src (PP2) it is shown that the non-receptor tyrosine kinases are involved in the surface CD25 expression. It is revealed that the PP-2-inhibitable expression of CD25 is timely associated with the initial stage of T cell activation, whereas WHI-P131-inhibitable expression was present during the whole G0/G1/S transition. These data indicate that at the early, antigen-dependent stage the expression of IL-2Ralpha is induced via Src-dependent signaling pathway, and prolonged increase in IL-2Ralpha expression is regulated by IL-2/IL-2 receptor interaction via JAK-dependent signaling pathway.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. II. The role of interleukin-2].

The expression of alpha-subunit of interleukin-2 receptor (IL-2Ralpha) was assessed by quantifying activation-induced upregulation of CD25 in human blood lymphocytes (HBL) stimulated by interleukin-2 (IL-2). It was established that exogenous IL-2 induced no surface expression of CD25 neither proliferation at 48 h of IL-2 action. In component HBL, pretreated by sub-mitogenic doses of phytohemagglutinin (PHA), 5-15 % of cell population was revealed to represent the CD2t+ cells, and in the competent cells only, exogenous IL-2 induced the surface expression of CD25 as well as the growth and the proliferative response, which was comparable with those to mitogenic doses of PHA. The JAK3 inhibitor WHI-P131 eliminated IL-2-dependent CD25 expression without influencing the CD25 expression in competent cells. Unlike, PP2 was found to inhibit the IL-2-dependent CD25 expression in a lesser extent than WHI-P131, however this drug was effectively inhibited CD25 expression in PHA-pretreated, competent HBL. These data suggest that Src-dependent signaling participate in the early IL-2Ralpha expression that precedes the IL-2-dependent cell cycle progression of activated HBL. It is concluded that in normal T cells, the IL-2Ralpha expression in firstly induced by antigen (mitogen) and thereafter it is held IL-2 through JAK-dependent signaling pathway.

[Specific function of STAT5 in regulation of proliferation of chronic leukemia K562 cells: inhibitory effect of WHI-P131].

In this study, we examined the possible role of JAK/STAT signaling pathway in regulation of proliferation of chronic leukemia cells K562. The thyrosine phosphorylation of STAT3 and STAT5 was used as a marker of an activation status of STAT proteins. We demonstrate that in growing culture of K562 both STAT3 and STAT5 are constitutively activated. To determine the significance of STATs activity in maintaining the high level of K562 proliferation we tested two JAK inhibitors: AG-490 (JAK2 and JAK3 inhibitor) and WHI-P131 (a new specific JAK3 inhibitor). We showed that in long-tern cultures (48 h) of K562 cells with AG-490 or WHI-P132 the cells remain viable. It was found that treatment with WHI-P131 (30-100 microM) decreased the thyrosine phosphorylation of STAT5 being without effect on the high level of STAT3 phosphorylation. In proliferating K562 cells, AG-490 (25-50 microM) did not influence STAT3 and STAT5 phosphorylation. The flow cytometry analysis revealed a dose-dependent decrease in G1 and S phases and an increase in G2/M phases in WHI-P131-treated K562 cell cultures and no changes in cell cycle structure in AG-490-treated cells. Thus, our findings indicate a preferential role for STAT5 (not constitutively active STAT3) in proliferation of leukemia to other JAK drugs which stimulate apoptosis and decrease proliferation, WHI-P131 prevents K562 cells growth by arresting in G2/M phases of cell cycle.

[The expression of CD25 in phytohemagglutinin- or interleukin-2-stimulated human peripheral blood lymphocytes].

The timely expression of a high affinity receptor for interleukin-2 (IL-2R) in human peripheral blood lymphocytes stimulated by various mitogens was examined by cytophotometric evaluation of the number of CD25 bearing cells (CD25+). In resting lymphocyte culture both phytohemagglutinin (PHA, 10 (microg/ml) and 12,13-phorbol dibutirate (PDBu, 10-8 M) and ionomycin (IM, 5 x 10(-7) M) induce the long-lasting increase (during 48 h) in the number of CD25+ cells. Only in competent (not in resting) lymphocytes, pretreated by submitogenic doses of PHA (1 microg/ml), interleukin-2 (IL-2) is capable to induce the time-dependent CD25 expression. When comparing the time course of CD25+ expression and the blasttransformation it was found that the CD25 markers were revealed on small stimulated lymphocytes and all the large blasts were the CD+ cells with high-affinity alphabetagamma(c) receptor for IL-2. In conclusion, the expression of alpha-subunit of IL-2R takes place during the IL-2-dependent stage of T cell proliferation and may be directly induced by IL-2 via IL-2R.

[Long-term regulation of Na+, K+-ATPase pump in human lymphocytes: the role of JAK/STAT- and MAPK-signaling pathways].

In interleukin-2 (IL-2)-induced human blood lymphocytes, the Na+/K+ pump function (assessed by ouabain-sensitive Rb+ influx), the abundance of Na+, K+-ATPase alpha1-subunit (determined by Western blotting) and the alpha1- and beta1-subunits mRNA of Na+, K+-ATPase (RT-PCR), as well as the phosphorylation of STAT5 and STAT3 family proteins and ERK1/2 kinase have been examined. A 3.5-4.0-fold increase in the expression of alpha1- and beta1-subunits mRNA of Na+, K+-ATPase was found at 24 h of IL-2 stimulation. The inhibitors of JAK3 kinase (B-42, WHI-P431) was shown to decrease both the phosphorylation of STATs and the rise in the oubain-sensitive rubidium influx as well as the increased abundance of Na+, K+-ATPase alpha1-subunit. The inhibition of the protein kinases ERK1/2 by PD98059 (20 microM) suppressed the alpha1-subunit accumulation. All the kinase inhibitors tested did not alter the intracellular content ofmonovalent cations in resting and IL-2-stimulated lymphocytes. It is concluded that MAPK and JAK/STAT signaling pathways mediate the IL-2-dependent regulation of the Na+, K+-ATPase expression during the lymphocyte transition from resting stage to proliferation.

[TRPV5 and TRPV6 calcium channels in human T cells].

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) provided the molecular base for the studying of new candidate of calcium influx in non-excitable cells. Using RT-PCR technique we obtained endogenous expression of the mRNAs trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in human blood primary T lymphocytes. Additionally, Western blot analysis showed TRPV5 proteins in both the whole lysate and in the crude membrane preparations from Jurkat cells and normal T lymphocytes. The using of the immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes TRPV6 was not detected. The expression pattern and the selective Ca2+ permeation properties of TRPV5 and TRPV6 channels indicate an important role of these channels in the Ca2+ homeostasis and probably in malignant transformation of blood cells.

[Activation of transcriptional factors stat5 and stat3 during human T-lymphocyte proliferation induced by different mitogenic stimuli].

The activation of closely related transcription factors STAT5alpha and STAT5beta is one of the key signalling events during interleukin-2-dependent stage of T-lymphocyte proliferation. Here, we investigate the activation of STAT5 and STAT3 family proteins on different stages of T-cell proliferative response to various mitogenic stimuli. The activation of STAT proteins was estimated according to their tyrosine phosphorylation. Isolated peripheral blood lymphocytes were stimulated by phytogemagglutinin (PHA), or by phorbol-12,13-dibutyrate (PDBu) with calcium ionophore--ionomycin, or by exogenous interleukin-2 (IL-2). After activation of T-cells by PHA as well as by PDBu with ionomycin we observed phosphorylation of STAT5 not earlier than in 5 h. The maximum phosphorylation (by 10 times and more, as compared to control resting lymphocytes) was detected following 24 h of mitogen stimulation. In the lymphocytes pretreated by PHA in submitogenic concentration, exogenous IL-2 induces a proliferative response. High phosphorylation level of STAT5 was determined 10 min after addition of IL-2 and kept for the next 24 h. In contrast to STAT5, some basal phosphorylation of STAT3 was found in resting and PHA-pretreated T-cells. The profile of phosphorylation STAT3 during mitogen-induced activation is more leveled. An immunosuppressant cyclosporine A taken at antiproliferative concentration decreased phosphorylation of both STAT5 and STAT3 by 1.5-2.0 times at early stages, as well as at late stages of activation. A correlation between alteration in tyrosine phosphorylation level of STAT5 and the expression of the high affinity IL-5 receptor was established. Taken together, we report that the increased STAT5 phosphorylation can serve as a marker of T-lymphocyte entrance into IL-2-dependent stage of proliferation after T-cell activation by different mitogens.

[Il-2-regulated expression of Na+,K(+)-ATPase in activated human lymphocytes].

Expression of Na+,K(+)-ATPase alfal-subunit and of oubain-sensitive rubidium influxes has been investigated in human peripheral blood lymphocytes. Isolated lymphocytes were stimulated by phytogemagglutinin (PHA) or interleukin-2 (IL-2). It has been shown that during the early stage of the PHA-activation the alfal-subunit abundance in the membrane fractions of the human blood lymphocytes does not change, whereas at the late stages of Go-->G1-->S transition (16-48 h) the alfa1 protein content increases. A translation inhibitor cycloheximide was found to prevent the late increase in alfa1-subunit expression. An immunosuppressant cyclosporin A decreases both IL-2-dependent T-lymphocyte progression and alfa1-subunit abundance by 48 h of PHA-induced lymphocyte activation. In the lymphocytes pretreated by PHA in submitogenic concentration (0.8-1.0 microg/ml) exogenous IL-2 (100 U/ml) induces a proliferative response as well as alfal-protein accumulation. A decrease in alfa1-protein accumulation in the presence of specific inhibitors of separate signal transduction pathways enables us to conclude that protein kinases ERK1/2 (MAPK pathway) and JAK3 (JAK-STAT pathway) mediate the IL-2-dependent regulation of Na+,K(+)-ATPase expression during lymphocyte transition from resting stage to proliferation. A correlation between changes in ouabain-sensitive rubidium influxes and the alfal-subunit amount has been demonstrated. It is concluded that IL-2-dependent-progression of normal human lymphocytes from quiescence to proliferation is accompanied by the increase in Na+,K(+)-ATPase alfa1-subunits expression, and the enhanced transport activity of a sodium pump during the prereplicative stage is provided by the increased number of functional pump units in plasma membrane.

[Porcine kidney extract contains a specific inhibitor of the ouabain-sensitive alpha2-isoform of Na, K-ATPase present in rat diaphragm fibres]. / Ekstrakt pochek svin'i soderzhit spetsificheskii ingibitor ouabain-chuvstvitel'noi alfa2 izoformy Na, K-ATFazy myshechnykh volokon diafragmy krysy.

In experiments on isolated rat diaphragm muscle, acetylcholine (100 nmol/l) hyperpolarized muscle fibres due to activation of the alpha 2 isoform of Na,K-ATPase. This hyperpolarization was blocked in a dose-dependent manner by ouabain (K0.5 = 8 +/- 4 nmol/l) as well as by a solution of porcine kidney extract (10 kDa cut-off filtration), with the K0.5 approximately equal to a 1:20,000-fold dilution. The inhibitory activity of the developed slowly over a period of 3 hours and, in contrast to ouabain, was still present after 1 hour of washing. Ouabain, but not the extract, inhibits Rb+ uptake in human erythrocytes that only express the alpha = 1 isoform of Na, K-ATPase. Our data suggest that in rat skeletal muscle the alpha 1 isoform of Na,K-ATPase is primarily responsible for ionic homeostasis, while the alpha 2 isoform provides a "regulatable" function and may be controlled by cholinergic stimulation and/or endogenous digitalis-like factors (EDLFs). Porcine kidney extract contains a factor (M. W. < 10 kDa) that selectively inhibits the rat alpha 2 isoform and differs from ouabain. Our experimental protocol can be used as a highly sensitive physiological assay for factors that selectively inhibit the alpha 2 isoform of Na,K-ATPase.

[Changes in sodium pump transport activity and Na+, K+-ATPase expression level following lymphocytes activation in humans]. / Izmenenie transportnoi aktivnosti natrievogo nasosa i urovnia ékspressii Na+, K+-ATFazy pri aktivatsii limfotsitov cheloveka.

Ouabain-inhibitable rubidium influxes, intracellular sodium content (Nai), and alpha 1-subunit abundance have been studied in human blood lymphocytes, stimulated by phytohemagglutinin (PHA) or by the phorbol 12,13-dibutyrate (PDBu), and calcium ionophore--ionomycin. It is shown that at early stages of PHA-induced activation, the Na/K pump expression (as determined by Wesrn blots of alpha 1 protein in membrane fractions of total cell lysates) does not change, and the increase in Rb influx is due to the increase in Nai and results from the enhanced transport activity of Na/K pumps present in plasma membrane. During the late stages of G0-->G1-->S transit (16-48 h), the increase in Rb influx occurs without changes in Nai, and monensin increases both Nai, and the Rb influx via the Na/K pump. To the end of the first day of mitogen activation, the alpha 1 protein content was found to increase by 5-7 times. A correlation was revealed between changes in ouabain-inhibitable Rb influxes, alpha 1 protein abundance, and the proliferation rate. It is concluded that blasttransformathion of normal human lymphocytes is accompanied by the increase in membrane-associated pool of alpha 1-subunit of Na+,K(+)-ATPase, and the enhanced activity of sodium pump during the G0-->G1-->S progression is provided by increased number of Na+,K(+)-ATPase pumps in plasma membrane.

[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.