A sodium channel opener inhibits stimulation of human peripheral blood mononuclear cells

Molecular Immunology,

Oft%5890/92 %5.00+0.00 Pergamon Press pit

Vol. 29,No.4,pp.517-524, 1992

Printed in Great Britain.

A SODIUM HUMAN

CHANNEL OPENER INHIBITS STIMULATION OF PERIPHE~L BLOOD MONONUCLEAR CELLS

CARLOPERI,RINARECCHIONI,FAUSTOMORONI,FIORELLA MARCHEBELLI, MARCOFALASCA,ZOLTAN KRASZNAI,*REZ~~GASPAR,* LASZL~MATYUS* and SANDORDAUIANOVICH*~ Cytology Center, Gerontolo~cal Research Department, I.N.R.C.A., Ancona, Italy and *Department of Biophysics, University Medical School of Debrecen, H-4012 Debrecen, Hungary (First received 13 May 1991; accepted in revised form 12 August 1991)

Abstract-The role of membrane potential changes in T cell activation was studied on human peripheral blood lymphocytes stimulated with phytohemagglutinin. Addition of bretylium tosylate,

a sodium channels opener, to PHA treated l~phocytes modified the membrane potential and consequently blocked cell activation in a dose-dependent fashion. BT was non-toxic even in long-term (72 hr) incubations. It was reversibly removable, and the removal restored the stimulatory effect of PHA. ‘H-thymidine incorporation was blocked if BT was present during the first 20-24 hr of the mitogenic activation. The later BT was added after PHA, the less inhibition of proliferation was observed. BT hyperpolarized the lymphocytes also in the presence of PHA. BT hindered the depolarizing effect of high extra~ll~ar ~tassi~ concns. The sustained polarized state of the lymphocytes did not influence the intracellular calcium increase upon PHA treatment. IL-2 and transferrin receptor expression was not hindered by BT during PHA stimulation of lymphocytes. Addition of rIL-2 did not abolish the inhibitory effect of BT. According to cell-cycle analysis BT arrested the majority of the cells in G, phase. It is suggested that cell activation demands the Aexibie maintenan~ of a relatively narrow membrane potential “window”. Any sustained and significant hyper-, or depolarization, may dramatically decrease the effectivity of transmembrane signalling.

after the ligand binding. The high number of conflicting data on the role and significance Activation of lymphocytes by mitogenic lectins involves of membrane potential changes in cell activation may a number of events that ultimately result in cell proliferpartially be attributed to the different techniques apation. The earliest events in the plasma membrane, are plied, as well as to the different target cells studied so direct consequences of lectin binding to cell membrane far (Gelfand et al., 1984, 1987a, 6; DeCoursey et al., receptors. Later ones in the cytosol are also regulated 1984; Cahalan et ai., 1985; Chandy et al., 1985; through second messengers. In the early phase of the Deutsch and Price, 1982; Matteson and Deutsch, 1984; lectin stimulation transmembrane ion fluxes and intraFukushima and Hagiwara, 1983; Schlichter ef al., cellular free calcium increase, the membrane potential 1986; Grinstein and Dixon, 1989). Recently it has been changes, and finally a number of biochemical steps, shown that the intracellular calcium increase was involving phosphatidylinositol breakdown products, severely damaged when completely depolarized human T lead to the activation and sequestration of the protein lymphocytes were treated with phytohemagglutinin kinase C (Berridge and Irvine, 1989). (Gelfand et af., 1987a). The lack of increase in the Though this is a common sequence of early stimu- intracellular calcium level may lead to impaired IL-2 latory events of lectins and antigens, it is not unique. production. Lymphokine-induced activation uses a different, yet Simultaneously with the discovery of calcium actiuncharacterized, pathway. IL-2 stimulates cells without vated and voltage gated potassium channels in lymphoCa2+ release or activating protein kinase C (Tigges ef al., cyte membranes their role in mitogenic activation 1989; Mills et al., 1988; Valge et al., 1988). was assumed (DeCoursey et al., 1984; Matteson and The membrane potential changes induced by mitoDeutsch, 1984; Fukushima and Hagiwara, 1983). Blockgens (or other agents), are among the earliest and ing of these channels inhibited cell proliferation after cell stimulation. However, the real causative role of these findings has not been proven yet. It has also been TAuthor to whom correspondence should be addressed. pointed out that the inhibitor effect of potassium A~~~ev~r~o~s: BT, b~tyIium tosylate; PHA, phytohemagchannel blockers may arise from hindered transport of glutinin; IL-2, interleukin-2; rIL-2, recombinant IL-2; essential metabolites (Schell et al., 1987). PBMC, peripheral blood mononuclear cells; Indo-l AM, Antigen triggered membrane potential changes were indo- 1 acetoxy methylester; PBS, phosphate buffered saline; TEA, tetraethyl ammonium chloride. found in IgE sensitized rat basophylic leukemia cells 517 MIMM 29,.+-F INTRODUCTION

most rapid changes

518

C. PIERIet al.

assuming a causative role in the cellular degranulation process of mast cells (Labrecque et al., 1989). Lectin stimulation of T lymphocytes induces an increase in the intracellular Na+ concn that is sufficient to activate the Na+-K+ pump. However, its direct participation in cell activation is not clear (Grinstein and Dixon, 1989). The principal aim of this study was to gain a deeper insight into the role of membrane potential changes and dynamic responsiveness of the molecular structures in the plasma membrane throughout cell stimulation. We provide data on the importance of the polarized state of the plasma membrane during pHA stimulation of human peripheral blood lymphocytes. Bretylium tosylatea quaternary ammonium ion, an opener of silent sodium channels-increases the membrane potential of partially depolarized human, rat and mouse T and B lymphocytes (Pieri et al., 1989; Tron et al., 1990). The repolarizing effect was explained by an enhanced activity of the electrogenic Na+-K+ ATP-ase, triggered by a BTinduced Na+ influx. This effect was utilized in the present study. We found that the BT initiated sustained polarization of the plasma membrane inhibited the PHA induced cell proliferation of human lymphocytes. This effect was reversible, and did not affect the calcium liberation and influx. IL-2 and transferrin receptor expressions were unperturbed. Addition of recombinant IL-2 to the medium did not abolish the effect of BT. It is suggested that only a relatively narrow membrane potential window is permissive for the events following the binding of stimulatory agents. MATERIALS

AND METHODS

Cells

Human lymphocytes were prepared from mononuclear cell rich plateletpheresis residues of healthy donors. The residue was diluted to 300 ml with Ca2+ and Mg2+ free Hank’s solution, centrifuged and washed twice in the same solution to remove platelets. Peripheral blood mononuclear cells were prepared by FicollHypaque gradient centrifugation (Boyum, 1968). 3H-thymidine incorporation

PBMC were incubated (1 x 105/we11) with PHA (10 pgg/ml) in the presence of different concns of BT. Five hr before the termination of the culture (72 hr altogether), 2 PCi ‘H-thymidine (6.7 Ci/mmol, Amersham, U.K.) was added to each well. Cell suspensions were collected on glass fiber filter (Labtek) by means of a cell harvester (Skatron AS). Filters were dried and radioactivity was measured in a liquid scintillation counter (Packard-Tricarb). Flow cytometric membrane potential measurements

Aliquots of the cell suspensions were incubated for 1 hr in RPMI-1640, containing 5% FCS to allow the recovery of eventual depolarization due to the preparation procedure. After washing, the cells were resus-

pended in PBS (pH 7.2, lO’cells/ml). Membrane potential was measured by applying 150 nM oxonol, di-BA-C4(3) (Molecular Probes, Eugen, OR) to 1 ml cell suspension containing lo6 cells, according to Wilson and Chused (1985). The measurements have been performed in a Coulter Epics V. flow cytometer. Viability of the cells was judged by their ability to exclude propidium iodide (PI) and retain fluorescein diacetate. Patch-clamp membrane potential measurements

Membrane potential has been measured in the wholecell configuration with an EPC-5 patch-clamp amplifier, working in current-clamp mode, in conjunction with an Axon Instruments TL- 1-125 computer interface (maximum sampling rate: 8 psec per point, low pass filtering: 3 kHZ). The patch-electrodes were made from GC 150 F- 15 glass capillaries (Clark Electromedical Instruments, Reading, U.K.). The cells were bathed in a solution containing 160 mM NaCl, 4.5 mM KCI, I mM MgCl,, 1 mM CaCl, and 10 mM HEPES, pH 7.3. The patchpipettes were filled with a solution containing 140 mM KCl, 2 mM MgCl,, 1 mM CaCl 11 mM EGTA and 10 mM HEPES, pH 7.3. The measurements were taken at room temp. Determination of cytoplasmic free calcium content

Cytoplasmic free calcium was measured by applying indo-l AM dye (Molecular Probes) as described earlier (Grynkiewicz et al., 1985; Damjanovich et al., 1987). As part of the procedure PBMC (lO’/ml) were loaded with 10pM indo-l AM in RPMI-1640 (60min at 37°C). Determination of IL-2 and transferrin receptor concns

Human peripheral blood lymphocytes were labelled with the respective monoclonal antibodies obtained from Becton and Dickinson (Mountain View, CA), directly tagged with fluorescein isothiocyanate. The cells were previously treated with PHA and BT as indicated in the legends to the figures. The fluorescence intensities were determined and analyzed in a Coulter Epics V flow cytometer (Hialeah, FL). Cell-cycle measurements

The relative cell cycle distribution of lymphocytes, stimulated by PHA in the presence or absence of 2 mM BT was determined by flow cytometric analysis of PI fluorescence using 514 nm excitation and 590 nm emission wavelength. Medium and stock solutions

RPM1 1640 medium supplemented with 2 mM glutamine, Ca*+ and Mg2+ free Hank’s balanced salt solution were from Gibco (Grand Island, NY). Foetal calf serum (Gibco) was present in lymphocyte cultures at a concn of 10% (v:v). Phytohemagglutinin (PHA, Difco, Detroit) was dissolved in distilled water (1 mg/ml) and used at a concn of 10 pg/ml. Buffered salt solution (PBS) contained (mM): 140 NaCl, 5 KCI, 1 Ca2+, 10 glucose, 10 HEPES, titrated to pH 7.4 at 37°C with NaOH. BT (Welcome Foundation, London, U.K.) was prepared as

Inhibition

519

of lymphocyte activation by a sodium channel opener

of 30 mM in RPM1 (to be added to the cell cultures) or in PBS (for membrane potential measurements). Oxonol, {b~s(l,3)dibutylbarbitu~c acid5-trimethine oxonol), (di-BA-~~-(3)) from Molecular Probes, was dissolved in dry DMSO. a stock solution

RESULTS The repolarizing effect of different doses of BT on the plasma membrane potential of human lymphocytes is shown in Fig. 1. The cells were slightly (10-15 mV) depolarized by elevating the extracellular potassium level from 5 to 25 mM in an isotonic way, prior to the addition of BT to the samples. Repolarization reached saturation in the vicinity of 1.5-Z mM BT. The inset shows primary data, i.e. flow cytometric histograms of oxonol fluorescence characterizing the membrane potential distribution of the cell population under different circumstances: (a) untreated cell population; (b) depolarand (c) bretylium tosylate ized cell population; (0.75 mM) treated cell population, The percentage membrane potential recovery was calculated by comparing the mean values of the individual histograms. The repolarizing effect of bretylium tosylate has been varyfied by patch-clamp measurement of the membrane potential of individual cells prior to the fluorescent treatment of the cell population using a similar treatment for depolarization. Figure 2 displays a characteristic membrane potential recovery curve caused by 2mM bretylium tosylate as detected by patch-clamp. The combined effect of PHA and BT on the membrane potential is shown in Fig. 3. The cells incubated with 10 pgg/ml of PHA for 3 min either slightly increased

80

0

t 0.5

1

b

1.5

1

e 2.5

I

2

s 3

”

3.5

4

9

4.5

’ 5

5.5

Time (min)

Fig. 2. Bretylium induced membrane potential recovery of a partially depolarized human lymphocyte measured by patchclamp. After entering the whole cell ~nfi~ration the cell was depolarized by increasing the external potassium concn from 4.5 to 14.5 mM (A), and following the stabilization of the membrane potential at a new level, bretylium tosylate (2 mM) was introduced into the bath (B).

their polarity or remained unaltered. However, a further 7 min incubation generally evoked the repolarizing effect of PHA. A more expressed repolarization was observed within 3 min after the introduction of PHA if the cells

0

10

20

30

40

50

so

70

80

30

80

30

1

Fluorescence (channel number)

Bretylium

tosylate

(mM)

Fig. 1. Effect of BT on the membrane potential of human l~phocytes. The cells were slightly depola~zed prior to the addition of BT by increasing the extracellular fK+] to 20 mM at the expense of [Na+]. The membrane potential was measured by oxonol uptake in the presence of BT concns, ranging from 0 to 2 mM, in a flow cytometer. Increasing intensity of oxonol fluorescence indicates a decrease in the resting potential of the cells. The plot was constructed from the mean values of the fluorescence intensity distributions of the cell populations under different conditions indicated below. The insert displays raw data of flow cytometric histograms for: (a) control, (b) slightly depolarized and (c) 0.75 mM BT treated lymphocytes.

-I

0

10

20

30

40

Fluorescence

50

80

(channel

70

10

number)

Fig. 3. Hyperpolarizing effect of PHA and BT on human lymphocytes as indicated by oxonol fluorescence. Panel A shows the effect of PHA alone: (a) untreated ceils, (b) slightly depofarized cells (by increasing the extracellular [K’] to 20 mM in an isotonic way) and (c) cells repolarized by approximately 15 mV, after treated by 10 pg/ml PHA for 3 min. Panel B displays the effect of 3 mM BT on 10 @g/ml PHA treated cells: (a) slightly depolarized, (b) PHA and (c) PHA and BT treated cells. Each histogram contains data of lo4 cells.

520

C. PIERIet al.

Fig. 4. Dose-dependent inhibition of lymphocyte response to PHA stimulation by BT detected via ‘H-thymidine incorporation and expressed in the percentage of maximum response. Human PBMC (2 x 105/well) were stimulated by 10 pgfml PHA in the presence of different BT eoncns up to 3 mM. The cells were cultured in RPMI-1640 with 10% FCS for 72 hr. Five hr before terminating the cultures 2 PC? H-thymidine was added to each well. The results are expressed as % of the control and represent the average of three experiments.

were in an elevated potassium chloride containing buffer depolarizing them by IO-15 mV. The hyperpolarizing effect of PHA was temperature and ouabain sensitive, while 4-amino pyridin, TEA and quinine had no influence on it (data not shown). A considerable factor in the enhancement of the effect of PHA on the membrane potential indicating fluorescence could have been the improved responsiveness of the oxonol dye in the membrane potential range provided by the elevated extracellular potassium concn (Wilson and Chused, 1985). However, patch-clamp evidence shows that the measured fluorescent intensity changes were due to membrane potential alterations indeed (see Fig. 2). In order to study the effect of BT on the proliferation of lymphocytes two different experimental strategies were followed. Figure 4 shows the data obtained with one of them, where a dose-dependent inhibition of lymphocyte proliferation is seen, after PHA stimulation in the presence of BT, i.e. when the two effecters were added simultaneously. There was a dramatic decrease in the incorporation of 3H-thymidine, at BT concns effectively repolarizing the lymphocytes (see in Fig. 1). A sharp decrease is observed in response to the mitogen added with BT in the concn range 0.5-l .75 mM.

Fig. 5. Effect of BT on the PHA stimulation of lymphocytes detected via 3H-thymidine incorporation and expressed in the percentage of the incorporation observed in the untreated cells. BT was added after the administration of PHA at different times indicated. The cells were cultured as described in the text to Fig. 4, with 0.5 (Cl), 1.0 (A), 1.75 (+) and 2mM (m) BT. Every point in the graph represents the average of three inde~ndent experiments.

In another set of experiments BT was added several hours after the stimulatory doses of PHA, as indicated in Fig. 5. Thus the stimulatory signal was allowed to be processed before applying the sustained hyperpolarization, imposed on the lymphocytes by BT. The longer processing time was allowed, the lower inhibition was obtained. The incorporation of 3H-thymidine was even enhanced at longer incubation times, when lower than 2 mM doses of BT has been applied. The lack of significant toxicity of BT is demonstrated in Tables l-3. The incubation of lymphocytes with 1.5 mM BT in primary lymphocyte culture practically did not change viability even after 72 hr (Table 1). When lymphocytes were co-cultured with IO pg/ml PHA with or without an inhibitory concn of BT (1.5 mM), and were washed after 6 hr, the cells retained their ability to proliferate without further addition of PHA, regardless of the presence or absence of BT (Table 2). However, coculturing the lymphocytes with 1.5 mM BT for 20 hr, resulted in a significant (approx. 60%) decrease in the 3H-thymidine incorporation. The proliferative response of the human lymphocytes to PHA applied with or without BT and recombinant IL-2 is presented in Table 3. Cells, stimulated under the indicated conditions showed a significant decrease in nucleotide incorporation in the presence of 1.5 mM BT

Table 1. Surviving ratio of PBMC in primary cultures with and without bretylium tosylate (1.5 mM) Time (hr)

0

Cells Cells + BT

95.5 + 1.4 94.4 * 1.7

% of surviving cells 24 48 93.3 + 2.3 91.5 f 1.9

87.8 f 2.8 89.1 rfi 2.3

72 75.1 + 2.0 76.7 + 2.6

1 x 106PBMC/ml were cultured in RPMI-1640 with 10% FCS. Cell viability was determined with the PI exclusion test. Data are mean f SEM from three experiments.

Inhibition

Table 2. Proliferative response of PBMC after simultaneous treatment with PHA and BT 1st Treatment PHA PHA PHA PHA PHA PHA PHA PHA

+ BT + BT

f BT -t BT

Time (hr)

2nd treatment after washing

6 6 6 6 20 20 20 20

PHA none PHA none PHA none PHA none

3H-th~idine incorp. (cpm x 103) 45.9 42.7 39.3 35.0 45.6 40.8 26.4 24.0

+ + + * + f f +

5.4 5.7 2.5 1.1 3.4 4.6 1.8 2.8

PBMC were incubated (1 x 105/well) for 6 or 20 hr in RPMI1640 with 10% FCS and PHA (10 pg/ml) in the presence or absence of 1.5 mM BT (1st treatment). The cells were then washed twice with Hank’s balanced solution, without calcium and magnesium, resuspended again in fresh medium (2nd treatment), and incubated up to a total of 72 hr. 3H-thymidine (2 pCi/well) was added for the last 5 hr of incubation. The counts represent mean * SEM of four samples.

and this effect was not mitigated by the presence of 20 U/ml rIL-2. The calcium influx and the increase of intracellular calcium level are prerequisites for PHA stim~ation. Thus intracellular calcium concns were determined upon PHA stimulation of cell samples in the presence and absence of proliferation inhibiting BT doses (Table 4).While the strong depolarization inhibited the increase in intracellular calcium (Gelfand et al., 1984), our data did not show a significant influence of BT treatment on the intracellular calcium as determined by the indo- 1-AM fluorescent calcium chelator. A quantitative determination of the expression of IL-2, as well as transferrin receptor levels at 24 hr of cell stimulation did not show significant changes in the presence of BT (Table 5). The respective receptors were labelled with fluorescein isothiocyanate conjugated

Table 3. Proliferative response of PBMC to PHA in the presence or absence of BT and/or IL-2

Treatment PHA -. PHA + BT PHA + rIL-2 PHA + rIL-2 + BT

521

of lymphocyte activation by a sodium channel opener

jH-thymidine incorp. (cpm x 10m3) 49.7 f 20.7 5 51.2 f 26.4 +

6.1 4.0 3.4 3.7

PBMC (1 x lo5 cells/well) were incubated with PHA (IOpg/ml); PHA + BT (1.5 mM); PHA + rIL-2 (20 U/ml); or PHA + rIL-2 + BT at 37°C for 72 hr. 3H-thymidine (2 pCi/well) was present during the last 5 hr of incubation. The results of four experiments (mean f SEM) are shown.

Table 4. Effect of BT on the PHA-induced changes of intracellular free Ca*+ [Ca*‘]i(nM) Treatment None BT

basal level

after PHA

16.5It 14 169 f 13

321 + 9 294 f 17

Indo-I-AM loaded PBMC were diluted to 5 x lO’/ml in PBS with or without 1.5 mM BT and the [Ca2+li was determined as described before (23). PHA was added to similar samples and the [Ca*+]i was determined after 10min of incubation at 37°C. Data shown are the mean f SEM of four experiments.

monoclonal antibodies and the distribution of the flourescence over the cell population was detected by flow cytometry. The ring-like labelling of ceils with the antibodies was controlled under a fluorescent microscope. Experiments, aimed to determine the number of effectively cycling cells in cell populations stimulated by PHA in the presence and absence of BT, were carried out by PI labelling the total DNA (Table 6). BT (2mM) decreased the number of cycling cells. Measurements taken after different length of incubation revealed that the BT confined the majority of cells to G, phase. DISCUSSION The role of membrane potential, ion-channel and ion-pump activities in cell activation is far from being understood (Grinstein and Dixon, 1989). We investigated the effects of some agents influencing cell activation at the level of plasma membrane (Pieri et al., 1989). The effect of BT, an opener of sodium channels, was investigated on the PHA caused activation of human peripheral mononuclear cells, since PHA stimulation is one of the best understood models of T-cell activation (Pieri et al., 1989; Ilani et nl., 1982). BT is a potent inhibitor of lymphocyte stimulation at concns causing sustained hype~ola~zation (Pieri et al., 1989). Table 5. Effect of BT on the induction of IL-2R and TFR by PHA stimulation Treatment

% of Tat posit&&y

% of TFR positivity

None PHA PHA + BT

2.5 f 2.0 75.1 -f 6.5 70.2 + 6.7

3.6 & 2.4 58.9 k 8.5 55.5 + 5.4

PBMC were cultured as described in the text to earlier tables in the presence and absence of 1.5 mM BT for 24 hr. The % of Tat and TFR positivity was determined by FITC labelled antibodies, directed against the receptors, in a flow cytometer. Data show the mean f SEM for five experiments. The number of cells counted for the individual histograms was 104.

522

C. PIERIet al.

Table 6. The effect of BT (2 mM) on the cell-cycle Time (hr) 0

Treatment none

(0;)

(2)

G/M W)

95 _t 2.7

4.5 f 2.3

0.5 f 0.2

24 24

PHA PHA + BT

82.3 rf: 2.5 81.6 + 0.6

16.1 f 1.1 15.2 f 0.8

2.7 + 0.8 3.1 f 1.3

32 32

PHA PHA + BT

77.1 f 3.2 83.9 + 3.1

19.7 + 2.7 13.3 1_2.9

3.1 + 1.1 2.7 + 1.0

48 48

PHA PHA + BT

67.2 &-6.0 85.9 + 3.3

23.0 + 6.7 9.7 f 1.3

9.8 + 0.7 4.4 f 1.9

Cells were incubated in the usual medium and aliquots were taken at indicated time points. After PI labelling the cells were analyzed in a Coulter Epics V flow cytometer using its cell cycle analysis program.

Among the earliest observable events after lectin binding are the changes of plasma membrane potential and ion channel activities (Grinstein and Dixon, 1989). These alterations have been observed by several authors, however the data are contradictory regarding the direction, role and significance of the membrane potential and ion channel activity changes (Grinstein and Dixon, 1989; Kieffer et al., 1980; Tsien et al., 1982; Felber and Brand, 1983a, b; Averdunk and Lauf, 1975). Our data indicate a possible new role of the sodium-potassium pump in cell activation. Sustained activation of this pump by opening otherwise silent Na+ channels can inhibit cell proliferation. Apparently, this is in conflict with the statement that mitogens increase Na+-influx and activate the Na+-K+ pump (Grinstein and Dixon, 1989). However, we emphasize the sustained nature of hy~~ola~zation. Lectins or antigens are internalized, thus any hyper, or depolarization caused by them should be transient. The inhibitory effect of BT on T cell activation evoked by PHA is not connected to the intracellular calcium level. The density of IL-2 and transferrin receptors, instrumental in T-cell activation, and the sequence of their appearance in the membrane after PHA stimulation remained unaltered when BT was applied. The expression of the latter receptors is known to be IL-2 dependent, thus not only the expression of the IL-2 receptors but the functional IL-2 secretion was also retained in the presence of BT. Recombinant IL-2 did not alter the inhibitory effect of BT. Its effectivity was not linked to the Na+ /H+ exchange. Ten times higher concn of amiloride was required to inhibit Na+H+ exchange than the effects of BT (Pieri et al., 1989; Tron er al., 1990). On the other hand the participation of the sodium-proton exchange in the cell activation can be questioned since Mills et al. (1986) inhibited the exchange without the impairment of the cell proliferation. PI labelling of total DNA in stimulated cells showed that the fraction of dividing cells was lower when BT was present. The majority of the cells were arrested in G, phase and did not cycle even after adding rIL-2. The presence of BT was essential for the inhibition during the first 20 hr of activation.

Essential events sufficient for DNA synthesis and morphological changes occur during the first two hours of mitogenic or antigen stimulation (Crabtree, 1989). Since the so-called immediate events occur even in an earlier period, the question arises what is the reason of the long period necessary for commitment to T-cell activation (Crabtree, 1989). A continuous contact with the antigen receptor-T3 complex, or mitogen might be essential to maintain the required concentration of a labile second messenger. The proposal that Ca2+ could serve this function is not relevant in our case since the effect of BT was independent of the extracellular calcium and the intracellular calcium level was increased upon PHA treatment when inhibitory doses of BT were present. On the other hand, the inhibitory effect of BT also demanded a sustained presence of the drug, and when it was removed, even after several hours, no irreversible changes were observed. Its lack of toxicity on primary lymphocyte cultures suggests that BT reversibly suspends some physiological activity necessary for cell activation. The inhibitory effect of BT was eliminated or even reversed into a slight enhancement of 3H-thymidine incorporation by simply separating the addition of PHA and BT in time. The former may indicate the decreasing significance of a reversibly blocked mechanism during the advancement of commitment to cell activation. The latter can easily be attributed to increased co-transport processes accompanying the increase in sodium influx. The following model may provide a framework for the observations. Similarly to regulation mechanisms in nerve and muscle cells, the proximity and mobility of membrane constituents, and the lipid domain dependent conformational changes of membrane proteins can up- or down-regulate immunological functions in lymphocytes (Lewis and Cahalan, 1988; Crabtree, 1989; Sardet et al., 1990; Matko et af., 1988; Damjanovich et al., 1983; Szijllijsi et al., 1987a, b, 1989; Edidin et al., 1988; Mittler et al., 1989; Edidin, 1990; Gorvel et al., 1989). The transient dipoles generated by transmembrane potential changes, regulate intermolecular interactions within the membrane. As a consequence, only a narrow membrane potential window suits membrane related intermolecular

Inhibition of l~ph~yte

activation by a sodium channel opener

interactions instrumental in evoking and propagating the transmembrane signal. Any significant alteration in membrane potential may severely impair the concerted action of the membrane bound molecules. The PHA hyperpolarizes the cells for a relatively short period that is followed by a depolarization, characterizing the activated cells in general (Grinstein and Dixon, 1989). The BT, hyperpolarizes the cells in a sustained fashion at this point, where the conditions for opening the voltage and ligand gated Na channels are given. The postponement of the addition of BT after triggering the activation by PHA causes a less and less effective inhibition. The reason why is the inhibition completely eliminate so late when the membrane bound processes are seemingly over is discussed in details by Crabtree (1989). A sustained and unimpaired trigger signal (which is impaired by the presence of the BT) is necessary for the late events of activation at genetic level. Taken together the permanent activation of the Na+-K+ pump by an agent that is not internalized within a short time, results in sustained hyperpolarization. Thus the altered plasma membrane potential can suspend intermolecular interactions essential for cell activation. This new regulatory role of Na+ accentuates that cell activation is influenced not only by potassium but also by sodium channels at more than one site. Alongside the Na+/H+ exchange influx may regulate

and

co-transport,

moreover

Na+

the Na+-K+ ATP-ase activity and thereby cell activation. Although, we emphasized that sustained hyperpolarization serves as an inhibitor of cell activation, it is likely that small alterations of the membrane potential might affect stimulatory responses in a more complex fashion. work was also supported by a grant from the Hungarian Academy of Sciences fOTKA 1492)to S. Damjanov~ch.

Acknowledgements-The

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