Ion contents of human lymphocytes

Experimental Cell Research 101 (1976) 3140

ION CONTENTS OFHUMAN The Effects of Concanavalin W. G. NEGENDANK Hematology-Oncology

Section.

of Pennsylvania,

Department

LYMPHOCYTES A and Ouabain

and C. R. COLLIER of Medicine.

Philadelphia,

Hospiral

PA 19174.

of the Universiry

USA

SUMMARY It has been suggested that mitogens may activate the Na,K-ATPase to cause an increased cell K’ which may then trigger metabolic events initiating DNA synthesis. To test this hypothesis, human lymphocytes were treated with ouabain and concanavalin A (ConA) and their ion contents measured directly by atomic absorption. Ouabain decreased cell K’ with a critical dose range, IOe8 to IO-’ F4, similar to that which inhibits mitogenesis, and induced a mole-for-mole replacement of K+ by Na’. ConA, in non-toxic, mitogenic doses, also caused a rapid and a sustained decrease in cell K+. but, unlike ouabain, did not induce replacement of the lost K’ by Na’, and the total K++Na’ was reduced in spite of a normal water content. Thus, although normal Na.K-ATPase function may be required-for mitogenesis, ConA does not affect cell-K+ and Na’ simply by activating the Na,K-ATPase. Stable-state K+ and Na+ contents were then determined over a wide range of sxternal K’ levels, and analyzed by the equivalent of a Hill plot. The normal cooperative uptake of K’ was inhibited in an allosteric manner by ouabain, while ConA failed to alter significantly the critical parameters of K+-Na’ exchange and the cooperativity in K’ uptake. We suggest that K+ is not a specific trigger in the initiation of mitogenesis, but that changes in K+ flux and content reflect a change within the physical state of an underlying macromolecular assembly that is poised to respond to the mitogenic stimulus in a critical cooperative fashion.

The mitogenic transformation of iymphocytes is accompanied by a variety of physiological and biochemical events. Many of these occur within minutes of exposure of lymphocytes to mitogens, and include increased influx of 4?K and of %a [l-6]. Normal levels of external K+ and Ca*+ are required for optimal mitogenic transformation [l-9], and it has been suggested that one or both of these may play a critical role in the triggering of the early events controlling DNA synthesis and cell division [g-12]. The ability of ouabain to reversibly inhibit mitogenesis [13, 141,a sigmoid relation between external K+ and DNA synthesis in phylymphocytes tohemagglutinin-stimulated 3-761806

[12], and the similarity in dose-response curves of the effects of various mitogens upon thymidine uptake and ‘?K influx [IS], led to the postulate that mitogens activate membrane Na,K-ATPase to increase cell K+ and decrease cell Nat levels, and that the increased cell K+ may trigger a key metabolic event initiating DNA synthesis P,

151.

The increased influx of 4?K and of %a into mitogenically-transformed cells has been determined by adding the labeled ion to the external medium, and then following its uptake by cells over a short period of time. This sort of study, however, involves two sets of variables; (a) isotopic exchange; Exp Cell Re.s 101 (1976)

32

Negendank and Collier

(b) changes in total ion influx and efflux. Thus, a change in the uptake of 42K or 4sCa does not necessarily reflect a change in total cell K+ or Na+ content. In this study, therefore, ion contents of lymphocytes were directly measured by atomic absorption spectrophotometry. In order to investigate the hypothesis that K+ and the Na,K-ATPase play a critical role in mitogenic transformation, we studied the effects of ouabain and of concanavalin A (ConA) upon K+ and Na+ contents of human lymphocytes.

METHODS AND MATERIALS Initial separation and incubation These were done under sterile conditions. 450 ml of heparinized peripheral blood from healthy adults aged 20-35 was diluted 1 : 4 in wash medium and incubated for 30 min at 37°C at 50 rotations/min with carbonyl iron suspension at 0.25 mg/ml (GAF Corp., New York, grade SF). The diluted blood was then layered over 60 ml diatrizoate-Ficoll at the snecific aravitv 1.077 in 250 ml centrifuge bottles at 4OOgfor S?min.-The lymphocyte-platelet layer at the interface of each bottle was washed in a 250 ml plastic bottle at 200 g for 15 min, then pooled into 34 bottles and washed again. Cells were resuspended in 10-12 ml of wash medium and layered over 12% sucrose in a 17X 100 mm polystyrene tube for 5 min at 200 g. The medium and the platelets, at the interface, were removed, and the pellet, containing lymphocytes and a few remaining platelets, was then incubated at 3-5~ 106lymphocytes/ ml in 50-70 ml of medium with either 10% autologous serum or 2 % human serum albumin in standing 250 ml plastic bottles. The wash medium contained 144.6mM Na+, 5.4 mM K+, 1.3 mM Ca2+, 0.9 mM Mg*+, 0.34 mM HPOd2-, 1.29 mM H,PO,-, 14.3 mM HCO,-, 5.6 mM glucose, 1.5 mgjl phenol red, 50 U/ml Na+-penicillin G, and 50 pg/ml streptomycin sulfate. The pH was 7.4 and stable in sealed containers. When necessary it was adjusted with 5 % CO* in air. The incubation medium was identical except for varying K+ and Na+, with total K++Na+ always 150 mM. Concanavalin A (ConA) (Miles-Yeda, Rehovot) and ouabain (Sigma, St Louis) were added directly at the beginning of incubation.

Final separation and ion extraction Following incubation the bottle was centrifuged at 200 g for 5 mitt, the pellet resuspended in l&12 ml of medium of similar K+ level and layered over 12% sucrose in a 17x 100 mm polystyrene tube at 200 g for 5 min. Cells were suspended in 0.4 ml of the sucrose Erp Cd RPS 101 (1976)

and packed in an 0.4 ml polyethylene mrcrotube at 6000 g for 30 min. The tip of the microtube was severed with a razor blade, weighed, placed in a 17X 100 mm polystyrene tube, and the pellet was dislodged with a Pasteur oioette. lons were extracted at room temperature for at-least 24 h in 5 or IO ml of 0.1 N HCl with 0.1 M LiCl and 0.05 M (NH&HPO,. K+ and Na+ were measured by atomic absorption spectrophotometry (Varian 1200) using standards of NaCl and KC1 made up in the same ion extraction fluid.

Water content, trapped space, viability, and counts Water was measured by transferring pellets from the severed microtube to 1x 1cm nieces of tared aluminum foil, weighing, and drying 12hat 100°C. Trapped space was determined bv addine 2 ma/ml (5 uCi/ml) WIpolyethylene glycol (New Engl&d Nuclear 73d-052) to the final 12% sucrose. Fifty ~1 aliquots of the fluid in the microtube and 100 ~1 aliquots of a 2 ml sample of medium into which the pellet was resuspended, were counted by liquid scintillation. Viability was tested by incubating three drops of lymphocyte suspension in 1 drop of 0.28% erythrosin B at room temperature for 10 min. Cell counts were done by hand using a standard hemocytometer and white blood cell dilution solution.

[3H]TdR incorporation One ml cultures of 0.5~ lo6 cells/ml were removed at about 72 h, 16-18 h after addition of 0.3 /.&i [3H]TdR, extracted in 5 ml of 5% TCA at 5°C for 1 h, spun at 1000 g for 10 min, washed in cold 5% TCA, spun again, and the remaining TCA allowed to run off by inverting tubes at 45” at 5°C. The precipitate was dissolved in 500 ~1 NH,OH and 250 ~1 were counted by liquid scintillation.

Use of radiolabeled ions to measure cell ion contents Cells prepared and incubated as above were preloaded with 4PK (100 PCi), or with %Cl (1.5 PCi), by adding the labeled ion without changing the total concentration of external ion. Full equilibration of *sK required about 8 h, determined in time courses in which cell K+ was flamed and the specific activity measured directly. Cells were incubated overnight and then divided into 3 ml aliquots in 12x75 mm polystyrene tubes at 2-3x 106cells/ml. ConA was added to- one-half of them, and duplicates removed at various times, packed for 3 min at loo0 g, washed twice in identical but non-labeled medium, and counted (4SK in 1 ml 0.1 N HCl in a gamma counter, and =Cl in 0.5 ml 0.1 N HNO.) by liquid scintillation.

Validation of techniques Fourhundredfifty ml of blood gave 34 final pellets weighing 5-15 mg each. After carbonyl iron incubation and 3Bcl in 0.5 ml 0.1 N HNO,) by liquid scintillation.

Lymphocyte

Table 1. Effect of ConA on cell K+ Cell K+ (mmoles/kg) Time (min) 0

16 39 98

By *?K

By flame

159 145 136 113

145 128 135 109

Results of a single experiment. Techniques are described in Methods. Cells were pre-incubated in @K overnight. ConA was added at time 0; samples were separated through 12% sucrose and packed in a microtube at 6 000 g.

ions

33

sucrose. The actual trapped space, under the conditions of the final 6 000 g spin, may be lower than 4.2 %, since simultaneous determinations with human red cells gave a trapped space of less than 1% with [“Clnolvethvlene glvcol. If the trapmd space were 1%, then the water-contents would be about 1% higher, and the ion contents about 5% lower than reported, while if trapped space were lo%, then the water content would be about 2% lower and the ion content about 7 % higher than reported. The values for K+ and for total K++Na+ are similar to those reported for normal human peripheral blood lymphocytes from other laboratories [ 16, 171. The use of radiolabeled ions to measure cell ion contents was partially validated in the following manner. Cells were preloaded overnight with 4qK and then incubated in the same labelled medium with ConA added. Cell K+ was estimated by determining the specific activity of the SK in the incubation medium and using the relation:

“K cpm/kg cells white cells were lymphocytes by morphologic criteria. After two washes at 200 g, 70% of lymphocytes were Cell K+ mmoles/kg= &K cpm/mmoles external K+ (1) retained and 70-90% of platelets lost. Remaining platelets were separated at the interface over the 12% sucrose. If there were more than 5% red cells at the The cells were later flamed for K+ content as well. The serial determinations are given in table 1. There is a final stage, the sample was discarded. rather close agreement between the two techniques, Erythrosin B dye exclusion after initial separation was lOO%, and after incubation at 37°C or at 37°C the pairs varying by an average of only 6 %. after preincubation at YC, for up to 48 h, was 86+ 8.5% in controls and 9Of3.9% (1 SD., n=6-8) with ouabain lo- M or ConA 20 pg/ml. Dye exclusion was RESULTS 50% (n=2) at ConA 50 fig/ml and at 100 &ml. Lymphocyte counts at the end of incubation were 9lf23% (1 SD., n=5) of those initially present; the Thymidine uptake: dose response wide deviation is attributed to variable amounts of Optimal [3H]TdR uptake at 72 h occurred microscopic clumping and adherence to containers. The pH was stable at 7.2 to 7.4 in all samples. within a range of ConA concentrations from Effective contact with 12% sucrose lasted about 15 m&nand caused a small amount of cell shrinkage as 2.5 to 20 ~/ml (2.5, 5.0, 7.5, 15, 15 and determined by four paired samples which were sep- 20 pg/ml in 7 individuals tested). Ouabain arated in either sucrose or in incubation medium inhibited mitogenesis with a half-maximal alone, and which had water contents of 74.8+1.2% and 77.1 il.8 % of wet weight, respectively. Similarly, effect between lo-’ and lops M (fig. 1). This pairs of samples from which platelets were nreviouslv removed by-a sucrose separation step befoie incubation, gave a difference in K+ contents of 130fl2 mmoles/kg (1 SD., n=4) in cells spun at 6000 gin the incubation medium, and 145+ 14 mmoles/kg (1 S.D., n=4) in cells spun at 6000 g in 12% sucrose. These differences in water and ion contents reflect a 9 % cell shrinkage in the 12% sucrose separation step. The data are corrected for this unless otherwise noted. Ion levels in the sucrose in the microtube were low, the maximum K+ being 0.94 mM and the average Na+ 4.2+ 1.5 mM (1 SD.. n=27). The trapped space in the pellet, determined by [Ylpolyethylene glycol, was 4.2f3.2% (1 S.D., n=5). The maximum amount of either ion in the pellet which could have been within the trapped space was negligible at 0.03 % of K+ and 0.4 % of Na+. Therefore, no corrections were made for ions, and the cell weight and water content were adjusted according to the 4.2% trapped space. It Fig. I. Abscissa: ouabain cont. (M): ordinate: [3H]should be noted that [W]polyethylene glycol was TdR uptake (% ot control). used to determine trapped space because considerable [3H]TdR uptake at 72 h at the optimal dose of ConA variability was obtained with [14C]inulin or with [“Clin 3 experiments. MeanfS.D. Exp Cell Res 101 (1976)

34

Negendank and Collier

I

5

OO

IO

15

20

Fig. 2. Abscissa: time (hours); ordinate:

cell K+ cont. (mmoles/kg wet weight). O-O, Control; I-m, ConA; O-O, ouabain. Time course of K+ equilibration. ConA was 20 pglml and ouabain was at doses giving a maximal effect (low6 M). Mean+S.D., n=4-6, uncorrected.

agrees with the data of Quastel & Kaplan [ 12-141, who have shown that the inhibition of mitogenesis by ouabain is reversible. To ensure that the effect of ouabain is upon blast transformation and not simply upon thymidine uptake, we examined morphology of cells under conditions described by Nowell [18]. In two sets of samples, ConA at 10 and at 20 pg/ml gave 30% and 37 % clearcut blasts, respectively, at 72 h, while ConA plus ouabain at 10m8or 10W6M gave less than 1% blasts. Effects of ouabain and ConA upon ceil K+, Na+, and water contents Control cells incubated at 37°C at an external K+ concentration of 5.4 mM lost a

small amount of K” and reached a stable state within 15-20 h (fig. 2). Over the same period of time the cell water contents were constant (table 2). Ouabain decreased cell K+ content, and K+ reached a new stable-state level within 15 h (fig. 2). ConA also decreased cell K+ content, and K+ reached a new stable level within 20 h (fig. 2). This effect of ConA on cell K+ content was verified by a different technique, in which cells were pre-loaded with 4?K for 24 h and then incubated in labeled media, as described in Methods. One experiment was done in which cell K+ was measured by both 4?K labelling and by flaming, after addition of ConA 20 kg/ml, and the results are given in table I. There was close agreement between the two techniques, with a drop in ceil K+ at 98 min of 29% and 25 %, respectively. The same experiment was done on six separate occasions using the labelling technique to measure cell K+ content, with simultaneous control and ConA-treated samples. This gave a ConA-induced drop in cell K+, relative to controls, of 21.7+12% (1 S.D., n=6) at 100 min. A dose response curve for ouabain is shown in fig. 3. The half-maximal effect upon K+ and Na+ is within the range of halfmaximal effect upon mitogenic transformation, as determined by C3H]TdR uptake, at

Table 2. Stable-state water and cation contents fin mmoles/liter cell water) Condition

Water (% wet weight)

Initial 37°C 5” to 37°C Ouabain 37°C ConA 37°C

78.7f2.0 (4) 77.4f 1.7 (4) 76.2+ 1.0 (4) 82.1f2.7 (4) 77.1+1.4(4)

P

K+

Na+

K++Na+

P

NS

153+ 15.6 (4) 135kl9.2 (6) 130f20.5 (4) 35.8+_10.2 (8) 85.8? 19.9 (8)

15.9f5.7 (4) 21.4f6.5 (6) 20.9f 12.9 (4) 118k30.8 (8) 38.6513.2 (8)

169+ 15.8 (4) 156+ 15.0 (6) 15Ok33.2 (4) 154f34.2 (8) 125f 19.8 (8)

NS

NS
NS NS co.025

Final K+,, varied between 5.4 and 6.4 mM. Cells were incubated for 2&24 h at 37°C. Those preincubated at 5°C were done so for 24 h. Meansf 1 S.D., n=4-10. Significance is determined by the r-test, relative to values obtained at straightforward 37°C incubation, for water content and for total K++Na+. Ouabain was 2X lo-* M and ConA 20 &ml. Exp CrllRes

101 (1976)

Lymphocyte ions

Fig. 3. Abscissa: ouabain cont. (M); ordinate: cell ion cont. (mmoles/kg wet weight). Dose-response curve for ouabain. Data from a single experiment, at external K+=5.4 mM, in the stable state at 21 h. In multiple experiments at 5x 1Om8M, K+ was 80.6f 15.7 (n=4) and Na+ 39.3f 14.7 (n=4) mmoles/kg. At 2~10~~ M, K+ was 29.4f8.4 (n=8) and Na+ 96.7f25.3 (n=8) mmoles/kg. At 10m2M, K+ was 20 mmoles/kg (n=2) and Na+ 104 mmoles/kg.

I

I

I

I

0

25

50

75

35

I

100 Fig. 4. Abscissa: ConA cont. (pg/ml); ordinate: cell ion cont. (mmoles/kg). 0, K+; 0, Na+. Doseresponse curve for ConA. Means of 2-4 samples, at external K+=5.4 mM, in the stable state at 20-24 h.

strate that this is actually associated with a rapid and a sustained decrease in cell K+ content. lo-’ to 10e8M. Ouabain induced a mole-formole replacement of cell K+ by Na+. The dose-response curve for ConA is shown in fig. 4. The decrease in cell K+ occurred throughout the mitogenic, non-toxic range, up to about 25 pg/ml, and extended into the toxic range as well. Unlike ouabain, ConA did not induce a replacement of lost cell K+ by Na+, and the total K++Na+ was decreased in spite of a normal water content. These data are summarized in table 2. Thus, ouabain affects the cell ion contents in a fashion that is predictable from its ability to inhibit Na,K-ATPase, and it does so with a critical dose range which approximates the critical dose range for inhibition of mitogenesis. This is consistent with the concept that normal Na,K-ATPase function is required for mitogenic transformation to occur. However, it does not prove that K+ content serves as a specific trigger in mitogenic stimulation. It should also be noted that although mitogens induce an increased influx of 4?K added to the external medium [ 1-3, 151,our studies demon-

Content of Cl- in transformed lymphocytes Because ConA induced a decrease in total cell K++Na+ without a change in cell water content, the nature of charge and solute balance in the transformed lymphocyte was examined. The loss of K+ was not accompanied by a loss of Cl- (fig. 5). In fact, there was a very low cell Cl- level, as confirmed by extraction of 3-10 mg samples into 0.1 M

36CI Prelood

00° 50 100 150 Fig. 5. Abscissa: time (min); ordinate: cum/sample (%). l , Control; 0, ConA. Cells were preloaded with 3BC1for 24 h, and ConA (20 &ml) added at time 0 without changing the specific activity of the medium. Exp CellRes lOl(1976)

36

Negendank and Collier

external K+ level (mM); ordinate: cell K+ cont. (mmoles/kg). -, Control; O-O, ConA; CO, ouabain. Stable-state distribution of cell K+ at 37°C. Control curve was that reported earlier (see text). Points are means+S.D., n=4-8, uncorrected. Ouabain is 5~ 1OF M and ConA 20 pg/ml.

Fig. 6. Abscissa:

HN03 for 24 h and titration with a BuchlerCotlove chloridometer; with this technique, almost no cell Cl- was measured. Stable-state ion distribution patterns In order to further define the mechanisms of action of ouabain and ConA, their effects upon cell K+ contents were determined over a wide range of external K+ levels. Lymphocytes incubated at 37°C at various external K+ levels reached a new stable level of cell K+ by 15-20 h, as shown in fig. 2. Incubation at 37°C for 2&24 h was sufficient to achieve a new stable state, at all external K+ levels, with ouabain or ConA. However, control cells at very low external K+ levels appeared to require up to 48 h to reach a stable-state internal K+ level. If preincubated at 5°C cells lost K+ much more quickly, and if then re-incubated at 37°C regained K+ to reach, within 6 h, the same stable-state level that would have been obtained at 37°C alone (table 2). Using this technique, we have Exp CellRes 101 (1976)

determined the distribution of cell K+ as a function of external K+ over a wide range of external K+ levels (submitted for publication). Part of the data accumulated in that study were obtained simultaneously with samples treated with ouabain or ConA, and will serve as the control. Subsequent experiments with ouabain or ConA were done at 37°C alone, and all the data were pooled together to generate the curves reported here. The stable-state levels of cell K+, as a function of external K+, are shown in fig. 6. There is a steeply-rising sigmoid relation between cell K+ and the external K+ level. By using a statistical-mechanical adsorption model [ 19-211, we have shown that a cooperative transition underlies K+ accumulation (ms in preparation). That is to say, sites involved in K+-Na+ exchange interact in such a manner as to increase the affinity for K+ as more and more K+ is adsorbed to them. The cooperative adsorption isotherm follows an equation which is analogous to the “Hill” plot: lnX,+/l -X,+ =n In K+ex/Na+ex+ n ln&E‘,,N,,

(2)

where X,+ is the mole fraction of cell K+, K+,, and Nafex are external K+ and Na+, n is Hill’s empirical coefficient, and ae,N. is the equilibrium constant of exchange of K+ for Na+. The major parameters characterizing the curve are xK,Na, a measure of the selectivity of K+ over Na+, and n, a measure of the steepness of the curve and of the cooperativity in K+ uptake. The “Hill” plots of the control, ouabain, and ConA data are shown in fig. 7, along with a tabulation of the key parameters of ion exchange. Ouabain shifts the K+ distribution curve to the right (fig. 6). At a ouabain con-

Lymphocyte -IO2

- IO'

- IO0

- lo-

c 16’

16’

lo”

I 16’

Fig. 7. Abscissa: ratio of external K+ to external Na+; ordinate: ratio of mole fraction of K+, XK+, to

1-XK+, in eq. (2). The “Hill” plot. The data is that of fig. 6, plotted according to es. (2). The slope through the midpoint determines -n, and the position along the abscissa represents KK,Na, the selectivity of K+ over Na+. N,,, is the saturable level for total K++Na+ (table 2).

centration of 5x lo-* M, kkpNa decreased greatly, reflecting a decreased selectivity of K+ over Na+ (fig. 7). The Hill coefficient, n, decreased from 3.7 to 2.4, reflecting a decreased cooperativity in K+ uptake. Thus, ouabain behaves in the manner expected if it were assumed to alter Na,K distribution in an allosteric manner. ConA shifts the K+ distribution downward (fig. 6), but only slightly decreases K+-Na+ selectivity, and perhaps slightly increases cooperativity (n=4.1) (fig. 7). It appears to decrease the total “sites” available for K+. Thus, ConA decreases cell K+ by a mechanism that is quite different from that of ouabain.

DISCUSSION There are four major conclusions from these data. (1) Ouabain inhibits mitogenesis in a critical dose-response range that approx-

ions

37

imates the critical dose-response range for its effect upon cell K+ and Na+; and it affects K+ and Na+ contents in a predictable manner assuming that it allosterically inhibits the K+-Na+ exchange process. Normal functioning of the Na,K-ATPase may be required for optimal mitogenic transformation to occur. There is, however, conflicting evidence whether PHA or ConA specifically alter the Na,K-ATPase enzymatic function [3, 15, 22, 231, although this is a small part of total cell ATPase [241. (2) ConA does not activate the Na,KATPase by a mechanism that simply opposes that of ouabain. Another mitogen, phytohemagglutinin (PHA) has, in preliminary reports, also been found to decrease the cell K+ content [16, 25, 261. In spite of the fact that cell K+ is reduced, the more critical parameters of ion accumulation-the selectivity of K+ over Na+ and the cooperativity in K+ uptake-are not substantially changed by mitogenic doses of ConA. (3) The transformed lymphocyte undergoes a loss of K+ without a change in the water content, and the apparent solute and charge deficit is not accounted for by an uptake of Na+, or by a movement of Cl-. (4) There is no significant change in the Na+ gradient between cell and external medium in transformed cells, and therefore the increased amino acid uptake [27, 281 is not energized by this mechanism. The loss of cell K+ might be explained by supposing that ConA induces an increased leakage of K+ outward by a nonspecific increase in membrane permeability. This might then result in a secondary activation of the Na,K-ATPase in order to partially offset the K+ leak, with the net result of increased leak and increased pump being a lower stable-state level of cell K+. ExpCdRes

101 (1976)

38

Negendank and Collier

In this case, however, one would expect a concomitant rise in cell Na+. This does not occur, and these data suggest that some more fundamental alteration in the ion-exchange mechanism must be occurring. It seems unlikely that K+ acts as a single specific “trigger” in the initiation of the early events of mitogenic transformation. On the other hand, it is also unlikely that the changes in K+ flux and content are merely coincidental to the effects of ouabain and of ConA upon mitogenesis, especially in view of the ability of increased external K+ to overcome the inhibition of mitogenesis by ouabain [12-141. We postulate, therefore, that K+ and Na+ associate with a macromolecule, or an assembly of macromolecules, that is critically affected by both ouabain and ConA and that may include, among other things, the Na,KATPase. K+ is then viewed as a physiologic probe that monitors the state of that assembly of macromolecules. Within this context, the following four hypotheses are suggested. (1) The parameter KK,Na, which relates explicitly to ion selectivity, reflects changes in anionic field strength of sites involved in K+ and Na+ exchange [2%31]. Changes in these sites, induced allosterically by chemical or physical stimuli, may shift the K+ distribution isotherm, and bring the curve within a critical range with respect to local ion activities. (2) The mechanism for K+/Na+ exchange, with its high selectivity for K+ and its high cooperativity in K+ uptake, is poised to respond to appropriate stimuli in a critical, all-or-none manner, and this may underly the rapid change in K+ flux that occurs within minutes of mitogenic stimulation. (3) Ouabain, by an allosteric effect upon the macromolecular assembly, shifts the E.~D Cell Res 101 (1976)

K+/Na+ exchange mechanism out of the critical range with respect to external, or local, K+ levels, and thus makes it insensitive to such stimuli. (4) The ability to respond to ConA is preserved as long as the mechanism for K+/Na+ exchange maintains its high K+ selectivity and cooperativity. However, ConA itself induces a change in this mechanism which decreases the selectivity for K+, not in favor of Na+, but in favor of some as yet unidentified cation. These conclusions, and the accompanying hypotheses, leave at least three fundamental questions: (1) What, and where, are the underlying macromolecules? (2) What accounts for the apparent solute and charge imbalance? (3) What is the relation between K+ and Ca2+ in the transforming lymphocyte? (1) The presence of a complex macromolecular assembly has been postulated to account for some of the effects of mitogens upon receptor mobility and the ability of colchicine to inhibit mitogenesis [32]. The assembly is thought to contain a membrane protein, microtubules, and microfilaments. The membrane proteins may be the glycoproteins that appear to aggregate during mitogenesis [33]. The assembly may either include the Na,K-ATPase or itself be involved in K+ and Na+ transport. Its precise nature, its relation to critical surface membrane events [34-361 or to lipid metabolism [37, 381, its cytoplasmic extent, and its possible interaction with the nuclear membrane, are undetermined. (2) The apparent charge deficit within the transforming lymphocyte might partially be accounted for by Ca2+, and perhaps by the positively-charged histone proteins which are released from binding to nucleic acids within 30 min of mitogen interaction [39]. However, these are unlikely to be of

Lymphocyte

sufficient magnitude to replace the loss of some 20% of cell solute. A similar discrepancy in total K++Na+ and cell water content occurs in synchronously dividing mouse leukemic lymphoblasts [40]. The major anions of the lymphocyte are not known. A higher total cell K++Na+, without a change in water content, has been described in human chronic lymphocytic leukemia cells [17], a finding we have confirmed (unpublished). One explanation for these discrepancies may be that a significant fraction of ions are closely associated with cellular macromolecules. Direct evidence for this is lacking, but there is some indication that a fraction of Na+ is associated with a nuclear component in rat liver and thymic nuclei [4 1,421. Finally, the relation between K+ and Na+, on the one hand, and Ca2+ and Mg*+, on the other, is unknown. The critical nature of changes in Ca*+ during mitogenesis is indicated by the requirement for external Ca*+ [7-91, the rapid influx of Ca*+ [4-6], and the mitogenic capacity of the ionophore A23187 [lo, 111. We postulate that changes in Ca*+, like changes in K+, reflect a more fundamental change within an underlying macromolecular assembly. There is conflicting evidence regarding the roles of CAMP and cGMP [43-46], but changes in Ca2+ and in cGMP may be related [47]. K+, Ca*+, and cyclic nucleotides closely interac.t in a number of cells [48], and may do so in the transforming lymphocyte as well.

ions

39

physio186 (1975) 327) suggested that lectin-induced decrease in cell K+ may be partially due to an artifact introduced during final washing procedures. To test this possibility we preloaded cells with “*K and incubated with and without ConA in labeled media as described above (Methods). Aliquots were simultaneouslv senarated through 12% sucrose and through a non-aqueous medium (dibutyl-phthalate-oil) sirnil& to that used bv Averdunk & Lauf Il51. ConA-induced decrease in cell K+ was similar with either sucrose or phthalate oil separation, and dropped 16f7.2%, n=9, at 4 h. Cell sample size, determined by [3H]Hz0, was eaual in control and ConA samnles. as was tKiDDed medium determined by [14C]polyethylene glycol. Thus. the ConA-induced decrease in cell K+ renorted here occurs during incubation and is not a washing artifact.

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