Effects of Cd2+ upon Ca2+ fluxes and proliferation in concanavalin A-stimulated lymphocytes

Experimental Cell Research 156 (1985) 191-197

Effects of Cd*+ upon Ca*+ Fluxes and Proliferation Concanavalin A-Stimulated Lymphocytes

in

IAN G. SCOTT,** ** C. HENRIK J. WOLFF, KARL E. 0. AKERMAN* and LEIF C. ANDERSSON Department of Pathology, University of Helsinki, SF-00290 Helsinki 29, Finland

The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) is inhibited by micromolar concentrations of CdC12.This inhibition is partially relieved by an increase in the external Ca*+ concentration (from 0.6 to 2.2 mM). The initial rate of conA-induced 4sCa2+ influx is unaltered by CdC12, although the level of 45Ca2+ accumulation increases. The basal rate of 45Ca2+ entry is not measurably disturbed by CdC12 (100 FM). The steady-state efthtx of 45Ca2+ and the calmodulinactivated (Ca*+ + Mg*+)-ATPase activity of erythrocyte ghosts are inhibited by CdC12 (10 PM). Thus, the mechanism behind the Cd*+-induced suppression of the mitogenic response to conA is not due to alteration of mitogen-stimulated Ca*+ influx. We suggest that Cd*+ competes with Ca*+ for intracellular Ca*+-binding molecules, such as calmodulin, essential for the induction of cell proliferation. @ 1985 Academic press. Inc.

An early increase in intracellular free Ca*+ is thought to be an essential factor in the mitogen-induced proliferation of T lymphocytes [l-3]. Mitogenic lectins cause an increase in cytosolic Ca*+ activity [4] within lymphocytes; an effect which has been linked to changes in Ca2+ flux across the plasma membrane [5-91. Removal of Ca*+ during the first 20 h of culture will block mitogeninduced DNA synthesis [IO-121 in a reversible manner [12]. Furthermore, a mitogenic effect of the Ca*+ ionophore A23 187 has been reported [7, 13-151. Mn*+, which blocks Ca*+ currents in excitable cells [16], inhibits mitogenic activation of human peripheral blood lymphocytes [17, 181if present during the initial 16 h of culture. This Mn*+ -induced inhibition of DNA synthesis may be reversed by increasing the Ca*+ concentration of the culture medium [18] and therefore it was suggested that Mn*+ blocks Ca*+ transport into lymphocytes, thus preventing the activation of Ca*+-dependent processes. Earlier studies have shown that metal cations including Co*+, Cd*+ and Mn2+ block DNA synthesis [ll] but do not affect Ca*+ uptake in resting lymphocytes [19]. These metal cations do, however, interact with the Ca*+-binding sites of the Ca*+-dependent regulator protein calmodulin [20-221. Cd*+ and Ca*+ have similar binding constants for calmodulin, the binding of these cations to pure calmodulin being * Present address: Department of Biochemistry and Pharmacy, Abe Academy, Porthansgatan 3, SF-20500 Turku 50, Finland. ** To whom correspondence and offprint requests should be addressed. 13-858331

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827185 $03.00

192 Scott et al. competitive with respect to one another 121, 221. Furthermore, the crystal ionic radii of the two cations are also similar [23]. The aim of the present study was to determine whether the inhibitory action upon lymphocyte proliferation of a heavy metal ion with physico-chemical similarities to Ca*+ is due to interference with the early rise in Ca*+ influx promoted by mitogenic stimulation, or attributable to a blocking of the intracellular triggering action of Ca*+.

MATERIALS

AND METHODS

Zsolation of Cells and Ceil Culture Human peripheral blood lymphocytes were isolated from buffy coats (generously supplied by the Finnish Red Cross Blood Transfusion Service) by Ficoll-Hypaque density gradient centrifugation 1241.Red blood cells were isolated from the blood of healthy donors by washing twice in 0.9% NaCl at 1000 g for 10 min and removing white cells layering at the top of the pellet. Erythrocyte ghosts were prepared by suspending the red cells in 10 mM Tris-HCl (pH 7.6) containing, in addition, 1 mM EGTA. Washing was repeated 3-4 times in the same medium until the red colour disappeared. The ghosts were then washed once with 10 mM Tris-HCl (pH 7.6) and stored at -20°C until required for use.

Measurement of Proliferative Response Lymphocytes were resuspended to 106 cells/ml in RPM1 1640 cell culture medium supplemented with 10% normal human serum plus 100 pg streptomycin/ml. The reagents tested (conA, CdCl*, supplementary Cat&) were added in small aliquots of aqueous solution to 1.5 ml volumes of lymphocyte suspension and 180 ul aliquots placed in the wells of cloning plates. After 66 h of incubation in a humidified atmosphere of 5 % CO* in air at 37.5”C, the cultures were pulsed with 2O+l volumes of 40 pCi/ml [‘Hlthymidine in RPM1 medium and, subsequent to a further 6-h incubation, harvested using an automatic cell harvester (Dynatech Automash.2). Cell-incorporated radioactivity was then measured.

Experimental Conditions in 45Ca2+Flux Studies The influx of 45Ca2+ was assayed using a protocol similar to that already described [9]. A suspension of 20-25 x 106cells/ml in RPM1 medium was supplemented with 20 mM Tes buffer, pH 7.4, and incubated for 15 min in order to reach a steady-state condition for ion distribution. CdC12was then added followed 3 min later by conA (pie-treated with 10 mM CaCl, in order to obviate 45Cabinding to the lectin) plus 45Ca(1 @ml), and samples taken over a time course. The measurement of 4sCa2+et&x was also conducted using a previously reported method [9]. 20-25x lo6 cells were preincubated with 4sCa2+ of high sp. act. (20 @i/ml) in a medium containing 137 mM NaCl, 5.4 mM KCI, 1.2 mM MgC12, 0.44 mM KH2P04. 4.2 mM NaHCOs, 5 mM glucose and 20 mM Tes (pH 7.4), at room temperature for 30 min. Thereafter the cells were washed twice, divided between two tubes, resuspended in the same medium (without 45Ca) containing either 0.4 mM CaC12 alone or in combination with 100 PM CdC12. incubated at 37.X and samples taken over a time course. ‘Hz0 (4 pCi/ml) was present during these assays in order to provide a relative measure of cell number/assay sample.

The Measurement of Erythrocyte (Ca*+ + Mg*+)-ATPase Activity The ATPase assay was performed in the manner described by Ronner et al. [25]. The assay medium contained 40 mM Hepes (pH 7.4, Tiis), 20 mM KCl, 4 mM MgCl, 0.05 mM CaC12, 1 pg/ml calmodulin, 0.1 mM ouabain, 100 mM NaCl, 0.2 mM NADH, 0.2 mM phosphoenolpyruvate, 1 IU/ml pyruvate kinase and 1 IU/ml lactate dehydrogenase. The ghosts were preincubated at 37°C for 15 min before initiation of the reaction by the addition of 0.5 mM ATP, and the absorbance at 340 nm was Exp Cell Res 156 (1985)

Lymphocyte Ca2+ flux and proliferation

inhibited by Cd2’

193

measured using a Labsystems (Helsinki, Finland) FP-9 automatic spectrophotometer. Calmodulin was isolated as described by Gopalkrishna & Anderson [26].

Materials RPMI-1640 medium is a product of Gibco Ltd, Paisley, Scotland. Radioisotopes were obtained from the Radiochemical Centre, Amersham, Bucks, UK. Concanavalin A was from Pharmacia AB, Uppsala, Sweden. The enzymes and substrates used in the ATPase assay were obtained from the Sigma Chemical Co. (Poole, Dorset, UK). All the other reagents employed were commercial products of the highest grade available.

RESULTS Effect of CdCl;! upon the Stimulation of Lymphocyte Proliferation by ConA The ability of CdC12 to inhibit conA-induced increase in L3H]thymidine incorporation is shown in table 1. CdC12 at micromolar concentrations partially inhibits and 100 PM completely abolishes the lectin-stimulated [3H]thymidine incorporation. The concentration-dependent inhibition of proliferation by Cd2+ appears to be proportionately stronger at sub-optimally mitogenic levels of conA. By increasing the external Ca2+ concentration from the normal level of 0.6-2.2 mM (table 2), a partial recovery of the proliferative response to conA is effected

Table 1. Effect of CdClz on conA-stimulated proliferation

of lymphocytes

For experimental procedures see Methods. [3H]Thymidine incorporation (cprn/well) expressed as the means + SE of six determinations per assay condition ConA cont. (i-d-4

CdCl* concentration (PM) 0

1

10

100

0.0 0.5 5.0 50.0

603&89 8 270+1 100 22 760+2 010 57 203f3 325

45lf225 5 012+1 730 18 25lfl 202 48 129?2 510

514+148 2 008f370 10 21151 450 47 233? 1 655

87+11 82+30 420+45 233+45

Table 2. Effect of an increase in culture medium Ca2+ on the inhibitory action of Cd2’ on conA (5 pug/ml)-stimulated lymphocyte proliferation Experimental conditions as described in Methods. 13H]Thymidine incorporation (cpmwell) pressed as mean + SE of six measurements/assay conditions Ca*+ cont. W)

0

30

100

0.6 2.2

10 82221 012 11 580+960

6 860+760 13 321f420

263+35 3 6305 150

ex-

CdC12concentration (PM)

Exp Cell Res 156 (1985)

194 Scott et al.

$,,1

‘. 25

50

tm? ml

Fig. I. The effect of CdC12upon 45Ca2+mflux into lymphocytes. Incubation conditions as described in Methods. At time 0, the following additions were made in combination with 45Ca: 0, None; 0, 50 ug conA/ml; A, none, to cells given 100 uM CdC12at time -3 mitt; A, 50 ug conA/ml to cells given 100 uM CdC12at time -3 min. Results are expressed as nmol Ca” taken up per ul ‘H20 space. The [3H]H20 volume per cell does not undergo significant change during the course of incubation with or without conA and/or CdC12. Data points represent the means of triplicate assays. SD from the mean of each triplicate assay was less than 10%. Fig. 2. The effect of CdC12 on 45Ca2+ efflux from lymphocytes. Experimental conditions as described in Methods. 45Ca2+-loaded cells resuspended in medium containing either 0, 0.4 mM CaC12alone, or 0, in combination with 100 @I CdC12.This experiment was repeated three times and gave qualitatively similar results on each occasion.

in 100 uM CdCl*-treated cells, while cells exposed to 30 uM CdC12, a submaximally inhibitory concentration, fully recover their DNA synthetic capacity. About 50% (average of three observations) of the cells, cultured either at 0.6 or 2.2 mM Ca in the presence of 100 uM CdCl*, excluded trypan blue at the end of the 3-day culture period, whereas about 80% of the control cells (no CdCl* present) excluded the dye. Effect of CdC12 upon the Stimulation of 45Ca2+ Uptake by ConA The rate of entry of 45Ca2+ions, either under basal conditions or in response to conA, is not measurably influenced by 100 uM CdC12(fig. 1). The heavy metal ion does, however, appear to amplify the increase in 45Ca2+ uptake caused by the lectin. Thus, provided that the outward flow of Ca*+ ions was not enhanced by some action of Cd*+, it was apparent that a net increase in the Ca*+ content of conA-stimulated cells might ensue. To test the possibility that Cd*+ might cause an increase in the accumulation of Ca*+ by actually blocking its active extrusion, the release of 45Ca*+ from pre-loaded cells was measured in the presence and absence of 100 yM CdC12(fig. 2). An inhibition in the unidirectional efflux of 45Ca2+ is caused by Cd*+; the rate constant, as calculated from the data of fig. 2, and that of two other similar experiments, decreasing from the control level of 0.0338~!10.0030per min to 0.0235f0.0024 per min (means ? SD). The conA-induced transitory increase in the rate of Ca*+ loss [9] is unaffected by Cd*+ (data not shown). Exp Cell Res 156 (1985)

Lymphocyte Ca*+ jlux and proliferation

inhibited by Cd*’

195

Effect of CdCl2 on Erythrocyte (Ca*+ + Mg*+ ATPase Activity The active extrusion of Ca*+ from cells is mediated by calmodulin-activated (Ca*+ + Mg*+) ATPase. An enzymatic activity having properties identical with that of erythrocyte ghosts, but with lower activity, has been found in plasma membrane from a variety of cells [27], including lymphocytes [28, 291. In order to investigate whether this activity is inhibited by Cd*+, red blood cell ghosts were used because of the absence of contaminating ATPases associated with the presence of mitochondria, endoplasmic reticulum or microfilaments in other plasma membrane preparations [30]. Table 3 shows that 10 yM CdClz blocks the calmodulin-activated (Ca*+ + Mg*+) ATPase activity of erythrocyte ghosts. If the ghost membranes are exposed to Ca*’ before addition of CdC12, the resultant inhibition is considerably less. DISCUSSION The results of the present study show that Cd*+ inhibits lymphocyte proliferation in a manner which is partially relieved by increasing the Ca*+ concentration of the culture medium, in agreement with the findings of Hart [18], regarding the relief by Ca2+ increment of Mn*+-suppressed proliferation. The protective role of Ca*+, together with the linear relationship between [3H]thymidine incorporation and proliferation of lymphocytes [31] renders it unlikely that thymidine transport could be rate-limiting during a 6-h period at sub-optimal CdC12concentrations. It should be noted that the free concentration of Cd*+ responsible for this inhibitory action is probably about an order of magnitude lower than the chemical strength of the added CdC&, due to the inevitable formation of complexes with certain anions (e.g., Cl-, PO:-) in the culture medium [32]. It is reasonable, however, to assume that Cd*+ is taken up by lymphocytes, since there are reports in the literature demonstrating its intracellular localization, both in human [33, 341 and murine tissue [35]. The effects of Cd*+ on [3H]thymidine incorporation are probably not attributable to an inhibition of DNA synthesis, per se, or to toxic effects, since the proliferation of an erythroblastoid leukemia cell line, K562, as well as several other types of transformed cells, is completely unaffected by CdC12 concentrations up to 100 uM (unpublished observations Table 3. Effect of Cd*’ on the calmodulin-activated activity of human erythrocyte ghosts

(Ca*+ + Mg*+)ATPase

For experimental conditions, see Methods. IO pM Cd was added to the assay medium either before or 10 min after the addition of ghosts. The results shown are an average of four separate determinations (+ SE) expressed as nmol/mg protein per min Control

Cd*+ before

Cd*+ after

40+3

1.3kO.6

15+1 Exp Cell Res 156 (1985)

196 Scott et al. from our laboratory). In addition, the ability of a large proportion of 100 ~I,M CdClz-treated lymphocytes to remain viable during the 3-day culture period, as indicated by the exclusion of the dye, trypan blue, suggests that toxic effects are unlikely to be the major reason for the failure of these cells to incorporate [3H]thymidine. The action of Ca2+ in enhancing the early rise in cell Ca2+ content caused by conA binding, whereas at a later stage, it inhibited the DNA synthesis response, suggests interference with some Ca2+-activated process(es) necessary for proliferation. In a wide variety of cells, calmodulin acts as a receptor for Ca*+ and the Ca2+-calmodulin complex subsequently activates a number of cellular functions [36, 371. The increase in lectin-stimulated Ca2’ accumulation caused by Cd2+, in association with its inhibitory effect upon 45Ca2+ efflux, suggests that the Ca2+ extrusion pump in the plasma membrane is one target for the action of heavy metal ions such as Cd2+. The inhibition, by Cd2+, of 45Ca2+efflux from lymphocytes may seem small. It should, however, be borne in mind that only a small part of this flux is assumed to be due to active Ca2+ extrusion-the rest must be due to Ca2’ exchange [36]. If Cd2+ merely interfered with the passive exchange of Ca2+ ions, one would expect to see effects upon both Ca2+ efflux and the resting inflow of the ion. Since Cd2+ does not affect the basal Ca2+ influx, it probably has no influence on the resting eMux either. The high permeability of the plasma membrane to Ca2+ in the presence of conA explains our inability to observe any inhibition of 45Ca2+ efflux induced by lectin, since any inhibition of Ca2+ extrusion would be masked by the high rate of 45Ca2+loss. In lymphocytes [28, 291 as in many other cell types [38, 391, the outwardly directed Ca2+ pump of the plasma membrane is calmodulin-activated (Ca2+ + Mg2+) ATPase. The inhibition of this enzyme in erythrocyte ghosts, by 10 PM CdC12, together with the findings that Cd2+ increases Ca2+ accumulation, decreases efflux and interacts with the Ca2+-binding sites of calmodulin [20-221, provides compelling circumstantial evidence to suggest that calmodulin may well be the target for the inhibitory action of these heavy metal cations in lymphocyte proliferation. The biological incompetence of the Cd2+-calmodulin complex has been demonstrated by Cox & Harrison [40] who have shown that Cd2+ inhibits calmodulin-activated phosphodiesterase activity. We cannot yet exclude that the possibility that the (Ca2+ + Mg2’) ATPase is inhibitable through interaction of Cd2+ with Ca2+-binding sites involved in the transport of Ca2+. In a more thorough kinetic analysis of the action of Cd2+ on (Ca2+ + Mg*+) ATPase activity we have found, however, that the effect of Cd2+ is very similar to that of the removal of calmodulin previously associated with the enzyme [41]. Thus the interaction of Cd2+ with calmodulin remains the most plausible explanation for inhibition of the Ca2+ extrusion mechanism, and perhaps also of the lymphocyte proliferative response. It is thus proposed that calmodulin or some other Ca2+-binding protein acts as a receptor for Ca2+ in lymphocyte activation, and that Cd2+ acts by blocking the response of intracelluEXF,CellRes 156 (1985)

Lymphocyte

Ca*+ jlux and proliferation

inhibited

by Cd*’

197

lar mechanisms to the enhanced cytosolic Ca*+ concentration promoted by the initial mitogenic signal. This study was aided by grants from the Finnish Academy, the Sigrid Juselius and the Finnish Medical Society (Finska Lakareslllskapet). I. G. S. is supported by a grant from the Royal Society European Science Exchange Programme. Appreciation is extended to MS Helena Nousiainen and MS Kaija Niva for technical assistance and MS Outi Rauanheimo and MS Maja-Leena Rissanen for typing the manuscript.

REFERENCES 1. Rasmussen, H & Goodman, D B P, Physiol rev 57 (1977) 421. 2. Metcalf, J C, Pozzan, T, Smith, G A & Hesketh, T R, Biochem sot symp 45 (1980) 1. 3. Akerman, K E 0, Med biol 60 (1982) 168. 4. Tsien, R Y, Pozzan, T & Rink, ‘f J, Nature 295 (1982) 68. 5. Freedman, M Y, Raff, M C & Gomperts, B, Nature 255 (1975) 378. 6. Hesketh, T R, Smith, G A, Houslay, M 0, Warren, G & Metcalfe, J C, Nature 267 (1977) 490. 7. Jensen P, Winger, L, Rasmussen, H & Howell, P, Biochim biophys acta 496 (1977) 374. 8. Mikkelsen, R B & Schmidt-Ullrich, R S, J biol them 255 (1980) 5177. 9. Wolff, C H J & Akerman, K E 0, Biochim biophys acta 693 (1982) 315. 10. Allwood, G, Asherson, G L, Davey, M J & Godford, P, J immunol21 (1971) 509. 1I. Whitney, R B & Sutherland, R M, Cell immunol 6 (1973) 137. 12. Hovi, T, Allison, A C & Williams, S C, Biochem biophys res commun 88 (1979) 1337. 13. Luckasen, J R, White, J G & Kersey, H H, Proc natl acad sci US 71 (1974) 4088. 14. Maino, V G, Green, N M & Crumpton, M J, Nature 251 (1974) 324. 15. Hovi, T, Allison, A C & Williams, S C, Exp cell res 96 (1976) 92. 16. Baker, P F, Progr biophys mol biol 24 (1972) 177. 17. Berger, N A 62 Skinner, A M, J cell biol 61 (1973) 49. 18. Hart, D A, Exp cell res 113 (1978) 139. 19. Parker, C W, Biochem biophys res commun 61 (1974) 1180. 20. Teo, W & Wang, J H, J biol them 247 (1973) 5950. 21. Forsen, S, Thulin, E, Drakenberg, T, Krebs, J & Seamon, K, FEBS lett 117 (1980) 189. 22. Andersson, A, Drakenberg, T, Forsen, S & Thulin, E, Eur j biochem 126 (1982) 501. 23. Weast, R C, Selby, S M & Hodgson, C D, Handbook of chemistry and physics, 46th edn, p. Fll7. Chemical Rubber Co., Cleveland, Ohio (1965). 24. Boyum, A, Stand j clin lab invest 21 (1968) 77. 25. Ronner, P, Gazotti, P & Carafoli, E, Arch biochem biophys 179 (1977) 578. 26. Gopalkrishna, R & Anderson, W B, Biochem biophys res commun 104 (1982) 830. 27. Vincenzi, F F & Hinds, T R, Calcium and cell function (ed W Y Cheung) vol. 1, pp. 127-165. Academic Press, New York (1980). 28. Lichtmann, A H, Segel, G B & Lichtmann, M A, J biol them 256 (1981) 6148. 29. Averdunk, R & Gunther, T, Biochem biophys res commun 97 (1980) 1146. 30. Schatzmann, H J, Current topics in membranes and transport (ed F Bonner & A Kleinzeller) vol. 6, p. 125. Academic Press, New York (1975). 31. Gunther G R, Wang J L & Edelman, G M, J cell bio162 (1974) 366. 32. Sill&t, L G & Martell, A E, Stability constants of metal-ion complexes. Special publication no. 17, The Chemical Society, London (1964). 33. Bakka, A, Eriksen, D @, Rugstad, H E & Bauer, R, FEBS lett 139 (1982) 57. 34. Rugstad, H E & Norseth, T, Nature 257 (1975) 136. 35. Bryan, S E & Hildago, H A, Biochem biophys res commun 68 (1976) 858. 36. Klee, C B, Crouch, T H & Richman, P G, Ann rev biochem 49 (1980) 489. 37. Cheung, W Y, Science 207 (1979) 19. 38. Borle, A B, Rev physiol biochem pharmacol90 (1981) 13. 39. Carafoli, E & Crompton, M, Curr top membr transp 10 (1977) 151. 40. Cox, J L & Harrison, S D, Biochem biophys res commun 115 (1983) 106. 41. Akerman, K E 0, Honaniemi, J, Scott, I G & Andersson, L. Submitted for publication. (1984). Received March 23, 1984 Revised version received June 21. 1984 Printed

in Sweden

fip

Cell Res 156 (1985)