110 Biochimica et Biophysica Acta, 1091 (1991) 110-114 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 016%4889/91/$03.50

ADONIS 0167488991000514

BBAMCR 12827

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization

in rat thymus lymphocytes

Francisco Martin, Antonio Gualberto, Francisco Sobrino and Elizabeth Pintado Departamento de Bioqulmica Mddica y Biologla Molecular, Facultad de Medicina. Seviila (Spain)

(Received 19 April 1990)

Key words: Thimerosal; Thymus lymphocyte; Calcium, intraccllular; pH, intracellular; Fructose 2,6-bisphosphate

The effect of thimerosal on intracellular calcium (ICa2+l,), pH (pH,) and fructose 2,6-bisphosphate (Fro 2,6-P 2) in thymus lymphecytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+h, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited 13H]thymidine incorporation when cells were challenged with PHA and ¢ostimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na+.containing buffer, thimerosal induced an initial acidification (0.05 + 0.01 pH units), followed by an alkalinization of 0.08 pH units/mish whereas in Na+-free media, pH i decreased 0.2 4- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pH i was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-in- duced acidification although pH returned to basal levels after 10 min. it was concluded that alkalinization induced by thimerosal is probably due to activation of the N a + / H + exchanger and that changes in internal Ca 2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.

Introduction

The activation of T lymphocytes in response to specific antigenic stimulation, growth factors or lectins leads to DNA synthesis and cell division. This activa- tion is rapidly transduced b5 ~ internal signals, which include the elevation of intraeelhlar free calcium [1-3], cytoplasmic alkalinization [4,5] and an enhanced glyco- lyric flux [6], among other pexameters.

Cytosolic alkalinization is possibly due to activation of the Na+/H + antiport that exists in the plasma membranes of thymic lymphocytes [4,7]. This exchanger is thought to play a mayor role in the regulation of cellular volume and in cytoplasmic pH homeostasis and it has been suggested that it may be involved in mito- genesis [8,9]. In thymocy~es, the activation of the Na+/H + antiport by mitogenic lectins is associated with an increase of intracelhlar Ca 2+ [10]. It has been reported that elevation og [Ca2+]i by means of iono-

Conmpondence: F.. Pintado, Departamento de Bioquimica M&lica y Biologia Molecular, Facultad de Medicina, Avenida Sanchez Pizjuan 4, 41009 Sevill~ Spain.

phores results in elevated metabolic H + production and increased H + uptake. However, these changes are over- come by a stimulated Na+ /H + antiport, which renders the cytoplasmic compartment alkaline [10]. In a~-ldition, phorbol esters induce activation of Na+/H + exchange through phosphorylation of the antiport by protein kinase C without any change in cytosolic Ca 2+ [.5].

Besides the changes in cytosolic ionic concentrations, glycolysis is also enhanced in malignantly transformed cells and in normal cells during proliferation [11]. A stimulation of the release of lactate can be observed following exposure of cultured mouse spleen cells and thymocytes to mitogens [12,13]. It has been suggested that this effect results from an increase of glucose transport in the plasma membrane [14].

Thimerosal is an ethylmercurithiosalicylate com- pound used as preservative in many chemical solutions. It has been reported that it may act as an hapten to elicit specific antibodies and cell mediated immunity [15]. In murine peritoneal macrophages, thimerosal in- hibits arachidonic acid reincorporation into cellular lipids concomitantly with a rise of the intraceUular calcium concentration [16].

In the present work we have investigated the action of thimerosal on thymic lymphocytes by measuring intracellular calcium concentration, intracellular pH and levels of Fru 2,~P 2 and lactate production, which give an indication o~ ° glycolytic rate [6]. The significance of these intracellular signals on cell proliferation was also investigated.

Materials and Methods

Chemicals Polymyxin B and TPA were obtained from Sigma

Chemical (St Louis, MO). BCECF acetoxymethyl ester (BCECF-AM), fura-2 acetoxymethyl ester (Fura-2/AM) and the acid form were purchased from Molecular Probes (Junction City, OR). Thimerosal was obtained from Fluka (Buchs, Switzerland). [5'-3H]Thymidine (1 # = 37 KBq) was obtained from Amersham Laborato- ries (Buckinghamshire, U.K.). Phytohaemagglutinin (PHA) and Concanavalin A (Con A) were purchased from Flow Laboratories (U.S.A.).

Solutions Na +-solution contained (in mM): 145 NaC1, 5 KCI, 1

CaCI 2, 0.5 MgCI 2, 10 glucose and 40 Hepes, BSA 1 mg/ml (pH 7.3). Choline solution and K+-solution were prepared by iso-osmotic replacement of Na + by choline or K +, respectively. To load the cells with BCECF-AM a Na+-solution (pH 7.2) was used. To load the cells with Fura-2/AM, the solution used contained (in mM): 145 NaCI, 5 KC1, 1 MgSO 4, 1 CaCI 2, 2.5 probenecid, 10 glucose and 25 Hepes, BSA 1 mg/ml (pH 7.4). Stock solutions of thimerosal (2 mM) and polymyxin B (40 mg/ml) were made in Na + solution and kept at -200C. Nigericin, digitonin, BCECF-AM and Fura-2/AM stocks were prepared in DMSO and stored at -20¢C. Probenecid was dissolved in NaOH solution buffered t6-pH 7.4.

Cell isolation Male Wistar rats weighing 130-200 g were used.

Thymic lymphocytes were isolated as described earlier [4].

pH measurement Thymocytes (2.107 cells/ml) were loaded with 4 ~M

BCECF-AM for 30 min at 37°C in Na+-solution, centrifuged and resuspended in different solutions to a concentration of 3.10 6 cells/ml, then pHi was mea- sured fluorimetrically as described [4], using a Perkin- Elmer LS-5 luminescence spectrometer, in a thermo- statically controlled cell kept at 37°C with continuous stirring. Calibration was performed using nigericin and K + and titrating the medium with concentrated Tris base.

111

Free [Ca: +] i determination Thymocytes (4.107 cells/ml) ':ere loaded with the

fluorescent indicator Fura-2 and incubated with 2 #M Fura-2/AM for 45 min at 37°C. IntraceUular Fura-2 concentration was calculated to be 40-60 #M. Loaded cells were washed and resuspended to a concentration of 1 • 107 cells/ml in the same medium used to load the cells but without CaCI 2 and BSA. Fluorescence mea- surements were performed as described in 'pH measure- ment'. Calibration was made lysing the cells with 50 mM digitonin, followed by quenching the fluorescence with 100 mM Mn 2+. [Ca2+]i levels were calculated as previously described [17].

Fru 2,6-P, assay The assay of Fru 2,6-P 2 was carried out by the

standard method slightly modified. The cells were treated with Hepes/Acetate (40/50 mM) (pH 8.5) and heated at 80°C for 10 min. The addition of hot NaOH produced a viscous and insoluble material. The proteins were eliminated by centrifugation and Fru 2,6-P 2 was measured in the supernatant as indicated in [18].

Proliferation assay Thymocytes in a number of 6.10 6 cells/ml were

cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 #g/ml penicillin-strepto- mycin, 250 btg/ml fungizone and 10 mM Hepes. The cultures were maintained in humidified atmosphere containing 5% CO 2 for 48 h. Cell proliferation was estimated by [5'-3H]thymidine (4.5 #Ci per well) incor- poration [19]. Costimulator was culture supernatant from thymocytes (1-107 cells/ ml) incubated for 18 h with 8 #g /ml Con A [20].

Viability Cell viability was determined either by measuring the

leakage of LDH from the cells [21] or by determination of the percentage of cells that excluded Trypan blue [221.

Results and Discussion

The effect of thimerosal on [Ca2+]i, pHi and Fru 2,6-P2 are shown in Fig. 1. Part A illustrates the re- sponses evoked by different concentrations of thimerosal on cytoplasmic free Ca 2+ in Fura-2-1oaded thymocytes. Basal calcium concentration was 70 + 7 nM. T!-dmerosal induced a dose-dependent elevation in [Ca2+]i with maximal effect at 20/~M. At this concentration, [Ca2+]i was 250 + 10 nM and remained at that level for the whole time of measurement (10 rain). Half-maximal response was obtained at 8 #M. It has been shown that thimerosal produces an enhanced prostanoid synthesis concomitantly with a significant rise of intracellular Ca 2+ in mouse peritoneal macrophages [16]. The K0.5

112

m

280 A

2 4 o - ~" 2oo! ~6o

Oil

80 ,u,

40-

0 ~ . 1 I I I

7.25 -

7,21 -

7,1!

7dl

7.01

701 - l I I I

i 30- C

o. 2 5

~ 20

~ ~o ,4 ~ s

u,,

0 - 1 I I .., I 0 10 20 30

[Thi,.,.~o.=°~ (,M) Fig, l, Dose-dependent effect of thimerosal, on cytoplasmic free Ca = + (A), cytoplasmic pH (B) and Fru 2,6-P: content (C) in rat thymocytes. Measurements were carried out 10 (A), 40 (B) and 60 (C) min, after the addition of thimerosal, Bars represent the mean:l: S.E. of three

experiments,

in these experiments was 03 #M, In both thymocytes and macrophages the change of calcium was not tran- sient but was maintained throughout the experiment. This persistant calcium elevation could be due either to an inhibition of the calcium-pump or to the increment of membrane-perturbing lysophospholipids induced by thimerosal [16].

Fig, 1B shows cytosolic pH, measured in BCECF- loaded cells, as a function of thimerosal concentration. Half-maximal response was attained at 3/~M and the maximal effect above 20 #M. At this concentration the alkalinization elicited was 0.2 :t: 0.02 units. A significant increase in pHi (almost 0.03 units), was observed with a concentration of thimerosal of I/~M. These changes in pHi are larger than those reported in thymocytes treated with maximal cor.centrations of Con A (0.1 pH units) or TPA (0.14 pH units) [23].

Finally, Fig. 1C shows the effect of different con- centrations of thimerosal on Fru 2,6-P2 level in thymo- cytes. Thimerosal progressively enhanced the content of Fru 2,6-P2, with a Ko.5 of 7 IaM. At 20/AM the levels of

Fru 2,6-P2 were 4-times those of untreated cells. It has been previously shown that Fru 2,6-1)2 may be used as a good index of glycolytic flux in liver [24]. In our experi- ments Fru 2,6-P2 levels were the same in the presence of 1 mM external Ca 2+ or in the absence of calcium with 100 vM EGTA (not shown), suggesting that Ca 2÷ does not play a significant role on glycolysis in thymocytes, in agreement with previous studies in hepatocytes [25]. A similar stimulatory effect of 20 #M thimerosal was observed when lactate in the medium was measured. Lactate production was 0.1 + 0.01/~mol/mg protein per h in cells incubated with thimerosal. The major function of enhanced glycolysis in activated lymphocytes is pos- sibly the maintainance of a high-steady state amount of intermediates necessary as precursors for macromole- cules synthesis [6].

The dependence of thimerosal-induced [Ca2+]i in- crease on external calcium is shown in Fig. 2. The upper trace (A) of the figure illustrates that in the presence of 1 mM external Ca 2+ thimerosai elicited a 3- to 4-fold increase in [Ca2+]~ which was maintained for more than 3 rain. When external calcium was removed, the same concentration of thimerosal produced only a slight in- crease in [Ca2+]i (Trace C). Similar results were ob- tained in the presence of 100 #M EGTA and without calcium added (Trace B). These results suggest that the rise in [Ca2+]i induced by thimerosal is mainly depen-

3 0 0 -

2 5 0 -

200--

150--

100--

50--

300-

2 5 0 -

E 2 0 0 - c:

- 150-

100-

5 0 -

T l l imerosol

L_____J 1rain

A

B

300-- C

250-

200- ~ '

150-

100-

5 0 -

Fig. 2. Effect of thimerosal on cytoplasmic free Ca 2 +, in the presence of 1 m M external Ca 2+ (A), in the absence of external Ca 2+ with 100 ~aM EGTA (B) and in a nominally Ca2+-free medium (C). Where indicated by the arrows 20 p M thimerosal was added. Traces are from

a representative experiment ( , = 6).

dent on calcium influx across the plasma membrane. Removal of external calcium could also produce a fast depletion of internal Ca 2+ stores. However, this possi- bility is very unlikely and most possibly thimerosal activates a calcium-pathway in the membrane, the na- ture of which remains to be clarified.

Intracellular alkalinization induced by thimerosal is probably due to activation of the Na+/H + antiport found in the plasma membrane of thymic lymphocytes, which plays a major role in cytoplasmic pH homeostasis [4]. Fig. 3 (A) shows a typical recording of changes in pH i evoked by the addition of 20 #M thimerosal on BCECF-loaded cells. After a transient acidification of about 0.05 units, pH i increased progressively at a rate of 0.08 pH units/min and in 30 min pH i was 0.2 + 0.02 units above the basal level. When external Na + was replaced by choline the initial acidification was en- hanced and pH i was maintained below the basal level for at least 40 min, but no alkalinization was observed (Fig. 3B). These results suggest that activation of the Na+/H + exchanger is involved in the alkalinizadon induced by thimerosal. The initial acidification and its enhancement in the absence of external Na + may be explained by an increase in H + uptake and/or H +

7.2-

7.1-

7.0-

6.9-

6.B-

Thin~e~OSol . i . / I ! 10rain

A

B 7.2--

7.1-

7.0-

6 . 9 -

6 .8-

J,

"L_ 7.2-

7.1-

7.0-

6.9-

6.8-

C

Fig. 3. Effect of thimerosal on cytoplasmic pH. (A) cells were resus- pended in Na+-solution. (B) cells were resuspended in choline solu- tion. (C) cells were resuspended in Na+-solution without Ca 2+ and containing 100/~M EGTA. Where indicated by the arrows 20 #M thimerosal was added. The traces are from a representative experi-

ment (n = 6).

113

69 t 6.8

TPA

• i

10 rain

A

=- r~

72" ,

7 .1 -

Z0-

69-

6.8-

TPA B

7.1--

ZO-

6 g -

t),Z:-

Tnimerosal C

Fig. 4. Effect of polymyxal~ !~ on thymocytes alkalinization induced by TPA and thimerosal. (A) control ,=ells. (B and C) cells incubated with 0.4 mg/ml polymyxin B. Where indicated hv the arrows 100 nM TPA o r 20 /~M thimerosal was added. Traces are i;om a representative

experiment (n = 4).

production that cannot be counterbalanced by stimula- tion of the Na+/H + antiport.

It has been observed that activation of the Na ~/H + antiport by serum growth factors is mediated by an increase in fret cytosolic Ca 2+ [26]. However, phorbol esters induce an increase in pH without any change in [Ca2+]i [5] and IL-1 activates the Na+/H + exchanger in a Murine T cell line by a mechamsi~, that is unrelated to changes in [Ca 2+ ] but may involve protein kinase C activation [27]. We studied the relationship between intracellular Ca 2 + and the activation of Na+/H + anti- port by thimerosal, by measuring pHi in the absence of external calcium. Almost no change in [Ca2+]i was observed (see above). In this situation thimerosal in- duced a transient decrease in pH i of about 0.08 units with no further change (Fig. 3C). These results suggest that activation of the Na +/H + exchanger by thimerosal is mediated by an increase in cytosolic Ca 2+

In thymocytes, TPA induces an intracellular alkalin- ization which is not dependent on external calcium [5]. Since it is generally accepted that the main target of the phorbol esters is Ca 2+ and phospholipid-dependent protein kinase C, the activation of the Na+/H + anti- port by TPA should be mediated by stimulation of the kinase and phosphorylation of the exchanger. Poly- myxin B inhibits intracellular alkalinization induced by

114

TABLE I

Effect of thimerosal on thymocyte proliferation

Fresh thymocytes were cultured at 6-106 cells/ml, and assayed for [3H]thymidine uptake rate at 91 h. Costimulator was culture super- natant from thymocytes (1.107 cells/ml) incubated for 18 h with 8 ~g/ml Con A. Final concentrations for the agents: thimerosal, 20 ~tM; costimulator, 1:3; PHA, 10 ~g/ml; Con A 5 ttg/ml. Results represent mean+ S.E. of four experiments.

Additions to thymocyte culture

Thimerosal Con A Costimulator Costimulator + PHA Costimulator + Thimerosal Costimulator + PHA + Thimerosal

3[H]Thymidine incorporation

2767+495 2 813 + 256 2 651 + 304

14 964 ± 392 18 300 + 900 3 000 ± 377 2 423 4- 204

phorbol esters in different cell preparations and it is considered to be an inhibitor of protein kinase C [28]. We have found that alkalinization induced by 100 nM TPA in thymoeytes was abolished by 0.4 mg/ml poly- myxyn B (Fig. 4A and B). The possibility that the alkalinization induced by thimerosal could be also mediated by protein kinase C was tested by studying the effect of thimerosal on pHi in the presence of polymyxin B (0.4 mg/ml). Fig. 4C shows that in this case, thimerosal (20/~M) induced the same initial tran- sient acidification but the subsequent elevation of pHi seen in control experiments was not observed (see Fig. 3A). These results indicate that the presumed activation of the Na+/H + antiport by thimerosal is mediated by both an increase of [Ca +]i and the activation of protein kinase C.

Since thimerosal activates the common internal sig- nals used by other agents that induce cell proliferation, we investigated the effect of thimerosal on [3H]thymi- dine incorporation on cultured thymocytes. A summary is given in Table I. Thimerosal had no appreciable effect on cells that could not be activated by Con A. However, in preparations activated with costimulator (with or without PHA) thimerosal clearly inhibited cell proliferation. Cell viability evaluated by measuring LDH activity and by Tripan Blue exclusion did not seem to be affected by thimerosal. In conclusion, thimerosal elicits dramatic alterations in [Ca2+]i and pHi as well as an enhancement of glycolytic flux. These changes are, however, not sufficient to induce cell proliferation since thimerosal strongly inhibits proliferation evoked by other agents. These results are consistent with recent work indicating that alkalinization is not sufficient to activate proliferation in lymphocytes [29]. The mecha- nism whereby thimerosal inhibits thymocytes prolifera-

tion needs to be investigated in future experimental work.

Acknowledgements

We wish to thank Dr. J. Lopez Barneo for helpful discussions and criticisms of the manuscript and Dr. A. Sanchez for helping in setting up the fluorescent tech- niques. This work was supported by Grant No. PB 164/86 from the CAICYT and by Grant No. 88/1472 from the FISSS. Francisco Martin is a recipient of a predoctoral fellowship from the CICYT.

References

1 Metcalfe, J.C., Pozzan, T., Smith, G.A. and Hesketh, T.R. (1980) Biochem. Soc. Symp. 45, 1.

2 Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) Nature 295, 68-70. 3 Imboden, J.B. and Weiss, A. (1987) Biochem. J. 24% 695-700. 4 Grinstein, S., Cohen, S. and Rothstein, A. (1984) J. Gen. Physiol.

83, 341-369. 5 Grinstein, S., Cohen, S., Goetz, J.D., Rothstein, A. and Geifand,

E.W. (1985) Proc. Natl. Acad. Sci. USA 82, 1429-1433. 6 Hume, D.A., Radik, J.L., Ferber, E. and Weidemann, M.J. (1978)

Biochem. J. 174, 703-709. 7 Grinstein, S., Clarke, C.A. and Rothstein, A. (1983) J. Gen.

Physiol. 82, 619-637. 8 L'Allemain, G., Franchi, A., Cragoe, E. and Pouyssrgur, J. (1984)

J. Biol. Chem. 259, 4133-4139. 9 Pouyssrgur, J., Sardet, C., Franchi, A., L'Allemain, G. and Paris,

J. (1984) Proc. Natl. Acad. Sci. USA 81, 4833-4837. 10 Grinstein, S. and Cohen, S. (1987) J. Gen. Physiol. 89, 185-213. 11 Warburg, O. (1956) Science 123, 309-314. 12 Hederkov, C.J. (1968) Biochem. J. 110, 373-380. 13 Wang, T., Marguadt, C. and Foker, J. (1976) Nature 261,702-705. 14 Weber, M.J. (1973) J. Biol. Chem. 248, 2973-2983. 15 Tosti, A. (1988) Contact Dermatitis 18, 268-273. 16 Kaever, V., Goppelt-Strube, M. and Resch, K. (1988) Prosta-

glandins 35, 885-902. 17 Sage, O.S., Pintado, E., Mahaut-Smith, M. and Merritt, J. (1990)

Biochem. J. 265, 915-918. 18 Gualberto, A., Molinero, P. and Sobrino, F. (1987) Biochem. J.

244, 137-142. 19 Cebrian, M., Yague, E., Rincbn, M., Lopez-Botet, M., Landazuri,

M. and Sanchez-Madrid, F. (1988) J. Exp. Med. 168, 1621-1637. 20 Paetkau, V., Mills, G., Gerhart, S. and Monticone, V. (1976) The

Journal of Immunology. 117, 1320-1324. 21 Seglen, P.O. (1976) Methods Cell Biol. 13, 29-83. 22 Moldeus, P., Hogberg, J. and Orrenius, S. (1978) Methods En-

zymol. 51, 60-71. 23 Metcalfe, J.C. (1984) Nature 317, 471-484. 24 Hers, H.G. and Hue, L. (1983) Annu. Rev. Biochem. 52, 617-653. 25 Becket, J. and Jakos, A. (1982) Eur. J. Biochem. 128, 293-296. 26 Owen, N.E. (1984) Am. J. Physiol. 247, C501-505. 27 Citivelli, R., Teitelbaum, S.L., Hkruska, K.A. and Lacey, D.L.

(1989) J. lmmunol. 143, 4000-4008. 28 Weisman, S.J., Punzo, A., Ford, Ch. and Sha'afi, R.I. (1987) J.

Leukocyte Biol. 41, 25-32. 29 Grinste~:,, S., Smith, J.D., Onizuka, R., Cheung, R.K., Gelfand,

E.W. and Benedict, S. (1988) J. Biol. Chem. 263, 8658-8665.