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Toluene treatment in vitro and calcium-regulated protein phosphorylation in primary astroglial cell cultures from the rat striatum
Toxic. in Vitro Vol. 3, No. 3, pp. 235--240, 1989
0887-2333/89 $3.00+ 0.00
Printed in Oreat Britain
Maxwell Pergamon Macmillanplc
TOLUENE TREATMENT IN VITRO A N D CALCIUM-REGULATED PROTEIN PHOSPHORYLATION IN PRIMARY ASTROGLIAL CELL CULTURES FROM THE RAT STRIATUM G. VON EULER*, E. HANSSON~"and K. FUXE Department of Histology and Neurobiology, Karolinska Institute, Box 60400, S-10401 Stockholm, and ?Institute of Neurobiology, University of G6teborg, G6teborg, Sweden (Received 13 July 1988; revisions received 31 January 1989)
Abstract--Primary astroglial cell cultures from striata of newborn rats were exposed to toluene/n vitro at doses of between 4.7 and 150/~mol/ml. The protein content of the cell cultures, about I mg/ml, was not affected by the toluene treatment. Calcium-induced back-phosphorylation was used to detect changes in the phosphorylation state of calcium-regulated glial phosphoproteins. The 11 most backphosphorylated protein bands were separated by gel electrophoresis and analysed by autoradiography and computer-assisted image analysis. The back-phosphorylation of the 59-kD protein band was increased by 150% at 9.4/~mol/ml toluene, but no further increase was observed with increasing concentrations. This increase is believed to correspond to a toluene-induced decrease in calcium-regulated protein phosphorylation. Furthermore, there was a trend for decreased back-phosphorylation of the 140-kD band and for increased back-phosphorylation of the 110-kD band in response to the higher concentrations of toluene used. The back-phosphorylation of the 42-, 44-, 48-, 51-, 54., 63-, 74- and > 200-kD protein bands showed no dose-related changes in response to the toluene treatment. Taken together, the data indicate that the 59-kD protein in astrocytes from the rat striatum is specifically sensitive to toluene treatment/n vitro.
INTRODUCTION
MATERIALS AND M E T H O D S
Toluene has been shown to induce alterations in the central nervous system of the rat. Thus, toluene affects the binding characteristics of central monoamine receptors (Celani et al., 1983; Fuxe et al., 1987; yon Euler et al., 1987a and 1989a), central catecholamine levels and turnover rates (Andersson et al., 1980 and 1983; Arito et al., 1984 and 1985; Fuxe et al., 1982; R e a e t al., 1984; von Euler et al., 1988a,b and 1989b) and electro-encephalographic patterns (Naalsund, 1986). Even at doses as low as 80 ppm, inhaled toluene influences the phosphorylation levels of calcium and cyclic AMP-regulated phosphoproteins (yon Euler et al., 1987b and 1988b). Since the activity of many proteins seems to be regulated through phospborylation processes (Nestler and Greengard, 1983), the altered phosphorylation levels induced by toluene may reflect functional changes in the brain areas investigated. Based on morphological changes in primary cell cultures from rat striatum, it has been found that not only nerve cells but also astrogliai cells are sensitive to toluene treatment in vitro (Hansson et al., 1988). These studies utilized primary cell cultures from striata of newborn and foetal rats (Hansson et al., 1984; S6derback et al., 1989). In the present study, it was investigated whether toluene produces changes in calciumregulated protein phosphorylation in astrogliai primary cultures of rat striata. *To W h o m correspondence should be addressed. 235
Tissue culture Astroglial primary cultures were prepared from newborn rat striata (Sprague-Dawley strain; Alab, Stockholm, Sweden) as described previously (Hansson et aL, 1984). Briefly, the cells were grown in Eagle's minimum essential medium (Flow Laboratories), to which other substances were added to make up the following final composition: double concentrations o f amino acids, 2mM-glutamine, 7 mM-glueose and quadruple concentrations of vitamins; 250,000 IU penicillin/litre, 0.5% streptomycin and 20% (v/v) foetal calf-serum (GIBCO Europe Ltd, Paisley, UK) were also added. The cells were grown for 2 wk in a humidified atmosphere at pH 7.3. The culture medium was changed three times each week, the last time 1 hr before the exposure to toluene in the absence of foetal calf serum. Exposure to toluene Toluene (pro anaiysi; Merck, Darmstadt, F R O ) was added to the cell cultures in amounts of 1-16/~i/batch (4.7-150/amol/rnl) for 30 min at 37°C in the cell incubator. Four replicates were used for each concentration. The cultures were then rinsed twice in glucose-saline (pH7.4), followed by one rinse in Tris-buffer (50 raM, pH 7.4), also containing 0.1raM-ethylene glycol tetraacetate and 10mMMgCl2. Proteins were determined according to the method of Lowry et aL (1951) with bovine serum albumin as a standard.
236
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Fig. 1. Dose-response curves showing the effects of toluene /n vitro on nine of the most backphosphorylated protein bands from astroglial primary cultures of the rat striatum. Means + SEM are shown, taken as a percentage of the respective control-group mean value. Striped areas indicate the SEM limits of the control group, n = 4 in each group. For specific absorbance values, see text. P < 0.01 by Jonckhcere-Terpstra's test for ordered alternatives.
Protein phosphorylation Phosphorylation levels were measured indirectly by means of calcium-induced back-phosphorylation (Rudolph and Kreuger, 1979). By adding an excess of calcium, all calcium-regulated proteins in a de-phosphorylated state become phosphorylated by a radioactive phosphoryl group donated from adenosine 5'-b,-32p]triphosphate, tetra(triethylammonium) salt (32p-ATP). Thus, the greater the extent of phosphorylation induced by calcium, the less phosphorylated the protein was at the start of the incubation. The cells were sonicated by ultrasound for 5 rain on ice, and incubated with 1/ZM-32p-ATP (1.0 TBq/ mmol) (New England Nuclear, Boston, MA, USA) in the presence of 1 m~-CaCl2 for 30 sec at room temperature. The incubation was terminated by boiling
for 5min, followed by the addition of 50gl of a solution containing 62.SmM-Tris-HCl (pH6.8), 1% sodium dodeeyl sulphate, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue to the samples, which were then left overnight. The samples were separated by electrophoresis on 12% sodium dodecyl sulphate-polyacrylamide gels (Protean II; Bio-Rad, Richmond, CA, USA) using standard procedures (Laemmli, 1970; Rudolf and Kreuger, 1979). The gels were stained with Coomassie Brilliant Blue, destained, vacuum dried and exposed to tritium-sensitive films (Ultrofilm; LKB, Stockholm, Sweden). The films were analysed on an image analyser (IBAS; Zciss-Kontron, Munich, FRG), which yielded average grey values of encircled areas representing the nine most back-phosphorylated protein bands. The absorbance was calculated as
T o l u e n e (iJI) C C > 200 ~ ........ 140 J"
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Hate 1. Representative autoradiogram of a sodium dodecyl sulphate-polyacrylamide gel, showing the effects of toluene in vitro on the 11 most back-phosphorylated protein bands from astroglial primary cultures from the rat striatum. The specific absorbances of these bands were obtained by subtracting the absorbance of inter-band parts from the absorbance of the band. The back-phosphorylation was induced by excess amounts of calcium (1 mr,l). The estimated protein weights of the phosphoproteins are given on the left and the doses of toluene used for the 30-min incubation at the top.
237
Toluene, glia and protein phosphorylation 100 [1 - (object average grey value/film background average grey value)]. Thus, absorbance values reach from 0 (no darkening) to 100 (maximum darkening). The specific absorbance was obtained by subtracting the absorbance of inter-band parts from the absorbance of the band. The effect of toluene on inter-band parts was not analysed. Molecular weights were calculated from a protein standard run on the same gel. Jonckheere-Terpstra's test for ordered alternatives was used in the statistical analysis (Hollander and Wolfe, 1973).
DISCUSSION
The absence of a toluene-induced change in total protein content, together with the short incubation time used (30 rain) in this study, indicates that the increase in calcium-induced back-phosphorylation observed in the 59-kD band is due to decreased level of calcium-dependent phosphorylation and not to a change in protein content. However, it may also be due to a molecular modification of the 59-kD band that renders this band a better substrate for backphosphorylation. It should be noted that homogenate preparations were used and therefore it was not possible to deter-
140 kD
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RESULTS
The 11 most hack-phosphorylated protein bands were analysed (Plate 1). However, two of the bands (1 l0 and 140 kD) did not migrate as separate entities. The estimated molecular weights and specific absorbance values of the protein bands under control conditions were as follows: 42 kD, 2; 44 kD, 3; 48 kD, 6; 51kD, ll; 54kD, 13; 59kD, 3; 63kD, 6; 74kD, 3; l l 0 k D , 20; 140kD, 27; and > 2 0 0 k D , 25. The addition of toluene in vitro was found to increase selectively the calcium-induced back-phosphorylation of the 59-kD protein band (Fig. 1). The increase in back-phosphorylation was c. 150%, a level that was reached at a toluene dose of 2/zl. At higher concentrations of toluene, there was no further increase in back-phosphorylation. At the lowest dose of toluene used (1 pl), no protein band was significantly affected, although the 59-kD and perhaps also the 74-kD protein may have been influenced. In the higher concentration range, a trend for reduced back-phosphorylation of the 140-kD protein band was observed, whereas there was a trend for increased back-phosphorylation of the l l0-kD protein band (Fig. 2). However, the total extent of phosphorylation of these two bands did not change with increased concentrations of toluene. Hence the sums of the specific absorbance were 47 under control conditions and 45 with 16 pl toluene. The other protein bands analysed showed no concentration-related change in calcium-dependent phosphorylation levels on treatment with toluene (Fig. 1). The protein content (about l mg/ml) was not affected by the toluene treatment: 0.97 + 0.04 mg/ml (mean + SEM) (control), 1.17 + 0.05 (1 pl toluene), 1.03 _+ 0.05 (2/zl toluene), 0.99 + 0.05 (4 pl toluene), 0.96+0.06 (8pl toluene) and 1.12+0.13 (16/zl toluene).
239
110 kD 50 4O
10
Illllllll Toluene (l~l)
Fig. 2. Dose-response curves showing the effects of toluene in vitro on the two most back-phosphorylated protein bands from astroglial primary cultures of the rat striatum. Means + SEM of the specific absorbance values are shown. Striped areas indicate the SEM limits of the control group. n = 4 in each group. mine the intracellular location or function of the proteins analysed. However, the high degree of backphosphorylation of these protein bands indicates their role to be regulatory rather than structural elements in the astrocytes. It may be assumed that the lipophilic nature of toluene would lead to a maximum concentration of the organic solvent in the cellular membranes, indicating a localization of the 59-kD protein within or in association with glial membranes. In an earlier study, calcium-induced back-phosphorylation in membrane-enriched fractions of the rat striatum showed a different pattern of protein bands and protein phosphorylation, probably due to differences in tissue composition, but also to different exposure schedules (yon Euler et al., 1987b). The threshold concentration at which toluene became effective in decreasing the level of phosphorylation of the 59-kD protein (2 pl) is in the same range as the concentrations found to affect dopamine D-2 receptor binding (yon Euler et al., 1987a and 1989a) and neurotensin receptor binding (yon Euler et al., 1988c). However, this concentration is much lower than that observed to induce morphological changes in the same primary astroglial cultures of the striatum (Hansson et ai., 1988). The trend for an increase in calcium-induced back-phosphorylation of the 110-kD band following
G. VON EULERet al.
240
toluene treatment may be due to a decreased phosphorylation level. A similar change was found in a 110-kD band in nerve cells from primary cultures of the same region (unpublished results). In contrast, the 140-kD band appeared to have decreased backphosphorylation. Similar effects were seen following toluene treatment /n vivo o n a 134-kD band in membrane preparations from rat striatum (von Euler et al., 1987b). Although unknown, it is possible that one of these bands may represent the dopamine D-2 receptor (Nielsen et al., 1984; Strange, 1987), which is known to be present also on astrocytes (Henn et al., 1977; H6sli and H6sli, 1986). In conclusion, toluene treatment in vitro was found to decrease selectively the calcium-regulated phosphorylation level of a 59-kD protein in astroglial primary cultures from the rat striatum.
Acknowledgements--The skilful work of Ingela Berrum is highly appreciated. This work was supported by Grant 84-1300 from the Swedish Work Environmental Health Fund and by the Swedish Medical Research Council (grant No. 12X-06812). REFERENCES Andersson K., Fuxe K., Toft~rd R., Nilsen O. G., Eneroth P. and Gustafsson J.-A. (1980) Toluene-induced activation of certain hypothulamic and median eminence cateeholamine nerve terminal systems of the male rat and its effects on anterior pituitary hormone secretion. Toxicology Lett. 5, 393. Andersson K., Nilsen O. G., Toftg~trd R., Eueroth P., Gustafsson J.-A., Battistini N. and Calza L. (1983) Increased amine turnover in several hypothulamic noradrenaline terminal systems and changes in GH, LH and prolactin secretion in the male rat by low concentrations of toluene. Neurotoxicology 4, 43. Arito H., Tsuruta H. and Nakagaki K. (1984) Acute effects of toluene on circadian rhythms of sleep-wakefulness and brain monoamine metabolism in rats. Toxicology 33, 291. Arito H., Tsuruta H., Nakagaki K. and Tanaka S. (1985) Partial insomnia, hyperactivity and hyperdipsia induced by repeated administration of toluene in rats: their relation to brain monoamine metabolism. Toxicology 37, 99. Celani M. F., Fuxe K., Agnati L. F., Andersson K., Hanseon T., Gustafsson J.-A., Battistini N. and Eneroth P. (1983) Effects of subacute treatment with toluene on central monoamine receptors in the rat. Reduced affinity in 3H-5-hydroxytryptamine binding sites and in 3H-spiperone binding sites linked to dopamine receptors. Toxicology Lett. 17, 275. Fuxe K,, Andersson K., Nilsen O. G., ToftgArd R., Eneruth P. and Gustafsson J.-A. (1982) Toluene and telencephalic dopamiue: selective reduction of amine turnover in discrete DA nerve terminal systems of the anterior caudate nucleus by low concentrations of toluene. Toxicology Lett. 12, 115. Fuxe K., Martire M., yon Euler G., Agnati L. F., Hansson T., Andersson K., Gustafsson J.-A. and Htiffstrand A. (1987) Effects of subacute treatment with toluene on cerebrocortical ~- and/3-adrenergic receptors in the rat. Evidence for an increased number and a reduced affinity of ~-adrenergic receptors. Acta physiol. scand. 130, 307. Hanuon E., R6nnbick L., Persson L. J., Lowenthal A., Noppe M., Ailing C. and Karlsson B. (1984) Cellular composition of primary cultures from cerebral cortex, striatum, hippocampus, brainstem and cerebellum. Brain Res. 300, 9. Hausson E., yon Euler G., Fuxe K. and Hansson T. (1988)
Toluene induces changes in the morphology of astrogiia and neurons in striatal primary cell cultures. Toxicology 49, 155. Henn F., Anderson D. and Sellstr6m .~. (1977) Possible relationship between giiul cells, dopamine and the effects of antipsychotic drugs. Nature, Lond. 266, 637. Hollander M. and Wolfe D. A. (1973) Nonparametric Statistical Methods. p. 120-123. John Wiley, New York. H6sli E. and H6sli L. (1986) Binding sites for (3H)dopamine and dopamine-antagonists on cultured astrocytes of rat striatum and spinal cord: an autoradiographical study. Neuroscience Lett. 65, 177. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, Lond. 227, 680. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. Naalsund L. U. (1986) Hippocampal EEG in rats after chronic toluene inhalation. Acta pharmac, fox. 59, 325. Nestler E. J. and Greengard P. (1983) Protein phosphorylation in the brain. Nature, Lond. 305, 583. Nielsen M., Klimek V. and Hyttel J. (1984) Distinct target size of dopamine D-I and D-2 receptors in rat striatum. Life Sci. 35, 325. Rea T. M., Nash J. F., Zabik J. E., Born G. S. and Kessler W. V. (1984) Effects of toluene inhalation on brain biogenic amines in the rat. Toxicology 31, 143. Rudolph S. A. and Kreuger B. K. 0979) Endogenous protein phosphorylation and dephosphorylation. In Advances in Cyclic Nucleotide Research. Edited by G. Brooker, P. Greengard and G. A. Robinson. Vol. 10. p. 107. Raven Press, New York. Strange P. G. (1987) Isolation and molecular characterisation of dopamine receptors. In Dopamine Receptors. Edited by I. Creese and C. M. Fraser. p. 29. Alan R. Liss, New York. S6derbick M., Hansson E., Tottmar O. and R6nnbiick L. (1989) Neurons in primary cultures from five defined brain regions--cellular composition and morphological appearance. Cell molec. Biol. 35, 1. yon Euler G., F uxe K., Agnati L. F., Hansson T. and Gustafsson J.-A. (1987a) Ganglioside GM~ treatment prevents the effects of subacute exposure to toluene on N-(3H)propylnorapomorphine binding characteristics in rat striatal membranes. Neuroscience Lett. 82, 181. yon Euler G., Fuxe K., Benfenati F., Hansson T., Agnati L. F. and Gustafsson J.-A. (1989a) Neurotensin modulates the characteristics of 3H-N-propylnorapomorphine binding sites in rat striatai membranes also following treatment with toluene. Acta physiol, scand. In press. yon Euler G., Fuxe K. and Hansson T. (1987b) Effects of subacute toluene exposure on protein phosphorylation levels in rat frontoparietal and striatal membranes. Acta physiol, scand. 131, 113. yon Euler G., Fuxe K., Hansson T., Agnati L. F., Hiirfstrand A., Ogren S.-O., Eneroth P. and Gustafsson J.-.~. (1988b) Effects of chronic toluene exposure on central monoamiue and peptide receptors and their interactions in the adult male rat. Toxicology 52, 103. yon Euler G., Fuxe K., Hansson T., Eneroth P., Agnati L. F., H~irfstrand A. and Gustafsson J.-A,. (1988a) Effects of acute haloperidol treatment on regional catecholamine levels and utilization in rats exposed to toluene. Acta physiol, scand. 132, 199. von Euler G., Fuxe K., Hansson T., Eneroth P. and Gustafsson J.-A. (1988b) Persistent effects of neonatal toluene exposure on regional brain catecholamine levels and turnover in the adult male rat. Toxicology 54, 1. yon Euler G., Fuxe K., Hansson T. and Gustafsson J.-.~. (1988c) Effects of toluene treatment in vivo and/n vitro on the binding characteristics of 3H-neurotensin in rat striatai membranes. Toxicology 49, 149.
0887-2333/89 $3.00+ 0.00
Printed in Oreat Britain
Maxwell Pergamon Macmillanplc
TOLUENE TREATMENT IN VITRO A N D CALCIUM-REGULATED PROTEIN PHOSPHORYLATION IN PRIMARY ASTROGLIAL CELL CULTURES FROM THE RAT STRIATUM G. VON EULER*, E. HANSSON~"and K. FUXE Department of Histology and Neurobiology, Karolinska Institute, Box 60400, S-10401 Stockholm, and ?Institute of Neurobiology, University of G6teborg, G6teborg, Sweden (Received 13 July 1988; revisions received 31 January 1989)
Abstract--Primary astroglial cell cultures from striata of newborn rats were exposed to toluene/n vitro at doses of between 4.7 and 150/~mol/ml. The protein content of the cell cultures, about I mg/ml, was not affected by the toluene treatment. Calcium-induced back-phosphorylation was used to detect changes in the phosphorylation state of calcium-regulated glial phosphoproteins. The 11 most backphosphorylated protein bands were separated by gel electrophoresis and analysed by autoradiography and computer-assisted image analysis. The back-phosphorylation of the 59-kD protein band was increased by 150% at 9.4/~mol/ml toluene, but no further increase was observed with increasing concentrations. This increase is believed to correspond to a toluene-induced decrease in calcium-regulated protein phosphorylation. Furthermore, there was a trend for decreased back-phosphorylation of the 140-kD band and for increased back-phosphorylation of the 110-kD band in response to the higher concentrations of toluene used. The back-phosphorylation of the 42-, 44-, 48-, 51-, 54., 63-, 74- and > 200-kD protein bands showed no dose-related changes in response to the toluene treatment. Taken together, the data indicate that the 59-kD protein in astrocytes from the rat striatum is specifically sensitive to toluene treatment/n vitro.
INTRODUCTION
MATERIALS AND M E T H O D S
Toluene has been shown to induce alterations in the central nervous system of the rat. Thus, toluene affects the binding characteristics of central monoamine receptors (Celani et al., 1983; Fuxe et al., 1987; yon Euler et al., 1987a and 1989a), central catecholamine levels and turnover rates (Andersson et al., 1980 and 1983; Arito et al., 1984 and 1985; Fuxe et al., 1982; R e a e t al., 1984; von Euler et al., 1988a,b and 1989b) and electro-encephalographic patterns (Naalsund, 1986). Even at doses as low as 80 ppm, inhaled toluene influences the phosphorylation levels of calcium and cyclic AMP-regulated phosphoproteins (yon Euler et al., 1987b and 1988b). Since the activity of many proteins seems to be regulated through phospborylation processes (Nestler and Greengard, 1983), the altered phosphorylation levels induced by toluene may reflect functional changes in the brain areas investigated. Based on morphological changes in primary cell cultures from rat striatum, it has been found that not only nerve cells but also astrogliai cells are sensitive to toluene treatment in vitro (Hansson et al., 1988). These studies utilized primary cell cultures from striata of newborn and foetal rats (Hansson et al., 1984; S6derback et al., 1989). In the present study, it was investigated whether toluene produces changes in calciumregulated protein phosphorylation in astrogliai primary cultures of rat striata. *To W h o m correspondence should be addressed. 235
Tissue culture Astroglial primary cultures were prepared from newborn rat striata (Sprague-Dawley strain; Alab, Stockholm, Sweden) as described previously (Hansson et aL, 1984). Briefly, the cells were grown in Eagle's minimum essential medium (Flow Laboratories), to which other substances were added to make up the following final composition: double concentrations o f amino acids, 2mM-glutamine, 7 mM-glueose and quadruple concentrations of vitamins; 250,000 IU penicillin/litre, 0.5% streptomycin and 20% (v/v) foetal calf-serum (GIBCO Europe Ltd, Paisley, UK) were also added. The cells were grown for 2 wk in a humidified atmosphere at pH 7.3. The culture medium was changed three times each week, the last time 1 hr before the exposure to toluene in the absence of foetal calf serum. Exposure to toluene Toluene (pro anaiysi; Merck, Darmstadt, F R O ) was added to the cell cultures in amounts of 1-16/~i/batch (4.7-150/amol/rnl) for 30 min at 37°C in the cell incubator. Four replicates were used for each concentration. The cultures were then rinsed twice in glucose-saline (pH7.4), followed by one rinse in Tris-buffer (50 raM, pH 7.4), also containing 0.1raM-ethylene glycol tetraacetate and 10mMMgCl2. Proteins were determined according to the method of Lowry et aL (1951) with bovine serum albumin as a standard.
236
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VON EULER
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42 kD 200,
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5 9 kO 300.
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Fig. 1. Dose-response curves showing the effects of toluene /n vitro on nine of the most backphosphorylated protein bands from astroglial primary cultures of the rat striatum. Means + SEM are shown, taken as a percentage of the respective control-group mean value. Striped areas indicate the SEM limits of the control group, n = 4 in each group. For specific absorbance values, see text. P < 0.01 by Jonckhcere-Terpstra's test for ordered alternatives.
Protein phosphorylation Phosphorylation levels were measured indirectly by means of calcium-induced back-phosphorylation (Rudolph and Kreuger, 1979). By adding an excess of calcium, all calcium-regulated proteins in a de-phosphorylated state become phosphorylated by a radioactive phosphoryl group donated from adenosine 5'-b,-32p]triphosphate, tetra(triethylammonium) salt (32p-ATP). Thus, the greater the extent of phosphorylation induced by calcium, the less phosphorylated the protein was at the start of the incubation. The cells were sonicated by ultrasound for 5 rain on ice, and incubated with 1/ZM-32p-ATP (1.0 TBq/ mmol) (New England Nuclear, Boston, MA, USA) in the presence of 1 m~-CaCl2 for 30 sec at room temperature. The incubation was terminated by boiling
for 5min, followed by the addition of 50gl of a solution containing 62.SmM-Tris-HCl (pH6.8), 1% sodium dodeeyl sulphate, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue to the samples, which were then left overnight. The samples were separated by electrophoresis on 12% sodium dodecyl sulphate-polyacrylamide gels (Protean II; Bio-Rad, Richmond, CA, USA) using standard procedures (Laemmli, 1970; Rudolf and Kreuger, 1979). The gels were stained with Coomassie Brilliant Blue, destained, vacuum dried and exposed to tritium-sensitive films (Ultrofilm; LKB, Stockholm, Sweden). The films were analysed on an image analyser (IBAS; Zciss-Kontron, Munich, FRG), which yielded average grey values of encircled areas representing the nine most back-phosphorylated protein bands. The absorbance was calculated as
T o l u e n e (iJI) C C > 200 ~ ........ 140 J"
I
1
2
2
4
4
8
8
16
16
.........
!
110
ii!
74
59]'54~ 51~"
........ T
48 tJ
_o o
:E
44},42
Hate 1. Representative autoradiogram of a sodium dodecyl sulphate-polyacrylamide gel, showing the effects of toluene in vitro on the 11 most back-phosphorylated protein bands from astroglial primary cultures from the rat striatum. The specific absorbances of these bands were obtained by subtracting the absorbance of inter-band parts from the absorbance of the band. The back-phosphorylation was induced by excess amounts of calcium (1 mr,l). The estimated protein weights of the phosphoproteins are given on the left and the doses of toluene used for the 30-min incubation at the top.
237
Toluene, glia and protein phosphorylation 100 [1 - (object average grey value/film background average grey value)]. Thus, absorbance values reach from 0 (no darkening) to 100 (maximum darkening). The specific absorbance was obtained by subtracting the absorbance of inter-band parts from the absorbance of the band. The effect of toluene on inter-band parts was not analysed. Molecular weights were calculated from a protein standard run on the same gel. Jonckheere-Terpstra's test for ordered alternatives was used in the statistical analysis (Hollander and Wolfe, 1973).
DISCUSSION
The absence of a toluene-induced change in total protein content, together with the short incubation time used (30 rain) in this study, indicates that the increase in calcium-induced back-phosphorylation observed in the 59-kD band is due to decreased level of calcium-dependent phosphorylation and not to a change in protein content. However, it may also be due to a molecular modification of the 59-kD band that renders this band a better substrate for backphosphorylation. It should be noted that homogenate preparations were used and therefore it was not possible to deter-
140 kD
.o t J 3O
i
10
'°t 0
1
i
2
i
4
i
8
•
16
Toluene (pl)
RESULTS
The 11 most hack-phosphorylated protein bands were analysed (Plate 1). However, two of the bands (1 l0 and 140 kD) did not migrate as separate entities. The estimated molecular weights and specific absorbance values of the protein bands under control conditions were as follows: 42 kD, 2; 44 kD, 3; 48 kD, 6; 51kD, ll; 54kD, 13; 59kD, 3; 63kD, 6; 74kD, 3; l l 0 k D , 20; 140kD, 27; and > 2 0 0 k D , 25. The addition of toluene in vitro was found to increase selectively the calcium-induced back-phosphorylation of the 59-kD protein band (Fig. 1). The increase in back-phosphorylation was c. 150%, a level that was reached at a toluene dose of 2/zl. At higher concentrations of toluene, there was no further increase in back-phosphorylation. At the lowest dose of toluene used (1 pl), no protein band was significantly affected, although the 59-kD and perhaps also the 74-kD protein may have been influenced. In the higher concentration range, a trend for reduced back-phosphorylation of the 140-kD protein band was observed, whereas there was a trend for increased back-phosphorylation of the l l0-kD protein band (Fig. 2). However, the total extent of phosphorylation of these two bands did not change with increased concentrations of toluene. Hence the sums of the specific absorbance were 47 under control conditions and 45 with 16 pl toluene. The other protein bands analysed showed no concentration-related change in calcium-dependent phosphorylation levels on treatment with toluene (Fig. 1). The protein content (about l mg/ml) was not affected by the toluene treatment: 0.97 + 0.04 mg/ml (mean + SEM) (control), 1.17 + 0.05 (1 pl toluene), 1.03 _+ 0.05 (2/zl toluene), 0.99 + 0.05 (4 pl toluene), 0.96+0.06 (8pl toluene) and 1.12+0.13 (16/zl toluene).
239
110 kD 50 4O
10
Illllllll Toluene (l~l)
Fig. 2. Dose-response curves showing the effects of toluene in vitro on the two most back-phosphorylated protein bands from astroglial primary cultures of the rat striatum. Means + SEM of the specific absorbance values are shown. Striped areas indicate the SEM limits of the control group. n = 4 in each group. mine the intracellular location or function of the proteins analysed. However, the high degree of backphosphorylation of these protein bands indicates their role to be regulatory rather than structural elements in the astrocytes. It may be assumed that the lipophilic nature of toluene would lead to a maximum concentration of the organic solvent in the cellular membranes, indicating a localization of the 59-kD protein within or in association with glial membranes. In an earlier study, calcium-induced back-phosphorylation in membrane-enriched fractions of the rat striatum showed a different pattern of protein bands and protein phosphorylation, probably due to differences in tissue composition, but also to different exposure schedules (yon Euler et al., 1987b). The threshold concentration at which toluene became effective in decreasing the level of phosphorylation of the 59-kD protein (2 pl) is in the same range as the concentrations found to affect dopamine D-2 receptor binding (yon Euler et al., 1987a and 1989a) and neurotensin receptor binding (yon Euler et al., 1988c). However, this concentration is much lower than that observed to induce morphological changes in the same primary astroglial cultures of the striatum (Hansson et ai., 1988). The trend for an increase in calcium-induced back-phosphorylation of the 110-kD band following
G. VON EULERet al.
240
toluene treatment may be due to a decreased phosphorylation level. A similar change was found in a 110-kD band in nerve cells from primary cultures of the same region (unpublished results). In contrast, the 140-kD band appeared to have decreased backphosphorylation. Similar effects were seen following toluene treatment /n vivo o n a 134-kD band in membrane preparations from rat striatum (von Euler et al., 1987b). Although unknown, it is possible that one of these bands may represent the dopamine D-2 receptor (Nielsen et al., 1984; Strange, 1987), which is known to be present also on astrocytes (Henn et al., 1977; H6sli and H6sli, 1986). In conclusion, toluene treatment in vitro was found to decrease selectively the calcium-regulated phosphorylation level of a 59-kD protein in astroglial primary cultures from the rat striatum.
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