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Effects of ethanol on phosphoinositide hydrolysis and muscarinic acetylcholine receptor number in SH-SY5Y cells
Life Sciences 67 (2000) 447Ð456
Effects of ethanol on phosphoinositide hydrolysis and muscarinic acetylcholine receptor number in SH-SY5Y cells Murielle Caron*, Amelie Andersson, Christer Alling Institute of Laboratory Medicine, Dept. of Medical Neurochemistry, Lund University Hospital, 221 85 Lund, Sweden
Abstract Previous studies suggest that the effects of ethanol on carbachol-stimulated I(1,4,5)P3 formation and on the number of mAChRs may be independent of each other. The aim of this work was to further study this hypothesis. Human neuroblastoma SH-SY5Y cells were used as a model system. Acute exposure of the cells to 100 mM ethanol induced a decrease in [3H]N-methylscopolamine ([3H]NMS) binding at 30 seconds which was of lower magnitude and of shorter duration than the previously described ethanol-induced inhibition of the peak of carbachol-stimulated I(1,4,5)P3 formation. Longterm ethanol treatment of the cells induced a time- and concentration-dependent increase in [3H]NMS binding. Three hours of 100 mM ethanol treatment were sufÞcient to increase the number of mAChRs at the cell surface but these receptors were not immediately functionally active, suggesting that they may be newly synthesized. Furthermore, the ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation, after two days, was, for all ethanol concentrations tested, of higher magnitude than the ethanol-induced increase in mAChR number. Together, these data indicate that both acute and chronic ethanol-induced changes in carbachol-stimulated I(1,4,5)P3 formation may not only be explained by changes in mAChR density at the cell surface but may rather be the consequence of actions of ethanol down-stream of the receptor. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Muscarinic acetylcholine receptors; Ethanol; Phosphoinositides
Introduction Both acute and chronic ethanol abuse have deleterious consequences on several important cerebral functions, like learning, memory, arousal and control of movements (1Ð7). All of these functions are at least partly regulated by central muscarinic cholinergic pathways (8Ð 10) and a possible mechanism for ethanol-induced cognitive impairments and motor distur-
* Corresponding author. Tel.: 46-46-175-375; fax: 46-46-149-870. E-mail address: [email protected] (M. Caron) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 4 1 -X
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bances may therefore be changes in cholinergic functions in the brain. As a matter of fact, both in vivo (11Ð13) and in vitro (14Ð17) studies have described changes in cholinergic functions in brain or brain tissue following exposure to ethanol but any consensus about the exact molecular mechanisms underlying these changes has not yet been obtained and further studies are therefore necessary. We have previously shown that acute exposure of human SH-SY5Y neuroblastoma cells to 100 mM ethanol induced an inhibition of the peak of carbachol-stimulated inositol(1,4,5)trisphosphate [I(1,4,5)P3] formation, with a maximal 20% inhibition after 30 seconds of ethanol treatment and an effect duration of 5Ð10 minutes (18). On the other hand, long-term ethanol treatment dose- and time-dependently potentiated the peak of carbachol-stimulated I(1,4,5)P3 formation and increased the number of M1 mAChRs (19). The ethanol-induced up-regulation of mAChRs in these cells was sensitive to increased PKC activity, while ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation was not (20). This suggested that ethanol might act at several differentially regulated steps in mAChR-coupled signal transduction pathway and that the observed ethanol-mediated effects on phosphoinositide metabolism might not be a direct consequence of ethanol-induced changes in mAChR density on the cell surface. The aim of this work was to test the hypothesis that ethanol-induced effects on carbacholstimulated I(1,4,5)P3 formation is not directly coupled to changes in mAChR density. Human neuroblastoma SH-SY5Y cells were used as a model system and both acute and chronic effects of ethanol were studied. Our results show that both acute and chronic effects of ethanol on carbachol-stimulated I(1,4,5)P3 formation can not simply be explained by changes in mAChR number but may rather be the consequence of speciÞc actions of ethanol downstream of the receptors or on the function of the receptors. Furthermore, the up-regulated mAChRs following long-term ethanol treatment were not immediately functional and may therefore be newly synthesized receptors.
Materials & Methods Materials Cell culture solutions were from Gibco and cell culture dishes were from Costar. D-[3H]inositol 1,4,5-trisphosphate (15Ð30 Ci/mmol) and [3H]N-methylscopolamine ([3H]NMS) (84 Ci/ mmol) were from New England Nuclear. Carbamylcholine chloride (carbachol) was from Sigma. Ethanol (95%) was from Kemetyl. Ecoscint A scintillation cocktail was from CR Hintze AB. All other chemicals were from Merck. Culture of SH-SY5Y neuroblastoma cells Passages 17Ð59 were used. SH-SY5Y cells were cultivated in 60-mm-diameter plastic dishes containing 4 mL EagleÕs minimal essential medium with L-glutamine and EarlÕs salts, supplemented with 10% fetal calf serum, penicillin 100 IU/mL and streptomycin 100 mg/mL, in a humidiÞed atmosphere with 5% CO2 at 378C. Cells were seeded at 280000 cells/ 60 mm dish and allowed to grow for one week prior to any treatment. Cell culture medium was changed every 2Ð3 days.
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Acute exposure of SH-SY5Y cells to ethanol Fifteen minutes prior to ethanol treatment, the complete culture medium was exchanged for Hepes-buffered EagleÕs minimal essential medium (pH 7.4) without any further supplements. Cells were then exposed to 100 mM ethanol for 10, 20, 30, 40, 60, 300 and 600 seconds. All incubations were performed at 37oC. Exposure was stopped by rapidly aspirating the ethanol-containing medium and replacing it with cold ethanol-free Hepes-buffered EagleÕs minimal essential medium. Long-term exposure of SH-SY5Y cells to ethanol One week after passage, medium was changed and increasing concentrations of ethanol were added to the dishes which were put inside polystyrene boxes. In order to keep a constant ethanol concentration in culture medium, the atmosphere within the box was saturated with ethanol by adding a beaker Þlled with 10 mM, 25 mM, 50 mM or 100 mM ethanol solutions in water to boxes containing the corresponding concentrations of ethanol in culture medium. Total liquid volume inside the box was 112 mL. Moreover, a dish Þlled with 2 mL 435 mM ethanol was added to polystyrene boxes containing 50 mM ethanol in culture medium and a dish Þlled with 4 mL 870 mM ethanol was added to polystyrene boxes containing 100 mM ethanol in culture medium. Cells were cultured in the presence or absence of ethanol for 1, 3, 6, 24, 48 or 96 hours. Stimulation of SH-SY5Y cells Fifteen minutes prior to stimulation, the complete medium was exchanged for HEPESbuffered EagleÕs minimal essential medium (pH 7.4) without any further supplements. Incubations were performed at 37oC. Cells were stimulated for 10 seconds with 100 mM carbachol. Analysis of I(1,4,5)P3 levels Stimulations were ended by aspiration of carbachol-containing medium and addition of 0.5 mL 0.5 M trichloroacetic acid (TCA). Cells were scraped and centrifuged at 2000 3 g for 15 minutes at 4oC. The pellet was mixed in 250 mL 1 M NaOH and stored for protein analysis (21). Fifty microliters 10 mM EDTA and 200 mL 1,1,2-trichloro-trißuoro-ethane:trioctylamine (1:1) were added to 200 mL of the supernatant. The solution was mixed and centrifuged at 12000 3 g for 5 minutes. The supernatant (120 mL) containing the water-soluble cell extract was transferred to a new tube and neutralized with 10 mL 250 mM Tris. A modiÞcation of a membrane binding assay (22) was used to measure I(1,4,5)P3 concentration. Brießy, 50 mg of I(1,4,5)P3 receptor rich-bovine cerebellar membranes, 5000Ð6000 cpm [3H]I(1,4,5)P3 and 25 mL buffer B (50 mM Tris, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 8.6) were added to the cell extract to be analyzed or to standard solutions of I(1,4,5)P3. The mixture was incubated in a shaker for 30 minutes at 14oC. Displaced [3H]-I(1,4,5)P3 was removed by aspiration in a SKATRON cell harvester, leaving membrane-bound [3H]-I(1,4,5)P3 on Þlterdiscs (FilterMat, 1.0 mm retention), which were mixed with 3 mL Ecoscint scintillation cocktail and taken for scintillation counting.
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Binding of [3H]N-methylscopolamine to intact cells SH-SY5Y cells were detached from the dish by washing each 60 mm dish with 2 mL cold HEPES-buffered medium. Cells were centrifuged at 1500 3 g for 1 minute at 14oC and the pellet was resuspended in 2 mL cold HEPES-buffered medium. Eighty microliters of the cell suspension were added to microplate wells containing [3H]NMS in a Þnal concentration of 1.5 nM. Following incubation for 1 hour at 37oC in a shaking water bath, unbound ligand was separated by Þltration in a cell harvester (SKATRON cell harvester) and bound [3H]NMS was analyzed by scintillation counting. Non-speciÞc binding was assayed in the presence of 10 mM atropine. Data analysis Means were compared with paired studentÕs t-test in the SPSS computer program for windows. The level of signiÞcance was set at 0.05. Bmax, Kd and n values were calculated using the curve-Þtting function in the SigmaPlotTM 1.02 computer program for windows. Values were Þtted to the hyperbolic function y5a/(11(b/x)n) where a was the maximal response (Bmax), b was the drug concentration that induced 50% of maximal effect (Kd), n was the slope factor and y the response induced by the concentration x of the drug.
Results Effects of acute ethanol treatment on [3H]N-methylscopolamine binding to SH-SY5Y cells In order to investigate whether the previously described inhibition of the peak of carbacholstimulated I(1,4,5)P3 formation induced by acute ethanol treatment was due to a decreased number of cell surface muscarinic receptors, SH-SY5Y cells were exposed to 100 mM ethanol for 10, 20, 30, 40, 60, 300 or 600 seconds and [3H]NMS binding to intact cells was analyzed (Fig. 1). Thirty seconds of ethanol treatment induced a maximal 11% decrease in [3H]NMS binding compared to control cells. This effect was transient and following 40 seconds of ethanol treatment, the amount of [3H]NMS binding sites had returned to control levels. Effects of long-term ethanol treatment on [3H]N-methylscopolamine binding to SH-SY5Y cells In order to investigate whether the previously described long-term ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation was due to an increased number of available receptors on the cell surface, SH-SY5Y cells were exposed to 100 mM ethanol for 1, 3, 6, 24, 48 and 96 hours and [3H]NMS binding to intact cells was analyzed (Fig. 2A). Three hours of ethanol treatment were sufÞcient to induce a statistically signiÞcant 8% increase in [3H]NMS binding. This increase was further potentiated when cells were exposed to ethanol for longer time-periods and reached 50% after 48 hours and 61% after 96 hours. This timedependency of ethanol-induced increase in [3H]NMS binding was also observed when cells were treated with 10 mM ethanol but 48 hours of ethanol treatment were then necessary to induce any (7%) signiÞcant effect. As shown in Fig. 2B, treatment of the cells with increas-
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Fig. 1. Effects of acute ethanol treatment on [3H]N-methylscopolamine binding in SH-SY5Y cells. Fifteen minutes prior to ethanol treatment, the complete medium was exchanged for Hepes-buffered EagleÕs minimal essential medium (pH 7.4) without any further supplements. Cells were then exposed to 100 mM ethanol for 10, 20, 30, 40, 60, 300 or 600 seconds. Ethanol treatment was stopped by rapidly aspirating the ethanol-containing medium and replacing it with cold ethanol-free Hepes-buffered EagleÕs minimal essential medium. Results are speciÞc binding of [3H]NMS/mg protein expressed as a percentage of speciÞc binding/mg protein in control cells. Data are mean 6 SEM from 2 (20, 40 and 300 seconds), 4 (10 and 600 seconds), 6 (30 seconds) and 8 (60 seconds) separate experiments (N511Ð33).
ing ethanol concentrations for 48 hours induced a concentration-dependent increase in [3H]NMS binding. The observed increase in [3H]NMS binding following ethanol treatment could be due to increased afÞnity of the ligand to the receptor and not to an increased amount of binding sites for the ligand. In order to control that this was not the case, a saturation curve for [3H]NMS binding was performed in cells treated with 100 mM ethanol for 3 hours and in control cells (Fig. 3). In one representative out of two experiments performed in duplicates, the Bmax value was 29.14 6 1.21 fmol/mg (mean 6 SEM) in ethanol-treated cells and 23.18 6 1.89 (mean 6 SEM) fmol/mg in control cells, expressed as speciÞc binding of [3H]NMS/mg protein. The Kd (0.23 6 0.028 nM in ethanol-treated cells and 0.19 6 0.039 nM in control cells) and n (1.74 6 0.50 in ethanol-treated cells and 2.00 6 1.51 in control cells) values were not affected by ethanol treatment, indicating that the observed increase in [3H]NMS binding was not due to an increased afÞnity of the ligand for the receptor but corresponded to an increased amount of binding sites for the ligand. The protocol used for binding studies involved removal of culture medium and replacement with HEPES-buffered medium without any supplements. Since this medium did not contain ethanol, the observed increase in [3H]NMS binding could be due to a rebound effect due to removal of ethanol. In order to investigate whether cells maintained in 100 mM etha-
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Fig. 2. (A) Time-course of 10 mM and 100 mM ethanol-induced increase in [3H]N-methylscopolamine binding in SH-SY5Y cells. Cells were exposed to 10 mM (j) or 100 mM (d) ethanol for 1, 3, 6, 24, 48 or 96 hours prior to [3H]NMS binding analysis. Results are speciÞc binding of [3H]NMS/mg protein expressed as a percentage of speciÞc binding/mg protein in control cells. Data are mean 6 SEM from 2 (10 & 100 mM, 96h), 3 (10 mM, 24h), 4 (100 mM, 1, 6 & 24h), 5 (100 mM, 3 & 48h) and 10 (10 mM, 48h) separate experiments (N518Ð30). (B) Concentration-response curve for ethanol-induced increase in [3H]N-methylscopolamine binding in SH-SY5Y cells. Cells were exposed to increasing concentrations of ethanol for 2 days prior to [3H]NMS binding analysis. Results are speciÞc binding of [3H]NMS expressed as a percentage of speciÞc binding in control cells. Data are mean 6 SEM from 4 (25 mM, 50 mM and 100 mM) and 8 (10 mM) separate experiments (N59Ð15).
nol throughout the binding study also exhibited an increased number of [3H]NMS binding sites, cells that had been exposed to ethanol for 2 days were kept in HEPES-buffered medium supplemented with 100 mM ethanol throughout the binding study. In cells treated according to this protocol, [3H]NMS binding was 122 6 8.66% (mean 6 SEM from 4 separate experiments performed in triplicates) expressed as a percentage of [3H]NMS binding/mg protein in control cells. In cells that had been exposed to ethanol and in which binding was analyzed in the absence of ethanol, [3H]NMS binding was 135 6 7.09% (mean 6 SEM from 4 separate experiments performed in triplicates) expressed as a percentage of [3H]NMS binding in control cells. No signiÞcant difference was found (P50.256) between these two groups when data were analyzed with paired studentÕsÕt-test. These results demonstrate that removal of ethanol prior to binding studies did not account for the observed increase in mAChR number in cells exposed to ethanol for several days. Effects of long-term ethanol treatment on carbachol-stimulated I(1,4,5)P3 levels The chronic ethanol-induced potentiation of carbachol- stimulated I(1,4,5)P3 formation and increase in [3H]NMS binding appeared to be uncoupled in time. In order to further deÞne this phenomenon, the concentration-dependency of ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation after 48 hours of ethanol treatment was investigated and compared to results of similar ethanol exposure on [3H]NMS binding. As shown in Fig. 4, treatment of the cells with 10 mM ethanol for 48 hours were sufÞcient to induce a signiÞcant
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Fig. 3. Saturation curve for [3H]N-methylscopolamine binding to SH-SY5Y cells following exposure to ethanol for 3 hours. Cells were cultured for 3 hours in the absence (d) or presence (j) of 100 mM ethanol and the speciÞc binding of increasing concentrations of [3H]NMS to the intact cells was determined. Results are speciÞc binding of [3H]NMS/mg protein expressed as a percentage of speciÞc binding of 0,15 nM [3H]NMS/mg protein in control cells and are mean 6 SEM from 2 separate experiments carried out in duplicates.
(17%) potentiation of receptor-stimulated I(1,4,5)P3 formation and higher ethanol concentrations further potentiated this effect. For all ethanol concentrations tested, the potentiation of carbachol-stimulated I(1,4,5)P3 formation was 40-50% more pronounced than the increase in [3H]NMS binding (Fig. 2B). Discussion The results of this study describe the relationship between both acute and long-term ethanolinduced effects on the number of cell surface mAChRs and carbachol-stimulated I(1,4,5)P3 formation in human SH-SY5Y neuroblastoma cells. We have previously described the effects of acute ethanol treatment on carbachol-stimulated I(1,4,5)P3 formation in SH-SY5Y cells (18). Acute exposure of the cells to 100 mM ethanol induced an inhibition of the peak of carbachol-stimulated I(1,4,5)P3 formation with a maximal 20% inhibition after 30 seconds of ethanol treatment. This effect was long-lasting with a duration of 5-10 minutes. In the present work, the effects of similar ethanol treatment on [3H]NMS binding to the cells were studied in order to investigate whether the ethanolinduced inhibition of carbachol-stimulated I(1,4,5)P3 formation could be due to a decreased number of available mAChRs. An 11% decrease in [3H]NMS binding was observed following 30 seconds of ethanol treatment, but this effect was transient and within 1 minute of etha-
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Fig. 4. Concentration-dependency of ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation in SH-SY5Y cells. SH-SY5Y cells were exposed to increasing concentrations of ethanol for 2 days. Fifteen minutes prior to stimulation, the complete medium was exchanged for HEPES-buffered EagleÕs minimal essential medium and cells were stimulated for 10 seconds with 100 mM carbachol. Results are expressed as a percentage of I(1,4,5)P3 levels/mg protein obtained in stimulated control cells. Data are mean 6 SEM from 6 (10 mM) and 4 (25 mM, 50 mM and 100 mM) separate experiments carried out in triplicates.
nol treatment, [3H]NMS binding had returned to control levels and remained there for up to 10 minutes of ethanol exposure. Thus, even though acute treatment of SH-SY5Y cells with 100 mM ethanol induced a decrease in [3H]NMS binding, this decrease was about 50% less pronounced and of much shorter duration than the inhibition of carbachol-stimulated stimulated I(1,4,5)P3 formation observed following the same ethanol treatment and could therefore not account for the observed inhibition of second messenger formation. This indicates that acute ethanol-induced effects on carbachol-stimulated I(1,4,5)P3 formation cannot just be explained by changes in the number of cell surface mAChRs in these cells. An effect of ethanol on the function of available cell surface receptors or down-stream of the receptors is therefore likely. The effects of long-term treatment of SH-SY5Y cells with 100 mM has been shown to induce a potentiation of carbachol-stimulated I(1,4,5)P3 formation and 48 hours of ethanol treatment were necessary to produce any signiÞcant effect (19). In order to investigate whether this effect could be related to an increased number of mAChRs on the cell surface, [3H]NMS binding to the cells was studied following the same ethanol treatment scheme. Ethanol time-dependently increased the number of [3H]NMS binding sites and three hours of ethanol treatment were sufÞcient to induce a signiÞcant (8%) effect. Thus, there was a latency of several hours between the appearance of mAChRs at the cell surface and their ability, when stimulated by carbachol, to activate phospholipase C. A lower functional activity, lasting for up to 23 hours has been described for mAChRs following their new-synthesis and appearance on the cell surface in embryonic chicken heart cells (23,24) and in stably trans-
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fected Y1 adrenal cells (25). This diminished response to agonist treatment was not due to decreased afÞnity for carbachol (23) nor to alterations in GTP-binding protein-coupling to effector enzymes nor to changes in molecular weight or isoelectric points for these receptors (24). Even though these studies were performed in cells expressing M2 subtypes of mAChRs while the receptors involved in carbachol-stimulated I(1,4,5)P3 formation in our cells have been described to be of the M1 subtype (19), the explanation to the observed lower functionality of mAChRs in our cells after several hours of 100 mM ethanol treatment might be that these receptors are newly synthesized, not immediately functional receptors. However, when SH-SY5Y cells were exposed to a low concentration of ethanol (10 mM), 2 days were necessary to induce a signiÞcant increase in mAChR number but these receptors appeared to be immediately fully functional as their stimulation with carbachol induced an elevation of I(1,4,5)P3 levels without any latency. A possible explanation to this observation is that our [3H]NMS binding analysis method may not be sensitive enough to discover minimal changes in receptor number. Thus, a small increase in [3H]NMS binding after only a few hours of treatment with 10 mM ethanol might have been missed, explaining the observed immediate functionality of the receptors. When the effects of 2 days exposure to different concentrations of ethanol on [3H]NMS binding and carbachol-stimulated I(1,4,5)P3 formation were compared, one could note that the ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation was 40Ð50% more pronounced than the increase in [3H]NMS binding observed at the same ethanol concentrations. These differences may be within the range of error of the assays involved but the fact that they were of similar degree and could be observed at all ethanol concentrations tested renders the hypothesis of an inter-assay variation less plausible. We have also been considering the eventuality of a sigmoidal relationship between receptor occupancy and second messenger formation as a possible explanation to our Þndings but have come to the conclusion that such a relationship is unlikely in the present case. A sigmoidal relationship would mean that the number of cell surface receptors would not be directly proportional to receptor-stimulated second messenger formation, thereby explaining the discrepancy between receptor number and I(1,4,5)P3 levels observed following both acute and chronic ethanol treatment in our cells. However, the 10% increase in mAChR number following 6 hours of 100 mM ethanol treatment was not paralleled by a similar increase in second messenger formation while the 10% increase in receptor number following 10 mM ethanol treatment for two days was paralleled by an even higher increase in second messenger levels. This indicates that the observed receptor-second messenger discrepancy in our cells is not caused by a sigmoidal relationship between receptor occupancy and second messenger formation but is rather the consequence of more complex mechanisms. In conclusion, the results of this study show that the effects of both acute and chronic ethanol treatment on carbachol-stimulated I(1,4,5)P3 formation in SH-SY5Y cells cannot only be explained by changes in cell surface mAChR density. Effects of ethanol down-stream of the receptor cannot be excluded. Several lines of evidence suggest that ethanol may act at the level of G proteins in NG108-15 cells (26Ð30), in human permeabilized platelets (31), in turkey erythrocyte ghosts (32), in mice striatal membranes (33) and in mice cerebral cortex (34,35). Effects of ethanol at this level may explain our Þndings but this remains to be demonstrated. Furthermore, long-term ethanol treatment of these cells caused an up-regulation of
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mAChRs which were not immediately functional, suggesting that these receptors might be newly synthesized. Acknowledgments The authors are grateful for excellent technical assistance by Monica Mihailescu and Berit FŠrjh. Financial support for this work was obtained from the Swedish Medical Research Council (05249), The Swedish Alcohol Research Fund, The Swedish Society for Medical Research, The Albert PŒhlsson and Crafoord Foundations and the Medical Faculty at Lund University. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
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Effects of ethanol on phosphoinositide hydrolysis and muscarinic acetylcholine receptor number in SH-SY5Y cells Murielle Caron*, Amelie Andersson, Christer Alling Institute of Laboratory Medicine, Dept. of Medical Neurochemistry, Lund University Hospital, 221 85 Lund, Sweden
Abstract Previous studies suggest that the effects of ethanol on carbachol-stimulated I(1,4,5)P3 formation and on the number of mAChRs may be independent of each other. The aim of this work was to further study this hypothesis. Human neuroblastoma SH-SY5Y cells were used as a model system. Acute exposure of the cells to 100 mM ethanol induced a decrease in [3H]N-methylscopolamine ([3H]NMS) binding at 30 seconds which was of lower magnitude and of shorter duration than the previously described ethanol-induced inhibition of the peak of carbachol-stimulated I(1,4,5)P3 formation. Longterm ethanol treatment of the cells induced a time- and concentration-dependent increase in [3H]NMS binding. Three hours of 100 mM ethanol treatment were sufÞcient to increase the number of mAChRs at the cell surface but these receptors were not immediately functionally active, suggesting that they may be newly synthesized. Furthermore, the ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation, after two days, was, for all ethanol concentrations tested, of higher magnitude than the ethanol-induced increase in mAChR number. Together, these data indicate that both acute and chronic ethanol-induced changes in carbachol-stimulated I(1,4,5)P3 formation may not only be explained by changes in mAChR density at the cell surface but may rather be the consequence of actions of ethanol down-stream of the receptor. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Muscarinic acetylcholine receptors; Ethanol; Phosphoinositides
Introduction Both acute and chronic ethanol abuse have deleterious consequences on several important cerebral functions, like learning, memory, arousal and control of movements (1Ð7). All of these functions are at least partly regulated by central muscarinic cholinergic pathways (8Ð 10) and a possible mechanism for ethanol-induced cognitive impairments and motor distur-
* Corresponding author. Tel.: 46-46-175-375; fax: 46-46-149-870. E-mail address: [email protected] (M. Caron) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 4 1 -X
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bances may therefore be changes in cholinergic functions in the brain. As a matter of fact, both in vivo (11Ð13) and in vitro (14Ð17) studies have described changes in cholinergic functions in brain or brain tissue following exposure to ethanol but any consensus about the exact molecular mechanisms underlying these changes has not yet been obtained and further studies are therefore necessary. We have previously shown that acute exposure of human SH-SY5Y neuroblastoma cells to 100 mM ethanol induced an inhibition of the peak of carbachol-stimulated inositol(1,4,5)trisphosphate [I(1,4,5)P3] formation, with a maximal 20% inhibition after 30 seconds of ethanol treatment and an effect duration of 5Ð10 minutes (18). On the other hand, long-term ethanol treatment dose- and time-dependently potentiated the peak of carbachol-stimulated I(1,4,5)P3 formation and increased the number of M1 mAChRs (19). The ethanol-induced up-regulation of mAChRs in these cells was sensitive to increased PKC activity, while ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation was not (20). This suggested that ethanol might act at several differentially regulated steps in mAChR-coupled signal transduction pathway and that the observed ethanol-mediated effects on phosphoinositide metabolism might not be a direct consequence of ethanol-induced changes in mAChR density on the cell surface. The aim of this work was to test the hypothesis that ethanol-induced effects on carbacholstimulated I(1,4,5)P3 formation is not directly coupled to changes in mAChR density. Human neuroblastoma SH-SY5Y cells were used as a model system and both acute and chronic effects of ethanol were studied. Our results show that both acute and chronic effects of ethanol on carbachol-stimulated I(1,4,5)P3 formation can not simply be explained by changes in mAChR number but may rather be the consequence of speciÞc actions of ethanol downstream of the receptors or on the function of the receptors. Furthermore, the up-regulated mAChRs following long-term ethanol treatment were not immediately functional and may therefore be newly synthesized receptors.
Materials & Methods Materials Cell culture solutions were from Gibco and cell culture dishes were from Costar. D-[3H]inositol 1,4,5-trisphosphate (15Ð30 Ci/mmol) and [3H]N-methylscopolamine ([3H]NMS) (84 Ci/ mmol) were from New England Nuclear. Carbamylcholine chloride (carbachol) was from Sigma. Ethanol (95%) was from Kemetyl. Ecoscint A scintillation cocktail was from CR Hintze AB. All other chemicals were from Merck. Culture of SH-SY5Y neuroblastoma cells Passages 17Ð59 were used. SH-SY5Y cells were cultivated in 60-mm-diameter plastic dishes containing 4 mL EagleÕs minimal essential medium with L-glutamine and EarlÕs salts, supplemented with 10% fetal calf serum, penicillin 100 IU/mL and streptomycin 100 mg/mL, in a humidiÞed atmosphere with 5% CO2 at 378C. Cells were seeded at 280000 cells/ 60 mm dish and allowed to grow for one week prior to any treatment. Cell culture medium was changed every 2Ð3 days.
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Acute exposure of SH-SY5Y cells to ethanol Fifteen minutes prior to ethanol treatment, the complete culture medium was exchanged for Hepes-buffered EagleÕs minimal essential medium (pH 7.4) without any further supplements. Cells were then exposed to 100 mM ethanol for 10, 20, 30, 40, 60, 300 and 600 seconds. All incubations were performed at 37oC. Exposure was stopped by rapidly aspirating the ethanol-containing medium and replacing it with cold ethanol-free Hepes-buffered EagleÕs minimal essential medium. Long-term exposure of SH-SY5Y cells to ethanol One week after passage, medium was changed and increasing concentrations of ethanol were added to the dishes which were put inside polystyrene boxes. In order to keep a constant ethanol concentration in culture medium, the atmosphere within the box was saturated with ethanol by adding a beaker Þlled with 10 mM, 25 mM, 50 mM or 100 mM ethanol solutions in water to boxes containing the corresponding concentrations of ethanol in culture medium. Total liquid volume inside the box was 112 mL. Moreover, a dish Þlled with 2 mL 435 mM ethanol was added to polystyrene boxes containing 50 mM ethanol in culture medium and a dish Þlled with 4 mL 870 mM ethanol was added to polystyrene boxes containing 100 mM ethanol in culture medium. Cells were cultured in the presence or absence of ethanol for 1, 3, 6, 24, 48 or 96 hours. Stimulation of SH-SY5Y cells Fifteen minutes prior to stimulation, the complete medium was exchanged for HEPESbuffered EagleÕs minimal essential medium (pH 7.4) without any further supplements. Incubations were performed at 37oC. Cells were stimulated for 10 seconds with 100 mM carbachol. Analysis of I(1,4,5)P3 levels Stimulations were ended by aspiration of carbachol-containing medium and addition of 0.5 mL 0.5 M trichloroacetic acid (TCA). Cells were scraped and centrifuged at 2000 3 g for 15 minutes at 4oC. The pellet was mixed in 250 mL 1 M NaOH and stored for protein analysis (21). Fifty microliters 10 mM EDTA and 200 mL 1,1,2-trichloro-trißuoro-ethane:trioctylamine (1:1) were added to 200 mL of the supernatant. The solution was mixed and centrifuged at 12000 3 g for 5 minutes. The supernatant (120 mL) containing the water-soluble cell extract was transferred to a new tube and neutralized with 10 mL 250 mM Tris. A modiÞcation of a membrane binding assay (22) was used to measure I(1,4,5)P3 concentration. Brießy, 50 mg of I(1,4,5)P3 receptor rich-bovine cerebellar membranes, 5000Ð6000 cpm [3H]I(1,4,5)P3 and 25 mL buffer B (50 mM Tris, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 8.6) were added to the cell extract to be analyzed or to standard solutions of I(1,4,5)P3. The mixture was incubated in a shaker for 30 minutes at 14oC. Displaced [3H]-I(1,4,5)P3 was removed by aspiration in a SKATRON cell harvester, leaving membrane-bound [3H]-I(1,4,5)P3 on Þlterdiscs (FilterMat, 1.0 mm retention), which were mixed with 3 mL Ecoscint scintillation cocktail and taken for scintillation counting.
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Binding of [3H]N-methylscopolamine to intact cells SH-SY5Y cells were detached from the dish by washing each 60 mm dish with 2 mL cold HEPES-buffered medium. Cells were centrifuged at 1500 3 g for 1 minute at 14oC and the pellet was resuspended in 2 mL cold HEPES-buffered medium. Eighty microliters of the cell suspension were added to microplate wells containing [3H]NMS in a Þnal concentration of 1.5 nM. Following incubation for 1 hour at 37oC in a shaking water bath, unbound ligand was separated by Þltration in a cell harvester (SKATRON cell harvester) and bound [3H]NMS was analyzed by scintillation counting. Non-speciÞc binding was assayed in the presence of 10 mM atropine. Data analysis Means were compared with paired studentÕs t-test in the SPSS computer program for windows. The level of signiÞcance was set at 0.05. Bmax, Kd and n values were calculated using the curve-Þtting function in the SigmaPlotTM 1.02 computer program for windows. Values were Þtted to the hyperbolic function y5a/(11(b/x)n) where a was the maximal response (Bmax), b was the drug concentration that induced 50% of maximal effect (Kd), n was the slope factor and y the response induced by the concentration x of the drug.
Results Effects of acute ethanol treatment on [3H]N-methylscopolamine binding to SH-SY5Y cells In order to investigate whether the previously described inhibition of the peak of carbacholstimulated I(1,4,5)P3 formation induced by acute ethanol treatment was due to a decreased number of cell surface muscarinic receptors, SH-SY5Y cells were exposed to 100 mM ethanol for 10, 20, 30, 40, 60, 300 or 600 seconds and [3H]NMS binding to intact cells was analyzed (Fig. 1). Thirty seconds of ethanol treatment induced a maximal 11% decrease in [3H]NMS binding compared to control cells. This effect was transient and following 40 seconds of ethanol treatment, the amount of [3H]NMS binding sites had returned to control levels. Effects of long-term ethanol treatment on [3H]N-methylscopolamine binding to SH-SY5Y cells In order to investigate whether the previously described long-term ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation was due to an increased number of available receptors on the cell surface, SH-SY5Y cells were exposed to 100 mM ethanol for 1, 3, 6, 24, 48 and 96 hours and [3H]NMS binding to intact cells was analyzed (Fig. 2A). Three hours of ethanol treatment were sufÞcient to induce a statistically signiÞcant 8% increase in [3H]NMS binding. This increase was further potentiated when cells were exposed to ethanol for longer time-periods and reached 50% after 48 hours and 61% after 96 hours. This timedependency of ethanol-induced increase in [3H]NMS binding was also observed when cells were treated with 10 mM ethanol but 48 hours of ethanol treatment were then necessary to induce any (7%) signiÞcant effect. As shown in Fig. 2B, treatment of the cells with increas-
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Fig. 1. Effects of acute ethanol treatment on [3H]N-methylscopolamine binding in SH-SY5Y cells. Fifteen minutes prior to ethanol treatment, the complete medium was exchanged for Hepes-buffered EagleÕs minimal essential medium (pH 7.4) without any further supplements. Cells were then exposed to 100 mM ethanol for 10, 20, 30, 40, 60, 300 or 600 seconds. Ethanol treatment was stopped by rapidly aspirating the ethanol-containing medium and replacing it with cold ethanol-free Hepes-buffered EagleÕs minimal essential medium. Results are speciÞc binding of [3H]NMS/mg protein expressed as a percentage of speciÞc binding/mg protein in control cells. Data are mean 6 SEM from 2 (20, 40 and 300 seconds), 4 (10 and 600 seconds), 6 (30 seconds) and 8 (60 seconds) separate experiments (N511Ð33).
ing ethanol concentrations for 48 hours induced a concentration-dependent increase in [3H]NMS binding. The observed increase in [3H]NMS binding following ethanol treatment could be due to increased afÞnity of the ligand to the receptor and not to an increased amount of binding sites for the ligand. In order to control that this was not the case, a saturation curve for [3H]NMS binding was performed in cells treated with 100 mM ethanol for 3 hours and in control cells (Fig. 3). In one representative out of two experiments performed in duplicates, the Bmax value was 29.14 6 1.21 fmol/mg (mean 6 SEM) in ethanol-treated cells and 23.18 6 1.89 (mean 6 SEM) fmol/mg in control cells, expressed as speciÞc binding of [3H]NMS/mg protein. The Kd (0.23 6 0.028 nM in ethanol-treated cells and 0.19 6 0.039 nM in control cells) and n (1.74 6 0.50 in ethanol-treated cells and 2.00 6 1.51 in control cells) values were not affected by ethanol treatment, indicating that the observed increase in [3H]NMS binding was not due to an increased afÞnity of the ligand for the receptor but corresponded to an increased amount of binding sites for the ligand. The protocol used for binding studies involved removal of culture medium and replacement with HEPES-buffered medium without any supplements. Since this medium did not contain ethanol, the observed increase in [3H]NMS binding could be due to a rebound effect due to removal of ethanol. In order to investigate whether cells maintained in 100 mM etha-
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Fig. 2. (A) Time-course of 10 mM and 100 mM ethanol-induced increase in [3H]N-methylscopolamine binding in SH-SY5Y cells. Cells were exposed to 10 mM (j) or 100 mM (d) ethanol for 1, 3, 6, 24, 48 or 96 hours prior to [3H]NMS binding analysis. Results are speciÞc binding of [3H]NMS/mg protein expressed as a percentage of speciÞc binding/mg protein in control cells. Data are mean 6 SEM from 2 (10 & 100 mM, 96h), 3 (10 mM, 24h), 4 (100 mM, 1, 6 & 24h), 5 (100 mM, 3 & 48h) and 10 (10 mM, 48h) separate experiments (N518Ð30). (B) Concentration-response curve for ethanol-induced increase in [3H]N-methylscopolamine binding in SH-SY5Y cells. Cells were exposed to increasing concentrations of ethanol for 2 days prior to [3H]NMS binding analysis. Results are speciÞc binding of [3H]NMS expressed as a percentage of speciÞc binding in control cells. Data are mean 6 SEM from 4 (25 mM, 50 mM and 100 mM) and 8 (10 mM) separate experiments (N59Ð15).
nol throughout the binding study also exhibited an increased number of [3H]NMS binding sites, cells that had been exposed to ethanol for 2 days were kept in HEPES-buffered medium supplemented with 100 mM ethanol throughout the binding study. In cells treated according to this protocol, [3H]NMS binding was 122 6 8.66% (mean 6 SEM from 4 separate experiments performed in triplicates) expressed as a percentage of [3H]NMS binding/mg protein in control cells. In cells that had been exposed to ethanol and in which binding was analyzed in the absence of ethanol, [3H]NMS binding was 135 6 7.09% (mean 6 SEM from 4 separate experiments performed in triplicates) expressed as a percentage of [3H]NMS binding in control cells. No signiÞcant difference was found (P50.256) between these two groups when data were analyzed with paired studentÕsÕt-test. These results demonstrate that removal of ethanol prior to binding studies did not account for the observed increase in mAChR number in cells exposed to ethanol for several days. Effects of long-term ethanol treatment on carbachol-stimulated I(1,4,5)P3 levels The chronic ethanol-induced potentiation of carbachol- stimulated I(1,4,5)P3 formation and increase in [3H]NMS binding appeared to be uncoupled in time. In order to further deÞne this phenomenon, the concentration-dependency of ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation after 48 hours of ethanol treatment was investigated and compared to results of similar ethanol exposure on [3H]NMS binding. As shown in Fig. 4, treatment of the cells with 10 mM ethanol for 48 hours were sufÞcient to induce a signiÞcant
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Fig. 3. Saturation curve for [3H]N-methylscopolamine binding to SH-SY5Y cells following exposure to ethanol for 3 hours. Cells were cultured for 3 hours in the absence (d) or presence (j) of 100 mM ethanol and the speciÞc binding of increasing concentrations of [3H]NMS to the intact cells was determined. Results are speciÞc binding of [3H]NMS/mg protein expressed as a percentage of speciÞc binding of 0,15 nM [3H]NMS/mg protein in control cells and are mean 6 SEM from 2 separate experiments carried out in duplicates.
(17%) potentiation of receptor-stimulated I(1,4,5)P3 formation and higher ethanol concentrations further potentiated this effect. For all ethanol concentrations tested, the potentiation of carbachol-stimulated I(1,4,5)P3 formation was 40-50% more pronounced than the increase in [3H]NMS binding (Fig. 2B). Discussion The results of this study describe the relationship between both acute and long-term ethanolinduced effects on the number of cell surface mAChRs and carbachol-stimulated I(1,4,5)P3 formation in human SH-SY5Y neuroblastoma cells. We have previously described the effects of acute ethanol treatment on carbachol-stimulated I(1,4,5)P3 formation in SH-SY5Y cells (18). Acute exposure of the cells to 100 mM ethanol induced an inhibition of the peak of carbachol-stimulated I(1,4,5)P3 formation with a maximal 20% inhibition after 30 seconds of ethanol treatment. This effect was long-lasting with a duration of 5-10 minutes. In the present work, the effects of similar ethanol treatment on [3H]NMS binding to the cells were studied in order to investigate whether the ethanolinduced inhibition of carbachol-stimulated I(1,4,5)P3 formation could be due to a decreased number of available mAChRs. An 11% decrease in [3H]NMS binding was observed following 30 seconds of ethanol treatment, but this effect was transient and within 1 minute of etha-
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Fig. 4. Concentration-dependency of ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation in SH-SY5Y cells. SH-SY5Y cells were exposed to increasing concentrations of ethanol for 2 days. Fifteen minutes prior to stimulation, the complete medium was exchanged for HEPES-buffered EagleÕs minimal essential medium and cells were stimulated for 10 seconds with 100 mM carbachol. Results are expressed as a percentage of I(1,4,5)P3 levels/mg protein obtained in stimulated control cells. Data are mean 6 SEM from 6 (10 mM) and 4 (25 mM, 50 mM and 100 mM) separate experiments carried out in triplicates.
nol treatment, [3H]NMS binding had returned to control levels and remained there for up to 10 minutes of ethanol exposure. Thus, even though acute treatment of SH-SY5Y cells with 100 mM ethanol induced a decrease in [3H]NMS binding, this decrease was about 50% less pronounced and of much shorter duration than the inhibition of carbachol-stimulated stimulated I(1,4,5)P3 formation observed following the same ethanol treatment and could therefore not account for the observed inhibition of second messenger formation. This indicates that acute ethanol-induced effects on carbachol-stimulated I(1,4,5)P3 formation cannot just be explained by changes in the number of cell surface mAChRs in these cells. An effect of ethanol on the function of available cell surface receptors or down-stream of the receptors is therefore likely. The effects of long-term treatment of SH-SY5Y cells with 100 mM has been shown to induce a potentiation of carbachol-stimulated I(1,4,5)P3 formation and 48 hours of ethanol treatment were necessary to produce any signiÞcant effect (19). In order to investigate whether this effect could be related to an increased number of mAChRs on the cell surface, [3H]NMS binding to the cells was studied following the same ethanol treatment scheme. Ethanol time-dependently increased the number of [3H]NMS binding sites and three hours of ethanol treatment were sufÞcient to induce a signiÞcant (8%) effect. Thus, there was a latency of several hours between the appearance of mAChRs at the cell surface and their ability, when stimulated by carbachol, to activate phospholipase C. A lower functional activity, lasting for up to 23 hours has been described for mAChRs following their new-synthesis and appearance on the cell surface in embryonic chicken heart cells (23,24) and in stably trans-
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fected Y1 adrenal cells (25). This diminished response to agonist treatment was not due to decreased afÞnity for carbachol (23) nor to alterations in GTP-binding protein-coupling to effector enzymes nor to changes in molecular weight or isoelectric points for these receptors (24). Even though these studies were performed in cells expressing M2 subtypes of mAChRs while the receptors involved in carbachol-stimulated I(1,4,5)P3 formation in our cells have been described to be of the M1 subtype (19), the explanation to the observed lower functionality of mAChRs in our cells after several hours of 100 mM ethanol treatment might be that these receptors are newly synthesized, not immediately functional receptors. However, when SH-SY5Y cells were exposed to a low concentration of ethanol (10 mM), 2 days were necessary to induce a signiÞcant increase in mAChR number but these receptors appeared to be immediately fully functional as their stimulation with carbachol induced an elevation of I(1,4,5)P3 levels without any latency. A possible explanation to this observation is that our [3H]NMS binding analysis method may not be sensitive enough to discover minimal changes in receptor number. Thus, a small increase in [3H]NMS binding after only a few hours of treatment with 10 mM ethanol might have been missed, explaining the observed immediate functionality of the receptors. When the effects of 2 days exposure to different concentrations of ethanol on [3H]NMS binding and carbachol-stimulated I(1,4,5)P3 formation were compared, one could note that the ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation was 40Ð50% more pronounced than the increase in [3H]NMS binding observed at the same ethanol concentrations. These differences may be within the range of error of the assays involved but the fact that they were of similar degree and could be observed at all ethanol concentrations tested renders the hypothesis of an inter-assay variation less plausible. We have also been considering the eventuality of a sigmoidal relationship between receptor occupancy and second messenger formation as a possible explanation to our Þndings but have come to the conclusion that such a relationship is unlikely in the present case. A sigmoidal relationship would mean that the number of cell surface receptors would not be directly proportional to receptor-stimulated second messenger formation, thereby explaining the discrepancy between receptor number and I(1,4,5)P3 levels observed following both acute and chronic ethanol treatment in our cells. However, the 10% increase in mAChR number following 6 hours of 100 mM ethanol treatment was not paralleled by a similar increase in second messenger formation while the 10% increase in receptor number following 10 mM ethanol treatment for two days was paralleled by an even higher increase in second messenger levels. This indicates that the observed receptor-second messenger discrepancy in our cells is not caused by a sigmoidal relationship between receptor occupancy and second messenger formation but is rather the consequence of more complex mechanisms. In conclusion, the results of this study show that the effects of both acute and chronic ethanol treatment on carbachol-stimulated I(1,4,5)P3 formation in SH-SY5Y cells cannot only be explained by changes in cell surface mAChR density. Effects of ethanol down-stream of the receptor cannot be excluded. Several lines of evidence suggest that ethanol may act at the level of G proteins in NG108-15 cells (26Ð30), in human permeabilized platelets (31), in turkey erythrocyte ghosts (32), in mice striatal membranes (33) and in mice cerebral cortex (34,35). Effects of ethanol at this level may explain our Þndings but this remains to be demonstrated. Furthermore, long-term ethanol treatment of these cells caused an up-regulation of
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mAChRs which were not immediately functional, suggesting that these receptors might be newly synthesized. Acknowledgments The authors are grateful for excellent technical assistance by Monica Mihailescu and Berit FŠrjh. Financial support for this work was obtained from the Swedish Medical Research Council (05249), The Swedish Alcohol Research Fund, The Swedish Society for Medical Research, The Albert PŒhlsson and Crafoord Foundations and the Medical Faculty at Lund University. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
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