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Effects of ethanol on cAMP production in murine embryonic palate mesenchymal cells
Life Sciences, Vol. 49, pp. 489-494 Printed in the U.S.A.
Pergamon Press
E F F E C T S OF E T H A N O L ON CAMP PRODUCTION IN MURINE EMBRYONIC PALATE M E S E N C H Y M A L C E L L S Wayde M. Weston and Robert M. Greene Department of Anatomy, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (Received in final form June 7, 1991)
Summary Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolinstimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that, in contrast to what has been observed in other systems, ethanol does not affect the production or activity of Gets in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome. Fetal alcohol syndrome (FAS) is a major public health problem in the United States. Recent epidemiological studies identify FAS as the leading known cause of mental retardation, ahead of conditions such as Down's syndrome and spina bifida (1). FAS is characterized by growth retardation, central nervous system dysfunction, and craniofacial defects, with certain characteristic craniofacial features being the means by which the syndrome is diagnosed (2, 3). Exposure of pregnant mice to ethanol resulted in a constellation of defects including a phenocopy of the craniofacial defects seen in human FAS (4). Whole embryos exposed to ethanol in vitro evidenced dose-dependent growth retardation and retarded differentiation (5). These observations demonstrated that ethanol could act directly on the developing embryo to impair development and provided the rationale for studies designed to examine the effect of ethanol on cellular metabolism in the embryo. Several recent studies on the biological effects of ethanol have focused on cyclic AMp-mediated signal transduction. Acute exposure of rat neuroblastoma cells to ethanol stimulates receptor-mediated production of cAMP, while chronic exposure has the opposite effect (6). Similar responses to ethanol treatment have been observed in rat brain cortex (7) and pinealocytes (8), human platelets (9) and lymphocytes (10,11), and in mouse hippocampus (12). Although the mechanisms underlying the effects of acute ethanol exposure are poorly understood, the effects of chronic ethanol exposure are believed to be due, at least in part, to specific alterations in the synthesis and activity of various G protein subunits. Alterations in the levels of expression of both Gas and Gai have been demonstrated in rat neuroblastoma cells chronically exposed to ethanol (13,14). There is substantial evidence suggesting that cAMP plays a critical role in normal growth and development of the mammalian secondary palate (reviewed in ref. 15). Endogenous levels of cAMP in palate tissue rise transiently during the period of palatal shelf elevation and closure (16, 17). cAMP has also been shown to regulate cell proliferation (18) and extracellular matrix synthesis (19) in palatal tissue. Both of these processes are vital to normal development of the palate (15). Since normal growth and differentiation of embryonic palatal tissue appear to be critically dependent on cAMP, and ethanol can affect cAMp-mediated signal transduction (see 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press olc
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above), it is reasonable to hypothesize that alterations in receptor-mediated production in cAMP could be the basis for ethanol-induced craniofacial abnormalities. We have therefore begun to investigate the effect of ethanol on cAMP synthesis in murine embryonic palatal mesenchyme (MEPM) ceils. The data presented here demonstrate that ethanol affects the cAMP signal transduction pathway in embryonic palatal ceils in a manner similar to that observed in other systems; however, it appears that alterations in cAMP production are not due to decreased expression of Gas but are most likely produced through a different mechanism. Methods Cell culture
Murine embryonic palatal mesenchymal (MEPM) cells were derived from palatal tissue dissected from day 13 ICR mouse embryos (date of vaginal plug detection = day 0 of gestation). The palates were minced and dissociated with 0.25% trypsin 1:250/0.1% EDTA in phosphate-buffered saline (PBS) for 10 minutes at 37~C with constant shaking. Trypsin was inhibited by the addition of Opti-MEM medium (Gibco) containing 5% fetal bovine serum (FBS, Sigma). Cells were plated in 35 mm tissue culture dishes at an initial density of 1.25x104cells/cm 2 in Opti-MEM containing Earle s salts and 25 mM Hepes buffer and supplemented with 2 mM glutamine, 5% FBS, 100 mg/ml streptomycin, and 100 units/ml penicillin and grown to confluency at 37-°C in a 95% air/5% CO2 atmosphere, with media replaced every other day. Acute ethanol treatment
Media was removed from dishes of confluent cells and replaced with media containing hormones or other agents to stimulate cAMP production. Cells were treated with either isoproterenol (1 mM), prostaglandin E2 (1 mM), forskolin (20 mM), or cholera toxin (2.5 mg/ml) in Opti-MEM/5% FBS/2 mM 3-isobutyl-l-methylxanthine in the presence or absence of 200 mM ethanol. After treatment for 30 minutes at 37~C, media was removed and cells scraped into ice-cold 10% trichloroacetic acid (TCA), sonicated, and incubated overnight at 4~2. Samples were then centrifuged at 1000 RPM for 20 minutes. Acid-soluble supernatants were transferred to fresh tubes. Acid-insoluble material was washed with 10% TCA and this wash combined with the original supernatants. The acid-insoluble material was then dissolved in 1N NaOH and protein determined by the method of Lowry (20). The acid-soluble fraction was extracted with water-saturated ether, lyophilized, and resuspended in 0.05 M acetate buffer, pH 6.2. Levels of cAMP were determined by radioimmunoassay. Chronic ethanol treatment
Media was removed from dishes of confluent cells and replaced with fresh media with or without 200 mM ethanol. Dishes were sealed with Parafilm to inhibit evaporation of ethanol and incubated at 37-°C in 5% CO2 for 24 hours, after which media with or without ethanol was replenished. After 48 hours of exposure to ethanol, cells were treated and processed as described under "Acute ethanol treatment." Results Effect of ethanol on hormone-stimulated cAMP production
Confluent MEPM cells were treated with 1 mM isoproterenol or 1 mM prostaglandin E2 (PGE2) in the presence or absence of 200 mM ethanol for 30 minutes. Both of these agents are normally present in the developing palate (21, 22), and have been shown to elevate cAMP in MEPM cells (23, 24). The results of this experiment are presented in Table I. Isoproterenol, in the absence of ethanol, produced an approximately 10-fold increase in intracellular cAMP levels. When ethanol was administered along with the hormone, a significantly larger amount of cAMP was produced. PGE2 treatment increased cAMP levels by approximately 40-fold. This response also appeared to be enhanced in the presence of ethanol; however, the difference
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between ethanol-treated and nontreated cells was not found to be statistically significant. Basal (nonstimulated) levels of cAMP were not affected by treatment with ethanol in this and in all other experiments. TABLE I Effect of Acute Ethanol Treatment on Hormone-Stimulated c A M P Synthesis
Treatment
No ethanol
Media Isoproterenol (lmM) Prostaglandin E2 (lmM)
0.424 4-/- 0.073 5.648 +/- 0.576 32.048 +/- 5.771
200 mM ethanol 0.440 +/- 0.067 7.859 4-/- 1.037 * 40.291 +/- 7.458
Confluent cells were treated with hormones and cAMP levels determined as described in "Materials and Methods." In this and all other tables, values are presented as pmol cAMP/mg protein and represent an average of 3 separate determinations. An asterisk (*) indicates a statistically significant difference in value from non-ethanol treated cells (p<0.05, Student's "t" test). Effect of chronic ethanol treatment on hormone-stimulated c A M P production
Confluent MEPM cells were treated for 48 hours with 200 mM ethanol in culture medium and then treated with either 1 mM isoproterenol or 1 mM PGE2 in the continuing presence or absence of ethanol. The effect of this treatment on cAMP production is shown in Table II. In the continued presence of ethanol, hormone-treated cells produced cAMP in amounts similar to those observed in non-ethanol treated cells when stimulated with isoproterenol or PGE2. When ethanol was removed, however, the cells produced significantly less cAMP in response to hormone treatment than the non-ethanol treated controls. These results are in agreement with those obtained in other systems (6-12) and indicate that these cells respond to long-term ethanol treatment by heterologous desensitization to hormone stimulation. TABLE II Effect of Chronic Ethanol Treatment on Hormone-Stimulated c A M P Synthesis Treatment
No ethanol pretreatment
Media Isoproterenol (lmM) Prostaglandin E2 (lmM)
0.424 4-/- 0.073 5.648 +/- 0.576 32.048 +/- 5.771
Chronic ethanol pretreatment/stimulate in absence of ethanol 0.446 +/- 0.026 4.568 +/- 0.119" 16.506 +/- 2.755*
Chronic ethanol treatment/stimulate in presence of ethanol 0.406 +/- 0.055 9.310 +/- 3.818 32.007 +/- 8.079
Confluent cells were treated with 200 mM ethanol in media for 48 hours prior to stimulation with hormones. Hormone stimulation was performed in the presence or absence of 200 mM ethanol as described in Materials and Methods. Asterisks (*) denote statistically significant differences from non-ethanol treated control values. Effect of ethanol on forskolin and cholera toxin-stimulated c A M P production
To investigate how ethanol might affect specific steps in cAMP production, ethanoltreated and control cells were treated with agents that act at defined points on the cAMP biosynthetic pathway. Forskolin, for example, stimulates cAMP production by passing directly through the cell membrane and activating adenylate cyclase (25, 26). The effects of ethanol on
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forskolin-stimulated cAMP production are shown in Table 3. In non-ethanol treated ceils, 20 mM forskolin produced an almost 85-fold increase in cAMP levels. Ceils treated with ethanol in an acute or chronic manner produced significantly less cAMP in response to forskolin than did controls. Both acute and chronic ethanol treatments produced a similar decrease in forskolin-stimulated cAMP levels. To assess the effect of ethanol on G proteins in MEPM cells, cells were treated with cholera toxin. Cholera toxin enters cells after binding to its receptor, the ganglioside GM1, and catalyzes the ADP-ribosylation of the Gas subunit, "locking" this subunit into an active conformation and causing persistent activation of adenylate cyclase. Changes in the ceils' ability to respond to cholera toxin could thus reflect changes in the amount or activity of Gas. TABLE III Effects of Acute and Chronic Ethanol Treatment on Forskolin and Cholera Toxin-Stimulated cAMP Synthesis
Media Forskolin
0.569 +/- 0.221 0.459 +/- 0.104 48.397 +/- 9.126 30.452 +/- 1,594"
0.515 +/- 0.120 27.794 +/- 5.907*
Chronic ethanol pretreatment/200 mM ethanol present 0.465 +/- 0.097 25.631 +/-3.390*
Media Cholera toxin (2.5mg/ml)
0.424 +/- 0.073 0.440 +/-0.067 73.784 +/- 9.410 86.874 +/- 13.447
0.446 +/- 0.026 90.668 +/- 19.355
0.406 +/- 0.055 105.541 +/- 9.098
Treatment
(20mM)
No ethanol
200 mM ethanol
Chronic ethanol pretreatment/no ethanol present
Confluent cell's were either maintained in regular media or treated with 200 mM ethanol in media for 48 hours prior to stimulation with forskolin or cholera toxin. Stimulation wascarried out in the presence or absence of 200 mM ethanol as described in Materials and Methods. Asterisks (*) denote statistically significant differences from non-ethanol treated control values. When treated with 2.5 mg/ml cholera toxin in the absence of ethanol and without prior ethanol treatment, MEPM cells display an approximately 175-fold increase in cAMP levels, compared to basal levels (Table III), Neither acute nor chronic ethanol treatment produced any significant differences in the ability of MEPM cells to produce cAMP in response to cholera toxin treatment. The data show an apparent trend towards increased sensitivity to cholera toxin, but this is not statistically significant. Discussion The data presented here clearly demonstrate that ethanol affects cAMP production in cells of craniofacial origin. The observed effects are similar to those seen in other systems: acute treatment with ethanol leads to increased cAMP synthesis following hormone stimulation, while treatment with ethanol in a chronic manner leaves MEPM cells less able to produce cAMP in response to hormone treatment in the absence of ethanol. This desensitization is not specific to an individual hormone or receptor type, as sensitivity to both isoproterenol and PGE2 is decreased by chronic ethanol treatment. When MEPM cells treated chronically with ethanol are treated with hormone in the continued presence of ethanol, cAMP levels are increased over those observed in chronically treated ceils in the absence of ethanol and are similar to those observed in non-ethanol treated cells in the absence of ethanol. This indicates that the effects of acute and chronic ethanol treatments are carried out by different mechanisms.
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493
The response to forskolin by ethanol-treated cells may reflect a decrease in cellular levels of adenylate cyclase. Alternatively, since ethanol has been shown in vitro to inhibit the activation of adenylate cyclase by forskolin (27), the observed effects could be attributed to decreased adenylate cyclase activity as opposed to a decreased amount of adenylate eyclase. In addition, forskolln has been reported to stabilize the interaction of adenylate cyclase and Gas (28), raising the possibility that this result may reflect changes at the G protein level. Desensitization to hormone stimulus in cells chronically treated with ethanol has been demonstrated in rat neuroblastoma cells to be due to altered expression of G protein ct subunits (13, 14). In most cases, expression of the stimulatory G protein, Gets, was observed to be decreased following chronic ethanol treatment. Stimulation of these cells with cholera toxin showed decreased sensitivity to this agent (14), and Northern and Western blot analysis directly demonstrated decreased amounts of Gas in these cells at both the protein and mRNA levels (13, 14). In one cell type, N1E-115, increased expression of the inhibitory G protein et subunit, Gai, was observed by Western blot analysis. When ethanol treatment was extended, however, decreased expression of Gas was also observed (14). The data presented here, however, indicate that palate cells respond to ethanol treatment in a different manner. The lack of sensitivity of the cholera toxin response to ethanol treatment provides evidence that Gets expression in MEPM cells is not decreased by ethanol treatment and that chronic ethanol treatment affects cAMP production in palate cells via an alternative mechanism. Quantification of G protein subunits in ethanol-treated palate cells should aid in confn'ming this observation and determining the means by which ethanol affects cAMP production in palate cells. While ethanol has long been recognized as a human and animal teratogen, the mechanisms underlying ethanol-induced birth defects are largely unknown. The data presented here indicate apossible means by which ethanol may affect craniofaciai development. Because cAMP appears to play such a crucial role in normal craniofacial development (15), it is reasonable to propose that agents that affect cAMP-mediated signal transduction may also affect developmental processes regulated by cAMP. This assumption is supported by the observation that some agents known to adversely affect palate development have been shown to cause alterations in palatal cAMP levels (17). If the in vivo effects of ethanol on cAMp-mediated signal transduction in the craniofacial region are similar to those observed in cultured MEPM cells, the perturbation of receptor-mediated cAMP production by ethanol may lead to the craniofacial abnormalities seen in FAS. Further study of the in vivo effects of ethanol on adenylate cyclase activity, cAMP levels, and G protein synthesis and activity in the palate should help clarify this situation. /~¢knowledg$ments This work supported in part by NIH grants DE 08199 and DE 09540. WMW is supported in part by NIH training grant HD07326 and NRSA DE 05593.
References 1. 2. 3. 4. 5. 6.
K.R. WARREN and R.J. BAST, Public Health Reports 103 638-642 (1988). S.K. CLARREN and D.W. SMITH, New Eng.J. Med. 298 1063-1067 (1978). K.K. SULIK, M.C. JOHNSTON and M.A. WEBB, Science 214 936-938 (1981). K.K. SULIK and M.C. JOHNSTON, Am. J. Anat. 166 257-269 (1983). N.A. BROWN, E.H. GOULDING and S. FABRO, Science 206 573-575 (1979). A.S, GORDON, K. COLLIER and I. DIAMOND, Proc. Natl. Acad. Sci. USA 83 21052108 (1986). 7. F. BAUCHE, A.M. BOURDEAUX-JAUBERT, Y. GIUDICELLI and R. NORDMANN, FEBS Lett. 219 296-300 (1987), 8. C.L. CHIK, A.K. HO and D.C. KLEIN, Biochem. Biophys. Res. Commun. 147 145151 (1987). 9. B. TABAKOFF, P.L. HOFFMAN, J.M. LEE, T. SAITO, B. WILLARD and F. DE LEON-JONES, New Eng. J. Med. 318 134-139 (1988).
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10. L.E. NAGY,, I. DIAMOND, and A. GORDON, Proc. Natl. Acad. Sci. USA 85 6973-6976 (1988). 11. I. DIAMOND, B. WRUBEL, W. ESTRIN and A. GORDON, Proc. Natl. Acad. Sci. USA 84 1413-1416 (1987). 12. P. VALVERIUS, P.L. HOFFMAN and B. TABAKOFF, J. Neurochem. 52 492-497 (1987). 13. D. MOCHLY-ROSEN, F-H. CHANG, L. CHEEVER, M. KIM, I. DIAMOND and A.S. GORDON, Nature 333 848-850 (1988). 14. M,E. CHARNESS, L.A. QUEREMIT and M. HENTELEFF, Biochem. Biophys. Res. Commun. 155 138-143 (1988). 15. R.M. GREENE, Crit. Rev. Toxicol. 20 137-152 (1989). 16. R. GREENE and R. J. PRAq~F, Histochem. Cytochem. 27 924-931 (1979). 17. F. OLSON and E. MASSARO, Teratology 22 155-166 (1980). 18. M.PISANO, M. SCHNEIDERMAN and R. GREENE, J. Cell. Physiol. 126 84-92 (1986). 19. R. GREENE, V. MCANDREW and M. LLOYD, Biochem. Biophys. Res. Commun. 130 232-238 (1982). 20. O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR, and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 21. M. PISANO, and R. GREENE, IRCS Med. Sci. 13 900-901 (1984). 22. R. GREENE, M. LLOYD and K. NICOLAU, J. Craniofacial Genet. Dev. Biol. 1 261272 (1981). 23. M. GARBARINO, and R. GREENE, Biochem. Biophys. Res. Commun. 119 193-202 (1984). 24. J. JONES and R. GREENE, Prostaglandins Leukotrienes Med. 22 139-151 (1986). 25. H, METZGER and E. LINDNER, ICRS Biochem. 9 99 (1981). 26.J.W. DALY, W.L. PADGETF and K.B. SEAMON, Fed. Proc. 40 626 (1981). 27. R-D. HUANG, M.F.SMITH and W.L. ZAHLER, J. Cyc. Nuc. Res. 8 385-394 (1982). 28. P.L. HOFFMAN, and B. TABAKOFF, FASEB Journal 4 2612-2622 (1990).
Pergamon Press
E F F E C T S OF E T H A N O L ON CAMP PRODUCTION IN MURINE EMBRYONIC PALATE M E S E N C H Y M A L C E L L S Wayde M. Weston and Robert M. Greene Department of Anatomy, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (Received in final form June 7, 1991)
Summary Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolinstimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that, in contrast to what has been observed in other systems, ethanol does not affect the production or activity of Gets in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome. Fetal alcohol syndrome (FAS) is a major public health problem in the United States. Recent epidemiological studies identify FAS as the leading known cause of mental retardation, ahead of conditions such as Down's syndrome and spina bifida (1). FAS is characterized by growth retardation, central nervous system dysfunction, and craniofacial defects, with certain characteristic craniofacial features being the means by which the syndrome is diagnosed (2, 3). Exposure of pregnant mice to ethanol resulted in a constellation of defects including a phenocopy of the craniofacial defects seen in human FAS (4). Whole embryos exposed to ethanol in vitro evidenced dose-dependent growth retardation and retarded differentiation (5). These observations demonstrated that ethanol could act directly on the developing embryo to impair development and provided the rationale for studies designed to examine the effect of ethanol on cellular metabolism in the embryo. Several recent studies on the biological effects of ethanol have focused on cyclic AMp-mediated signal transduction. Acute exposure of rat neuroblastoma cells to ethanol stimulates receptor-mediated production of cAMP, while chronic exposure has the opposite effect (6). Similar responses to ethanol treatment have been observed in rat brain cortex (7) and pinealocytes (8), human platelets (9) and lymphocytes (10,11), and in mouse hippocampus (12). Although the mechanisms underlying the effects of acute ethanol exposure are poorly understood, the effects of chronic ethanol exposure are believed to be due, at least in part, to specific alterations in the synthesis and activity of various G protein subunits. Alterations in the levels of expression of both Gas and Gai have been demonstrated in rat neuroblastoma cells chronically exposed to ethanol (13,14). There is substantial evidence suggesting that cAMP plays a critical role in normal growth and development of the mammalian secondary palate (reviewed in ref. 15). Endogenous levels of cAMP in palate tissue rise transiently during the period of palatal shelf elevation and closure (16, 17). cAMP has also been shown to regulate cell proliferation (18) and extracellular matrix synthesis (19) in palatal tissue. Both of these processes are vital to normal development of the palate (15). Since normal growth and differentiation of embryonic palatal tissue appear to be critically dependent on cAMP, and ethanol can affect cAMp-mediated signal transduction (see 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press olc
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above), it is reasonable to hypothesize that alterations in receptor-mediated production in cAMP could be the basis for ethanol-induced craniofacial abnormalities. We have therefore begun to investigate the effect of ethanol on cAMP synthesis in murine embryonic palatal mesenchyme (MEPM) ceils. The data presented here demonstrate that ethanol affects the cAMP signal transduction pathway in embryonic palatal ceils in a manner similar to that observed in other systems; however, it appears that alterations in cAMP production are not due to decreased expression of Gas but are most likely produced through a different mechanism. Methods Cell culture
Murine embryonic palatal mesenchymal (MEPM) cells were derived from palatal tissue dissected from day 13 ICR mouse embryos (date of vaginal plug detection = day 0 of gestation). The palates were minced and dissociated with 0.25% trypsin 1:250/0.1% EDTA in phosphate-buffered saline (PBS) for 10 minutes at 37~C with constant shaking. Trypsin was inhibited by the addition of Opti-MEM medium (Gibco) containing 5% fetal bovine serum (FBS, Sigma). Cells were plated in 35 mm tissue culture dishes at an initial density of 1.25x104cells/cm 2 in Opti-MEM containing Earle s salts and 25 mM Hepes buffer and supplemented with 2 mM glutamine, 5% FBS, 100 mg/ml streptomycin, and 100 units/ml penicillin and grown to confluency at 37-°C in a 95% air/5% CO2 atmosphere, with media replaced every other day. Acute ethanol treatment
Media was removed from dishes of confluent cells and replaced with media containing hormones or other agents to stimulate cAMP production. Cells were treated with either isoproterenol (1 mM), prostaglandin E2 (1 mM), forskolin (20 mM), or cholera toxin (2.5 mg/ml) in Opti-MEM/5% FBS/2 mM 3-isobutyl-l-methylxanthine in the presence or absence of 200 mM ethanol. After treatment for 30 minutes at 37~C, media was removed and cells scraped into ice-cold 10% trichloroacetic acid (TCA), sonicated, and incubated overnight at 4~2. Samples were then centrifuged at 1000 RPM for 20 minutes. Acid-soluble supernatants were transferred to fresh tubes. Acid-insoluble material was washed with 10% TCA and this wash combined with the original supernatants. The acid-insoluble material was then dissolved in 1N NaOH and protein determined by the method of Lowry (20). The acid-soluble fraction was extracted with water-saturated ether, lyophilized, and resuspended in 0.05 M acetate buffer, pH 6.2. Levels of cAMP were determined by radioimmunoassay. Chronic ethanol treatment
Media was removed from dishes of confluent cells and replaced with fresh media with or without 200 mM ethanol. Dishes were sealed with Parafilm to inhibit evaporation of ethanol and incubated at 37-°C in 5% CO2 for 24 hours, after which media with or without ethanol was replenished. After 48 hours of exposure to ethanol, cells were treated and processed as described under "Acute ethanol treatment." Results Effect of ethanol on hormone-stimulated cAMP production
Confluent MEPM cells were treated with 1 mM isoproterenol or 1 mM prostaglandin E2 (PGE2) in the presence or absence of 200 mM ethanol for 30 minutes. Both of these agents are normally present in the developing palate (21, 22), and have been shown to elevate cAMP in MEPM cells (23, 24). The results of this experiment are presented in Table I. Isoproterenol, in the absence of ethanol, produced an approximately 10-fold increase in intracellular cAMP levels. When ethanol was administered along with the hormone, a significantly larger amount of cAMP was produced. PGE2 treatment increased cAMP levels by approximately 40-fold. This response also appeared to be enhanced in the presence of ethanol; however, the difference
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491
between ethanol-treated and nontreated cells was not found to be statistically significant. Basal (nonstimulated) levels of cAMP were not affected by treatment with ethanol in this and in all other experiments. TABLE I Effect of Acute Ethanol Treatment on Hormone-Stimulated c A M P Synthesis
Treatment
No ethanol
Media Isoproterenol (lmM) Prostaglandin E2 (lmM)
0.424 4-/- 0.073 5.648 +/- 0.576 32.048 +/- 5.771
200 mM ethanol 0.440 +/- 0.067 7.859 4-/- 1.037 * 40.291 +/- 7.458
Confluent cells were treated with hormones and cAMP levels determined as described in "Materials and Methods." In this and all other tables, values are presented as pmol cAMP/mg protein and represent an average of 3 separate determinations. An asterisk (*) indicates a statistically significant difference in value from non-ethanol treated cells (p<0.05, Student's "t" test). Effect of chronic ethanol treatment on hormone-stimulated c A M P production
Confluent MEPM cells were treated for 48 hours with 200 mM ethanol in culture medium and then treated with either 1 mM isoproterenol or 1 mM PGE2 in the continuing presence or absence of ethanol. The effect of this treatment on cAMP production is shown in Table II. In the continued presence of ethanol, hormone-treated cells produced cAMP in amounts similar to those observed in non-ethanol treated cells when stimulated with isoproterenol or PGE2. When ethanol was removed, however, the cells produced significantly less cAMP in response to hormone treatment than the non-ethanol treated controls. These results are in agreement with those obtained in other systems (6-12) and indicate that these cells respond to long-term ethanol treatment by heterologous desensitization to hormone stimulation. TABLE II Effect of Chronic Ethanol Treatment on Hormone-Stimulated c A M P Synthesis Treatment
No ethanol pretreatment
Media Isoproterenol (lmM) Prostaglandin E2 (lmM)
0.424 4-/- 0.073 5.648 +/- 0.576 32.048 +/- 5.771
Chronic ethanol pretreatment/stimulate in absence of ethanol 0.446 +/- 0.026 4.568 +/- 0.119" 16.506 +/- 2.755*
Chronic ethanol treatment/stimulate in presence of ethanol 0.406 +/- 0.055 9.310 +/- 3.818 32.007 +/- 8.079
Confluent cells were treated with 200 mM ethanol in media for 48 hours prior to stimulation with hormones. Hormone stimulation was performed in the presence or absence of 200 mM ethanol as described in Materials and Methods. Asterisks (*) denote statistically significant differences from non-ethanol treated control values. Effect of ethanol on forskolin and cholera toxin-stimulated c A M P production
To investigate how ethanol might affect specific steps in cAMP production, ethanoltreated and control cells were treated with agents that act at defined points on the cAMP biosynthetic pathway. Forskolin, for example, stimulates cAMP production by passing directly through the cell membrane and activating adenylate cyclase (25, 26). The effects of ethanol on
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forskolin-stimulated cAMP production are shown in Table 3. In non-ethanol treated ceils, 20 mM forskolin produced an almost 85-fold increase in cAMP levels. Ceils treated with ethanol in an acute or chronic manner produced significantly less cAMP in response to forskolin than did controls. Both acute and chronic ethanol treatments produced a similar decrease in forskolin-stimulated cAMP levels. To assess the effect of ethanol on G proteins in MEPM cells, cells were treated with cholera toxin. Cholera toxin enters cells after binding to its receptor, the ganglioside GM1, and catalyzes the ADP-ribosylation of the Gas subunit, "locking" this subunit into an active conformation and causing persistent activation of adenylate cyclase. Changes in the ceils' ability to respond to cholera toxin could thus reflect changes in the amount or activity of Gas. TABLE III Effects of Acute and Chronic Ethanol Treatment on Forskolin and Cholera Toxin-Stimulated cAMP Synthesis
Media Forskolin
0.569 +/- 0.221 0.459 +/- 0.104 48.397 +/- 9.126 30.452 +/- 1,594"
0.515 +/- 0.120 27.794 +/- 5.907*
Chronic ethanol pretreatment/200 mM ethanol present 0.465 +/- 0.097 25.631 +/-3.390*
Media Cholera toxin (2.5mg/ml)
0.424 +/- 0.073 0.440 +/-0.067 73.784 +/- 9.410 86.874 +/- 13.447
0.446 +/- 0.026 90.668 +/- 19.355
0.406 +/- 0.055 105.541 +/- 9.098
Treatment
(20mM)
No ethanol
200 mM ethanol
Chronic ethanol pretreatment/no ethanol present
Confluent cell's were either maintained in regular media or treated with 200 mM ethanol in media for 48 hours prior to stimulation with forskolin or cholera toxin. Stimulation wascarried out in the presence or absence of 200 mM ethanol as described in Materials and Methods. Asterisks (*) denote statistically significant differences from non-ethanol treated control values. When treated with 2.5 mg/ml cholera toxin in the absence of ethanol and without prior ethanol treatment, MEPM cells display an approximately 175-fold increase in cAMP levels, compared to basal levels (Table III), Neither acute nor chronic ethanol treatment produced any significant differences in the ability of MEPM cells to produce cAMP in response to cholera toxin treatment. The data show an apparent trend towards increased sensitivity to cholera toxin, but this is not statistically significant. Discussion The data presented here clearly demonstrate that ethanol affects cAMP production in cells of craniofacial origin. The observed effects are similar to those seen in other systems: acute treatment with ethanol leads to increased cAMP synthesis following hormone stimulation, while treatment with ethanol in a chronic manner leaves MEPM cells less able to produce cAMP in response to hormone treatment in the absence of ethanol. This desensitization is not specific to an individual hormone or receptor type, as sensitivity to both isoproterenol and PGE2 is decreased by chronic ethanol treatment. When MEPM cells treated chronically with ethanol are treated with hormone in the continued presence of ethanol, cAMP levels are increased over those observed in chronically treated ceils in the absence of ethanol and are similar to those observed in non-ethanol treated cells in the absence of ethanol. This indicates that the effects of acute and chronic ethanol treatments are carried out by different mechanisms.
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The response to forskolin by ethanol-treated cells may reflect a decrease in cellular levels of adenylate cyclase. Alternatively, since ethanol has been shown in vitro to inhibit the activation of adenylate cyclase by forskolin (27), the observed effects could be attributed to decreased adenylate cyclase activity as opposed to a decreased amount of adenylate eyclase. In addition, forskolln has been reported to stabilize the interaction of adenylate cyclase and Gas (28), raising the possibility that this result may reflect changes at the G protein level. Desensitization to hormone stimulus in cells chronically treated with ethanol has been demonstrated in rat neuroblastoma cells to be due to altered expression of G protein ct subunits (13, 14). In most cases, expression of the stimulatory G protein, Gets, was observed to be decreased following chronic ethanol treatment. Stimulation of these cells with cholera toxin showed decreased sensitivity to this agent (14), and Northern and Western blot analysis directly demonstrated decreased amounts of Gas in these cells at both the protein and mRNA levels (13, 14). In one cell type, N1E-115, increased expression of the inhibitory G protein et subunit, Gai, was observed by Western blot analysis. When ethanol treatment was extended, however, decreased expression of Gas was also observed (14). The data presented here, however, indicate that palate cells respond to ethanol treatment in a different manner. The lack of sensitivity of the cholera toxin response to ethanol treatment provides evidence that Gets expression in MEPM cells is not decreased by ethanol treatment and that chronic ethanol treatment affects cAMP production in palate cells via an alternative mechanism. Quantification of G protein subunits in ethanol-treated palate cells should aid in confn'ming this observation and determining the means by which ethanol affects cAMP production in palate cells. While ethanol has long been recognized as a human and animal teratogen, the mechanisms underlying ethanol-induced birth defects are largely unknown. The data presented here indicate apossible means by which ethanol may affect craniofaciai development. Because cAMP appears to play such a crucial role in normal craniofacial development (15), it is reasonable to propose that agents that affect cAMP-mediated signal transduction may also affect developmental processes regulated by cAMP. This assumption is supported by the observation that some agents known to adversely affect palate development have been shown to cause alterations in palatal cAMP levels (17). If the in vivo effects of ethanol on cAMp-mediated signal transduction in the craniofacial region are similar to those observed in cultured MEPM cells, the perturbation of receptor-mediated cAMP production by ethanol may lead to the craniofacial abnormalities seen in FAS. Further study of the in vivo effects of ethanol on adenylate cyclase activity, cAMP levels, and G protein synthesis and activity in the palate should help clarify this situation. /~¢knowledg$ments This work supported in part by NIH grants DE 08199 and DE 09540. WMW is supported in part by NIH training grant HD07326 and NRSA DE 05593.
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