Neurochemical Research

, Volume 16, Issue 11, pp 1235–1240 | Cite as

Time-, concentration-, and age-dependent inhibition of muscarinic receptor-stimulated phosphoinositide metabolism by ethanol in the developing rat brain

  • Walter Balduini
  • Stefano M. Candura
  • Luigi Manzo
  • Flaminio Cattabeni
  • Lucio G. Costa
Original Articles


We have previously reported that administration of ethanol (EtOH; 4 g/Kg/day) to rats from postnatal day 4 to day 10 causes microencephaly and decreases muscarinic receptor-stimulated inositol metabolism on days 7 and 10 (1). An identical exposure to EtOH of adult rats, which resulted in similar blood EtOH concentrations, did not have any effect on the same system. Initial in vitro studies have shown the presence of a differential sensitivity to EtOH of the phosphoinositide system coupled to muscarinic receptors during development (2). In the present study we have expanded these findings by investigating the concentration-, time-, and age-dependent effects of EtOH on accumulation of [3H]inositol phosphates ([3H]InsPs) in brain slices. EtOH caused a dose-dependent inhibition of carbachol-stimulated phosphoinositide metabolism in cerebral cortex slices from 7 day-old rats. When the time of incubation with EtOH was increased to 90 minutes, concentrations as low as 50 mM, which are reached following in vivo administration of EtOH, significantly inhibited the muscarinic response. The effect of EtOH was rather specific for the muscarinic receptors, since, even with longer incubation times, the accumulation of [3H]InsPs induced by norepinephrine or serotonin was inhibited only at concentrations of 150–500 mM. The effect of EtOH was more pronounced in cerebral cortex, hippocampus and cerebellum, and less in the brainstem. The potency of EtOH in inhibiting carbachol-stimulated phosphoinositide metabolism was also dependent on the age of the animals. Its effect was maximal in the 7 day-old rat and less pronounced in younger and older animals. These results confirm that the phosphoinositide system coupled to muscarinic receptors might represent a relevant target for the developmental neurotoxicity of EtOH.

Key Words

Ethanol developmental neurotoxicity phosphoinositide metabolism muscarinic receptor 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Balduini, W., and Costa, L. G. 1989. Effects of ethanol on muscarinic receptor-stimulated phosphoinositide metabolism during brain development. J. Pharmacol. Exp. Ther. 250:541–547.Google Scholar
  2. 2.
    Balduini, W., and Costa, L. G. 1990. Developmental neurotoxicity of ethanol: in vitro inhibition of muscarinic receptor-stimulated phosphoinositide metabolism in brain from neonatal but not adult rats. Brain Research 512:248–252.Google Scholar
  3. 3.
    Balduini, W., Murphy, S. D., and Costa, L. G. 1987. Developmental changes in muscarinic receptor-stimulated phosphoinositide metabolism in rat brain. J. Pharmacol. Exp. Ther. 241:421–427.Google Scholar
  4. 4.
    Heacock, A. M., Fisher, S. K., and Agranoff, B. W. 1987. Enhanced coupling of neonatal muscarinic receptors in rat brain to phosphoinositide turnover. J. Neurochem. 48:1904–1911.Google Scholar
  5. 5.
    Rooney, T. A., and Nahorski, S. R. 1987. Postnatal ontogeny of agonist-and depolarization-induced phosphoinositide hydrolysis in rat cerebral cortex. J. Pharmacol. Exp. Ther. 243:333–341.Google Scholar
  6. 6.
    Dobbing, J., and Sands, J. 1979. Comparative aspects of the brain growth spurt. Early Human Develop. 3:79–83.Google Scholar
  7. 7.
    Diaz, J., and Samson, H. H. 1980. Impaired brain growth in neonatal rats exposed to ethanol. Science (Wash. DC) 208:757–753.Google Scholar
  8. 8.
    Burns, E. M. 1986. Effects of ethanol on synaptic membrane biochemistry during the brain growth spurt. Curr. Topics Res. Synapses 3:29–76.Google Scholar
  9. 9.
    Burns, E. M., Kruckeberg, T. W., Stibler, H., Cerven, E., and Borg, S. 1984. The effects of ethanol exposure during the brain growth spurt in rats. Teratology 29:251–258.Google Scholar
  10. 10.
    Kelly, S. J., Pierce, D. R., and West, J. R. 1987. Microencephaly and hyperactivity in adult rats can be induced by neonatal exposure to high blood alcohol concentrations. Exp. Neurol. 96:580–593.Google Scholar
  11. 11.
    Berridge, M. J., Downes, C. P., and Hanley, M. R. 1982. Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206:587–592.Google Scholar
  12. 12.
    Costa, L. G., Kaylor, G., and Murphy, S. D. 1986. Carbachol- and norepinephrine-stimulated phosphoinositide metabolism in rat brain: effect of chronic cholinesterase inhibition. J. Pharmacol. Exp. Ther. 239:32–37.Google Scholar
  13. 13.
    Balduini, W., Murphy, S. D., and Costa, L. G. 1990. Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats. J. Pharmacol. Exp. Ther. 253:573–579.Google Scholar
  14. 14.
    Snedecor, G. W., and Cochran, W. G. 1980. Statistical Methods, Iowa State University Press, Ames, Iowa.Google Scholar
  15. 15.
    West, J. R., and Pierce, D. R. 1986. Perinatal alcohol exposure and neuronal damage. Pages 120–157,in West, J. R. (ed.), Alcohol and Brain Development, Boston University Press, New York.Google Scholar
  16. 16.
    Bode, D. C., and Molinoff, P. B. 1988. Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system. J. Pharmacol. Exp. Ther. 246:1040–1047.Google Scholar
  17. 17.
    Saffey, K., Gillman, M. A., and Cantrill, R. C. 1988. Chronic in vivo ethanol administration alters the sensitivity of adenylate cyclase coupling in homogenates of rat brain. Neurosci. Lett. 84:317–322.Google Scholar
  18. 18.
    Mochly-Rosen, D., Chang, F. H., Cheever, L., Kim, M., Diamond, I., and Gordon, A. S. 1988. Chronic ethanol causes heterologous desensitization of receptors by reducing αs messenger RNA. Nature 333:848–850.Google Scholar
  19. 19.
    Valverius, P., Hoffmann, P. L., and Tabakoff, B. 1989. Brain forskolin binding in mice dependent on and tolerant to ethanol. Brain Research 503:38–43.Google Scholar
  20. 20.
    Hoek, J. B., Thomas, A. P., Rubin, R., and Rubin, E. 1987. Ethanol-induced mobilization of calcium by activation of phosphoinositide specific phospholipase C in intact hepatocytes. J. Biol. Chem. 262:682–691.Google Scholar
  21. 21.
    Rubin, R., and Hoek, J. B. 1988. Alcohol-induced stimulation of phospholipase C in human platelets requires G-protein activation. Biochem. J. 254:147–153.Google Scholar
  22. 22.
    Hoffman, P. L., Moses, F., Luthin, G. R., and Tabakoff, B. 1986. Acute and chronic effects of ethanol on receptor-mediated phosphatidylinositol 4,5-biphosphate breakdown in mouse brain. Mol. Pharmacol. 30:13–18.Google Scholar
  23. 23.
    Gonzales, R. A., Theiss, C., and Crews, F. T. 1986. Effects of ethanol on stimulated inositol phospholipid hydrolysis in rat brain. J. Pharmacol. Exp. Ther. 237:92–98.Google Scholar
  24. 24.
    Rand, M. L., Vickers, J. D., Kinlough-Rathbone, R. L., Packham, M. A., and Mustard, J. F. 1988. Thrombin-induced inositol triphosphate production by rabbit platelets in inhibited by ethanol. Biochem. J. 251:279–284.Google Scholar
  25. 25.
    Simonsson, P., Sun, G. Y., Vecsei, L., and Alling, C. 1989. Ethanol effects on bradykinin-stimulated phosphoinositide hydrolysis in NG 108-15 neuroblastoma-glioma cells, Alcohol 6:475–479.Google Scholar
  26. 26.
    Ashkenazi, A., Dramachandran, J., and Capon, D. J. 1989. Acetylcholine analogue stimulates DNA synthesis in brain-derived cells via specific muscarinic receptor subtypes. Nature 340:146–150.Google Scholar
  27. 27.
    Van Hoof, C. O. M., De Graan, P. N. E., Oestreicher, A. B., and Gispen, W. H. 1989. Muscarinic receptor activation stimulates B50/GAP43 phosphorylation in isolated nerve growth cones. J. Neurosci. 9:3753–3759.Google Scholar
  28. 28.
    Moon, K. H., Lee, S. Y., and Rhee, S. G. 1989. Developmental changes in the activities of phospholipase C, 3-kinase and 5-phosphatase in rat brain. Biochem. Biophys. Res. Comm. 164:370–374.Google Scholar
  29. 29.
    Sposi, N. M., Bottero, L., Cossu, G., Russo, G., Testa, U., and Peschile, C. 1989. Expression of protein kinase C genes during ontogenic development of the central nervous system. Mol. Cell Biol. 9:2284–2288.Google Scholar
  30. 30.
    Ashkenazi, A., Peralta, E. G., Winslow, J. W., Ramachandran, J., and Capon, D. J. 1989. Functional diversity of muscarinic receptor subtypes in cellular signal transduction and growth. Trends Pharmacol. Sci. 10 (Suppl.):16–21.Google Scholar
  31. 31.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.Google Scholar

Copyright information

© Plenum Publishing Corporation 1991

Authors and Affiliations

  • Walter Balduini
    • 1
  • Stefano M. Candura
    • 1
  • Luigi Manzo
    • 2
  • Flaminio Cattabeni
    • 3
  • Lucio G. Costa
    • 1
  1. 1.Department of Environmental HealthUniversity of WashingtonSeattle
  2. 2.Department of Internal Medicine and Therapeutics, Pharmacology and Toxicology DivisionUniversity of PaviaPaviaItaly
  3. 3.Institute of Pharmacological SciencesUniversity of MilanoMilanoItaly

Personalised recommendations