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Down-regulation of brain muscarinic cholinergic receptor promoted by diacylglycerols and phorbol ester

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Abstract

Sustained agonist stimulation induces an asymmetric down-regulation of brain muscarinic acetylcholine receptor (mAChR): 43±2% in the right and 26±2% in the left cerebral hemisphere, respectively (Ref. 1). In order to determine the possible involvement of endogenous diacylglycerols produced under muscarinic stimulation in the down-regulation phenomenon, here we have studied the effects of synthetic diacylglycerols and a phorbol ester on cells dissociated from rat cerebral cortex. Oleoylacetylglycerol decreased the amount of cell-surface mAChR by 37±2% and 25±2% in right and left cerebral cortex, respectively. Long-term treatment with phorbol dibutyrate also produced internalization of the mAChR (25±1.5% and 33±2% in right and left cortical cells, respectively). These changes occurred without modification of the Kdapp for the selective antagonist pirenzepine. The action of calcium ions was also studied using incubation of cells with the ionophore A23187. No changes were observed in the amount of mAChR detected at the plasma membrane with the ionophore alone, but when used in combination with phorbol dibutyrate and the agonist carbamylcholine a sinergistic decrease in mAChR was apparent. It is concluded that long-term exposure to exogenously added diacyglycerols and phorbol ester significantly reduces the amount of mAChR detected at the plasma membrane and abolishes the asymmetry of the down-regulation phenomenon observed under specific muscarinic stimulation, suggesting that diacylglycerols may be one of the factors responsible for such asymmetry.

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Abbreviations

A23187:

ionophore A23187

ATRO:

atropine

CARB:

carbamoylcholine

DAG:

diacylglycerol

DMEM:

Dulbecco's modified Eagle's medium

DMSO:

dimethylsulfoxide

HEPES:

4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer

PZ:

pirenzepine

LCC:

left cerebral cortex

mAChR:

muscarinic acetylcholine receptor

OAG:

oleoylacetylglycerol

PDB:

phorbol dibutyrate

RCC:

right cerebral cortex

References

  1. Pediconi M. F., Roccamo de Fernández, A. M., and Barrantes F. J. 1993. Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex. Neurochem. Res. 18:565–572.

    Google Scholar 

  2. González, R. A., and Crews, F. T. 1984. Characterization of the cholinergic stimulation of phosphoinositide hydrolysis in rat brain slices. J. Neurosci. 4:4120–4127.

    Google Scholar 

  3. Gil, D. W., and Wolfe, B. D. 1985. Pirenzepine distinguishes between muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase. J. Pharm. Exp. Ther. 232:608–616.

    Google Scholar 

  4. Lazareno, S., Kendall, D. A., and Nahorski, S. R. 1985. Pirenzepine indicates heterogeneity of muscarinic receptor linked to cerebral inositol phospholipid metabolism. Neuropharmacol. 24:593–595.

    Google Scholar 

  5. Fisher, S. K., and Snider, R. M. (1987). Differential receptor occupancy requirements for muscarinic cholinergic stimulation of inositol lipid hydrolysis in brain and in neuroblastoma. Mol. Pharmacol. 32:81–90.

    Google Scholar 

  6. Bonner, T. I. 1989. The molecular basis of muscarinic receptor diversity. Trends Neurosci. 12:148–151.

    Google Scholar 

  7. Nathanson, N. M., Klein, W. L., and Niremberg, M. 1978. Regulation of adenylate cyclase activity mediated by muscarinic acetylcholine receptors. Proc. Nat. Acad. Sci. 75:1788–1791.

    Google Scholar 

  8. Sibley, D. R., Benovic, J. L., Caron, M. G., and Lefkowitz, R. 1987. Regulation of transmembrane signaling by receptor phosphorylation. Cell 48:913–922.

    Google Scholar 

  9. Richelson, E. 1984. Studying neurotransmitter receptors: binding and biological assays. Pages 4–19, in Monogr. Neural Sci., Karger, Basel.

    Google Scholar 

  10. Bennett, J. P., and Yamamura, H. I. 1985. Neurotransmitters, hormones, or drug receptor binding methods. Pages 61–89,in Yamamura, Enna, and Kuhar (eds.), Neurotransmitter receptor binding, 2nd. edition. Raven Press, N. Y.

    Google Scholar 

  11. Lai, W. S., and El-Fakahany, E. E. 1987. Phorbol ester-induced inhibition of cyclic GMP formation mediated by muscarinic receptors in murine neuroblastoma cells. J. Pharmacol. Exp. Ther. 241:366–373.

    Google Scholar 

  12. Xu, J., and Chuang, D. M. 1987. Muscarinic acetylcholine receptor-mediated phosphoinositide turnover in culture cerebellar granule cells: desensitization by receptor agonist. J. Pharmacol. Exp. Ther. 242:238–244.

    Google Scholar 

  13. El-Fakahany, E. E., Alger, B. E., Lai, W. S., Worley, P. F., and Baraban, J. M. 1988. Neuronal muscarinic responses: role of protein kinase C. FASEB J. 2:2575–2583.

    Google Scholar 

  14. Liles, W. C., Hunter, D. D., Meier, K. E., and Nathanson, N. M. 1986. Activation of protein kinase C induces rapid internalization and subsequent degradation of muscarinic acetylcholine receptors in neuroblastoma cells. J. Biol. Chem. 261:5307–5313.

    Google Scholar 

  15. Cioffi, C. L., and Fisher, S. K. 1990. Reduction of muscarinic receptor density and of guanine nucleotide-stimulated phosphoinositide hydrolysis in human SH-SY5Y neuroblastoma cells following long-term treatment with 12-O-tetradecanoylphorbol 13-acetate or mezerein. J. Neurochem. 54:1725–1734.

    Google Scholar 

  16. Sierra, M., Smith, T. L., and Yamamura, H. J. 1986. Phorbol esters alter muscarinic receptor binding and inhibit polyphosphoinositide breakdown in human neuroblastoma (SH-SY5Y) cells. Biochem. Biophys. Res. Comm. 140:160–166.

    Google Scholar 

  17. Pediconi, M. F., and Barrantes, F. J. 1993. Phospholipid metabolism under muscarinic cholinergic stimulation exhibits brain asymmetry. Neurochem. Res. 18:559–563.

    Google Scholar 

  18. Farooqui, A. A., Farooqui, T., Yates, A. J., and Horrocks, L. A. 1988. Regulation of protein kinase C by various lipids. Neurochem. Res. 13:499–511.

    Google Scholar 

  19. Watson, M., Yamamura, H. Y., and Roeske, W. R. 1983. A unique regulatory profile and regional distribution of [3H]pirenzepine binding in the rat provide evidence for distinct M1 and M2 muscarinic receptor subtypes. Life Sci. 32:3001–3011.

    Google Scholar 

  20. Taylor, J. E., Yaksh, T. L., and Richelson, E. 1982. Agonist regulation of muscarinic acetylcholine receptors in rat spinal cord. J. Neurochem. 39:521–527.

    Google Scholar 

  21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. S. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.

    Google Scholar 

  22. Labarca, R., Janoswky, A., Patel, J., and Paul, S. M. 1984. Phorbol esters inhibit agonist-induced [3H]inositol-1-phosphate accumulation in rat hippocampal slices. Biochim. Biophys. Res. Comm. 123:703–709.

    Google Scholar 

  23. Fisher, S. K., and Agranoff, B. W. 1980. Calcium and the musuarinic synaptosomal phospholipid labelling effect. J. Neurochem. 34:1231–1240.

    Google Scholar 

  24. Fisher, S. K., and Agranoff, B. W. 1981. Enhancement of the muscarinic synaptosomal phospholipid labelling effect by ionophore A23187. J. Neurochem. 37:968–977.

    Google Scholar 

  25. Berridge, M. 1984. Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220:345–360.

    Google Scholar 

  26. Van Rooijen, L. A. A., and Traber, J. 1986. Muscarinic cholinergic enhancement of inositide turnover in cerebral nerve endings is not mediated by calcium uptake. Biochem. Pharmacol. 35:2715–2720.

    Google Scholar 

  27. Eichberg, J., de Graan, P. N. E., Shrama, L. H., and Gispen, W. H. 1986. Dioctanoylglycerol and phorboldiesters enhance phosphorylation of phosphoprotein B-50 in native synaptic plasma membrane. Biochem. Biophys. Res. Commun. 136:1007–1012.

    Google Scholar 

  28. Dekker, L. V., de Graan, P. N. E., De Wit, M., Hens, J. J. H., and Gispen, W. H. 1990. Depolarization-induced-phosphorylation of the protein B-50 (GAP-43) in rat cortical synaptosomes. J. Neurochem. 54:1645–1652.

    Google Scholar 

  29. Welsh, C. J., and Cabot, M. C. 1987. Second messenger diacylglycerols and analogs: metabolic fate in cell culture. Pages 225–235, in Mechanisms of signal transduction by hormones and growth factors, Alan Liss Inc., New York.

    Google Scholar 

  30. Chandler, L. J., and Crews, F. T. 1990. Calcium-versus G proteinmediated phosphoinositide hydrolysis in rat cerebral cortical synaptoneurosomes. J. Neurochem. 55:1022–1030.

    Google Scholar 

  31. Boess, F. G., Balasubramanian, M. K., Brammer, M. J., and Campbell, I. C. 1990. Stimulation of muscarinic acetylcholine receptors increases synaptosomal free calcium concentration by protein kinase dependent opening of L-type calcium channels. J. Neurochem. 55:230–236.

    Google Scholar 

  32. Anderson, C. W. 1993. DNA damage and the DNA-activated protein kinase. Trends Biochem. Sci. 18:433–437.

    Google Scholar 

  33. Hug, H. and Sarre T. F. 1993. Protein kinase C isoenzymes: divergence in signal transduction? Biochem. J. 291:329–343.

    Google Scholar 

  34. Dekker, L. V., and Parker P. J. 1994. Protein kinase C—a question of specificity. Trends Biochem. Sci. 19:73–77.

    Google Scholar 

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  1. Instituto de Investigaciones Bioquímicas, Consejo Nacional de Investigaciones Científicas y Técnicas and Universidad Nacional del Sur, C. C. 857, 8000, Bahía Blanca, Argentina

    M. F. Pediconi & F. J. Barrantes

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  1. M. F. Pediconi
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Pediconi, M.F., Barrantes, F.J. Down-regulation of brain muscarinic cholinergic receptor promoted by diacylglycerols and phorbol ester. Neurochem Res 20, 1225–1231 (1995). https://doi.org/10.1007/BF00995387

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