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. 1992 Jun 1;89(11):5035–5038. doi: 10.1073/pnas.89.11.5035

Regulation of muscarinic acetylcholine receptor mRNA expression by activation of homologous and heterologous receptors.

B A Habecker 1, N M Nathanson 1
PMCID: PMC49223  PMID: 1594610

Abstract

Muscarinic acetylcholine receptors (mAChR) in the embryonic chicken heart undergo agonist-induced internalization and a subsequent decrease in receptor number (downregulation). Cloning studies have identified two subtypes of mAChR expressed in the embryonic chicken heart, the cm2 and cm4 receptors. We report here that persistent activation of the mAChR in cultured chicken heart cells with the cholinergic agonist carbachol causes significant decreases in the levels of both cm2 and cm4 mRNA, as measured by solution hybridization analyses. The half-lives of the cm2 and cm4 mRNAs are not altered by agonist treatment, indicating that agonist most likely regulates mRNA levels by regulating the rate of gene transcription. Activation of mAChR in chicken heart causes both inhibition of adenylate cyclase activity and stimulation of phospholipase C activity. To test whether changes in the levels of intracellular second messengers were involved in the changes in mAChR mRNAs observed following agonist exposure, we determined the effects of incubation with agonists for the A1 adenosine receptors (which inhibit adenylate cyclase in chicken heart) and angiotensin II receptors (which stimulate phospholipase C) on mAChR receptor number and mRNA levels. Activation of these pathways together through heterologous receptors resulted in decreased mAChR number and mRNA levels, although these changes were not as large as those seen with direct activation of the mAChR. These results suggest that regulation of adenylate cyclase and phospholipase C activities may be involved in the regulation of mAChR gene expression.

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Selected References

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  1. Baker K. M., Aceto J. A. Characterization of avian angiotensin II cardiac receptors: coupling to mechanical activity and phosphoinositide metabolism. J Mol Cell Cardiol. 1989 Apr;21(4):375–382. doi: 10.1016/0022-2828(89)90648-2. [DOI] [PubMed] [Google Scholar]
  2. Belardinelli L., Vogel S., Linden J., Berne R. M. Antiadrenergic action of adenosine on ventricular myocardium in embryonic chick hearts. J Mol Cell Cardiol. 1982 May;14(5):291–294. doi: 10.1016/0022-2828(82)90207-3. [DOI] [PubMed] [Google Scholar]
  3. Blair T. A., Parenti M., Murray T. F. Development of pharmacological sensitivity to adenosine analogs in embryonic chick heart: role of A1 adenosine receptors and adenylyl cyclase inhibition. Mol Pharmacol. 1989 May;35(5):661–670. [PubMed] [Google Scholar]
  4. Bonner T. I., Buckley N. J., Young A. C., Brann M. R. Identification of a family of muscarinic acetylcholine receptor genes. Science. 1987 Jul 31;237(4814):527–532. doi: 10.1126/science.3037705. [DOI] [PubMed] [Google Scholar]
  5. Bonner T. I., Young A. C., Brann M. R., Buckley N. J. Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron. 1988 Jul;1(5):403–410. doi: 10.1016/0896-6273(88)90190-0. [DOI] [PubMed] [Google Scholar]
  6. Bouvier M., Collins S., O'Dowd B. F., Campbell P. T., de Blasi A., Kobilka B. K., MacGregor C., Irons G. P., Caron M. G., Lefkowitz R. J. Two distinct pathways for cAMP-mediated down-regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level. J Biol Chem. 1989 Oct 5;264(28):16786–16792. [PubMed] [Google Scholar]
  7. Collins S., Altschmied J., Herbsman O., Caron M. G., Mellon P. L., Lefkowitz R. J. A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP. J Biol Chem. 1990 Nov 5;265(31):19330–19335. [PubMed] [Google Scholar]
  8. Collins S., Bouvier M., Bolanowski M. A., Caron M. G., Lefkowitz R. J. cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4853–4857. doi: 10.1073/pnas.86.13.4853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Durnam D. M., Palmiter R. D. A practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem. 1983 Jun;131(2):385–393. doi: 10.1016/0003-2697(83)90188-4. [DOI] [PubMed] [Google Scholar]
  10. Feigenbaum P., El-Fakahany E. E. Regulation of muscarinic cholinergic receptor density in neuroblastoma cells by brief exposure to agonist: possible involvement in desensitization of receptor function. J Pharmacol Exp Ther. 1985 Apr;233(1):134–140. [PubMed] [Google Scholar]
  11. Fong H. K., Hurley J. B., Hopkins R. S., Miake-Lye R., Johnson M. S., Doolittle R. F., Simon M. I. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2162–2166. doi: 10.1073/pnas.83.7.2162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fujimoto J., Gershengorn M. C. Evidence for dual regulation by protein kinases A and C of thyrotropin-releasing hormone receptor mRNA in GH3 cells. Endocrinology. 1991 Dec;129(6):3430–3432. doi: 10.1210/endo-129-6-3430. [DOI] [PubMed] [Google Scholar]
  13. Fujimoto J., Straub R. E., Gershengorn M. C. Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect. Mol Endocrinol. 1991 Oct;5(10):1527–1532. doi: 10.1210/mend-5-10-1527. [DOI] [PubMed] [Google Scholar]
  14. Fukamauchi F., Hough C., Chuang D. M. Expression and agonist-induced down-regulation of mRNAs of m2- and m3-muscarinic acetylcholine receptors in cultured cerebellar granule cells. J Neurochem. 1991 Feb;56(2):716–719. doi: 10.1111/j.1471-4159.1991.tb08210.x. [DOI] [PubMed] [Google Scholar]
  15. Galper J. B., Dziekan L. C., Miura D. S., Smith T. W. Agonist-induced changes in the modulation of K+ permeability and beating rate by muscarinic agonists in cultured heart cells. J Gen Physiol. 1982 Aug;80(2):231–256. doi: 10.1085/jgp.80.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Galper J. B., Smith T. W. Agonist and guanine nucleotide modulation of muscarinic cholinergic receptors in cultured heart cells. J Biol Chem. 1980 Oct 25;255(20):9571–9579. [PubMed] [Google Scholar]
  17. Hadcock J. R., Malbon C. C. Down-regulation of beta-adrenergic receptors: agonist-induced reduction in receptor mRNA levels. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5021–5025. doi: 10.1073/pnas.85.14.5021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hadcock J. R., Ros M., Malbon C. C. Agonist regulation of beta-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants. J Biol Chem. 1989 Aug 15;264(23):13956–13961. [PubMed] [Google Scholar]
  19. Halvorsen S. W., Nathanson N. M. In vivo regulation of muscarinic acetylcholine receptor number and function in embryonic chick heart. J Biol Chem. 1981 Aug 10;256(15):7941–7948. [PubMed] [Google Scholar]
  20. Hough C., Chuang D. M. Differential down-regulation of beta 1- and beta 2-adrenergic receptor mRNA in C6 glioma cells. Biochem Biophys Res Commun. 1990 Jul 16;170(1):46–52. doi: 10.1016/0006-291x(90)91238-n. [DOI] [PubMed] [Google Scholar]
  21. Hunter D. D., Nathanson N. M. Decreased physiological sensitivity mediated by newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3582–3586. doi: 10.1073/pnas.81.11.3582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Izzo N. J., Jr, Seidman C. E., Collins S., Colucci W. S. Alpha 1-adrenergic receptor mRNA level is regulated by norepinephrine in rabbit aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6268–6271. doi: 10.1073/pnas.87.16.6268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klein W. L., Nathanson N., Nirenberg M. Muscarinic acetylcholine receptor regulation by accelerated rate of receptor loss. Biochem Biophys Res Commun. 1979 Sep 27;90(2):506–512. doi: 10.1016/0006-291x(79)91264-6. [DOI] [PubMed] [Google Scholar]
  24. Kubo T., Fukuda K., Mikami A., Maeda A., Takahashi H., Mishina M., Haga T., Haga K., Ichiyama A., Kangawa K. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature. 1986 Oct 2;323(6087):411–416. doi: 10.1038/323411a0. [DOI] [PubMed] [Google Scholar]
  25. Kubo T., Maeda A., Sugimoto K., Akiba I., Mikami A., Takahashi H., Haga T., Haga K., Ichiyama A., Kangawa K. Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence. FEBS Lett. 1986 Dec 15;209(2):367–372. doi: 10.1016/0014-5793(86)81144-9. [DOI] [PubMed] [Google Scholar]
  26. McKnight G. S., Uhler M. D., Clegg C. H., Correll L. A., Cadd G. G. Application of molecular genetic techniques to the cAMP-dependent protein kinase system. Methods Enzymol. 1988;159:299–311. doi: 10.1016/0076-6879(88)59030-4. [DOI] [PubMed] [Google Scholar]
  27. Peppel K., Baglioni C. A simple and fast method to extract RNA from tissue culture cells. Biotechniques. 1990 Dec;9(6):711–713. [PubMed] [Google Scholar]
  28. Peralta E. G., Ashkenazi A., Winslow J. W., Smith D. H., Ramachandran J., Capon D. J. Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J. 1987 Dec 20;6(13):3923–3929. doi: 10.1002/j.1460-2075.1987.tb02733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peralta E. G., Winslow J. W., Peterson G. L., Smith D. H., Ashkenazi A., Ramachandran J., Schimerlik M. I., Capon D. J. Primary structure and biochemical properties of an M2 muscarinic receptor. Science. 1987 May 1;236(4801):600–605. doi: 10.1126/science.3107123. [DOI] [PubMed] [Google Scholar]
  30. Sakaue M., Hoffman B. B. cAMP regulates transcription of the alpha 2A adrenergic receptor gene in HT-29 cells. J Biol Chem. 1991 Mar 25;266(9):5743–5749. [PubMed] [Google Scholar]
  31. Subers E. M., Nathanson N. M. Muscarinic acetylcholine receptor function in chick heart cells cultured in serum-free medium. J Mol Cell Cardiol. 1988 Feb;20(2):131–140. doi: 10.1016/s0022-2828(88)80026-9. [DOI] [PubMed] [Google Scholar]
  32. Tietje K. M., Goldman P. S., Nathanson N. M. Cloning and functional analysis of a gene encoding a novel muscarinic acetylcholine receptor expressed in chick heart and brain. J Biol Chem. 1990 Feb 15;265(5):2828–2834. [PubMed] [Google Scholar]
  33. Tietje K. M., Nathanson N. M. Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity for pirenzepine. J Biol Chem. 1991 Sep 15;266(26):17382–17387. [PubMed] [Google Scholar]
  34. Wang S. Z., Hu J. R., Long R. M., Pou W. S., Forray C., el-Fakahany E. E. Agonist-induced down-regulation of m1 muscarinic receptors and reduction of their mRNA level in a transfected cell line. FEBS Lett. 1990 Dec 10;276(1-2):185–188. doi: 10.1016/0014-5793(90)80538-t. [DOI] [PubMed] [Google Scholar]
  35. Zhu S. Z., Wang S. Z., Abdallah E. A., el-Fakahany E. E. DFP-induced regulation of cardiac muscarinic receptor mRNA in vivo measured by DNA-excess solution hybridization. Life Sci. 1991;48(26):2579–2584. doi: 10.1016/0024-3205(91)90615-i. [DOI] [PubMed] [Google Scholar]

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