Skip to main content
SpringerLink
Log in

Agonist-induced down-regulation of the m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels

  • Original Articles
  • Published:
Naunyn-Schmiedeberg's Archives of Pharmacology Aims and scope Submit manuscript

Abstract

The regulation of m4 muscarinic acetylcholine receptor mRNA expression by receptor activation was studied in NIE-115 neuroblastoma and AtT-20 pituitary cells that endogeneously express the m4 muscarinic receptor. Receptor concentration was measured by binding of the muscarinic receptor radioligand [3H]quinuclidinyl benzilate, and RNA-RNA solution hybridization/RNase protection assay with a m4 receptor-specific [32P]-cRNA probe was used to evaluate the levels of receptor mRNA.

Treatment of both cell lines with a receptor-saturating concentration of the agonist carbachol decreased receptor number. However, there was no change in steady-state levels of m4 mAChR mRNA in both cell lines. Determination of mRNA stability in the presence of the transcription blocker actinomycin D revealed that carbachol treatment increased half-life of receptor mRNA in N1E-115 cells, but not in AtT-20 cells, suggesting that receptor activation can regulate m4 receptor mRNA stability dependently on cell type. Analysis of receptor degradation kinetics in the presence of the protein synthesis inhibitor cycloheximide showed that receptor down-regulation in N1E-115 and AtT-20 cells is sufficiently accounted for by increased receptor degradation.

These results indicate than m4 muscarinic receptor down-regulation is substantially different from that of the muscarinic receptor subtypes m2 and m3 which is reported to be associated with agonist-induced reduction in receptor mRNA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akiyama K, Vickroy TW, Watson W, Roeske WR, Reisine TD, Smith TL, Yamamure HI (1986) Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16–16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover. J Pharmacol Exp Ther 236:653–661

    Google Scholar 

  • Ashkenazi A, Peralta EG, Winslow JW, Ramachandran J, Capon DJ (1988) Functional role of muscarinic acetylcholine receptor subtype diversity. Cold Spring Harbor Symp Quant Biol 53:263–272

    Google Scholar 

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current protocols in molecular biology. Green Publishing Associates and John Wiley & Sons, New York

    Google Scholar 

  • Buckley NJ, Bonner TI, Brann MR (1988) Localization of a family of muscarinic receptor mRNAs in rat brain. J Neurosci 8:4646–4652

    Google Scholar 

  • Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biological active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299

    CAS  PubMed  Google Scholar 

  • Collins S, Caron MG, Lefkowitz RJ (1988) β2-Adrenergic receptors in hamster smooth muscle cells are transcriptionally regulated by glucocorticoids. J Biol Chem 263:9067–9070

    Google Scholar 

  • Dörjé F, Levey AI, Brann MR (1991) Immunological detection of muscarinic receptor subtype proteins (m1–m5) in rabbit peripheral tissues. Mol Pharmacol 40:459–462

    Google Scholar 

  • Fukamauchi F, Saunders PA, Hough C, Chuang DM (1993) Agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells. Mol Pharmacol 44:940–949

    Google Scholar 

  • Fukuda K, Higashida H, Kubo T, Maeda A, Akiba I, Bujo H, Mishina M, Numa S (1988) Selective coupling with K+ currents of muscarinic acetylcholine receptor subtypes in NG108–15 cells. Nature 335:355–358

    Google Scholar 

  • Habecker BA, Nathanson NM (1992) Regulation of muscarinic acetylcholine receptor mRNA expression by activation of homologous and heterologous receptors. Proc Natl Acad Sci USA 89:5035–5038

    Google Scholar 

  • Habecker BA, Wang H, Nathanson NM (1993) Multiple second-messenger pathways mediate agonist regulation of muscarinic receptor mRNA expression. Biochemistry 32:4986–4990

    Google Scholar 

  • Hadcock JR, Wang H, Malbon CC (1989) Agonist-induced destabilization of β-adrenergic receptor mRNA. Attenuation of glucocorticoid-induced up-regulation of β-adrenergic-receptors. J Biol Chem 264:19928–19933

    Google Scholar 

  • Hook VYH, Heisler S, Sabol SL, Axelrod J (1980) Corticotropin releasing factor stimulates adrenocorticotropin and β-endorphin release from AtT-20 mouse pituitary tumor cells. Biochem Biophys Res Commun 106:1364–1371

    Google Scholar 

  • Hough C, Chuang DM (1990) Differential down-regulation of β1- and β2-adrenergic receptor mRNA in C6 glioma cells. Biochem Biophys Res Commun 170:46–52

    Google Scholar 

  • Jones SVP (1992) m4 Muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT20 cells. Neurosci Lett 147:125–130

    Google Scholar 

  • Levey AI, Kitt CA, Simonds WF, Price DL, Brann MR (1991) Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies. J Neurosci 11: 3218–3226

    Google Scholar 

  • Liles WC, Nathanson NM (1987) Regulation of muscarinic acetylcholine receptor number in cultured neuronal cells by chronic membrane depolarization. J Neurosci 7:2556–2563

    Google Scholar 

  • Maeda A, Kubo T, Mishina M, Numa S (1988) Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes. FEBS Lett 239:339–342

    Google Scholar 

  • McKinney M, Stenstrom S, Richelson E (1985) Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Mediation of separate responses by high affinity and low affinity agonist-receptor conformations. Mol Pharmacol 27:223–235

    Google Scholar 

  • McKinney M, Robbins M (1992) Chronic atropine administration up-regulates rat cortical muscarinic m1 receptor mRNA molecules: assessment with the RT/PCR. Mol Brain Res 12:39–45

    Google Scholar 

  • Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl Acids Res 12:7035–7056

    Google Scholar 

  • Nathanson NM (1987) Molecular properties of the muscarinic acetylcholine receptor. Ann Rev Neurosci 10:195–236

    Google Scholar 

  • Nathanson NM, Goldman PS, Habecker BA, Migeon JC, Morton ME, Tietje KM, Van Koppen CJ (1992) Analysis of muscarinic acetylcholine receptor expression and function. Methods Neurosci 9:116–134

    Google Scholar 

  • Peralta EG, Ashkenazi A, Winslow JW, Smith DH, Ramachandran J, Capon DJ (1987) Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J 6:3923–3929

    Google Scholar 

  • Peralta EG, Ashkenazi A, Winslow JW, Ramachandran J, Capon DJ (1988) Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature 334:434–437

    Google Scholar 

  • Schimke RT (1975) Methods for analysis of enzyme synthesis and degradation in animal tissues. Methods Enzymol 40:241–266

    Google Scholar 

  • Steel MC, Buckley NJ (1993) Differential regulation of muscarinic receptor mRNA levels in neuroblastoma cells by chronic agonist exposure: a comparitive polymerase chain reaction study. Mol Pharmacol 43:694–701

    Google Scholar 

  • Thomas RF, Holt BD, Schwinn DA, Liggett SB (1992) Long-term agonist exposure induces upregulation of β3-adrenergic receptor expression via multiple cAMP response elements. Proc Natl Acad Sci USA 89:4490–4494

    Google Scholar 

  • Van Koppen CJ, Nathanson NM (1990) Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function. J Biol Chem 265:20887–20892

    Google Scholar 

  • Van Koppen CJ, Lenz W, Nathanson NM (1993) Isolation, sequence and functional expression of the mouse m4 muscarinic acetylcholine receptor gene. Biochim Biophys Acta 1173:342–344

    Google Scholar 

  • Yasuda RP, Ciesla W, Flores LR, Wall SJ, Li M, Satkus SA, Weisstein JS, Spagnola BV, Wolfe BB (1993) Development of antisera selective for m4 and m5 muscarinic cholinergic receptors: distribution of m4 and m5 receptors in rat brain. Mol Pharmacol 43:149–157

    Google Scholar 

  • Zhao D, Yang J, Jones KE, Gerald C, Suzuki Y, Hogan PG, Chin WW, Tashjian AH (1992) Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells. Endocrinology 130:3529–3536

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Pharmakologie, Universität GH Essen, Hufelandstrasse 55, D-45122, Essen, Germany

    Wolfgang Lenz, Cornelia Petrusch, Karl H. Jakobs & Chris J. van Koppen

Authors
  1. Wolfgang Lenz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cornelia Petrusch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karl H. Jakobs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chris J. van Koppen
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Correspondence to: C. J. van Koppen at the above address

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lenz, W., Petrusch, C., Jakobs, K.H. et al. Agonist-induced down-regulation of the m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels. Naunyn-Schmiedeberg's Arch Pharmacol 350, 507–513 (1994). https://doi.org/10.1007/BF00173020

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00173020

Key words

Search

Navigation