title: Comparison of subcellular distribution and functions between exogenous and endogenous M1 muscarinic acetylcholine receptors.
abstract: OBJECTIVE
  Recombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors.
  
  
  METHODS
  Recombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7).
  
  
  RESULTS
  M1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca(2+) mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca(2+) mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs.
  
  
  CONCLUSIONS
  Exogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin.
authors.affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Morishima
authors.affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan.
authors.forename: Abu Syed Md
authors.initials: ASM
authors.lastname: Anisuzzaman
authors.affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan; Organization for Life Science Advancement Programs, Graduate School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan.
authors.forename: Junsuke
authors.initials: J
authors.lastname: Uwada
authors.affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan.
authors.forename: Hatsumi
authors.initials: H
authors.lastname: Yoshiki
authors.affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan; Organization for Life Science Advancement Programs, Graduate School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan; Child Development Research Center, Graduate School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan. Electronic address: muramatu@u-fukui.ac.jp.
authors.forename: Ikunobu
authors.initials: I
authors.lastname: Muramatsu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001285:Atropine; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D051379:Mice; D018613:Microscopy, Confocal; D019832:N-Methylscopolamine; D015388:Organelles / Q000378:metabolism*; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014316:Tritium
chemical_list: D043643:Receptor, Muscarinic M1; D011994:Recombinant Proteins; D014316:Tritium; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D001285:Atropine; D002118:Calcium; D019832:N-Methylscopolamine