title: Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells.
abstract: Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.
authors: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, and Organization for Life Science Advancement Programs, School of Medicine, University of Fukui, Eiheiji, Fukui, Japan.
authors: Junsuke
authors: J
authors: Uwada
authors: Abu Syed Md
authors: AS
authors: Anisuzzaman
authors: Atsushi
authors: A
authors: Nishimune
authors: Hatsumi
authors: H
authors: Yoshiki
authors: Ikunobu
authors: I
authors: Muramatsu
mesh_terms: D000818:Animals; D001285:Atropine / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D004546:Elapid Venoms / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D007150:Immunohistochemistry; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D019832:N-Methylscopolamine / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000494:pharmacology; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects
chemical_list: D004546:Elapid Venoms; D007295:Inositol Phosphates; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010456:Peptides, Cyclic; D047428:Protein Kinase Inhibitors; D043643:Receptor, Muscarinic M1; D011956:Receptors, Cell Surface; C475455:YM-254890; C419876:muscarinic toxin 7; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D001285:Atropine; D002217:Carbachol; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium; D019832:N-Methylscopolamine