Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells

L. Mei, J. Lai, W. R. Roeske, C. M. Fraser, J. C. Venter, H. I. Yamamura

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Abstract

The muscarinic receptors in a B82 cell line which were transfected with the rat m1 muscarinic receptor gene (cTB10 cells) were studied by using radioligand binding assays. Their possible coupling to the hydrolysis of inositol lipids and cyclic AMP formation were also investigated. [(-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] binding to the intact cTB10 cells was saturable and displaceable by 1 μM atropine sulfate. The K(d) and maximum binding values of (-)-[3H]QNB from saturation studies were 12 pM and 17 fmol/106 cells, respectively. Inhibition studies of (-)-[3H]QNB binding to intact cTB10 cells suggested that these muscarinic receptors are of the M1 type defined by their high affinity for pirenzepine and low affinity for AF-DX 116 {11-[2-diethylamino methyl-1-piperidinylacetyl]-5,11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepine-6-one}. The muscarinic agonist carbachol stimulated [3H]inositol monophosphate accumulation in the cTB10 cells, which could be reversed by the muscarinic antagonists atropine, pirenzepine or AF-DX 116. The rank order of potency of the muscarinic antagonists in inhibiting carbachol-stimulated [3H]inositol monophosphate accumulation was atropine > pirenzepine > AF-DX 116, in agreement with that from ligand/(-)-[3H]QNB competition experiments. Pertussis toxin and 4β-phorbol,12-β-myristate, 13-α-acetate reduced carbachol-stimulated [3H]inositol monophosphate accumulation. Prostaglandin E1 stimulated cyclic AMP formation in the cTB10 cells. Carbachol at the concentration of 10 mM exhibited no stimulatory or inhibitor effect on the basal or prostaglandin E1-stimulated cyclic AMP formation. These results suggest that the muscarinic receptors encoded by the transfected m1 gene in the cTB10 cells are of the M1 type and are coupled to the hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein.

Original languageEnglish (US)
Pages (from-to)661-670
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Volume248
Issue number2
StatePublished - Jan 1 1989

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Muscarinic M1 Receptors
Fibroblasts
Inositol
Pharmacology
Carbachol
Pirenzepine
Atropine
Cyclic AMP
Muscarinic Antagonists
Alprostadil
Pertussis Toxin
Muscarinic Receptors
Hydrolysis
Quinuclidinyl Benzilate
Lipids
Muscarinic Agonists
Radioligand Assay
GTP-Binding Proteins
Genes
Acetates

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

Mei, L., Lai, J., Roeske, W. R., Fraser, C. M., Venter, J. C., & Yamamura, H. I. (1989). Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells. Journal of Pharmacology and Experimental Therapeutics, 248(2), 661-670.

Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells. / Mei, L.; Lai, J.; Roeske, W. R.; Fraser, C. M.; Venter, J. C.; Yamamura, H. I.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 248, No. 2, 01.01.1989, p. 661-670.

Research output: Contribution to journalArticle

Mei, L, Lai, J, Roeske, WR, Fraser, CM, Venter, JC & Yamamura, HI 1989, 'Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells', Journal of Pharmacology and Experimental Therapeutics, vol. 248, no. 2, pp. 661-670.
Mei, L. ; Lai, J. ; Roeske, W. R. ; Fraser, C. M. ; Venter, J. C. ; Yamamura, H. I. / Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells. In: Journal of Pharmacology and Experimental Therapeutics. 1989 ; Vol. 248, No. 2. pp. 661-670.
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abstract = "The muscarinic receptors in a B82 cell line which were transfected with the rat m1 muscarinic receptor gene (cTB10 cells) were studied by using radioligand binding assays. Their possible coupling to the hydrolysis of inositol lipids and cyclic AMP formation were also investigated. [(-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] binding to the intact cTB10 cells was saturable and displaceable by 1 μM atropine sulfate. The K(d) and maximum binding values of (-)-[3H]QNB from saturation studies were 12 pM and 17 fmol/106 cells, respectively. Inhibition studies of (-)-[3H]QNB binding to intact cTB10 cells suggested that these muscarinic receptors are of the M1 type defined by their high affinity for pirenzepine and low affinity for AF-DX 116 {11-[2-diethylamino methyl-1-piperidinylacetyl]-5,11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepine-6-one}. The muscarinic agonist carbachol stimulated [3H]inositol monophosphate accumulation in the cTB10 cells, which could be reversed by the muscarinic antagonists atropine, pirenzepine or AF-DX 116. The rank order of potency of the muscarinic antagonists in inhibiting carbachol-stimulated [3H]inositol monophosphate accumulation was atropine > pirenzepine > AF-DX 116, in agreement with that from ligand/(-)-[3H]QNB competition experiments. Pertussis toxin and 4β-phorbol,12-β-myristate, 13-α-acetate reduced carbachol-stimulated [3H]inositol monophosphate accumulation. Prostaglandin E1 stimulated cyclic AMP formation in the cTB10 cells. Carbachol at the concentration of 10 mM exhibited no stimulatory or inhibitor effect on the basal or prostaglandin E1-stimulated cyclic AMP formation. These results suggest that the muscarinic receptors encoded by the transfected m1 gene in the cTB10 cells are of the M1 type and are coupled to the hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein.",
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