Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line

Ildiko Kovacs, Henry I. Yamamura, Sue L. Waite, Eva V. Varga, William R Roeske

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Abstract

The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with K(d) values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and B(max) values of 97 ± 4 and 338 ± 16 fmol/106 cells, respectively. Inhibition of [3H](-)-N- methyl-3-quinuclidlnyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11- dihidro-6H-pyrido-[2,3-b](1,4)-benzodiazepine-6-one > hexahydro-sila- difenidolhydrochloride > pirenzepine. Correlation analysis of the pK(l) values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin- stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 μM (0.2-2.8) and 1.1 μM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2- B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms.

Original languageEnglish (US)
Pages (from-to)500-507
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume284
Issue number2
StatePublished - Feb 1998

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Muscarinic M2 Receptors
Fibroblasts
Pharmacology
Cell Line
Genes
Carbachol
Dexetimide
Benzilates
Pirenzepine
Colforsin
Inositol
Atropine
Pharmaceutical Preparations
Ligands
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Pharmacology

Cite this

Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line. / Kovacs, Ildiko; Yamamura, Henry I.; Waite, Sue L.; Varga, Eva V.; Roeske, William R.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 284, No. 2, 02.1998, p. 500-507.

Research output: Contribution to journalArticle

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abstract = "The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with K(d) values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and B(max) values of 97 ± 4 and 338 ± 16 fmol/106 cells, respectively. Inhibition of [3H](-)-N- methyl-3-quinuclidlnyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11- dihidro-6H-pyrido-[2,3-b](1,4)-benzodiazepine-6-one > hexahydro-sila- difenidolhydrochloride > pirenzepine. Correlation analysis of the pK(l) values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin- stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 μM (0.2-2.8) and 1.1 μM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2- B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms.",
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AU - Roeske, William R

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