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 language||English (US)|
|Number of pages||8|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - Feb 1998|
ASJC Scopus subject areas
- Molecular Medicine