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 withKd values of 243 pM (155–352 pM) and 345 pM (234–539 pM) and Bmax values of 97 ± 4 and 338 ± 16 fmol/106 cells, respectively. Inhibition of [3H](−)-N-methyl-3-quinuclidinyl 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-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi 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.
Footnotes
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Send reprint requests to: William R. Roeske, M.D., Department of Medicine, The University of Arizona, Health Sciences Center, Tucson, AZ 85724.
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↵1 This work was supported by Grant HL20984 from the National Institutes of Health.
- Abbreviations:
- MQNB
- N-methyl-3-quinuclidinyl benzilate
- EC50
- half-effective concentration
- Emax
- maximal effective concentration
- IC50
- half-inhibitory concentration
- Kd
- dissociation constant
- Bmax
- maximal binding
- Ki
- inhibition constant
- pKi
- negative logarithm ofKi
- nH
- Hill coefficient
- IBMX
- 3-isobutyl-methylxanthine
- PI
- phosphoinositide
- IP1
- inositol monophosphate
- CHO
- Chinese hamster ovary
- IMDM
- Iscove’s modified Dulbecco’s medium
- PZ
- pirenzepine
- 4-DAMP
- 4-diphenylacetoxy-N-methylpiperidine methiodide
- mAChR
- muscarinic acetylcholine receptor
- HHSiD
- hexahydro-sila-difenidol hydrochloride
- PCR
- polymerase chain reaction
- dNTP
- deoxynucleotidetriphosphate
- ANOVA
- analysis of variance
- Received July 7, 1997.
- Accepted October 22, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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