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

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.