Inverse agonist activity of pirenzepine at M 2 muscarinic acetylcholine receptors

The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M 2 receptors in pig atrial sarcolemma. Competition binding experiments were performed with [ 3 H]‐oxotremorine‐M to assess the affinity of receptors coupled to G proteins (R*), with [ 3 H]‐N‐methylscopolamine ([ 3 H]‐NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [ 3 H]‐NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). The ranking of K i values for the agonist carbachol was R*<R*+R>R (174, 155, 115 n M ), suggesting inverse agonism. The V max of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt‐2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K M value (0.26–0.33 μ M ) was not modified by mastoparan or GPAnt‐2. Carbachol increased the V max of GTP hydrolysis (EC 50 8.1±0.3 μ M ), whereas atropine and AF‐DX 116, up to 1 m M , did not modify it. Pirenzepine decreased the V max of GTP hydrolysis (EC 50 77.5±10.3 μ M ). This effect was enhanced when KCl was substituted for NaCl (EC 50 11.0±0.8 μ M ) and was antagonized by atropine and AF‐DX 116 (IC 50 0.91±0.71 and 197±85 n M ). Pirenzepine is proposed as an inverse agonist and atropine and AF‐DX 116 as neutral antagonists at the muscarinic M 2 receptor.British Journal of Pharmacology (1999) 126 , 1246–1252; doi: 10.1038/sj.bjp.0702407