Inverse agonist activity of pirenzepine at M-2 muscarinic acetylcholine receptors

1 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. 2 Competition binding experiments were performed with [H-3]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [H-3]- N-methylscopolamine ([H-3]-NMS) to estimate the affinities of coupled and u ncoupled receptors (R*+R) and with [H-3]-NMS in the presence of GppNHp to a ssess the affinity of uncoupled receptors (R). 3 The ranking of K-i values for the agonist carbachol was R*<<R*+R<<R (0.95 , 124 and 1017 nM). K-i values for atropine and AF-DX 116 were similar for the three binding conditions (0.34, 0.42, 0.41 and 19, 22, 32 nM). The rank ing of K-i values for pirenzepine was R*>R*+R>R (174, 155, 115 nM), suggest ing inverse agonism. 4 The V-max of the basal high affinity GTPase activity of pig atrial sarcol emma was increased by mastoparan and decreased by GPAnt-2 indicating the re levance of this activity to G proteins coupled to receptors (R*). The K-M v alue (0.26-0.33 mu M) was not modified by mastoparan or GPAnt-2. 5 Carbachol increased the V-max of GTP hydrolysis (EC50 8.1+/-0.3 mu M), wh ereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine de creased the V-max of GTP hydrolysis (EC50 77.5+/-10.3 mu M). This effect wa s enhanced when KCl was substituted for NaCl (EC50 11.0+/-0.8 mu M) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6 Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M-2 receptor.