PHARMACOLOGICAL CHARACTERIZATION OF GUANINE-NUCLEOTIDE EXCHANGE-REACTIONS IN MEMBRANES FROM CHO CELLS STABLY TRANSFECTED WITH HUMAN MUSCARINIC RECEPTORS M1-M4

We have studied muscarinic agonist stimulated [S-35]GTPgammaS binding and [gammaP-32]GTP hydrolysis (GTPase) in membranes from CHO cells stably transfected with human muscarinic m1-m4 receptors. 'Full' agonists were at least 10-fold more potent at m2 & m4 receptors than at ml & m3. This pattern was less marked with 'partial' agonists, which had a greater maximal effect at m2 & m4 than at m1 & m3. McN-A343 uniquely was more potent and efficacious at m4 than at m2 receptors, Antagonist affinity constants were estimated by fitting the data from inhibition curves directly to the Schild model. Antagonist affinity estimates were very similar to those measured earlier in binding studies using animal tissues, and confirmed a small degree of m4 selectivity for tropicamide and secoverine. The receptor subtypes activated more than one G-protein subtype, m2 & m4 receptors activated only pertussis (PTX) sensitive G-proteins, while ml & m3 coupled to both PTX sensitive and insensitive G-proteins. Acetylcholine (ACh) was more potent in stimulating guanine nucleotide exchange in PTX treated ml cells than in controls.