Identification of rat brain muscarinic M4 receptors coupled to cyclic AMP using the selective antagonist muscarinic toxin 3

In membranes of olfactory tubercle and striatum, the selective muscarinic M4 receptor antagonist muscarinic toxin 3 completely antagonized the acetylcholine-induced inhibition of forskolin- and dopamine D1 receptor-stimulated cyclic AMP formation with Ki values of 7 and 4 nM, respectively. In olfactory bulb, where acetylcholine stimulated basal adenylyl cyclase activity and inhibited forskolin-stimulated enzyme activity, muscarinic toxin 3 caused a partial antagonism of both acetylcholine effects with high potencies (Ki values = 4-6 nM). In frontal cortex, muscarinic toxin 3 counteracted the acetylcholine-induced potentiation of corticotropin-releasing hormone-stimulated cyclic AMP with a Ki of 58 nM, which is close to the toxin affinity for the muscarinic M1 receptor. In the same brain region, the acetylcholine inhibition of forskolin-stimulated enzyme activity was not affected by muscarinic toxin 3. In microdissected regions of the hippocampus, a significant portion (33-48%) of the acetylcholine inhibition of forskolin-stimulated adenylyl cyclase activity was blocked by muscarinic toxin 3 with Ki values (6-8 nM) consistent with the involvement of muscarinic M4 receptors. These data show that muscarinic toxin 3 discriminates between adenylyl cyclase-coupled muscarinic receptors and demonstrate the utility of the toxin in identifying the relative contribution by the muscarinic M4 receptor subtype.