A unique regulatory profile and regional distribution of [3H]pirenzepine binding in the rat provide evidence for distinct M1 and M2 muscarinic receptor subtypes.

We recently demonstrated that the non-classical muscarinic receptor antagonist [3H]pirenzepine ([3H]PZ) identifies a high affinity population of muscarinic sites in the rat cerebral cortex. We now report that cortical muscarinic sites to which [3H]PZ binds with high affinity are modulated by ions but not guanine nucleotides. We also have examined equilibrium [3H]PZ binding in homogenates of various rat tissues using a new rapid filtration assay. All regional saturation isotherms yielded a similar high affinity dissociation constant (Kd = 2-8 nM) in 10 mM sodium-potassium phosphate buffer. Receptor density (Bmax in fmol/mg tissue) varied as follows: corpus striatum = 154.5, cerebral cortex = 94.6, hippocampus = 94.3, ileum = 1.3, cerebellum = 1.0, and heart = 0.45. The cerebral cortex and hippocampus possess 61 percent of striatal binding sites, while the ileum, cerebellum and heart contain only 0.84 percent, 0.65 percent and 0.29 percent of striatal sites respectively. The [3H]PZ sites in heart, ileum, and cerebellum represent 3.1 percent, 9.6 percent, and 10.4 percent of the sites obtained by using [3H] (-)quinuclidinyl benzilate. Thus, [3H]PZ labels high affinity muscarinic receptor binding sites with a tissue distribution compatible with the concept of distinct M1 and M2 receptor subtypes. Accordingly, regions such as heart, cerebellum, and ileum would be termed M2, though each have an extremely small population of the M1 high affinity [3H]PZ site. [3H]PZ therefore appears to be a useful ligand for M1 receptor identification. Furthermore, the inability to demonstrate a significant effect of guanine nucleotides upon high affinity [3H]PZ binding to putative M1 receptors suggests that M1 sites may be independent of a guanine regulatory protein.