Antibodies selective for subtypes of muscarinic cholinergic receptors: Studies on receptor distribution and regulation

The availability of selective probes for each of the five genetically-defined muscarinic receptor proteins (m1-m5) would greatly facilitate studies on muscarinic receptor-mediated physiological responses. Towards that end, antisera were raised against unique domains of the m1 and m3 receptor. These antibodies were able to quantitatively immunoprecipitate m1 and m3 receptors, respectively, while not precipitating m2, m4 or m5 receptors. The distributions of m1 and m3 receptors in rat brain were determined by immunoprecipitation. The m1 subtype predominates in cortex, hippocampus and striatum, (constituting 30-50% of expressed receptors), whereas the m3 subtype is found at lower levels in these regions (5-12%). When used in conjunction with m2, m4, and m5 selective antisera, $\ge$90% of total receptors in brain were precipitated. In peripheral tissues the m2 muscarinic receptor predominates in smooth-muscle containing organs, while m3 receptors were found in glandular tissues. In addition, the m3 receptor is expressed at high levels in several human clonal cell lines. The regulation of muscarinic receptor subtypes was examined in three experimental paradigms. (1) The ontogenic patterns for muscarinic receptor subtypes in forebrain were similar, with receptor densities increasing three to four-fold in the initial 21 days postpartum. (2) Chronic antagonist was found to upregulate total muscarinic receptor density by 24%. The density of high-affinity pirenzepine sites (M1) did not change, while low affinity sites (M2) doubled in number. Interestingly, receptor subtypes which couple preferentially to phosphoinositide turnover were upregulated by 32%, while the density of receptors coupled to adenylyl cyclase inhibition increased by only 10%. (3) The receptor density in the dorsal hippocampus increased by 17% following fimbria-fornix lesioning. The m1 receptor density increased by 17%, m4 receptors by 29%, and m3 receptors by 77%. In agreement with its hypothesized role as an autoreceptor, the m2 subtype density decreased by 22%. The direction of these changes suggest that subtypes possess different pre- and postsynaptic locations. Cumulatively, these data confirm that muscarinic receptor subtypes are subject to differential regulation within the central nervous system