We have carried out an extensive pharmacological characterization of
muscarinic binding sites in rabbit lung and chicken heart in parallel with M1, M2, and M3 sites, [3H]
Pirenzepine, a selective antagonist at M1 receptors, bound saturably and reversibly to membranes from chicken heart and rabbit lung. These binding sites were not M1 receptors, however, because the cardioselective antagonist
himbacine had 10-fold higher affinity at these sites than at [3H]
pirenzepine sites in rat and rabbit cortex (true M1 sites). We measured the inhibitory potency of 28 antagonists at [3H]
N-methylscopolamine-labeled sites in chicken heart, rabbit lung, rat heart (M2 sites), and rat submandibular gland (M3 sites) and at M1 sites in rat cortex. The sites in rabbit lung were different from M1, M2, and M3 sites, because they had moderate to high affinity for M1-selective compounds (
pirenzepine and
telenzepine), M2-selective compounds (
himbacine and
methoctramine), and M3-selective compounds (hexahydrosiladifenidol and 4-diphenylacetoxy-N-methylpiperidine methiodide). The sites in chicken heart resembled most those in rabbit lung, with similar high affinity for
secoverine, but they were not the same because
tropicamide, diphenylacetoxybutynyl
dimethylamine, and [3H]-
N-methylscopolamine were more potent in rabbit lung. In a further series of experiments, we compared the affinity of six of the most discriminating antagonists in membranes from rabbit lung and NG108-15 cells, a
neuroblastoma-
glioma cell line reported to express the
muscarinic m4 receptor gene. The antagonists had very similar affinities in the two tissues, the largest discrepancy being that
pirenzepine was twice as potent in rabbit lung as in NG108-15 cells. Northern blots using probes designed to discriminate between five species of
muscarinic receptor RNA detected only m4
mRNA in rabbit lung. We conclude that rabbit lung contains a
muscarinic M4 binding site with a quite distinctive pharmacology and that chicken heart contains a receptor with similarities to the M4 sites. This is the first report to characterize native M4 binding sites in a nonneuronal mammalian tissue.