Computer-assisted analysis of the pirenzepine competition binding curves yielded the amount of high affinity (M1) and low affinity sites (M2) as well as their respective K_i value for this ligand.

The neocortex contained a mixed population of 67% M1 and 33% M2 sites, without locoregional heterogeneity. The muscarinic receptors of the caudate nucleus, putamen, pallidum, hippocampus and nucleus amygdalis were predominantly of the M1 type as well. The cerebellum and the brain-stem are examples of regions containing over 90% M2 sites. White matter structures like the centrum ovale appeared to contain low concentrations of [³H]QNB binding sites, predominantly of the M1 subtype.

The data for the frontal cortex were compared with data obtained in 3 patients who died from Alzheimer's disease with very early onset: the muscarinic receptor concentrations were somewhat lower but their M1/M2 ratio remained unchanged.

GTP caused an appreciable rightward shift and steepening of the carbachol competition binding curve in M2 predominant regions such as the pons, whereas only a slight shift was observed in the cortex. In the presence of GTP, the alkylating reagent N-ethylmaleimide caused a 35-fold increase of the affinity for carbachol in M2 predominant regions. In contrast, regions where the receptors are predominantly of the M1 type, N-ethylmaleimide caused only a 5-fold increase in agonist affinity. These findings confirm our previously formulated hypothesis that the ability of N-ethylmaleimide to modulate the agonist affinity is an additional criterion for the characterization of M1 and M2-type receptors.