Autoradiographic analyses of agonist binding to muscarinic receptor subtypes

The binding of four muscarinic receptor agonists to regions of rat brain was examined through quantitative autoradiographic techniques. Oxotremorine, arecoline, pilocarpine and bethanechol were chosen based on their different potencies and efficacies in muscarinic second messenger systems. Overall, the order of potency for inhibition of [ 3 H]-l- quinuclidinyl benzilate ([ 3 H]-l- QNB) binding to rat brain slices was oxotremorine > pilocarpine = arecoline > bethanecol. Regional assays of agonist potency indicated that all agonists were more selective for brainstem and thalamic regions than for hippocampal and cortical regions. The high selectivity of agonists for areas such as the paraventricular thalamus and the superior colliculus, which also display low affinity for pirenzepine, suggests that muscarinic agonists bind with higher affinity to M 2 receptors. Of the four agonists examined, pilocarpine displayed the lowest selectivity for M 2 receptors in that ic 50 values for pilocarpine were only 3-fold higher in the hippocampal and striatal regions (e.g. CA3: 40.6 ± 9.4 μM) than in thalamic and brainstem regions (e.g. paraventricular thalamus: 14.9 ± 6.2 μM). Oxotremorine was 8-fold more potent in the brainstem and thalamus, while arecoline and bethanechol were, respectively, 19- and 100-fold more selective for brainstem and thalamic receptors. Scatchard analyses revealed heterogeneous binding profiles for some agonists within single brain regions, suggesting that multiple agonist sites exist even within regions of predominately M 1 or M 2 receptors. For example, arecoline displayed curved Scatchard plots within the external layers of the cerebral cortex, layer CA1 of the hippocampus (predominantly M 1 subtype), and the paraventricular thalamus (predominantly M 2 subtype). The ability of agonists to recognize multiple sites within a single region may reflect the ability to recognize receptors coupled or uncoupled to second messenger systems through G-proteins