Agonist-induced muscarinic acetylcholine receptor down-regulation in intact rat brain cells

Intact brain cells were prepared by dissociating whole adult rat brains without the cerebellum using a sieving technique. It has been found that preincubation of these cells with the muscarinic acetylcholine receptor agonist, carbamylcholine, results in a significant reduction in the specific binding of [³H]N-methylscopolamine to the receptors after the agonist was washed away. This agonist-mediated receptor down-regulation increased with prolongation of the exposure period to the agonist, and a steady state was achieved after 3 h at 37°C. This effect of agonist was concentration-dependent, reaching a 30-35% decline in subsequent ligand binding upon preincubation with 1 mM carbamylcholine for 3 h. Carbamylcholine-induced receptor down-regulation was not apparent when exposure to the agonist was performed at 15°C. In addition, it was abolished when the receptors were blocked by atropine. The decline in [³H]N-methylscopolamine binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that 'true' receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist [³H]quinuclidinyl benzilate instead of the hydrophilic ligand [³H]N-methylscopolamine, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggests that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded. These results are discussed in relation to previous findings regarding muscarinic receptor regulation in clonal cell lines.