The effects of brief incubation with
carbamylcholine on subsequent binding of [3H]
N-methylscopolamine were investigated in mouse
neuroblastoma cells (clone N1E-115). This treatment demonstrated that the
muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both
pirenzepine and
carbamylcholine interacted with high- and low-affinity subsets of
muscarinic receptors. Computer-assisted analysis of the competition between
pirenzepine and
carbamylcholine with [3H]
N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of
pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various
muscarinic receptor agonists to induce a decrease in consequent [3H]
N-methylscopolamine binding and their efficacy in stimulating
cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in
cyclic GMP levels were capable of diminishing
ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the
pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of
muscarinic receptor-mediated
cyclic GMP formation.