Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115): Cyclic GMP formation is mediated by a low affinity agonist-receptor conformation and cyclic AMP reduction is mediated by a high affinity agonist-receptor conformation

Murine neuroblastoma cells (clone N1E-115) possess two subtypes of the muscarinic receptor each of which separately mediates a cyclic nucleotide response. The formation of cyclic GMP is postulated to involve a low affinity agonist-receptor conformation, whereas the reduction of prostaglandin E₁-stimulated cyclic AMP formation appears to involve a high affinity conformation. Further evidence supporting this hypothesis was obtained in experiments measuring the equilibrium dissociation constants for the full agonist carbachol by the method of partial receptor inactivation. Quinuclidinyl benzilate (QNB) was employed to occlude muscarinic receptors; measurements with [³H] QNB ensured that the amount of QNB appearing in the assay after washout had only a minimal effect on the determination of the equilibrium dissociation constants. Carbachol mediated cyclic GMP formation with an equilibrium dissociation constant (K(D)) of 325 μM and cyclic AMP reductions with a K(D) value of 13 μM. These K(D) values are similar to but somewhat higher than those determined by direct binding at 15°, and they are strong evidence in support of the view that a low affinity conformation mediates cyclic GMP formation, whereas a highly affinity conformation mediates cyclic AMP reductions.