Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Selective loss with subculturing of the low-affinity agonist site mediating cyclic GMP formation

Cells of the murine neuroblastoma clone N1E-115 possess muscarinic receptors that influence the intracellular level of cyclic nucleotides. The stimulation of [³H]cyclic GMP levels occurs only with intact cells and has an EC₅₀ near the 'low-affinity' agonist equilibrium dissociation constant (K(L)) determined by radioligand binding assays. The inhibition of prostaglandin E₁-stimulated [³H]cyclic AMP formation has an EC₅₀ close to the value for the 'high-affinity' agonist equilibrium dissociation constant (K(H)). During sequential subculturing in medium supplemented with newborn bovine serum, the inhibition of [³H]cyclic AMP was maintained, but the [³H]cyclic GMP response declined dramatically, and after 7 subculturings it was essentially absent. The time course for [³H]cyclic GMP formation in a late subculture with an 88% loss of the response was identical with the time course in early subcultures. A normal [³H]cyclic GMP response to bradykinin and histamine was demonstrated to be present in cells that had lost the [³H]cyclic GMP response to carbachol. The EC₅₀ and K(D) values for the two muscarinic responses and binding sites increased 3- to 4-fold after several subculturings. A 90% loss of low-affinity binding sites was closely correlated with a similar loss of the [³H]cyclic GMP response. High-affinity binding sites did not decline significantly in concentration until the 11th subculture, where the total number of muscarinic sites was only 6% of the earliest subculture. In all subcultures, however, the ability of the muscarinic receptor to decrease [³H]cyclic AMP levels was maintained. These data, which show that the subculturing of N1E-115 cells in medium supplemented with newborn calf serum results in a selective loss of one muscarinic function, strongly support the hypothesis that these cells contain two separate muscarinic receptor-effector systems. One receptor subtype or conformation has a low affinity for the agonist and mediates cyclic GMP formation. The other receptor subtype or conformation has a higher affinity for the agonist and mediates an inhibition of prostaglandin E₁-stimulated cyclic AMP formation.