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 (KL) 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 (KH). 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 KD 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.
D000818:Animals; D001667:Binding, Competitive; D001920:Bradykinin / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002999:Clone Cells; D006152:Cyclic GMP / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D019832:N-Methylscopolamine; D009447:Neuroblastoma / Q000378:metabolism*; D010276:Parasympatholytics / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism
D010276:Parasympatholytics; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D011813:Quinuclidinyl Benzilate; D006632:Histamine; D002217:Carbachol; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D001920:Bradykinin; D019832:N-Methylscopolamine