title: Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover.
abstract: (-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems.
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001570:Benzodiazepinones / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D010890:Pirenzepine; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000032:analysis / Q000378:metabolism*; D014316:Tritium
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D001570:Benzodiazepinones; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D014316:Tritium; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D001285:Atropine; D002217:Carbachol; D000242:Cyclic AMP