KINETIC-STUDIES OF COOPERATIVITY AT ATRIAL MUSCARINIC M(2) RECEPTORS WITH AN INFINITE DILUTION PROCEDURE

The effects of two competitive antagonists and two allosteric ligands on the rate of dissociation of [H-3]N-methylscopolamine ([H-3]NMS) wer e studied at atrial muscarinic acetylcholine M(2) receptors by the tec hnique of ''infinite dilution.'' The dissociation rate for [H-3]NMS, i nitiated by diluting the incubation mixture in a 100-fold volume of bu ffer, was 0.61 +/- 0.10 min(-1). Addition of the competitive antagonis ts, atropine or NMS, to the dilution medium did not alter the observed [H-3]NMS dissociation rate. In contrast, gallamine and the bisquatern ary, ne-1,7-bis-(dimethyl-3'-phthalimidopropyl-ammonium bromide) (C-7/ 3'-phth), produced a concentration-dependent slowing of the dissociati on rate of [H-3]NMS, with IC50 values of 7.5 mu M and 196 nM, respecti vely. Gallamine exhibited an increased modulatory potency when equilib ration with the tissue was allowed prior to dilution. The findings sho wed that the influence of low concentrations of allosteric modulators on the [H-3]NMS dissociation rate may be demonstrated separately from any effects on association rate, and that the contact time with the al losteric ligand may influence the extent of these effects. (C) 1997 El sevier Science Inc.