C. Trankle et al., M-2 RECEPTOR-BINDING OF THE SELECTIVE ANTAGONIST AF-DX-384 - POSSIBLEINVOLVEMENT OF THE COMMON ALLOSTERIC SITE, Molecular pharmacology, 53(2), 1998, pp. 304-312
The hypothesis was tested that M-2-selective antagonists partially uti
lize the allosteric site of muscarinic WI, receptors. The interactions
of the allosteric agent W84 ane-1,6-bis[dimethyl-3'-phthalimidopropyl
-ammonium bromide]) were studied with the M-2/M-4-selective AF-DX 384
onyl}-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the nonselective N-
methylscopolamine (NMS), and a number of other muscarinic antagonists.
In isolated paced guinea pig atria, the antagonistic effect of W84 ag
ainst oxotremorine-and arecaidine propargyl ester-induced negative ino
tropic actions reached a limiting value at higher W84 concentrations,
revealing negative cooperativity (factors of cooperativity alpha = 311
and alpha = 495, respectively). The antagonistic potency of VV84 in t
his M-2 receptor model (W84 binding constant K-A similar to 160 nM) wa
s higher than at M-1/M-4-like receptors of rabbit vas deferens (K-B si
milar to 800 nM) and at M-3 receptors of guinea pig ileum (K-B similar
to 4,000 nM). In paced atria, combinations of W84 with muscarinic ant
agonists yielded more-than-additive antagonistic effects against oxotr
emorine in case of conventional antagonists such as NMS (alpha = 18) b
ut less-than-additive effects with the M-2-preferring AF-DX 384 (alpha
= 444). In guinea pig heart homogenates, the equilibrium binding of [
H-3]NMS was only partially inhibited by W84 (alpha = 2.4), whereas [3H
]AF-DX 384 binding could be suppressed completely (alpha = 194). The d
ifference in cooperativity reflects that W84 inhibits PH]NMS dissociat
ion with a similar to 40-fold higher potency (ECdiss = 900 nM) than [H
-3]AF-DX 384 dissociation (ECdiss = 33,300 nM). [H-3]NMS dissociation
also could be retarded by AF-DX 384 (ECdiss = 22,000 nM), probably via
an interaction with the site used by W84. The results suggest that th
e binding domain of AF-DX 384 partially overlaps with the common allos
teric site of the M-2 receptor protein.