Analysis of the muscarinic receptor subtype mediating inhibition of the neurogenic contractions in rabbit isolated vas deferens by a series of polymethylene tetra-amines

1 The pharmacological characteristics of the presynaptic muscarinic recepto r subtype, which mediates inhibition of the neurogenic contractions in the prostatic portion of rabbit vas deferens, have been investigated by using a series of polymethylene tetra-amines, which were selected for their abilit y to differentiate among muscarinic receptor subtypes. 2 It was found that all tetra-amines antagonized McN-A-343-induced inhibiti on in electrically stimulated rabbit vas deferens in a competitive manner a nd with affinity values (pA(2)) ranging between 6.27+/-0.09 (spirotramine) and 8.51+/-0.02 (AM170). 3 Competition radioligand binding studies, using native muscarinic receptor s from rat tissues (M-1, cortex; M-2, heart; M-3, submaxillary gland) or fr om NG 108-15 cells (M-4) and human cloned muscarinic M-1-M-4 receptors expr essed in CHO-K1 cells, were undertaken with the same tetraamines employed i n functional assays. All antagonists indicated a one-site fit. 4 The affinity estimates (pk;) of tetra-amines calculated in binding assays using native receptors were similar to those obtained using cloned recepto rs. Among these compounds some displayed selectivity between muscarinic rec eptor subtypes, indicating that they may be valuable tools in receptor char acterization. Spirotramine was selective for Mi receptors versus all other subtypes (pK(i) native: M-1, 7.32+/-0.10; M-2, 6.50+/-0.11; M-3, 6.02+/-0.1 3; M-4, 6.28+/-0.16; pK(i) cloned: M-1, 7.69+/-0.08; M-2, 6.22+/-0.14; M-3, 6.11+/-0.16; 6.35+/-0.11) whereas CC8 is highly selective for M-2 receptor s versus the other subtypes (pK(i) native: M-1, 7.50+/-0.04; M-2, 9.01+/-0. 12; M-3, 6.70+/-0.08; M-4, 7.56+/-0.04; pK(i) cloned: M-1, 7.90+/-0.20; M-2 , 9.04+/-0.08; M-3, 6.40+/-0.07; M-4, 7.40+/-0.04). Furthermore, particular ly relevant for this investigation were tetra-amines dipitramine and AM172 for their ability to significantly differentiate MI and Mg receptors. 5 The apparent affinity values (pA(2)) Obtained for tetra-amines in functio nal studies using the prostatic portion of rabbit vas deferens correlated m ost closely with the values (pK(i)) obtained at either native or human reco mbinant muscarinic M-4 receptors. This supports the view that the muscarini c receptor mediating inhibition of neurogenic contractions of rabbit vas de ferens may not belong to the M-1 type but rather appears to be of the M-4 s ubtype.