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 receptor 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 ability to differentiate among muscarinic receptor subtypes. 2. It was found that all tetra-amines antagonized McN-A-343-induced inhibition in electrically stimulated rabbit vas deferens in a competitive manner and with affinity values (pA2) ranging between 6.27 ± 0.09 (spirotramine) and 8.51 ± 0.02 (AM170). 3. Competition radioligand binding studies, using native muscarinic receptors from rat tissues (M1, cortex; M2, heart; M3, submaxillary gland) or from NG 108-15 cells (M4) and human cloned muscarinic M1-M4 receptors expressed in CHO-K1 cells, were undertaken with the same tetraamines employed in functional assays. All antagonists indicated a one-site fit. 4. The affinity estimates (pKi) of tetra-amines calculated in binding assays using native receptors were similar to those obtained using cloned receptors. Among these compounds some displayed selectivity between muscarinic receptor subtypes, indicating that they may be valuable tools in receptor characterization. Spirotramine was selective for M1 receptors versus all other subtypes (pKi native: M1, 7.32 ± 0.10; M2, 6.50 ± 0.11; M3, 6.02 ± 0.13; M4, 6.28 ± 0.16; pKi cloned: M1, 7.69 ± 0.08; M2, 6.22 ± 0.14; M3, 6.11 ± 0.16; 6.35 ± 0.11) whereas CC8 is highly selective for M2 receptors versus the other subtypes (pKi native: M1 7.50 ± 0.04; M2, 9.01 ± 0.12; M3, 6.70 ± 0.08; M4, 7.56 ± 0.04; pKi cloned: M1, 7.90 ± 0.20; M2, 9.04 ± 0.08; M3, 6.40 ± 0.07; M4, 7.40 ± 0.04). Furthermore, particularly relevant for this investigation were tetra-amines dipitramine and AM172 for their ability to significantly differentiate M1 and M4 receptors. 5. The apparent affinity values (pA2) obtained for tetra-amines in functional studies using the prostatic portion of rabbit vas deferens correlated most closely with the values (pKi) obtained at either native or human recombinant muscarinic M4 receptors. This supports the view that the muscarinic receptor mediating inhibition of neurogenic contractions of rabbit vas deferens may not belong to the M1 type but rather appears to be of the M4 subtype.