Mansfield_2003_Eur.J.Pharmacol_482_101

Characteristics of muscarinic receptors were investigated in circular muscle from normal human colon. In saturation studies (n=18), binding of [3H]quinuclidinyl benzylate (QNB) was of high affinity (K(d) 87.3 pM) and capacity (B(max) 362+/-27 fmol/mg protein), with no differences between ascending and sigmoid colon. Kinetic studies gave a K(d) of 55 pM. Methoctramine and darifenacin displayed biphasic binding profiles, the high affinity components being compatible with a population of approximately 80+/-5% M(2) and 13+/-2% M(3) muscarinic receptors, respectively. Pirenzepine, mamba toxin 1 and mamba toxin 3 were very weak competitors, indicating negligible expression of muscarinic M(1) and M(4) receptors. Six other subtype-preferring antagonists exhibited K(i) values typical of those reported at cloned human muscarinic M(2) receptors. In the presence of methoctramine, pre-treatment with alkylating agent 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard) inhibited [3H]quinuclidinyl benzylate binding to 26% of sites. Following alkylation of muscarinic M(3) receptors, darifenacin bound to a single low affinity site, indicating binding to muscarinic M(2) receptors.