Muscarinic receptor subtypes in the human colon: lack of evidence for atypical subtypes

Characteristics of muscarinic receptors were investigated in circular muscle from normal human colon. In saturation studies (n = 18), binding of [H-3]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 [H-3]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. (C) 2003 Elsevier B.V. All rights reserved.