Binding of [3H]quinuclidinyl benzilate to intestinal mucus: An artifact in identification of epithelial cell muscarinic receptors

The widely used muscarinic receptor ligand ³H]quinuclidinyl benzilate (³H]QNB) was found to bind in a site-specific but artifactual manner to rat intestinal mucus, obscuring specific binding to muscarinic receptors on intestinal epithelial cells. Atropine inhibited ³H]QNB binding to mucus with an apparent IC₅₀ of 2.1 × 10^?7 M, compared to an IC₅₀ of 1.4 × 10^?8 M obtained with a homogenate of intestinal epithelial cells. Unlabeled QNB also inhibited binding of ³H]QNB to mucus but the apparent IC₅₀(4 × 10^?7 M) was about 300-fold greater than the IC₅₀ determined with a control tissue, heart muscle (IC₅₀, 1.2 × 10^?9M). ³H]QNB binding was saturable over the concentration range of 1–7 nM in the heart, with an apparent k_D of 0.76 nM. As expected from the high IC₅₀ for QNB in the mucus binding experiments, binding to mucus was not saturable over the 1–15 nM concentration range. Based on pH profiles and temperature dependency of binding, it seems unlikely that mucin, the primary component of mucus, was responsible for ³H]QNB binding to the mucus. The findings have implications for studies which involve binding of ³H]QNB in particular and other ligands in general to mucus-secreting epithelial tissues.