(a) Antagonism of M3 muscarinic receptors by the μ-opioid agonist methadone in a direct binding assay. Competition binding curves with [3H]quinuclidinyl benzilate in membrane fractions from CHO cells stably transfected with the human M3 muscarinic receptor. Each data point represents the mean and standard error of 4 conducted in duplicate or quadruplicate. Competition curves represent the best fit to a single-component logistic equation (GraphPad Prism 4.0). Two-site models did not yield a better fit. Membranes were incubated for 60 min at 25 °C with 0.5 nM [3H]quinuclidinyl benzilate and increasing concentrations of competing drug. Incubations were terminated by rapid vacuum filtration. Nonspecific binding was defined in the presence of 1.0 μM atropine and represented less than 10% of total binding. (b) Methadone antagonism of M3 muscarinic receptors by functional assay. Either methadone (10 μM final concentration) or vehicle was added at T = 20 s (1st addition), and then at T = 50 s (2nd addition) 1 μM carbachol was added to CHO-M3 cells and intracellular Ca2+ mobilization was measured, as previously described34. Dose-response curves (not shown) indicated that methadone was a competitive antagonist at M3-muscarinic receptors. (c) Loperamide antagonism of neurokinin NK2 receptors. Dose responses of CHO cells expressing Neurokinin NK2 receptors treated with [β-Ala8]-Neurokinin A were measured following administration of either DMSO vehicle or 10 μM loperamide. (d,e) Emetine antagonism of adrenergic receptors. Dose response of clonidine treatment of MDCK cells expressing either (d) alpha 2a adrenergic or (e) alpha 2c adrenergic receptors after incubation with DMSO vehicle or 10 μM emetine. Shown are representative curves, mean values ± s.e.m., of intracellular calcium release experiments performed in quadruplicate for each drug concentration per pre-treatment condition as described in Methods.