Differential inverse agonism at the human muscarinic M₃ receptor

Human muscarinic M₃ receptors (hM₃Rs) induce smooth muscle contraction and mucus gland secretion in response to parasympathetic stimulation. As a consequence of hM₃R function, muscarinic antagonists have wide therapeutic use to treat overactive bladder, abdominal pain (irritable bowel syndrome), and chronic obstructive pulmonary disease (COPD). In this chapter, we describe the set up and results obtained with different in vitro assays to monitor hM₃R activation (agonist-dependent and constitutive) and evaluate functional potencies of different anticholinergics in CHO cells. Given the G(q) coupling of hM₃R, assays measuring the second messengers inositol phosphates (InsP) and an AP-1-driven reporter luciferase were developed. In our hands, the reporter gene assay shows advantages: firstly, thanks to the longer incubation times, it allows reaching of pseudo-equilibrium also for ligands with slower receptor dissociation kinetics (e.g., tiotropium). Secondly, the AP-1-driven luciferase detects significant constitutive activity of the hM₃R, which allows characterizing the different anticholinergics for their inverse agonist properties. Given the potential for inverse agonists to cause changes in receptor expression, monitoring hM₃R upregulation is another important pharmacological parameter. Here, we describe how to measure the effect of chronic exposure to anticholinergics on the expression levels of hM₃R, with particular attention to ensure full antagonist removal from receptor pool before hM₃R quantification. Taken together, our results indicate that anticholinergics exhibit differential pharmacological behaviors, which are dependent on the pathway investigated, and therefore provide evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.