Differential Inverse Agonism at the Human Muscarinic M3 Receptor

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