Genomic crosstalk between carbachol, a muscarinic receptor agonist, and the long-acting β2-adrenoceptor agonist, indacaterol, in human airway epithelial cells.

Many patients with chronic obstructive pulmonary disease are susceptible to recurrent exacerbations. In this study, we hypothesized that endogenous acetylcholine (ACh) may act as a proinflammatory mediator because long-acting muscarinic receptor antagonists protect against exacerbations, which have an inflammatory basis. This possibility was explored by determining if carbachol (CCh), a stable ACh analog, was a genomic stimulus in BEAS-2B bronchial epithelial cells. The ability of CCh to interact with indacaterol (Ind), a long-acting β₂-adrenoceptor agonist, was also assessed given that (1) sympathomimetic bronchodilators can promote adverse gene expression changes in airway structural cells, and (2) crosstalk between β₂-adrenoceptor and Gq-coupled muscarinic receptor agonists is well described. Unlike Ind, which induced 624 unique genes, CCh was a relatively weak genomic stimulus, implying that ACh may not behave as a proinflammatory mediator as hypothesized. Nevertheless, checkerboard assays using BEAS-2B cells expressing a cAMP-response element luciferase reporter determined that CCh interacted with Ind in a supra-additive manner and that this interaction was replicated on 39 Ind-regulated genes. Functional annotation of the Ind-regulated transcriptomes identified "transcription" and "signalling" as the dominant themes, with gene ontology terms associated with "inflammation" and "immune processes" being highly represented. A comparable gene ontology signature was obtained when Ind and CCh were combined; however, the number, magnitude and duration of gene expression changes were significantly enhanced. If genomic interactions occur between a long-acting β₂-adrenoceptor agonist and ACh in vivo, then they may enhance the expression of adverse-effect genes that could maintain, or even augment, features of lung pathology in chronic obstructive pulmonary disease. SIGNIFICANCE STATEMENT: Long-acting muscarinic receptor antagonists reduce exacerbation risk in chronic obstructive pulmonary disease, implying the etiology could have an inflammatory basis mediated by acetylcholine. However, in BEAS-2B cells, carbachol was a weak genomic stimulus, although it enhanced changes in indacaterol-regulated gene expression. Functional annotation of carbachol + indacaterol-regulated genes identified gene ontology terms associated with several themes, including inflammation. Interaction between a long-acting β₂-adrenoceptor agonist and endogenous acetylcholine could, paradoxically, augment airway inflammation in patients with chronic obstructive pulmonary disease.