Thompson, M.

Abstract Rationale: Asthma involves airway inflammation, hyperresponsiveness (AHR), and remodeling (cell proliferation and fibrosis), with both aspects not fully alleviated by current therapies. GPCR-based drugs represent a major aspect of current asthma therapy. Asthmatic airways express several orphan receptors with unknown physiological roles, highlighting a potentially novel and significant area for therapeutic exploration. Herein, we focus on orphan retinoic acid (RA)-induced Class C GPCR members GPRC5A (RAIG1; 5A) and GPRC5B (RAIG2; 5B). We leveraged recently identified 5A agonists to test the hypothesis that these GPCRs have alleviating effects in airways. Methods: Primary human airway smooth muscle cells (ASM) originating from non-asthmatic (NA) and asthmatic (Asth) individuals were obtained post-surgery from lung tissues after approval by the Mayo Clinic Institutional Review Board. PRESTO-Tango assay was used to determine 5A and 5B activity. cADDis assay was used to assess cAMP levels. Fluorescence imaging was used to determine calcium regulation. Recently identified 5A agonists [tryptamine, 7 fluoro-tryptamine (7FT), L-tryptophan, N-methyltryptamine, and indole-3-acetic acid] were used in assays, along with RA and 5A vs. 5B siRNAs. Results: PRESTO-Tango assay showed that tryptamine (10/100 µM) and 7FT (1/10/100 µM) increased 5A activity while high concentrations of 7FT (100 µM) increased 5B activity as well. cADDis assay showed 100 µM tryptamine increased cAMP more than 1/10 µM tryptamine and that 10 µM 7FT increased cAMP more than 1/100 µM 7FT in NA ASM.Calcium imaging showed that 5A agonist blunts responses to histamine, especially in Asth ASM. Calcium responses were higher in Asth ASM and 5A siRNA-treated ASM while 5B siRNA had an alleviating effect. Conclusions: Class C GPCRs 5A and 5B interestingly have opposing effects in human ASM towards blunting calcium responses to bronchoconstrictor agonists in the context of asthma. Thus, 5A agonists and 5B antagonists may represent novel GPCR-based approaches in airway diseases.