A2A Adenosine Receptor Deficiency Leads to Impaired Tracheal Relaxation via NADPH Oxidase Pathway in Allergic Mice

A 2A adenosine receptor (A 2A AR) has been shown to suppress superoxide generation in leukocytes via the cAMP-protein kinase A (PKA) pathway. However, no study has yet explored the role of A 2A AR in relation to NADPH oxidase in murine tracheas in vitro, which may lead to altered smooth muscle relaxation in asthma. Therefore, the present study evaluated the effects of A 2A AR deficiency on the NADPH oxidase pathway in tracheas of A 2A wild-type (WT) and A 2A knockout (KO) mice. A 2A WT mice were sensitized with ovalbumin (30 μg i.p.) on days 1 and 6, followed by 5% ovalbumin aerosol challenge on days 11, 12, and 13. A 2A AR (gene and protein expression), cAMP, and phosphorylated PKA (p-PKA) levels were decreased in A 2A WT sensitized mice compared with controls. A 2A KO mice also showed decreased cAMP and p-PKA levels. A 2A WT sensitized and A 2A KO control mice had increased gene and protein expression of NADPH oxidase subunits (p47phox and gp91phox) compared with the controls. Tracheal relaxation to specific A 2A AR agonist, 4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9 H -purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680), decreased in A 2A WT sensitized mice compared with the controls, although it was absent in A 2A KO mice. Pretreatment with NADPH oxidase inhibitors apocyanin/diphenyliodonium reversed the attenuated relaxation to CGS 21680 in A 2A WT sensitized tracheas, whereas specific PKA inhibitor (9 S ,10 S ,12 R )-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1 H -diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4- i ] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) blocked CGS 21680-induced relaxation. Tracheal reactive oxygen species (ROS) generation was also increased in A 2A WT sensitized and A 2A KO control mice compared with the controls. In conclusion, this study shows that A 2A AR deficiency causes increased NADPH oxidase activation leading to decreased tracheal relaxation via altered cAMP-PKA signaling and ROS generation.