Abstract
A2A adenosine receptor (A2AAR) 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 A2AAR 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 A2AAR deficiency on the NADPH oxidase pathway in tracheas of A2A wild-type (WT) and A2A knockout (KO) mice. A2AWT 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. A2AAR (gene and protein expression), cAMP, and phosphorylated PKA (p-PKA) levels were decreased in A2AWT sensitized mice compared with controls. A2AKO mice also showed decreased cAMP and p-PKA levels. A2AWT sensitized and A2AKO control mice had increased gene and protein expression of NADPH oxidase subunits (p47phox and gp91phox) compared with the controls. Tracheal relaxation to specific A2AAR agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680), decreased in A2AWT sensitized mice compared with the controls, although it was absent in A2AKO mice. Pretreatment with NADPH oxidase inhibitors apocyanin/diphenyliodonium reversed the attenuated relaxation to CGS 21680 in A2AWT sensitized tracheas, whereas specific PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-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 A2AWT sensitized and A2AKO control mice compared with the controls. In conclusion, this study shows that A2AAR deficiency causes increased NADPH oxidase activation leading to decreased tracheal relaxation via altered cAMP-PKA signaling and ROS generation.
Footnotes
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This work was supported in part by the National Institutes of Health [Grants HL027339, HL094447]; and by Bridge Grant Funding from the Health Sciences Center of West Virginia University.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.151613.
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ABBREVIATIONS: ROS, reactive oxygen species; AR, adenosine receptor; β2-AR, β2-adrenergic receptor; KO, knockout; PKA, protein kinase A; 7-TSR, seven transmembrane-spanning receptor; WT, wild-type; CRC, concentration response curve; PCR, polymerase chair reaction; DCF, dichlorofluorescein; DHE, dihydroethidium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DPI, diphenyliodonium; ZM 241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; KT 5720, (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester; CGS 21680, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride; PEG-SOD, polyethylene glycol-conjugated superoxide dismutase; Tx, Triton-X.
- Received January 29, 2009.
- Accepted April 23, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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