Limonene restores A2AAR mediated‐vascular relaxation in asthmatic mice via nitric oxide

Abstract

Limonene, a mono‐terpene flavonoid, has anti‐inflammatory effects in asthma by activating A2A adenosine receptors (A2AAR). Our objective was to identify the mechanism by which limonene restored A2A mediated aortic relaxation in asthmatic mice. Ex‐vivo tissue bath was used to study the effects of inhaled d‐limonene on isolated aortic rings from asthmatic mice. We used mice in which the A2A receptor was genetically knocked out (A2AKO) and C57BLJ/6 (wild‐type; WT) for the study. The mice were divided into control (CON) and allergen‐induced sensitized (SEN), control mice treated with limonene through aerosol route (CON+LIM) and allergen‐induced sensitized mice treated with limonene (SEN+LIM). Mice were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Control groups were given an i.p injection of vehicle (1,6 days) and the aerosol challenge of normal saline (11–13 days). SEN +LIM, CON +LIM groups were given 0.01% v/v aerosolized limonene via inhalation 90 min prior to challenges. Aorta were isolated on day 14 for further experiments. Tissue responses were studied in the form of contraction and relaxations in all groups. Concentration‐response curves (CRC) for selective A2Aagonist CGS‐21680 (10‐11‐10‐6M) were obtained for all groups. eNOS inhibitor L‐NAME (10‐4M) or prostaglandin inhibitor indomethacin (10‐5M) was added 30 min prior to starting CRC to study effect of nitric oxide and prostaglandins on vascular response. CGS‐21680 induced aortic relaxation was absent in WT SEN (5.62±3.72% WT SEN Vs 66.91 ± 6.8% CON, *p<0.05). Limonene restored CGS 21680‐induced relaxation in asthmatic mice (63.8 ± 6.32% WT SEN+LIM Vs 5.62± 3.72% WT SEN). L‐NAME blocked CGS‐21680 response in CON, CON+ LIM and SEN+LIM, while indomethacin had minimal effect. In conclusion, it appears that limonene restores A2AAR‐mediated aortic relaxation induced through nitric oxide in asthmatic mice.