20-hydroxyecdysone dilates muscle arterioles in a nitric oxide-dependent, estrogen ER-β receptor-independent manner

Abstract

Background20-hydroxyecdysone is an ecdysteroid which is abundant in plants and insects and has anabolic potentials in mammals. It was recently shown to have affinity for estrogen ER-β receptor, which could potentially make it vasodilatory. Yet this possibility has not been previously investigated. Such an activity in muscle arterioles could have huge implications for muscle blood flow and performance. Hypothesis/PurposeWe hypothesized that 20-hydroxyecdysone would dilate muscle arterioles by activating estrogen ER-β receptors. To test this, we investigated its vasodilatory properties in ovine muscle arterioles and further explored the mechanisms in human tissues and cells. Study DesignThe study was carried out experimentally, employing functional recording of arteriolar reactivity in intact ovine muscle arterioles and gene and protein expression analysis in human tissues and cells. MethodsDirect effects of the compound on arteriolar tone were assessed by wire myography in abdominal muscle and mesenteric arterioles isolated from samples obtained from male sheep. The roles of endothelial nitric oxide synthase (NOS3), cyclooxygenase (COX) and estrogen ER-β receptor (ER-β), and their effects were determined with specific blockers. The NOS3 mRNA and protein expressions were analyzed in human coronary artery endothelial cells (HCAECs) and humanized liver of uPA+/+-SCID mice, before and after 20-hydroxyecdysone administration. ResultsComparable dose-dependent relaxations were recorded for 20-hydroxyecdysone in both muscle and mesenteric arterioles with maximum relaxations of 46.94 ± 5.84% and 56.88 ± 7.04% respectively, which were not statistically different. Similar relaxation was recorded for β-estradiol in both arterioles. NOS inhibition with 100 µM L-NAME attenuated the relaxation to 20-hydroxyecdysone (p < 0.001) and β-estradiol (p < 0.001) in muscle arterioles. Neither COX inhibition with 10 µM indomethacin nor ER blockade with 1 µM PHTPP or 1 µM ICI182780 had any noticeable effect on 20-hydroxyecdysone relaxation in these arterioles. Transcriptome analysis revealed elevated NOS3 gene in the humanized liver of 20-hydroxyecdysone-treated mice, and, elevation of both NOS3 mRNA and protein in HCAECs treated with 20-hydroxyecdysone. ConclusionThe data suggest that 20-hydroxyecdysone has a nitric oxide-dependent, but ERβ-independent, vasodilatory property in muscle arterioles. The benefits to muscle blood flow would however be dependent on the impact of its effects on other vascular beds.