2.6. Vascular reactivity studies and NADPH oxidase activity
Descending thoracic aortic rings were dissected to assess obesity-associated vascular dysfunction by measuring acetylcholine vasorelaxant ability and NADPH oxidase activity.
For the vascular reactivity study, the organ chamber was filled with Krebs solution (composition in mM: NaCl 118, KCl 4.75, NaHCO3 25, MgSO4 1.2, CaCl2 2, KH2PO4 1.2 and glucose 11) at 37 °C and gassed with 95% O2 and 5% CO2 (pH 7.4). Length-tension characteristics were obtained via the myograph software (Myodaq 2.01 (Danish Myotechnologies, Denmark)) and the aortae were loaded to a tension of 5 mN. After the stabilization period, cumulative concentration-response curves to acetylcholine (10−9 M-10-5 M) were performed in intact rings pre-contracted by U46619 (10-8 M). Relaxant responses to acetylcholine were expressed as a percentage of pre-contraction.
The evaluation of NADPH oxidase activity in aortic rings was performed by lucigenin-enhanced chemiluminescence assay. Aortic rings were incubated for 30 min at 37 °C in HEPES-containing physiological salt solution (pH 7.4; in mM: NaCl 119, HEPES 20, KCl 4.6, MgSO4 1, Na2HPO4 0.15, KH2PO4 0.4, NaHCO3 1, CaCl2 1.2 and glucose 5.5). To stimulate the aortic production of O2− , the rings were incubated with NADPH (100 μM). Consequently, the aortic rings were then placed in tubes containing physiological salt solution, with or without NADPH and lucigenin was injected automatically at a final concentration of 5 μmol/L. NADPH oxidase activity were determined by measuring luminescence over 200 s in a scintillation counter (Lumat LB 9507, Berthold, Germany) in 5-s intervals and was calculated by subtracting the basal values from those in the presence of NADPH. Vessels were then dried, and dry weight was determined. NADPH oxidase activity is expressed as relative luminescence units (RLU)/min/mg dry aortic ring.