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.