Quantitative Measurement of Nitric Oxide and Superoxide in Arterial Tissues

Aortas, carotid arteries and SMA were dissected and incubated for NO production for 30 min in Krebs–Hepes buffer containing: BSA (20.5 g/L), CaCl₂ (3 mM) and L-Arginine (0.8 mM) (Sigma-Aldrich). NaDETC (1.5 mM) and FeSO₄.7H₂O (1.5 mM) (Sigma-Aldrich) were separately dissolved under argon gas bubbling in 10 mL volumes of ice-cold Krebs–Hepes buffer. These were rapidly mixed to obtain a pale yellow-brown opalescent colloid Fe(DETC)₂ solution (0.4 mM), which was used immediately. The colloid Fe(DETC)₂ solution was added to vessels and incubated for 45 minutes at 37°C. Then, arteries were immediately frozen in plastic tubes using liquid N₂. NO measurement was performed on a table-top x-band spectrometer Miniscope (Magnettech, MS200; Berlin, Germany). Recordings were made at 77°K, using a Dewar flask. Instrument settings were 10 mW of microwave power, 1 mT of amplitude modulation, 100 kHz of modulation frequency, 150 s of sweep time and 3 scans. Signals were quantified by measuring the total amplitude, after correction of baseline as done previously [23] (link). Values are expressed in unit/mg weight of dried tissue.
For O₂⁻ spin-trapping, aortas, carotid arteries and SMA were dissected and allowed to equilibrate in deferoxamine-chelated Krebs-Hepes solution containing 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidin (CMH, Noxygen; Denzlingen, Germany) (500 µM), deferoxamin (25 µM, Sigma-Aldrich) and DETC (5 µM, Sigma-Aldrich) under constant temperature (37°C) for 60 minutes. Then, they were frozen in liquid N₂ and analyzed in a Dewar flask by EPR. Values are expressed in unit/mg weight of dried tissue.