Isolated organ bath experiments

Vascular reactivity was evaluated according to Cifuentes, Paredes et al.^19,40. Following the sacrifice of the animals by cervical dislocation, the aorta was separated and transferred to a Krebs–Ringer bicarbonate buffer (KRB) solution at 4 °C, (mM): 4.2 KCl, 1.19 KH₂PO₄, 120 NaCl, 25 NaHCO₃, 1.2 MgSO₄, 1.3 CaCl₂, and 5 D-glucose (pH7.4). 3–4 mm rings were prepared, and cleaned of connective tissue, taking special care to avoid endothelial damage. After 30 min period of equilibration, the aortic rings were stabilized with KCl (60 mM) near-maximum contractions for 10 min. We maintained a passive tension of 1.0 g on the aorta, as determined to be the optimal resting tension for obtaining maximum active tension in our laboratory. For dose–response curves, cumulative concentrions of PE (10^–9 to 10^–5 M) was used, and for relaxation experiments, the aortic rings were pre-contracted with PE 10^–6 M. In addition, the aortic tissue was pre-incubated for 20 min with l-NAME 10^–4 M, ODQ 10^–6 M or indomethacin 10^–5 M.