Isometric tension recording

Mouse mesenteric arteries (150 to 300 μm in diameter) were dissected and cleaned of connective tissues. The arteries were cut into 1.5 to 1.8 mm rings and mounted in a four-chamber wire myograph (Danish MyoTechnology A/S) (Gauthier et al., 2011, Siangjong et al., 2013). The arteries were maintained in physiological saline solution (PSS, in mM: 119 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.17 MgSO₄, 24 NaHCO₃, 1.18 KH₂PO₄, 0.026 EDTA and 5.5 glucose), at 37°C, supplied with 95% O₂/5% CO₂. After 30 min, tension recording was performed as previously described (Gauthier et al., 2011, Siangjong et al., 2013). Briefly, the arteries were stretched to a tension of 0.80 mN, where optimum isometric length-tension was achieved (Gauthier et al., 2011). The arteries were challenged with KCl (60 mM) and the thromboxane mimetic, U46619 (100 nM) 3–4 times until the maximum active tension was established. Thereafter, U46619 or phenylephrine (in the presence of the COX inhibitor indomethacin (10 μM) and the nitric oxide synthase inhibitor, nitro-L-arginine (30 μM)) was added to partially constrict the arteries to approximately 50–70% of maximum active tension. Upon a stable constriction, increasing concentrations of test compounds were added and tension was recorded. In some experiments, the arteries were pretreated with AUDA (1 μM), a soluble epoxide hydrolase (sEH) inhibitor, for 30 min prior to the constriction (Chawengsub et al., 2009b, Chawengsub et al., 2008). Results are expressed as percent relaxation with basal tension representing 100% relaxation. In other experiments, arteries were treated with vehicle or trioxilin C₃ (10 μM) for 30 min. Then, constrictor responses to U46619 (10⁻⁸-10⁻⁶M) were determined. U46619 constriction responses were expressed as % constriction with maximum active tension being 100%.