Wire myography

Vascular function was assessed as previously described^21,22 with the following modifications. Briefly, first order mesenteric arteries and abdominal aortae were cut into two mm long rings and mounted on a multi-wire myograph system 620 M (Danish Myo Technology, Aarhus, Denmark) and allowed to stabilise at zero tension for 15 min followed by normalisation. All experiments were performed at 37 °C and continuously bubbled with carbogen (95% O₂ and 5% CO₂). Changes in isotonic tension were recorded using Powerlab/ Lab Chart data acquisition system (AD Instruments, Bella Vista, NSW, Australia). Firstly, mesenteric arteries were maximally contracted with high K⁺ physiological salt solution (KPSS, 100 mM), followed by the determination of endothelium integrity. Cumulative dose-response curves to vasoconstrictors such as Ang II (0.1 nM–0.3 μM), PE (1 nM–30 μM), U46619 (0.1 nM–1 μM) and ET-1 (0.1 nM–0.1 μM) were produced to evaluate vascular smooth muscle reactivity. Similarly, cumulative dose-response curves to endothelium-dependent dilator, ACh (0.1 nM–10 μM) and -independent dilator, SNP (0.1 nM–10 μM) were constructed. In order to determine the mechanisms of endothelium-dependent relaxation in the mesenteric artery, responses to ACh were evaluated in the presence and/ or absence of a COX inhibitor, indomethacin (indo, 1 μM), a NOS inhibitor, L-NAME (200 μM), TRAM-34 (1 μM) and apamin (1 μM). ACh-evoked relaxation was analysed using AUC to determine the relative contributions of NO, EDH and PGI₂-mediated relaxation in the mesenteric artery. Briefly, the contribution of NO was calculated by subtracting AUC of control curve from AUC in the presence of L-NAME. Similarly, the contribution of PGI₂ was calculated by subtracting AUC obtained in the presence of L-NAME from AUC in the presence of indomethacin + L-NAME. The remaining relaxation after blockade with indomethacin + L-NAME was attributed to EDH-type relaxation.

Aortic rings were maximally contracted with U46619 (1 μM), followed by cumulative dose-response curves to ACh (0.1 nM–10 μM) in the presence or absence of indomethacin (1 μM). In a separate set of experiments, mesenteric artery and aorta were sub-maximally pre-contracted using titrated concentrations of U46619, followed by the addition of L-NAME (200 μM) to determine basal NOS activity. Dose-response curves were fitted to a sigmoidal curve using non-linear regression (Prism version 6.0, GraphPad Software, San Diego, CA, USA) to calculate the sensitivity (pEC₅₀) of each agonist. Maximal relaxation (R_max) was measured as a percentage of U46619 contraction. Maximal contraction (C_max) was measured as a percentage of KPSS.