Measurement of Vascular Tension

Isometric tension of aortic rings was recorded by Multi Myograph System (Danish Myo Technology A/S, Denmark). Briefly, after euthanasia, the mouse thoracic aorta was quickly dissected out and cut into 2 mm-long rings in ice-cold and oxygenated Krebs buffer, which contained in mmol/L: 119 NaCl, 4.7 KCl, 2.5 CaCl₂, 1 MgCl₂, 25 NaHCO₃, 1.2 KH₂PO₄, and 11 D-glucose. The vascular rings were mounted onto two thin stainless steel holders in myograph filled with Krebs solution bubbled with 95% O₂ and 5% CO₂ at 37°C. The optimum baseline tension was set at 3 mN. After an equilibration period of 60 min, each endothelium-intact ring was contracted with 60 mmol/L K⁺ at 30-min intervals until two consecutive contractions were similar in amplitude (<10%). Thereafter, the contractile response to phenylephrine (Phe, 1 μmol/L), angiotensin II (Ang-II, 50 nmol/L) or endothelin-1 (ET-1, 50 nmol/L) was measured. The rings were subsequently challenged with acetylcholine to verify the integrity of endothelium.