Preparation and tension recording of rat aortic rings

Male Sprague-Dawley rats weighing 240–280 g (60–70 days old) were provided by Vital River Laboratories, Beijing, China. The animals were housed in plastic cages under controlled humidity (50%) and temperature (25 °C), and they were exposed to a 12 h light/dark cycle with free access to purified water and a standard diet. The animal care and handling were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the Laboratories Institutional Animal Care and Use Committee of Chinese Academy of Medical Science and Peking Union Medical College.

The descending thoracic aorta was isolated rapidly after rats were euthanized by cervical dislocation. The adherent connective tissue was cleaned carefully, and then the aorta was cut into ring segments (3-4 mm in length). The endothelial layer of the aorta was destroyed by gently rubbing the luminal surface with a moist cotton swab as necessary. Two stainless steel triangles were passed through the lumen of each ring. One triangle was connected to an isometric force transducer connected to a BIOPAC polygraph (MP100WSW, Biopac Systems, Inc, Goleta, CA, USA) to measure tension in the vessels^16,17. The rings were mounted in organ baths containing 10 mL Krebs-Henseleit (K-H) solution of the following composition (mmol/L): NaCl 120, KCl 4.8, MgSO₄ 1.4, KH₂PO₄ 1.2, glucose 11.0, CaCl₂ 2.5, NaHCO₃ 25.0, and EDTA 0.01. The K-H solution was kept at 37 °C and continuously bubbled with a 95% O₂/5% CO₂ gas mixture¹⁸. The aortic preparations were stretched until they reached a resting tension of 1.2 g. Then, they were equilibrated for 60 min; during this time, the bath fluid was changed every 20 min¹⁹.

After the equilibration period, the aortic rings were constricted with a high K⁺ (60 mmol/L) K-H solution to stimulate the tissue and to verify its availability. Then, the rings were washed with normal K-H solution to restore the basic tension of 1.2 g. To confirm the integrity of the endothelium, acetylcholine (10 μmol/L) was added to the bath following stabilized contraction induced by NE (1 μmol/L). When the relaxant response to ACh was less than 10%, the endothelium was considered to be completely removed. When the relaxant response was greater than 90%, the endothelium was considered to be intact. Suitable aortic rings were chosen for the following experiments.

Subsequently, Ang II was used to stimulate the tension of the aortic rings. DL0805-2 were added to the organ bath, and the aortic rings were incubated with various concentrations of DL0805-2 for 30 min. Then, a single dose of Ang II (100 nmol/L) was added to induce a transient vasoconstriction. In this experiment, both endothelium-intact and -denuded rings were used to verify the influence of the endothelium. In a separate experiment of endothelium-denuded aortic rings, Ang II ranging from 1×10⁻¹⁰ mol/L to 1×10⁻⁷ mol/L was applied cumulatively to obtain concentration-response curves. An interval time of 3 min was used to ensure that the next concentration of Ang II was added when the last maximum tension was obtained. In other experiments, KCl ranging from 10 to 60 mmol/L or NE ranging from 1×10⁻⁹ mol/L to 1×10⁻⁶ mol/L was applied cumulatively to obtain concentration-response curves. The interval time was 5 min.