Preparation of rat aortic rings

Rats were sacrificed by cervical dislocation. The thoracic aorta was carefully removed, and placed directly into ice-cold Krebs-Henseleit bicarbonate solution (117.8 mM NaCl, 6.0 mM KCl, 1.6 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 24.2 mM NaHCO₃, 11.0 mM glucose, 0.027 mM EDTA), equilibrated with 95% O₂ and 5% CO₂, pH 7.4. Then, periadventitial fat was removed. Care was taken during the complete procedure to ensure the integrity of the vascular endothelium. For experiments with endothelium-denuded aortic rings, the endothelium was removed by gently rubbing the intimal surface of the vessels. Aortic rings (3-4 mm wide) were mounted in 5 mL water-jacketed organ baths at 37°C and equilibrated for 1.5 to 2.0 h. A maximum of two aortic rings from either 3- or 9-week-old rats were used simultaneously. Basal tone was set at 1.5 and 2.0 g, respectively. Changes in tension were measured using a pressure transducer (TSD125) connected to a computerized data acquisition system (MP150-BIOPAC, BIOPAC Systems, USA). This procedure was determined to produce optimal conditions for reproducible isometric force development.