Immediately after the animals were killed with an overdose of isoflurane at 20 hours after resuscitation, the chest was opened and thoracic aortas were rapidly removed. Each thoracic aorta was immediately immersed in Krebs-Ringer bicarbonate (HCO3) solution (composition, 118.3mM sodium chloride, 4.7mM potassium chloride, 2.5mM calcium chloride, 1.2mM magnesium sulfate, 1.2mM potassium phosphate, 25.0mM sodium bicarbonate, 0.026mM calcium-EDTA, and 1.11mM glucose),13,14 which was aerated with a mixture of 95% oxygen and 5% carbon dioxide (pH, 7.4; PO2, 580 mm Hg). The thoracic aorta was dissected with care to prevent any damage to vascular endothelial cells and was cut into rings approximately 2.5 mm in length. The aorta rings were carefully mounted on 2 specimen holders and placed in glass organ chambers containing 20 mL of aerated Krebs-Ringer HCO3 solution at a temperature of 37°C. One holder was stationary and the other was connected to an isometric force-displacement transducer (model FT03; Astro-Med, Inc, West Warwick, RI) coupled to a polygraph (model 7D; Astro-Med, Inc). The vessels were incubated for 60 minutes at a tension of 1000 mg, during which the organ chamber was rinsed every 15 minutes with the aerated Krebs-Ringer HCO3 solution. When basal tension was stable, a submaximal contraction (approximately 75% of the maximal contraction) was induced by 2 ×10 −7M norepinephrine bitartrate (Sigma-Aldrich Corp, St Louis, Mo). An endothelium-dependent vasodilator, acetylcholine chloride (concentration range, 10 −8M to 10 −5M) (Sigma-Aldrich Corp), or an endothelium-independent vasodilator, nitroglycerine (concentration range, 5 ×10−9M to 5 ×10−6M) (American Regent Laboratories, Shirley, NY), was applied cumulatively thereafter. After each series of agent additions, the ring preparations were washed with Krebs-Ringer HCO3 solution and allowed to reequilibrate for at least 30 minutes.