Preparation of aortic rings for tension measurement was performed as previously described [14 (link)]. Male Sprague-Dawley rats weighing 250-300 g were anesthetized by intramuscular injection of zoletil (15 mg/kg, Virbac Laboratories, Carros, France). The descending thoracic aorta was dissected free, and surrounding connective tissues and fat were removed under microscopic guidance in a Krebs solution bath of the following composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.4 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose. The aorta was then cut into 2.5-mm rings, suspended on Grass isometric transducers (FT-03, Grass Instrument, Quincy, MA, USA) under a 3.0-g resting tension in a 10-ml Krebs bath at 37℃, and aerated continuously with 95% O2 and 5% CO2 to maintain pH values within the range of 7.35-7.45. The rings were equilibrated at a 3.0-g resting tension for 120 min, and the bath solution was changed every 30 min. The endothelium was removed from the aortic rings by inserting a 25-gauge needle tip into the lumen of the rings and gently rubbing the ring for a few seconds. After contractions induced by 10-8 M phenylephrine stabilized, endothelial denudation of aortic ring was confirmed by observation of less than 15% relaxation in response to acetylcholine (10-5 M). The contractile response induced by an isotonic 60 mM KCl solution was measured for all aortic rings to check vascular smooth muscle viability and was used as a reference value (100%). The isotonic 60 mM KCl solution was prepared by replacing the NaCl in Krebs solution with an equimolar amount of KCl. After washing out KCl from the organ bath and allowing the return of isometric tension to baseline, the main experiments were performed according to experimental protocols described below. Each ring was used for only one concentration-response curve induced by bupivacaine alone or a local anesthetic (bupivacaine, ropivacaine, lidocaine and mepivacaine) plus lipid emulsion. Because this in vitro study used endothelium-denuded aorta, the Krebs solution contained the nitric oxide synthase inhibitor NW-nitro-L-arginine methyl ester (L-NAME, 10-4 M) to prevent the release of endogenous nitric oxide from residual endothelium [14 (link)].