Reactive oxygen species, endothelium, lidocaine, procaine is sufficient to induce ROS injury [8]. However, 5 minutes of EL with 15 mA constant current caused too much injury in our model, so we used a 35-second treatment, indicating that there are species differences in the degree of injury by ROS. Lecour et al. [18] verified that the function of the left ventricle declined when ROS was generated by 1 minute of EL in the K-H solution with a constant current of 1, 1.5, 3, 5, 7.5, and 10 mA, as measured by the Langendorff perfusion method. Superoxide radicals and hydroxyl radicals were also generated by the reaction of the 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO)- OH signal, the adduct of DMPO EL, with superoxide dismutase (SOD : 100 IU/ml) and mannitol (50 mM), followed by a quantitative analysis with electron spin resonance. The DMPO- OH signal occurred as early as 10 seconds after starting EL and was maintained for 60 seconds, with diminishing levels after EL stopped. SOD pretreatement reduced the DMPO-OH signal by 60%, which showed that the hydroxyl radical generation process was partially by the Fenton reaction of a superoxide radical. Mannitol (50 mM) blocked the DMPO-OH signal but did not change cardiac parameters after EL [18]. Similarly, pretreatment of mannitol a scavenger of hydroxyl radicals, did not prevent endothelial dysfunction in our model. ROS is also generated via cyclooxygenase during an inflammatory response [19]. Endotheliocytes, Kuffer cells, neutrophils, and macrophages also produce ROS and reactive nitrogen species (RNS) during an inflammatory response via antagonizing pathogenic bacteria in those cells. Antioxidant defenses are depleted by chronic exposure to ROS or RNS, leading to cancers, rheumatoid arthritis, or organ transplantation rejection. Large inflammatory responses also produce ROS, including peroxyl radical (ROO.), hydroxyl radical, superoxide radical, and hydrogen peroxide, and RNS, including .NO and peroxynitrite anion (ONOO-). Vascular relaxation induced by ACh is caused by inducing NO release in endotheliocytes, and ROS inactivates NO [20,21]. In addition, ROS generated by ischemia or reperfusion causes endothelial damage [15, 22]. We generated ROS with 35 seconds of EL and then replaced the K-H solution for measurements after 15 minutes of equilibrium. Thus, ACh should induce vascular relaxation via changes in endothelial injury and not from inactivation of NO [20]. The endothelial monolayer is easily damaged and dysfunctional during chronic hypertension, even to the point of detachment [23]. Smooth muscle may also be directly injured by ROS. During oxygen depletion, such as ischemia, ATP in the endothelium is rapidly consumed, causing failure of the Na+-K+ ATPase pump and an increase of intracellular [Na+] i and [H+]i by inhibiting the removal of the metabolites and mitochondrial NADH oxidation. Increased intracellular [H+] i leads to an increase in [Na+]i by facilitating Na+-H+ exchange 108 Vol. 59, No. 2, August 2010 to maintain constant [pH]i and thus triggers the Na+-Ca2+ exchange, increasing [Ca2+]i as well [24]. Increased [Ca 2+] i deforms proteins and phospholipids [25] via activation of phospholipase A2 to generate arachidonic acid (AA), a free fatty acids. AA is then converted to prostanoids such as prostacyclin (PGI2), a strong smooth muscle relaxant, and prostaglandin G, a constrictor, to thromboxane A2 (TXA2) or to leukotrienes (LT), which cause the synechia of leukocytes to endotheliocytes. In addition, it facilitates the conversion of xanthine dehydrogenase to xanthine oxygenase (XO) and increases the intracellular superoxide radical (O2.) [23]. Oxygen re-supply after ischemia increases ROS: O2., OH., and LOO. [26]. These ROS that are generated mainly in mitochondria of neutrophil [25] denature lipids and proteins and damage the cell membrane and mitochondria (mitochondrial permeability transition pore, MPTP) [27], and denature enzymes [25]. O2. is the first ROS is generated by oxygen re-supply; it is produced by XO or by LT from neutrophils and nerve cells in the microvascular structure. Superoxide radical is converted by SOD to hydrogen peroxide, which eventually becomes water (H2O) by catalase. Here, catalase (1,000 U/ml) pretreatment prevented endothelial injury by ROS and maintained endothelial-dependent aorta relaxation. However, excess O 2. is converted by the Fenton reaction and Haber-Weiss reaction to a hydroxyl radical, which causes the severe injury and eventually causes lipids peroxidation and cell and tissue injury by LOO. generation [26]. Pretreatment with mannitol (5 mM) as the OH. scavenger did not prevent endothelial injury, potentially because ROS sensitivity may shows species variation and higher doses may be needed. Lidocaine protects against brain ischemia by reducing energy demand [28]. Anoxia-induced depolarization in the mouse hippocampus pretreated with lidocaine 1 μM and 0.25 μM 15 minutes before the ischemia-induction started after 49 seconds and 44 seconds, respectively, indicating a depolarization delay compared to the 27-second interval in the control group. The amount of ATP in the lidocaine pretreated group increased to 165% and 212% of controls at 1 minute and 2 minutes after ischemia, respectively. The activity of Na+-K+ ATPase and the activity of Ca2+ ATPase were not changed, and [Ca2+]i did not increase [28]. These findings imply that lidocaine maintains energy by delaying neuronal depolarization. In addition, [Ca2+] i increased in the left ventricle of the excised mouse heart where dysfunction was induced by hydrogen peroxide, but pretreatment with lidocaine 10 μM blocked this increase [29]. In human microvascular endothelial cells (HMVEC), lipo- polysacharide in the culture solution caused necrosis of the endotheliocytes; amide local anesthetic pretreatment pre- vented this necrosis, but tetracaine and procaine, the esters, could not [30]. The protection by amide local anesthetics, www.ekja.org