Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats

Background: Nicorandil, an anti-angina agent, reportedly improves outcomes even in angina patients with diabetes. However, the precise mechanism underlying the beneficial effect of nicorandil on diabetic patients has not been examined. We investigated the protective effect of nicorandil on endothelial function in diabetic rats because endothelial dysfunction is a major risk factor for cardiovascular disease in diabetes. Methods: Male Sprague-Dawley rats (6 weeks old) were intraperitoneally injected with streptozotocin (STZ, 40 mg/kg, once a day for 3 days) to induce diabetes. Nicorandil (15 mg/kg/day) and tempol (20 mg/kg/day, superoxide dismutase mimetic) were administered in drinking water for one week, starting 3 weeks after STZ injection. Endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anaesthetised rats. Cultured human coronary artery endothelial cells (HCAECs) were treated with high glucose (35.6 mM, 24 h) and reactive oxygen species (ROS) production with or without L-NAME (300 mu M), apocynin (100 mu M) or nicorandil (100 mu M) was measured using fluorescent probes. Results: Endothelial function as evaluated by FMD was significantly reduced in diabetic as compared with normal rats (diabetes, 9.7 +/- 1.4%; normal, 19.5 +/- 1.7%; n = 6-7). There was a 2.4-fold increase in p47(phox) expression, a subunit of NADPH oxidase, and a 1.8-fold increase in total eNOS expression in diabetic rat femoral arteries. Nicorandil and tempol significantly improved FMD in diabetic rats (nicorandil, 17.7 +/- 2.6%; tempol, 13.3 +/- 1.4%; n = 6). Nicorandil significantly inhibited the increased expressions of p47(phox) and total eNOS in diabetic rat femoral arteries. Furthermore, nicorandil significantly inhibited the decreased expression of GTP cyclohydrolase I and the decreased dimer/monomer ratio of eNOS. ROS production in HCAECs was increased by high-glucose treatment, which was prevented by L-NAME and nicorandil suggesting that eNOS itself might serve as a superoxide source under high-glucose conditions and that nicorandil might prevent ROS production from eNOS. Conclusions: These results suggest that nicorandil improved diabetes-induced endothelial dysfunction through antioxidative effects by inhibiting NADPH oxidase and eNOS uncoupling.