Aim: To examine whether iNOS contributes to endothelial dysfunction in aged rats. Methods: Male Sprague Dawley rats were divided into three groups: young rats, aged rats treated with vehicle and aged rats treated with N-[3-(Aminomethyl) benzyl] acetamidine (1400W, 1 mg/kg, ip). Vasorelaxation was measured in isolated thoracic aorta. iNOS expression of thoracic aortic arteries was detected using immunohistochemistry and Western blot. Nitrotyrosine (a marker for peroxynitrite formation) content and expression in thoracic aortic tissue were determined using enzyme linked immunosorbent assay and immunohistochemistry. Results: Maximal relaxation induced by acetylcholine (10 -9 to 10 -5 mol/L) in the aged rats treated with vehicle was significantly decreased (70%±15%, P<0.01), as compared with the young rats (95%±8%). However, the maximal relaxation induced by acidified NaNO2 (an endothelium-independent vasodilator) had no significant difference between the two groups. Moreover, iNOS and nitrotyrosine expression increased in the vessels of the aged rats. In the aged rats treated with 1400W (a highly selective iNOS inhibitor) nitrotyrosine expression was reduced and acetylcholine-induced vasorelaxation was markedly improved (maximal relaxation was increased to 87%±8%, P<0.05), but the acidified NaNO 2 -induced vasorelaxation had no significant change. Conclusion: Our study demonstrated that inhibition of iNOS by 1400W increased endothelium-dependent vasodilation in aged rats. The mechanism was related with attenuation of peroxynitrite formation.Keywords: aging; nitric oxide synthase; endothelium; vasodilation; 1400W Acta Pharmacologica Sinica (2010Sinica ( ) 31: 1324Sinica ( -1328 doi: 10.1038/aps.2010 published online 13 Sep 2010 Original Article * To whom correspondence should be addressed. [17,18] , respectively. Thirty minutes after 1400W treatment [18,19] young and aged rats were sacrificed to obtain vessel tissues (n=7 rats per group) for nitrotyrosine content and immunohistochemical studies of iNOS and nitrotyrosine.
Determination of endothelial functionEndothelial function was determined by comparing the vasorelaxation response to acetylcholine (ACh), an endothelium-dependent vasodilator, with that of acidified NaNO 2 , an endothelium-independent vasodilator, as described previously [20] . Briefly, thoracic aortic rings were mounted onto hooks, suspended in organ chambers filled with Krebs buffer (mmol/L: NaCl 118.3, KCl 4.7, CaCl 2 2.5, MgSO 4 1.2, KH 2 PO 4 1.2, NaHCO 3 25.0, glucose 11.0, pH=7.4) [21] and aerated with 95% O 2 and 5% CO 2 at 37 °C, and connected to force transducers to record changes in tension via a Chart 5.3 data acquisition system. After equilibration for 60 min at a preload of 1 g, the rings were precontracted with norepinephrine (NE, 1 μmol/L). Once a stable contraction was achieved, the rings were exposed to cumulative concentrations of ACh (10 -9 to 10 -5 mol/L). After the cumulative response was stabilized, the rings were washed and allowed to equilibrate to baseline. The procedur...