Szöcs Katalin, Lassègue Bernard, Wenzel Philip, Wendt Maria, Daiber Andreas, Oelze Matthias, Meinertz Thomas, Münzel Thomas, Baldus Stephan
Department of Cardiology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
J Mol Cell Cardiol. 2007 Jun;42(6):1111-8. doi: 10.1016/j.yjmcc.2007.03.904. Epub 2007 Mar 30.
Chronic administration of nitroglycerin (NTG) induces nitrate tolerance. Among possible underlying mechanisms, increased vascular production of reactive oxygen species (ROS) has emerged as a principal mechanism. Using cell culture and animal models of nitrate tolerance, we aimed to assess the impact of nitrates on NAD(P)H oxidases and aldehyde dehydrogenase 2 (ALDH2) expression. Rats and vascular smooth muscle cells were treated with NTG. Vascular reactivity was assessed by isometric tension studies. Superoxide was detected by dihydroethidium staining. Gene expression was measured by real-time polymerase chain reaction. NAD(P)H oxidase activity was measured using lucigenin-enhanced chemiluminescence. ALDH activity was measured biochemically, and NO consumption electrochemically. Nitrate tolerance was induced in rats by treatment with NTG for 3 days, and detected as impaired endothelium-dependent and -independent relaxation of aortic segments. Although superoxide production was increased in all aortic layers, expression of nox1, nox2 and nox4 was significantly decreased. Similarly, in vascular smooth muscle cells exposed to NTG for 6-24 h, NAD(P)H oxidase activity was increased, in spite of nox1 downregulation. In addition, expression and activity of ALDH-2 was decreased in nitrate-tolerant rings. Furthermore, exogenous addition of ALDH decreased superoxide generation in vitro and attenuated NO consumption in vascular smooth muscle cell homogenates. Our data suggest that in nitrate tolerance, activation of nox enzymes more than compensates for their downregulation, resulting in a net increase in superoxide and NO consumption. Furthermore, reduced ALDH-2 activity and expression leads to decreased NTG bioconversion. Therefore, both mechanisms reduce NO availability and impair vasorelaxation.
长期服用硝酸甘油（NTG）会导致硝酸盐耐受性。在可能的潜在机制中，血管活性氧（ROS）生成增加已成为主要机制。我们利用硝酸盐耐受性的细胞培养和动物模型，旨在评估硝酸盐对NAD（P）H氧化酶和醛脱氢酶2（ALDH2）表达的影响。用NTG处理大鼠和血管平滑肌细胞。通过等长张力研究评估血管反应性。用二氢乙锭染色检测超氧化物。通过实时聚合酶链反应测量基因表达。使用光泽精增强化学发光法测量NAD（P）H氧化酶活性。通过生化方法测量ALDH活性，并用电化学方法测量NO消耗。用NTG处理大鼠3天可诱导硝酸盐耐受性，并检测到主动脉段内皮依赖性和非内皮依赖性舒张受损。尽管所有主动脉层的超氧化物生成均增加，但nox1、nox2和nox4的表达均显著降低。同样，在暴露于NTG 6 - 24小时的血管平滑肌细胞中，尽管nox1下调，但NAD（P）H氧化酶活性仍增加。此外，在硝酸盐耐受性环中，ALDH - 2的表达和活性降低。此外，外源性添加ALDH可减少体外超氧化物生成，并减弱血管平滑肌细胞匀浆中的NO消耗。我们的数据表明，在硝酸盐耐受性中，nox酶的激活超过了对其下调的补偿，导致超氧化物和NO消耗净增加。此外，ALDH - 2活性和表达降低导致NTG生物转化减少。因此，这两种机制都会降低NO的可用性并损害血管舒张。
