Contrasting effects of thiol-modulating agents on endothelial NO bioactivity
Contrasting effects of thiol-modulating agents on endothelial NO bioactivity
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DOI：
10.1152/ajpcell.2001.281.2.c719
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发表时间：
2001-08-01
影响因子：
5.5
通讯作者：
Keaney, JF
中科院分区：
文献类型：
--
作者：
Huang, AN;Xiao, H;Keaney, JF
内皮源性一氧化氮（NO）的生物活性是血管稳态的重要组成部分，对细胞内氧化还原状态敏感。由于谷胱甘肽（GSH）是细胞内氧化还原状态的主要决定因素，我们试图确定其在调节内皮NO生物活性的作用。在猪主动脉内皮细胞（PAEC）中，我们使用1）丁硫堇-（S，R）-磺酰亚胺（BSO）耗尽细胞内的GSH（> 70%），它抑制GSH合成; 2）二胺，它氧化硫醇;或3）1-氯-2，4-二硝基苯（CDNB），推测通过谷胱甘肽S-转移酶活性耗尽GSH。用BSO耗竭细胞GSH对A-23187诱导的cGMP积累测定的内皮NO生物活性无影响。相比之下，用二酰胺氧化细胞内硫醇可抑制A-23187诱导的cGMP蓄积和cGMP对外源性NO的反应。PAEC、PAEC膜组分或纯化的内皮型一氧化氮合酶（eNOS）的二酰胺处理导致eNOS催化活性显著抑制（类似于75%），测定为L-[H-3]精氨酸转化为L-[H-3]瓜氨酸。这种作用似乎与eNOS硫醇的氧化有关，因为它被二硫苏糖醇完全逆转。谷氨酸耗尽与CDNB抑制A-23187刺激cGMP的积累，但不是cGMP对外源性NO的反应。相反，CDNB治疗受损的eNOS催化活性在完整的PAEC，这种效果被逆转过量的NADPH在分离纯化的eNOS测定。与这些结果一致，我们发现了光谱证据表明CDNB与NADPH反应，并使其作为eNOS或谷胱甘肽还原酶的辅因子失活。因此，巯基调节剂发挥多效性作用的内皮细胞NO的生物活性，这些数据可能有助于解决一些相互矛盾的先前的研究连接谷胱甘肽状态与内皮细胞NO的生物活性。
The bioactivity of endothelium-derived nitric oxide (NO) is an important component of vascular homeostasis that is sensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define its role in modulating endothelial NO bioactivity. In porcine aortic endothelial cells (PAECs), we depleted intracellular GSH (>70%) using 1) buthionine-(S, R)-sulfoximine (BSO), which inhibits GSH synthesis; 2) diamide, which oxidizes thiols; or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putatively depletes GSH through glutathione S-transferase activity. Cellular GSH depletion with BSO had no effect on endothelial NO bioactivity measured as A-23187-induced cGMP accumulation. In contrast, oxidation of intracellular thiols with diamide inhibited both A-23187-induced cGMP accumulation and the cGMP response to exogenous NO. Diamide treatment of either PAECs, PAEC membrane fractions, or purified endothelial nitric oxide synthase (eNOS) resulted in significant inhibition (similar to 75%) of eNOS catalytic activity measured as L-[H-3] arginine- to-L-[H-3] citrulline conversion. This effect appeared related to oxidation of eNOS thiols as it was completely reversed by dithiothreitol. Glutathione depletion with CDNB inhibited A-23187-stimulated cGMP accumulation but not the cGMP response to exogenous NO. Rather, CDNB treatment impaired eNOS catalytic activity in intact PAECs, and this effect was reversed by excess NADPH in isolated purified eNOS assays. Consistent with these results, we found spectral evidence that CDNB reacts with NADPH and renders it inactive as a cofactor for either eNOS or glutathione reductase. Thus thiol-modulating agents exert pleiotropic effects on endothelial NO bioactivity, and these data may help to resolve a number of conflicting previous studies linking GSH status with endothelial cell NO bioactivity.