Shah C M, Bell S E, Locke I C, Chowdrey H S, Gordge M P
Department of Biomedical Science, University of Westminster, 115 New Cavendish Street, London, UK.
Nitric Oxide. 2007 Feb;16(1):135-42. doi: 10.1016/j.niox.2006.08.001. Epub 2006 Aug 11.
In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.
在本研究中，我们调查了蛋白质二硫键异构酶（PDI）在S-亚硝基谷胱甘肽（GSNO）和S-亚硝基白蛋白（albSNO）快速代谢以及这些化合物向细胞内递送一氧化氮（NO）过程中的作用。将GSNO或albSNO（1微摩尔）与巨核细胞系MEG-01一起孵育，导致细胞介导的对每种化合物的清除，这种清除可被用5,5'-二硫代双（2-硝基苯甲酸）（DTNB）（100微摩尔）封闭细胞表面硫醇或用杆菌肽（5毫摩尔）抑制PDI所抑制。GSNO而非albSNO能迅速抑制血小板聚集并刺激环磷酸鸟苷（cGMP）积累（用作细胞内NO进入的指标）。用杆菌肽或DTNB处理MEG-01可抑制对GSNO（1微摩尔）的cGMP积累，提示PDI和表面硫醇在NO递送中起作用。PDI活性存在于MEG-01条件培养基中，并被高浓度的GSNO（500微摩尔）抑制。使用不可渗透的硫醇特异性探针3-（N-马来酰亚胺丙酰基）生物素（MPB）标记了许多细胞表面含硫醇的蛋白质。用GSNO预处理细胞导致部分而非全部蛋白质硫醇反应性丧失，提示细胞表面硫醇的选择性修饰。免疫沉淀实验表明，GSNO导致PDI硫醇反应性呈浓度依赖性丧失。我们的数据表明，PDI参与GSNO的快速代谢和细胞内NO递送，并且在此过程中PDI自身通过硫醇修饰而改变。相比之下，PDI介导的albSNO代谢与NO信号传导的相关性尚不确定。
