Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole.
Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole.
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DOI：
10.1152/ajpheart.00642.2008
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发表时间：
2008-11
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影响因子：
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通讯作者：
Somshuvra Mukhopadhyay;Jason E. Lee;P. Sehgal
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作者：
Somshuvra Mukhopadhyay;Jason E. Lee;P. Sehgal
血管相关蛋白调控s -亚硝基化和脱硝基化的研究是血管生物学的一个新兴领域。我们之前的研究表明，单罗塔碱吡罗（MCTP）诱导的肺动脉内皮细胞（PAECs）巨细胞增生是肺动脉高压的基础，与高尔基阻滞有关，其特征是高尔基体中多种囊泡系链、可溶性n -乙基酰亚胺敏感因子（NSF）附着蛋白受体（SNAREs）和可溶性NSF附着蛋白（SNAPs）被捕获；减少了高尔基体向质膜输送小室蛋白-1 （cav-1）和内皮一氧化氮合成酶（eNOS）；空洞NO减少。我们研究了参与所有SNARE分解的atp酶NSF是否可能是MCTP的上游靶点，以及MCTP是否可能通过s -亚硝基化调节NSF。免疫荧光显微镜和高尔基体纯化技术显示，MCTP后高尔基膜中NSF减少了约50%，尽管α - snap、cav-1、eNOS和syntaxin-6增加。用生物素开关法测定，NO清除剂（4-羧基苯基）-4,4,5,5-四甲基咪唑啉-1-氧-3-氧化物虽然能降低4,5-二氨基荧光素双乙酸酯的荧光，抑制eNOS的s -亚硝基化，但对MCTP巨细胞增多症的发生或进展没有影响。此外，后者不仅显示paec中NSF、eNOS、cav-1和网格蛋白重链（CHC）的组成性s -亚硝基化，而且MCTP后NSF、eNOS、cav-1和CHC的s -亚硝基化显著降低70-95%。这些数据表明，高尔基膜中NSF的耗竭是MCTP后高尔基阻断的机制，血管相关蛋白的脱硝基化对内皮细胞巨细胞增多症的发展至关重要。
Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by approximately 50% in Golgi membranes after MCTP despite increases in alpha-SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70-95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.