Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging
Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging
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DOI：
10.1152/ajplung.00086.2009
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发表时间：
2009-10-01
影响因子：
4.9
通讯作者：
Sehgal, Pravin B.
中科院分区：
文献类型：
--
作者：
Lee, Jason;Reich, Reuben;Sehgal, Pravin B.
Lee J，赖希R，Xu F，Sehgal PB.暴露于野百合碱吡咯和NO清除的肺动脉内皮细胞中的高尔基体、运输和有丝分裂功能障碍。美国生理学杂志肺细胞分子生理学297：L715-L728，2009年。首次发表于2009年7月31日; doi：10.1152/ajplung.00086.2009。虽然野百合碱（MCT）给药到实验动物中被广泛使用的肺动脉高压（PAH）的调查，今天的基础细胞和亚细胞机制，最终在血管重塑不完全理解。暴露于野百合碱吡咯（MCTP）的培养物中的牛肺动脉内皮细胞（PAEC）在18-24小时后发展为“巨细胞症”，其特征在于具有增大的高尔基体的增大的超倍体细胞、远离质膜的内皮一氧化氮合酶的错误定位、减少的细胞表面/小窝一氧化氮（NO）以及小窝蛋白-1、网格蛋白重链、和N-乙基马来酰亚胺敏感因子。我们调查了MCTP是否确实影响功能性细胞内运输。NO清除剂（4-羧基苯基）-4，4，5，5-四甲基咪唑啉-1-氧基-3-氧化物（c-PTIO）和NO供体二乙胺NONOate用于比较。MCTP和c-PTIO在24-48小时内产生了明显的4 - 5倍放大的PAEC，高尔基体明显放大/分散，如高尔基体系链/基质蛋白giantin，GM 130和p115的免疫染色所示。活细胞摄取的高尔基体标记物C-5神经酰胺揭示了一个紧凑的非核高尔基体在未经处理的PAEC，明亮的标记扩大后MCTP核周围的高尔基体，但最低限度地标记后的高尔基体元件c-PTIO。NONOate减少了这些高尔基体的变化。在第一天的初始抑制后，MCTP和c-PTIO在接下来的4天内显著增强了可溶性货物（外源性载体表达的重组辣根过氧化物酶）的顺行分泌。使用荧光标记配体的活细胞内化测定显示MCTP和c-PTIO均抑制乙酰化低密度脂蛋白、转铁蛋白和霍乱毒素B的逆行摄取。此外，MCTP和c-PTIO在不同程度上降低了所有测定受体（LDLR、TfnR、BMPR、Tie-2和PECAM-1/CD 31）的细胞表面密度。在一个重要的区别，c-PTIO增强PAEC有丝分裂，但MCTP抑制有丝分裂，即使是由于c-PTIO，尽管明显夸大高尔基体分散。总之，这些数据定义了一个广谱高尔基体和亚细胞运输功能障碍综合征的内皮细胞暴露于MCTP或NO清除。
Lee J, Reich R, Xu F, Sehgal PB. Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging. Am J Physiol Lung Cell Mol Physiol 297: L715-L728, 2009. First published July 31, 2009; doi:10.1152/ajplung.00086.2009.-Although the administration of monocrotaline (MCT) into experimental animals is in widespread use today in investigations of pulmonary arterial hypertension (PAH), the underlying cellular and subcellular mechanisms that culminate in vascular remodeling are incompletely understood. Bovine pulmonary arterial endothelial cells (PAECs) in culture exposed to monocrotaline pyrrole ( MCTP) develop "megalocytosis" 18-24 h later characterized by enlarged hyperploid cells with enlarged Golgi, mislocalization of endothelial nitric oxide synthase away from the plasma membrane, decreased cell-surface/caveolar nitric oxide ( NO), and hypo-S-nitrosylation of caveolin-1, clathrin heavy chain, and N-ethylmaleimidesensitive factor. We investigated whether MCTP did in fact affect functional intracellular trafficking. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and the NO donor diethylamine NONOate were used for comparison. Both MCTP and c-PTIO produced distinctive four- to fivefold enlarged PAECs within 24-48 h with markedly enlarged/dispersed Golgi, as visualized by immunostaining for the Golgi tethers/matrix proteins giantin, GM130, and p115. Live-cell uptake of the Golgi marker C-5 ceramide revealed a compact juxtanuclear Golgi in untreated PAECs, brightly labeled enlarged circumnuclear Golgi after MCTP, but minimally labeled Golgi elements after c-PTIO. These Golgi changes were reduced by NONOate. After an initial inhibition during the first day, both MCTP and c-PTIO markedly enhanced anterograde secretion of soluble cargo ( exogenous vector-expressed recombinant horseradish peroxidase) over the next 4 days. Live-cell internalization assays using fluorescently tagged ligands showed that both MCTP and c-PTIO inhibited the retrograde uptake of acetylated low-density lipoprotein, transferrin, and cholera toxin B. Moreover, MCTP, and to a variable extent c-PTIO, reduced the cell-surface density of all receptors assayed ( LDLR, TfnR, BMPR, Tie-2, and PECAM-1/CD31). In an important distinction, c-PTIO enhanced mitosis in PAECs but MCTP inhibited mitosis, even that due to c-PTIO, despite markedly exaggerated Golgi dispersal. Taken together, these data define a broad-spectrum Golgi and subcellular trafficking dysfunction syndrome in endothelial cells exposed to MCTP or NO scavenging.