Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging
Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging
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DOI：
10.1152/ajpheart.00990.2006
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发表时间：
2007-03-01
影响因子：
4.8
通讯作者：
Sehgal, Pravin B.
中科院分区：
文献类型：
--
作者：
Mukhopadhyay, Somshuvra;Xu, Fang;Sehgal, Pravin B.
我们之前报道过，小窝/筏的破坏、高尔基体系链、N-乙基马来酰亚胺敏感因子附着蛋白（SNARE）和SNAP的功能障碍，以及暴露于野百合碱吡咯（MCTP）的培养物和大鼠肺中内皮细胞顺行运输的抑制是肺动脉高压发生的前奏。我们现在研究了 1) 这种运输阻断是否影响内皮一氧化氮 (NO) 合酶 (eNOS) 的亚细胞定位和功能，以及 2) 在缺氧和衰老后是否观察到高尔基体阻断和 eNOS 隔离。免疫荧光数据显示，MCTP 诱导的肺动脉内皮细胞 (PAEC) 的“巨细胞增多”伴随着质膜 eNOS 的损失，并且细胞质中的积累增加。这种细胞质 eNOS 被隔离在异质区室中，并部分与高尔基体和内质网 (ER) 标记物、caveolin-1、NOSTRIN 和 ER Tracker 共定位，但不与 Lyso Tracker 共定位。缺氧和衰老也会导致 PAEC 增大，导致高尔基体功能失调，质膜 eNOS 丢失，并被隔离在细胞质中。以 4,5-二氨基荧光素二乙酸酯 (DAF-2DA) 作为探针对小凹和细胞质 NO 进行活细胞成像，结果显示在 MCTP、缺氧和衰老后小凹 NO 显着损失。虽然离子霉素刺激对照 PAEC 中的 DAF-2DA 荧光，但这种离子载体降低了 MCTP 处理和衰老 PAEC 中的 DAF-2DA 荧光，表明 eNOS 定位在异常的细胞质区室中，该区室很容易通过 Ca2+ 诱导的胞吐作用排出。因此，野百合碱、缺氧和衰老会在 PAEC 中产生高尔基体阻断，导致 eNOS 远离其功能性小窝位置，并为实验性肺动脉高压中经常报道的肺动脉 NO 水平降低提供了一种机制，尽管 eNOS 蛋白水平持续存在。
We previously reported the disruption of caveolae/rafts, dysfunction of Golgi tethers, N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptor proteins (SNAREs), and SNAPs, and inhibition of anterograde trafficking in endothelial cells in culture and rat lung exposed to monocrotaline pyrrole (MCTP) as a prelude to the development of pulmonary hypertension. We have now investigated 1) whether this trafficking block affects subcellular localization and function of endothelial nitric oxide (NO) synthase (eNOS) and 2) whether Golgi blockade and eNOS sequestration are observed after hypoxia and senescence. Immunofluorescence data revealed that MCTP-induced "megalocytosis" of pulmonary arterial endothelial cells (PAEC) was accompanied by a loss of eNOS from the plasma membrane, with increased accumulation in the cytoplasm. This cytoplasmic eNOS was sequestered in heterogeneous compartments and partially colocalized with Golgi and endoplasmic reticulum (ER) markers, caveolin-1, NOSTRIN, and ER Tracker, but not Lyso Tracker. Hypoxia and senescence also produced enlarged PAEC, with dysfunctional Golgi and loss of eNOS from the plasma membrane, with sequestration in the cytoplasm. Live-cell imaging of caveolar and cytoplasmic NO with 4,5-diaminofluorescein diacetate (DAF-2DA) as probe showed a marked loss of caveolar NO after MCTP, hypoxia, and senescence. Although ionomycin stimulated DAF-2DA fluorescence in control PAEC, this ionophore decreased DAF-2DA fluorescence in MCTP-treated and senescent PAEC, suggesting localization of eNOS in an aberrant cytoplasmic compartment that was readily discharged by Ca2+-induced exocytosis. Thus monocrotaline, hypoxia, and senescence produce a Golgi blockade in PAEC, leading to sequestration of eNOS away from its functional caveolar location and providing a mechanism for the often-reported reduction in pulmonary arterial NO levels in experimental pulmonary hypertension, despite sustained eNOS protein levels.