首页> 外文期刊>American Journal of Physiology >Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging.
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Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging.

机译:单芥子碱,缺氧和衰老后内皮细胞中eNOS的胞质异常隔离:活细胞海绵体和胞质NO成像。

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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 Ca(2+)-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.
机译:我们之前曾报道过小窝/筏,高尔基束缚,N-乙基马来酰亚胺敏感因子附着蛋白(SNAP)受体蛋白(SNARE)和SNAP的功能紊乱,以及在培养的和暴露于大鼠肺的内皮细胞中抑制顺行运输用单芥子酚吡咯(MCTP)作为肺动脉高压发展的前奏。我们现在研究了1)这种运输阻滞是否影响亚细胞定位和内皮型一氧化氮(NO)合酶(eNOS)的功能,以及2)在缺氧和衰老后是否观察到高尔基阻滞和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”伴随着质膜中eNOS的丢失,并增加了细胞质中的积累。该细胞质eNOS被隔离在异质区室中,并与高尔基体和内质网(ER)标记,caveolin-1,NOSTRIN和ER Tracker局部共定位,但与Lyso Tracker无关。缺氧和衰老还导致PAEC增大,高尔基功能异常,质膜中eNOS丢失,并被细胞质隔离。用4,5-二氨基荧光素二乙酸酯(DAF-2DA)作为探针的海绵体和细胞质NO的活细胞成像显示,MCTP,缺氧和衰老后海绵体NO明显减少。尽管离子霉素在对照PAEC中刺激DAF-2DA荧光,但该离子载体在MCTP处理的和衰老的PAEC中降低了DAF-2DA荧光,表明eNOS定位于异常的细胞质区室,该区很容易被Ca(2+)诱导的胞吐作用排出。因此,单肾上腺素,低氧和衰老会在PAEC中产生高尔基阻滞,导致eNOS隔离在其功能性小窝位置,并提供了一种机制,尽管持续存在eNOS蛋白水平,但在实验性肺动脉高压中肺动脉NO水平经常减少。 。

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