Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells
Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells
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DOI：
10.1074/jbc.m308160200
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发表时间：
2004-04-30
影响因子：
4.8
通讯作者：
Eichler, DC
中科院分区：
文献类型：
--
作者：
Goodwin, BL;Solomonson, LP;Eichler, DC
尽管精氨酸的细胞水平大大超过内皮一氧化氮合酶的表观 K-m，但目前的证据表明，大部分精氨酸可能无法用于一氧化氮 (NO) 的产生。我们提出精氨酸再生，即瓜氨酸再循环回精氨酸，定义了 NO 产生的精氨酸的重要来源。为了支持这一提议，使用RNA干扰分析来选择性降低精氨基琥珀酸合酶（AS）的表达，因为AS在内皮细胞中唯一已知的代谢作用是从L-瓜氨酸再生L-精氨酸。蛋白质印迹分析表明，尽管培养基中的精氨酸水平达到饱和，但由于 AS 小干扰 RNA 处理，AS 蛋白显着且呈剂量依赖性减少，并相应降低了产生基础或刺激水平 NO 的能力。然而，出乎意料的是，与对照细胞相比，AS 小干扰 RNA 处理的内皮细胞的活力显着降低。台盼蓝排除分析表明活力丧失并非由于坏死。 Bcl-2 表达减少和 caspase 活性增加这两个指标与 AS 表达减少直接相关，表明活力丧失是由于细胞凋亡所致。将细胞暴露于 NO 供体可防止与 AS 表达减少相关的细胞凋亡。总的来说，这些结果证明了 AS 对于内皮 NO 产生和细胞活力的重要作用。
Although cellular levels of arginine greatly exceed the apparent K-m for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide ( NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase ( AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of L-arginine from L-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.