Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium
Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium
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DOI：
10.1152/ajpheart.00222.2004
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发表时间：
2004-09-01
影响因子：
4.8
通讯作者：
Schnitzer, JE
中科院分区：
文献类型：
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作者：
Czarny, M;Schnitzer, JE
最近，我们发现中性鞘磷脂酶（N-SMase）集中在小窝中的内皮细胞表面，并通过大鼠肺脉管系统中流速和压力的增加而被激活以急性和瞬时方式产生神经酰胺（Czarny M，Liu J，Oh P，and Schnitzer JE，J Biol Chem 278：4424 - 4430，2003）。在这里，我们进一步报告我们的调查，这一新的急性机械传导途径。我们采用三种实验模型来探索N-SMase和神经酰胺在机械信号传导中的作用：1）使用大鼠肺内皮的分离的管腔质膜的无细胞体外模型; 2）使用培养的完整牛主动脉内皮细胞单层的流体剪切应力模型;和3）使用大鼠肺脉管系统的受控灌注的原位模型。Scyphostatin，特异性抑制N-SMase，但不酸性SMase活性，防止机械活化的N-SMase以及下游酪氨酸和丝裂原活化蛋白激酶。细胞可渗透的神经酰胺类似物（N-乙酰鞘氨醇、C2-神经酰胺和N-己酰鞘氨醇、C6-神经酰胺），但非无活性的二氢衍生物D2-神经酰胺和D 6-神经酰胺（分别为N-乙酰-二氢鞘氨醇和N-己酰二氢鞘氨醇）模拟细胞表面蛋白的快速机械诱导的酪氨酸磷酸化以及Src样激酶的机械活化和细胞外调节激酶途径。神经酰胺和机械应力共同的反应被抑制染料木素，herbamycin A，和PP 2，但不PP 3，这表明一个专性的作用Src样激酶在神经酰胺介导的mechanotransduction。神经酰胺还诱导丝氨酸/苏氨酸磷酸化以激活Akt/内皮型一氧化氮合酶途径。因此，N-SMase在质膜在小窝可能是一个上游启动mechanosensor，它急性触发mechanosensor通过产生的脂质第二信使神经酰胺。
Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny M, Liu J, Oh P, and Schnitzer JE, J Biol Chem 278: 4424 - 4430, 2003). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited N-SMase but not acid SMase activity, prevented mechanoactivation of N-SMase as well as downstream tyrosine and mitogen-activated protein kinases. Cell-permeable ceramide analogs (N-acetylsphingosine, C2-ceramide, and N-hexanoylsphingosine, C6-ceramide) but not the inactive dihydroderivatives D2-ceramide and D6-ceramide (N-acetyl-sphinganine and N-hexanoylsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins as well as mechanoactivation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3, which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induced serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger ceramide.