Palytoxin-induced cell death cascade in bovine aortic endothelial cells.
Palytoxin-induced cell death cascade in bovine aortic endothelial cells.
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DOI：
10.1152/ajpcell.00063.2006
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发表时间：
2006-10
期刊：
影响因子：
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通讯作者：
W. Schilling;D. Snyder;W. Sinkins;M. Estacion
中科院分区：
文献类型：
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作者：
W. Schilling;D. Snyder;W. Sinkins;M. Estacion
质膜Na（+）-K（+）-ATPase（NKA）泵是海洋毒素Palytoxin（PTX）的受体。PTX与NKA结合，并将泵转化为对Ca（2+）表现出轻微渗透性的单价阳离子通道。然而，PTX通过Na（+）泵通道直接增加胞质游离Ca（2+）浓度（[Ca（2+）]（i））和启动Ca（2+）过载诱导的肿瘤细胞死亡的能力尚未被检测。因此，本研究的目的是确定PTX对牛主动脉内皮细胞[Ca（2+）]（i）和与细胞死亡相关的下游事件的影响。PTX（3-100 nM）产生依赖于细胞外Ca（2+）的[Ca（2+）]（i）的分级增加。100 nM PTX引起的[Ca（2+）]（i）增加可被哇巴因预处理阻断，IC（50）< 1 μ M。[Ca（2+）]（i）的升高可以通过在PTX后的不同时间加入哇巴因来逆转，但这需要更高浓度的哇巴因（0.5 mM）。这些结果表明PTX诱导的[Ca（2+）]（i）升高通过Na（+）泵发生。在[Ca（2+）]（i）升高之后，PTX还引起活体染料溴化乙锭（EB）摄取的浓度依赖性增加，但不引起YO-PRO-1摄取的浓度依赖性增加。在PTX之前或之后加入哇巴因也阻断EB摄取。延时视频显微镜显示，PTX最终导致细胞溶解，如瞬时表达的绿色荧光蛋白（分子量27 kDa）的释放和碘化丙啶的快速摄取所示。细胞溶解1）通过去除细胞外Ca（2+）或在PTX后加入哇巴因大大延迟，2）被细胞保护性氨基酸甘氨酸阻断，3）伴随着戏剧性的膜起泡。这些结果表明，PTX启动细胞死亡级联特征的Ca（2+）超载。
The plasmalemmal Na(+)-K(+)-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca(2+). However, the ability of PTX to directly increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via Na(+) pump channels and to initiate Ca(2+) overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca(2+)](i) and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca(2+)](i) that was dependent on extracellular Ca(2+). The increase in [Ca(2+)](i) initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC(50) < 1 microM. The elevation in [Ca(2+)](i) could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca(2+)](i) occurs via the Na(+) pump. Subsequent to the rise in [Ca(2+)](i), PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca(2+) or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca(2+) overload.