Rate, extent and concentration dependence of histamine-evoked Weibel-Palade body exocytosis determined from individual fusion events in human endothelial cells
Rate, extent and concentration dependence of histamine-evoked Weibel-Palade body exocytosis determined from individual fusion events in human endothelial cells
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DOI：
10.1113/jphysiol.2007.132993
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发表时间：
2007-08-15
影响因子：
5.5
通讯作者：
Carter, T.
中科院分区：
文献类型：
--
作者：
Erent, A.;Meli, A.;Carter, T.
用时间分辨荧光显微镜观察了表达WPB靶向增强绿色荧光蛋白嵌合体（EGFP）的人脐静脉内皮细胞组胺诱导的韦贝尔-帕拉德体（WPB）胞外分泌的速率、浓度依赖性和程度。单个WPB的胞吐表现为EGFP荧光增强、形态改变和WPB含量释放。荧光增强是由于wpb内pH从静息水平上升，估计pH为5.45 +/- 0.26（标准差，n = 144），至pH 7.40。它与细胞外Alexa-647的摄取一致，表明在荧光内容物丢失之前形成融合孔。组胺引起的细胞内游离钙离子浓度升高与第一次融合事件之间的延迟时间为0.3 μ m组胺时10.0 +/- 4.42 s （n = 9个细胞），而在100 μ m组胺时为1.57 +/- 0.21 s （n = 15个细胞），表明组胺引起的WPB胞外分泌存在一个或多个缓慢过程。组胺浓度为0.3 μ m时最大胞吐率为1.20 +/- 0.16 WPB s(-1) (n = 9)，组胺浓度为100 μ m时最大胞吐率为3.66 +/- 0.45 WPB s(-1) （n = 15）。在加入组胺后2-5 s出现，并随着持续刺激下降到更低的水平。胞吐的初始延迟和最大速率不受去除外部Ca2+的影响，这表明最初的分泌爆发是由内部储存的Ca2+释放驱动的，但持续的胞吐需要外部Ca2+。将数据与最强促分泌离子霉素最大浓度（1 μ M）引起的胞吐进行比较，其中钙升高与分泌之间的延迟时间为1.67 +/- 0.24 s(n = 6)，峰值融合率类似于10 WPB s（-1）。
The rate, concentration dependence and extent of histamine-evoked Weibel-Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45 +/- 0.26 (S.D., n = 144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 +/- 4.42 s (n = 9 cells) at 0.3 mu m histamine and 1.57 +/- 0.21 s (n = 15 cells) at 100 mu m histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 +/- 0.16 WPB s(-1) (n = 9) at 0.3 mu m and 3.66 +/- 0.45 WPB s(-1) at 100 mu m histamine (n = 15). These occurred 2-5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 mu M), for which there was a delay between calcium elevation and secretion of 1.67 +/- 0.24 s (n = 6), and a peak fusion rate of similar to 10 WPB s(-1).