Zupancic G, Ogden D, Magnus C J, Wheeler-Jones C, Carter T D
University of Ljubljana, Department of Biology, Vec na Pot 111, POB 2995, Slovenia.
J Physiol. 2002 Nov 1;544(3):741-55. doi: 10.1113/jphysiol.2002.027490.
Endothelial cells secrete a range of procoagulant, anticoagulant and inflammatory proteins by exocytosis to regulate blood clotting and local immune responses. The mechanisms regulating vesicular exocytosis were studied in human umbilical vein endothelial cells (HUVEC) with high-resolution membrane capacitance (C(m)) measurements. The total whole-cell C(m) and the amplitudes and times of discrete femtoFarad (fF)-sized C(m) steps due to exocytosis and endocytosis were monitored simultaneously. Intracellular calcium concentration Ca(2+) was elevated by intracellular photolysis of calcium-DM-nitrophen to evoke secretion and monitored with the low-affinity Ca(2+) indicator furaptra. Sustained elevation of Ca(2+) to > 20 microM evoked large, slow increases in C(m) of up to 5 pF in 1-2 min. Exocytotic and endocytotic steps of amplitude 0.5-110 fF were resolved, and accounted on average for ~33 % of the total C(m) change. A prominent component of C(m) steps of 2.5-9.0 fF was seen and could be attributed to exocytosis of von-Willebrand-factor-containing Weibel-Palade bodies (WPb), based on the near-identical distributions of capacitance step amplitudes, with calculated estimates of WPb capacitance from morphometry, and on the absence of 2.5-9.0 fF C(m) steps in cells deficient in WPb. WPb secretion was delayed on average by 23 s after Ca(2+) elevation, whereas total C(m) increased immediately due to the secretion of small, non-WPb granules. The results show that following a large increase of Ca(2+), corresponding to strong stimulation, small vesicular components are immediately available for secretion, whereas the large WPb undergo exocytosis only after a delay. The presence of events of magnitude 9-110 fF also provides evidence of compound secretion of WPb due to prior fusion of individual granules.
内皮细胞通过胞吐作用分泌一系列促凝血、抗凝血和炎症蛋白，以调节血液凝固和局部免疫反应。利用高分辨率膜电容（C(m)）测量技术，在人脐静脉内皮细胞（HUVEC）中研究了调节囊泡胞吐作用的机制。同时监测全细胞总C(m)以及由于胞吐和胞吞作用导致的离散飞法（fF）大小的C(m)阶跃的幅度和时间。通过钙-DM-硝基苯酚的细胞内光解升高细胞内钙浓度Ca(2+)以引发分泌，并使用低亲和力Ca(2+)指示剂呋喃普拉进行监测。Ca(2+)持续升高至>20 microM会在1-2分钟内引起C(m)大幅缓慢增加，增幅高达5 pF。分辨出幅度为0.5-110 fF的胞吐和胞吞步骤，平均占总C(m)变化的约33%。观察到2.5-9.0 fF的C(m)步骤有一个突出成分，基于电容阶跃幅度的近乎相同分布、形态测量学计算的Weibel-Palade小体（WPb）电容估计值，以及WPb缺陷细胞中不存在2.5-9.0 fF的C(m)步骤，可将其归因于含血管性血友病因子的WPb的胞吐作用。Ca(2+)升高后，WPb分泌平均延迟23秒，而由于小的非WPb颗粒的分泌，总C(m)立即增加。结果表明，在Ca(2+)大幅增加（对应于强烈刺激）后，小囊泡成分可立即用于分泌，而大的WPb仅在延迟后才进行胞吐作用。9-110 fF大小事件的存在也提供了由于单个颗粒先前融合导致WPb复合分泌的证据。
