Kerper L E, Hinkle P M
Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Toxicol Appl Pharmacol. 1997 Sep;146(1):127-33. doi: 10.1006/taap.1997.8234.
The mechanism by which lead crosses the blood-brain barrier is not known. Brain capillary endothelial cells, which form tight junctions with each other, are an important component of the blood-brain barrier. Lead must traverse these cells to reach the brain. In the present study, uptake of lead was followed in primary cultures of bovine brain capillary endothelial cells. Lead uptake into cells was measured by monitoring the fluorescence of cells loaded with indo-1 at a wavelength where indo-1 fluorescence is independent of calcium but quenched by binding of lead. Lead uptake was visualized with digital images recorded with a fluorescence microscope. Lead added to the extracellular medium caused fluorescence quench over time which was reversed upon addition of a membrane permeant heavy metal chelator. Lead uptake by cells in suspension, measured by fluorescence spectroscopy, exhibited time and concentration dependence. Lead uptake was enhanced following depletion of intracellular Ca2+ stores by the addition of thapsigargin, cyclopiazonic acid, or tert-butylhydroquinone, inhibitors of the sarco/endoplasmic reticulum calcium ATPase. SK&F 96365, which blocks store-operated calcium channels, inhibited the stimulation of lead uptake by thapsigargin. These results indicate that indo-1 fluorescence quench is a useful method for investigation of lead uptake in brain capillary endothelial cells. Furthermore, entry of lead into these cells is activated by the depletion of intracellular Ca2+ stores and may occur via store-operated cation channels.
铅穿过血脑屏障的机制尚不清楚。脑毛细血管内皮细胞相互形成紧密连接，是血脑屏障的重要组成部分。铅必须穿过这些细胞才能到达大脑。在本研究中，对牛脑毛细血管内皮细胞的原代培养物中铅的摄取进行了跟踪。通过监测加载indo-1的细胞在indo-1荧光与钙无关但因铅结合而淬灭的波长下的荧光来测量细胞对铅的摄取。用荧光显微镜记录的数字图像观察铅的摄取。添加到细胞外培养基中的铅会导致荧光随时间淬灭，加入膜通透性重金属螯合剂后荧光会逆转。通过荧光光谱法测量悬浮细胞对铅的摄取，显示出时间和浓度依赖性。通过添加毒胡萝卜素、环匹阿尼酸或叔丁基对苯二酚（肌浆网/内质网钙ATP酶抑制剂）耗尽细胞内Ca2+储存后，铅的摄取增加。阻断储存操纵性钙通道的SK&F 96365抑制了毒胡萝卜素对铅摄取的刺激作用。这些结果表明，indo-1荧光淬灭是研究脑毛细血管内皮细胞铅摄取的一种有用方法。此外，细胞内Ca2+储存的耗尽激活了铅进入这些细胞的过程，并且可能通过储存操纵性阳离子通道发生。
