Guillot F L, Audus K L, Raub T J
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66045.
Microvasc Res. 1990 Jan;39(1):1-14. doi: 10.1016/0026-2862(90)90055-v.
Blood-brain barrier (BBB) fluid-phase endocytosis was examined in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. By fluorescence spectroscopy. Lucifer yellow (LY, a fluorescent, soluble molecule used as a marker for pinocytosis) accumulation by BMEC was observed to be linear over a concentration range of 0.05 to 1.0 mg/ml. Time-dependent uptake of LY exhibited curvilinear kinetics composed of an initially rapid uptake rate of 1338 ng of LY/mg protein per hour at 0.5 mg/ml LY. Within 20 min, the rate of LY accumulation slowed to a steady-state rate of 23 ng of LY/mg protein per hour. Accumulation of LY was inhibited in the presence of metabolic inhibitors, potassium cyanide or 2-deoxyglucose, and was decreased, but not completely inhibited, at 4 degrees. Pulse-chase experiments revealed that efflux of LY was very rapid with at least 80% of the accumulated LY being lost within 2 min and was not sensitive to low temperature. Only 3-5% of the LY initially accumulated by BMEC remained cell-associated after a 30-min chase. The calculated turnover of the endocytic compartment's total volume (per hour) is 8- to 20-fold less than values for fibroblasts and macrophages, respectively. We have interpreted these data to suggest that the efflux of most of the LY involves loss from a rapidly recycled compartment of finite volume, possibly caveolae, that had sequestered marker during accumulation and suggest that these results are consistent with the present understanding of BBB pinocytosis in vivo.
在牛脑微血管内皮细胞（BMEC）单层原代培养物中检测血脑屏障（BBB）液相内吞作用。通过荧光光谱法，观察到BMEC对荧光素黄（LY，一种用作胞饮作用标记的荧光可溶性分子）的积累在0.05至1.0 mg/ml的浓度范围内呈线性。LY的时间依赖性摄取呈现曲线动力学，在0.5 mg/ml LY时，初始摄取速率为每小时1338 ng LY/mg蛋白质。在20分钟内，LY积累速率减缓至每小时23 ng LY/mg蛋白质的稳态速率。在代谢抑制剂、氰化钾或2-脱氧葡萄糖存在的情况下，LY的积累受到抑制，并且在4℃时积累减少但未完全抑制。脉冲追踪实验表明，LY的外排非常迅速，至少80%积累的LY在2分钟内丢失，并且对低温不敏感。在30分钟的追踪后，最初由BMEC积累的LY中只有3-5%仍与细胞相关。计算得出的内吞区室总体积（每小时）的周转率分别比成纤维细胞和巨噬细胞的值低8至20倍。我们对这些数据的解释表明，大部分LY的外排涉及从有限体积的快速循环区室（可能是小窝）中丢失，该区室在积累过程中隔离了标记物，并表明这些结果与目前对体内BBB胞饮作用的理解一致。
