FLUID-PHASE ENDOCYTOSIS BY PRIMARY CULTURES OF BOVINE BRAIN MICROVESSEL ENDOTHELIAL-CELL MONOLAYERS
FLUID-PHASE ENDOCYTOSIS BY PRIMARY CULTURES OF BOVINE BRAIN MICROVESSEL ENDOTHELIAL-CELL MONOLAYERS
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DOI：
10.1016/0026-2862(90)90055-v
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发表时间：
1990-01-01
影响因子：
3.1
通讯作者：
RAUB, TJ
中科院分区：
文献类型：
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作者：
GUILLOT, FL;AUDUS, KL;RAUB, TJ
在牛脑微血管内皮细胞（BMEC）单层原代培养物中检查了血脑屏障（BBB）液相内吞作用。通过荧光光谱，观察到 BMEC 的荧光黄（LY，一种用作胞饮作用标记的荧光可溶性分子）积累在 0.05 至 1.0 mg/ml 的浓度范围内呈线性。 LY 的时间依赖性摄取表现出曲线动力学，由 0.5 mg/ml LY 下的初始快速摄取速率组成，每小时 1338 ng LY/mg 蛋白质。 20 分钟内，LY 积累速率减慢至每小时 23 ng LY/mg 蛋白质的稳态速率。在代谢抑制剂、氰化钾或2-脱氧葡萄糖存在下，LY的积累受到抑制，在4°C时，LY的积累有所减少，但并未完全受到抑制。脉冲追踪实验表明，LY的流出速度非常快，至少80%的积累的LY在2分钟内丢失，并且对低温不敏感。经过 30 分钟的追踪后，BMEC 最初积累的 LY 中只有 3-5% 仍与细胞相关。计算得出的内吞室总体积（每小时）的周转率分别比成纤维细胞和巨噬细胞的值小 8 至 20 倍。我们对这些数据的解释表明，大部分 LY 的外流涉及有限体积的快速回收隔室（可能是凹穴）的损失，这些隔室在积累过程中隔离了标记，并表明这些结果与目前对体内 BBB 胞饮作用的理解一致。
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.degree.. 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.