Breen Christopher M, Sykes Destiny B, Fricker Gert, Miller David S
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Am J Physiol Renal Physiol. 2002 May;282(5):F877-85. doi: 10.1152/ajprenal.00171.2001.
We used confocal microscopy and quantitative image analysis to follow the movement of the fluorescent organic anion fluorescein (FL) from bath to cell and cell to blood vessel in intact rat lateral choroid plexus. FL accumulation in epithelial cells and underlying vessels was rapid, concentrative, and reduced by other organic anions. At steady state, cell fluorescence exceeded bath fluorescence by a factor of 3-5, and vessel fluorescence exceeded cell fluorescence by a factor of approximately 2. In cells, FL distributed between diffuse and punctate compartments. Cell and vessel accumulation of FL decreased when metabolism was inhibited by KCN, when bath Na(+) was reduced from 130 to 26 mM, and when the Na(+) gradient was collapsed with ouabain. Cell and vessel accumulation increased by >50% when 1-10 microM glutarate was added to the bath. Finally, transport of FL and carboxyfluorescein (generated intracellularly from carboxyfluorescein diacetate) from cell to blood vessel was greatly diminished when medium K(+) concentration ([K(+)]) was increased 10-fold. These results 1) validate a new approach to the study of choroid plexus function, and 2) indicate a two-step mechanism for transepithelial organic anion transport: indirect coupling of uptake to Na(+) at the apical membrane and electrical potential-driven efflux at the basolateral membrane.
我们利用共聚焦显微镜和定量图像分析技术，追踪了荧光有机阴离子荧光素（FL）在完整大鼠外侧脉络丛中从浴液到细胞以及从细胞到血管的移动过程。FL在上皮细胞和其下方血管中的积累迅速、呈浓缩状态，并且会被其他有机阴离子所减少。在稳态时，细胞荧光比浴液荧光高3至5倍，而血管荧光比细胞荧光高约2倍。在细胞中，FL分布于弥散和点状区室之间。当代谢被KCN抑制、浴液中Na(+)从130 mM降至26 mM以及用哇巴因使Na(+)梯度消失时，FL在细胞和血管中的积累减少。当向浴液中添加1至10 μM戊二酸时，细胞和血管中的积累增加超过50%。最后，当培养基K(+)浓度（[K(+)]）增加10倍时，FL和羧基荧光素（由羧基荧光素二乙酸酯在细胞内生成）从细胞到血管的转运大大减少。这些结果1）验证了一种研究脉络丛功能的新方法，并且2）表明了一种跨上皮有机阴离子转运的两步机制：在顶端膜处摄取与Na(+)的间接偶联以及在基底外侧膜处由电势驱动的流出。
