Lowy R J, Spring K R
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Membr Biol. 1990 Jul;117(1):91-9. doi: 10.1007/BF01871568.
MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450-500 nm excitation/greater than 510 nm emission) or rhodamine (514 nm excitation/greater than 530 emission) type dyes. A second 5- to 10-fold brighter emission for 450-500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled "domes" and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 microM riboflavin resulted in accumulation in domes of 565 +/- 80 microM. The transport rate was calculated to be 189 +/- 30 pmol/min-cm2. One mM probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10 mM nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatible with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.
当通过微光水平视频显微镜检查时，MDCK细胞显示出具有两种不同模式的内源性荧光。在通常用于观察荧光素（450 - 500 nm激发/大于510 nm发射）或罗丹明（514 nm激发/大于530发射）型染料的发射和激发条件下，观察到一种点状的低强度发射，其与细胞器相关。对于450 - 500 nm激发，观察到第二种亮度高5至10倍的发射，其不同寻常之处在于每个细胞似乎都有轮廓。从光谱学以及使用成分改变的培养基获得的证据支持了这样的结论，即水溶性维生素核黄素积聚在基底外侧空间和充满液体的“穹顶”中，并且是这种荧光发射的来源。定量测量表明，将培养物暴露于10 microM核黄素会导致在穹顶中积聚565 +/- 80 microM。计算得出转运速率为189 +/- 30 pmol/min-cm2。1 mM丙磺舒是体内已知的核黄素转运抑制剂，可将转运降低至对照的54%，而10 mM几乎完全消除了摄取。结果表明，去除核黄素会将MDCK细胞荧光降低到与微光水平成像兼容的水平。此外，这些细胞主动转运核黄素，并为该过程提供了一种新的体外模型。
