Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy

Abstract

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.