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.

Multidrug Transporter ABCG2/Breast Cancer Resistance Protein Secretes Riboflavin (Vitamin B2) into Milk

“…3A). This is in concordance with the accumulation of riboflavin in basolateral spaces and fluid-filled "domes" of MDCK-II cells (15) and may reflect the mechanism(s) responsible for reabsorption of riboflavin in the kidney and possibly in the intestine (9,20). Bcrp1 is apically located and should transport its substrates to the apical side of the monolayer, possibly counteracting the endogenous absorptive process (19).…”

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

“…In addition, the subject of riboflavin excretion has gained increasing attention. Jusko et al (18,19) and others (7,25,40,45) demonstrated saturable renal reabsorption and accumulation of riboflavin. Therefore, riboflavin transporters are thought to be essential for the maintenance of riboflavin homeostasis in the intestine and kidney.…”

Single‐ and two‐photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes

“…Although AF originating from the cytoplasmic region of mammalian cells has been studied by many researchers (Andersson et al, 1998;Aubin, 1979;Benson et al, 1979;Bondza-Kibangou et al, 2001;Croce et al, 1999;DaCosta et al, 2005;Georgakoudi et al, 2002;Matsui et al, 1998;Mayeno et al, 1992;Reinert et al, 2004), AF from plasma membranes of viable intact cells, less known and not thoroughly investigated, has been mentioned only in a few older reports (Lowy and Spring, 1990;Nokubo et al, 1988Nokubo et al, , 1989. These early reports did not provide detailed information about the spectral properties of the described AF.…”

“…It has been postulated that membrane AF was due to the presence of riboflavin transported to cells (Lowy and Spring, 1990). However, it is difficult to understand how a constant concentration of free riboflavin would be maintained by the cell in the region close to the plasma membrane.…”

“…It has been previously suggested that the fluorophore responsible for membrane AF was riboflavin transported to cells (Lowy and Spring, 1990). In order to verify that hypothesis, we altered riboflavin availability in the culture medium.…”