Mechanism of riboflavine uptake by Caco-2 human intestinal epithelial cells

Abstract

The cellular and molecular regulation of intestinal absorption of the water-soluble vitamin riboflavine (RF) is poorly understood. The availability of a suitable in vitro cultured system that possesses the transport characteristics of the native intestinal absorptive cells would provide a powerful means to address this issue. In this study, we examined RF uptake by the human-derived cultured Caco-2 intestinal epithelial cells. RF uptake was Na+ and pH independent and occurred without metabolic alterations of the transported RF. Initial rate of RF uptake was temperature dependent and saturable as a function of concentration at 37 degrees C but not at 4 degrees C (apparent Michaelis constant = 0.30 +/- 0.03 microM, maximal velocity = 209.90 +/- 24.40 pmol.mg protein-1.3 min-1). Unlabeled RF, lumiflavine, 8-amino-riboflavine, isoriboflavine, and lumichrome in the incubation solution caused significant inhibition of RF uptake. RF uptake was also energy dependent and was sensitive to the inhibitory effect of sulfhydryl group reagents. The membrane transport inhibitor amiloride, but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid, furosemide, or probenecid, inhibited RF uptake in a competitive (inhibitory constant = 0.48 mM) and reversible manner. Growing Caco-2 monolayers in a RF-deficient and oversupplemented media caused significant up- and downregulation of RF uptake, respectively. These results demonstrate the existence of a carrier-mediated system for RF uptake by Caco-2 cells and provide new information regarding amiloride sensitivity, involvement of sulfhydryl groups, and up- and downregulation by the substrate level and clarify the controversy regarding the role of Na+ in the uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of High Concentration of Thiamin, Riboflavin and Pyridoxine across Intestinal Epithelial Cells Caco-2

“…The results confirmed the dual mechanism of transport for the studied vitamins and are compatible for the three vitamins and with previous published results ( [23][24][25][26][27][28][29][30][31]; however the experiments for thiamin should be widened to include different concentrations. For low concentrations, normally observed in the human diet, the active mechanism of transport is suggested.…”

“…However, human regulation of intes- tinal absorption is poorly understood. At low vitamin concentration, the results of the experiments showed that riboflavin uptake involves a specialized, carriermediated mechanism of absorption in the alimentary canal (22)(23)(24)(25). In vitro studies using rat everted jejunal sacs have shown that absorption of the vitamin is a saturable and energy-dependent process at physiological concentrations and a simple diffusion takes place at higher concentrations (26).…”

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

“…7, B-D) and was completely inhibited by riboflavin analogues (Fig. 7E) as previously reported and discussed (36). These results and previous reports suggested that hRFT1 could play, at least in part, a role in the absorption of riboflavin in the intestine as a high affinity transporter.…”

Functional Characterization of the Carrier-Mediated Transport System for Glycerol in Everted Sacs of the Rat Small Intestine

“…Since glycerol uptake was also inhibited by pCMBS, a thiol-modifying reagent, cysteine residues, which have a thiol group, seem to play an important role in the function of the carrier, as suggested for Na ϩ -dependent D-glucose transporter 1 (SGLT1) 8) and several other carriers. [9][10][11][12][13] At the higher concentration of 40 mM (Tables 1, 2), glycerol uptake was not altered by NaCl substitution or DNP, consistent with the suggestion that the contribution of passive transport is predominant.…”