MECHANISM OF RIBOFLAVINE UPTAKE BY CACO-2 HUMAN INTESTINAL EPITHELIAL-CELLS

Mechanism of riboflavine uptake by Caco-2 human intestinal epithelial cells. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G15-G21, 1994.-The cellular and molecular regulation of intestinal absorption of the water-soluble vitamin riboflavine (RF) is poorly understood. Th e availability of a suitable in vitro cultured system that possesses t he 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 wi thout metabolic alterations of the transported RF. Initial rate of RF uptake was temperature dependent and saturable as a function of concen tration at 37 degrees C but not at 4 degrees C (apparent Michaelis con stant = 0.30+/-0.03 mu M, maximal velocity = 209.90+/-24.40 pmol.mg pr otein(-1).3 min-l). Unlabeled RF, lumiflavine, 8-amino-riboflavine, is oriboflavine, and lumichrome in the incubation solution caused signifi cant inhibition of RF uptake. RF uptake was also energy dependent and was sensitive to the inhibitory effect of sulfhydryl group reagents. T he membrane transport inhibitor amiloride, but not 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid, -acetamide-4'-isothiocyanostilbene-2,2' -disulfonic acid, furosemide, or probenecid, inhibited RF uptake in a competitive (inhibitory constant = 0.48 mM) and reversible manner. Gro wing Caco-2 monolayers in a RF-deficient and oversupplemented media ca used significant up- and downregulation of RF uptake, respectively. Th ese results demonstrate the existence of a carrier-mediated system for RF uptake by Caco-2 cells and provide new information regarding amilo ride sensitivity, involvement of sulfhydryl groups, and up- and downre gulation by the substrate level and clarify the controversy regarding the role of Na+ in the uptake process. These results also demonstrate the suitability of Caco-2 cells as an in vitro cultured model system f or studying the regulation of RF intestinal transport.