Riboflavin uptake by human-derived colonic epithelial NCM460 cells

Normal microflora of the large intestine synthesize a number of water-solub le vitamins including riboflavin (RF). Recent studies have shown that colon ic epithelial cells posses an efficient carrier-mediated mechanism for abso rbing some of these micronutrients. The aim of the present study was to det ermine whether colonic cells also posses a carrier-mediated mechanism for R F uptake and, if so, to characterize this mechanism and study its cellular regulation. Confluent monolayers of the human-derived nontransformed coloni c epithelial cells NCM460 and [H-3] RF were used in the study. Uptake of RF was found to be 1) appreciable and temperature and energy dependent; 2) Na + independent; 3) saturable as a function of concentration with an apparent K-m of 0.14 mu M and V-max of 3.29 pmol.mg protein(-1).3 min(-1); 4) inhib ited by the structural analogs lumiflavin and lumichrome (K-i of 1.8 and 14 .1 mu M, respectively) but not by the unrelated biotin; 5) inhibited in a c ompetitive manner by the membrane transport inhibitor amiloride (K-i = 0.86 mM) but not by furosemide, DIDS, or probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and d ownregulation in RF uptake in RF-deficient and oversupplemented conditions, respectively; and 7) modulated by an intracellular Ca2+/calmodulin-mediate d pathway. These studies demonstrate for the first time the existence of a specialized carrier-mediated mechanism for RF uptake in an in vitro cellula r model system of human colonocytes. This mechanism appears to be regulated by extracellular substrate level and by an intracellular Ca2+/calmodulin-m ediated pathway. It is suggested that the identified transport system may b e involved in the absorption of bacterially synthesized RF in the large int estine and that this source of RF may contribute toward RF homeostasis, esp ecially that of colonocytes.