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Veterans Affairs Medical Center, Long Beach 90822; Veterans Affairs Medical Center, Sepulveda 91343; Departments of Medicine and Physiology/Biophysics, University of California at Irvine, Irvine 92697; Department of Medicine, University of California at Los Angeles, Los Angeles, California 90024; and INCELL Corporation, San Antonio, Texas 78249
Normal microflora of
the large intestine synthesize a number of water-soluble vitamins
including riboflavin (RF). Recent studies have shown that colonic
epithelial cells posses an efficient carrier-mediated mechanism for
absorbing some of these micronutrients. The aim of the present study
was to determine whether colonic cells also posses a carrier-mediated
mechanism for RF uptake and, if so, to characterize this mechanism and
study its cellular regulation. Confluent monolayers of the
human-derived nontransformed colonic epithelial cells NCM460 and
[3H]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 Km
of 0.14 µM and Vmax of 3.29 pmol · mg
protein
1 · 3 min1; 4) inhibited by the structural analogs
lumiflavin and lumichrome (Ki of 1.8 and 14.1 µM,
respectively) but not by the unrelated biotin; 5) inhibited in
a competitive manner by the membrane transport inhibitor amiloride
(Ki = 0.86 mM) but not by furosemide, DIDS, or
probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF
uptake in RF-deficient and oversupplemented conditions, respectively;
and 7) modulated by an intracellular
Ca2+/calmodulin-mediated pathway. These studies demonstrate
for the first time the existence of a specialized carrier-mediated
mechanism for RF uptake in an in vitro cellular model system of human
colonocytes. This mechanism appears to be regulated by extracellular
substrate level and by an intracellular
Ca2+/calmodulin-mediated pathway. It is suggested that the
identified transport system may be involved in the absorption of
bacterially synthesized RF in the large intestine and that this source
of RF may contribute toward RF homeostasis, especially that of colonocytes.
riboflavin transport; human colonocytes in culture; membrane transport mechanism; transport regulation; normal colonic epithelial cells
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