Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid.
Previous studies showed that the normal microflora of the large intestine synthesizes biotin and that the colon is capable of absorbing intraluminally introduced free biotin. Nothing, however, is known about the mechanism of biotin absorption in the large intestine and its regulation. To address these issues, we used the human-derived, nontransformed colonic epithelial cell line NCM460. The initial rate of biotin uptake was found to be 1) temperature and energy dependent, 2) Na+ dependent (coupling ratio of 1:1), 3) saturable as a function of concentration [apparent Michaelis constant (Km) of 19.7 microM], 4) inhibited by structural analogs with a free carboxyl group at the valeric acid moiety, and 5) competitively inhibited by the vitamin pantothenic acid (inhibition constant of 14.4 microM). Pretreatment with the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) and 1, 2-dioctanoyl-sn-glycerol significantly inhibited biotin uptake. In contrast, pretreatment with the PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediated via a marked decrease in maximal uptake velocity and a slight increase in apparent Km. Pretreatment of cells with modulators of the protein kinase A-mediated pathway, on the other hand, showed no significant effect on biotin uptake. These results demonstrate, for the first time, the functional existence of a Na+-dependent, specialized carrier-mediated system for biotin uptake in colonic epithelial cells. This system is shared with pantothenic acid and appears to be under the regulation of an intracellular PKC-mediated pathway.
Said
H M
HM
Veterans Affairs Medical Center, Long Beach 90822, California, USA.
Ortiz
A
A
McCloud
E
E
Dyer
D
D
Moyer
M P
MP
Rubin
S
S
D000470:Alkaloids; D053119:Benzophenanthridines; D001692:Biological Transport / Q000187:drug effects; D001710:Biotin / Q000031:analogs & derivatives / Q000493:pharmacokinetics* / Q000494:pharmacology; D002460:Cell Line; D003106:Colon; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D010205:Pantothenic Acid / Q000493:pharmacokinetics*; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D004075:Diglycerides; D004791:Enzyme Inhibitors; D010617:Phenanthridines; C044295:1,2-dioctanoylglycerol; D010205:Pantothenic Acid; D001710:Biotin; C016299:chelerythrine; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate