HUMAN INTESTINAL-CELL LINE CACO-2 - A USEFUL MODEL FOR STUDYING CELLULAR AND MOLECULAR REGULATION OF BIOTIN UPTAKE

The mechanisms of enterocyte and molecular regulation of biotin uptake are poorly understood. An intestinal cell line possessing the transpo rt characteristics of native intestinal cells is highly desirable to i nvestigate the finer details of the cellular processing and molecular regulation of biotin transport. In the present study, we investigated the uptake of the water-soluble vitamin biotin by a human intestinal c ell line Caco-2. Uptake of both low (4 nM) and high (20 mu M) concentr ations of biotin by confluent monolayers of Caco-2 cells was appreciab le and linear for up to 10 min of incubation. Replacement of Na+ in th e incubation medium with other monovalent cations - K+, choline, Li+ a nd NH4+ - caused a significant inhibition of biotin uptake; a relative ly lesser inhibition was seen with Li+. Initial rate of uptake of biot in was temperature-dependent and saturable as a function of concentrat ion at 37 degrees C but not at 4 degrees C. The V-max and apparent K-m of the temperature-dependent saturable process were 520 pmol/mg prote in per min and 9.5 mu M, respectively. The addition of unlabeled bioti n and the structural analogue desthiobiotin to the incubation media ca used a significant inhibition of the uptake of [H-3]biotin. The inhibi tory effect of desthiobiotin was competitive in nature with an inhibit ion constant (K-i) of 41 mu M. Biocytin, on the other hand, was a weak inhibitor and biotin methyl ester and diaminobiotin did not have any effect. Pretreatment of Caco-2 cells with the monovalent cation ionoph ore gramicidin and the Na+,K+-ATPase inhibitor ouabain caused signific ant inhibition of biotin uptake. Pretreatment with the K+ ionophore va linomycin did not affect biotin uptake. Using the 'Activation Method', the stoichiometric ratio of biotin(-) to Na+ coupling was found to be 1:1. Growing confluent Caco-2 cells in a biotin-deficient environment resulted in rapid up-regulation of biotin transport with a marked inc rease (258%) in the V-max of biotin uptake. These findings demonstrate that biotin uptake by Caco-2 cells is via a carrier-mediated system. This system is temperature-dependent, driven by Na+-gradient and is re gulated by the substrate level. These in-vitro findings are very simil ar to and further confirm previous findings in human and animal studie s and dispute other findings previously reported for Caco-2 cells; the present study also demonstrates the suitability of this system for fu rther characterization of the cellular and molecular regulation of bio tin uptake.