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Volume 272, Number 10, Issue of March 7, 1997 pp. 6226-6231
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

(Received for publication, July 9, 1996, and in revised form, October 24, 1996)

Chandira K. Kumar , Mary Pat Moyer ^§ , Pradeep K. Dudeja ^¶ and Hamid M. Said

From the  Veterans Administration Medical Center, Long Beach, California 90822,  University of California School of Medicine, Irvine, California 92717, ^§ Center for Human Cell Biotechnology, The University of Texas Health Science Center, San Antonio, Texas 78284, and the ^¶ University of Illinois and Westside Veterans Administration Medical Center, Chicago, Illinois 60612

A significant proportion of the bacterially synthesized folate in the large intestine exists in the form of folate monoglutamate. Recent studies in our laboratory using human colonic apical membrane vesicles have shown the existence of an efficient carrier-mediated system for folate uptake. Nothing, however, is known about the cellular regulation of the colonic uptake process. In this study, we used a recently established human normal colonic epithelial cell line NCM460 to address this issue. Uptake of folic acid by NCM460 cells was: 1) linear with time for 4 min of incubation and occurred with minimal metabolic alterations, 2) temperature- and pH- (but not Na⁺) dependent, 3) saturable as a function of concentration (apparent K_m of 1.4 µM), 4) inhibited by structural analogs and anion transport inhibitors, and 5) energy-dependent. These characteristics of folic acid uptake by NCM460 cells are similar to those seen with apical membrane vesicles derived from human native colonic tissue. Using these cells, we found that protein kinase C- and Ca²⁺/calmodulin-mediated pathways have no role in regulating folic acid uptake. On the other hand, cAMP (through a mechanism independent of protein kinase A) and protein-tyrosine kinase-mediated pathways were found to play a role in the regulation of folic acid uptake by these cells. These results establish the suitability of NCM460 cells as an in vitro model system for investigating the details of the mechanism of colonic folate uptake and its regulation. Folic acid uptake by these cells appears to involve a carrier-mediated system, which is temperature-, pH-, and energy-dependent and appears to be under the regulation of cAMP and protein tyrosine kinase.

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