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

Abstract

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 M), 4) inhibited by structural analogs and anion transport inhibitors, and 5) energydependent. 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 Cand Ca 2؉ /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.Folate is an essential micronutrient, which acts as a coenzyme in the synthesis of DNA and RNA and the interconversion and degradation of several amino acids (1-4). An adequate supply of folate is therefore necessary for normal cellular function, growth, and development. Folate deficiency has been suggested as one of the most common vitamin deficiencies in the Western Hemisphere (5, 6). Humans and other mammals cannot synthesize folate and rely on exogenous sources to meet their metabolic requirements. Folates are presented to the host from the diet and are also synthesized in the large intestine by normal microflora. The mechanism of absorption of dietary folate has been intensively examined over the past two decades at the tissue, cellular, subcellular, and more recently, molecular levels (6 -15). Absorption of dietary folate has been shown to occur mainly in the proximal small intestine and involves a specialized, carrier-mediated system (6 -12).As to the bacterially synthesized folate in the large intestine, a significant amount of that folate exists in the monoglutamate, i.e. the absorbable form. Using [ 3 H]p-aminobenzoic acid to label the newly synthesized folate by the intestinal flora, Rong et al. (16) have shown that a portion of this folate is indeed absorbed by the rat and is incorporated into its various tissues. ...

Mechanism and regulation of folate uptake by pancreatic acinar cells: effect of chronic alcohol consumption

“…We focused on possible role of the cAMP/PKA-and the PTK-mediated pathways since these two pathways were reported to play a role in regulating folate uptake by other cells (11,17). The results showed that whereas modulators of cAMP/PKA-mediated pathway cause significant inhibition in folate uptake (and at both buffer pH 7.4 and 6.0), modulators of the PTK-mediated pathway failed to affect the uptake process.…”

A Reduced Folate Carrier Mutation Produces Substrate-dependent Alterations in Carrier Mobility in Murine Leukemia Cells and Methotrexate Resistance with Conservation of Growth in 5-Formyltetrahydrofolate

“…Folate receptors, anchored in the plasma membrane through a glycosylphosphatidylinositol moiety, translocate folates unidirectionally into cells via an endocytotic process (3)(4)(5)(6). A transport pathway with optimal activity at low pH has been described in murine leukemia L1210 cells (7,8), and a low pH route dominates transport of folates in intestinal cells (9,10).…”

Pancreatic beta cells and islets take up thiamin by a regulated carrier-mediated process: studies using mice and human pancreatic preparations

“…Possible regulation of thiamin uptake by pancreatic beta cells by intracellular regulatory pathways was also examined in this investigation with special focus on pathways that have been shown to regulate uptake of other nutrients and substrates in other cellular systems (i.e., PKA-, Ca 2ϩ /calmodulin-, and PTK-mediated pathways) (5,15,31). The results showed that although modulators of the PKA-mediated pathway failed to affect thiamin uptake, modulators (inhibitors) of the Ca 2ϩ / calmodulin-mediated pathway caused significant inhibition in the vitamin uptake process.…”

“…Mouse-derived pancreatic beta-TC-6 insulinoma cells (passages [10][11][12][13][14][15][16][17][18][19][20][21][22] were obtained from American Type Tissue Collection (ATCC; Rockville, MD) and maintained in DMEM growth medium in the presence of 15 nM thiamin. Uptake was examined (unless otherwise stated) at 37°C in Krebs-Ringer buffer (in mM: 133 NaCl, 4.93 KCl, 1.23 MgSO 4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES; pH 8.0) by a rapid filtration technique (14), as described by us previously (10,30).…”