Evidence for the existence of a carrier-mediated folate uptake mechanism in human colonic luminal membranes

Abstract

Colonic bacteria synthesize significant amounts of folate. The current studies were undertaken to examine the presence of a possible folate transporter and its characterization in apical membranes of the human colon. Apical membrane vesicles were purified from mucosal scrapings of proximal organ donor colons using a differential centrifugation and divalent cation precipitation technique. [3H]folate (PteGlu) uptake was measured by a rapid filtration technique. Our results demonstrate that [3H]PteGlu uptake into these vesicles 1) was significantly increased with decreasing pH of the incubation buffer, 2) was markedly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by acetazolamide, amiloride, bumetanide, furosemide, and diphenyl 2-carboxylic acid, 3) was sensitive to temperature and osmolarity of the incubation medium, 4) was inhibited by structural analogs methotrexate and 5-methyltetrahydrofolate, 5) exhibited trans-stimulation phenomenon, 6) was potential insensitive, and 7) exhibited saturation kinetics with an apparent Michaelis constant for PteGlu of 8.2 +/- 2.4 microM and a maximal enzyme reaction rate of 19.8 +/- 2.9 pmol.mg protein-1.10 s-1. Studies on distal colonic membranes also demonstrated the presence of a folate transporter with similar kinetic characteristics. These results demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral carrier-mediated mechanism for folate absorption in the human colon.