Intracellular regulation of intestinal folate uptake: studies with cultured IEC-6 epithelial cells

Abstract

Although the mechanism of folate uptake in the small intestine has been well characterized, very little is known about the intracellular regulation of the uptake process. Using mature confluent monolayers of the intestinal epithelial cell line IEC-6 as an in vitro intestinal epithelial cell model, we have found the uptake of folic acid to be similar to that of the native small intestine in that it is 1 ) temperature, energy, and pH dependent, 2) Na+ independent, 3) inhibited by structural analogs and anion transport inhibitors, and 4) saturable as a function of substrate concentration [apparent Michaelis constant (Km) = 0.45 +/- 0.06 microM; maximal velocity (Vmax) = 3.08 +/- 0.14 pmol x mg protein(-1) x 5 min(-1)]. Furthermore, IEC-6 cells were found by Northern blot analysis to lack the expression of the membrane folate-binding protein. Pretreatment of IEC-6 monolayers with specific protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin A25 caused a significant inhibition in folic acid uptake. On the other hand, their negative controls, genistin and tyrphostin A1, respectively, had no effect. The inhibitory effect of genistein was mediated through inhibition in the Vmax of the folate uptake process with no change in the apparent Km. Pretreatment of IEC-6 monolayers with compounds that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level (e.g., dibutyryl cAMP) also resulted in a significant (though modest) inhibition in folic acid uptake; however, specific inhibitors of protein kinase A did not affect the uptake process. Specific modulators of protein kinase C and Ca2+/calmodulin-mediated pathways did not significantly affect folic acid uptake. These results demonstrate the suitability of IEC-6 monolayers as an intestinal epithelial model to study folate transport and demonstrate for the first time that uptake of folic acid is regulated by a PTK- and a cAMP-mediated pathway.

Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications

“…This transport activity has properties similar to what has been observed for folate intestinal absorption in vivo, in intestinal loops (64,65), in brush-border intestinal vesicles (66,67), isolated intestinal cells (68,69), and in tumor cell lines of intestinal origin (70). This transport activity was shown to be independent of RFC.…”

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function

“…In one case, the carrier was present but mutated; in the other case, the basis for impaired carrier function was not clear (12,30). The transport activity identified in these cells has a pattern of structural specificity and energy requirement similar to findings in IEC-6 cells (23,26). Low-pH folate transport activity has also been detected in freshly isolated hepatocytes, primary cultured rat astrocytes, and primary cultured cerebellar granule cells, as well as in a few cancer cell lines (2, 3, 14 -16).…”

“…The characteristics of this process have been studied using a variety of experimental systems, including intact intestinal segments, everted intestinal sacs, intestinal loops, freshly isolated intestinal cells, and brush-border membrane vesicles (20). Rat intestinal epithelial (IEC-6) cells have been an important model for characterization of intestinal folate transport at the cellular level because folate transport properties in this cell line resemble those of intact intestine in many respects (19,23,26,31).…”

“…However, while RFC is expressed in intestinal cells and a small folate transport component is detected at physiological pH in IEC-6 cells, the major transport component has a low-pH optimum, and transfection of mRFC into these cells produced low-pH activity (19) or low-pH activity together with much more prominent high-pH activity (23). 2) While RFC has a very low affinity for folic acid in leukemia cells that is one or two orders of magnitude below that for 5-CH 3 -THF, 5-formyltetrahydrofolate (5-CHO-THF), and methotrexate (MTX), the affinity of transport in cells of intestinal origin and in IEC-6 cells at low pH is comparable for all these folates (11,25,26). 3) Influx of MTX mediated by RFC in leukemia cells is minimally affected, or increased, by energy inhibitors, while influx in IEC-6 cells is partially suppressed under these conditions (23).…”

Rescue of Embryonic Lethality in Reduced Folate Carrier-deficient Mice by Maternal Folic Acid Supplementation Reveals Early Neonatal Failure of Hematopoietic Organs

“…Prior to birth, high folate concentrations achieved by folic acid injection, with transport or diffusion across the placenta barrier where high levels of folate receptor are expressed (23), provides adequate folates for the developing embryo. However, possibly due to low concentrations of folic acid in the mother's milk and inactivation of intestinal RFC1 that is involved in folate absorption across the pup's small intestine (21,35), where folate receptors are not expressed (36), there is apparently insufficient folate available to sustain the life of pups long after birth.…”