INTRACELLULAR REGULATION OF INTESTINAL FOLATE UPTAKE - STUDIES WITH CULTURED IEC-6 EPITHELIAL-CELLS

Although the mechanism of folate uptake in the small intestine has bee n well characterized, very little is known about the intracellular reg ulation of the uptake process. Using mature confluent monolayers of th e intestinal epithelial cell line IEC-6 as an in vitro intestinal epit helial cell model, we have found the uptake of folic acid to be simila r to that of the native small intestine in that it is 1) temperature, energy, and pH dependent, 2) Na+ independent, 3) inhibited by structur al analogs and anion transport inhibitors, and 4) saturable as a funct ion of substrate concentration [apparent Michaelis constant (K-m) = 0. 45 +/- 0.06 mu M; maximal velocity (V-max) = 3.08 +/- 0.14 pmol . mg p rotein(-1). 5 min(-1)]. Furthermore, IEC-6 cells were found by Norther n blot analysis to lack the expression of the membrane folate-binding protein. Pretreatment of IEC-6 monolayers with specific protein tyrosi ne kinase (PTK) inhibitors genistein and tyrphostin A25 caused a signi ficant inhibition in folic acid uptake. On the other hand, their negat ive controls, genistin and tyrphostin A1, respectively, had no effect. The inhibitory effect of genistein was mediated through inhibition in the V-max of the folate uptake process with no change in the apparent K-m. Pretreatment of IEC-6 monolayers with compounds that increase in tracellular adenosine 3',5'-cyclic monophosphate (cAMP) level (e.g., d ibutyryl cAMP) also resulted in a significant (though modest) inhibiti on in folic acid uptake; however, specific inhibitors of protein kinas e A did not affect the uptake process. Specific modulators of protein kinase C and Ca2+/calmodulin-mediated pathways did not significantly a ffect folic acid uptake. These results demonstrate the suitability of IEC-6 monolayers as an intestinal epithelial model to study folate tra nsport and demonstrate for the first time that uptake of folic acid is regulated by a PTK- and a cAMP mediated pathway.