Intracellular regulation of intestinal folate uptake: studies with
cultured iec-6 epithelial cells.
Said, Hamid M., Thomas Y. Ma, Alvaro Ortiz, Alicia Tapia, and Claudia
K. Valerio.
Medical Research Service, VA Medical Center, Long Beach, CA 90822
and Departments of Medicine and Physiology/Biophysics, University of
California-School of Medicine, Irvine, CA 92717
APStracts 3:0297C, 1996.
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 being: 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 Km =
0.45 +/- 0.06 [mu]M; Vmax = 3.08 + 0.14 pmol/mg protein/5 min).
Furthermore, IEC-6 cells were found by Northern blot analysis to lack
the expression of the membrane folate binding protein (mFBP).
Pretreatment of IEC-6 monolayers with specific protein tyrosine
kinase (PTK) inhibitors genistein and Tyr-A-25 caused a significant
inhibition in folic acid uptake. On the other hand, their negative
controls, genistin and Tyr-A-1, 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 cAMP level [e.g., dibutyryl cAMP (dB cAMP)] also
resulted in a significant (though modest) inhibition in folic acid
uptake; however, specific inhibitors of PKA did not affect the uptake
process. Specific modulators of protein kinase C (PKC) and
Ca2+/calmoduline 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 pathways.
Received 10 September 1996; accepted in final form 10 September
1996.
APS Manuscript Number C407-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 7 October 1996