Posttranslational alanine trans-stimulation of zwitterionic amino acid transport systems in human intestinal Caco-2 cells.
BACKGROUND
Neutral dietary amino acids, such as alanine, are transported across the gut lumen by both Na(+)-dependent (System B) and Na(+)-independent (System L) carriers, but the nature of the acute phase of substrate-induced uptake is unknown. This study examined the effects of acute amino acid substrate exposure on the rapid modulation of apical membrane alanine transport in cultured human intestinal cells.


METHODS
System B and System L transport activity kinetics, as well as ATB(0) mRNA levels, were measured in confluent Caco-2 monolayers treated with various metabolic agents during short-term and extended time periods.


RESULTS
Depleting the incubation medium of alanine attenuated both System B and System L uptake activities within 30 mins, with a complete return to baseline values within 3 h. Extracellular alanine added to depleted Caco-2 cells rapidly (within 5 min) increased alanine transport activities. Kinetic analysis showed that acute alanine exposure increased both K(m) and V(max) of each transport system, indicative of a trans-stimulation effect. Augmenting intracellular alanine levels using the cytosolic alanine aminotransferase inhibitor, aminooxyacetic acid, increased alanine uptake activity. Acute exposure to other substrates of Systems B and L also increased the uptake of alanine, while nonsubstrates did not affect alanine uptake. Cycloheximide or actinomycin did not affect substrate acute activation of System B, and the steady-state level of ATB(0) mRNA was not altered by amino acid exposure.


CONCLUSIONS
Increasing alanine availability to intestinal cells, by either exogenous substrate exposure or inhibition of intracellular catabolism, acutely and reversibly increases apical membrane alanine transport activity via a posttranslation trans-stimulation mechanism.
Pan
Ming
M
Department of Surgery, Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
Souba
Wiley W
WW
Wolfgang
Christopher L
CL
Karinch
Anne M
AM
Stevens
Bruce R
BR
D000409:Alanine / Q000493:pharmacokinetics / Q000494:pharmacology*; D026921:Amino Acid Transport Systems, Neutral / Q000378:metabolism*; D000596:Amino Acids / Q000172:deficiency; D001692:Biological Transport; D018938:Caco-2 Cells; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000473:pathology; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D013997:Time Factors
D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D000409:Alanine