Specific Induction of Amino Acid Alanine Uptake in Intestinal Cells
Abstracts
|
Background: In contrast to cells of most organs that down-regulate amino acid transport activity in response to substrates, the small intestinal epithelia selectively increase uptake ability in the presence of substrates. This study was to elucidate the substrate specific regulation mechanism(s) involved in the in vitro transport of the neutral amino acid alanine. Methods: 3H L-alanine (50 µM) transporter activity and mRNA levels were measured in intestinal Caco-2 cells incubated in amino acid free medium, various amino acids, actinomycin-D (Act-D), and cycloheximide (CHX). Data were analyzed by ANOVA. Results: Alanine was transported by transport Systems B (85 %) and L (15 %). This alanine transport activity decreased in the absence of amino acids in the incubation medium, an effect noted by 30 minutes. Prolong exposure (> 24 hours) of L-alanine in the incubation medium persistently augmented both System B and L activity. This augmented transport was abolished by Act-D and CHX (Fig. 1). Alanine exposure increased the System B Vmax by 3.5 fold (2.25 ? 0.1 vs. 0.67 ? 0.05 nmole mg protein-1 min-1 control, p < 0.01) without altering Km (152 ? 10 vs. 163 ? 10 uM control, p = NS) (Fig. 2). System B transporter gene ATB0 mRNA levels were not affected by the prolonged alanine inhibition. Conclusion: Prolong substrate exposure stimulates intestinal brush border membrane alanine transport via a post-transcriptional mechanism that leads to an increase in functional transport units with no change in carrier configuration. |
