The regulation of sodium-dependent L-alanine transport is described for the first time in intestinal cells. Substrate analogue inhibition patterns and Dixon analyses indicated that uptake occurred via transport system B, an epithelial cell variant of systems B and ASC. System B served >95% of the Na-dependent alanine uptake in both undifferentiated (2 days postpassaging) and differentiated (>9 days postpassaging) states of the human Caco-2 cultured intestinal cell line. (Methylamino)isobutyric acid-inhibitable system A transport accounted for <5% of total alanine uptake. System B activity was greater in undifferentiated cells compared with the differentiated state, and activity at any differentiation state was stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA). The maximal stimulation, determined by TPA dose-response/exposure time data, was attributable to a change in cell transport capacity (V), with K unaffected. The V of system B was greater in 2-day-old cells (2.79 ± 0.21 nmol min mg of protein; K = 164 ± 26 µM alanine), decreasing to V = 0.51 ± 0.03 nmol min mg of protein (K= 159 ± 14 µM) in 9-day-old cells. Regardless of differentiation status, the sodium-activation Hill coefficient was 1.06 ± 0.10, and the alanine passive diffusion permeability coefficient was 0.53 ± 0.08 µl min mg of protein. Phorbol ester up-regulated the V of system B in 2-day-old cells to V = 6.32 ± 0.37 nmol min mg of protein (K = 169 ± 18 µM), and in 9-day-old cells to V = 1.42 ± 0.05 nmole min mg of protein (K = 180 ± 10 µM). Phorbol ester stimulation of transport occurred after at least 6 h of continual exposure, and was blocked by the protein kinase C inhibitors chelerythrine or photoactivated calphostin C. Protein synthesis inhibitors cycloheximide and actinomycin D each blocked the phorbol ester up-regulation of system B activity. It is concluded that Caco-2 cells regulate carrier-mediated sodium-dependent transport of L-alanine by changing the membrane capacity to transport alanine via system B and that this regulation involves de novo protein synthesis under the control of protein kinase C.

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