Differentiation- and protein kinase C-dependent regulation of alanine transport via system B.
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 B0,+ 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 (Vmax), with Km unaffected. The Vmax of system B was greater in 2-day-old cells (2.79 +/- 0.21 nmol min-1 mg of protein-1; Km = 164 +/- 26 microM alanine), decreasing to Vmax = 0.51 +/- 0.03 nmol min-1 mg of protein-1 (Km = 159 +/- 14 microM) 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 microliter min-1 mg of protein-1. Phorbol ester up-regulated the Vmax of system B in 2-day-old cells to Vmax = 6.32 +/- 0.37 nmol min-1 mg of protein-1 (Km = 169 +/- 18 microM), and in 9-day-old cells to Vmax = 1.42 +/- 0.05 nmole min-1 mg of protein-1 (Km = 180 +/- 10 microM). 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.
Pan
M
M
Department of Physiology, College of Medicine, University of Florida, Gainesville 32610.
Stevens
B R
BR
D000409:Alanine / Q000378:metabolism*; D001692:Biological Transport; D002454:Cell Differentiation; D002460:Cell Line; D016922:Cellular Senescence; D006801:Humans; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
D015091:beta-Alanine; C012924:2,2-dimethyl-beta-alanine; D015240:Phorbol 12,13-Dibutyrate; D012964:Sodium; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine