J. Membrane Biol. 52, 83 92 (1980) The Journal af Membrane Biology Pathways for Alanine Transport in Intestinal Basal Lateral Membrane Vesicles Austin K. Mircheff*, Carel H. van Os**, and Ernest M. Wright Department of Physiology, University of California Medical Center, Los Angeles, California 90024 Summary. Membrane vesicles obtained from the basal lateral membranes of the rat intestinal epithelium were used to study the pathways for neutral amino acid transport. In the absence of sodium there was a stereospecific uptake of L-alanine which exhibited saturation ki- netics (Kin0.73 mM and Vmax 5.3 nmol/mg min at 22 ~ The activation energy for this process was 8.1 kcal/mole between 5 and 25 ~ Preloading the vesicles with alanine increased the unidirectional in- flux of alanine into the vesicle. Competition experi- ments indicated that the affinity of the sodium-inde- pendent transport system was glutamine>threo- nine > alanine > phenylalanine > valine > methio- nine > glycine > histidine > proline, N-MeAIB. These are the characteristics of the classical "L" transport system. External sodium increased the rate of the stereo- specific L-alanine uptake. The Na-dependent flux had a K~ of 0.04 mM and a lima x of 0.26 nmol/mg min at 22 ~ and an activation energy of 9.1 kcal/mole between 5 and 25 ~ Competition experiments sug- gest the existence of three separate pathways for ala- nine transport in the presence of sodium. A major pathway is shared by all other amino acids tested (i.e., threonine, glutamine, methionine, phenylala- nine, valine, proline and N-MeAIB). This resembles the classical "A " system. A second pathway is un- available to either phenylalanine or N-MeAIB; this is reminiscent of the classical "ASC" system; and the third is a novel pathway which is shared by N- MeAIB but not phenylalanine. The sodium-independent and the sodium-depen- dent transport of L-alanine was blocked by PCMBS * Presant address: Department of Physiology & Biophysics, School of Medicine, University of Southern California, Los Angeles, California 90033. ** Present address: Department of Physiology, University of Nij- megen, Nijmegen, The Netherlands. and significantly inhibited by DTP and NEM. It is concluded that the sodium-independent system (the "L "-like system) accounts for the efflux of neutral amino acids from the epithelium to the blood during the absorption of amino acids from the gut, and that the sodium-dependent transport processes may play an important role in the supply of amino acids to the epithelium in the absence of amino acids from the gut lumen. Neutral amino acids are actively transported across the intestinal epithelium from the gut lumen into the blood by a two-stage process: (i) entry into the epithe- lium across the brush border membrane and (ii) trans- port from cell to blood across the basal lateral mem- brane. Studies of uptake into intact epithelium (Schultz & Curran, 1970) and isolated brush-border membrane vesicles (Sigrist-Nelson, Murer & Hopfer, 1975; Evers, Murer & Kinne, 1976; Hopfer etaI.; 1976, Fass, Hammerman & Sacktor, 1977; Hammer- man & Sacktor, 1977, 1978; Slack etaI., 1977) have shown the first step to be ~ accumulation by sodium-coupled mechanisms. In principle, facilitated diffusion driven by the amino acid concentration gra- dients would be sufficient for the second, basal lateral membrane exit step. It has been impossible to obtain direct information about the exit step from studies with the intact epithe- lium, since the serosal surface is obscured by muscle and connective tissue layers. However, studies with isolated plasma membrane vesicles have provided a preliminary view of amino acid transport by renal and intestinal basal lateral membranes. Hopfer et al. (1976) observed that uptake of valine by intestinal basal lateral membranes was saturable, stimulated by preloading, and independent of sodium. Similarly, Evers et al. (1976) and Slack et al. (1977) obtained evidence for mediated uptake of phenylalanine and 0022-2631/80/0052-0083 $02.00 9 1980 Springer-Verlag New York Inc.