Neutral amino acid transport. Characterization of the A and L systems in isolated rat hepatocytes

Abstract

Hepatocytes isolated from adult rat liver by enzymatic dispersion were used to investigate amino acid transport. Steady state and influx experiments were carried out with alpha-amino[1-14C]isobutyric acid and [1-14C]cycloleucine in the presence and absence of sodium under various experimental conditions. Hepatocytes concentrated alpha-aminoisobutyric acid to a 3-fold higher degree than cycloleucine. At low external alpha-aminoisobutyric acid levels (2 to 5 mM), about 25% and 75% of entry were accounted for by nonsaturable and saturable processes, respectively. The nonsaturable component was sodium-independent, and had the properties of passive diffusion. The saturable transport was dependent on external sodium; the rate of transport reached its maximal value with sodium greater than or equal to 75 mM. Sodium increased the apparent Vmax of transport without changing the apparent Km. This component was largely dependent on energy supplies and was strongly reduced at pH less than or equal to 6.5. The value for activation energy (Ea approximately equal to 15 kcal/mol, calculated from the Arrhenius plot) favors a mediated active transport. The Na+-dependent influx of alpha-aminoisobutyric acid was competitively inhibited by N-methyl-alpha-aminoisobutyric acid (Ki approximately equal to 9.3 mM) and alanine (Ki approximately equal to 2 mM) to the extent of 70% and 100%, respectively. The N-methyl-alpha-aminoisobutyric acid-sensitive part of alpha-aminoisobutyric acid influx represents transport through the "A" system, whereas the N-methyl-alpha-aminoisobutyric acid-insensitive part of transport is believed to occur through the "ASC" system. No evidence was obtained to suggest that alpha-aminoisobutyric acid is transported by the "L" system. Cycloleucine transport was a composite phenomenon involving at least two saturable processes, one of which was sodium-dependent and inhibited by alpha-aminoisobutyric acid, and probably represents entry through the A and ASC systems. The sodium-independent component was completely and competitively inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (Ki approximately equal to 2 mM). This component exhibited accelerative exchange-diffusion and was pH-insensitive, properties which suggest a facilitated diffusion process. However, the weak inhibition exerted by oligomycin and cyanide along with the concentrative effect observed indicated that uphill transport was also operative. These data are in good agreement with those reported for the L system. We conclude that, as in Ehrlich ascites tumor cells and in embryonic heart cells, the A, ASC, and L systems are operative in isolated hepatocytes for the transport of amino acids.