Energization of amino acid uptake by system A in cultured human fibroblasts

• Published in: The Journal of biological chemistry Vol. 266; no. 3; pp. 1591 - 1596
• Main Authors: DALL'ASTA, V, BUSSOLATI, O, GUIDOTTI, G. G, GAZZOLA, G. C
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.01.1991
• Subjects: amino acids; Amino Acids - metabolism; beta-Alanine - analogs & derivatives; beta-Alanine - metabolism; Biological and medical sciences; Biological Transport, Active; Cell Line; Cell physiology; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Membrane and intracellular transports; Membrane Potentials; Molecular and cellular biology; Sodium - physiology; system A; Human; Cell culture; Regulation(control); Sodium; Transportation system; Aminoacid; Electrochemical gradient; Kinetics; Membrane potential; Fibroblast; Energy transfer
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52335-8

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.