Energization of amino acid uptake by system A in cultured human fibroblasts.
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.
Dall'Asta
V
V
Istituto di Patologia Generale, Università di Parma, Italy.
Bussolati
O
O
Guidotti
G G
GG
Gazzola
G C
GC
D000596:Amino Acids / Q000378:metabolism*; D001693:Biological Transport, Active; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D008564:Membrane Potentials; D012964:Sodium / Q000502:physiology*; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
D000596:Amino Acids; D015091:beta-Alanine; C012924:2,2-dimethyl-beta-alanine; D012964:Sodium