Adaptive increase of amino acid transport system A requires ERK1/2 activation

Amino acid starvation markedly stimulates the activity of system A, a widely distributed transport route for neutral amino acids. The involvement of MAPK (mitogen-activated protein kinase) pathways in this adaptive increase of transport activity was studied in cultured human fibroblasts. In these cells, a 3-fold stimulation of system A transport activity required a 6-h amino acid-free incubation. However, a rapid tyrosine phosphorylation of ERK (extracellular regulated kinase) 1 and 2, and JNK (Jun N-terminal kinase) 1, but not of p38, was observed after the substitution of complete medium with amino acid-free saline solution. ERK1/2 activity was 4-fold enhanced after a 15-min amino acid-free incubation and maintained at stimulated values thereafter. A transient, less evident stimulation of JNK1 activity was also detected, while the activity of p38 was not affected by amino acid deprivation. PD98059, an inhibitor of ERK1/2 activation, completely suppressed the adaptive increase of system A transport activity that, conversely, was unaffected by inhibitors of other transduction pathways, such as rapamycin and wortmannin, as well as by chronic treatment with phorbol esters. In the presence of either L-proline or 2-(methylaminoisobutyric) acid, two substrates of system A, the transport increase was prevented and no sustained stimulation of ERK1/2 was observed. To identify the stimulus that maintains MAPK activation, cell volume was monitored during amino acid-free incubation. It was found that amino acid deprivation caused a progressive cell shrinkage (30% after a 6-h starvation). If proline was added to amino acid-starved, shrunken cells, normal values of cell volume were rapidly restored. However, proline-dependent volume rescue was hampered if cells were pretreated with PD98059. It is concluded that (a) the triggering of adaptive increase of system A activity requires a prolonged activation of ERK1 and 2 and that (b) cell volume changes, caused by the depletion of intracellular amino acid pool, may underlie the activation of MAPKs

Response of human fibroblasts to hypertonic stress. Cell shrinkage is counteracted by an enhanced active transport of neutral amino acids

Regulatory volume increase (RVI) has been studied in cultured human fibroblasts (CHF) incubated in a complete hypertonic growth medium (400 mosmol/kg). After the initial cell shrinkage induced by hypertonic treatment, cells recover their volume almost completely within 3 h. This RVI response is associated with a marked increase of the cell content of free amino acids. The cell content of potassium increases only slightly. Chromatographic analysis of the intracellular amino acid pool shows that the RVI-associated increase in cell amino acids is mainly a result of changes in the L-glutamine content. The intracellular accumulation of the analog 2-methylaminoisobutyric acid, a specific substrate of transport system A, is increased in CHF undergoing RVI. Hypertonic treatment causes an immediate and sustained cell hyperpolarization, as demonstrated by changes in the trans-membrane distribution ratio of L-arginine and in the fluorescence of the potential-sensitive dye bis-1,3-diethylthiobarbiturate-trimethineoxonol. Because of cell hyperpolarization, at the end of RVI the trans-membrane gradient of the sodium electrochemical potential is higher than that of the control. The increase in the extracellular potassium concentration ([K+]out = 40 mM) abolishes the hyperpolarization induced by hypertonic treatment and delays volume recovery. Cycloheximide suppresses RVI at a high but not at physiologic [K+]out. It is proposed that CHF counteract hypertonic shrinkage through an enhanced accumulation of substrates of transport system A sustained, initially, by an increase in the energy available for transport and, subsequently, also by the synthesis of new site A carriers