No abstract

Nutritional stress caused by amino acid starvation involves a coordinated cellular response that includes the global decrease of protein synthesis and the increased production of cell defense proteins. Part of this response is the induction of transport system A for neutral amino acids that leads to the recovery of cell volume and amino acid levels once extracellular amino acid availability is restored. Hypertonic stress also increases system A activity as a mechanism to promote a rapid recovery of cell volume. Both a starvation-dependent and a hypertonic increase of system A transport activity are due to the induction of SNAT2, the ubiquitous member of SLC38 family. The molecular mechanisms underlying SNAT2 induction were investigated in tissue culture cells. We show that the increase in system A transport activity and SNAT2 mRNA levels upon amino acid starvation were blunted in cells with a mutant eIF2␣ that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2␣ phosphorylation. The translational control of the SNAT2 mRNA during amino acid starvation was also investigated. It is shown that the 5-untranslated region contains an internal ribosome entry site that is constitutively active in amino acid-fed and -deficient cells and in a cell-free system. We also show that amino acid starvation caused a 2.5-fold increase in mRNA and protein expression from a reporter construct containing both the SNAT2 intronic amino acid response element and the SNAT2-untranslated region. We conclude that the adaptive response of system A activity to amino acid starvation requires eukaryotic initiation factor 2␣ phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2␣ phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.The severe nutritional stress caused by amino acid starvation triggers several adaptive changes. One of these is the stimulation of transport system A for neutral amino acids. Adaptive regulation of system A has been described in most amino acid-starved animal cells (1-3). Work from several laboratories (4 -6) has demonstrated that this regulatory mechanism is associated with an increase in the mRNA for SNAT2, the ubiquitously expressed member of the sodium-coupled neutral amino acid transporter family (7). The induction of SNAT2 transcription leads to the increased synthesis of SNAT2 carriers, which are found in greater amounts on the membranes of starved cells (5,8). Recently, an amino acid-responsive element has been identified in the first intron of the mouse and human SNAT2 genes (9), supporting the hypothesis that transcriptional activation contributes to the adaptive regulation of system A.Unlike transcriptional activation, the translation of SNAT2 during amino acid starvation has received little attention ...

No abstract

It has been known for several years that the triggering of cell proliferation is associated with an increase of the activity of Na,K,Cl cotransport and of transport system A for neutral amino acids. These systems are also enhanced during the volume recovery of hypertonically shrunk cells. We demonstrate here that during the cell cycle of NIH3T3 cells, an increase in cell volume is associated with an enhanced cell content of potassium and amino acids. Bumetanide delays cell cycle progression and hampers volume increase. The nonmetabolizable analog 2-methylamino-isobutyric acid, a specific substrate of system A, can partially substitute natural amino acids accumulated during the cell cycle as intracellular osmolytes. It is therefore proposed that the stimulation of Na,K,Cl cotransport and of system A, observed in proliferating cells, causes an expansion of cell volume through an enhanced intracellular accumulation of both inorganic and organic osmolytes and the concurrent, osmotically obliged uptake of water.