Pathways of L-glutamic acid transport in cultured human fibroblasts.

The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax

The non-proteinogenic amino acids l-methionine sulfoximine and dl-phosphinothricin activate mTOR

l-Methionine sulfoximine (MSO) and dl-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators

Synthesis, structural characterisation and solution chemistry of ruthenium(III) triazole-thiadiazine complexes

Two ruthenium(III) complexes structurally similar to the anticancer compound NAMI were prepared: Na[RuCl4(DMSO)(L1)] (1) and Na[RuCl4(DMSO)(L2)] (2), where L1 and L2 are differently functionalised triazole-thiadiazine ligands. To facilitate the crystallisation of the complex anions, Na+ was substituted with the [bis(triphenylphosphoranylidene)ammonium] cation (PPN+), allowing the X-ray characterisation of PPN[RuCl4(DMSO)(L1)]·2H2O (1a·2H2O) and PPN[RuCl4(DMSO)(L2)]·3H2O (2a·3H2O), respectively. The two compounds undergo stepwise hydrolytic processes, as assessed by means of UV-vis and 1H NMR spectroscopy. The first hydrolytic step consists of the replacement of a chloride anion with a water molecule, with a half-life of 50 min (1) and 110 min (2), while the subsequent hydrolytic steps are more complicated to describe since more than one product is generated at the same time. The redox potential of the Ru(III)/Ru(II) couple (0.31 V for 1 and 0.28 V for 2) suggests that these complexes can be reduced in the intracellular environment, in agreement with the “activation by reduction” mechanism proposed for NAMI and NAMI-A. 1 and 2 were tested on a human cancer cell line derived from a fibrosarcoma (HT1080), and on non-cancerous primary human fibroblasts (HF), where they showed a modest inhibitory effect

The regulation of sodium-dependent transport of anionic amino acids in cultured human fibroblasts

AbstractIn cultured human fibroblasts the transport of anionic amino acids through the sodium-dependent system X−AG is stimulated rapidly and transiently by phorbol 12,13-dibutyrate. Transport stimulation is consistent with an effect due to the activation of protein kinase C. Bradykinin (1 μM) and PDGF-AA (100 ng/ml) also stimulate the activity of system X−AG. The bradykinin effect appears to be fully dependent upon PKC activation whereas the stimulation of aspartate transport by PDGF-AA is also due to PKC-independent mechanisms

SNAT2 silencing prevents the osmotic induction of transport system A and hinders cell recovery from hypertonic stress

Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two si- RNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress