Valeria Dall’Asta

In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function

AIM Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells. METHODS AND RESULTS Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor alpha (TNFalpha). After a 24 h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity, and lactate dehydrogenase release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNFalpha-dependent E-selectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay and western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability. CONCLUSION A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.

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 eitherl-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.

Amino acids are compatible osmolytes for volume recovery after hypertonic shrinkage in vascular endothelial cells

The response to chronic hypertonic stress has been studied in human endothelial cells derived from saphenous veins. In complete growth medium the full recovery of cell volume requires several hours and is neither associated with an increase in cell K+ nor hindered by bumetanide but depends on an increased intracellular pool of amino acids. The highest increase is exhibited by neutral amino acid substrates of transport system A, such as glutamine and proline, and by the anionic amino acid glutamate. Transport system A is markedly stimulated on hypertonic stress, with an increase in activity roughly proportional to the extent and the duration of the osmotic shrinkage. Cycloheximide prevents the increase in transport activity of system A and the recovery of cell volume. It is concluded that human endothelial cells counteract hypertonic stress through the stimulation of transport system A and the consequent expansion of the intracellular amino acid pool.

Inhibition of Glutamine Synthetase Triggers Apoptosis in Asparaginase-Resistant Cells

The resistance to L-asparaginase (ASNase) has been associated to the overexpression of asparagine synthetase (AS), although the role played by other metabolic adaptations has not been yet defined. Both in ASNase-sensitive Jensen rat sarcoma cells and in ARJ cells, their ASNase-resistant counterparts endowed with a five-fold increased AS activity, ASNase treatment rapidly depletes intracellular asparagine. Under these conditions, cell glutamine is also severely reduced and the activity of glutamine synthetase (GS) is very low. After 24h of treatment, while sensitive cells have undergone massive apoptosis, ARJ cells exhibit a marked increase in GS activity, associated with overexpression of GS protein but not of GS mRNA, and a partial restoration of glutamine and asparagine. However, when ARJ cells are treated with both ASNase and L-methionine-sulfoximine (MSO), an inhibitor of GS, no restoration of cell amino acids occurs and the cell population undergoes a typical apoptosis. No toxicity is observed upon MSO treatment in the absence of ASNase. The effects of MSO are not referable to depletion of cell glutathione or inhibition of AS. These findings indicate that, in the presence of ASNase, the inhibition of GS triggers apoptosis. GS may thus constitute a target for the suppression of ASNase-resistant phenotypes.

Oxidative stress induced by copper and iron complexes with 8-hydroxyquinoline derivatives causes paraptotic death of HeLa cancer cells.

Here, we report the antiproliferative/cytotoxic properties of 8-hydroxyquinoline (8-HQ) derivatives on HeLa cells in the presence of transition metal ions (Cu(2+), Fe(3+), Co(2+), Ni(2+)). Two series of ligands were tested, the arylvinylquinolinic L1-L8 and the arylethylenequinolinic L9-L16, which can all interact with metal ions by virtue of the N,O donor set of 8-HQ; however, only L9-L16 are flexible enough to bind the metal in a multidentate fashion, thus exploiting the additional donor functions. L1-L16 were tested for their cytotoxicity on HeLa cancer cells, both in the absence and in the presence of copper. Among them, the symmetric L14 exhibits the highest differential activity between the ligand alone (IC50 = 23.7 μM) and its copper complex (IC50 = 1.8 μM). This latter, besides causing a significant reduction of cell viability, is associated with a considerable accumulation of the metal inside the cells. Metal accumulation is also observed when the cells are incubated with L14 complexed with other late transition metal ions (Fe(3+), Co(2+), Ni(2+)), although the biological response of HeLa cells is different. In fact, while Ni/L14 and Co/L14 exert a cytostatic effect, both Cu/L14 and Fe/L14 trigger a caspase-independent paraptotic process, which results from the induction of a severe oxidative stress and the unfolded protein response.

Role of Amino Acid Transport System A in the Control of Cell Volume in Cultured Human Fibroblasts

Human fibroblasts shrink and are unable to recover their initial volume when incubated in hypertonic saline solutions, whereas an efficient volume restoration takes place in hypertonic media containin

Hypertonicity Induces Injury to Cultured Human Endothelium: Attenuation by Glutamine

BACKGROUND Although most preservation solutions as well as some cardioplegic solutions used for organ storage and transplantation are hypertonic, the effects of extracellular hypertonicity on endothelium are not well established. Aims of this study were to evaluate the response of cultured human saphenous vein endothelial cells to extracellular hypertonicity and to investigate the role of the amino acid glutamine in preventing endothelial damage in vitro. METHODS Eight distinct strains of human saphenous vein endothelial cells were studied. Hypertonic (350 and 400 mosm/kg) media were obtained by supplementing culture medium with sucrose. Cell viability was assessed in the absence or the presence of glutamine through the determination of cell number and protein content of the cultures. Confocal microscopy of cells loaded with the fluorescent dye calcein was also performed. RESULTS Exposure of human saphenous vein endothelial cells to hypertonic media without glutamine caused significant cell loss within 30 minutes. Cell loss progressed steadily during incubation and after 6 hours reached 50% at 350 mosm/kg and 65% at 400 mosm/kg. In the presence of 2 mmol/L glutamine, endothelial damage was completely prevented at 350 mosm/kg and significantly lessened at 400 mosm/kg compared with glutamine-free media. Confocal microscopy showed that most hypertonicity-treated cells exhibited the typical features of an apoptotic death and confirmed the osmoprotective effect of glutamine. CONCLUSIONS These results indicate that the supplementation of hypertonic storage solutions with glutamine might exert a partial osmoprotective effect and suggest that the relationship between endothelial damage and tonicity of storage and cardioplegic solutions should be carefully investigated.

The transport of cationic amino acids in human airway cells: expression of system y+L activity and transepithelial delivery of NOS inhibitors

The transport of arginine has been characterized in human airway Calu‐3 cells. As assessed with RT‐PCR, Calu‐3 cells express the genes for several transporters, such as the system y+‐related SLC7A1, SLC7A2, and SLC7A4; the system y+L‐related SLC7A6, SLC7A7, and SLC3A2; and the system B0,+‐related SLC6A14. In polarized Calu‐3 cell monolayers, apical arginine influx has a leucine‐sensitive, sodium‐dependent component and a leucine‐ and lysine‐resistant sodium‐independent fraction. At the basolateral membrane, arginine transport was fully sodium‐independent and partially inhibited by leucine provided that sodium was present in the extracellular medium. Moreover, extracellular leucine trans‐stimulated arginine efflux from the basolateral membrane in the presence, but not in the absence, of sodium. The transepithelial, apical to basolateral, arginine transport strictly depended on the presence of sodium and was markedly inhibited by apical leucine, but significantly trans‐stimulated by the neutral amino acid added at the basolateral side. When added at the apical side, the NOS‐inhibitors NMMA and NIL, CAA analogs with a free carboxyl group, markedly inhibited the apical arginine influx and the transepithelial flux of the cationic amino acid. The same compounds trans‐stimulated basolateral arginine efflux. None of these effects were observed in the presence of the methyl ester analog NAME. The basolateral medium of Calu‐3 cell monolayers, obtained after incubation in the presence of the three inhibitors at the apical side, inhibited the production of NO by activated murine macrophages. The inhibitory effect of the Calu‐3 cell conditioned medium was time‐dependent and markedly higher with NMMA and NIL than with NAME. Moreover, the NOS‐inhibitory effect of the medium was significantly enhanced if NMMA and NIL, at the apical side, and basolateral leucine were simultaneously present during the conditioning procedure. These results indicate that 1) human airway epithelial cells express a functional system y+L at the basolateral membrane; 2) in this model, transepithelial arginine transport involves apical influx through system B0,+ and basolateral efflux through system y+L, and 3) the same transporters also perform an efficient transepithelial transport of amino acid‐like NOS inhibitors.

Glutamine stimulates mTORC1 independent of the cell content of essential amino acids

Glutamine and leucine are important mTORC1 modulators, although their roles are not precisely defined. In HepG2 and HeLa cells glutamine-free incubation lowers mTORC1 activity, although cell leucine is not decreased. mTORC1 activity, suppressed by amino acid-free incubation, is completely rescued only if essential amino acids (EAA) and glutamine are simultaneously restored, although cell leucine is higher in the absence than in the presence of glutamine. Thus, glutamine stimulates mTORC1 independent of cell leucine, suggesting the existence of two distinct stimulatory signals from either glutamine or EAA.

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 siRNAs 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.