Gian C. Gazzola

The cluster-tray method for rapid measurement of solute fluxes in adherent cultured cells

Abstract The use of an inexpensive and simple modification of Costar 24-well cluster trays is described in a rapid and reproducible method for measuring substrate fluxes in adherent cultured eukaryotic cells.

Regulation of amino acid transport in chick embryo heart cells: I. Adaptive system of mediation for neutral amino acids

Abstract The regulation of amino acid transport systems in chick embryo heart cells has been studied. Experiments were designed to investigate activity and kinetics of transport for analogue and natural amino acids as a function of time under various in vitro conditions (active and inhibited protein and RNA synthesis, amino acid dependence, transinhibition). Conclusions based on the proposed experimental approach include the following: 1. 1. The transport activity of a group of neutral amino acids (corresponding to those ascribed to the A mediation by Christensen and co-workers, J. Biol. Chem. , 238(1963)3686; Adv. Enzymol. , 32(1969)1) increases with time when cells or intact hearts are incubated in the absence of added amino acids. This increase is abolished in the presence of puromycin, cycloheximide and actinomycin D. 2. 2. The increase in activity of the A transport system (investigated by aminoisobutyrate and proline as representative amino acids) is prevented by the addition to the incubation medium of amino acids assigned to the same system of mediation on the basis of competition studies. It is not substantially altered by the addition of amino acids pertaining to the L system of mediation or basic amino acids. 3. 3. Transinhibition does not appear of major importance in the regulation of the activity of the A transport system. 4. 4. Kinetic analyses of aminoisobutyrate uptake in which the transport of the analogue by the A and L systems of mediation could be separated stress further that a time-dependent regulation is effective for the sole A transport system (affecting the maximal velocity without substantial changes in K m ) and provide a comprehensive explanation for previously reported changes with time and intracellular substrate concentration of the kinetic parameters governing the uptake of the analogue 9 . 5. 5. Provisional mechanisms for the observed adaptive regulation of the activity of the A transport system for neutral amino acids have been considered.

Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy

SUMMARY Although morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate.

Amino acid starvation induces the SNAT2 neutral amino acid transporter by a mechanism that involves eukaryotic initiation factor 2α phosphorylation and cap-independent translation

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 eIF2α that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2α 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 2α phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2α phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.

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.

The adaptive regulation of amino acid transport system A is associated to changes in ATA2 expression

The activity of transport system A for neutral amino acids is adaptively stimulated upon amino acid starvation. In cultured human fibroblasts this treatment causes an increase in the expression of the ATA2 system A transporter gene. ATA2 mRNA increase and transport stimulation are suppressed by system A substrates, but they are unaffected by other amino acids. Supplementation of amino acid‐starved cells with substrates of system A causes a decrease in both ATA2 mRNA and system A transport activity. These results suggest a direct relationship between ATA2 expression and system A transport activity.

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 acid starvation induces the SNAT2 neutral amino acid transporter by a mechanism that involves eIF2alpha phosphorylation and cap-independent translation

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 eIF2α that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2α 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 2α phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2α phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.

Amino Acid Transporters: Systematic Approach and Principles of Control

All living cells, from autonomous protists to constrained components of the complex multicellular mammalian organisms, require amino acids for replacing their structure, synthesizing essential proteins and supplying sources of metabolic energy. Transport of these molecules across the membranes that fix the boundaries of the cell and generate intracellular discontinuities is one of the determinants of cell survival.

The role of the neutral amino acid transporter SNAT2 in cell volume regulation

Sodium‐dependent neutral amino acid transporter‐2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti‐SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short‐term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.