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Biomedical subjects

G C Gazzola

Publications and source records attributed to G C Gazzola.

At least 19 recordsLinked to original sources
PubMed2002

Employment of confocal microscopy for the dynamic visualization of domes in intact epithelial cell cultures.

Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 microM secretin or 10 microM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.

Arylsulfonates↗
PubMed2001

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.

Amino Acid Transport Systems↗
PubMed2001

Amino acid depletion activates TonEBP and sodium-coupled inositol transport.

The expression of the osmosensitive sodium/myo-inositol cotransporter (SMIT) is regulated by multiple tonicity-responsive enhancers (TonEs) in the 5'-flanking region of the gene. In response to hypertonicity, the nuclear abundance of the transcription factor TonE-binding protein (TonEBP) is increased, and the transcription of the SMIT gene is induced. Transport system A for neutral amino acids, another osmosensitive mechanism, is progressively stimulated if amino acid substrates are not present in the extracellular compartment. Under this condition, as in hypertonicity, cells shrink and mitogen-activated protein kinases are activated. We demonstrate here that a clear-cut nuclear redistribution of TonEBP, followed by SMIT expression increase and inositol transport activation, is observed after incubation of cultured human fibroblasts in Earle's balanced salts (EBSS), an isotonic, amino acid-free saline. EBSS-induced SMIT stimulation is prevented by substrates of system A, although these compounds do not compete with inositol for transport through SMIT. We conclude that the incubation in isotonic, amino acid-free saline triggers an osmotic stimulus and elicits TonEBP-dependent responses like hypertonic treatment.

Adolescent↗
PubMed2000

Arginine transport through system y(+)L in cultured human fibroblasts: normal phenotype of cells from LPI subjects.

In lysinuric protein intolerance (LPI), impaired transport of cationic amino acids in kidney and intestine is due to mutations of the SLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic amino acid (CAA) transport in human fibroblasts obtained from LPI patients and a normal subject. In both cell types the bidirectional fluxes of arginine are due to the additive contributions of two Na(+)-independent, transstimulated transport systems. One of these mechanisms, inhibited by N-ethylmaleimide (NEM) and sensitive to the membrane potential, is identifiable with system y(+). The NEM- and potential-insensitive component, suppressed by L-leucine only in the presence of Na(+), is mostly due to the activity of system y(+)L. The inward and outward activities of the two systems are comparable in control and LPI fibroblasts. Both cell types express SLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) as well as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). We conclude that LPI fibroblasts exhibit normal CAA transport through system y(+)L, probably referable to the activity of SLC7A6/y+LAT2.

Adolescent↗
PubMed2000

Secretin increases the paracellular permeability of CAPAN-1 pancreatic duct cells.

The effects of secretin, the physiological secretagogue for pancreatic ducts, were studied in CAPAN-1 pancreatic duct carcinoma cells. When grown to confluence on plastic dishes, CAPAN-1 cells form domes and exhibit marked increases in culture content of Na+ and urea distribution space (UDS). This parameter is measured as an index of both intracellular and dome compartments under the conditions adopted. Both Na increase and dome formation are inhibited by long term incubation with phorbols, DIDS, DPC, EIPA, H2DIDS, and brefeldin. Short term treatment with secretin or 8-Br-cAMP/teophylline causes dome collapse and a marked decrease in UDS and culture content of Na. Secretin-induced sodium decrease is not abolished by ion channel inhibitors, suggesting that diffusion routes other than ion channels are involved in hormone effects. This hypothesis is also in agreement with data obtained on CAPAN-1 cells cultured on permeable inserts, where no change in Na content or UDS is detected upon secretin treatment. Confluent monolayers exhibit a high transepithelial resistance (Rms) which is markedly and reversibly decreased by secretin. The hormone also decreases the transepithelial voltage (Vms) and raises the monolayer permeability to mannitol. It is concluded that secretin enhances the paracellular permeability of pancreatic duct cells. This effect of secretin, unknown thus far, may be involved in the mechanism of pancreatic secretion in vivo.

Cell Membrane Permeability↗
PubMed1999

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.

Amino Acids↗
PubMed1999

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.

Amino Acids↗
PubMed1998

Comparison of annexin V and calcein-AM as early vital markers of apoptosis in adherent cells by confocal laser microscopy.

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. (J Histochem Cytochem 46:895-900, 1998)

3T3 Cells↗
PubMed1997

Membrane potential changes visualized in complete growth media through confocal laser scanning microscopy of bis-oxonol-loaded cells.

Confocal laser scanning microscopy (CLSM) was employed to visualize and measure membrane potential changes in several types of cultured adherent cells, such as human fibroblasts, mouse mammary tumor C127 cells, and human saphenous vein endothelial cells, preloaded with the anionic dye bis-1, 3,-diethylthiobarbituratetrimethineoxonol (bis-oxonol). The fluorescence of cell-associated bis-oxonol was detected in a single confocal plane. An original flow-chamber apparatus was employed to replace the extracellular medium, avoiding alterations of the plane selected for observation. In all the cell types and the experimental situations tested the intracellular distribution of the dye was typical; perinuclear zones accumulated the dye which, conversely, was excluded by the nucleus. Fluorescence was calibrated versus the membrane potential by varying the extracellular concentration of sodium in the presence of gramicidin. With this approach membrane potential was measured (i) in cultured human fibroblasts incubated under anisotonic conditions, (ii) in heterogeneous cell populations which respond unevenly to potential perturbing conditions, and (iii) in human macrovascular endothelial cells maintained in high-serum, complete growth medium. The results obtained indicate that CLSM can be successfully employed to measure changes of membrane potential in single, bis-oxonol-loaded adherent cells under experimental conditions which severely hinder conventional spectrofluorimetric approaches.

Animals↗
PubMed1997

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.

Aged↗
PubMed1996

CFTR expression in C127 cells is associated with enhanced cell shrinkage and ATP extrusion in Cl(-)-free medium.

In this study we have employed three lines of C127 murine cells. C127 CFTR w/t, C127 CFTR delta F508 and C127 mock, transfected with, respectively, wild type, delta F508 mutant human CFTR cDNA or the vector only. In the first 10 minutes of a Cl(-)-free incubation the three cell lines exhibit a significant shrinkage due to a loss of K+ and Cl-. However, C127 CFTR w/t cells shrink more than C127 CFTR delta F508 and the mock cells. The supplementation of Cl(-)-free medium with ATP causes a marked decrease in the cell volume of C127 CFTR delta F508 and of the mock cells but not of C127 CFTR w/t cells. ATP effect is mimicked by adenosine 5'-O-(3-thiotriphosphate), but neither by adenosine nor by UTP. Measurements of extracellular ATP indicate that during the Cl(-)-free incubation C127 CFTR w/t cells extrude more ATP than the other two cell lines. The results are consistent with the hypothesis that CFTR enhances K+ and Cl- permeabilities by promoting the extrusion of ATP.

Adenosine Triphosphate↗
PubMed1996

Involvement of protein kinase Cepsilon in the stimulation of anionic amino acid transport in cultured human fibroblasts.

Protein kinase C (PKC) activation stimulates transport system X-AG for anionic amino acids in cultured human fibroblasts (Franchi-Gazzola, R., Visigalli, R., Bussolati, O., and Gazzola, G. C. (1994) FEBS Lett. 352, 109-112). To identify which PKC isoform is responsible for this effect, aspartate transport through system X-AG, PKC activity, and the subcellular distribution of PKC isoforms have been studied before and after treatment with phorbol 12, 13-dibutyrate (PDBu) in fibroblasts maintained at low serum for 1 (control cells) or 7 days (quiescent cells). In control cells aspartate transport and PKC activity in the particulate fraction were stimulated by short term PDBu treatment; both stimulatory effects were down-regulated by a prolonged exposure to the phorbol. In contrast, in quiescent cells aspartate transport and particulate PKC activity were higher than control under basal conditions, unaffected by a short term PDBu treatment, and lowered by a prolonged incubation with the phorbol. In both control and quiescent cells a short term PDBu treatment modified PKCalpha distribution, increasing its membrane-associated fraction. PKCdelta was mostly in the soluble fraction and scarcely sensitive to PDBu. A brief exposure to PDBu increased membrane-associated PKCepsilon in control but not in quiescent cells. In these cells epsilon isoform was found exclusively in the particulate fraction even in PDBu-untreated cells. A prolonged PDBu treatment caused a partial down-regulation of membrane-associated PKCepsilon in control cells and its marked decrease in quiescent cells. It is concluded that PKC-dependent changes in system X-AG activity parallel the behavior of PKCepsilon, thus suggesting a specific role for this isoform in system X-AG regulation.

Amino Acid Transport Systems↗
PubMed1996

The stimulation of Na,K,Cl cotransport and of system A for neutral amino acid transport is a mechanism for cell volume increase during the cell cycle.

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.

3T3 Cells↗
PubMed1995

Suppression of anionic amino acid transport impairs the maintenance of intracellular glutamate in Ha-ras-expressing cells.

When the expression of a Ha-ras oncogene is triggered in NIH3T3 cells, a progressive inhibition of sodium dependent transport of anionic amino acids through system X-AG is observed. After 48 h of ras expression the transport activity of system X-AG is almost abolished, while other transport systems involved in anionic amino acid transport are unaffected or even stimulated. In the presence of high extracellular concentrations of glutamine, the intracellular concentration of glutamate is comparable in ras expressing and non-expressing cells. On the contrary, when the extracellular pool of glutamine is depleted by the enzyme L-asparaginase, intracellular glutamate decreases at a much faster rate in ras expressing, low-transport cells. These results suggest that transport system X-AG significantly contributes to the homeostasis of intracellular glutamate under conditions of glutamine deprivation.

3T3 Cells↗
PubMed1995

Characterization of apoptotic phenomena induced by treatment with L-asparaginase in NIH3T3 cells.

The treatment of NIH3T3 cells with L-asparaginase causes a complete and reversible growth arrest with a decrease of cell number in the first 2 days. The enzyme induces impressive morphological changes that have been studied exploiting eosin in fixed cells and calcein in intact cells as sources of fluorescence for confocal microscopy. The first changes are observed after 12 h of treatment and the process is complete after 48 h. Both nucleus and cytoplasm shrink, while cells round and lose processes. Eventually most cells break; several debris include strongly hematoxylinic bodies negative for eosin fluorescence. Some cells neither round nor break in fragments. Throughout the process cells and fragments retain calcein fluorescence, thus indicating the integrity of the cell membrane. A rapid depletion of the intracellular pools of both glutamine and glutamate occurs in treated cells, followed by a decrease in DNA and protein syntheses, while the cell content of ATP, the transmembrane gradient of sodium, and the active transport of amino acids are scarcely affected. It is concluded that (i) L-asparaginase induces an apoptotic process in NIH3T3 cells that is forerun by a marked intracellular depletion of glutamate and glutamine; and (ii) although the enzyme completely suppresses cell proliferation, only a subset of cells undergoes apoptosis upon treatment. These findings provide a model for the characterization of factors that determine cell sensitivity to the effects of L-asparaginase.

3T3 Cells↗
PubMed1994

CFTR protein is involved in the efflux of neutral amino acids.

Trans-membrane fluxes of leucine were measured in mouse C127i cells transfected with the wild type (C127 CFTRw/t) or the delta F508 CF gene (C127 CFTR delta F508). Leucine efflux was significantly faster in C127 CFTRw/t cells. On the contrary, leucine influx was comparable in the two cell lines and referable to a "L-type" transport system. No significant differences in leucine content were detected among the two cell lines when maintained in complete growth medium; in contrast, after prolonged incubation in amino-acid-free saline solution, the amount of intracellular leucine was significantly smaller in C127 CFTRw/t than in C127 CFTR delta F508 cells. Leucine behavior was shared by other neutral amino acids with non polar side chains. These results suggest that the expression of normal CFTR increases the efflux of a subgroup of neutral amino acids.

Animals↗