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O Bussolati

Publications and source records attributed to O Bussolati.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1994

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

In 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 microM) 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.

Adolescent↗
PubMed1994

Response of human fibroblasts to hypertonic stress. Cell shrinkage is counteracted by an enhanced active transport of neutral amino acids.

Regulatory volume increase (RVI) has been studied in cultured human fibroblasts (CHF) incubated in a complete hypertonic growth medium (400 mosmol/kg). After the initial cell shrinkage induced by hypertonic treatment, cells recover their volume almost completely within 3 h. This RVI response is associated with a marked increase of the cell content of free amino acids. The cell content of potassium increases only slightly. Chromatographic analysis of the intracellular amino acid pool shows that the RVI-associated increase in cell amino acids is mainly a result of changes in the L-glutamine content. The intracellular accumulation of the analog 2-methylaminoisobutyric acid, a specific substrate of transport system A, is increased in CHF undergoing RVI. Hypertonic treatment causes an immediate and sustained cell hyperpolarization, as demonstrated by changes in the trans-membrane distribution ratio of L-arginine and in the fluorescence of the potential-sensitive dye bis-1,3-diethylthiobarbiturate-trimethineoxonol. Because of cell hyperpolarization, at the end of RVI the trans-membrane gradient of the sodium electrochemical potential is higher than that of the control. The increase in the extracellular potassium concentration ([K+]out = 40 mM) abolishes the hyperpolarization induced by hypertonic treatment and delays volume recovery. Cycloheximide suppresses RVI at a high but not at physiologic [K+]out. It is proposed that CHF counteract hypertonic shrinkage through an enhanced accumulation of substrates of transport system A sustained, initially, by an increase in the energy available for transport and, subsequently, also by the synthesis of new site A carriers.

Adolescent↗
PubMed1994

Regulatory volume decrease of cultured human fibroblasts involves changes in intracellular amino-acid pool.

Regulatory volume decrease (RVD) has been studied in cultured human fibroblasts incubated in a complete growth medium at low osmolality (215 mosmolal). After the initial swelling induced by hypotonic treatment, cells recover their volume almost completely within about 60 min. This RVD is associated with comparable losses of cell potassium and amino acids. After an initial increase, cell content of sodium is kept at values close to control. Chromatographic analysis of intracellular amino-acid pool has shown that RVD-associated decrease in cell amino acids is due for the most part to changes in the intracellular concentration of L-glutamine. RVD-exerting cells undergo a rapid and marked depolarization that is maintained after cell volume recovery. This change in membrane potential has been detected with measurements of both the transmembrane distribution ratios of L-arginine and of fluorescence of potential-sensitive dye bis-oxonol. Due to depolarization, the trans-membrane gradient of sodium electrochemical potential is lowered. It is proposed that cell depolarization concurs to keep the intracellular concentration of amino acids low by inhibiting sodium-coupled uptake through system A.

Adolescent↗
PubMed1993

The relationship between sodium-dependent transport of anionic amino acids and cell proliferation.

The relationship between the transport of anionic amino acids and the proliferative status of the cell population has been studied in NIH-3T3 cells. Proliferative quiescence, verified by determinations of growth-rate quotient and incorporation of thymidine, is associated with a marked increase of the influx of L-aspartate. After 7-10 days of serum starvation, the initial influx of L-aspartate increases by 8-10-times with respect to the transport activity determined in growing cells. The operational properties of the influx of L-aspartate are similar in growing and quiescent cells; in particular, the influx of the anionic amino acid is mostly Na(+)-dependent and completely suppressed by an excess of L-glutamate and D-aspartate, but not of D-glutamate. These features suggest that, in both cases, aspartate uptake occurs through system X(-)AG. The quiescence-related increase in aspartate transport is gradual, sensitive to the inhibition of protein synthesis and referable to the enhanced maximal capacity of transport system X(-)AG. Restoration of serum concentration in the culture medium of serum-starved cells causes a decrease in aspartate transport that is maximal in correspondence to late G1/S phases. It is concluded that the X(-)AG system for anionic amino-acid uptake is sensitive to the proliferative status of the cell population and that, in particular, its transport activity is stimulated by the establishment of proliferative quiescence.

3T3 Cells↗
PubMed1993

Characterization of amino acid transport in human endothelial cells.

The transport of amino acids has been studied in human umbilical vein endothelial cells. Neutral amino acids enter human umbilical vein endothelial cells through three distinct agencies endowed with the characteristics of systems A, ASC, and L. Each system has been studied by evaluating the influx of preferential substrates. The influx of L-proline and 2-methylaminoisobutyric acid occurs through an Na(+)-dependent adaptively regulated trans-inhibited agency identifiable with system A. L-Threonine influx occurs mainly through a distinct Na(+)-dependent trans-stimulated pathway corresponding to system ASC. System L accounts for Na(+)-independent influx of L-leucine. These systems cooperate for the transport of L-glutamine, which is due mainly to system ASC, whereas the component due to the operation of system A increases upon amino acid starvation. No clear evidence was found for a glutamine-specific system ("system N"). Two systems, one Na+ dependent (system XAG-) and the other Na+ independent (system xc-), transport anionic amino acids. L-Arginine influx exhibits a poor dependence on extracellular Na+, whereas it is sensitive to conditions known to change membrane potential and to trans-stimulation by intracellular amino acids. These features are consistent with a process mediated by system y+ and may be of significance for the regulation of the intracellular concentration of L-arginine.

Amino Acids↗
PubMed1993

Anionic amino acid transport in ras- transformed fibroblasts.

The sodium-dependent transport of anionic amino acids is suppressed in NIH3T3 cells that constitutively express ras oncogenes. In a model of NIH3T3 cells in which ras expression is triggered in the presence of dexamethasone, aspartate transport decreases gradually upon dexamethasone treatment and is almost completely suppressed after two days of incubation in the presence of the steroid. In the same cell model, lovastatin, an inhibitor of beta hydroxy-beta methyl-glutaryl-CoA-reductase and, hence, of farnesylation of p21ras, partially protects aspartate transport from the inhibition observed upon steroid treatment. Determinations of cell glutamate in ras-expressing and non expressing cells indicate that in both cell models glutamate decreases when extracellular medium is depleted of glutamine. However, this decrease is much faster in cells expressing ras (either constitutively or conditionally). It is proposed i) that cell production of oncogenic p21ras hinders sodium-dependent transport of anionic amino acids and ii) that the transport alteration impairs the maintenance of cell levels of glutamate in ras-expressing cells.

Amino Acids↗
PubMed1992

Transport system ASC for neutral amino acids. An electroneutral sodium/amino acid cotransport sensitive to the membrane potential.

The influx of L-threonine through system ASC does not influence the membrane potential in cultured human fibroblasts although comparable fluxes of amino acids through another Na(+)-dependent agency, system A, effectively depolarize the cells. The membrane potential, however, stimulates the influx of amino acids through system ASC with a maximal effect at -50 mV. The sensitivity of amino acid influx through system ASC to the membrane potential is not constant, but rather, is dependent on intracellular and extracellular concentrations of the substrates, Na+ and amino acids, of the system. Conditions which favor the loading of the ASC carrier at the external surface reduce the sensitivity of ASC-mediated amino acid influx to the membrane potential; in contrast, the sensitivity of this amino acid influx increases under conditions which favor loading of the carrier at the internal surface. Trans-stimulation, a well-known characteristic of system ASC, also varies with the concentrations of the substrates of the system and, in fact, this characteristic is not observed when external Na+ is low. These data may be accommodated by a model in which an electrically silent mode of operation of the transporter is dominant. The influence of the membrane potential on the transport system is dependent on the extent to which a charge-translocating step in the cycling of the carrier is rate limiting (relative rate limitance).

Adolescent↗
PubMed1991

The preferential interaction of L-threonine with transport system ASC in cultured human fibroblasts.

The transport of L-threonine was studied in cultured human fibroblasts. A kinetic analysis of L-threonine transport in a range of extracellular concentrations from 0.01 to 20 mM indicated that this amino acid enters cells through both Na(+)-independent and Na(+)-dependent routes. These routes are: (1) a non-saturable, Na(+)-independent route formally indistinguishable from diffusion; (2) a saturable, Na(+)-independent route inhibitable by the analog BCH and identifiable with system L; (3) a low-affinity, Na(+)-dependent component (Km = 3 mM) which can be attributed to the activity of system A since it is adaptively enhanced by amino acid starvation and suppressed by the characterizing analog MeAIB and (4) a high-affinity, Na(+)-dependent route (Km = 0.05 mM). This latter route is identifiable with system ASC since it is insensitive to adaptive regulation, uninhibited by MeAIB, trans-stimulated by intracellular substrates of system ASC, markedly stereoselective, and relatively insensitive to changes in external pH. At an external concentration of 0.05 mM more than 90% of L-threonine transport is referrable to the activity of system ASC; in these conditions, the transport of the amino acid exhibits typical ASC-features even in the absence of inhibitors of other transport agencies, and, therefore, it can be employed as a reliable indicator of the activity of transport system ASC in cultured human fibroblasts.

Amino Acids↗
PubMed1991

Energization of amino acid uptake by system A in cultured human fibroblasts.

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.

Amino Acids↗
PubMed1991

Membrane potential and amino acid transport in a mutant Chinese hamster ovary cell line.

The bioenergetics of amino acid transport system A was studied in two Chinese hamster ovary (CHO) cell lines, the parent line CHO-PEOT/1 and CHY-1, a mutant of the former exhibiting a low activity of the same transport system. The steady-state transmembrane distribution ratio of the cationic amino acid L-arginine (RARG) was employed as an indicator of membrane potential (delta psi). Evidence for the reliability of RARG to measure delta psi can be summarized as follows: (1) L-arginine transmembrane distribution increased under conditions of cell hyperpolarization and decreased under conditions of cell depolarization; (2) L-arginine distribution conformed closely to that expected for a probe of delta psi in conditions in which delta psi depends largely on the transmembrane potassium gradient; and (3) the value of delta psi obtained through a valinomycin null point experiment (-72.7 mV) was very similar to the value calculated from L-arginine distribution using the Nernst equation (-73.4 mV). The transmembrane gradient of sodium electrochemical potential (delta mu Na), the driving force for the operation of system A, was slightly higher in the mutant cell line CHY-1. In the same line, the intracellular level of the specific system A substrate MeAIB at steady state was also higher. Studies of the rheogenicity of system A in the two lines indicated that the depolarization associated with the entry of substrates of system A was proportional to the amount of amino acid taken up by the cells. Kinetic analysis showed that the low activity of system A in the mutant cell line was referrable to a decrease in transport Vmax. It is concluded that neither a decrease in energy available for the operation of system A nor a decreased efficiency of coupling of the system to delta psi is responsible for the defect observed in the mutant line.

Alanine↗