C6 glioma cells differentiated by retinoic acid overexpress the glutamate transporter excitatory amino acid carrier 1 (EAAC1).

The transport of excitatory amino acids (EAA) in CNS is performed by a family of high affinity, sodium dependent carriers. One of these transporters, excitatory amino acid carrier 1 (EAAC1), is known to be regulated by several mechanisms that modify carrier abundance on the plasma membrane. Much less is known on EAAC1 regulation at the level of gene expression. Here we report that, in C6 rat glioma cells, a line recently described to contain neural stem-like cells, EAAC1 is markedly induced by all trans-retinoic acid (ATRA), a well known differentiating agent. Consistently, ATRA stimulates EAA transport, with the maximal effect observed at concentrations>or=1 microM. After 4 days of treatment with 10 microM ATRA, the transport Vmax is fivefold enhanced, Slc1a1 mRNA is increased by 400% compared with control, EAAC1 carrier is sixfold overexpressed and the C6 culture is greatly enriched of cells with bipolar morphology strongly positive for EAAC1 immunoreactivity. Compared with untreated cells, ATRA-treated C6 cells express less Slc1a3 mRNA, for the transporter GLAST, but significantly higher levels of Slc1a2 mRNA, for the transporter GLT-1, although no expression of either protein is detected with Western blot in both untreated and ATRA-treated cells. Consistently, the inhibition pattern of aspartate transport and its stimulation by phorbol esters are indicative of a transport process due to EAAC1 operation. Under the conditions adopted, ATRA treatment causes the induction of proteolipid protein, an oligodendrocytic marker. These results indicate that, in C6 cells, ATRA stimulates the expression of EAAC1, possibly as a step toward oligodendrocytic differentiation, and constitute the first demonstration of the induction of this transporter by a differentiating agent.

The inhibition of glutamine synthetase sensitizes human sarcoma cells to L-asparaginase.

PURPOSE: To evaluate the activity of the antitumor enzyme L: -asparaginase (ASNase) on tumor cells of mesenchymal origin and the contribution of glutamine synthetase (GS) to the adaptation to the metabolic stress caused by the anti-tumor enzyme. METHODS: We studied the effects of ASNase in six human sarcoma cell lines: HT1080 (fibrosarcoma); RD (rhabdomyosarcoma); SW872 (liposarcoma); HOS, SAOS-2, and U2OS (osteosarcoma) in the absence or in the presence of the GS inhibitor methionine L: -sulfoximine (MSO). RESULTS: HT1080 and SW872 cells were highly sensitive to ASNase-dependent cytotoxicity. In contrast, RD, SAOS-2, HOS, and U2OS cells exhibited only a partial growth suppression upon treatment with the anti-tumor enzyme. In these cell lines ASNase treatment was associated with increased levels of GS. When ASNase was used together with MSO, the proliferation of the poorly sensitive cell lines was completely blocked and a significant decrease in the IC(50) for ASNase was observed. Moreover, when ASNase treatment was carried on in the presence of MSO, HOS and U2OS osteosarcoma cells exhibited a marked cytotoxicity, with increased apoptosis. CONCLUSIONS: In human sarcoma cells (1) GS markedly contributes to the metabolic adaptation of tumor cells to ASNase and (2) the inhibition of GS activity enhances the antiproliferative and cytotoxic effects of ASNase. The two-step interference with glutamine metabolism, obtained through the combined treatment with ASNase and MSO, may provide a novel therapeutic approach that should be further investigated in human tumors of mesenchymal origin.

Comparison of efficacy, safety and immunologic effects of subcutaneous and sublingual immunotherapy in birch pollinosis: a randomized study.

BACKGROUND: Sublingual immunotherapy (SLIT) is currently considered a valid option to subcutaneous immunotherapy (SCIT), but only a few studies made a direct comparison of their effectiveness. The aim of this study was to compare the clinical and immunological effects of SCIT and SLIT in pollinosis induced by Betulaceae. METHODS: Forty-seven adult patients were randomized to receive SCIT or SLIT, performed by Betulaceae (alder, birch, and hazel) extracts from Stallergenes (Antony, France) standardized in index of reactivity (IR) with the treatment schedules proposed by the producer. The clinical effects were established by symptom-medication scores recorded during the month of March. Side effects were reported directly by the physicians for SCIT and were registered in diary cards by the patients for SLIT. Immunologic evaluation was done by measuring specific IgE and IgG4 to Bet v 1. RESULTS: Thirty-four patients (19 for SCIT and 15 for SLIT) completed the registration of symptoms and drug consumption during pollen period of Betulaceae. Mean cumulative doses of respectively 50.65 IR by SCIT and 4653.1 IR by SLIT were administered, with a SLIT/SCIT ratio of 92. There was no significant difference in mean symptom-medication score between SCIT and SLIT. Systemic reactions occurred in 16% of SCIT treated but in none of SLIT treated. As to immunologic evaluation, Bet v 1 specific IgE did not rise after the pollen season in SCIT treated, while increased non significantly in SLIT treated. Bet v 1 specific IgG4 increased in both treatment, buy only the increase with SCIT was significant (p = 0.001). CONCLUSION: SLIT and SCIT with a ratio of about 100 are equally effective in controlling rhinoconjunctivitis caused by tree pollen allergy. SLIT is safer than SCIT, but does not show the same immunologic effects on serum specific IgE and lgG4 antibodies.

Adaptive regulation of amino acid transport across the cell membrane in avian and mammalian tissues.

The regulation of amino acid transport across the cell membrane by adaptive mechanisms has been studied in a variety of mesenchymal and epithelial cells and tissues of avian and mammalian origin. Changes in transport activity as a function of time under various in vitro conditions (amino acid dependence, active and inhibited protein synthesis) have been evaluated by measurements of initial entry rates with representative amino acids. Results and conclusions based on the adopted experimental approach include the following. (1) An adaptive control mechanism for the transport of neutral amino acids corresponding to the typical substrates of the A mediation is operative in (a) mesenchymal cells (fibroblasts, chondroblasts, osteoblasts and myoblasts) from embryonic tissues of avian (chick embryo) origin and (b) mesenchymal cells from immature rat uterus (fibroblasts and smooth muscle cells) and other mammalian tissues (cardiac cells from newborn mouse and rat heart). (2) Adaptive regulation is restricted to a discrete subgroup of amino acids (L-proline, glycine and the analogue alpha-aminoisobutyric acid) in rat peritoneal macrophages and thymic lymphocytes. (3) Adaptive regulation is absent in erythroid cells (human erythrocytes, rabbit erythrocytes and reticulocytes, avian erythrocytes) which lack the A mediation and are incapable of active gene transcription. (4) Adaptive regulation is absent in the epithelial kidney cortex tissue and possibly absent in the epithelial component of liver tissue from adult rats; it is fully operative in the chick embryo crystalline lens, i.e. an epithelial preparation of embryonic origin. (5) These observations indicate that adaptive control mechanisms of amino acid transport across the cell membrane are quite common among tissues and species and emphasize their broad biological significance in eukaryotes.

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.

Perturbation of Na+ and K+ gradients in human fibroblasts incubated in unsupplemented saline solutions.

Changes in the intracellular concentrations of Na+ and K+ of fetal human fibroblasts have been followed after replacement of serum-containing growth media with unsupplemented and serum-supplemented saline solution (Earle's balanced salt solution). Incubation in unsupplemented salt solution was followed by a progressive increase of the internal Na+ counterbalanced by a decrease of internal K+, without major alterations of the internal osmolarity. After 3 h incubation the intracellular Na+ and K+ concentrations were 120 mM and 50 mM, respectively. These intracellular ion derangements were not associated with a failure of the (Na+ + K+)-ATPase pump, whose activity actually increased with enhanced intracellular Na+ concentration. Ion changes did not take place when serum (in excess of 0.5%, final concentration) was present in the saline solution and a complete restoration to normal of the Na+ and K+ gradients occurred upon addition of serum to cells previously incubated in plain saline solution. The effects of serum were mimicked by furosemide, thus suggesting that channels sensitive to this diuretic are involved in the movement of Na+ and K+ following fibroblast incubation in unsupplemented saline solution.

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.

The transport of L-glutamine into cultured human fibroblasts.

The transport of L-glutamine has been studied in diploid human fibroblasts in culture. Mathematical discrimination by nonlinear regression, competition analysis, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of this amino acid. The adopted criteria showed that L-glutamine enters the fibroblast by the Na(+)-dependent systems ASC and A and by a Na(+)-independent route identified as system L. The relative contribution of these agencies to the total saturable uptake of glutamine varied with the concentration of the amino acid and with the nutritional state of the cell. At amino acid concentrations approaching those encountered in human plasma: (1) system ASC represented the primary mediation for entry of L-glutamine in human fibroblasts; (2) the contribution of system A was lower, though significant, in unstarved repressed cells and became predominant in starved derepressed cells; (3) the Na(+)-dependent system L accounted for less than one-fifth of glutamine uptake in either nutritional condition. The changes in the relative contribution of the various systems to the uptake of glutamine as a function of its concentration may have implications in pathophysiology under conditions associated with enhanced glutamine concentrations in the extracellular fluids.

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.

Effect of insulin on the activity of amino acid transport systems in cultured human fibroblasts.

The regulation of amino acid transport by insulin has been studied in cultured human fibroblasts. Among the six amino acid transport systems operating in cultured human fibroblasts, two systems (A and X-C) are strongly stimulated by insulin and four (ASC, X-AG, y+ and L) are essentially not sensitive to the presence of the hormone in the incubation medium. The hormonal stimulation of system A and system X-C became significant after 3 h of incubation and increased up to 12 h. The stimulatory effect was related to insulin concentration, with a half-maximal stimulation at 10(-9) M hormone concentration. Insulin enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in Km values.

Effect of extracellular potassium on amino acid transport and membrane potential in fetal human fibroblasts.

The distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) has been used to estimate the electrical potential difference across the plasma membrane in cultured human fibroblasts. These cells exhibit a membrane potential markedly influenced by the diffusion potential of K+. High extracellular potassium concentrations depolarize human fibroblasts and depress the activity of transport systems A, ASC (both serving for zwitterionic amino acids), X-AG (for anionic amino acids), and y+ (for cationic amino acids). High doses (100 microM) of the K+-ionophore valinomycin hyperpolarize the cells. This condition enhances the activity of systems A, ASC and y+. Transport systems L (for neutral amino acids) and x-C (for anionic amino acids) are insensitive to changes in extracellular K+ or to valinomycin. System X-AG is inhibited by the addition of 100 microM valinomycin, but the effect of the ionophore appears to be potential-independent. These results indicate that: (a) the activity of systems L and x-C is potential-independent and (b) the activity of systems A, ASC, X-AG and y+ is sensitive to alterations of external [K+] associated to changes in membrane potential.

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.

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.

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)

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

When grown on permeable supports, pancreatic duct adenocarcinoma CAPAN-1 cells establish very high values of transepithelial resistance (TER). The addition of ethanol produced a dose-related, reversible drop in the TER of these cells, ranging from 15% (with 1% ethanol) to 65% (with 10% ethanol). The ethanol effect was rapid and reversible. The resistance decrease was associated with an increase in monolayer permeability to mannitol. No significant decrease in cell ATP was detected for ethanol concentrations lower than 7%. Confocal vertical sections of calcein-loaded monolayers of CAPAN-1 cells, grown on plasticware, showed a progressive deflation of domes detectable after 5 min of treatment with 2% ethanol. Incubation in an ethanol-free medium caused a progressive dome restoration. Immunocytochemical analysis of ethanol-treated cells indicated that ZO-1 and occludin exhibited clear cut distribution changes while the perijunctional actin pattern was slightly modified. Electron microscopy showed that a discrete intercellular space was detectable between adjacent ethanol-treated cells but not between control cells. These data indicate that ethanol is a tight junction barrier opener in pancreatic duct cells.

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.

Immunological patterns in monoarticular juvenile rheumatoid arthritis.

Seven pediatric patients with monoarticular arthritis, three of whom had a recent onset form and the remaining four a disease of longer duration, were examined for possible modifications of their immunological parameters. The diagnosis of JRA was made on all these patients according to the ARA criteria after a follow-up of at least two years. Humoral and cellular abnormalities of the immune system were searched for in peripheral blood, synovial fluid and synovial membrane. No evidence for complement consumption and for increased levels of immune-complexes was found in the sera and in the synovial fluids of these patients, who were all seronegative. Some patients had antinuclear antibodies in their sera and synovial fluids. With regard to the lymphocyte distribution, whereas only some patients had an increased number of circulating B cells, the majority had a decreased CD4+/CD8+ ratio in the synovial fluid compared to the ratio found in the peripheral blood. A massive infiltration of CD4+ cells and macrophages and the presence of a substantial number of OKT9+ cells was found in the synovial membranes.