G. C. Gazzola

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.

Platelet gel in the treatment of cutaneous ulcers: the experience of the Immunohaematology and Transfusion Centre of Parma

BACKGROUND Platelet gel is being ever more frequently used to promote healing of cutaneous ulcers. However, the factors that determine the often variable clinical outcome of this procedure are still incompletely understood. AIMS The aims of this study were to demonstrate that platelet gel, even when obtained under strictly controlled conditions, produces highly variable outcomes in patients with cutaneous ulcers and to propose a method for in vitro standardisation of the biological properties of platelet gel. MATERIAL AND METHODS.: Patients were enrolled on the basis of a pre-defined protocol. Platelet concentrate was produced with standard methods, with a variability in platelet count among the different samples of less than 10%. The platelet gel for clinical use was obtained, under strictly standardized conditions, by adding thrombin and calcium gluconate to the concentrates. For in vitro studies, platelet gel, obtained from platelet-rich plasma from four donors, was frozen and thawed twice so as to increase gel contraction. The supernatant was used to modify cell proliferation, protein synthesis, and the expression of selected genes in cultures of human diploid fibroblasts. RESULTS Seventeen patients (aged 44-78 years) with ulcers (4 diabetic, 11 vascular, 1 post-traumatic, 1 decubitus) were treated with platelet gel (4 autologous, 13 homologous). Complete re-epithelialisation of four ulcers (1 diabetic, 1 post-traumatic, 2 vascular) was obtained after applications of platelet gel (2 autologous, 2 homologous); in 11 other cases there was a greater than 50% reduction in the size of the ulcer. Two patients had no benefit. The supernatant of the platelet gel was able to promote dose-dependent proliferation and changes in gene expression as well as in metabolic activities related to protein synthesis. CONCLUSIONS Although the use of platelet gel in the treatment of cutaneous ulcers is increasing, and conditions for its production are better standardised, very considerable variability of clinical outcomes is still observed, even within single centres, suggesting that there are differences in biological properties of platelet concentrates from individual patients which cannot be readily controlled with current techniques. The biological effects of the platelet gel supernatant described in this article may provide the basis for a simple biological validation of platelet preparations before their clinical use, so as to reduce this potentially important source of variability.

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

PurposeTo 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.MethodsWe 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).ResultsHT1080 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 IC50 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.ConclusionsIn 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.

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 µM secretin or 10 µM 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.

Emerging roles for sodium dependent amino acid transport in mesenchymal cells.

SummaryThe functional aspects of sodium dependent amino acid transport in mesenchymal cells are the subject of this contribution. In a survey of the cross-talk existing among the various transport mechanisms, particular attention is devoted to the role played by substrates shared by several transport systems, such as L-glutamine. Intracellular levels of glutamine are determined by the activity of System A, the main transducer of ion gradients built on by Na,K-ATPase into neutral amino acid gradients. Changes in the activity of the System are employed to regulate intracellular amino acid pool and, hence, cell volume. System A activity has been found increased in hypertonically shrunken cells and in proliferating cells. Under both these conditions cells have to increase their volume; therefore, System A can be employed as a convenient mechanism to increase cell volume both under hypertonic and isotonic conditions. Although less well characterized, the uptake of anionic amino acids performed by System X−AG may be involved in the maintenance of intracellular amino acid pool under conditions of limited availability of neutral amino acids substrates of System A.

The glutamate transporter excitatory amino acid carrier 1 associates with the actin-binding protein α-adducin

Excitatory amino acid carrier 1 (EAAC1) belongs to the family of the Na(+)-dependent glutamate carriers. Although the association between defective EAAC1 function and neurologic disease has been repeatedly studied, EAAC1 regulation is not yet fully understood. We have reported that in C6 glioma cells both the activity and membrane targeting of EAAC1 require the integrity of actin cytoskeleton. Here we show that, in the same model, EAAC1 partially co-localizes with actin filaments at the level of cell processes. Moreover, perinuclear spots in which EAAC1 co-localizes with the actin binding protein alpha-adducin are observed in some cells and, consistently, faint co-immunoprecipitation bands between EAAC1 and alpha-adducin are detected. Co-localization and partial co-immunoprecipitation of EAAC1 and adducin are still detectable after cell treatment with phorbol esters, a condition that leads to a protein kinase C (PKC)-dependent increase of EAAC1 expression on the membrane and to the phosphorylation of adducin. A co-immunoprecipitation band was also detected in protein extracts of rat hippocampus. The amount of adducin co-immunoprecipitated with EAAC1 increases after the treatment of C6 cells with retinoic acid, a differentiating agent that induces EAAC1 overexpression in this cell model. Moreover, in clones of C6 cells transfected with a hemagglutinin (HA)-tagged adducin, the bands of EAAC1 immunoprecipitated by an anti-HA antiserum were proportional to EAAC1 expression. These results suggest the existence of a pool of EAAC1 transporters associated with the actin binding protein alpha-adducin in a PKC-insensitive manner.

Chlorpromazine, clozapine and olanzapine inhibit anionic amino acid transport in cultured human fibroblasts

Summary.We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X−AG (EAAT transporters) in cultured human fibroblasts. With 30 µM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (>60%), and referable to a decrease in Vmax. Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X−AG since transport systems A and y+ are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients.

Changes in neutral amino acid efflux and membrane potential associated with the expression of CFTR protein

SummaryThe expression of wild type CFTR facilitates the efflux of neutral amino acids (Rotoli et al., Biochem. Biophys. Res. Commun. 204: 653–658, 1994); as a result, after an extensive depletion of intracellular amino acid pool obtained through an incubation in saline solution, the intracellular leucine levels were lower in murine C127 cells transfected with the wild type CF gene (C127 CFTRw/t) than in cells transfected with either mutant CF (C127 CFTRΔF508 cells) or mock vector only. No change in amino acid efflux was detected when C127 CFTRw/t and C127 CFTRw/t and C127 CFTRΔF508 cells were studied under conditions known to activate protein kinase A. Upon an incubation in Cl− free medium, a permeant analogue of cAMP caused a marked cell depolarization of C127 CFTRw/t cells but not of C127 CFTRΔF508 cells, thus showing a functional expression of CFTR protein in the former cell line. However, we found that, upon a Cl− free incubation and in the absence of exogenous cAMP, C127 CFTRw/t cells developed a marked hyperpolarization that was not detected in C127 CFTRΔF508 cells. It is concluded that the expression of normal CFTR accelerates amino acid efflux and enhances cell hyperpolarization in Cl− free media; both these effects appear to be independent from PKA stimulation of CFTR.

Derangements of Cationic Amino Acid Transport in Fibroblasts from Human Desmoid Tumor

An increased rate of amino acid uptake by System A has been considered a consistent feature of tumor cells1. This inference is based on the following facts: (a) enhancements in the transport rates of site A-reactive amino acids are among the early events associated with cell transformation in vitro 2; (b) increased uptake of some amino acid substrates of System A has been reported for virus- and chemically-transformed cell lines3,4 and (c) rat fibroblasts made tumorigenic by ras transfection exhibit a somewhat faster uptake of 2-(methyl)aminoisobutyric acid (a transport-specific substrate of System A) than non-transfected cells5. However, tumor cell lines have been described that do not exhibit this metabolic feature6,7 and little is known on amino acid transport changes in cells from spontaneously occurring tumors8. We therefore devised experiments to investigate the amino acid transport in cells from a spontaneous human tumor (desmoid tumor fibroblasts) as compared with their normal counterpart (skin-derived fibroblasts). To avoid variability among individuals for transport, the comparison was made between cell cultures obtained from the same donor. The present study concerns the six transport systems for amino acids so far characterized in human fibroblasts9–13.