Anna Dybel

Increased expression of a glutamine transporter SNAT3 is a marker of malignant gliomas.

Glutamine (Gln) is a growth determinant in neoplastic tissues. We analysed by RT-PCR the expression of mRNAs coding for the human variants of Gln transporters: ASCT2 (system ASC), SNAT1 [ATA1] (system A), SNAT3 [SN1] and SNAT5 [SN2] (system N), in samples of human malignant gliomas WHO grades III/IV (anaplastic astrocytoma and glioblastoma), glioma-derived cell cultures, brain metastases from peripheral organs, and control brain tissue. (Transporters designated according to the nomenclature recently proposed by Mckenzie and Ericksson [1]. Original designations in square brackets.) SNAT3 mRNA showed a 3–5 times stronger expression in gliomas than in metastases or control tissue, and was virtually absent from glioma cultures. Native glioblastoma immunostained positively with anti-SNAT3 antibody. The expression of ASCT2 mRNA, but not SNAT5 or SNAT1 mRNAs, was increased in all neoplastic tissues studied. Hence, increased expression of SNAT3 is a marker of primary malignant gliomas in situ.

Glutamine transport in C6 glioma cells shows ASCT2 system characteristics.

Previous studies from this laboratory have shown that glutamine (Gln) uptake in a rat astrocytoma-derived C6 cell line shows characteristics similar with the uptake of a model ASC system substrate, threonine, whose pH-dependence and partial tolerance of Li(+) substitution for Na(+) resemble the ASCT2 variant of the system. In support of the previous findings, RT-PCR analysis revealed that C6 cells strongly express ASCT2 mRNA, but not at all GlnT mRNA or NAT2 mRNA, the A and N system variants specifically engaged in Gln transport in normal CNS. Other features of Gln transport in C6 cells indicating the involvement of ASCT2 system included its resistance to ouabain and stimulation of Gln efflux from the cells in the presence of excess Gln or cysteine (Cys), demonstrating that the system operates in the exchange mode. Replacement of NaCl in the incubation medium with isoosmotic sucrose did neither significantly affect the kinetics, nor any other major characteristics of Gln or Thr transport, including its pH-dependence, inhibition by ASCT system substrates or resistance to the model system A substrate-N-methylamino-isobutyric acid (MeAiB).

Demonstration of kynurenine aminotransferases I and II and characterization of kynurenic acid synthesis in cultured cerebral cortical neurons

The present study characterizes the synthesis of kynurenic acid (KYNA) from exogenously added kynurenine and its regulation by extrinsic factors, in cultured cerebral cortical neurons and, for comparison, in astrocytes incubated under identical conditions. The neuronal culture showed positive immunostaining for both kynurenic acid aminotransferase (KAT) isoforms I and II. Neurons synthesized KYNA at a rate about 2.3 times higher than astrocytes. Neuronal, but not astrocytic, KYNA synthesis was lowered ∼30% by ionotropic glutamate receptor agonists [(R,S)‐3‐hydroxy‐5‐methoxyloxasole‐4‐propionic acid (AMPA; 100 μM) and N‐methyl‐D‐aspartic acid (NMDA; 100 μM)] and depolarizing agents [KCl (50 mM) and 4‐aminopyridine (4‐AP; 10 μM)]. Neuronal and astrocytic synthesis alike were vulnerable to inhibition exerted by the aminotransferase inhibitor aminooxyacetic acid (AOAA), glutamate (IC50: 31 and 85 μM, respectively), substrates of the L‐amino transport system [leucine (Leu); IC50: 19 and 42 μM, respectively] and 2‐aminobicyclo[2,2,1]heptane‐2‐carboxylic acid (BCH; IC50: 19 and 28 μM, respectively). Glutamine (Gln), which is a metabolic precursor of glutamate in astrocytes and L‐system substrate in both cell types, inhibited KYNA synthesis both in neurons and in astrocytes (IC50: 268 and 318 μM, respectively). α‐Ketoisocaproic acid (KIC), a Leu transamination product that is produced mainly in astrocytes and shuttled to neurons to modulate intraneuronal concentration of glutamate, stimulated KYNA synthesis in neurons but did not affect the synthesis in astrocytes. In conclusion, this study is the first to demonstrate active, regulation‐prone KYNA synthesis in neurons.

Delayed induction of apoptosis by ammonia in C6 glioma cells.

Ammonia is a neurotoxin whose administration in large doses causes coma and death of the exposed animals, but whether and in what degree these whole body effects are related to the death of CNS cells is not known. Since the downstream effects of ammonia in cultured CNS cells appear to be partly mediated by overactivation of several putative signalling mechanisms characteristic for the apoptotic program, we speculated that ammonia neurotoxicity may be apoptogenic. In this study, C6 glioma cells grown in 2% serum were exposed to 5 mM or 10 mM NH(4)Cl (ammonia) for 96 h and tested for the appearance of apoptosis by (a) Hoechst staining, (b) TUNEL reaction and (c) DNA ladder, at different times of exposure. In cultures exposed to either 5 mM or 10 mM ammonia, about 10% of the cells were found to enter apoptosis at 48 h of exposure, and the number of apoptotic cells rose to 30% at 72 h, and to 50% at 96 h of exposure, respectively. The first transduction signal purportedly involved in apoptosis, activation of PKCalphabeta, was transient and appeared already after 3-6 h of treatment. Coincident with pronounced manifestation of apoptosis (at 72 h and even more at 96 h of exposure) was an increased transfer of the transcription factor NFkappaB from cytoplasmto nucleus as revealed by EMSA assay. The number of cells affected by ammonia-induced apoptosis was markedly reduced by incubation with a NOS inhibitor, L-NAME at 100 microM concentration. The results indicate that ammonia-induced apoptosis is a result of a complex interplay of at least three signalling molecules: NO, PKC and the transcription factor NFkappaB, with NFkappaB being possibly involved in the induction of iNOS and generation of toxic levels of NO in the cells.

Regulation of kynurenic acid synthesis in C6 glioma cells

Studies with brain slices have provided evidence that synthesis of kynurenic acid (KYNA) from kynurenine (KYN), which occurs in astrocytes, is modulated by changes in the ionic composition of the medium and the presence of depolarizing agents or the excitatory amino acid glutamate (Glu). The present study analyzed the effects of changes in incubation medium on KYNA synthesis in cultured C6 glioma cells. The synthesis was not affected by omission of Na+ and raising K+ concentration to 50 mM, conditions that in brain slices stimulate or inhibit KYNA formation, respectively. KYNA synthesis in C6 cells was inhibited by the absence of Ca2+, which contrasts with its Ca2+ independence in brain slices. Also, lack of Mg2+ and addition of a chloride channel blocker, 4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulfonate (SITS), did not affect the synthesis. KYNA synthesis in C6 cells was dose dependently inhibited by Glu. The inhibitory effect of Glu was not affected by GDPβS, an antagonist of metabotropic Glu receptors, the receptor class prevailing in C6 cells, suggesting that Glu acted intracellularly. NH4Cl and veratridine decreased KYNA production, mirroring the effects noted in brain slices. KYNA synthesis was strongly reduced in the presence of leucine (Leu), and the uptake of [14C]Leu was inhibited by the KYNA precursor KYN, which points to Leu as a potential endogenous modulator of KYNA formation in CNS cells.

Glutamine transport in C6 glioma cells: Substrate specificity and modulation in a glutamine deprived culture medium

A previous study has shown that glutamine (Gln) uptake in C6 cells grown in a standard medium containing 2 mM Gln, is predominantly mediated by a sodium‐dependent system that is inhibited by ASC system substrates alanine (Ala), serine (Ser), cysteine (Cys) and threonine (Thr), shows pH sensitivity and partial tolerance to substitution of Na+ by Li+, features compatible with system ASCT2 that is strongly expressed in cultured astrocytes. The uptake was not inhibited by the model system A substrate α‐(methylamino)isobutyric acid (MeAiB), and glycine (Gly) or proline (Pro), indicating that the substrate‐regulated system A as defined by routine criteria is relatively inactive in these cells (Dolińska et al., 2000 ). In this study we compared the uptake of radiolabeled Gln and a model ASC substrate ‐Thr in cells grown to the same density in Gln‐containing and Gln‐deprived media. Cells grown in the absence of Gln showed a reduced activity of system ASC‐mediated Gln uptake, and the system lost tolerance for Li+ and became somewhat more resistant to lowering pH of the medium. In contrast to cultured astrocytes deprived of Gln, the overall Gln uptake activity in C6 cells adapted to grow in a medium without Gln was lower than in cells grown in a Gln containing medium, and the uptake by system A remained inactive. C6 cells cultured both in the presence and absence of Gln expressed ASCT2 mRNA, indicating that system ASCT2‐mediated Gln uptake is modulated at a posttranscriptional level. In contrast to Gln uptake, Thr uptake was more active in cells cultured in the absence of Gln and showed neither pH dependence nor lithium tolerance in either medium, which is typical of an uptake mediated by the widespread ASCT1 isoform of system ASC. In C6 cells grown in the presence or absence of Gln alike, ≈20% of the sodium‐dependent Gln uptake was resistant to MeAiB+Thr, indicating contribution of system N. The N system‐mediated uptake in C6 cells grown in the absence, but not in the presence of Gln was not inhibited by glutamate (Glu) that conforms to the characteristics of the glial N system variant, SN1.

Glutamine transport in C6 glioma cells.

Glutamine transport across the cell membranes of a variety of mammalian tissues is mediated by at least four transport systems: a sodium-independent system L, and sodium-dependent systems A, ASC and N, the latter occurring in different tissue-specific variants. In this study we assessed the contribution of these systems to the uptake of [(3)H]glutamine in C6 rat glioma cells. The sodium-dependent uptake, which accounted for more than 80% of the total uptake, was not inhibited by 2-methylaminoisobutyric acid (MeAIB), indicating that system A was inactive, possibly being depressed by glutamine present in the culture medium. About 80% of the sodium-dependent uptake was mediated by system ASC, which differed from system ASC common to other CNS- and non-CNS tissues by its pH-dependence and partial lithium tolerance. The residual 20% of sodium-dependent uptake appeared to be mediated by system N, which was identified as a component resistant to inhibition by MeAIB+threonine. The system N in C6 cells appeared to be neither fully compatible with the neuronal system Nb, nor with the N system described in astrocytes: it differed from the former in being strongly inhibited by histidine and showing fair tolerance for lithium, and from the latter in its pH-insensitivity and strong inhibition by glutamate. The sodium-independent glutamine uptake differed from the astrocytic or neuronal uptake in its relatively weak inhibition by system L substrates and a strong inhibition by system ASC substrates, indicating a possible contribution of a variant of the ASC system.

The role of protein kinase C and cyclic AMP in the ammonia-induced shift of the taurine uptake/efflux balance towards efflux in C6 cells.

A previous study showed that treatment of C6 glioma cells with 10 mM ammonium chloride (“ammonia”) for 24 h decreases taurine uptake and evokes sodium-dependent taurine efflux, indicating reversal of the taurine transporter (TauT)-mediated transport as an underlying mechanism. Consistent with the involvement of TauT we now show that the ammonia-induced changes in Tau uptake and efflux are inhibited by the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). Ammonia treatment of C6 cells resulted in increased intracellular accumulation of cAMP. Incubation of the cells with dibutyryl cAMP (dbcAMP) mimicked the effects of ammonia on both taurine uptake and efflux. The effects of dbcAMP on taurine uptake and efflux were additive to the effects of ammonia. Collectively, the results suggest that the effects of ammonia on taurine uptake and efflux may be partly mediated by cAMP. Consistent with this mechanism, the adenyl cyclase inhibitor, miconazole reduced the stimulation of efflux by ammonia.