Attenuation of initial pilocarpine-induced electrographic seizures by methionine sulfoximine pretreatment tightly correlates with the reduction of extracellular taurine in the hippocampus.

OBJECTIVE: Initiation and development of early seizures by chemical stimuli is associated with brain cell swelling resulting in edema of seizure-vulnerable brain regions. We previously reported that pretreatment with a nonconvulsive dose of glutamine (Gln) synthetase inhibitor methionine sulfoximine (MSO) mitigates the intensity of initial pilocarpine (Pilo)-induced seizures in juvenile rats. We hypothesized that MSO exerts its protective effect by preventing the seizure-initiating and seizure-propagating increase of cell volume. Taurine (Tau) is an osmosensitive amino acid, whose release reflects increased cell volume. Therefore, we tested whether the poststimulus rise of amplitude of Pilo-induced electrographic seizures and their attenuation by MSO are correlated with the release of Tau from seizure-affected hippocampus. METHODS: Lithium-pretreated animals were administered MSO (75 mg/kg ip) 2.5 h before the induction of convulsions by Pilo (40 mg/kg ip). Electroencephalographic (EEG) power was analyzed during 60 min post-Pilo, at 5-min intervals. Extracellular accumulation of Tau (eTau) served as a marker of cell swelling. eTau, extracellular Gln (eGln), and extracellular glutamate (eGlu) were assayed in the microdialysates of the ventral hippocampal CA1 region collected at 15-min intervals during the whole 3.5-h observation period. RESULTS: The first EEG signal became apparent at ~10 min post-Pilo. The EEG amplitude across most frequency bands peaked at ~40 min post-Pilo, and showed strong (r ~ .72-.96) temporal correlation with eTau, but no correlation with eGln or eGlu. MSO pretreatment delayed the first EEG signal in Pilo-treated rats by ~10 min, and depressed the EEG amplitude across most frequency bands, to values that remained strongly correlated with eTau (r > .92) and moderately correlated (r ~ -.59) with eGln, but not with eGlu. SIGNIFICANCE: Strong correlation between attenuation of Pilo-induced seizures and Tau release indicates that the beneficial effect of MSO is due to the prevention of cell volume increase concurrent with the onset of seizures.

Perturbation of astroglial Slc38 glutamine transporters by NH4+ contributes to neurophysiologic manifestations in acute liver failure.

Ammonia is considered the main pathogenic toxin in hepatic encephalopathy (HE). However, the molecular mechanisms involved have been disputed. As altered glutamatergic and GABAergic neurotransmission has been reported in HE, we investigated whether four members of the solute carrier 38 (Slc38) family of amino acid transporters-involved in the replenishment of glutamate and GABA-contribute to ammonia neurotoxicity in HE. We show that ammonium ion exerts multiple actions on the Slc38 transporters: It competes with glutamine for the binding to the system N transporters Slc38a3 and Slc38a5, consequently inhibiting bidirectional astroglial glutamine transport. It also competes with H+ , Na+ , and K+ for uncoupled permeation through the same transporters, which may perturb astroglial intracellular pH, membrane potential, and K+ -buffering. Knockdown of Slc38a3 in mice results in cerebral cortical edema and disrupted neurotransmitter synthesis mimicking events contributing to HE development. Finally, in a mouse model of acute liver failure (ALF), we demonstrate the downregulation of Slc38a3 protein, impeded astroglial glutamine release, and cytotoxic edema. Altogether, we demonstrate contribution of Slc38 transporters to the ammonia-induced impairment of glutamine recycling between astrocytes and neurons, a phenomenon underlying acute ammonia neurotoxicity in the setting of ALF.

The Role of Nrf2 Transcription Factor and Sp1-Nrf2 Protein Complex in Glutamine Transporter SN1 Regulation in Mouse Cortical Astrocytes Exposed to Ammonia.

Ammonia toxicity in the brain primarily affects astrocytes via a mechanism in which oxidative stress (OS), is coupled to the imbalance between glutamatergic and GABAergic transmission. Ammonia also downregulates the astrocytic N system transporter SN1 that controls glutamine supply from astrocytes to neurons for the replenishment of both neurotransmitters. Here, we tested the hypothesis that activation of Nrf2 is the process that links ammonia-induced OS formation in astrocytes to downregulation and inactivation of SN1 and that it may involve the formation of a complex between Nrf2 and Sp1. Treatment of cultured cortical mouse astrocytes with ammonia (5 mM NH4Cl for 24 h) evoked Nrf2 nuclear translocation, increased its activity in a p38 MAPK pathway-dependent manner, and enhanced Nrf2 binding to Slc38a3 promoter. Nrf2 silencing increased SN1 mRNA and protein level without influencing astrocytic [3H]glutamine transport. Ammonia decreased SN1 expression in Nrf2 siRNA treated astrocytes and reduced [3H]glutamine uptake. In addition, while Nrf2 formed a complex with Sp1 in ammonia-treated astrocytes less efficiently than in control cells, treatment of astrocytes with hybrid-mode inactivated Sp1-Nrf2 complex (Nrf2 silencing + pharmacological inhibition of Sp1) did not affect SN1 protein level in ammonia-treated astrocytes. In summary, the results document that SN1 transporter dysregulation by ammonia in astrocytes involves activation of Nrf2 but does not require the formation of the Sp1-Nrf2 complex.

Decreased Expression and Uncoupling of Endothelial Nitric Oxide Synthase in the Cerebral Cortex of Rats with Thioacetamide-Induced Acute Liver Failure.

Acute liver failure (ALF) is associated with deregulated nitric oxide (NO) signaling in the brain, which is one of the key molecular abnormalities leading to the neuropsychiatric disorder called hepatic encephalopathy (HE). This study focuses on the effect of ALF on the relatively unexplored endothelial NOS isoform (eNOS). The cerebral prefrontal cortices of rats with thioacetamide (TAA)-induced ALF showed decreased eNOS expression, which resulted in an overall reduction of NOS activity. ALF also decreased the content of the NOS cofactor, tetrahydro-L-biopterin (BH4), and evoked eNOS uncoupling (reduction of the eNOS dimer/monomer ratio). The addition of the NO precursor L-arginine in the absence of BH4 potentiated ROS accumulation, whereas nonspecific NOS inhibitor L-NAME or EDTA attenuated ROS increase. The ALF-induced decrease of eNOS content and its uncoupling concurred with, and was likely causally related to, both increased brain content of reactive oxidative species (ROS) and decreased cerebral cortical blood flow (CBF) in the same model.

Inhibition of Glutamate Release, but Not of Glutamine Recycling to Glutamate, Is Involved in Delaying the Onset of Initial Lithium-Pilocarpine-Induced Seizures in Young Rats by a Non-Convulsive MSO Dose.

Initial seizures observed in young rats during the 60 min after administration of pilocarpine (Pilo) were delayed and attenuated by pretreatment with a non-convulsive dose of methionine sulfoximine (MSO). We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was administered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (short period) or 15 min before sacrifice (long period). Their conversion to Glu and Gln in the hippocampus and entorhinal cortex was followed using [13C] gas chromatography-mass spectrometry. Release of in vitro loaded Glu surrogate, [3H]d-Asp from ex vivo brain slices was monitored in continuously collected superfusates. [3H]d-Asp uptake was tested in freshly isolated brain slices. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]d-Asp release, but did not affect [3H]d-Asp uptake. The results indicate that MSO at a non-convulsive dose delays the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.

Selected Molecular Targets for Antiepileptogenesis.

The term epileptogenesis defines the usually durable process of converting normal brain into an epileptic one. The resistance of a significant proportion of patients with epilepsy to the available pharmacotherapy prompted the concept of a causative treatment option consisting in stopping or modifying the progress of epileptogenesis. Most antiepileptic drugs possess only a weak or no antiepileptogenic potential at all, but a few of them appear promising in this regard; these include, for example, eslicarbazepine (a sodium and T-type channel blocker), lamotrigine (a sodium channel blocker and glutamate antagonist) or levetiracetam (a ligand of synaptic vehicle protein SV2A). Among the approved non-antiepileptic drugs, antiepileptogenic potential seems to reside in losartan (a blocker of angiotensin II type 1 receptors), biperiden (an antiparkinsonian drug), nonsteroidal anti-inflammatory drugs, antioxidative drugs and minocycline (a second-generation tetracycline with anti-inflammatory and antioxidant properties). Among other possible antiepileptogenic compounds, antisense nucleotides have been considered, among these an antagomir targeting microRNA-134. The drugs and agents mentioned above have been evaluated in post-status epilepticus models of epileptogenesis, so their preventive efficacy must be verified. Limited clinical data indicate that biperiden in patients with brain injuries is well-tolerated and seems to reduce the incidence of post-traumatic epilepsy. Exceptionally, in this regard, our own original data presented here point to c-Fos as an early seizure duration, but not seizure intensity-related, marker of early epileptogenesis. Further research of reliable markers of early epileptogenesis is definitely needed to improve the process of designing adequate antiepileptogenic therapies.

The Status of Bile Acids and Farnesoid X Receptor in Brain and Liver of Rats with Thioacetamide-Induced Acute Liver Failure.

Acute liver failure (ALF) leads to neurological symptoms defined as hepatic encephalopathy (HE). Although accumulation of ammonia and neuroinflammation are generally accepted as main contributors to HE pathomechanism, a buildup of bile acids (BA) in the blood is a frequent component of liver injury in HE patients. Recent studies have identified the nuclear farnesoid X receptor (FXR) acting via small heterodimer partner (SHP) as a mediator of BA-induced effects in the brain of ALF animals. The present study investigated the status of the BA-FXR axis in the brain and the liver, including selective changes in pertinent genes in thioacetamide (TAA)-induced ALF in Sprague-Dawley rats. FXR was found in rat neurons, confirming earlier reports for mouse and human brain. BA accumulated in blood but not in the brain tissue. Expression of mRNAs coding for Fxr and Shp was reduced in the hippocampus and of Fxr mRNA also in the cerebellum. Changes in Fxr mRNA levels were not followed by changes in FXR protein. The results leave open the possibility that mobilization of the BA-FXR axis in the brain may not be necessarily pathognomonic to HE but may depend upon ALF-related confounding factors.

TNFα increases STAT3-mediated expression of glutaminase isoform KGA in cultured rat astrocytes.

Glutamate related excitotoxicity and excess of cerebral levels of tumor necrosis factor alpha (TNFα) are interrelated and well documented abnormalities noticed in many central nervous system diseases. Contribution of kidney type glutaminase (KGA) and shorter alternative splicing form (GAC) to glutamine degradation in astrocytes has been recently a matter of dispute and extensive study but the regulation of the GLS isoforms by inflammatory factors is still not well known. Here we show that treatment of cultured rat cortical astrocytes with pathophysiologically relevant (50 ng/ml) concentration of TNFα specifically increases the expression of KGA but not GAC and increases activity of GLS. No changes in the expression of either of two GLS isoforms were observed following treatment with other tested cytokines IL-1ß and IL-6. The TNFα mediated KGA expression was associated with increased phosphorylation of signal transducer and activator of transcription 3 (STAT3). Stimulatory effect of TNF-α on KGA expression was reduced by selective inhibition of (STAT3) but not by inhibition of STAT1 nor nuclear transcription factor kappa. Additionally, the role of miRNA in TNFα-induced expression of KGA in astrocytes was excluded, since the expression of miR-23a/b and miR-200c, potential regulators of KGA expression, was unchanged. This study documents increased KGA expression in the astrocytes under inflammatory stimulation, identifying TNFα as a cytokine mediating this response, and demonstrates the specific and selective involvement of STAT3.

NMDA Receptors in Astrocytes: In Search for Roles in Neurotransmission and Astrocytic Homeostasis.

Studies of the last two decades have demonstrated the presence in astrocytic cell membranes of N-methyl-d-aspartate (NMDA) receptors (NMDARs), albeit their apparently low abundance makes demonstration of their presence and function more difficult than of other glutamate (Glu) receptor classes residing in astrocytes. Activation of astrocytic NMDARs directly in brain slices and in acutely isolated or cultured astrocytes evokes intracellular calcium increase, by mutually unexclusive ionotropic and metabotropic mechanisms. However, other than one report on the contribution of astrocyte-located NMDARs to astrocyte-dependent modulation of presynaptic strength in the hippocampus, there is no sound evidence for the significant role of astrocytic NMDARs in astrocytic-neuronal interaction in neurotransmission, as yet. Durable exposure of astrocytic and neuronal co-cultures to NMDA has been reported to upregulate astrocytic synthesis of glutathione, and in this way to increase the antioxidative capacity of neurons. On the other hand, overexposure to NMDA decreases, by an as yet unknown mechanism, the ability of cultured astrocytes to express glutamine synthetase (GS), aquaporin-4 (AQP4), and the inward rectifying potassium channel Kir4.1, the three astroglia-specific proteins critical for homeostatic function of astrocytes. The beneficial or detrimental effects of astrocytic NMDAR stimulation revealed in the in vitro studies remain to be proven in the in vivo setting.

Mechanisms of Excessive Extracellular Glutamate Accumulation in Temporal Lobe Epilepsy.

There is compelling evidence that initiation and maintenance of epileptic seizures in temporal lobe epilepsy (TLE) is facilitated by excessive accumulation in the extracellular (perisynaptic) space of the excitatory neurotransmitter glutamate (Glu). This review discusses the mechanisms underlying this phenomenon. Glu released from neurons is taken up by astrocytes and activated there by glutamine synthetase (GS) to form glutamine (Gln) which upon entry to neurons is degraded back to Glu by phosphate-activated glutaminase (PAG): this chain of reactions has been defined as the glutamine/glutamate/cycle (GGC). In the initial phase of epileptogenesis, increased Glu supply is a consequence of activation of its turnover in GGC by Glu released by a primary chemical or physical stimulus. In chronic TLE, profound astrogliosis and demise of neurons which culminate in hippocampal sclerosis, are associated with changes in GGC which act in concert towards increasing the extracellular Glu concentration. Deficiency of GS and of the astrocytic Glu transporter, GLT-1, impede Glu inactivation, whereas Glu release from neurons appears facilitated by activation of PAG and increased activity of the neuronal Glu transporter EAAC1. Conclusions derived from measurements of activities/expression patterns of the GGC enzymes and transporter moieties find support in metabolic studies employing 13C labeled Glu precursors. Glu reuptake by astrocytes is additionally impeded by unfavorable ion gradients resulting from ion and water dyshomeostasis, and extracellular Glu concentration is further increased by reduction of extracellular space due to edema and altered cytoarchitecture of the hippocampus. Missing links in the scenario are discussed in concluding comments.

Ammonia Reduces Intracellular Asymmetric Dimethylarginine in Cultured Astrocytes Stimulating Its y⁺LAT2 Carrier-Mediated Loss.

Previously we had shown that ammonia stimulates nitric oxide (NO) synthesis in astrocytes by increasing the uptake of the precursor amino acid, arginine via the heteromeric arginine/glutamine transporter y⁺LAT2. Ammonia also increases the concentration in the brain of the endogenous inhibitor of nitric oxide synthases (NOS), asymmetric dimethylarginine (ADMA), but distribution of ADMA surplus between the intraastrocytic and extracellular compartments of the brain has not been studied. Here we tested the hypothesis that ammonia modulates the distribution of ADMA and its analog symmetric dimethylarginine (SDMA) between the two compartments of the brain by competition with arginine for the y⁺LAT2 transporter. In extension of the hypothesis we analyzed the ADMA/Arg interaction in endothelial cells forming the blood-brain barrier. We measured by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) technique the concentration of arginine, ADMA and SDMA in cultured cortical astrocytes and in a rat brain endothelial cell line (RBE-4) treated with ammonia and the effect of silencing the expression of a gene coding y⁺LAT2. We also tested the expression of ADMA metabolism enzymes: protein arginine methyltransferase (PRMT) and dimethylarginine dimethyl aminohydrolase (DDAH) and arginine uptake to astrocytes. Treatment for 48 h with 5 mM ammonia led to an almost 50% reduction of ADMA and SDMA concentration in both cell types, and the effect in astrocytes was substantially attenuated by silencing of the Slc7a6 gene. Moreover, the y⁺LAT2-dependent component of ammonia-evoked arginine uptake in astrocytes was reduced in the presence of ADMA in the medium. Our results suggest that increased ADMA efflux mediated by upregulated y⁺LAT2 may be a mechanism by which ammonia interferes with intra-astrocytic (and possibly intra-endothelial cell) ADMA content and subsequently, NO synthesis in both cell types.

System N transporters are critical for glutamine release and modulate metabolic fluxes of glucose and acetate in cultured cortical astrocytes: changes induced by ammonia.

Glutamine (Gln) is synthesized in astrocytes from glutamate (Glu) and ammonia, whereupon it can be released to be transferred to neurons. This study evaluated the as yet not definitely established role of the astrocytic Gln transporters SN1 and SN2 (Slc38a3 and Slc38a5 respectively) in Gln release and metabolic fluxes of glucose and acetate, the canonical precursors of Glu. Cultured neocortical astrocytes were grown in the absence or presence of ammonia (5 mM NH4 Cl, 24 h), which deregulates astrocytic metabolism in hyperammonemic encephalopathies. HPLC analyses of cell extracts of SN1/SN2 siRNA-treated (SN1/SN2-) astrocytes revealed a ~ 3.5-fold increase in Gln content and doubling of glutathione, aspartate, alanine and glutamate contents, as compared to SN1/SN2+ astrocytes. Uptake and efflux of preloaded [(3) H]Gln was likewise significantly decreased in SN1/SN2- astrocytes. The atom percent excess (13) C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the SN1/SN2- cells were incubated with [1-(13) C] glucose, while Gln consumption was not changed. No difference was seen in M + 1 values in SN1/SN2- cells incubated with [2-(13) C] acetate, which were not treated with ammonia. In SN1/SN2- astrocytes, the increase in Gln content and the decrease in radiolabeled Gln release upon exposure to ammonia were found abrogated, and glutamate labeling from [2-(13) C]acetate was decreased as compared to SN1/SN2+ astrocytes. The results underscore a profound role of SN1 and/or SN2 in Gln release from astrocytes under physiological conditions, but less so in ammonia-overexposed astrocytes, and appear to manifest dependence of astrocytic glucose metabolism to Glu/Gln on unimpaired SN1/SN2- mediated Gln release from astrocytes. The astrocytic N system transporters SN1 and SN2 show preponderance to mediate glutamine (Gln) efflux. Under hyperammonemic conditions, accumulation of Gln, a direct product of ammonia detoxification, may deregulate astrocytic metabolism and seems to be responsible for astrocytic swelling. This study evaluated not definitely established role of SN1 and SN2 in Gln release and metabolic fluxes of radiolabeled glucose and acetate. Simultaneous silencing of SN1/SN2 transporters increase Gln, glutathione, aspartate, alanine and glutamate contents (Panel B; marked in red) as compare to non-silenced astrocytes (Panel A). The atom percent excess (13) C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the cells with silenced transporters were incubated with [1-(13) C]glucose, whereas no difference was seen in M + 1 values when those cells were incubated with [2-(13) C]acetate. Ammonia abrogated the increase in Gln content and decrease in radiolabeled Gln release in astrocytes with silenced transporters, but caused a decrease in glutamate labeling from [2-(13) C]acetate.

Downregulation of GLS2 in glioblastoma cells is related to DNA hypermethylation but not to the p53 status.

Human phosphate-activated glutaminase (GA) is encoded by two genes: GLS and GLS2. Glioblastomas (GB) usually lack GLS2 transcripts, and their reintroduction inhibits GB growth. The GLS2 gene in peripheral tumors may be i) methylation- controlled and ii) a target of tumor suppressor p53 often mutated in gliomas. Here we assessed the relation of GLS2 downregulation in GB to its methylation and TP53 status. DNA demethylation with 5-aza-2'-deoxycytidine restored GLS2 mRNA and protein content in human GB cell lines with both mutated (T98G) and wild-type (U87MG) p53 and reduced the methylation of CpG1 (promoter region island), and CpG2 (first intron island) in both cell lines. In cell lines and clinical GB samples alike, methylated CpG islands were detected both in the GLS2 promoter (as reported earlier) and in the first intron of this gene. CpG methylation of either island was absent in GLS2-expressing non-tumoros brain tissues. Screening for mutation in the exons 5-8 of TP53 revealed a point mutation in only one out of seven GB examined. In conclusion, aberrant methylation of CpG islands, appear to contribute to silencing of GLS2 in GB by a mechanism bypassing TP53 mutations. © 2015 Wiley Periodicals, Inc.

Expression of RNAs Coding for Metal Transporters in Blood of Patients with Huntington's Disease.

Recent studies have demonstrated elevated levels of iron (Fe) in brains of patients with Huntington's disease (HD). Striatal cells carrying mutated Huntingtin presented increased sensitivity to cadmium (Cd) toxicity, decreased sensitivity to manganese (Mn) toxicity and deficits in Mn uptake. The hypothesis arose that the observed alterations result from the altered expression and/or activity of proteins engaged in the transport of these metals, that is: transferrin (TF), transferrin receptor (TFR), divalent metal transporter 1 (DMT1) and ZIP8 protein. Here we examined the expression levels of genes encoding these proteins in blood of HD patients and control subjects. A decreasing tendency in the level of TF transcript and increasing tendency of SLC11A2 mRNA encoding DMT1 was observed in the blood of HD patients compared to the control subjects, but neither attained statistical significance. No changes were found in the levels of TFRC coding for TFR and SLC39A8 coding for ZIP8 between HD patients and controls. The results indicate that HD-associated changes in metal homeostasis occur are not related to mechanisms other than the expression level of the here analyzed metal transporters.

Carnosine Reduces Oxidative Stress and Reverses Attenuation of Righting and Postural Reflexes in Rats with Thioacetamide-Induced Liver Failure.

Cerebral oxidative stress (OS) contributes to the pathogenesis of hepatic encephalopathy (HE). Existing evidence suggests that systemic administration of L-histidine (His) attenuates OS in brain of HE animal models, but the underlying mechanism is complex and not sufficiently understood. Here we tested the hypothesis that dipeptide carnosine (ß-alanyl-L-histidine, Car) may be neuroprotective in thioacetamide (TAA)-induced liver failure in rats and that, being His metabolite, may mediate the well documented anti-OS activity of His. Amino acids [His or Car (100 mg/kg)] were administrated 2 h before TAA (i.p., 300 mg/kg 3× in 24 h intervals) injection into Sprague-Dawley rats. The animals were thus tested for: (i) brain prefrontal cortex and blood contents of Car and His, (ii) amount of reactive oxygen species (ROS), total antioxidant capacity (TAC), GSSG/GSH ratio and thioredoxin reductase (TRx) activity, and (iii) behavioral changes (several models were used, i.e. tests for reflexes, open field, grip test, Rotarod). Brain level of Car was reduced in TAA rats, and His administration significantly elevated Car levels in control and TAA rats. Car partly attenuated TAA-induced ROS production and reduced GSH/GSSG ratio, whereas the increase of TRx activity in TAA brain was not significantly modulated by Car. Further, Car improved TAA-affected behavioral functions in rats, as was shown by the tests of righting and postural reflexes. Collectively, the results support the hypothesis that (i) Car may be added to the list of neuroprotective compounds of therapeutic potential on HE and that (ii) Car mediates at least a portion of the OS-attenuating activity of His in the setting of TAA-induced liver failure.

Glutaminases in slowly proliferating gastroenteropancreatic neuroendocrine neoplasms/tumors (GEP-NETs): Selective overexpression of mRNA coding for the KGA isoform.

Glutamine (Gln) is a crucial metabolite in cancer cells of different origin, and the expression and activity of different isoforms of the Gln-degrading enzyme, glutaminase (GA), have variable implications for tumor growth and metabolism. Human glutaminases are encoded by two genes: the GLS gene encodes the kidney-type glutaminases, KGA and GAC, while the GLS2 gene encodes the liver-type glutaminases, GAB and LGA. Recent studies suggest that the GAC isoform and thus high GAC/KGA ratio, are characteristic of highly proliferating tumors, while GLS2 proteins have an inhibitory effect on tumor growth. Here we analyzed the expression levels of distinct GA transcripts in 7 gastroenteropancreatic neuroendocrine tumors (GEP-NETs) with low proliferation index and 7 non-neoplastic tissues. GEP-NETs overexpressed KGA, while GAC, which was the most abundant isoform, was not different from control. The expression of the GLS2 gene showed tendency towards elevation in GEP-NETs compared to control. Collectively, the expression pattern of GA isoforms conforms to the low proliferative capacity of GEP-NETs encompassed in this study.

Induction of inducible nitric oxide synthase expression in ammonia-exposed cultured astrocytes is coupled to increased arginine transport by upregulated y(+)LAT2 transporter.

One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-κB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-κB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism involving activation (nuclear translocation) of the transcription factor nuclear factor-Nuclear factor-κB (Nf-κB-p65). Up-regulation of y(+)LAT2 transporter is coupled with increased inducible nitric oxide synthase (iNOS) expression, which leads to increase nitric oxide (NO) synthesis and protein nitration.

Changes of the thioredoxin system, glutathione peroxidase activity and total antioxidant capacity in rat brain cortex during acute liver failure: modulation by L-histidine.

Glutathione and thioredoxin are complementary antioxidants in the protection of mammalian tissues against oxidative-nitrosative stress (ONS), and ONS is a principal cause of symptoms of hepatic encephalopathy (HE) associated with acute liver failure (ALF). We compared the activities of the thioredoxin system components: thioredoxin (Trx), thioredoxin reductase (TrxR) and the expression of the thioredoxin-interacting protein, and of the key glutathione metabolizing enzyme, glutathione peroxidase (GPx) in the cerebral cortex of rats with ALF induced by thioacetamide (TAA). ALF increased the Trx and TrxR activity without affecting Trip protein expression, but decreased GPx activity in the brains of TAA-treated rats. The total antioxidant capacity (TAC) of the brain was increased by ALF suggesting that upregulation of the thioredoxin may act towards compensating impaired protection by the glutathione system. Intraperitoneal administration of L-histidine (His), an amino acid that was earlier reported to prevent acute liver failure-induced mitochondrial impairment and brain edema, abrogated most of the acute liver failure-induced changes of both antioxidant systems, and significantly increased TAC of both the control and ALF-affected brain. These observations provide further support for the concept of that His has a potential to serve as a therapeutic antioxidant in HE. Most of the enzyme activity changes evoked by His or ALF were not well correlated with alterations in their expression at the mRNA level, suggesting complex translational or posttranslational mechanisms of their modulation, which deserve further investigations.

Silencing of GLS and overexpression of GLS2 genes cooperate in decreasing the proliferation and viability of glioblastoma cells.

Glutamine (Gln) metabolism, initiated by its degradation by glutaminases (GA), is elevated in neoplastic cells and tissues. In malignant glia-derived tumors, GA isoforms, KGA and GAC, coded by the GLS gene, are overexpressed, whereas the GLS2-coded GAB and LGA isoforms, are hardly detectable in there. Our previous study revealed that transfection of T98G glioblastoma cells with GAB reduced cell proliferation and migration, by a yet unknown mechanism not related to Gln degradation. The question arose how simultaneous overexpression of GAB and inhibition of KGA would affect glioblastoma cell growth. Here, we used siRNA to silence the expression of Gls in T98G cells which were or were not stably transfected with GAB (TGAB cells). In both T98G and TGAB cell lines, silencing of Gls with siRNAs targeted at different sequences decreased cell viability and proliferation in a different, sequence-dependent degree, and the observed decreases were in either cell line highly correlated with increase of intracellular Gln (r > 0.9), a parameter manifesting decreased Gln degradation. The results show that combination of negative modulation of GA isoforms arising from GLS gene with the introduction of the GLS2 gene product, GAB, may in the future provide a useful means to curb glioblastoma growth in situ. At the same time, the results underscore the critical role of Gln degradation mediated by KGA in the manifestations of aggressive glial tumor phenotype.

Roles of changes in active glutamine transport in brain edema development during hepatic encephalopathy: an emerging concept.

Excessive glutamine (Gln) synthesis in ammonia-overloaded astrocytes contributes to astrocytic swelling and brain edema, the major complication of hepatic encephalopathy (HE). Much of the newly formed Gln is believed to enter mitochondria, where it is recycled to ammonia, which causes mitochondrial dysfunction (a "Trojan horse" mode of action). A portion of Gln may increase osmotic pressure in astrocytes and the interstitial space, directly and independently contributing to brain tissue swelling. Here we discuss the possibility that altered functioning of Gln transport proteins located in the cellular or mitochondrial membranes, modulates the effects of increased Gln synthesis. Accumulation of excess Gln in mitochondria involves a carrier-mediated transport which is activated by ammonia. Studies on the expression of the cell membrane N-system transporters SN1 (SNAT3) and SN2 (SNAT5), which mediate Gln efflux from astrocytes rendered HE model-dependent effects. HE lowered the expression of SN1 at the RNA and protein level in the cerebral cortex (cc) in the thioacetamide (TAA) model of HE and the effect paralleled induction of cerebral cortical edema. Neither SN1 nor SN2 expression was affected by simple hyperammonemia, which produces no cc edema. TAA-induced HE is also associated with decreased expression of mRNA coding for the system A carriers SAT1 and SAT2, which stimulate Gln influx to neurons. Taken together, changes in the expression of Gln transporters during HE appear to favor retention of Gln in astrocytes and/or the interstitial space of the brain. HE may also affect arginine (Arg)/Gln exchange across the astrocytic cell membrane due to changes in the expression of the hybrid Arg/Gln transporter y(+)LAT2. Gln export from brain across the blood-brain barrier may be stimulated by HE via its increased exchange with peripheral tryptophan.