Brain Glutamine Synthetase Research Articles

Metabolic changes of the reduction of manganese intake in the hepatic encephalopathy rat: NMR- and MS-based metabolomics study.

To investigate the metabolic changes in type C hepatic encephalopathy (CHE) rats after reducing manganese (Mn) intake. A total of 80 Sprague-Dawley rats were divided into control group and CHE groups (induced by intraperitoneal injection of thioacetamide at a dose of 250mg/kg of body weight twice a week for 6 weeks). CHE rats were subdivided into 1Mn group (fed a standard diet, with 10mg Mn/kg feed), 0.5Mn group (half-Mn diet), 0.25Mn group (quarter-Mn diet) and 0Mn group (no-Mn diet) for 4 to 8weeks. Morris water maze (MWM), Y maze and narrow beam test (NBT) were used to evaluate cognitive and motor functions. Blood ammonia, brain Mn content, the number of GS-positive cells, and glutamine synthetase (GS) activity were measured. The metabolic changes of CHE rats were investigated using hydrogen-nuclear magnetic resonance and mass spectrometry. Multivariate statistical analysis was used to analyze the results. Significantly decreased numbers of entries in target area of MWM and Y maze, longer NBT latency and total time, higher blood ammonia, brain Mn content and GS activity were found in CHE rats. After reducing Mn intake, CHE rats had better behavioral performance, significantly lower blood ammonia, brain Mn content and GS activity. The main up-regulated metabolites were Ala, GABA, Glu, Gln, Lac, Tyr, Phe in 1Mn rats. After reducing Mn intake, metabolites returned to normal level at different degrees. Reducing Mn intake could reduce brain Mn content and blood ammonia, regulate GS activity and amino acid metabolism, ultimately improve behavioral performance in CHE rats.

Immunohistochemical study of the brain glutamine synthetase expression in the rat septic model

Severe sepsis is accompanied by multiple organ dysfunction where acute liver failure (ALF) plays one of the most critical roles. The principal sign of acute hepatic encephalopathy accompanying ALF is ammonia induced edema of astrocytes. In case of ALF in sepsis, one can suppose increase in systemic and brain ammonia. Astroglia are target cells for ammonia metabolism as they are principal source of glutamine-synthetase (GS). Previous studies have reported that ALF stimulates increase in astroglial GS which correlates with deterioration of the animal state. The aim of the study was to determine the level of glutamine synthetase expression in different brain regions of rats in the conditions of experimental sepsis. Materials and methods. The study was conducted in Wistar rats: 5 sham-operated (control) animals and 20 rats with cecum ligation and puncture (CLP) septic model. Immunohistochemical study of GS expression was performed in the cortex, white matter, hippocampus, thalamus, caudate nucleus/putamen in the period of 20–48 h after CLP. Results. Starting at 12th h after CLP, operated animals displayed progressive impairment finished by profound lethargy and respiratory failure. Between 20–38 h, 9 animals expressed final mentioned symptoms and were euthanized (CLP-B, non-survived), 11 rats displayed less expressed suffering up to 48 h (CLP-A, survived). At 23 h in CLP-B and 48 h in CLP-A rats, liver tissue displayed morphological signs of the focal irreversible damage which was aggravated with time after CLP which could be observed dynamically in non-survived group. Both CLP-A and CLP-B rats showed gradual elevation of GS in all studied brain regions. From 24 to 38 h after CLP, non-survived animals showed significant region-specific dynamic increase in GS expression: in the cortex – by 69.35 %, hippocampus – by 53.6 %, thalamus – by 50.0 %; with the most substantive elevation in the cortex – 1.69-fold increase compared to control. Conclusions. In CLP model, 24 h after operation there is significant dynamic increase in GS level in the cortical, hippocampal and thalamic regions of the rat brain with the most prominent in the cortex. Heterogeneous increase in GS indicates regions more or less vulnerable for incoming systemic agents as well as region-specific reactiveness of the brain tissue, including local astroglia, to these factors in septic conditions. Morphological signs of sepsis-associated liver damage preceding significant increase in the brain GS by 1 h, might suppose addition of the liver failure to the course of sepsis which is accompanied by increased level of hepatogenic toxins (presumably including ammonia) in the blood and in the brain parenchyma by 23 h after CLP. The latter might propose an active implication of hepatogenic detrimental agents in sepsis pathophysiology and involvement of reactively increased brain GS levels in the complex mechanisms of sepsis-associated encephalopathy.

Тhe role of the glutamatergic system in the mechanism of development of hyperbaric oxygen seizures

The purpose of the scientific article was to study the neurophysiological mechanisms of the occurrence of convulsive activity when breathing oxygen under high pressure under conditions of hyperbaric oxygen and the role of the glutamatergic system in this process.Research methods: Wistar rats, males, weighing 200–250 g. in the amount of 20 were used in the work. The animals were placed in a pressure chamber and compressed with medical oxygen up to 5 ATА. Behavioral reactions and signs of convulsive activity were recorded in animals during compression and during subsequent exposure. Two experimental groups were previously injected with a glutamine synthetase inhibitor at various concentrations 6 hours before compression.The main results showed that irreversible inhibition of glutamine synthetase in the rat brain under the action of increased oxygen pressure led to a significant decrease in the latent period of convulsions.

Acute Liver Toxicity Modifies Protein Expression of Glutamate Transporters in Liver and Cerebellar Tissue.

Glutamate is the main excitatory amino acid acting at the level of pre and postsynaptic neurons, as well as in glial cells. It is involved in the coordinated modulation of energy metabolism, glutamine synthesis, and ammonia detoxification. The relationship between the functional status of liver and brain has been known for many years. The most widely recognized aspect of this relation is the brain dysfunction caused by acute liver injury that manifests a wide spectrum of neurologic and psychiatric abnormalities. Inflammation, circulating neurotoxins, and impaired neurotransmission have been reported in this pathophysiology. In the present contribution, we report the effect of a hepatotoxic compound like CCl4 on the expression of key proteins involved in glutamate uptake and metabolism as glutamate transporters and glutamine synthetase in mice liver, brain, and cerebellum. Our findings highlight a differential expression pattern of glutamate transporters in cerebellum. A significant Purkinje cells loss, in parallel to an up-regulation of glutamine synthetase, and astrogliosis in the brain have also been noticed. In the intoxicated liver, glutamate transporter 1 expression is up-regulated, in contrast to glutamine synthetase which is reduced in a time-dependent manner. Taken together our results demonstrate that the exposure to an acute CCl4 insult, leads to the disruption of glutamate transporters expression in the liver-brain axis and therefore a severe alteration in glutamate-mediated neurotransmission might be present in the central nervous system.

Chronic Valproic Acid Administration Increases Plasma, Liver, and Brain Ammonia Concentration and Suppresses Glutamine Synthetase Activity.

Asymptomatic valproic acid (VPA)-induced hyperammonemia in the absence of liver impairment is fairly common. However, the underlying mechanisms through which VPA causes elevation in plasma ammonia (NH4) remains under investigation. Male Sprague Dawley rats (n = 72) were randomly allocated to receive VPA 400 mg/kg, 200 mg/kg, or vehicle IP daily for either 8, 14, or 28 consecutive days. The behavioral effects of VPA were assessed. Plasma, liver, and prefrontal cortex (PFC), striatum (Str), and cerebellum (Cere) were collected 1 h post last injection and assayed for NH4 concentration and glutamine synthetase (GS) enzyme activity. Chronic VPA treatment caused attenuation of measured behavioral reflexes (p < 0.0001) and increase in plasma NH4 concentration (p < 0.0001). The liver and brain also showed significant increase in tissue NH4 concentrations (p < 0.0001 each) associated with significant reduction in GS activity (p < 0.0001 and p = 0.0003, respectively). Higher tissue NH4 concentrations correlated with reduced GS activity in the liver (r = −0.447, p = 0.0007) but not in the brain (r = −0.058, p = 0.4). Within the brain, even though NH4 concentrations increased in the PFC (p = 0.001), Str (p < 0.0001), and Cere (p = 0.01), GS activity was reduced only in the PFC (p < 0.001) and not in Str (p = 0.2) or Cere (p = 0.1). These results suggest that VPA-induced elevation in plasma NH4 concentration could be related, at least in part, to the suppression of GS activity in liver and brain tissues. However, even though GS is the primary mechanism in brain NH4 clearance, the suppression of brain GS does not seem to be the main factor in explaining the elevation in brain NH4 concentration. Further research is urgently needed to investigate brain NH4 dynamics under chronic VPA treatment and whether VPA clinical efficacy in treating seizure disorders and bipolar mania is impacted by its effect on GS activity or other NH4 metabolizing enzymes.

Interaction of Manganese and Ammonia in the Brain of Hepatic Encephalopathy Rats

Background: Both ammonia and manganese (Mn) play a key role in the pathogenesis of hepatic encephalopathy (HE) and cause similar morphological and functional changes in astrocytes. Objectives: To investigate the interaction between brain Mn and ammonia in HE rats. Methods: Three rat models of minimal HE (MHE), chronic manganism (CHM), and chronic hyperammonemia (CHA) were constructed. A total of 48 Sprague-Dawley rats were divided into one control group (n = 6), MHE groups (n = 18, among which six rats were used to evaluate the MHE model), CHM groups (n = 12), and CHA groups (n = 12). The CHM, CHA, and the rest of MHE rats were randomly divided further into two subgroups, according to the MgSO4 treatment (oral administration of 496 mg/kg/day for seven weeks): MHE-7W and MHE + Mg-7W; CHM-7W and CHM + Mg-7W; and CHA-7W and CHA + Mg-7W, respectively. Rats’ blood ammonia, brain Mn, glutamine synthetase (GS), and glutamine (GLN) levels were measured and compared among groups. Results: Significantly higher brain Mn content in MHE-7W and CHM-7W rats, higher blood ammonia levels, brain GS activity, and GLN content were observed in MHE-7W, CHM-7W, and CHA-7W rats than in control rats. After MgSO4 treatment for seven weeks, significantly lower brain Mn content, blood ammonia levels, and GLN content were observed in MHE, CHM, and CHA rats. Conclusions: Our study showed that brain Mn accumulation could increase brain ammonia levels, while the accumulation of brain ammonia had no effect on the content of brain Mn.

The alterations of glutamate transporter 1 and glutamine synthetase in the rat brain of a learned helplessness model of depression.

Although glutamate transmission via astrocytes has been proposed to contribute to the pathophysiology of depression, the precise mechanisms are unknown. Herein, we investigated the levels of glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) of astrocytes in learned helplessness (LH) rats (an animal model of depression) and non-LH rats (an animal model of resilience). We administered inescapable mild electric shock to rats and then discriminated the LH and non-LH rats by a post-shock test. Almost 55% of the rats acquired LH. We then measured the expressions of GLT-1 and GS in several brain regions of LH and non-LH rats by Western blot analysis. The levels of GLT-1 and GS in the CA-1, CA-3, dentate gyrus (DG), medial prefrontal cortex (mPF), and nucleus accumbens (NAc) of the LH group were significantly higher than those of the control group. The GS levels in the amygdala of the LH rats were significantly decreased compared to the controls. There were significant differences in GLT-1 and GS levels between the non-LH and LH rats in the CA-1 and CA-3. These results suggest that the LH rats experienced up-regulations of GLT-1 and GS in the CA-1, CA-3, DG, mPF, and NAc and a down-regulation of GS in the amygdala. It is possible that the effects of the GLT-1 and GS levels on astrocytes in the CA-1 and CA-3 are critical for the differentiation of resilience from vulnerability.

CHAPERONES AS MOLECULAR TARGET FOR ONCOLOGICAL AND NEUROLOGICAL DISEASES

Brain manganese deposition is led by liver dysfunction and/or portal-systemic shunting in minimal hepatic encephalopathy (MHE). Manganese is toxic and can cause cognitive disorders and extrapyramidal symptoms. Thus, reduction of manganese intake might be considered as a potential treatment strategy for MHE. In this study we aimed to investigate whether low- or no-manganese feed can improve the neuropsychological manifestations in MHE rats. Rats with MHE were established by partially ligating the portal vein and fed a manganese diet (MHE-M, 10 mg per kg feed; n = 24), a no-manganese diet (MHE-N; n = 24) and a half-manganese diet (MHE-H; n = 24) for 2, 4, 6 and 8 weeks, with six rats in each subgroup. Morris water maze (MWM), open-field test and narrow beam test (NBT) were used to evaluate the cognitive and locomotor situations. Fasted blood ammonia, manganese content and glutamine synthetase (GS) activity in basal ganglia and cortex were measured. A significantly longer MWM escape latency, less locomotor activity, longer NBT latency and total time, higher blood ammonia, higher brain manganese content and GS activity were found in MHE-M rats. However, a significantly shorter MWM escape latency, increased locomotor activity, shorter NBT latency and total time, lower blood ammonia, lower brain manganese content and lower GS activity were found in MHE-N rats after no-manganese feed treatment. Partial improvements were found in MHE rats with half-manganese feed treatment. Reduction of manganese intake can significantly improve the cognitive and locomotor situations in MHE rats by reducing brain manganese content and regulating GS activity.

Ферменты глутаматного обмена в лобной, лимбической коре и мозжечке: аномалии при шизофрении

Aim: to compare distributions of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) activities and amounts of their immunoreactive forms amongst frontal, anterior, posterior cingulate, and cerebellar cortices in schizophrenia cases and controls. Material and methods: group of patients with schizophrenia: 4 men and 4 women (36–80 years old) diagnosed according ICD-10 (F20.00, F20.02, F20.31, and F20.50 — 3, 3, 1 and 1 patient). Control group: 8 men and 1 woman (29–79 years old). No significant between-group differences were found in age and postmortem interval (p &gt; 0,05). Non-parametric statistics was used. Results: no signifcant between-group difference was observed in GS activity in investigated brain structures (p &gt; 0,05). Following differences were found in schizophrenia compared with controls: amount of immunoreactive GS was decreased in frontal cortex (p &lt; 0,001), it was increased in posterior cingulate cortex and cerebellum (p &lt; 0,001 and p &lt; 0,004), but unchanged in anterior cingulate cortex, whereas level of GS-like protein (GSLP) was signifcantly elevated in all four brain structures (р &lt; 0,004, р &lt; 0,02, р &lt; 0,04, and р &lt; 0,001). When compared with controls, schizophrenia cases displayed elevated GDH activity in frontal, posterior cingulate cortex and cerebellum (p &lt; 0,004, p &lt; 0,05, and p &lt; 0,002), but unchanged in anterior cingulate cortex. When compared with control, following differences were found in schizophrenia: elevation in levels of immunoreactive GDHI, GDHII, and GDHIII in frontal cortex (р &lt; 0,008, р &lt; 0,003, and р &lt; 0,001); elevation in levels of immunoreactive GDHI, GDHII, and GDHIII in posterior cingulate cortex (р &lt; 0,004, р &lt; 0,001, and р &lt; 0,02); elevation in levels of immunoreactive GDHII and GDHIII in cerebellum (р &lt; 0,02 and р &lt; 0,001). No between-group differences in levels of immunoreactive GDH forms were found in anterior cingulate cortex. Conclusion: alteration in levels of GS, GSLP, and GDH forms in brain of patients with schizophrenia is one of causes of glutamate metabolism disturbances in these brain structures and an important aspect of schizophrenia pathogenesis.

The acute effect of cannabis on plasma, liver and brain ammonia dynamics, a translational study

Recent reports of ammonia released during cannabis smoking raise concerns about putative neurotoxic effects. Cannabis (54mg) was administered in a double-blind, placebo-controlled design to healthy cannabis users (n=15) either orally, or through smoking (6.9%THC cigarette) or inhalation of vaporized cannabis (Volcano®). Serial assay of plasma ammonia concentrations at 0, 2, 4, 6, 8, 10, 15, 30, and 90min from onset of cannabis administration showed significant time (P=0.016), and treatment (P=0.0004) effects with robust differences between placebo and edible at 30 (P=0.002), and 90min (P=0.007) and between placebo and vaporized (P=0.02) and smoking routes (P=0.01) at 90min. Furthermore, plasma ammonia positively correlated with blood THC concentrations (P=0.03). To test the hypothesis that this delayed increase in plasma ammonia originates from the brain we administered THC (3 and 10mg/kg) to mice and measured plasma, liver, and brain ammonia concentrations at 1, 3, 5 and 30min post-injection. Administration of THC to mice did not cause significant change in plasma ammonia concentrations within the first 5min, but significantly reduced striatal glutamine-synthetase (GS) activity (P=0.046) and increased striatal ammonia concentration (P=0.016). Furthermore, plasma THC correlated positively with striatal ammonia concentration (P<0.001) and negatively with striatal GS activity (P=0.030). At 30min, we found marked increase in striatal ammonia (P<0.0001) associated with significant increase in plasma ammonia (P=0.042) concentration.In conclusion, the results of these studies demonstrate that cannabis intake caused time and route-dependent increases in plasma ammonia concentrations in human cannabis users and reduced brain GS activity and increased brain and plasma ammonia concentrations in mice.

Reduction of manganese intake improves neuropsychological manifestations in rats with minimal hepatic encephalopathy

Brain manganese deposition is led by liver dysfunction and/or portal-systemic shunting in minimal hepatic encephalopathy (MHE). Manganese is toxic and can cause cognitive disorders and extrapyramidal symptoms. Thus, reduction of manganese intake might be considered as a potential treatment strategy for MHE. In this study we aimed to investigate whether low- or no-manganese feed can improve the neuropsychological manifestations in MHE rats. Rats with MHE were established by partially ligating the portal vein and fed a manganese diet (MHE-M, 10mg per kg feed; n=24), a no-manganese diet (MHE-N; n=24) and a half-manganese diet (MHE-H; n=24) for 2, 4, 6 and 8weeks, with six rats in each subgroup. Morris water maze (MWM), open-field test and narrow beam test (NBT) were used to evaluate the cognitive and locomotor situations. Fasted blood ammonia, manganese content and glutamine synthetase (GS) activity in basal ganglia and cortex were measured. A significantly longer MWM escape latency, less locomotor activity, longer NBT latency and total time, higher blood ammonia, higher brain manganese content and GS activity were found in MHE-M rats. However, a significantly shorter MWM escape latency, increased locomotor activity, shorter NBT latency and total time, lower blood ammonia, lower brain manganese content and lower GS activity were found in MHE-N rats after no-manganese feed treatment. Partial improvements were found in MHE rats with half-manganese feed treatment. Reduction of manganese intake can significantly improve the cognitive and locomotor situations in MHE rats by reducing brain manganese content and regulating GS activity.

Chronotherapeutic effect of fisetin on expression of urea cycle enzymes and inflammatory markers in hyperammonaemic rats

BackgroundElevated blood ammonia leads to hyperammonaemia that affects vital central nervous system (CNS) functions. Fisetin, a naturally occurring flavonoid, exhibits therapeutic benefits, such as anti-cancer, anti-diabetic, anti-oxidant, anti-angiogenic, neuroprotective and neurotrophic effects. MethodsIn this study, the chronotherapeutic effect of fisetin on ammonium chloride (AC)-induced hyperammonaemic rats was investigated, to ascertain the time point at which the maximum drug effect is achieved. The anti-hyperammonaemic potential of fisetin (50mg/kg b.w. oral) was analysed when administered to AC treated (100mg/kg b.w. i.p.) rats at 06:00, 12:00, 18:00 and 00:00h. Amelioration of pathophysiological conditions by fisetin at different time points was measured by analysing the levels of expression of liver urea cycle enzymes (carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS)), nuclear transcription factor kappaB (NF-κB p65), brain glutamine synthetase (GS) and inducible nitric oxide synthase (iNOS) by Western blot analysis. ResultsFisetin increased the expression of CPS-I, OTC, ASS and GS and decreased iNOS and NF-κB p65 in hyperammonaemic rats. Fisetin administration at 00:00h showed more significant effects on the expression of liver and brain markers, compared with other time points. ConclusionsFisetin could exhibit anti-hyperammonaemic effect owing to its anti-oxidant and cytoprotective influences. The temporal variation in the effect of fisetin could be due to the (i) chronopharmacological, chronopharmacokinetic properties of fisetin and (ii) modulations in the endogenous circadian rhythms of urea cycle enzymes, brain markers, redox enzymes and renal clearance during hyperammonaemia by fisetin. However, future studies in these lines are necessitated.

Possible treatment of end-stage hyperammonemic encephalopathy by inhibition of glutamine synthetase

Glutamine synthetase (GS) is highly active in astrocytes, and these cells are physiologically and morphologically compromised by hyperammonemia. Hyperammonemia in end-stage acute liver failure (ALF) is often associated with cerebral edema and astrocyte pathology/swelling. Many studies of animal models of hyperammonemia, and, more recently, nuclear magnetic resonance studies of liver disease patients, have shown that cerebral glutamine is elevated in hyperammonemia, contributing to the edema and encephalopathy. The GS inhibitor L-methionine-S,R-sulfoximine (MSO) is protective in animal models against acute ammonia intoxication. MSO is also an inhibitor of glutamate cysteine ligase, is converted to metabolic products, and causes convulsions at high doses. However, the susceptibility to MSO-induced convulsions is species dependent, with primates being relatively resistant. Moreover, it is possible to chronically maintain cerebral GS activity in mice at low levels by MSO treatment without any obvious untoward effects. Furthermore, MSO is protective in a mouse model of ALF. Extreme caution would be needed in administering MSO to patients. Nevertheless, inhibition of brain GS by MSO (or other GS inhibitors) may have therapeutic benefit in ALF.

Comparative enzymology of (2S,4R)4-fluoroglutamine and (2S,4R)4-fluoroglutamate

Many cancer cells have a strong requirement for glutamine. As an aid for understanding this phenomenon the (18)F-labeled 2S,4R stereoisomer of 4-fluoroglutamine [(2S,4R)4-FGln] was previously developed for in vivo positron emission tomography (PET). In the present work, comparative enzymological studies of unlabeled (2S,4R)4-FGln and its deamidated product (2S,4R)4-FGlu were conducted as an adjunct to these PET studies. Our findings are as follows: Rat kidney preparations catalyze the deamidation of (2S,4R)4-FGln. (2,4R)4-FGln and (2S,4R)4-FGlu are substrates of various aminotransferases. (2S,4R)4-FGlu is a substrate of glutamate dehydrogenase, but not of sheep brain glutamine synthetase. The compound is, however, a strong inhibitor of this enzyme. Rat liver cytosolic fractions catalyze a γ-elimination reaction with (2S,4R)4-FGlu, generating α-ketoglutarate. Coupling of a deamidase reaction with this γ-elimination reaction provides an explanation for the previous detection of (18)F(-) in tumors exposed to [(18)F](2S,4R)4-FGln. One enzyme contributing to this reaction was identified as alanine aminotransferase, which catalyzes competing γ-elimination and aminotransferase reactions with (2S,4R)4-FGlu. This appears to be the first description of an aminotransferase catalyzing a γ-elimination reaction. The present results demonstrate that (2S,4R)4-FGln and (2S,4R)4-FGlu are useful analogues for comparative studies of various glutamine- and glutamate-utilizing enzymes in normal and cancerous mammalian tissues, and suggest that tumors may metabolize (2S,4R)4-FGln in a generally similar fashion to glutamine. In plants, yeast and bacteria a major route for ammonia assimilation involves the consecutive action of glutamate synthase plus glutamine synthetase (glutamate synthase cycle). It is suggested that (2S,4R)4-FGln and (2S,4R)4-FGlu will be useful probes in studies of ammonia assimilation by the glutamate synthase pathway in these organisms. Finally, glutamine transaminases are conserved in mammals, plants and bacteria, and probably serve to close the methionine salvage pathway, thus linking nitrogen metabolism to sulfur metabolism and one-carbon metabolism. It is suggested that (2S,4R)4-FGln may be useful in studies of the methionine salvage pathway in a variety of organisms.

Cerebral Glutamine Metabolism under Hyperammonemia Determined in vivo by Localized 1H and 15N NMR Spectroscopy

Brain glutamine synthetase (GS) is an integral part of the glutamate-glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. (15)N MRS is an alternative approach to (13)C MRS. Incorporation of labeled (15)N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate-glutamine cycle. To measure (15)N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel (15)N pulse sequence to simultaneously detect, for the first time, [5-(15)N]Gln and [2-(15)N]Gln+Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized (1)H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of (1)H and (15)N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30±0.050 μmol/g per minute), apparent neurotransmission (0.26±0.030 μmol/g per minute), glutamate dehydrogenase (0.029±0.002 μmol/g per minute), and net glutamine accumulation (0.033±0.001 μmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification.

Phenotypic differences between fast and slow methionine sulfoximine-inbred mice: Seizures, anxiety, and glutamine synthetase

Seizures induced by the convulsant methionine sulfoximine (MSO) resemble human "grand mal" epilepsy, and brain glutamine synthetase is inhibited. We recently selected two inbred lines of mice: sensitive to MSO (MSO-Fast) and resistant (MSO-Slow). In the present study, the selection pressure was increased and consanguinity established. To gain insight into the mechanisms of epileptogenesis, we studied the behaviour of MSO-Fast and MSO-Slow mice based on their responses to various convulsants and anticonvulsants, and also the kinetics of glutamine synthetase. The results show that increasing the number of generations of sib-crossings resulted in an increase in the differences between MSO-Fast and MSO-Slow mice. The dose-response curve of MSO-dependent seizures demonstrated that the MSO-Slow mice were highly insensitive to MSO-dependent seizures compared with MSO-Fast inbred mice that were highly sensitivity. The MSO-Slow were resistant to convulsions induced by various convulsants having different mechanisms of action, whereas those in the MSO-Fast line were more sensitive to kainic acid-induced seizures. These data, in addition to the effects of anticonvulsant, strongly suggest that glutamatergic pathways are most likely involved in MSO-dependent seizures, rather than GABAergic ones. This hypothesis is corroborated by the glutamine synthetase activity, which is more elevated in the MSO-Slow line. Behaviour tests showed that MSO-Slow were less anxious than MSO-Fast. Collectively, these results showed that glutamatergic pathways could be involved in the epileptogenic action of MSO, which may be related to the glutamate/glutamine cycle in the brain.

Altered expression of brain monocarboxylate transporter 1 in models of temporal lobe epilepsy

Monocarboxylate transporter 1 (MCT1) facilitates the transport of monocarboxylate fuels (lactate, pyruvate and ketone bodies) and acidic drugs, such as valproic acid, across cell membranes. We recently reported that MCT1 is deficient on microvessels in the epileptogenic hippocampal formation in patients with medication-refractory temporal lobe epilepsy (TLE). To further define the role of MCT1 in the pathophysiology of TLE, we used immunohistochemistry and stereological analysis to localize and quantify the transporter in the hippocampal formation in three novel and highly relevant rat models of TLE and in nonepileptic control animals. One model utilizes methionine sulfoximine to induce brain glutamine synthetase deficiency and recurrent limbic seizures, while two models employ an episode of perforant pathway stimulation to cause epilepsy. MCT1 was lost on microvessels and upregulated on astrocytes in the hippocampal formation in all models of TLE. Notably, the loss of MCT1 on microvessels was not due to a reduction in microvessel density. The similarities in MCT1 expression among human subjects with TLE and several animal models of the disease strongly suggest a critical role of this molecule in the pathogenesis of TLE. We hypothesize that the downregulation of MCT1 may promote seizures via impaired uptake of ketone bodies and antiepileptic drugs by the epileptogenic brain. We also propose that the overexpression of MCT1 on astrocytes may lead to increased uptake or release of monocarboxylates by these cells, with important implications for brain metabolism and excitability. These hypotheses can now be rigorously tested in several animal models that replicate key features of human TLE.

Circumstantial evidence for a role of glutamine-synthetase in suicide

Suicide occurs during depression, schizophrenia, diabetes and epilepsy. A common denominator of these disorders is the presence of inflammation. Inflammatory cytokines affect function and expression of the glial enzyme glutamine synthetase and post mortem studies indicate that brain glutamine synthetase function is suppressed in mood disorders and epilepsy. In a study of schizophrenia brains, the expression of glutamine synthetase was reduced in those cases where the cause of death was suicide. The glycogen synthase kinase 3 (GSK3) inhibitor, lithium, which has a proven efficacy against suicide, increased in an animal experiment the expression of glutamine synthetase. Based on these data one could reason that suicide may be prevented by centrally acting GSK3 inhibitors. However, since inhibition of glutamine synthetase may lead to a deficit in glutamine and as consequence a GABA and glutamate deficit, even simple food supplementation with glutamine might help to reduce suicide.

Inactivation of astrocytic glutamine synthetase by hydrogen peroxide requires iron

The specific activity of brain glutamine synthetase (GS) is lowered in several neurodegenerative diseases that involve iron-mediated oxidative stress. The present study has investigated whether H 2O 2 directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H 2O 2 reacts with ferrous iron. Exposure of purified sheep brain GS to supraphysiological concentrations of H 2O 2 (1 mM for 30 min) reduced its specific activity by only 41%, indicating that the enzyme is fairly resistant to oxidation by peroxide. However, the enzyme was completely inactivated when co-incubated with H 2O 2, iron and ascorbate, indicating a vulnerability to oxidation by conditions that favour the production of hydroxyl radicals. Similarly, specific GS activity in cultured mouse astrocytes was resistant to supraphysiological concentrations of H 2O 2, with approximately 37% of activity remaining 3 h after incubation with 1 mM H 2O 2. This inactivation was prevented by the iron chelators 2,2′-dipyridyl or 1,10-phenanthroline, but not by their non-chelating analogues. These data suggest that inactivation of astrocytic GS is caused by H 2O 2 indirectly via the Fenton reaction as it required the presence of chelatable intracellular iron.

Astrocyte Glutamine Synthetase: Importance in Hyperammonemic Syndromes and Potential Target for Therapy

Many theories have been advanced to explain the encephalopathy associated with chronic liver disease and with the less common acute form. A major factor contributing to hepatic encephalopathy is hyperammonemia resulting from portacaval shunting and/or liver damage. However, an increasing number of causes of hyperammonemic encephalopathy have been discovered that present with the same clinical and laboratory features found in acute liver failure, but without liver failure. Here, we critically review the physiology, pathology, and biochemistry of ammonia (i.e., NH3 plus NH4+) and show how these elements interact to constitute a syndrome that clinicians refer to as hyperammonemic encephalopathy (i.e., acute liver failure, fulminant hepatic failure, chronic liver disease). Included will be a brief history of the status of ammonia and the centrality of the astrocyte in brain nitrogen metabolism. Ammonia is normally detoxified in the liver and extrahepatic tissues by conversion to urea and glutamine, respectively. In the brain, glutamine synthesis is largely confined to astrocytes, and it is generally accepted that in hyperammonemia excess glutamine compromises astrocyte morphology and function. Mechanisms postulated to account for this toxicity will be examined with emphasis on the osmotic effects of excess glutamine (the osmotic gliopathy theory). Because hyperammonemia causes osmotic stress and encephalopathy in patients with normal or abnormal liver function alike, the term "hyperammonemic encephalopathy" can be broadly applied to encephalopathy resulting from liver disease and from various other diseases that produce hyperammonemia. Finally, the possibility that a brain glutamine synthetase inhibitor may be of therapeutic benefit, especially in the acute form of liver disease, is discussed.