S100B Protein but Not 3-Nitrotyrosine Positively Correlates with Plasma Ammonia in Patients with Inherited Hyperammonemias: A New Promising Diagnostic Tool?

Individuals with inherited hyperammonemias often present developmental and intellectual deficiencies which are likely to be exaggerated by hyperammonemia episodes in long-term outcomes. In order to find a new, systemic marker common to the course of congenital hyperammonemias, we decided to measure the plasma level of S100 calcium-binding protein B (S100B), which is associated with cerebral impairment. Further, we analyzed three mechanistically diverged but linked with oxidative-nitrosative stress biochemical parameters: 3-nitrotyrosine (3-NT), a measure of plasma proteins' nitration; advanced oxidation protein products (AOPP), a measure of protein oxidation; and glutathione peroxidase (GPx) activity, a measure of anti-oxidative enzymatic capacity. The plasma biomarkers listed above were determined for the first time in congenital hyperammonemia. Also, the level of pro- and anti-inflammatory mediators (i.e., IL-12, IL-6, IL-8, TNF-α, IL-1ß, and IL-10) and chemokines (IP-10, MCP-1, MIG, and RANTES) were quantified. S100B was positively correlated with plasma ammonia level, while noticeable levels of circulating 3-NT in some of the patients' plasma did not correlate with ammonia concentration. Overall, the linear correlation between ammonia and S100B but not standard oxidative stress-related markers offers a unique perspective for the future identification and monitoring of neurological deficits risk-linked with hyperammonemia episodes in patients with inherited hyperammonemias. The S100B measure may support the development of therapeutic targets and clinical monitoring in these disorders.

The contribution of astrocytes to obesity-associated metabolic disturbances.

Obesity is a worldwide health, economic and social concern, despite efforts made to counteract the spreading wave of eating and nourishment-associated disorders. The review aims to show how the glial cells, astrocytes, contribute to the central regulation of appetite and energy metabolism. The hypothalamus is the brain center responsible for nutrients and nutritional hormone sensing, signal processing, and execution of metabolic and behavioral responses, directed at sustaining energy homeostasis. The astrocytes are endowed with receptors, transporters and enzymatic machinery responsible for glucose, lactate, fatty acids, ketone bodies, as well as leptin or ghrelin transport and metabolism, and that render them supporters and partners for neurons in governing the brain and body energy intake and expenditure. However, the role of astrocytes associated with brain energy metabolism reaches far beyond simple fuel contingent-they contribute to cognitive performance. The cognitive decline which often accompanies high fat- and/or high-calorie diets and correlates with neuroinflammation and astrogliosis, is a major concern. The last two decades of research enabled us to acknowledge the astroglia in obesity-associated dysfunctions and to investigate astrocytes as contributors to the pathology, as well as targets for therapy.

The tryptophan metabolism, kynurenine pathway and oxidative stress - implications for glioma pathobiology.

The kynurenine pathway receives increasing attention due to its involvement in central nervous system pathologies, i.a. neurodegenerative and psychiatric disorders, but also due to the contribution to the pathomechanism of neoplasms, including brain tumors. The present review focuses on kynurenine pathway activity in gliomas, brain tumors of glial origin. The upregulation of kynurenine pathway enzyme, indoleamine 2,3-dioxygenase (IDO), resulting in a decreased level of tryptophan and augmented kynurenine synthesis (increased (KYN/Trp ratio) are the most recognised hallmark of malignant transformation, characterised with immunomodulatory adaptations, providing an escape from defence mechanisms of the host, growth-beneficial milieu and resistance to some therapeutics. The review addresses, however, the oxidative/nitrosative stress-associated mechanisms of tryptophan catabolism, mainly the kynurenine pathway activity, linking them with glioma pathobiology.

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.

Pretreatment with a glutamine synthetase inhibitor MSO delays the onset of initial seizures induced by pilocarpine in juvenile rats.

The contribution of glutamatergic transmission to generation of initial convulsive seizures (CS) is debated. We tested whether pretreatment with a glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), affects the onset and progression of initial CS by cholinergic stimulus in juvenile rats. Male rats (24 days old, Sprague Dawley) sequentially received i.p. injections of lithium-carbonate, MSO, methyl-scopolamine, and pilocarpine (Pilo). Pilo was given 150 min after MSO. Animals were continuously monitored using the Racine scale, EEG/EMG and intrahippocampal glutamate (Glu) biosensors. GS activity as measured in hippocampal homogenates, was not altered by MSO at 150 min, showed initial, varied inhibition at 165 (15 min post-Pilo), and dropped down to 11% of control at 60 min post-Pilo, whereas GS protein expression remained unaltered throughout. Pilo did neither modulate the effect of MSO on GS activity nor affect GS activity itself, at any time point. MSO reduced from 32% to 4% the number of animals showing CS during the first 12 min post-Pilo, delayed by ~6 min the appearance of electrographic seizures, and tended to decrease EMG power during ~15 min post-Pilo. The results indicate that MSO impairs an aspect of glutamatergic transmission involved in the transition from the first cholinergic stimulus to the onset of seizures. A continuous rise of extracellular Glu lasting 60 min was insignificantly affected by MSO, leaving the nature of the Glu pool(s) involved in altered glutamatergic transmission undefined.

Targeting Glutamine Addiction in Gliomas.

The most common malignant brain tumors are those of astrocytic origin, gliomas, with the most aggressive glioblastoma (WHO grade IV) among them. Despite efforts, medicine has not made progress in terms of the prognosis and life expectancy of glioma patients. Behind the malignant phenotype of gliomas lies multiple genetic mutations leading to reprogramming of their metabolism, which gives those highly proliferating cells an advantage over healthy ones. The so-called glutamine addiction is a metabolic adaptation that supplements oxidative glycolysis in order to secure neoplastic cells with nutrients and energy in unfavorable conditions of hypoxia. The present review aims at presenting the research and clinical attempts targeting the different metabolic pathways involved in glutamine metabolism in gliomas. A brief description of the biochemistry of glutamine transport, synthesis, and glutaminolysis, etc. will forego a detailed comparison of the therapeutic strategies undertaken to inhibit glutamine utilization by gliomas.

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.

Persistent Overexposure to N-Methyl-D-Aspartate (NMDA) Calcium-Dependently Downregulates Glutamine Synthetase, Aquaporin 4, and Kir4.1 Channel in Mouse Cortical Astrocytes.

Astrocytes express N-methyl-D-aspartate (NMDA) receptor (NMDAR) but its functions in these cells are not well defined. This study shows that the sustained exposure (8-72 h) of mouse astrocytes to NMDA decreases the expression of the functional astroglia-specific proteins, glutamine synthetase (GS), and the water channel protein aquaporin-4 (AQP4) and also reduces GS activity. Similar to rat astrocytes (Obara-Michlewska et al. Neurochem Int 88:20-25, 2015), the exposure of mouse astrocytes to NMDA also decreased the expression of the inward rectifying potassium channel Kir4.1. NMDA failed to elicit the effects in those cells incubated in the absence of Ca2+ and in those in which the GluN1 subunit of the NMDAR was silenced with GluN1 siRNA. The downregulation of GS, AQP4, and Kir4.1 observed in vitro may reflect NMDAR-mediated alterations of astrocytic functions noted in central nervous system pathologies associated with increased glutamate (Glu) release and excitotoxic tissue damage.

Interstitial ion homeostasis and acid-base balance are maintained in oedematous brain of mice with acute toxic liver failure.

Acute toxic liver failure (ATLF) rapidly leads to brain oedema and neurological decline. We evaluated the ability of ATLF-affected brain to control the ionic composition and acid-base balance of the interstitial fluid. ATLF was induced in 10-12 weeks old male C57Bl mice by single intraperitoneal (i.p.) injection of 100 µg/g azoxymethane (AOM). Analyses were carried out in cerebral cortex of precomatous mice 20-24 h after AOM administration. Brain fluid status was evaluated by measuring apparent diffusion coefficient [ADC] using NMR spectroscopy, Evans Blue extravasation, and accumulation of an intracisternally-injected fluorescent tracer. Extracellular pH ([pH]e) and ([K+]e) were measured in situ with ion-sensitive microelectrodes. Cerebral cortical microdialysates were subjected to photometric analysis of extracellular potassium ([K+]e), sodium ([Na+]e) and luminometric assay of extracellular lactate ([Lac]e). Potassium transport in cerebral cortical slices was measured ex vivo as 86Rb uptake. Cerebral cortex of AOM-treated mice presented decreased ADC supporting the view that ATLF-induced brain oedema is primarily cytotoxic in nature. In addition, increased Evans blue extravasation indicated blood brain barrier leakage, and increased fluorescent tracer accumulation suggested impaired interstitial fluid passage. However, [K+]e, [Na+]e, [Lac]e, [pH]e and potassium transport in brain of AOM-treated mice was not different from control mice. We conclude that in spite of cytotoxic oedema and deregulated interstitial fluid passage, brain of mice with ATLF retains the ability to maintain interstitial ion homeostasis and acid-base balance. Tentatively, uncompromised brain ion homeostasis and acid-base balance may contribute to the relatively frequent brain function recovery and spontaneous survival rate in human patients with ATLF.

Roles of Glutamate and Glutamine Transport in Ammonia Neurotoxicity: State of the Art and Question Marks.

BACKGROUND: Excessive accumulation of ammonia in the brain is a causative factor of an array of neurological manifestations of hyperammonemic encephalopathies ("hyperammonemias", HA) among which hepatic encephalopathy (HE) is a major epidemiologic and therapeutic challenge. While ammonia neurotoxicity is symptomatically and mechanistically very complex, there is a consensus with regard to the leading role in its pathogenesis of: i) astrocytes being the primary cellular target of ammonia toxicity; ii) alterations of glutamate (Glu)-dependent neurotransmission (over-excitation followed by inhibition of glutamatergic tone) being the cornerstone of its neurophysiological manifestations; and iii) brain edema, an often lethal consequence of astrocytic swelling, being among other factors caused by the retention of glutamine (Gln) in these cells. OBJECTIVE: This article critically evaluates the present literature attempting to relate manifestations of HA to changes in astrocytic Glu and Gln transport as observed in different in vivo and in vitro HA and/or HE models. Emphasis is put on two disproportions in the state of the art: i) the paucity of available data regarding ammonia-dependent changes in Glu transport activity vs the relative abundance of information on the expression of astrocytic Glu transporters (GLT-1/EAAT2 and GLAST/EAAT1); ii) the just emerging still not very conclusive knowledge on the response of astrocytic Gln transporters SN1 and SN2. CONCLUSION: The review on the above issues is complemented by own recent data which fill some of the many gaps in the knowledge. A brief account is included on the roles of heteromeric cell membrane Glu/arginine (Arg) exchanger y+LAT2 and on the mitochondrial Gln transport.

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.

Astroglial NMDA receptors inhibit expression of Kir4.1 channels in glutamate-overexposed astrocytes in vitro and in the brain of rats with acute liver failure.

Astroglial inward rectifying Kir4.1 potassium channels are fundamental for the maintenance of ion and water homeostasis in the central nervous system (CNS). Down-regulation of Kir4.1 expression is observed in CNS disorders associated with excessive extracellular glutamate (Glu) accumulation, including hepatic encephalopathy related to acute liver failure (ALF). Here we demonstrate that prolonged (3 days) treatment of cultured rat cortical astrocytes with 2 mM Glu or 100 µM NMDA decreases the expression of Kir4.1 mRNA and protein. Inhibition by Glu of Kir4.1 mRNA expression was reversed by NMDA receptor antagonists MK-801 and AP-5 (each at 50 µM), and by a non-transportable inhibitor of Glu uptake TBOA (100 µM). MK-801 reversed the inhibitory effect of Glu on Kir4.1 protein expression. In contrast, transcription of Kir4.1 channels was not affected by: (i) a transportable Glu uptake inhibitor PDC (100 µM); (ii) by group I mGluR antagonist MTEP (100 µM); (iii) by antagonists of oxidative-nitrosative stress (ONS) in astrocytes, including the neuroprotective amino acid taurine (Tau; 10 mM), the NADPH oxidase inhibitor apocyanine (APO; 300 µM), the nitric oxide synthase inhibitor, L-NNA (100 µM), and a membrane permeable glutathione precursor, glutathione-diethyl ester (GEE; 3 mM). Down-regulation of Kir4.1 transcription in rats with ALF was attenuated by intraperitoneal administration of a competitive NMDA receptor antagonist memantine, but not by histidine, which reverses ONS associated with ALF. Collectively, the results indicate that over-activation of astroglial NMDA receptors, aided by as yet undefined effects of Glu entry to astrocytes, is a primary cause of the reduction of Kir4.1 expression in CNS disorders associated with increased exposure to Glu.

Modulation by kynurenine of extracellular kynurenate and glutamate in cerebral cortex of rats with acute liver failure.

BACKGROUND: Kynurenic acid (KYNA) modulates the glutamatergic tone by controlling neuronal glutamate (GLU) release. The present study tested the potential of the KYNA precursor, kynurenine (KYN) to counter increased extracellular GLU associated with the pathogenesis of hepatic encephalopathy accompanying acute liver failure (ALF). METHODS: ALF was induced in adult rats by administration of a hepatotoxin, thioacetamide. KYNA and GLU were measured in the cerebral cortical microdialysates of control (saline-treated) and ALF rats using HPLC. The expression of mRNA coding for kynurenine aminotransferase II (KAT-II), the astrocytic enzyme converting KYN to KYNA, was assayed by real-time PCR. RESULTS: Cerebral cortical extracellular KYNA was increased in ALF rats not treated with KYN, consistent with a previously observed increase of cerebral cortical KATII activity in this ALF model. Single intraperitoneal administration of KYN (50 mg/kg, 120 min before microdialysate collection), produced a further substantial increase of extracellular KYNA, paralleled by a decrease of extracellular GLU. In cultured cerebral cortical astrocytes, the cells which in situ are the primary target of blood-derived ammonia and other toxins liberated due to ALF, elevation of KAT-II mRNA expression was noted upon their incubation with KYN and the KYN precursor, tryptophan (Trp), which is normally elevated by ALF. CONCLUSIONS: Administration of exogenous KYN to stimulate KYNA synthesis may help correcting excessive extracellular accumulation of GLU in cerebral cortex caused by ALF. The therapeutic potential of KYN in ALF appears to be fostered by increased expression of KAT-II in astrocytes upon exposure to KYN or Trp.

Citrulline uptake in rat cerebral cortex slices: modulation by Thioacetamide -Induced hepatic failure.

L-citrulline (Cit) is a co-product of NO synthesis and a direct L-arginine (Arg) precursor for de novo NO synthesis. Acute liver failure (ALF) is associated with increased nitric oxide (NO) and cyclic GMP (cGMP) synthesis in the brain, indirectly implicating a role for active transport of Cit. In the present study we characterized [(3)H]Cit uptake to the cortical brain slices obtained from control rats and rats with thioacetamide (TAA)-induced ALF ("TAA slices"). In both control and TAA slices the uptake was partially Na(+)-dependent and markedly inhibited by substrates of systems L and N, including L-glutamine (Gln), which accumulates in excess in brain during ALF. Cit uptake was not affected by Arg, the y(+)/y(+)L transport system substrate, nor by amino acids taken up by systems A, xc (-)or XAG. The Vmax of the uptake in TAA slices was ~60 % higher than in control slices. Chromatographic (HPLC) analysis revealed a ~30 % increase of Cit concentration in the cerebral cortical homogenates of TAA rats. The activity of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), the two enzymes of Cit-NO cycle catalyzing synthesis of Arg, showed an increase in TAA rats, consistent with increased ASS and ASL protein expression, by ~30 and ~20 %, respectively. The increased Cit-NO cycle activity was paralleled by increased expression of mRNA coding for inducible nitric oxide synthase (iNOS). Taken together, the results suggest a role for Cit in the activation of cerebral NO synthesis during ALF.

Ammonia upregulates kynurenine aminotransferase II mRNA expression in rat brain: a role for astrocytic NMDA receptors?

Kynurenine aminotransferase II (KAT-II) is the astrocytic enzyme catalyzing the synthesis of kynurenic acid (KYNA), an endogenous inhibitor of the α7-nicotinic receptor and the NMDA receptor (NMDAr). A previous study demonstrated an increase of KYNA synthesis in the brain of rats with thioacetamide (TAA)-induced acute liver failure. Here we show that TAA administration increases KAT-II expression in the rat cerebral cortex and the effect is mimicked in cerebral cortical astrocytes in culture treated with high (5 mM) concentration of ammonia. KAT-II expression in control and TAA-treated rats was increased by NMDAr antagonist memantine, and the effects of TAA and memantine appeared additive. In astrocytes, the NMDAr antagonist MK-801 raised KAT-II expression as well, while NMDA added alone had no effect. Glutamate decreased KAT-II mRNA level, which was attenuated by MK-801. The results suggest that stimulation of KAT-II expression during hepatic encephalopathy may be associated with a partial inactivation of astrocytic NMDAr by ammonia.

Transfection of a human glioblastoma cell line with liver-type glutaminase (LGA) down-regulates the expression of DNA-repair gene MGMT and sensitizes the cells to alkylating agents.

O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA-repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth-depressing transfection of a T98G glioma cell line with liver-type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA-transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non-transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100-1000 µM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 µM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.

Down-regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy.

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

Synergistic action of hypoosmolarity and glutamine in inducing acute swelling of retinal glial (Müller) cells.

High blood ammonia, elevated glutamine, and hyponatremia are pathogenic factors contributing to astrocytic swelling and brain edema in liver failure. We investigated the effects of hypoosmolarity, ammonia, and glutamine on the induction of glial cell swelling in freshly isolated slices of the rat retina. Glutamine, but not ammonia or hypoosmolarity per se, evoked a rapid (within one minute) swelling of retinal glial (Müller) cell bodies under hypoosmotic conditions. Under isoosmotic conditions, glutamine evoked a delayed swelling after 10 min of exposure. The effect of glutamine was concentration-dependent, with half-maximal and maximal effects at ∼ 0.1 and 0.5 mM. Glutamine in hypoosmotic solution induced a dissipation of the mitochondrial membrane potential. The effects on the mitochondrial membrane potential and the glial soma size were reduced by (i) agents which inhibit the transfer of glutamine into mitochondria and its hydrolysis there, (ii) inhibition of the mitochondrial permeability transition, (iii) inhibitors of oxidative-nitrosative stress, and (iv) inhibitors of phospholipase A(2) and cyclooxygenase. Glutamine-induced glial swelling was also prevented by ATP and adenosine, acting at adenosine A(1) receptors. The data suggest that hypoosmolarity accelerates the swelling-inducing effect of glutamine on retinal glial cells, and that swelling induction by glutamine is mediated by inducing oxidative-nitrosative stress, inflammatory lipid mediators, and mitochondrial dysfunction.

Gain of function of Kir4.1 channel increases cell resistance to changes of potassium fluxes and cell volume evoked by ammonia and hypoosmotic stress.

The Kir4.1 channel is an inward rectifying potassium channel involved in the control of potassium and water movement in mammalian cells. To evaluate independently the role of Kir4.1 alone and without interaction with other cellular effectors, we compared (86)Rb fluxes and cell volume in Kir4.1 transfected cells (Kir4.1(+)) with cells transfected with an empty vector (Kir4.1(-)). Transfection with Kir4.1 neither increased (86)Rb uptake nor (86)Rb efflux from cells in isotonic medium. Pretreatment with ammonia (5 mM ammonium chloride) in isotonic medium produced a pronounced increase of (86)Rb uptake and a moderate decrease of cell volume in Kir4.1(-) but not in Kir4.1(+) cells. However, pretreatment evoked no change in (86)Rb efflux in either cell type. Hypotonic treatment (HT) markedly increased (86)Rb efflux in Kir4.1(-) cells and increased cell volume in both cell types. Although pretreatment with ammonia did not alter the effect of HT on (86)Rb efflux in either Kir4.1(+) or Kir4.1(-) cells, it potentiated the effect of hypotonic treatment in increasing cell volume in Kir4.1(-) cells. The results demonstrate that the presence of Kir4.1 in cells increases their resistance to alterations of potassium fluxes and/or cell volume imposed by ammonia and hypotonicity.