Magdalena Zielińska

Roles of glutamine in neurotransmission

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor-product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.

Changes in the extracellular profiles of neuroactive amino acids in the rat striatum at the asymptomatic stage of hepatic failure

Rats were treated with a hepatotoxin thioacetamide (TAA) and examined 21 days later, when they showed moderate fatty metamorphosis of the liver and morphological changes in brain indicative of excitotoxic neuronal damage, but no evident biochemical or neurophysiological symptoms of hepatic encephalopathy (HE). High‐performance liquid chromatography (HPLC) analysis of extracellular amino acids in striatal microdialysates of TAA‐treated rats revealed a significant increase in the excitatory amino acids glutamate (Glu) and aspartate (Asp) and their amino acid metabolites glutamine (Gln) and alanine (Ala). Microdialysis in the presence of 50 mM K+ triggered in TAA‐treated rats an accumulation of Asp and Glu, and diminished the accumulation of Gln. These effects were virtually absent in control rats. None of the treatments affected the accumulation of the nontransmitter amino acid leucine (Leu). The above changes mirror those previously described in symptomatic HE and are likely to contribute to excitotoxic damage. The basal microdialysate content of taurine (Tau), an amino acid with antioxidant and volume regulatory properties, was 60% lower in TAA‐treated rats than in control rats despite its increased blood‐to‐brain transport. The decrease in extracellular Tau may thus reflect Tau redistribution to adjacent central nervous system (CNS) cells manifesting a cell‐protective response. Stimulation with 50 mM K+ increased extracellular Tau in control rats by 182% and in TAA‐treated rats by 322%. Stimulation with 100 μM N‐methyl‐D‐aspartate (NMDA) increased extracellular Tau in control rats by 27% and in TAA‐treated rats by as much as 250%. The increase of K+‐ or NMDA‐dependent Tau release may reflect improved cell volume regulation and neuroprotection and contribute to attenuation of neurologic symptoms in rats with liver failure. J. Neurosci. Res. 56:76–84, 1999.

AMMONIA INCREASES PARACELLULAR PERMEABILITY OF RAT BRAIN ENDOTHELIAL CELLS BY A MECHANISM ENCOMPASSING OXIDATIVE/NITROSATIVE STRESS AND ACTIVATION OF MATRIX METALLOPROTEINASES

J. Neurochem. (2012) 121, 125–134.

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

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

Role of N-Methyl-d-aspartate Receptors in the Neuroprotective Activation of Extracellular Signal-regulated Kinase 1/2 by Cisplatin

Neurons are exposed to damaging stimuli that can trigger cell death and subsequently cause serious neurological disorders. Therefore, it is important to define defense mechanisms that can be activated in response to damage to reduce neuronal loss. Here we report that cisplatin (CPDD), a neurotoxic anticancer drug that damages DNA, triggered apoptosis and activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in cultured rat cortical neurons. Inhibition of ERK1/2 activation using either pharmacological inhibitors or a dominant-negative mutant of the ERK1/2 activator, mitogen-activated protein kinase kinase 1, increased the toxicity of CPDD. Interestingly, N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonists reduced the ERK1/2 activation and exacerbated apoptosis in CPDD-treated neurons. Pre-treatment with CPDD increased ERK1/2 activation triggered by exogenous NMDA, suggesting that CPDD augmented NMDAR responsiveness. CPDD-enhanced response of NMDAR and CPDD-mediated ERK1/2 activation were both decreased by inhibition of poly(ADP-ribose) polymerase (PARP). Interestingly, PARP activation did not produce ATP depletion, suggesting involvement of a non-energetic mechanism in NMDAR regulation by PARP. Finally, CPDD toxicity was reduced by brain-derived neurotrophic factor, and this protection required ERK1/2. In summary, our data identify a novel compensatory circuit in central nervous system neurons that couples the DNA injury, through PARP and NMDAR, to the defensive ERK1/2 activation.

Ammonia-Induced Extracellular Accumulation of Taurine in the Rat Striatum In Vivo: Role of Ionotropic Glutamate Receptors

Accumulation of taurine (Tau), glutamate (Glu) and glutamine (Gln) was measured in vivo in microdialysates of the rat striatum following a direct application to the microdialysis tube of 60 mM ammonium chloride which renders the final ammonia concentration in the extracellular space to ∼5 mM. The following compounds were coadministered with ammonia to distinguish between the different mechanisms that may underlie the accumulation of amino acids: ion transport inhibitors, diisothiocyanostilbene-2,28′-disulfonate (DIDS) and furosemide, a Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an NMDA receptor antagonist dizocilpine (MK-801) and an 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Ammonia stimulated Tau accumulation in the microdialysates to ∼250% of the basal value. Furosemide did not significantly affect the stimulation by ammonia and DIDS only moderately depressed the effect. The ammonia-dependent Tau accumulation was increased by ∼50% in the presence of PDC and reduced by ∼35% in the presence dizocilpine and DNQX. In the microdialysates ammonia stimulated Glu and Gln accumulation somewhat less than Tau accumulation. Except for stimulation of Gln accumulation by DNQX, the effects were not modified by any of the cotreatments. The results are consistent with the assumption that ammonia stimulates Tau efflux mainly via activation of ionotropic Glu receptors.

Stimulation of natriuretic peptide receptor C attenuates accumulation of reactive oxygen species and nitric oxide synthesis in ammonia-treated astrocytes

J. Neurochem. (2010) 115, 1068–1076.

Extracellular Concentrations of Taurine, Glutamate, and Aspartate in the Cerebral Cortex of Rats at the Asymptomatic Stage of Thioacetamide-Induced Hepatic Failure: Modulation by Ketamine Anesthesia

Subclinical hepatic encephalopathy (SHE) was produced in rats by two intraperitoneal injections of TAA at 24 h intervals and the animals were examined 21 days later. Concentrations of the neuroactive amino acids taurine (Tau), glutamate (Glu) and aspartate (Asp), were measured in the cerebral cortical microdialysates of thioacetamide (TAA)-treated and untreated control rats. During microdialysis some animals were awake while others were anesthetized with ketamine plus xylazine. There was no difference in the water content of cerebral cortical slices isolated from control and SHE rats, indicating a recovery from cerebral cortical edema that accompanies the acute, clinical phase of hepatic encephalopathy in this model. When microdialysis was carried out in awake rats, dialysate concentrations of all the three amino acids were 30% to 50% higher in SHE rats than in control rats. Ketamine anesthesia caused a 2.2% increase of water content of cerebral cortical slices and increased Asp, Glu, and Tau concentration in microdialysates of control rats. In SHE rats, ketamine anesthesia produced a similar degree of cerebral edema, however, it did not alter Asp and Glu concentrations in the microdialysates. These data may reflect on one hand a neuropathological process of excitotoxic neuronal damage related to increased Glu and Asp, on the other hand neuroprotection from neuronal swelling indicated by Tau redistribution in the cerebral cortex. The reduction of the effects of SHE on Glu and Asp content in ketamine-anesthesized rats is likely to be due to interference of ketamine with the NMDA receptor-mediated component of the SHE-evoked excitatory neurotransmitter efflux and/or reuptake of the two amino acids. By contrast, the SHE-related increase of Tau content was not affected by ketamine anesthesia, indicating that the mechanism(s) underlying SHE-evoked accumulation of Tau must be different from the mechanism causing release of excitatory amino acids. The results with ketamine advocate caution when using this anesthetic in studies employing the cerebral microdialysis technique for measurement of extracellular amino acids.

Roles of Changes in Active Glutamine Transport in Brain Edema Development During Hepatic Encephalopathy: An Emerging Concept

Abstract 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.

Hyperammonemia increases the expression and activity of the glutamine/arginine transporter y+ LAT2 in rat cerebral cortex: implications for the nitric oxide/cGMP pathway.

The pathogenesis of hepatic encephalopathy (HE) is associated with hyperammonemia (HA) and subsequent exposure of the brain to excess of ammonia. Alterations of the NO/cGMP pathway and increased glutamine (Gln) content are collectively responsible for many HE symptoms, but how the two events influence each other is not clear. Previously we had shown that Gln administered intracerebrally inhibited the NO/cGMP pathway in control rats and even more so in rats with HA, and we speculated that this effect is due to inhibition by Gln of arginine (Arg) transport (Hilgier et al., 2009). In this study we demonstrate that a 3-day HA in the ammonium acetate model increases the expression in the brain of y(+)LAT2, the heteromeric transporter which preferentially stimulates Arg efflux from the cells in exchange for Gln. The expression of the basic amino acid transporter CAT1, transporting Arg but not Gln remained unaffected by HA. Multiple parameters of Arg or Gln uptake and/or efflux and their mutual dependence were altered in the cerebral cortical slices obtained from HA rats, in a manner indicating enhanced y(+)LAT2-mediated transport. HA elevated Gln content and decreased cGMP content as measured both in the cerebral cortical tissue and microdialysates. Intracortical administration of 6-diazo-5-oxo-L-norleucine (DON), which inhibits Gln fluxes between different cells of the CNS, attenuated the HA-induced decrease of cGMP in the microdialysates of HA rats, but not of control rats. The results suggest that, reduced delivery of Arg due to enhanced y(+)LAT2-mediated exchange of extracellular Gln for intracellular Arg may contribute to the decrease of NO/cGMP pathway activity evoked in the brain by HA.