Kinetics of glial glutamine efflux and the mechanism of neuronal uptake studied in vivo in mildly hyperammonemic rat brain

Kinetics of glial glutamine (GLN) transport to the extracellular fluid (ECF) and the mechanism of GLN(ECF) transport into the neuron--crucial pathways in the glutamine-glutamate cycle--were studied in vivo in mildly hyperammonemic rat brain, by NMR and microdialysis to monitor intra- and extracellular GLN. The minimum rate of glial GLN efflux, determined from the rate of GLN(ECF) increase during perfusion of alpha-(methylamino)isobutyrate (MeAIB), which inhibits neuronal GLN(ECF) uptake by sodium-coupled amino-acid transporter (SAT), was 2.88 +/- 0.22 micromol/g/h at steady-state brain [GLN] of 8.5 +/- 0.8 micromol/g. Our previous study showed that the rate of glutamine synthesis under identical experimental conditions was 3.3 +/- 0.3 micromol/g/h. At steady-state glial [GLN], this is equal to its efflux rate to the ECF. Comparison of the two rates suggests that SAT mediates at least 87 +/- 8% (= 2.88/3.3 x 100%) of neuronal GLN(ECF) uptake. While MeAIB induced > 2-fold elevation of GLN(ECF), no sustained elevation was observed during perfusion of the selective inhibitor of LAT, 2-amino-bicyclo[1,1,2]heptane-2-carboxylic acid (BCH), or of d-threonine, a putative selective inhibitor of ASCT2-mediated GLN uptake. The results strongly suggest that SAT is the predominant mediator of neuronal GLN(ECF) uptake in adult rat brain in vivo.     

Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

Quantitative determination of extracellular glutamine concentration in rat brain, and its elevation in vivo by system A transport inhibitor, alpha-(methylamino)isobutyrate

The basal concentration of glutamine in the extracellular fluid, [GLN(ECF)], was determined to be 385 +/- 16 microm in the cortico-striatal region of awake rats. This in vivo concentration was determined by measuring glutamine concentrations in dialysates collected at several flow rates (0.2-4 microL/min), and extrapolating to the concentration at zero flow-rate. Dialysate glutamine concentrations in the somatosensory cortex, hippocampus and thalamus showed no statistically significant difference. In these brain regions, [GLN(ECF)] was elevated 1.5- to 1.8-fold upon perfusion of 50-250 mmalpha-(methylamino)isobutyrate (MeAIB), a competitive inhibitor of glutamine uptake by system A amino acid transporter. The results show, for the first time, that MeAIB causes elevation of brain GLN(ECF)in vivo. The MeAIB-induced elevation of [GLN(ECF)] provides additional support for the current view that system A GLN transporter (Gln T/SAT 1) is the major pathway for the uptake of GLN(ECF) by neurons, while GLN release from glia is mainly mediated by a system N transporter (SN1) which is not inhibitable by MeAIB. The steady-state GLN(ECF) concentration and the effectiveness of MeAIB in inhibiting neuronal GLN uptake in vivo, reported in this study, will be useful, when combined with the known in vitro kinetic properties of the GLN transporters, for study of GLN transport in the intact brain.     

Effects of CGP 28014 on the in vivo release and metabolism of dopamine in the rat striatum assessed by brain microdialysis

The effects on rat striatal dopamine (DA) metabolism of systemic and local administration of CGP 28014, an inhibitor of catechol-O-methyl-transferase (COMT), were studied by in vivo microdialysis. CGP 28014 (30 mg/kg i.p.) significantly reduced the levels of homovanillic acid (HVA), but did not modify DA and 3,4-dihydroxyphenylacetic acid (DOPAC). The intrastriatal administration (via the microdialysis probe) of 5, 7.5, 10, and 20 mM of CGP 28014 elicited a concentration-dependent, several-fold increase in extracellular DA but did not alter the levels of HVA and DOPAC. Thus, the effects of CGP 28014 observed after i.p. injection (decrease in HVA levels) are different from those measured after intrastriatal administration (increase in DA release). Therefore, the inhibition of COMT is likely to be due to the action of a metabolite of CGP 28014 formed in the periphery and not in the brain.     

Role of manganese accumulation in increased brain glutamine of the cirrhotic rat

Cirrhosis promotes increases of both manganese and glutamine in brain. Manganese is a modulator and glutamine is the product of glutamine synthetase. This work studies the relationship between manganese and glutamine synthetase in a model of cirrhosis in the rat. We administered manganese (1 g/L) in the drinking water of sham-operated and bile-duct obstructed rats. We evaluated the manganese and glutamine accumulation and the glutamine synthetase activity in frontal cortex, striatum, and pallidum after 2, 4, and 6 weeks of biliary obstruction or sham surgery. Cirrhotic rats receiving manganese increased their brain content of metal about 400%–600% after 4 weeks of treatment (P < .05) and also remarkably accumulated glutamine through time in the three regions studied (P < .05 at week 6). Interestingly, bile-duct obstructed rats treated with manganese showed no effect on glutamine synthetase activity. Results from this study suggest that manganese induces increases of brain glutamine independently of its synthesis.     

In vivo 31P NMR spectroscopy of energy rich phosphates in the brain of the hyperammonemic rat

Hyperammonemia is a major contributing factor to the neurological abnormalities observed in hepatic encephalopathy and in congenital defects of ammonia detoxication. In rats variable changes in labile energy rich phosphates in the brain have been observed in hyperammonemia using biochemical methods. Using 31P-NMR spectroscopy however no significant changes of the relative concentrations of the energy rich phosphates alpha, beta and gamma-ATP, phosphocreatine, inorganic phosphate and the pH were found in the fronto parietal cortex of the urease treated hyperammonemic rat. Alterations in the metabolites of these compounds do not appear to be a major pathomechanism of ammonia toxicity in this brain area.     

Evaluation of monoaminergic neurotransmitters in the acute focal ischemic human brain model by intracerebral in vivo microdialysis

The release of neurotransmitters principally glutamate during cerebral ischemia has been extensively studied. It is well recognized that ischemia induced release of glutamate plays a key role in “excitotoxic” neuronal death. The role of monoaminergic neurotransmitters is however unclear. The purpose of this study was to evaluate the extracellular norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA) and serotonin (5-HT) under varied degrees of ischemia in the acute focal ischemic model of the human brain by in-vivo microdialysis. The ischemic response of these amines was correlated with the glutamate levels. Our study concludes that these amines and metabolites can be detected in the human “stroke” model. No marked fluctuations were noted in the levels of norepinephrine and DOPAC. However, significant changes to partial and total ischemia were noted in the extracellular levels of 5-HIAA and 5-HT. These compounds showed a dramatic increase with the onset of ischemia with higher detectable levels in the partial ischemic state in comparison to the total ischemic dialysate levels. The exact role played by the differential increase in the levels of 5-HT to the other catecholamines in the pathogenesis of ischemic neuronal damage remains unclear and warrants further study.     

Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been suggested to play a facilitatory role in kainate seizure expression. Furthermore, mRNA levels for the N/OFQ precursor are increased following kainate seizures, while its receptor (NOP) density is decreased. These data suggest increased N/OFQ release. To obtain direct evidence that this is the case, we have developed a microdialysis technique, coupled with a sensitive radioimmunoassay, that allows measurement of N/OFQ release from the hippocampus and thalamus of awake, freely moving animals. In both these brain areas, the spontaneous N/OFQ efflux decreased by approximately 50% and 65% when Ca2+ was omitted and when tetrodotoxin was added to the perfusion medium, respectively. Perfusion of the dialysis probe with high K+ increased N/OFQ release (approximately threefold) in a Ca2+-dependent and tetrodotoxin-sensitive manner. Kainate seizures caused a twofold increase in N/OFQ release followed, within 3 h, by a return to baseline levels. Approximately 5 h after kainate, a late increase in N/OFQ release was observed. On the following day, when animals were having only low grade seizures, N/OFQ release was not significantly different from normal. These phenomena were observed with similar patterns in the hippocampus and in the thalamus. The present data indicate that acute limbic seizures are associated with increased N/OFQ release, which may prime the molecular changes described above, i.e. cause down-regulation of NOP receptors and activation of N/OFQ biosynthesis.     

Effect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions: Role of glutamic acid

We studied the effect of local administration of nicotine on the release of monoamines in striatum, substantia nigra, cerebellum, hippocampus, cortex (frontal, cingulate), and pontine nucleus and on the release of glutamic acid in striatum of rats in vivo, using microdialysis for nicotine administration and for measuring extracellular amine and glutamic acid levels. Following nicotine administration the extracellular concentration of dopamine, increased in all regions except cerebellum; serotonin increased in cingulate and frontal cortex; and norepinephrine increased in substantia nigra, cingulate cortex, and pontine nucleus. Cotinine, the major nicotine metabolite, had no effect at similar concentrations. The cholinergic antagonists mecamylamine and atropine, the dopaminergic antagonists haloperidol and sulpiride, and the excitatory amino acid antagonist kynurenic acid all inhibited the nicotine-induced increase of extracellular dopamine in the striatum. The fact that kynurenic acid almost completely prevented the effects of nicotine, and nicotine at this concentration produced a 6-fold increase of glutamic acid release, suggests that the effect of nicotine is mainly mediated via glutamic acid release.     

Analysis of acetylcholine from extracellular fluid in brain by in vivo microdialysis and LC-ESI-MS/MS with the stable isotope-labeled internal standard

Acetylcholine (ACh) associated with Alzheimer's and Parkinson's disease is the major neurotransmitter in vertebrates. In support of clinical studies on the mechanism of the illnesses and development of medicines for these diseases, the LC-ESI-MS/MS method was developed and validated for the direct quantification of ACh in dialysate samples with acetylcholine-D(9) bromide (IS) as the isotope-labeled internal standard. The analytes were separated on the Waters Hilics C(18) Column (2.1 mm×100 mm, 3 μm) on LC with mobile phase ultrapure water-200 mM ammonium formate (pH 3.04)-acetonitrile (30:5:65, vol/vol/vol) at a flow rate of 300 μL/min, and monitored with a fragment ion of m/z 87 formed from a molecular ion of m/z 146 for ACh and that of m/z 87 from m/z 155 for IS during multiple reaction monitoring (MRM) positive ion mode. The lower limit of quantitation (LLOQ) of ACh was lower than 0.1 nmol/L in dialysate samples, equivalent to 0.2fmol injected on-column. The developed method could be utilized in the analysis of ACh in dialysate samples and these results were in good agreement with the gradient elution study.     

Transport of alovudine (3'-fluorothymidine) into the brain and the cerebrospinal fluid of the rat, studied by microdialysis

Extracellular unbound concentrations of alovudine were sampled by microdialysis in order to study the transport of alovudine between the blood and the brain and the cerebrospinal fluid (CSF) in the rat. The AUC (area under the curve) ratio CSF/blood was higher than the brain/blood ratio after i.v. infusion of alovudine 25mg/kg/hr after a loading dose of 25 mg/kg in 5 minutes (n=4). Neither i.v. infusion of thymidine (25 mg/kg/hr, n=5; 100 mg/kg/hr, n=2) nor acetazolamide (50 mg/kg i.p. bolus followed by 25 mg/kg i.p. every second hour, n=3) influenced the brain/blood AUC ratio after alovudine 25 mg/kg s.c. injection compared to controls (n=5). Finally, perfusion through the microdialysis probe with thymidine (1000 microM, n=3) had also no effect on the brain/blood AUC ratio after alovudine 25 mg/kg s.c. Because neither thymidine nor acetazolamide has significant influence on the ability of alovudine to penetrate the blood-brain barrier in the rat, neither thymidine transport nor carboanhydrase dependent CSF production appear to be major determinants of the blood-brain concentration gradient. Thus, it is concluded that alovudine reaches the extracellular fluid of the brain not by cerebrospinal fluid, but via the cerebral capillaries and that the existence of a concentration gradient over both blood-brain and CSF-brain barrier can probably be explained by the presence of an active process pumping alovudine out from the brain.     

NMDA-mediated release of glutamate and GABA in the subthalamic nucleus is mediated by dopamine: an in vivo microdialysis study in rats

The present study investigated the effects of N-methyl-D-aspartic acid.H2O (NMDA) on the dopamine, glutamate and GABA release in the subthalamic nucleus (STN) by using in vivo microdialysis in rats. NMDA (100 micromol/L) perfused through the microdialysis probe evoked an increase in extracellular dopamine in the STN of the intact rat of about 170%. This coincided with significant increases in both extracellular glutamate (350%) and GABA (250%). The effect of NMDA perfusion on neurotransmitter release at the level of the STN was completely abolished by co-perfusion of the selective NMDA-receptor antagonist MK-801 (10 micromol/L), whereas subthalamic perfusion of MK-801 alone had no effect on extracellular neurotransmitter concentrations. Furthermore, NMDA induced increases in glutamate were abolished by both SCH23390 (8 micromol/L), a selective D1 antagonist, and remoxipride (4 micromol/L), a selective D2 antagonist. The NMDA induced increase in GABA was abolished by remoxipride but not by SCH23390. Perfusion of the STN with SCH23390 or remoxipride alone had no effect on extracellular neurotransmitter concentrations. The observed effects in intact animals depend on the nigral dopaminergic innervation, as dopamine denervation, by means of 6-hydroxydopamine lesioning of the substantia nigra, clearly abolished the effects of NMDA on neurotransmitter release at the level of the STN. Our work points to a complex interaction between dopamine, glutamate and GABA with a crucial role for dopamine at the level of the STN.     

Modulation of ventral pallidal dopamine and glutamate release by the intravenous anesthetic propofol studied by in vivo microdialysis

Summary. The intravenous anesthetic propofol is reported to have various psychological side effects as hallucinations, sexual disinhibition, or euphoria. Hedonic and rewarding states like these are modulated by the dopaminergic system in the nucleus accumbens, prefrontal cortex and also in the ventral pallidum and by the glutamatergic system in the neocortex and limbic system. In the present study, propofol was administered either alone or in combination with the GABAA receptor antagonist bicuculline via reverse microdialysis into the ventral pallidum of freely moving rats. Dialysis fractions were taken every 20min and analyzed for dopamine and glutamate using high performance liquid chromatography. Application of propofol decreased dopamine levels in the ventral pallidum. This effect seems to be mainly mediated through GABAA receptors, since it was compensated by the GABAA receptor antagonist bicuculline. Propofol and propofol plus bicuculline exerted no effect on glutamate release in this brain region. The reduced dopamine release in ventral pallidum was most probably mediated through a GABAergic feedback loop from the ventral pallidum via the nucleus accumbens to the dopaminergic neurons of the ventral tegmental area or by long loop feedback. As an increase but not a decrease of dopamine release in the ventral pallidum is involved in hedonic and rewarding properties, similar symptoms induced by propofol seem to be unrelated to an action of propofol in the ventral pallidum.     

Induction of glutamine synthetase by 8-bromo cyclic AMP in primary cultures of rat brain astrocytes

Glutamine synthetase (GS) is the key enzyme in cerebral glutamine production. Understanding the regulation of the expression of GS is important for definition of the control of glutamine metabolism in brain. Therefore, we studied the control of GS expression by 8-bromo cyclic AMP in primary cultures of astrocytes prepared from brains of neonatal rats. GS activity was increased by 8-bromo cyclic AMP in a dose- and time-dependent manner. This increase was associated with a corresponding increase in the steady-state level of GS mRNA.     

An update on the role of brain glutamine synthesis and its relation to cell-specific energy metabolism in the hyperammonemic brain: further studies using NMR spectroscopy

Ammonia is a neurotoxin that is implicated in the pathogenesis of hepatic encephalopathy due to acute and chronic liver failure. However, its relation to neurological damage and brain edema is poorly understood. During the last decades, it has been the prevailing hypothesis that an osmotic disturbance induced by the astrocytic accumulation of glutamine leads to brain edema. However, various findings are at variance with this hypothesis. The present review will discuss: (a) correlation of ammonia with encephalopathy and brain edema in HE; (b) glutamine synthesis and astrocyte swelling; (c) glutamine synthesis and the glutamine-cycle: relation to brain energy metabolism; (d) glutamine synthesis and the glutamate-glutamine cycle and its relation to anaplerotic activity; (e) evidence favouring the "glutamine hypothesis"; (f) evidence contradicting the "glutamine hypothesis"; (g) glutamine synthesis and osmoregulation; (h) glutamine synthesis in chronic liver failure; (i) impaired brain energy metabolism in acute liver failure (ALF) and its relation to astrocytic glutamine synthesis. Taken together, the precise role of glutamine in the development of brain edema in ALF remains unclear. Astrocytic changes due to glutamine accumulation may lead secondarily to effects on brain energy metabolism. However, the relation between impaired energy metabolism and glutamine accumulation has not been well established. It is noteworthy that no single biochemical factor appears to be responsible for the many symptoms of HE. For example, brain glutamine accumulation and low-grade brain edema occur in chronic liver failure (CLF) suggesting common mechanisms are responsible for the neurological dysfunction in CLF and ALF. Recent NMR spectroscopic studies have provided considerably new information in this area. Future NMR studies using the stable isotope 13C may be useful in the study of the dynamics of brain metabolism in patients with ALF so as to better elucidate the precise role of glutamine accumulation and of glutamine-independent components to brain edema in ALF.     

Direct measurement of oxidative metabolism in the living brain by microdialysis: a review

This review summarizes microdialysis studies that address the question of which compounds serve as energy sources in the brain. Microdialysis was used to introduce ¹⁴C-labeled glucose, lactate, pyruvate, glutamate, glutamine, and acetate into the interstitial fluid of the brain to observe their metabolism to ¹⁴CO₂. Although glucose uptake from the systemic system supplies the carbon source for these compounds, compounds synthesized from glucose by the brain are subject to recycling including complete metabolism to CO₂. Therefore, the brain utilizes multiple compounds in its domain to provide the energy needed to fulfill its function. The physiological conditions controlling metabolism and the contribution of compartmentation into different brain regions, cell types, and subcellular spaces are still unresolved. The aconitase inhibitor fluorocitrate, with a lower inhibition threshold in glial cells, was used to identify the proportion of lactate and glucose that was oxidized in glial cells versus neurons. The fluorocitrate data suggest that glial and neuronal cells are capable of utilizing both lactate and glucose for energy metabolism.     

Homocysteine-evoked 45Ca release in the rabbit hippocampus is mediated by both NMDA and group I metabotropic glutamate receptors: in vivo microdialysis study

This in vivo microdialysis study compared the effects of NMDA and d,l-homocysteine (Hcy) administered via dialysis medium on ⁴⁵Ca efflux from prelabeled rabbit hippocampus. Application of these agonists evoked dose-dependent, and sensitive to MK-801, opposite effects: NMDA decreased the ⁴⁵Ca radioactivity in the dialysate, whereas Hcy induced the release of ⁴⁵Ca. The latter effect was potentiated by glycine, inhibited by the antagonist of group I metabotropic glutamate receptors (mGluR) LY367385, and mimicked by t-ADA, an agonist of these receptors. Electron microscopic examination of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of the microdialysis probe after NMDA application demonstrated swelling of mitochondria, which was prevented by cyclosporin A. This study shows, for the first time, Hcy-induced activation of both group I mGluR and NMDA receptors, which may play a role in acute Hcy neurotoxicity. We present new applications of brain microdialysis in studies on excitotoxicity and neuroprotection.     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

NMR evidence for different conformations of the bioactive region of rat CCK-8 and CCK-58

Sulfated CCK-58 and CCK-8 have identical bioactive C-terminal primary sequences but distinct C-terminal solution structures and different bioactivities. To examine structural differences in greater detail, rat CCK-58 and -8 were synthesized with isotopic enrichment of C-terminal residues with (15)N at alpha-amino nitrogens. Proton and nitrogen chemical shift assignments of peptide solutions were obtained by homo- and heteronuclear NMR methods. These data show that the tertiary structure ensembles of C-terminal CCK-8 and CCK-58 differ significantly. Thus, distinct solution conformations may explain differences in CCK(A) and CCK(B) receptor interactions of large and small molecular forms of CCK.