Brain-Specific Modulation of Kynurenic Acid Synthesis in the Rat

Abstract: This study was designed to investigate modulatory mechanisms that control the synthesis of the neuroprotective endogenous excitatory amino acid receptor antagonist kynurenate. De novo kynurenate formation was examined in vitro using tissue slices from rat brain, liver, and kidney. In slices from adult cerebral cortex, veratridine, quisqualate, and l -α-aminoadipate decreased kynurenate synthesis substantially. Glucose removal or changes in the ionic milieu, too, influenced kynurenate formation significantly, suggesting that demands on cellular energy interfere with kynurenate production in the adult rat brain. The effects of quisqualate and l -α-aminoadipate were also observed in the immature brain, in the quinolinate-lesioned adult striatum, and, to a lesser extent, in peripheral organs. In contrast, the effect of veratridine was not seen in the lesioned brain or in kidney and liver tissue, indicating its dependency on intact neuron-glia interactions. Compared with the normal adult brain, ionic manipulations yielded qualitatively distinct results in the developing brain and in the periphery, but their effects remained unchanged in the lesioned striatum. Glucose deprivation was less consequential in the immature than in the adult brain and was entirely ineffective in the lesioned striatum and in the periphery. These results further link cellular, especially astrocytic, energy metabolism to kynurenate formation in the brain. More generally, the existence of brain-specific mechanisms for the regulation of kynurenate production is suggestive of a modulatory role of this metabolite in excitatory amino acid receptor function and dysfunction.     

On the Production and Disposition of Quinolinic Acid in Rat Brain and Liver Slices

Abstract: The de novo production and subsequent disposition of the endogenous excitotoxin quinolinic acid (QUIN) was investigated in vitro in tissue slices from rat brain and liver. Incubation of tissue with QUIN's immediate bioprecursor 3-hydroxyanthranilic acid (3-HANA) in oxygenated Krebs-Ringer buffer yielded measurable amounts of QUIN both in the tissue and in the incubation medium. Saturation was reached between 16 and 64 μM 3-HANA (166 pmol of QUIN formed per milligram of protein after a 60-min incubation with 64 μM 3-HANA). In the brain, more QUIN was recovered from the tissue than from the incubation medium at all time points examined (5 min to 4 h). In contrast, the tissue-to-medium ratio for QUIN in parallel experiments with hepatic slices was ? 1. The disposition of newly synthesized QUIN was further elaborated in tissue slices that had been preincubated for 60 min with 64 μM 3-HANA. Subsequent incubation of brain tissue in fresh buffer revealed a steady but relatively slow efflux of QUIN from the cellular compartment, with >30% remaining in the tissue after a 90-min incubation. Analogous experiments with liver slices showed that >93% of newly synthesized QUIN had entered the extracellular compartment within 30 min. Striatal and nigral slices obtained 7 days after an intrastriatal ibotenic acid injection showed severalfold increases in QUIN production compared with control tissues, in all likelihood due to astrogliosis and associated large increases in 3-hydroxyanthranilic acid oxygenase activity. In addition, the apparent tissue-to-medium ratio was markedly reduced in striatal slices from lesioned animals. Taken together, these data indicate that both brain and liver cells have a rather limited capacity to retain QUIN, and that 3-hydroxyanthranilic acid oxygenase activity is a critical determinant controlling extracellular QUIN concentrations in both organs. Changes in the activity of QUIN's biosynthetic enzyme in the brain can therefore be expected to influence the possible function of QUIN as an endogenous agonist at the N-methyl-D-aspartate receptor in health and disease.     

Metabolism of [5-3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-³H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-³H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µ M [5-³H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µ M [5-³H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [³H]KYNA (5.0 vs. 1.8%), [³H]3-HK (20.9 vs. 4.5%), [³H]XA (1.5 vs. 0.4%), and [³H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.     

Regulation of kynurenic acid levels in the developing rat brain

Summary Several brain-specific mechanisms control the formation of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the adult rat brain. Two of these, dopaminergic neurotransmission and cellular energy metabolism, were examined in the brain of immature (postnatal day 7) rats. The results indicate that during the early postnatal period cerebral KYNA synthesis is exceptionally amenable to modulation by dopaminergic mechanisms but rather insensitive to fluctuations in cellular energy status. These findings may be of relevance for the role of KYNA in the function and dysfunction of the developing brain.     

Presence of 3-Hydroxyanthranilic Acid in Rat Tissues and Evidence for Its Production from Anthranilic Acid in the Brain

As assessed by HPLC with electrochemical detection, 3-hydroxyanthranilic acid (3-HANA) was found to be present in the rat brain and peripheral organs. The highest concentrations were measured in the kidney (86 fmol/mg of tissue) and spleen (56 fmol/mg of tissue), whereas the adrenal gland, liver, heart, and several forebrain areas (hippocampus, striatum, parietal cortex, thalamus, amygdala/pyriform cortex, and frontal cortex) contained less 3-HANA (between 15 and 22 fmol/mg of tissue). Slightly lower concentrations of 3-HANA were found in the brainstem and the cerebellum. The metabolic disposition of 3-HANA was examined in tissue slices which were incubated in Krebs-Ringer buffer at 37 degrees C in vitro. Incubation for up to 2 h did not affect 3-HANA concentration in brain tissue. However, inhibition of 3-HANA degradation by the specific 3-hydroxyanthranilic acid oxygenase blocker 4-chloro-3-hydroxyanthranilic acid (4-Cl-3-HANA; 10 microM) resulted in a rapid (within 2.5 min) doubling of 3-HANA levels in slices from cerebral cortex. No further increases were observed after incubations of up to 120 min. Exposure of cortical slices to 3-HANA's putative bioprecursors, 3-hydroxykynurenine (3-HK) and anthranilic acid (ANA), in the absence of 4-Cl-3-HANA resulted in rapid, transient increases in 3-HANA production. Maximal 3-HANA synthesis from ANA exceeded the maximal effect of 3-HK by approximately 11-fold.2+ In the presence of 4-Cl-3-HANA, 1 mM ANA produced 9.0 +/- 0.3 and 89.0 +/- 9.3 (5 min) or 51.6 +/- 7.9 and 187.5 +/- 11.2 (120 min) fmol of newly synthesized 3-HANA/mg of brain tissue, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L-kynurenine (KYN) and by determining the irreversible conversion of [3H]KYN to [3H]KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2-oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT-rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate-specific. Thus, of the amino acids tested, only alpha-aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3-hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 microM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 microM for KYN with 1 mM 2-oxoglutarate, but a much higher Km (910 microM) with 1 mM pyruvate. Km values for 2-oxoglutarate and pyruvate were 150 and 160 microM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some metabotropic glutamate receptor ligands reduce kynurenate synthesis in rats by intracellular inhibition of kynurenine aminotransferase II

Some metabotropic glutamate receptor (mGluR) ligands, such as quisqualate, L-(+)-2-amino-4-phosphonobutyric acid (L-AP4), 4-carboxy-3-hydroxyphenylglycine (4C3HPG), and L-serine-O:-phosphate (L-SOP), reduced the formation of the endogenous excitatory amino acid receptor antagonist kynurenate in brain and liver slices. The use of novel, subtype-selective mGluR agonists and antagonists excluded a role for any known mGluR subtype in this effect. The reduction of kynurenate formation was no longer observed when slices were incubated with the active mGluR ligands in the absence of extracellular Na(+). trans-Pyrrolidine-2,4-dicarboxylate (trans-PDC), a broad-spectrum ligand of Na(+)-dependent glutamate transporters, was also able to reduce kynurenate formation. Quisqualate, 4C3HPG, L-AP4, and L-SOP did not further reduce kynurenate formation in the presence of trans-PDC, suggesting that the two classes of drugs may share the same mechanism of action. Hence, we hypothesized that the active mGluR ligands are transported inside the cell and act intracellularly to reduce kynurenate synthesis. We examined this possibility by assessing the direct effect of mGluR ligands on the activity of kynurenine aminotransferases (KATs) I and II, the enzymes that transaminate kynurenine to kynurenate. In brain tissue homogenates, KAT II (but not KAT I) activity was inhibited by quisqualate, 4C3HPG, L-AP4, L-SOP, and trans-PDC. Drugs that were unable to reduce kynurenate formation in tissue slices were inactive. We conclude that some mGluR ligands act intracellularly, inhibiting KAT II activity and therefore reducing kynurenate formation. This effect should be taken into consideration when novel mGluR ligands are developed for the treatment of neurological and psychiatric diseases.     

Synthesis of Quinolinic Acid by 3-Hydroxyanthranilic Acid Oxygenase in Rat Brain Tissue In Vitro

In mammalian peripheral organs, 3-hydroxyanthranilic acid oxygenase (3HAO), catalyzing the conversion of 3-hydroxyanthranilic acid to quinolinic acid, constitutes a link in the catabolic pathway of tryptophan to NAD. Because of the possible involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we examined the presence and characteristics of 3HAO in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy-14C]3-hydroxyanthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chromatographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed a Km of 3.6 +/- 0.5 microM for 3-hydroxyanthranilic acid and a Vmax of 73.7 +/- 9.5 pmol quinolinic acid/h/mg tissue. The enzyme showed pronounced selectivity for its substrate, since several substances structurally and metabolically related to 3-hydroxyanthranilic acid caused less than 25% inhibition of activity at 500 microM. Both the Fe2+ dependency and the distinct subcellular distribution (soluble fraction) of brain 3HAO indicated a close resemblance to 3HAO from peripheral tissues. Examination of the regional distribution in the brain demonstrated a 10-fold variation between the region of highest (olfactory bulb) and lowest (retina) 3HAO activity. The brain enzyme was present at the earliest age tested (7 days postnatum) and increased to 167% at 15 days before reaching adult levels. Enzyme activity was stable over extended periods of storage at -80 degrees C. Taken together, these data indicate that measurements of brain 3HAO may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.     

Rat Brain Slices Produce and Liberate Kynurenic Acid upon Exposure to l-Kynurenine

The incorporation of L-kynurenine (L-KYN) into kynurenic acid (KYNA) was examined in rat brain slices. KYNA was measured in the slices and in the incubation medium after purification by ion-exchange and HPLC chromatography. In pilot experiments, the formation of KYNA was confirmed by gas chromatography. KYNA was produced stereoselectively from L-KYN, and approximately 90% of the newly synthesized KYNA was recovered from the incubation medium. Intracellular KYNA was not actively retained by the tissue and was lost from the cells upon repeated washes. Thus, regulation of the levels of extracellular KYNA appears to occur at the level of L-KYN uptake and/or kynurenine transaminase, the biosynthetic enzyme of KYNA. KYNA production from L-KYN was linear up to 4 h and reached a plateau at a L-KYN concentration of 250 microM. The process was effectively inhibited by the transaminase inhibitor aminooxyacetic acid (IC50, approximately 25 microM), and showed pronounced regional distribution (hippocampus greater than cortical areas greater than thalamus much greater than cerebellum). The conversion of L-KYN to KYNA was dependent on oxygenation and on the presence of glucose in the incubation medium. Neither deletion of Ca2+ or Mg2+ nor addition of 20 mM Mg2+ had any effect. However, KYNA production was significantly attenuated in the absence of Cl- or in the presence of 50 mM K+ in the incubation medium. In Na+-free medium, the production of KYNA from L-KYN was increased by 30%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat 3-Hydroxyanthranilic Acid Oxygenase: Purification from the Liver and Immunocytochemical Localization in the Brain

3-Hydroxyanthranilic acid oxygenase (3HAO; EC 1.13.11.6), the biosynthetic enzyme of the endogenous excitotoxin quinolinic acid, was purified to homogeneity from rat liver and partially purified from rat brain. The pure enzyme is a single subunit protein with a molecular weight of 37-38,000. Kinetic analyses of both pure liver and partially purified brain 3HAO revealed an identical Km of 3 microM for the substrate 3-hydroxyanthranilic acid. Evidence for the identity of liver and brain 3HAO was further provided by physicochemical (electrophoretic behavior, heat sensitivity) and biochemical (pH dependency, activation by Fe2+) means. Antibodies were produced against the pure liver enzyme and the identity of liver and brain 3HAO substantiated immunologically in immunotitration and Ouchterlony double-diffusion experiments. Immunohistochemical studies using purified anti-rat 3HAO antibodies were performed on tissue sections of perfused brains and demonstrated a preferential staining of astroglial cells. Notably, the cellular localization of 3HAO in the brain appears to be in part distinct from that of quinolinic acid phosphoribosyltransferase, the catabolic enzyme of quinolinic acid. Pure rat 3HAO and its antibodies can be expected to constitute useful tools for the further elucidation of the brain's quinolinic acid system.     

Presence of Kynurenic Acid in the Mammalian Brain

Kynurenic acid, a tryptophan metabolite able to antagonize the actions of the excitatory amino acids, has been identified and measured for the first time in the brain of mice, rats, guinea pigs, and humans by using an HPLC method. Its content was 5.8 +/- 0.9 in mouse brain, 17.8 +/- 2.0 in rat brain, 16.2 +/- 1.5 in guinea pig brain, 26.8 +/- 2.9 in rabbit brain, and 150 +/- 30 in human cortex (pmol/g wet wt. mean +/- SE). The regional distribution of this molecule was uneven. In rats, guinea pigs, and rabbits, the brainstem was the area richest in this compound. Tryptophan administration (100-300 mg/kg, i.p.) to rats resulted in a significant increase of the brain content of kynurenic acid. Similarly, 1 h after probenecid administration (200 mg/kg, i.p.), the brain content of kynurenate increased by fourfold, thus suggesting that its turnover rate is relatively fast.     

Analysis of Content of 2-Oxoacids in Rat Brain Extracts Using High-Performance Liquid Chromatography

Biochemistry (Moscow) - 2-Oxoacids are involved in a number of important metabolic processes and can be used as biomarkers in some human diseases. A new optimized method for quantification of...     

Fructose 2, 6-Bisphosphate in Hypoglycemic Rat Brain

Abstract: Fructose 2,6-bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6-bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6-bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6-bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6-phosphofructo-2-kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6-phosphate). These results suggest that fructose 2,6-bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6-phosphofructo-l-kinase and glycolysis.     

Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine

Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography-mass spectrometry or HPLC. The enzyme indoleamine 2,3-dioxygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin-induced accumulation of quinolinic acid while potentiating the accumulation of kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.     

Inhibition of Large Neutral Amino Acid Transporters Suppresses Kynurenic Acid Production Via Inhibition of Kynurenine Uptake in Rodent Brain

The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor and N-methyl-d-aspartic acid receptor at endogenous brain concentrations. Recent studies have suggested that increases of brain KYNA levels are involved in psychiatric disorders such as schizophrenia and depression, and regulation of KYNA production has become a new target for treatment of these diseases. Kynurenine (KYN), the immediate precursor of KYNA, is transported into astrocytes via large neutral amino acid transporters (LATs). In the present study, the effect of LATs regulation on KYN uptake and KYNA production was investigated in vitro and in vivo using an LATs inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). In the in vitro study, cortical slices of rat brain were incubated with a physiological concentration of KYN and 3 µmol/L–3 mmol/L BCH. BCH inhibited KYNA production and KYN uptake in a dose-dependent manner, and their IC₅₀ values were 90.7 and 97.4 µmol/L, respectively. In the in vivo study, mice were administered KYN (50 mg/kg BW) orally and BCH (200 mg/kg BW) intravenously. Administration of KYN increased brain KYN and KYNA levels compared with the mice treated with vehicle, whereas additional administration of BCH suppressed KYN-induced elevations in KYN and KYNA levels to 50 and 70 % in the brain. These results suggest that inhibition of LATs prevented the increase of KYNA production via blockade of KYN uptake in the brain in vitro and in vivo. LATs can be a target to modulate brain function by regulation of KYNA production in the brain.     

Nicotinylalanine Increases the Formation of Kynurenic Acid in the Brain and Antagonizes Convulsions

Kynurenic acid (KYNA) was quantified in the extracellular spaces of the rat hippocampus using microdialysis and HPLC (fluorimetric detection) to study the possible role of this tryptophan metabolite in the modulation of the function of the N-methyl-D-aspartate (NMDA) receptor. Addition of probenecid (1 mM), which is an inhibitor of the organic acid transport system, to the Ringer's solution perfusing the dialysis probe increased the KYNA concentration in the dialysate from 10.4 +/- 0.9 to 48 +/- 6 nM. Addition of 2 mM aminooxyacetic acid, a nonspecific inhibitor of KYNA synthesis, reduced this concentration by 50%. These data suggest that KYNA is continuously synthesized in the rat hippocampus. Nicotinylalanine (NAL), 200-400 mg/kg i.p., an analogue of kynurenine that is able to direct the flow of tryptophan metabolites toward the synthesis of KYNA, significantly increased the KYNA concentration in the hippocampal dialysate and significantly potentiated the effect of tryptophan on the accumulation of KYNA in the brain and other organs. This increase resulted in pharmacological actions compatible with an antagonism of the NMDA receptors. In fact, NAL antagonized sound-induced seizures and prevented death in DBA/2 mice. Pretreatment of the mice with D-serine (100 micrograms intracerebroventricularly), a glycine agonist and a competitive antagonist of KYNA, completely prevented the anticonvulsive action of NAL. These data suggest that changes in the extracellular concentration of KYNA in the brain are associated with a modulation of NMDA receptor function.     

Anti-ischemic Effect of Curcumin in Rat Brain

Turmeric has been in use since ancient times as a condiment and due to its medicinal properties. Curcumin, the yellow colouring principle in turmeric, is polyphenolic and major active constituent. Besides anti-inflammatory, thrombolytic and anticarcinogenic activities, curcumin also possesses strong antioxidant property. In view of the novel combination of properties, neuroprotective efficacy of curcumin was studied in rat middle cerebral artery occlusion (MCAO) model. Rats were subjected to 2 h of focal ischemia followed by 72 h of reperfusion. They were pre-treated with curcumin (100 mg/kg, po) for 5 days prior to MCAO and for another 3 days after MCAO. The parameters studied were behavioural, biochemical and histological. Treatment with curcumin could significantly improve neurobehavioral performance compared to untreated ischemic rats as judged by its effect on rota-rod performance and grid walking. A significant inhibition in lipid peroxidation and an increase in superoxide dismutase (SOD) activity in corpus striatum and cerebral cortex was observed following treatment with curcumin in MCAO rats as compared to MCAO group. Intracellular calcium levels were decreased following treatment with curcumin in MCAO rats. Histologically, a reduction in the infarct area from 33% to 24% was observed in MCAO rats treated with curcumin. The study demonstrates the protective efficacy of curcumin in rat MCAO model.     

Partial Inhibition of Brain Succinate Dehydrogenase by 3-Nitropropionic Acid Is Sufficient to Initiate Striatal Degeneration in Rat

Abstract: Chronic inhibition of succinate dehydrogenase (SDH) by systemic injection of the selective inhibitor 3-nitropropionic acid (3NP) has been used as an animal model for Huntington's disease (HD). However, the mechanisms by which 3NP produces lesions in the striatum are not fully characterized. A quantitative histochemical method was developed to study the level of regional SDH inhibition resulting from intraperitoneal injection of 3NP or chronic intoxication using osmotic pumps. The results showed that (a) 3NP was an irreversible SDH inhibitor in vivo, (b) the level of SDH inhibition in the striatum (the brain region most vulnerable to 3NP) was similar to that observed in other brain regions not affected by the toxin, such as the cerebral cortex, and (c) the neurotoxic threshold of SDH inhibition in the brain was 50–60% of control levels. The present study demonstrates that the selective degeneration in the striatum observed after chronic 3NP administration cannot be ascribed to a preferential inhibition of SDH in this particular brain region. This work also suggests that the partial decrease in the activity of the respiratory chain complex II–III reported in HD patients may be sufficient to induce the selective striatal degeneration observed in this disorder.     

Excitatory Amino Acid Synthesis in Hypoxic Brain Slices: Does Alanine Act as a Substrate for Glutamate Production in Hypoxia?

Abstract: Excitatory amino acids are an important cause of cell death in the hypoxic and ischaemic brain. Neuronal glutamate stores are depleted rapidly in hypoxia, but alanine production rises under such conditions and has been suggested to be a potential precursor of glutamate. To test this hypothesis, we have investigated amino acid metabolism using ¹³C NMR with superfused guinea pig cortical slices subjected to varying degrees of hypoxia. During severe hypoxia, brain slices metabolising 5 m M [2-¹³C]pyruvate exported [2-¹³C]alanine into the superfusion fluid. The metabolic fate of alanine during normoxia and hypoxia was tested by superfusion of brain slices with 10 m M glucose and 2 m M [2-¹³C, ¹⁵N]alanine. Metabolism of exogenous alanine leads to the release of aspartate into the superfusion fluid. The pattern of labelling of aspartate indicated that it was synthesised via the glial-specific enzyme pyruvate carboxylase. ¹³C-labelled glutamate was produced with both normoxia and hypoxia, but concentrations were 30-fold lower than for labelled aspartate. Thus, although substantial amounts of glutamate are not synthesised from alanine in hypoxia, there is significant production of aspartate, which also may have deleterious effects as an excitatory amino acid.     

Changes in Hexokinase Isoenzymes in Regions of Rat Brain During Insulin-Induced Hypoglycemia

Activities of hexokinase isoenzymes were determined during insulin-induced hypoglycemia in soluble and total particulate fractions from three regions of rat brain. Type I hexokinase isoenzyme activity showed a small decrease in both soluble and particulate fractions from the cerebral hemispheres. In cerebellum and brain stem, however, Type I isoenzyme showed a decrease only in the soluble fraction. A significant increase was observed in hexokinase Type II isoenzyme from both the fractions, in all the three brain regions 1 h after insulin administration.