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.     

Quinolinic Acid Phosphoribosyltransferase in Rat Brain

Because of the possible participation of quinolinic acid in brain function and/or dysfunction, the characteristics of its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRTase; EC 2.4.2.19), were examined in rat brain tissue. For this purpose, a sensitive radiochemical assay method, based on the conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN), was developed. For brain QPRTase, the Mg2+ dependency, substrate specificity, and optimal assay conditions were virtually identical to those of the liver enzyme. Kinetic analyses of brain QPRTase revealed a Km of 3.17 +/- 0.30 microM for quinolinic acid and Km = 65.13 +/- 13.74 microM for the cosubstrate phosphoribosylpyrophosphate. The respective Vmax values were: 0.91 +/- 0.08 pmol NAMN/h/mg tissue for quinolinic acid and 11.65 +/- 1.55 fmol NAMN/h/mg tissue for phosphoribosylpyrophosphate. All kinetic parameters measured for the brain enzyme were significantly different from those determined for liver QPRTase, indicating structural differences or distinct regulatory processes for the brain and liver enzymes. Phthalic acid was a potent competitive inhibitor of brain QPRTase. Examination of the regional distribution of QPRTase in the rat CNS and retina indicated a greater than 20-fold difference between the area displaying the highest activity (olfactory bulb) and those of only moderate activity (frontal cortex, striatum, retina, hippo-campus). Enzyme activity was present at the earliest age tested, 2 days, and tended to increase in older animals. Brain QPRTase activity was preferentially located in the nerve-ending (synaptosomal) fraction. Enzyme activity was stable over extensive periods of storage at -80 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of indoleamine dioxygenase-1 deficiency and kynurenine pathway inhibition on murine cerebral malaria

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. In this study, two different approaches were used to examine the role of indoleamine 2,3-dioxygenase-1 (IDO-1) and its metabolites in the development of murine CM. Mice genetically deficient in IDO-1 were not protected against CM, but partial protection was observed in C57BL/6 mice treated with Ro 61-8048, an inhibitor of kynurenine-3-hydroxylase. This protection was associated with suppressed levels of picolinic acid (PA) within the brain, but not with changes in the levels of kynurenic acid (KA) or quinolinic acid (QA). These data suggest that although IDO-1 is not directly involved in the pathogenesis of CM in C57BL/6 mice, the production of the kynurenine pathway metabolite PA may contribute to the development of murine CM.     

Content of Quinolinic Acid and of Other Tryptophan Metabolites Increases in Brain Regions of Rats Used as Experimental Models of Hepatic Encephalopathy

The content of the tryptophan metabolites quinolinic acid (QUIN), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) was measured in various brain areas of rats bearing a portocaval anastomosis (PCA) for 4 weeks, using mass fragmentography or HPLC. In these animals, the content of the excitotoxic compound QUIN increased by 75% in the cortex and 125% in the cerebellum. The content of 5-HT increased by 27% in the brainstem. No changes occurred in other brain areas. On the other hand, the content of 5-HIAA increased by 66% in the cortex, 65% in the caudate, 64% in the hippocampus, 120% in the diencephalon, and 185% in the brainstem. Probenecid administration caused a larger increase of 5-HIAA accumulation in various brain areas of PCA-bearing rats than in those of sham-operated controls. The cortical content of QUIN and 5-HIAA increased after administration of ammonium acetate (7 mmol/kg), whereas an equimolar amount of sodium acetate was inactive. These results confirm that profound changes in the disposition of tryptophan occur in the brains of experimental animals used as models of hepatic encephalopathy. Furthermore, this study adds the excitotoxic compound QUIN to the list of molecules possibly involved in the pathogenesis of this brain disorder.     

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)     

Increased Cerebrospinal Fluid Quinolinic Acid, Kynurenic Acid, and L-Kynurenine in Acute Septicemia

Increases in brain quinolinic acid have been implicated in neurodegeneration and convulsions that may accompany infectious diseases. In three rhesus macaques (Macaca mulatta) with septicemia, both CSF and serum quinolinic acid concentrations were markedly elevated and were accompanied by increases in CSF kynurenic acid levels that were of a smaller magnitude. Elevated serum and CSF L-kynurenine concentrations also occurred and are consistent with activation of indoleamine-2,3-dioxygenase and increased substrate flux through the kynurenine pathway. Although it is probable that the marked increases in CSF quinolinic acid and kynurenic acid concentrations are reflected in the extracellular fluid space of brain, it remains to be determined whether the magnitude of such increases influences the activity of excitatory amino acid receptors in brain to produce excitotoxic pathology or noncytolytic disruption of functions mediated by excitatory amino acid receptors.     

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.     

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.     

Brain Quinolinic Acid in Huntington''s Disease

Concentrations of the endogenous neurotoxic tryptophan metabolite, quinolinic acid (QA), were measured in postmortem brain tissue obtained from patients with Huntington's disease (HD) and matched controls, using a gas chromatography/mass spectrometry method. There was no significant difference in either the putamen or the frontal cortex between the HD and control groups. These results do not support the hypothesis that increased QA is responsible for neuronal degeneration in HD.     

Evolution of Vertebrate Indoleamine 2,3-Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the same reaction, the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed among life-forms, being found not only in eukaryotes but also in bacteria. In contrast, IDO has been found only in mammals and yeast to date. However, recent genome and EST projects have identified IDO homologues in non-mammals and found an IDO paralogue that is expressed in mice. In this study, we cloned the frog and fish IDO homologues and the mouse IDO paralogue, and characterized their enzymatic properties using recombinants. The IDOs of lower vertebrates and the mouse IDO paralogue had IDO activity but had 500–1000 times higher K _m values and very low enzyme efficiency compared with mammalian IDOs. It appears that L-Trp is not a true substrate for these enzymes in vivo, although their actual function is unknown. On the phylogenetic tree, these low-activity IDOs, which we have named “proto-IDOs,” formed a cluster that was distinct from the mammalian IDO cluster. The IDO and proto-IDO genes are present tandemly on the chromosomes of mammals, including the marsupial opossum, whereas only the proto-IDO gene is observed in chicken and fish genomes. These results suggest that (mammalian) IDOs arose from proto-IDOs by gene duplication that occurred before the divergence of marsupial and eutherian (placental) mammals in mammalian evolutionary history.     

Brain and Plasma Quinolinic Acid in Profound Insulin-Induced Hypoglycemia

Profound insulin-induced hypoglycemia is associated with early-onset neuronal damage that resembles excitotoxic lesions and is attenuated in severity by antagonists of N-methyl-D-aspartate receptors. Hypoglycemia increases L-tryptophan concentrations in brain and could increase the concentration of the L-tryptophan metabolite quinolinic acid (QUIN), an agonist of N-methyl-D-aspartate receptors and an excitotoxin in brain. Therefore, we investigated the effects of 40 min of profound hypoglycemia (isoelectric EEG) and 1-2 h of normoglycemic recovery on the concentrations of QUIN in brain tissue, brain extracellular fluid, and plasma in male Wistar rats. Plasma QUIN increased 6.5-fold by the time of isoelectricity (2 h after insulin administration). Regional brain QUIN concentrations increased two- to threefold during hypoglycemia and increased a further two- to threefold during recovery. However, no change in extracellular fluid QUIN concentrations in hippocampus occurred during hypoglycemia or recovery as measured using in vivo microdialysis. Therefore, the increases in brain tissue QUIN concentrations may reflect elevations of QUIN in the intracellular space or be secondary to the increases in QUIN in the vascular compartment in brain per se. L-Tryptophan concentrations increased more than twofold during recovery only. Serotonin decreased greater than 50% throughout the brain during hypoglycemia, while 5-hydroxyindoleacetic acid concentrations increased more than twofold during hypoglycemia and recovery. In striatum, dopamine was decreased 75% during hypoglycemia but returned to control values during recovery, while striatal 3,4-dihydroxyphenylacetic acid and homovanillic acid were increased more than twofold during both hypoglycemia and recovery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indoleamine 2,3-dioxygenase activity and l-tryptophan transport in human breast cancer cells

The activity and expression of indoleamine 2,3-dioxygenase together with l-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-γ (1000 units/ml). Accordingly, l-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-γ. 1-Methyl-dl-tryptophan (1 mM) inhibited interferon-γ induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-γ. l-Tryptophan transport into MDA-MB-231 cells was via a Na⁺-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-d,l-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, l-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.     

Kynurenine Pathway Measurements in Huntington''s Disease Striatum: Evidence for Reduced Formation of Kynurenic Acid

Recent evidence suggests that there may be overactivation of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16-sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p less than 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.     

Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.     

Increase in the Content of Quinolinic Acid in Cerebrospinal Fluid and Frontal Cortex of Patients with Hepatic Failure

Abstract: Quinolinic acid (QUIN), an excitotoxic tryptophan metabolite, has been identified and measured in human cerebrospinal fluid (CSF) using a mass-fragmentographic method. Furthermore, its content has been evaluated in frontal cortex obtained at autopsy from the cadavers of patients who died after hepatic coma. During the coma, the concentration of QUIN in the CSF was 152 ± 38 pmol ml^-1. In contrast, the concentration in control patients affected by different pathologies was 22 ± 7 pmol ml^-1. In the frontal cortex of patients who died after episodes of hepatic encephalopathy, the content of QUIN was three times higher than in controls (2.6 ± 0.6 versus 0.80 ± 0.08 nmol/g wet weight). As a result of these investigations we are now able to extend our previous observations on the increase of QUIN in the brains of rats used as experimental models of hepatic encephalopathy to man. QUIN should therefore be added to the list of compounds possibly involved in the pathogenesis and symptomatology of brain disorders associated with liver failure.     

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.     

Quinolinic Acid-induced Seizures Stimulate Glutamate Uptake into Synaptic Vesicles from Rat Brain: Effects Prevented by Guanine-based Purines

Glutamate uptake into synaptic vesicles is a vital step for glutamatergic neurotransmission. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. Guanine-based purines (GBPs) guanosine 5′-monophosphate (GMP and guanosine) have been shown to exert anticonvulsant effects against QA-induced seizures. The aims of this study were to investigate the effects of in vivo administration of several convulsant agents on glutamate uptake into synaptic vesicles and investigate the role of MK-801, guanosine or GMP (anticonvulsants) on glutamate uptake into synaptic vesicles from rats presenting QA-induced seizures. Animals were treated with vehicle (saline 0.9%), QA 239.2 nmoles, kainate 30 mg/kg, picrotoxin 6 mg/kg, PTZ (pentylenetetrazole) 60 mg/kg, caffeine 150 mg/kg or MES (maximal transcorneal electroshock) 80 mA. All convulsant agents induced seizures in 80–100% of animals, but only QA stimulated glutamate uptake into synaptic vesicle. Guanosine or GMP prevented seizures induced by QA (up to 52% of protection), an effect similar to the NMDA antagonist MK-801 (60% of protection). Both GBPs and MK-801 prevented QA-induced glutamate uptake stimulation. This study provided additional evidence on the role of QA and GBPs on glutamatergic system in rat brain, and point to new perspectives on seizures treatment.     

Quinolinic Acid Stimulates Somatostatin Gene Expression in Cultured Rat Cortical Neurons

Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphonovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p less than 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kynurenine Disposition in Blood and Brain of Mice: Effects of Selective Inhibitors of Kynurenine Hydroxylase and of Kynureninase

Abstract: To study the regulation of the synthesis of quinolinic and kynurenic acids in vivo, we evaluated (a) the metabolism of administered kynurenine by measuring the content of its main metabolites 3-hydroxykynurenine, anthranilic acid, and 3-hydroxyanthranilic acid in blood and brain of mice; (b) the effects of ( m -nitrobenzoyl)alanine, a selective inhibitor of kynurenine hydroxylase and of ( o -methoxybenzoyl)alanine, a selective inhibitor of kynureninase, on this metabolism; and (c) the effects of ( o -methoxybenzoyl)alanine on liver kynureninase and 3-hydroxykynureninase activity. The conclusions drawn from these experiments are (a) the disposition of administered kynurenine preferentially occurs through hydroxylation in brain and through hydrolysis in peripheral tissues; (b) ( m -nitrobenzoyl)alanine, the inhibitor of kynurenine hydroxylase, causes the expected changes in brain kynurenine metabolism, such as a decrease of 3-hydroxykynurenine, and an increase of kynurenic acid; and (c) ( o -methoxybenzoyl)alanine, the kynureninase inhibitor, increases brain concentration of the cytotoxic compound 3-hydroxykynurenine, and unexpectedly does not reduce brain concentration of 3-hydroxyanthranilic acid, the direct precursor of quinolinic acid. Taken together, the experiments suggest that the systemic administration of a kynurenine hydroxylase inhibitor is a rational approach to increase the brain content of kynurenate and to decrease that of cytotoxic kynurenine metabolites, such as 3-hydroxykynurenine and quinolinic acid.