Systemic Lipopolysaccharide and Pokeweed Mitogen Increase Quinolinic Acid Content of Mouse Cerebral Cortex

The effects of lipopolysaccharide and pokeweed mitogen on brain L-tryptophan and quinolinic acid (QUIN) concentrations were investigated in C57BL/6NCR mice. Twenty-four hours after an intraperitoneal injection of lipopolysaccharide (5 micrograms from Salmonella abortus equii) or pokeweed mitogen (500 micrograms), cortical QUIN concentrations were increased by 81 +/- 6% and 182 +/- 15%, respectively. Plasma QUIN was increased 175 +/- 7% of control in pokeweed-mitogen treated mice only. Brain L-tryptophan concentrations were increased, whereas plasma L-tryptophan concentrations were decreased. The consequences of increased QUIN concentrations during endotoxin and mitogen exposure remain to be determined.     

4-chloro-3-hydroxyanthranilate inhibits brain 3-hydroxyanthranate oxidase

Quinolinic acid is synthesized from 3-hydroxyanthranilic acid via 3-hydroxyanthranilic acid oxidase. In liver, 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase. To determine whether 4-chloro-3-hydroxyanthranilic acid also inhibits 3-hydroxyanthranilic acid oxidase in brain, 3-hydroxyanthranilic acid was injected into the cisterna magna of rats either with or without 4-chloro-3 hydroxyanthranilic acid. 3-Hydroxyanthranilic acid increased quinolinic acid concentrations throughout the brain. 4-Chloro-3-hydroxyanthranilic acid attenuated increases in brain quinolinic acid. These observations indicate that 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase in brain.Quinolinic acid is a well established systemic metabolite of l-tryptophan which has been shown to be present in brain (Wolfensberger et al., 1983; Heyes and Markey, 1988a). QUIN has proved to be a convulsant (Lapin, 1982), neurotoxin (Schwarcz et al., 1983) and agonist of N-methyl-D-aspartate receptors (Perkins and Stone, 1983) when injected directly into the central nervous system of experimental animals. Therefore increased concentrations of QUIN in brain may have neoropathologic consequences. l-Tryptophan is converted to QUIN via the kynurenine pathway. The precursor of QUIN, 2-amino-3-carboxymuconic semialdehyde is synthesized from 3-hydroxyanthranilic acid (3-HAA) by the action of 3-hydroxyanthranilic acid oxidase (3-HAA/OX) in liver and brain (Foster et al., 1986; Okuno et al., 1987). QUIN is then formed from 2-amino-3-carboxymuconic semialdehyde by a spontaneous, non-enzymatic reaction. In liver, 3-HAA/OX is inhibited by 4-chloro-3-hydroxyantranilic acid (CL-HAA; Parli et al., 1980). In the present study, rats were given an intracisternal injection of 3-HAA and the resultant increases in regional brain QUIN concentrations quantified by gas chromatography/mass spectrometry (Heyes and Markey, 1988a,b). To determine whether CL-HAA inhibit 3-hydroxyanthranilic acid oxidase in brain, CL-HAA was co-administered with 3-HAA to see whether increases in QUIN were attenuated.     

Quinolinic Acid In Vivo Synthesis Rates, Extracellular Concentrations, and Intercompartmental Distributions in Normal and Immune-Activated Brain as Determined by Multiple-Isotope Microdialysis

Abstract: Quinolinic acid (QUIN) kills neurons by activation of NMDA receptors that are accessed via the extracellular fluid (ECF). In vivo microdialysis was employed to quantify the dynamics of ECF QUIN levels. [¹³C₇]QUIN was perfused through the probe for in vivo calibration to accurately quantify ECF QUIN concentrations. Osmotic pumps infused [²H₃]QUIN subcutaneously to quantify blood contributions to ECF and tissue levels. Local QUIN production rates and influx and efflux rates across the blood-brain barrier were calculated from the extraction fraction of [¹³C₇]QUIN, probe geometry, tissue diffusion coefficients, the extracellular volume fraction, and [²H₃]QUIN/QUIN ratios in blood and dialysates. In normal brain, 85% of ECF QUIN levels (110 n M ) originated from blood, whereas 59% of tissue homogenate QUIN (130 pmol/g) originated from local de novo synthesis. During systemic immune activation (intraperitoneal injection of endotoxin), blood QUIN levels increased (10.2-fold) and caused a rise in homogenate (10.8-fold) and ECF (18.5-fold) QUIN levels with an increase in the proportions of QUIN derived from blood. During CNS inflammation (local infusion of endotoxin), increases in brain homogenate (246-fold) and ECF (66-fold) QUIN levels occurred because of an increase in local synthesis rate (146-fold) and a reduction in efflux/influx ratio (by 53%). These results demonstrate that brain homogenate measures are a reflection of ECF concentrations, although there are quantitative differences in the values obtained. The mechanisms that maintain ECF QUIN levels at low values cannot do so when there are large increases in local brain synthesis or when there are large elevations in blood QUIN concentrations.     

Mechanism of Delayed Increases in Kynurenine Pathway Metabolism in Damaged Brain Regions Following Transient Cerebral Ischemia

Abstract: Delayed increases in the levels of an endogenous N-methyl-D-aspartate receptor agonist, quinolinic acid (QUIN), have been demonstrated following transient ischemia in the gerbil and were postulated to be secondary to induction of indoleamine-2,3-dioxygenase (IDO) and other enzymes of the L-tryptophan-kynurenine pathway. In the present study, proportional increases in IDO activity and QUIN concentrations were found 4 days after 10 min of cerebral ischemia, with both responses in hippocampus > striatum > cerebral cortex > thalamus. These increases paralleled the severity of local brain injury and inflammation. IDO activity and QUIN concentrations were unchanged in the cerebellum of postischemic gerbils, which is consistent with the preservation of blood flow and resultant absence of pathology in this region. Blood QUIN and L-kynurenine concentrations were not affected by ischemia. Brain tissue QUIN levels at 4 days postischemia exceeded blood concentrations, minimizing a role for breakdown of the blood–brain barrier. Marked increases in the activity of kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase were also detected in hippocampus but not in cerebellum on day 4 of recirculation. In vivo synthesis of [¹³C₆]QUIN was demonstrated, using mass spectrometry, in hippocampus but not in cerebellum of 4-day postischemic animals 1 h after intracisternal administration of L-[¹³C₆]tryptophan. However, accumulation of QUIN was demonstrated in both cerebellum and hippocampus of control gerbils following an intracisternal injection of 3-hydroxyanthranilic acid, which verifies the availability of precursor to both regions when administered intracisternally. Notably, although IDO activity and QUIN concentrations were unchanged in the cerebellum of ischemic gerbils, both IDO activity and QUIN content were increased in cerebellum to approximately the same degree as in hippocampus, striatum, cerebral cortex, and thalamus 24 h after immune stimulation by systemic pokeweed mitogen administration, demonstrating that the cerebellum can increase IDO activity and QUIN content in response to immune activation. No changes in kynurenic acid concentrations in either hippocampus, cerebellum, or cerebrospinal fluid were observed in the postischemic gerbils compared with controls, in accordance with the unaffected activity of kynurenine aminotransferase activity. Collectively, these results support roles for IDO, kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase in accelerating the conversion of L-tryptophan and other substrates to QUIN in damaged brain regions following transient cerebral ischemia. Immunocytochemical results demonstrated the presence of macrophage infiltrates in hippocampus and other brain regions that parallel the extent of these biochemical changes. We hypothesize that increased kynurenine pathway metabolism after ischemia reflects the presence of macrophages and other reactive cell populations at sites of brain injury.     

Quantification of quinolinic acid in rat brain, whole blood, and plasma by gas chromatography and negative chemical ionization mass spectrometry: effects of systemic L-tryptophan administration on brain and blood quinolinic acid concentrations

A gas chromatography/mass spectrometry assay is described to quantify the endogenous neurotoxin quinolinic acid (QUIN) in brain, whole blood, and plasma. High specificity and high sensitivity were obtained by using negative chemical ionization and accuracy was achieved by using [18O]QUIN as internal standard. Neutralized perchloric acid extracts were washed with chloroform, applied to Dowex 1 x 8 (formate form), and eluted with 6 M formic acid. After lyophilization, QUIN and [18O]QUIN were esterified with hexafluoroisopropanol (to mass 467 and 471, respectively) using trifluoroacetylimidazole as catalyst. The esters were extracted into heptane and injected onto a gas chromatograph, DB-5 capillary column. QUIN and [18O]QUIN were quantified by selected ion monitoring of QUIN-specific anion currents from the molecular anions (m/z 467 and 471, respectively) and a specific anion fragment (m/z 316 from QUIN and m/z 320 from [18O]QUIN). Minimum sensitivity was 3 fmol, intraassay variability was 3.2%, and interassay variability was 8.1% QUIN concentrations in frontal cortex from over 200 rats ranged from 20 to 180 fmol/mg wet wt. Two hours after systemic L-tryptophan (L-Trp; 0.370 mmol/kg) administration, QUIN increased in whole blood 134.8-fold and in plasma, 74.3-fold. In frontal cortex, increases in QUIN (22.6-fold, corrected for QUIN in blood) exceeded increases in cortical L-Trp (2.54-fold), 5-HT (1.35-fold), and 5-HIAA (1.74-fold). These studies demonstrate that QUIN is present in brain and is sensitive to the availability of systemic L-Trp.     

Changes in quinolinic acid production and its related enzymes following D-galactosamine and lipopolysaccharide-induced hepatic injury

Increases in quinolinic acid (QUIN), a neurotoxic L-tryptophan metabolite, have been observed in human serum and cerebrospinal fluid and in animal models of severe hepatic injury. The aim of this study was to evaluate the changes in QUIN accumulation and its related enzymes after acute hepatic injury induced by D-galactosamine and endotoxin. Gerbils were given an intraperitoneal injection of pyrogen-free saline alone as control, lipopolysaccharide (LPS) alone (150 ng/kg), D-galactosamine alone (500 mg/kg) or a combination of D-galactosamine with LPS. Concentrations of QUIN, its related metabolites, and related enzyme activities were determined. D-Galactosamine treatment significantly decreased activities of hepatic aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) resulting in increased QUIN concentrations in serum and tissues. The magnitude of QUIN responses was markedly increased by endotoxin due to the increased availability of L-kynurenine, a rate-limiting substrate for QUIN synthesis. Further, infiltration of monocytes/macrophages, which is a possible major source of QUIN production in the liver, was shown by immunohistochemistry after hepatic injury induced by D-galactosamine and endotoxin. Increased serum QUIN concentrations are probably due to the increased substrate availability and the decreased activity of aminocarboxymuconate-semialdehyde decarboxylase in the liver, accompanying the increased monocyte/macrophage infiltration into the liver after hepatic injury.     

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.     

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.     

In Vivo Brain Dialysis of Amino Acids and Simultaneous EEG Measurements Following Intrahippocampal Quinolinic Acid Injection: Evidence for a Dissociation Between Neurochemical Changes and Seizures

The extracellular content of taurine, glutamate, glutamine, and glycine was measured by the novel method of brain dialysis in the acute phases following an intrahippocampal injection of the excitotoxic convulsant brain metabolite quinolinic acid (QUIN). Using bilaterally implanted depth electrodes physically combined with hollow fibers for dialysis, it was possible to collect continuously brain perfusates while simultaneously monitoring brain activity in the unanesthetized rat. In separate animals, hippocampal amino acid tissue levels were measured 2 h after an intracerebral injection of a convulsant dose (156 nmol) of QUIN. When compared with those in animals receiving the nonconvulsant decarboxylation product of QUIN, nicotinic acid, no differences in tissue levels were detected. In contrast, the same dose of QUIN caused a selective increase (2.24-fold) in taurine levels in perfusates from the injected hippocampus. These changes were apparent prior to the onset of electrographic seizures and did not occur in the contralateral hippocampus where seizure activity was equally severe. Thus, increases in extracellular taurine, triggered by the presence of QUIN in the hippocampus, may reflect a selective tissue response to the neurotoxic (rather than the convulsant) effects of this excitotoxin.     

Effect of Tryptophan on Extracellular Concentrations of Tryptophan and 5-Hydroxyindoleacetic Acid in the Striatum and Cerebellum

The effects of L-tryptophan (50 mg/kg i.p.) on extracellular concentrations of tryptophan and the 5-hydroxytryptamine (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in the rat striatum and cerebellum, regions with rich and poor 5-HT innervation, respectively. Determinations were on perfusates from dialysis probes in the brains of conscious, freely moving rats. The pharmacokinetic profiles of dialysate tryptophan after tryptophan load (peak concentration, time to peak concentration, area under curve, and half-life) in the two regions did not differ significantly. The dialysate 5-HIAA concentration in the striatum rose two- to threefold after the administration of tryptophan. Therefore, as 5-HIAA was undetectable in the cerebellum either before or after the administration of tryptophan, the increase of 5-HIAA in the striatum is unlikely to depend appreciably on its production within the cerebral vasculature or outside the brain or on its entering the striatum through a blood-brain barrier damaged by placement of the dialysis probe. Overall, the findings strengthen previous evidence that extracellular 5-HIAA concentrations determined by cerebral dialysis are a valid measure of the metabolism of 5-HT of brain neuronal origin.     

Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain

We describe here a gas chromatography-tandem mass spectrometry (GC/MS/MS) method for the sensitive and concurrent determination of extracellular tryptophan and the kynurenine pathway metabolites kynurenine, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN) in rat brain. This metabolic cascade is increasingly linked to the pathophysiology of several neurological and psychiatric diseases. Methodological refinements, including optimization of MS conditions and the addition of deuterated standards, resulted in assay linearity to the low nanomolar range. Measured in samples obtained by striatal microdialysis in vivo, basal levels of tryptophan, kynurenine, and QUIN were 415, 89, and 8 nM, respectively, but 3-HK levels were below the limit of detection (<2 nM). Systemic injection of kynurenine (100 mg/kg, i.p.) did not affect extracellular tryptophan but produced detectable levels of extracellular 3-HK (peak after 2-3 h: ~50 nM) and raised extracellular QUIN levels (peak after 2h: ~105 nM). The effect of this treatment on QUIN, but not on 3-HK, was potentiated in the N-methyl-D-aspartate (NMDA)-lesioned striatum. Our results indicate that the novel methodology, which allowed the measurement of extracellular kynurenine and 3-HK in the brain in vivo, will facilitate studies of brain kynurenines and of the interplay between peripheral and central kynurenine pathway functions under physiological and pathological conditions.     

Quinolinic acid, the inescapable neurotoxin

Over the last two decades, evidence for the involvement of quinolinic acid (QUIN) in neuroinflammatory diseases has been exponentially increasing. Within the brain, QUIN is produced and released by infiltrating macrophages and activated microglia, the very cells that are prominent during neuroinflammation. QUIN acts as an agonist of the N-methyl-D-aspartate receptor and as such is considered to be a brain endogenous excitotoxin. Since the discovery of the excitotoxic activity of QUIN in the early 1980s, several other cytotoxic mechanisms have been identified. We know today that QUIN acts as a neurotoxin, gliotoxin, proinflammatory mediator, pro-oxidant molecule and can alter the integrity and cohesion of the blood-brain barrier. This paper aims to review some of the most recent findings about the effects of QUIN and its mode of action.     

The neurotoxic actions of quinolinic acid in the central nervous system

Excitotoxins such as kainic acid, ibotenic acid, and quinolinic acid are a group of molecules structurally related to glutamate or aspartate. They are capable of exciting neurons and producing axon sparing neuronal degeneration. Quinolinic acid (QUIN), an endogenous metabolite of the amino acid, tryptophan, has been detected in brain and its concentration increases with age. The content of QUIN in the brain and the activity of the enzymes involved in its synthesis and metabolism show a regional distribution. The neuroexcitatory action of QUIN is antagonized by magnesium (Mg2+) and the aminophosphonates, proposed N-methyl-D-aspartate (NMDA) receptor antagonists, suggesting that QUIN acts at the Mg2+ -sensitive NMDA receptor. Like its excitatory effects, QUIN's neurotoxic actions in the striatum are antagonized by the aminophosphonates. This suggests that QUIN neurotoxicity involves the NMDA receptor and (or) another receptor sensitive to the aminophosphonates. The neuroexcitatory and neurotoxic effects of QUIN are antagonized by kynurenic acid (KYN), another metabolite of tryptophan. QUIN toxicity is dependent on excitatory amino acid afferents and shows a regional variation in the brain. Local injection of QUIN into the nucleus basalis magnocellularis (NBM) results in a dose-dependent reduction in cortical cholinergic markers including the evoked release of acetylcholine. A significant reduction in cortical cholinergic function is maintained over a 3-month period. Coinjection of an equimolar ratio of QUIN and KYN into the NBM results in complete protection against QUIN-induced neurodegeneration and decreases in cortical cholinergic markers. In contrast, focal injections of QUIN into the frontoparietal cortex do not alter cortical cholinergic function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper blocks quinolinic acid neurotoxicity in rats: contribution of antioxidant systems

Reactive oxygen species and oxidative stress are involved in quinolinic acid (QUIN)-induced neurotoxicity. QUIN, a N-methyl-D-aspartate receptor (NMDAr) agonist and prooxidant molecule, produces NMDAr overactivation, excitotoxic events, and direct reactive oxygen species formation. Copper is an essential metal exhibiting both modulatory effects on neuronal excitatory activity and antioxidant properties. To investigate whether this metal is able to counteract the neurotoxic and oxidative actions of QUIN, we administered copper (as CuSO(4)) intraperitoneally to rats (2.5, 5.0, 7.5, and 10.0 mg/kg) 30 min before the striatal infusion of 1 microliter of QUIN (240 nmol). A 5.0 mg/kg CuSO(4) dose significantly increased the copper content in the striatum, reduced the neurotoxicity measured both as circling behavior and striatal gamma-aminobutyric acid (GABA) depletion, and blocked the oxidative injury evaluated as striatal lipid peroxidation (LP). In addition, copper reduced the QUIN-induced decreased striatal activity of Cu,Zn-dependent superoxide dismutase, and increased the ferroxidase activity of ceruloplasmin in cerebrospinal fluid from QUIN-treated rats. However, copper also produced significant increases of plasma lactate dehydrogenase activity and mortality at the highest doses employed (7.5 and 10.0 mg/kg). These results show that at low doses, copper exerts a protective effect on in vivo QUIN neurotoxicity.     

Evidence for a serotonin transporter deficit in experimental acute liver failure

It has been suggested that alterations of serotonin transport may be implicated in the pathogenesis of the neuropsychiatric symptoms encountered in acute liver failure. In order to address this issue, microdialysate concentrations of serotonin, its precursor L-tryptophan and metabolite 5-hydroxyindoleacetic acid (5-HIAA) as well as brain regional distribution of serotonin transporter ([3H]-citalopram) sites were measured in rats with acute liver failure resulting from hepatic devascularization. A significant loss of [3H]-citalopram sites was observed in dorsal Raphe nucleus, in frontal and frontoparietal cortices as well as in substantia nigra of rats with severe encephalopathy resulting from acute liver failure. In frontal cortex, this loss of transporter binding sites was accompanied by significant increases of L-tryptophan, serotonin and 5-HIAA concentrations in extracellular fluid. Pharmacological manipulation of the brain serotonin system could afford a novel therapeutic approach to the prevention of the neuropsychiatric symptoms characteristic of acute liver failure in humans.     

Quantification of Local De Novo Synthesis Versus Blood Contributions to Quinolinic Acid Concentrations in Brain and Systemic Tissues

Abstract: The source of the neurotoxin quinolinic acid (QUIN) in brain and systemic tissues under normal and pathologic circumstances reflects either de novo synthesis from l -tryptophan and other precursors, or entry of QUIN itself from the blood. To quantify the relative contributions of blood- versus tissue-derived QUIN, [¹³C₇]QUIN was infused subcutaneously via osmotic pumps (0.55 µl/h, 30 mM) in gerbils, and the fraction of QUIN in tissue (Ti; measured in tissue homogenates) derived from blood (BI; measured in serum) was calculated by the formula ([¹³C₇]QUIN_Ti/QUIN_Ti)/([¹³C₇]QUIN_Bl/QUIN_Bl). In controls, blood QUIN contributed 38–49% of QUIN in brain, 70% in CSF, between 40 and 70% in kidney, heart, and skeletal muscle, but <5% in spleen, lung, liver, and intestine. Systemic endotoxin (450 µg/kg) increased blood, brain, CSF, and systemic tissue QUIN levels. Notably, the relative proportion of QUIN derived from blood in brain, spleen, lung, and intestine was unchanged by endotoxin, but increased in kidney, heart, and skeletal muscle. In contrast, cerebral ischemic injury (10 min of bilateral carotid artery occlusion) increased regional brain QUIN concentrations at 4 days post ischemia, with a proportional increase in the amount of QUIN derived from de novo synthesis by brain tissue. In the blood and systemic tissues of postischemic gerbils, there were no changes in systemic tissue or blood QUIN levels, or changes in the relative proportions of blood- versus systemic tissue-derived QUIN. These results establish that the brain normally synthesizes QUIN, that the blood is a significant source of QUIN in controls and during acute systemic immune activation, and that the rate of QUIN formation by brain tissue increases in conditions of brain and systemic immune activation.     

Cyclosporin A prevents calpain activation despite increased intracellular calcium concentrations,as well as translocation of apoptosis-inducing factor,cytochrome c and caspase-3 activation in neurons exposed to transient hypoglycemia

Blockade of mitochondrial permeability transition protects against hypoglycemic brain damage. To study the mechanisms downstream from mitochondria that may cause neuronal death, we investigated the effects of cyclosporin A on subcellular localization of apoptosis-inducing factor and cytochrome c, activation of the cysteine proteases calpain and caspase-3, as well as its effect on brain extracellular calcium concentrations. Redistribution of cytochrome c occurred at 30 min of iso-electricity, whereas translocation of apoptosis-inducing factor to nuclei occurred at 30 min of recovery following 30 min of iso-electricity. Active caspase-3 and calpain-induced fodrin breakdown products were barely detectable in the dentate gyrus and CA1 region of the hippocampus of rat brain exposed to 30 or 60 min of insulin-induced hypoglycemia. However, 30 min or 3 h after recovery of blood glucose levels, fodrin breakdown products and active caspase-3 markedly increased, concomitant with a twofold increase in caspase-3-like enzymatic activity. When rats were treated with neuroprotective doses of cyclosporin A, but not with FK 506, the redistribution of apoptosis-inducing factor and cytochrome c was reduced and fodrin breakdown products and active caspase-3 immuno-reactivity was diminished whereas the extracellular calcium concentration was unaffected. We conclude that hypoglycemia leads to mitochondrial permeability transition which, upon recovery of energy metabolism, mediates the activation of caspase-3 and calpains, promoting cell death.     

Role of amino acid metabolism in an air-breathing catfish,Clarias batrachus in response to exposure to a high concentration of exogenous ammonia

The air-breathing ureogenic walking catfish (Clarias batrachus) faces various environmental constraints throughout the year leading to the problem of accumulation of toxic ammonia. In the present study, the possible role of conversion of accumulated ammonia to various non-essential free amino acids (FAAs) was tested in this fish under hyper-ammonia stress caused by exposing the fish at 25 mM NH(4)Cl for 7 days. Significant accumulation of ammonia of approximately two- to threefold was observed in different tissues (except in the brain), which was accompanied with the significant accumulation of non-essential FAAs in the NH(4)Cl-exposed fish. There was approximately two- to threefold increase of non-essential FAAs in different tissues and in the plasma of the NH(4)Cl-exposed fish compared to the control fish after 7 days of exposure, which was mainly attributable to the increase of Asp, Ala, Gly, Glu, Gln and taurine (Tau) concentrations in general, with certain tissue-specific variations. This was also accompanied with significant increase of activity of certain amino acid metabolism-related enzymes such as the glutamine synthetase (approx. two- to threefold), glutamate dehydrogenase (ammonia utilizing direction) (approx. twofold), aspartate and alanine aminotransaminases (approx. twofold) mainly in the liver, kidney and muscle of the NH(4)Cl-exposed fish. Thus, it appears that the walking catfish has the capacity of active conversion of accumulated ammonia to non-essential FAAs under condition of high concentrations of external ammonia. However, the increase of urea excretion rate due to active conversion of ammonia to urea via the induced urea cycle appears to be quantitatively much more important pathway than the increase of tissue levels of FAAs in dealing with a severe ammonia load.     

Age- and Sex-Dependent Laterality of Rat Hippocampal Cholinergic System in Relation to Animal Models of Neurodevelopmental and Neurodegenerative Disorders

Studies suggest age- and sex-dependent structural and functional patterns of human cerebral lateralization underlie hemisphere specialization and its alterations in schizophrenia. Recent works report sexual dimorphism of neurons in the hippocampal formation and specialization of hemispheres in rats. Our experiments indicate for the first time functional lateralization of the high-affinity choline uptake (HACU) system directly associated with a synthesis of acetylcholine in the hippocampus of Wistar rats. The markedly increased HACU activity was found in the left compared to the right hippocampus of adult male but not female animals. Lineweaver-Burk plot analysis revealed a statistically significant increase of Vmax in the left hippocampus of 14-day-old when compared to 7-day-old males. It appears that laterality of HACU occurs during late postnatal maturation, and its degree is markedly enhanced after puberty and attenuated during aging. Quinolinic acid (QUIN), an endogenous agonist of N-methyl-D-aspartate type glutamate receptors, was used in this study to evaluate the neurodevelopmental hypothesis of schizophrenia. It is known that elevated levels of QUIN accompany viral infections, increasing the risk of developing schizophrenia. Bilateral intracerebroventricular application of QUIN (250 nmoles/ventricle) to pups aged 12 days significantly impaired the cholinergic hippocampal system of adolescent male and female rats and reversed lateralization of male HACU. Morphological analysis indicated marked changes in brain lesion sizes (extensive 24 h and moderate 38 days after the operation). Asymmetry of lesions was observed in the majority of cases, but the left hemisphere was not generally more vulnerable to QUIN effects than the right side. Moreover, no lateral differences were found between lesioned hippocampi in the specific binding of [3H]hemicholinium-3 (10%-15% loss of binding sites when compared to sham-operated animals). In summary, our results indicate a symmetrical drop in the number of choline carriers of lesioned male rats but a asymmetrical decrease in the activity of remaing carriers, suggesting defects in processes of sexual brain differentiation, leading under normal conditions to the higher activity of carriers in the left hippocampus. The data demonstrate viral infection-mediated alterations in normal patterns of brain asymmetry and are discussed in relation to animal models of neurodevelopmental and neurodegenerative diseases.     

Extracellular Acidic Sulfur-Containing Amino Acids and γ-Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin-Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.