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.     

Poliovirus induces indoleamine-2,3-dioxygenase and quinolinic acid synthesis in macaque brain.

Accumulation of the neurotoxin quinolinic acid within the brain occurs in a broad spectrum of patients with inflammatory neurologic disease and may be of neuropathologic significance. The production of quinolinic acid was postulated to reflect local induction of indoleamine 2,3-dioxygenase by cytokines in reactive cells and inflammatory cell infiltrates within the central nervous system. To test this hypothesis, macaques received an intraspinal injection of poliovirus as a model of localized inflammatory neurologic disease. Seventeen days later, spinal cord indoleamine 2,3-dioxygenase activity and quinolinic acid concentrations in spinal cord and cerebrospinal fluid were both increased in proportion to the degree of inflammatory responses and neurologic damage in the spinal cord, as well as the severity of motor paralysis. The absolute concentrations of quinolinic acid achieved in spinal cord and cerebrospinal fluid exceeded levels reported to kill spinal cord neurons in vitro. Smaller increases in indoleamine 2,3-dioxygenase activity and quinolinic acid concentrations also occurred in parietal cortex, a poliovirus target area. In frontal cortex, which is not a target for poliovirus, indoleamine 2,3-dioxygenase was not affected. A monoclonal antibody to human indoleamine 2,3-dioxygenase was used to visualize indoleamine 2,3-dioxygenase predominantly in grey matter of poliovirus-infected spinal cord, in conjunction with local inflammatory lesions. Macrophage/monocytes in vitro synthesized [13C6]quinolinic acid from [13C6]L-tryptophan, particularly when stimulated by interferon-gamma. Spinal cord slices from poliovirus-inoculated macaques in vitro also converted [13C6]L-tryptophan to [13C6]quinolinic acid. We conclude that local synthesis of quinolinic acid from L-tryptophan within the central nervous system follows the induction of indoleamine-2,3-dioxygenase, particularly within macrophage/microglia. In view of this link between immune stimulation and the synthesis of neurotoxic amounts of quinolinic acid, we propose that attenuation of local inflammation, strategies to reduce the synthesis of neuroactive kynurenine pathway metabolites, or drugs that interfere with the neurotoxicity of quinolinic acid offer new approaches to therapy in inflammatory neurologic disease.     

The anti-inflammatory effects of quinolinic acid in the rat

Quinolinic acid (QUIN) levels are elevated in patients and animals suffering from chronic infectious diseases. In the present study, male Sprague-Dawley rats were used to test the anti-inflammatory effects of QUIN using the carrageenan (CGN)-induced paw edema assay and the CGN sponge assay. Results of these studies indicate that QUIN (30, 100 or 300 mg/kg i.p.) caused a reduction of carrageenan-induced inflammation by as much as 80% at the highest dose. Moreover, QUIN reduced exudate volume and inhibited leukocyte migration in the sponge granuloma assay. In another experiment, the anti-inflammatory activity of QUIN was eliminated in adrenalectomized rats. QUIN did not reduce edema caused by arachidonic acid, bradykinin or compound 48/80. Neither morphine nor naloxone altered the anti-inflammatory activity of QUIN. These results may suggest that QUIN exerts its anti-inflammatory activity through a direct action on neutrophils or vascular permeability.     

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)     

Cholesteryl nitrolinoleate,a nitrated lipid present in human blood plasma and lipoproteins

Nitric oxide (*NO) and *NO-derived reactive species (e.g., peroxynitrite anion, nitrogen dioxide radical) react with lipids containing unsaturated fatty acids to generate nitrated species. In the present work, we synthesized, characterized, and detected a nitrated derivative of cholesteryl linoleate (Ch18:2) in human blood plasma and lipoproteins using a high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry method. It was synthesized by a reaction of Ch18:2 with nitronium tetrafluoroborate, yielding a species with m/z 711, which is characteristic of the cholesteryl nitrolinoleate (Ch18:2NO2) ammonium adduct. The presence of the nitro group was confirmed by using [15N]nitrite, which gave a product with m/z 712, with the same chromatographic and spectrometric characteristics of those of m/z 711. Furthermore, a C-NO2 structure was also demonstrated in Ch18:2NO2 by infrared analysis (Vmax 1549, 1374 cm-1). A stable product with m/z of 711, showing the same chromatographic characteristics and fragmentation pattern as those of synthesized standard, was found in human blood plasma and lipoproteins of normolipidemic subjects. The presence of this novel nitrogen-containing lipid product in human plasma and lipoproteins could represent a potential indicator of the oxidative/nitrative roles that *NO or its metabolites play during in vivo lipid oxidation, generating a compensatory mechanism of protection in vascular disease.     

Characterization of linoleic acid nitration in human blood plasma by mass spectrometry

Nitric oxide (*NO) is a pervasive free radical species that concentrates in lipophilic compartments to serve as a potent inhibitor of lipid and low-density lipoprotein oxidation processes. In this study, we synthesized, characterized, and detected nitrated derivatives of linoleic acid (18:2) in human blood plasma using high-pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry. While the reaction of nitronium tetrafluoroborate with 18:2 presented ions with a mass/charge (m/z) ratio of 324 in the negative ion mode, characteristic of nitrolinoleate (LNO(2)), the reaction of nitrite (NO(2)(-)) with linoleic acid hydroperoxide yielded nitrohydroxylinoleate (LNO(2)OH, m/z 340). Further analysis by MS/MS gave a major fragment at m/z 46, characteristic of a nitro group (-NO(2)) present in the parent ion. This was confirmed by using [(15)N]O(2), which gave products of m/z 325 and 341, that after fragmentation yielded a daughter ion at m/z 47. Moreover, a C-NO(2) structure was also demonstrated in LNO(2)OH by nuclear magnetic resonance spectroscopy ((15)N NMR, delta 375.9), as well as by infrared analysis in both LNO(2)OH (nu(max) = 3427, 1553, and 1374 cm(-1)) and LNO(2) (nu(max) = 1552 and 1373 cm(-1)). Stable products with m/z of 324 and 340, which possessed the same chromatographic characteristics and fragmentation pattern as synthesized standards, were found in human plasma of normolipidemic and hyperlipidemic donors. The presence of these novel nitrogen-containing oxidized lipid adducts in human plasma could represent "footprints" of the antioxidant action of *NO on lipid oxidation and/or a pro-oxidant and nitrating action of *NO-derived species.     

Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington''s Disease

Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50 = 6.1 microM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89-3.75 microM and Vmax values of 33.4-72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2-fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at -20 degrees C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.     

Collisionally induced dissociation of epoxyeicosatrienoic acids and epoxyeicosatrienoic acid-phospholipid molecular species.

Four isomers of epoxyeicosatrienoic acid (EET) can be formed by cytochrome P-450 oxidation of arachidonic acid: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. The collision-induced dissociation of the [M-H]- anion at m/z 319 from each of these isomers, using negative-ion fast atom bombardment ionization and a triple quadrupole mass spectrometer, resulted in a series of common ions as well as ions characteristic of each isomer. The common ions were m/z 301 [M-H2O]- and 257 [M-(H2O + CO2)]-. Unique ions resulted from cleavages alpha to the epoxide moiety to form either conjugated carbanions or aldehydes. Mechanisms involving charge site transfer are suggested for the origin of these ions. A distonic ion series that may involve a charge-remote fragmentation mechanism was also observed. The epoxyeicosatrienoic acids were also incorporated into cellular phospholipids following incubation of the free acid with murine mast cells in culture. Negative fast atom bombardment mass spectrometry of purified glycerophosphoethanolamine-EET species and glycerophosphocholine-EET species yielded abundant [M-H]- and [M-CH3]- ions, respectively. The collision-induced dissociation of these specific high-mass ions revealed fragment ions characteristic of the epoxyeicosatrienoic acids incorporated (m/z 319, 301, and 257) and the same unique ions as those seen with each isomeric epoxyeicosatrienoic acid. With this direct method of analysis, phospholipids containing the four positional isomers of EET, including the highly labile (5,6-EET), could be identified as unique molecular species in mast cells incubated with EET.     

Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

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.     

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.     

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.     

Quantitative analysis of 5-oxo-6,8,11,14-eicosatetraenoic acid by electrospray mass spectrometry using a deuterium-labeled internal standard

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachidonic acid formed by the 5-lipoxygenase pathway, is a potent eosinophil chemoattractant that may be an important mediator in asthma. To further investigate the physiological and pathological roles of 5-oxo-ETE we have developed a mass spectrometric assay employing a tetradeuterated analog (5-oxo-[11,12,14,15-(2)H]ETE) as an internal standard. Collision-induced dissociation of the quasimolecular anion of 5-oxo-[11,12,14,15-(2)H]ETE (m/z 321) resulted in the formation of a major ion at m/z 207 that retained all four deuterium atoms. Measurement of the ratio of ions at m/z 203 (endogenous 5-oxo-ETE) and m/z 207 permitted quantitation of this compound by liquid chromatography-mass spectrometry-mass spectrometry using multiple reaction monitoring. The resulting assay was highly sensitive (< or =20 pg/sample) and selective, enabling detection of the amount of 5-oxo-ETE produced by as few as 10,000 neutrophils. This assay should permit measurement of 5-oxo-ETE in biological fluids, enabling evaluation of its role in asthma and other inflammatory diseases.     

Effect of kynurenine loading on quinolinic acid production in the rat: studies in vitro and in vivo

In vitro and in vivo techniques were used to examine the production and subsequent fate of the endogenous excitotoxin quinolinic acid (QUIN) following administration of its bioprecursor L-kynurenine (KYN). Incubation of liver slices in the presence of 10-1000 microM KYN resulted in a dose- and time-dependent release of QUIN into the incubation medium. Less than 15% of total QUIN produced was recovered from the tissue. In vivo experiments, performed with a microdialysis probe inserted in the jugular vein of anesthetized rats, showed that injection of KYN (20-600 mg/kg, i.v.) causes rapid and dose-dependent increases in the serum level of QUIN. Peak QUIN concentrations in serum dialysates were reached 75 minutes following KYN administration. Longer lasting increases were detected following the administration of pyrazinamide (20 mg/kg, i.p.), an indirectly acting stimulator of QUIN biosynthesis in the periphery. The data demonstrate the feasibility of assessing the mechanisms of QUIN production and disposition in experimental paradigms which can be expected to allow insights into the function and possible dysfunction of QUIN in the brain.     

Transport of quinolinic acid into rabbit and rat brain

The transport metabolism of [³H]quinolinic acid in the central nervous system of rabbits and rats were studied. In vitro [³H]quinolinic acid was not readily accumulated by isolated choroid plexus. After the intraventricular injection of tracer quantities of [³H]quinolinic acid, the [³H]quinolinic acid did not enter the brain as readily as concurrently injected [¹⁴C]mannitol and was not metabolized, The permeability-surface area constant for [³H]quinolinic acid at the rat blood-brain barrier was 1.5±1.3×10^–5 sec^–1 compared to 2.8±0.4×10^–5 sec^–1 for [³H]mannitol. Our results suggest that: 1) [³H]quinolinic acid is transported in the CNS by passive diffusion and 2) is not metabolized.     

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.     

Apomorphine-induced upregulation of serotonin 5-HT2A receptors in male rats is independent from development of aggressive behaviour.

The [3H]ketanserin binding characteristics in the apomorphine-induced aggressive and nonaggressive adult male Wistar rats were studied. Repeated apomorphine (0.5 mg/kg, once daily) treatment gradually induced aggressive behaviour in sixteen animals from twenty. Thereafter the animals were retrospectively divided into apomorphine-induced aggressive and nonaggressive group. The maximal number of the [3H]ketanserin binding sites was increased in the apomorphine-treated animals in the frontal (233.9+/-26.5, 364.6+/-31.7, and 367.0+/-34.8 fmol/mg protein for the vehicle, apomorphine-nonaggressive, and apomorphine-aggressive group, respectively) and cerebral cortex (164.2+/-6.7, 289.7+/-29.3, and 249.0+/-15.4 fmol/mg protein for the vehicle, apomorphine-nonaggressive, and apomorphine-aggressive group, respectively). In conclusion, our experiments demonstrate that repeated apomorphine treatment upregulates the maximal number of the 5-HT2A receptors in rat frontal and cerebral cortex as measured by [3H]ketanserin binding and this phenomenon is independent from the development of aggressive behaviour.     

Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Cerebral Cortex: Effect of Quinolinic Acid Lesions of the Basal Forebrain

The effects of neurotensin (NT) on endogenous acetylcholine (ACh) release from basal forebrain, frontal cortex, and parietal cortex slices were tested. The results show that NT differentially regulates evoked ACh release from frontal and parietal cortex slices without altering either spontaneous or evoked ACh release from basal forebrain slices. In the frontal cortex, NT significantly inhibited evoked ACh release by a tetrodotoxin (TTX)-insensitive mechanism, suggesting an action directly on cholinergic terminals. In the parietal cortex, NT enhanced evoked ACh release by a TTX-sensitive mechanism, suggesting an action of NT on the cholinergic neuron or in close proximity to the cholinergic neuron. The effects of NT on ACh release were confined to evoked ACh release; that is, spontaneous ACh release was not affected. NT did not affect spontaneous or potassium-evoked ACh release from occipital cortex slices. The second set of experiments tested the effects of quinolinic acid (QUIN) lesions of the basal forebrain cell bodies on the NT-induced regulation of evoked ACh release in the cerebral cortex. QUIN lesions of basal forebrain cell bodies caused decreases in choline acetyltransferase activity (27 and 28%), spontaneous ACh release (14 and 21%), and evoked ACh release (38 and 44%) in frontal and parietal cortex, respectively. In addition, 11 days following QUIN lesions of basal forebrain cell bodies, the action of NT to regulate evoked ACh release in frontal cortex or parietal cortex was no longer observed. The results suggest that in the rat frontal and parietal cortex, NT differentially regulates the activity of cholinergic neurons by decreasing and increasing evoked ACh release, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of quinolinic acid on neurons in dissociated cultures of cells of different structures of the brain

Using neurohistological and cytochemical methods in the living cells, the peculiarities of the action of endogenous neurotoxin, quinolinic acid (QUIN), on the neurons developing in the cell cultures of the hippocamp, neocortex and septum have been investigated in 17-19-day-old mouse embryos. The addition of 500 microM of QUIN on the 21st--22nd day into the nutrition medium in vitro resulted in the rapid destruction of neurons localized in glioneuronal aggregates, while the isolated nervous cells as well as septal cholinergic neurons remained intact. At earlier stages of cultivation (up to 2 weeks) QUIN did not provoke degenerative changes in the cultivated neurons. The comparison of our results with the literary data suggests that in nervous cell cultures QUIN, having mature synaptic connections with afferent nervous fibers, causes destruction of neurons.     

L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen.

Previously proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO.) synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO. synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H2(18)/16O2. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry. Its mass spectrum matched that of L-citrulline generated in H2(16)O/16O2. The base fragment ion of m/z 99 was shown to contain the ureido carbonyl group by using L-[guanidino-13C]arginine as substrate. When the enzyme reaction was performed in H2(16)O/18O2, the base fragment ion shifted to m/z 101 with L-[guanidino-12C]arginine as the substrate and to m/z 102 with L-[guanidino-13C]arginine. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO.synthase derives from dioxygen and not from water.