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.     

[3H]Norepinephrine Release from Hippocampal Slices Is an In Vitro Biochemical Tool for Investigating the Pharmacological Properties of Excitatory Amino Acid Receptors

[3H]Norepinephrine ([3H]NE) efflux from preloaded rat hippocampal slices was increased in a dose-dependent manner by excitatory amino acids, with the following order of potencies: N-methyl-D-aspartate (NMDA) greater than kainic acid (KA) greater than L-glutamate greater than or equal to D,L-homocysteate greater than L-aspartate greater than quinolinic acid greater than quisqualic acid. The effect of the excitatory amino acids was blocked by physiological concentrations of Mg2+, with the exception of KA. D,L-2-Amino-7-phosphonoheptanoic acid dose-dependently inhibited the NMDA effect (ID50 = 69 microM), whereas at 1 mM it was ineffective versus KA. The release of [3H]-NE induced by quinolinic acid was blocked by 0.1 mM D,L-2-amino-7-phosphonohepatanoic acid. gamma-D-Glutamylglycine dose-dependently inhibited the KA effect with an ID50 of 1.15 mM. Tetrodotoxin (2 microM) reduced by 40 and 20% the NMDA and KA effects, respectively. The data indicate that [3H]NE release from hippocampal slices can be used as a biochemical marker for pharmacological investigations of excitatory amino acid receptors and their putative agonists and antagonists.     

Autoradiographic localization of γ-aminobutyric acidA receptors within the ventral tegmental area

Destruction of intrinsic neurons in the ventral tegmental area (VTA) with the excitotoxin, quinolinic acid produced a significant decrease (80%) in [³H]muscimol binding to GABA_A receptors within the parabrachial pigmented and paranigral nuclei of the VTA. Selective destruction of the dopaminergic neurons with 6-hydroxydopamine (6-OHDA) did not reduce [³H]muscimol binding within the VTA. However, the destruction of dopaminergic neurons did produce an increase (20%) in [³H]muscimol binding contralateral to the lesion, suggesting a reduction in the GABAergic innervation to this region. Additionally, destruction of the VTA afferents with quinolinic acid injections in the medial accumbens failed to produce alterations in [³H]muscimol binding within the VTA. These results are consistent with the predominant localization of GABA_A receptors to non-dopaminergic neurons intrinsic to the VTA.Special issue dedicated to Dr. Frederick E. Samson     

Experiments in marine biochemistry. Homarine metabolism in Penaeus duorarum.

A fractionation procedure has been developed which permits the isolation of 1 to 2 mg of homarine from a single shrimp. This procedure was used to show that homarine is endogenously synthesized by Penaeus duorarum in the free unbound form, and to study the metabolic precursors involved. Injected DL-[14C]tryptophan was not converted to [14C]homarine. However, [6-14C]quinolinic acid, a known catabolite of tryptophan, is an effective precursor. [2-14C]Acetate and [U-14C]glycerol are effectively converted to [14C]homarine while [14C]bicarbonate is poorly utilized. The injection of L-[U-14C]aspartate resulted in labeled homarine, but the quantity converted was less than expected. Since [14C]glycerol is an effective precursor there is a possibility that quinolinic acid may be formed in P. duorarum by a condensation similar to that of glyceraldehyde 3-phosphate with aspartic acid or a closely related metabolite. It is suggested that decarboxylation of quinolinic acid gives rise to picolinic acid which is methylated to yield homarine. L-[methyl-14C]Methionine efficiently provides the N-methyl carbon presumably via S-adenosylmethionine.     

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.     

Anticonvulsant properties of linalool in glutamate-related seizure models.

In order to investigate the pharmacodynamic basis of the previously-established anticonvulsant properties of linalool, we examined the effects of this compound on behavioral and neurochemical aspects of glutamate expression in experimental seizure models. Specifically, linalool effects were investigated to determine its inhibition of (i) L-[3H]glutamate binding at CNS (central nervous system membranes), (ii) N-methyl-D-aspartate (NMDA)-induced convulsions, (iii) quinolinic acid (QUIN)-induced convulsions, and the behavioral and neurochemical correlates of PTZ-kindling. The data indicate that linalool modulates glutamate activation expression in vitro (competitive antagonism of L-[3H]glutamate binding) and in vivo (delayed NMDA convulsions and blockage of QUIN convulsions). Linalool partially inhibited and significantly delayed the behavioral expression of PTZ-kindling, but did not modify the PTZ-kindling-induced increase in L-[3H]glutamate binding.     

Evidence that quinolinic acid severely impairs energy metabolism through activation of NMDA receptors in striatum from developing rats

In the present study we investigated the effect of intrastriatal administration of 150 nmol quinolinic acid to young rats on critical enzyme activities of energy production and transfer, as well as on 14CO2 production from [1-14C]acetate at distinct periods after quinolinic acid injection. We observed that quinolinic acid injection significantly inhibited complexes II (50%), III (46%) and II-III (35%), as well as creatine kinase (27%), but not the activities of complexes I and IV and citrate synthase in striatum prepared 12 h after treatment. In contrast, no alterations of these enzyme activities were observed 3 or 6 h after quinolinic acid administration. 14CO2 production from [1-14C]acetate was also significantly inhibited (27%) by quinolinic acid in rat striatum prepared 12 h after injection. However, no alterations of these activities were observed in striatum homogenates incubated in the presence of 100 microm quinolinic acid . Pretreatment with the NMDA receptor antagonist MK-801 and with creatine totally prevented all inhibitory effects elicited by quinolinic acid administration. In addition, alpha-tocopherol plus ascorbate and the nitric oxide synthase inhibitor l-NAME completely abolished the inhibitions provoked by quinolinic acid on creatine kinase and complex III. Furthermore, pyruvate pretreatment totally blocked the inhibitory effects of quinolinic acid injection on complex II activity and partially prevented quinolinic acid-induced creatine kinase inhibition. These observations strongly indicate that oxidative phosphorylation, the citric acid cycle and cellular energy transfer are compromised by high concentrations of quinolinic acid in the striatum of young rats and that these inhibitory effects were probably mediated by NMDA stimulation.     

Quinolinic acid lesion of nucleus accumbens reduces D1 but not D2 dopamine receptors: an autoradiographic study

Information concerning the cellular localization of dopamine receptor subtypes in the nucleus accumbens (NAcc) was obtained using receptor autoradiographic analysis. Unilateral, stereotaxic injection of the axon-sparing neurotoxin, quinolinic acid, into the NAcc resulted in a prominent loss of dopamine D1 receptors (as labeled by [3H]SCH 23390). Contrarily, no appreciable decrement in D2 receptors (labeled by [3H]raclopride) could be identified within the same region of the NAcc. The findings support the view that accumbens D1 receptors are located postsynaptically on neurons or their processes, while D2 receptors within this nucleus are primarily located on afferent terminals.     

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.     

Precursors of ricinine in the castor bean plant

Isotopic tracer experiments confirmed that glycerol and succinic acid are good precursors of the pyridine ring of ricinine in castor bean plants. Tritium from C-2 was lost from tritiated glycerol while tritium from C-1 was retained. Thus a derivative of dihydroxyacetone is likely to be intermediate. By simultaneous feeding of glycerol-1-(3)-[³H] and succinic acid-2(3)-[¹⁴C], it was hoped to find precursors of ricinine containing both labels, but none could be found. There was no evidence for the appearance of labeled quinolinic acid, which is presumed to be a precursor of ricinine.     

大肠杆菌来源的喹啉酸磷酸核糖转移酶和烟酸磷酸核糖转移酶的表达纯化及酶活性的初步检测

【目的】在原核表达体系中实现大肠杆菌来源的喹啉酸磷酸核糖转移酶(Quinolinic acid phosphoribosyl transferase,QPRT)和烟酸磷酸核糖转移酶(Nicotinic acidphosphoribosyl transferase,NaPPT)的表达与纯化,并利用酶的生物催化作用实现2,3-二羧酸喹啉的2位选择性脱羧得到烟酸【。方法】通过PCR扩增分别得到编码QPRT和NaPPT的基因片段,构建成原核表达质粒pET28a-NadC和pRSETB-PncB,在Escherichia coli(E.coli)中对其进行表达,在体外对目标蛋白进行纯化并利用高效液相色谱法(HPLC)检测酶催化反应的发生。【结果】成功表达纯化得到QPRT和NaPPT,检测结果表明在这两个酶的生物催化作用下可实现喹啉酸的2位选择性脱羧。     

The fidelity of protein synthesis: can mischarging by aspartyl-tRNA(Asp) synthetase lead to the formation of isoaspartyl residues in proteins?

We have tested the hypothesis that isoaspartic acid residues in proteins can arise via errors that occur during protein synthesis. One such error involves a mischarging step in which the aspartic acid side-chain beta-carboxyl group is linked to the tRNA(Asp) instead of the main chain alpha-carboxyl group. If this altered Asp-tRNA(Asp) is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or beta-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue. Using an ammonium sulfate fraction of aspartyl-tRNA(Asp) synthetase from Escherichia coli and [3H]aspartic acid, we have prepared [3H]aspartyl-tRNA(Asp) complexes and directly analyzed the linkage of the [3H]aspartate to the tRNA by identifying the products of ammonolysis. Normal attachment of the alpha-carboxyl group of aspartate to the tRNA produces [3H]isoasparagine, while the mischarging reaction leads to [3H]asparagine formation after ammonolysis. We have separated [3H]isoasparagine from [3H]asparagine and found an upper limit of 1 asparagine per 10,000 isoasparagines. These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the alpha- and beta-carboxyl groups of aspartic acid and suggest that only a very small fraction of the isoaspartic acid residues found to occur in cellular proteins may be the result of mischarging steps.     

Effect of Quinolinic Acid in the Nucleus Basalis Magnocellularis on Cortical High-Affinity Choline Uptake

A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific [3H]hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.     

Quinolinic acid induces NMDA receptor-mediated lipid peroxidation in rat brain microvessels

Quinolinic acid increased the generation of lipid peroxidation products by isolated rat brain microvessels in vitro. The effect was inhibited both by a specific NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid and by reduced glutathione (GSH). Furthermore, quinolinic acid displaced specific binding of [(3)H]-L-glutamate by cerebral microvessel membranes, particularly in the presence of NMDA receptor co-agonist (glycine) and modulator (spermidine). We conclude that quinolinic acid can cause potentially cytotoxic lipid peroxidation in brain microvessels via an NMDA receptor mediated mechanism.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.     

Effects of Undernutrition on Glutamatergic Parameters in Rat Brain

Early restriction of nutrients during the perinatal period has marked repercussions on CNS ontogeny, Leading to impaired functions. This study investigated the effects of pre- and postnatal (up to 75 days) undernutrition (diet: 8% protein; normonourished group: 25% protein) on some glutamatergic and behavioral parameters of rats. Undernutrition reduced: (i) seizures caused by ICV quinolinic acid (QA) administration; (ii) Na-independent [³H]glutamate binding in cell plasma membranes of cerebral cortex, and (ii) basal [³H]glutamate release from synaptosomal preparation. Behavioral parameters related to locomotion, anxiety, or memory were not affected. These results indicate that our model of undernutrition decreased the sensitivity to QA as convulsing agent and point to some putative glutamatergic parameters involved in this effect.     

On the mechanism of oxidation of cholesterol at C-7 in a lipoxygenase system.

Incubation of [7-2H2]cholesterol with soybean lipoxygenase and linoleic acid in the presence of oxygen gave a mixture of 5-cholestene-3 beta,7 alpha-diol, 5-cholestene-3 beta,7 beta-diol, 3 beta-hydroxy-5-cholesten-7-one,5 alpha,6 alpha-epoxycholestan-3 beta-ol, and 5 beta,6 beta-epoxycholestan-3 beta-ol. The conversion into the 7-oxygenated products was associated with a very high intermolecular isotope effect (KH/KD = 15-17), suggesting that the rate-limiting step in the overall conversion is likely to be the abstraction of hydrogen at C-7 in a radical reaction. Evidence that linoleic acid is to some extent directly involved was obtained with the use of [7-3H]cholesterol. Incubation of [7-3H]cholesterol resulted in a significant incorporation of 3H in the reisolated linoleic acid fraction. The isotope effect associated with conversion of [7 alpha-2H]cholesterol into 7-oxygenated products in the lipoxygenase system was 2-3, indicating that the extraction of hydrogen is nonstereospecific. Incubation of [7-2H2]cholesterol with 13-hydroperoxy-9,11-octadecadienoic acid gave the above 7-oxygenated products with relatively small isotope effects (KH/KD = 3-4). It is concluded that the most important mechanism for oxidation of cholesterol at C-7 in the lipoxygenase system involves participation of radicals and that a carbon-centered linoleic acid radical can extract hydrogen directly from cholesterol. Fatty acid hydroperoxides and their secondary products seem to be less important as initiators in connection with oxidation of cholesterol.     

Evidence for a lack of regulatory importance of the 12 alpha-hydroxylase in formation of bile acids in man: an in vivo study

The possibility that the 12 alpha-hydroxylase involved in formation of bile acids is of regulatory importance for the ratio between cholic acid and chenodeoxycholic acid in bile was studied with an in vivo technique. [4-14C]7 alpha-Hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one were synthesized, and a mixture of these two bile acid intermediates was administered intravenously in five healthy subjects and in one patient with severe liver cirrhosis. The patient with liver cirrhosis was included in the study because of a considerable reduction in biosynthesis of cholic acid. Since the [4-14C]-labeled steroid is an intermediate just proximal to and since the [6 beta-3H]-labeled steroid is an intermediate just distal to the 12 alpha-hydroxylase step, the 3H/14C ratio in the cholic acid formed should reflect the relative 12 alpha-hydroxylase activity. The 3H/14C ratio varied between 1.8 and 3.9 in the cholic acid isolated from the healthy subjects and was 3.6 in the cholic acid isolated from the patient with liver cirrhosis. The ratio between cholic acid and chenodeoxycholic acid varied between 0.6 and 3.9 in the bile from the control subjects and was only 0.4 in the bile from patients with liver cirrhosis. There was no correlation between the 3H/14C ratios and the ratios between cholic acid and chenodeoxycholic acid in bile.(ABSTRACT TRUNCATED AT 250 WORDS)