RESUMO
The synthetic compound 4-chlorokynurenine has been shown to be enzymatically transaminated to the selective glycine(B) receptor antagonist 7-chlorokynurenate. Since 4-chlorokynurenine, in contrast to 7-chlorokynurenate, readily penetrates the blood-brain barrier, the present study evaluated its neuroprotective properties after systemic administration in rats. Intrahippocampal injection of the NMDA receptor agonist quinolinate (15 nmol/l microl) was used as the neurotoxic paradigm. Serum and hippocampal tissue measurements confirmed that 4-chlorokynurenine serves as an effective pro-drug of 7-chlorokynurenate both in the periphery and in the brain. These studies and complementary hippocampal microdialysis experiments compared the effects of single and repeated injections of 4-chlorokynurenine (50 or 200 mg/kg, intraperitoneal (i.p.), 10 min prior to an intrahippocampal quinolinate injection; or 50 mg/kg, i.p., 10 min before and 30, 120 and 360 min after quinolinate). With the multiple-dosing regimen, extracellular 7-chlorokynurenate levels in the hippocampus reached a maximum of approximately 750 nM 7 h after quinolinate and gradually decreased with a half-life of about 3 h. In contrast, a single injection of 200 mg/kg 4-chlorokynurenine resulted in a considerably shorter rise in extracellular 7-chlorokynurenate without yielding higher peak levels. In separate animals, repeated treatment with 50 mg/kg 4-chlorokynurenine, but not a single injection of 200 mg/kg of the pro-drug, provided total protection against quinolinate-induced excitotoxicity. These data suggest that a prolonged and functionally relevant blockade of hippocampal glycine(B) receptors can be achieved after the systemic administration of 4-chlorokynurenine.
Assuntos
Hipocampo/efeitos dos fármacos , Cinurenina/análogos & derivados , Pró-Fármacos/farmacologia , Ácido Quinolínico/toxicidade , Animais , Encefalopatias/patologia , Encefalopatias/prevenção & controle , Soluções para Diálise/química , Hipocampo/patologia , Injeções Intraperitoneais , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/sangue , Cinurenina/sangue , Cinurenina/farmacologia , Masculino , Microdiálise , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of L-kynurenine to kynurenic acid. A combination of polymerase chain reaction techniques and hybridization screening was used to isolate a cDNA clone encompassing the entire coding region of KAT from rat kidney. Identification of the cDNA as coding for KAT was based both on the comparison of amino acid sequences obtained from purified rat KAT and on the expression of KAT activity in COS-1 cells transfected with the cDNA. RNA blot analysis indicated that KAT mRNA is widely expressed in rat tissues. Cultured cells transfected with the cDNA for KAT also showed glutamine transaminase K activity. Based mainly on sequence data, these results demonstrate that rat kidney KAT is identical with glutamine transaminase K.
Assuntos
Liases , Transaminases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar , Rim/enzimologia , Dados de Sequência Molecular , RatosRESUMO
The kynurenine pathway metabolites quinolinic acid and kynurenic acid have been hypothetically linked to the occurrence of seizure phenomena. The present immunohistochemical study reports the activation of astrocytes containing three enzymes responsible for the metabolism of quinolinic acid and kynurenic acid in a rat model of chronic epilepsy. Rats received 90 min of patterned electrical stimulation through a bipolar electrode stereotaxically positioned in one hippocampus. This treatment induces non-convulsive limbic status epilepticus that leads to chronic, spontaneous, recurrent seizures. One month after the status epilepticus, the rats showed neuronal loss and gliosis in the piriform cortex, thalamus, and hippocampus, particularly on the side contralateral to the stimulation. Astrocytes containing the kynurenic acid biosynthetic enzyme (kynurenine aminotransferase) and the enzymes for the biosynthesis and degradation of quinolinic acid (3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase, respectively) became highly hypertrophied in brain areas where neurodegeneration occurred. Detailed qualitative and quantitative analyses were performed in the hippocampus. In CA1 and CA3 regions, the immunostained surface area of reactive astrocytes increased up to five-fold as compared to controls. Enlarged cells containing the three enzymes were mainly observed in the stratum radiatum, whereas the stratum pyramidale, in which neuronal somata degenerated, showed relatively fewer reactive glial cells. Hypertrophied kynurenine aminotransferase- and 3-hydroxyanthranilic acid oxygenase-immunoreactive cells were comparable in their morphology and distribution pattern. In contrast, reactive quinolinic acid phosphoribosyl transferase-positive glial cells displayed diversified sizes and shapes. Some very large quinolinic acid phosphoribosyl transferase-immunoreactive cells were noticed in the molecular layer of the dentate gyrus. In the hippocampus, the number of immunoreactive glial cells increased in parallel to the hypertrophic responses. In addition, pronounced increases in immunoreactivities, associated with hypertrophied astrocytes, occurred around lesioned sites in the thalamus and piriform cortex. These findings indicate that kynurenine metabolites derived from glial cells may play a role in chronic epileptogenesis.
Assuntos
Astrócitos/enzimologia , Dioxigenases , Modelos Animais de Doenças , Epilepsia/enzimologia , Excitação Neurológica , Cinurenina/metabolismo , Sistema Límbico/fisiopatologia , Liases , Proteínas do Tecido Nervoso/análise , Oxigenases/análise , Pentosiltransferases/análise , Estado Epiléptico/complicações , Transaminases/análise , 3-Hidroxiantranilato 3,4-Dioxigenase , Animais , Astrócitos/patologia , Morte Celular , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Doença Crônica , Epilepsia/etiologia , Epilepsia/patologia , Gliose/etiologia , Hipocampo/enzimologia , Hipocampo/patologia , Sistema Límbico/enzimologia , Sistema Límbico/patologia , Masculino , Degeneração Neural , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Recidiva , Tálamo/enzimologia , Tálamo/patologiaRESUMO
The cellular localizations of the synthetic [3-hydroxyanthranilic acid oxygenase (3HAO)] and degradative [quinolinic acid phosphoribosyltransferase (QPRT)] enzymes of the endogenous excitotoxin quinolinic acid were studied in the adult rat main olfactory bulb by immunohistochemical techniques. 3HAO and QPRT were expressed only in astrocytes. The two enzymes were differentially expressed by astrocytes in a complementary pattern: 3HAO staining was strongest at the glomerular-external plexiform layer junction; QPRT staining was strongest at the glomerular-olfactory nerve layer junction. The complementary distributions of these metabolic enzymes suggests that there could be a gradient of quinolinic acid across the glomerular layer of the main olfactory bulb. Such a gradient could function to restrict the ingrowth of new olfactory axons to the glomeruli and/or to stabilize the formation of new synapses.
Assuntos
Astrócitos/enzimologia , Dioxigenases , Bulbo Olfatório/enzimologia , Oxigenases/metabolismo , Pentosiltransferases/metabolismo , Ácidos Quinolínicos/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenase , Animais , Astrócitos/citologia , Imuno-Histoquímica , Bulbo Olfatório/anatomia & histologia , Bulbo Olfatório/citologia , Ácido Quinolínico , Ratos , Ratos Endogâmicos , Coloração e RotulagemRESUMO
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)
Assuntos
Encéfalo/metabolismo , Ácido Cinurênico/metabolismo , Cinurenina/metabolismo , Ácido Amino-Oxiacético/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Cloretos/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/fisiologia , Hipocampo/metabolismo , Ácido Ibotênico/farmacologia , Cinética , Masculino , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Tálamo/metabolismo , Distribuição TecidualRESUMO
Serial ion-exchange and high-performance liquid chromatography separations were employed for the tissue extraction and purification of kynurenic acid (KYNA). Subsequently, the compound isolated from postmortem human brain tissue was unequivocally identified as KYNA by nuclear magnetic resonance and mass spectrometric analyses. Regional distribution analyses revealed the highest concentration of KYNA (1.58 +/- 0.43 pmol/mg tissue) in the caudate nucleus with lower levels in the thalamus, globus pallidus, hippocampus, parietal cortex and frontal cortex. Of the brain structures examined, the lowest concentration of KYNA (0.14 +/- 0.02 pmol/mg tissue) was found in the cerebellum.
Assuntos
Química Encefálica , Ácido Cinurênico/isolamento & purificação , Mudanças Depois da Morte , Idoso , Núcleo Caudado/análise , Humanos , Masculino , Pessoa de Meia-Idade , Tálamo/análiseRESUMO
Astroglial reactions to intrastriatal and intrahypothalamic injections of the endogenous excitotoxin quinolinic acid (50 micrograms in 1 microliter) were studied in adult rats, using immunohistochemistry with antiserum to glial fibrillary acidic protein. Animals were sacrificed 6 h, 24 h, 3, 7 and 30 days or 1 year after the injection. Six and 24 h after quinolinic acid, the amount of glial fibrillary acidic protein-like immunoreactivity in the injected striatum was lower than in controls but returned to a normal level at 3 days. Not until 7 days was a clear striatal gliosis apparent, as evidenced by an increased density of glial fibrillary acidic protein-positive structures and brightly fluorescent, clearly hypertrophic cells. This gliosis was even more developed in animals sacrificed 30 days postoperatively. A weak astrocytic reaction was also observed in the ipsilateral corpus callosum at 6 h after quinolinic acid. By 3 days, a marked gliosis restricted to the injected hemisphere was present throughout corpus callosum and cortex cerebri. In animals sacrificed 30 days after quinolinic acid the extrastriatal astrocytic reaction was clearly diminished, although the striatal gliosis was still prominent. One year postinjection, no obvious gliosis could be observed in cortex cerebri or corpus callosum while striatal tissue, now markedly reduced in volume, was clearly gliotic. Using neurofilament antiserum, increased fluorescence intensity was noted in striatal nerve bundles during the first day after an intrastriatal quinolinic acid injection and persisted 1 year postoperatively. Controls were similarly injected with an equimolar amount of nicotinic acid, the non-excitatory, non-neurotoxic decarboxylation product of quinolinic acid. No changes in immunoreactivity of glial fibrillary acidic protein or neurofilament were found in these animals. In animals treated intrahypothalamically, a spherical central area almost devoid of glial fibrillary acidic protein-immunoreactivity was noted around the injection site 7 days after quinolinic acid administration. Around this area, gliosis was observed. Apart from a very restricted gliotic reaction around the needle tract, no astrocytic reaction was observed in nicotinic acid-injected control animals. We conclude that quinolinic acid causes both reversible and long-lasting gliosis when injected into the rat striatum. As a natural brain metabolite, quinolinic acid may constitute a particularly valuable tool for the elucidation of a possible role of glia in neurodegenerative disorders.
Assuntos
Astrócitos/análise , Proteína Glial Fibrilar Ácida/metabolismo , Neurotoxinas/administração & dosagem , Piridinas/administração & dosagem , Ácidos Quinolínicos/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Corpo Estriado/análise , Corpo Estriado/efeitos dos fármacos , Feminino , Histocitoquímica , Hipotálamo/análise , Hipotálamo/efeitos dos fármacos , Filamentos Intermediários/análise , Filamentos Intermediários/efeitos dos fármacos , Masculino , Microinjeções , Neurotoxinas/farmacologia , Ácido Quinolínico , Ácidos Quinolínicos/farmacologia , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Bilateral intrahypothalamic injections of the brain metabolite quinolinic acid (QUIN) were made in an attempt to examine its effects on the secretion of LH, PRL, GH and TSH. Quin, a neuroexcitatory amino acid with close structural similarities to glutamate, kainate and N-methylaspartate, was infused into unanesthetized male rats, the animals sacrificed 7.5 min later, and serum hormone concentrations determined by radioimmunoassay. QUIN caused surges in LH, PRL and GH release (316, 607 and 1,134% of control, respectively, at 50 micrograms QUIN) without affecting the serum concentrations of TSH. At lower doses, a preferential effect of QUIN on PRL release was observed. All QUIN-induced hormonal changes were inhibited by concomitant administration of the specific antagonist (-)-2-amino-7-phosphonoheptanoic acid, indicating the presence of QUIN-sensitive receptors on neurons which are intimately associated with endocrine regulation. Moreover, because QUIN-treated animals exhibited behavioral signs of seizure activity and neuroendocrine dysfunction has been reported to occur in human convulsive disorders, the data are also of interest in view of a possible mechanistic link between epileptic phenomena and hormone secretion.
Assuntos
Sistema Hipotálamo-Hipofisário/fisiologia , Hipotálamo/fisiologia , Hormônios Adeno-Hipofisários/metabolismo , Piridinas/farmacologia , Ácidos Quinolínicos/farmacologia , Animais , Hormônio do Crescimento/sangue , Hormônio do Crescimento/metabolismo , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Cinética , Hormônio Luteinizante/sangue , Hormônio Luteinizante/metabolismo , Masculino , Prolactina/sangue , Prolactina/metabolismo , Ácido Quinolínico , Ácidos Quinolínicos/metabolismo , Ratos , Ratos Endogâmicos , Tireotropina/metabolismoRESUMO
Quinolinic acid (QUIN) is an endogenous, excitotoxic amino acid which is currently under investigation as a possible etiological factor in human neurodegenerative disorders such as Huntington's disease and epilepsy. We explored certain aspects of this hypothesis, using the adult rat as an experimental animal. After intrastriatal infusions of [3H]QUIN, radioactivity was cleared from the injected region with an apparent half-life of 22 min. To 2 h after injection, all radioactivity recovered from the striatum corresponded to unmetabolized QUIN. Consistent with these data was the lack of significant uptake of [3H]QUIN by slices or crude synaptosomes prepared from rat hippocampus or striatum. When applied intravenously, a high dose of QUIN (450 mg/kg) caused relatively minor seizure-related EEG changes and no signs of neuronal degeneration. Direct measurements indicated negligible penetration of the blood-brain barrier by QUIN. The lack of an effective inactivation mechanism for extracellular QUIN in the brain negates QUIN's proposed role as a classical neurotransmitter substance, but may be of significance for the postulated effects of this compound in neurodegenerative diseases. An important role of blood-borne QUIN or QUIN precursors in human disorders cannot be ruled out at present; although the brain appears to be well protected by the blood-brain barrier from an acute elevation of blood QUIN, a possible breakdown of the barrier under pathologic conditions and the effects of chronic elevations of blood QUIN remain to be examined.
Assuntos
Piridinas/administração & dosagem , Ácidos Quinolínicos/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Barreira Hematoencefálica , Corpo Estriado/metabolismo , Eletroencefalografia , Técnicas In Vitro , Injeções , Injeções Intra-Arteriais , Injeções Intravenosas , Masculino , Ácido Quinolínico , Ácidos Quinolínicos/metabolismo , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
Organotypic cultures of caudate nucleus and frontal cerebral cortex, either alone or in combination with each other, have been used to evaluate and compare the neurotoxic effects of two dicarboxylic amino acids, kainic acid (KA) and the tryptophan metabolite quinolinic acid (QUIN). Both of these agents can induce specific post-synaptic degeneration in cultures in which a complement of well-developed mature synapses exists. The neurotoxic effects of QUIN can be blocked by the synthetic anti-convulsant agent (-)2-amino-7-phosphonoheptanoic acid [( -]APH), but neurotoxicity of KA cannot. These studies support the candicacy of QUIN as an endogenous neurotoxin with properties similar to KA. Furthermore, the studies demonstrate the usefulness of the organotypic nerve tissue culture model as a research tool for examining certain neurodegenerative phenomena and for identifying neurotoxic amino acids as well as compounds which may antagonize amino acid neurotoxicity.