RESUMEN
Using sodium azide (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia in the hippocampal slice, we have evaluated the effects of the novel 5-HT(1A) partial agonist/5-HT(2) receptor antagonist adatanserin and the 5-HT(1A) receptor agonist BAYx3702 on the efflux of endogenous glutamate, aspartate and GABA. BAYx3702 (10-1000 nM) produced a significant (P<0.05) dose-related attenuation of ischemic efflux of both glutamate and GABA with maximum decrease being observed at 100 nM (73 and 69%, respectively). This attenuation was completely reversed by the addition of the 5-HT(1A) antagonist, WAY-100635 (100 nM). Similarly, adatanserin (10-1000 nM) produced a significant (P<0.05) dose-related attenuation in glutamate and GABA efflux with a maximum of 72 and 81% at 100 nM, respectively. This effect was completely reversed by the 5-HT(2A/C) receptor agonist, DOI but unaffected by WAY-100635. The 5-HT(2A) receptor antagonist MDL-100907 produced a comparable attenuation of glutamate when compared to adatanserin, while the 5-HT(2C) receptor antagonist, SB-206553, had no effect on ischemic efflux. None of these compounds significantly altered aspartate efflux from this preparation. In conclusion, the 5-HT(1A) receptor partial agonist 5-HT(2) receptor antagonist, adatanserin is able to attenuate ischemic amino acid efflux in a comparable manner to the full 5-HT(1A) agonist BAYx3702. However, in contrast to BAYx3702, adatanserin appears to produce it effects via blockade of the 5-HT(2A) receptor. This suggests that adatanserin may be an effective neuroprotectant, as has been previously demonstrated for full 5-HT(1A) receptor agonists such as BAYx3702.
Asunto(s)
Aminoácidos/metabolismo , Isquemia Encefálica/metabolismo , Hipocampo/irrigación sanguínea , Hipocampo/metabolismo , Piperazinas/farmacología , Pirimidinas/farmacología , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Animales , Fluorobencenos/farmacología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Indoles/farmacología , Ligandos , Masculino , Piperidinas/farmacología , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A , Receptor de Serotonina 5-HT2B , Receptores de Serotonina 5-HT1RESUMEN
Using sodium (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia, we have characterized the endogenous release of excitatory and inhibitory amino acids from superfused hippocampal slices. Chemical ischemia produced an azide (1-30 mM) dose-dependent increase in the efflux of glutamate, aspartate and GABA. These increases were attenuated to varying degrees by removal of Ca2+, or the addition of the voltage dependent Na+-channel blocker tetrodotoxin (TTX), the selective Ca2+ channel blockers conotoxin MVIIA, MVIIC, and nifedipine, the NMDA antagonist MK801, the AMPA antagonist GYKI-52466. Similarly, addition of the GLT-1 glutamate transport inhibitor dihydrokainate (DHK) and the anti-estrogen/anion channel blocker tamoxifen also attenuated the efflux of glutamate and GABA. It would therefore appear that the increases in amino acid efflux induced by chemical ischemia originates from both the neuronal pool, via conventional exocytotic release, and glial sources via reversal of the GLT-1 transporter and anion channel regulated cell swelling.
Asunto(s)
Aminoácidos/metabolismo , Glucosa/antagonistas & inhibidores , Hipocampo/irrigación sanguínea , Hipocampo/metabolismo , Isquemia/inducido químicamente , Isquemia/metabolismo , Azida Sódica , Animales , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-Dawley , Azida Sódica/administración & dosificaciónRESUMEN
Using middle cerebral artery occlusion (MCAO) and in vivo microdialysis, we have evaluated the changes in extracellular concentrations of the excitatory amino acids (EAA) glutamate and aspartate during varying periods of MCAO (0, 30, 60 min) in the striatum of spontaneously hypertensive rats (SHR). A positive correlation between occlusion time-dependent elevations in EAAs and the resulting ischemic injury was observed. This is the first demonstration of the temporal profile of EAA efflux during transient focal ischemia in SHRs. Possible sources and mechanisms of ischemia-induced EAA efflux were examined during 60 min of MCAO. Removal of Ca(2+) from the microdialysis infusion media significantly attenuated ischemia-induced increases in both glutamate (from ischemic peak of 4892 +/- 1298 to 1144 +/- 666% of preischemic values) and aspartate (from 2703 +/- 682 to 2090 +/- 599% of preischemic values). Similarly, infusion of the voltage dependent Na(+) channel blocker tetrodotoxin (TTX; 10 microM) significantly attenuated MCAO-induced increases in glutamate (to 1313 +/- 648%) and aspartate (to 359 +/- 114%). Infusion of the GLT-1 selective nontransportable inhibitor, dihydrokainate (DHK; 1 mM) also significantly attenuated the ischemia-induced increases in both EAAs (1285 +/- 508 and 1366 +/- 741% of the preischemic levels, respectively). These results indicate that during transient focal ischemia the increase in extracellular EAAs originates from both the neuronal pool, via conventional exocytotic release, and glial sources via the reversal of the GLT-1 transporter.
Asunto(s)
Ácido Aspártico/metabolismo , Isquemia Encefálica/metabolismo , Espacio Extracelular/metabolismo , Ácido Glutámico/metabolismo , Ácido Kaínico/análogos & derivados , Ratas Endogámicas SHR/metabolismo , Daño por Reperfusión/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Sistema de Transporte de Aminoácidos X-AG , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Isquemia Encefálica/fisiopatología , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Ácido Kaínico/farmacología , Masculino , Ratas , Daño por Reperfusión/fisiopatología , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
Because of the well-documented importance of glutamate uptake in protecting neurons against glutamate toxicity, we were interested in testing the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on rat cortical cultures. This compound is a substrate for glutamate transporters and is a potent glutamate transport inhibitor that does not interact significantly with glutamate receptors. Using a 30 min exposure, and assessing neuronal survival after 20-24 h, PDC was neurotoxic in conventional astrocyte-rich cortical cultures, with an EC50 in these cultures of 320 +/- 157 microM. In astrocyte-poor cultures, an EC50 for PDC of 50 +/- 5 microM was determined. The neurotoxicity of PDC in both astrocyte-rich and astrocyte-poor cultures was blocked by the NMDA antagonist MK-801, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). We tested the possibility that the neurotoxicity of PDC might be due to release of excitatory amino acids using several approaches. After pre-loading cells with the non-metabolizable analogue of glutamate, [3H]-D-aspartate, first we demonstrated that PDC caused significant efflux of [3H]-D-aspartate. This effect of PDC was dependent upon extracellular sodium. In contrast with glutamate neurotoxicity, PDC neurotoxicity was inhibited by removal of extracellular sodium. In the presence of 1 mM PDC, sodium caused neurotoxicity with an EC50 of 18 +/- 7.6 mM. Tetrodotoxin had no effect on either PDC neurotoxicity or on PDC-evoked [3H]-D-aspartate release. PDC-evoked release of [3H]-D-aspartate was demonstrable in astrocyte cultures with no neurons present. PDC also evoked release of endogenous glutamate. Finally, the neurotoxicity of PDC was blocked by coincubation with glutamate-pyruvate transaminase plus pyruvate to degrade extracellular glutamate. These results demonstrate the neurotoxicity of PDC, and suggest that the mechanism of this toxicity is the glutamate transporter-dependent accumulation of glutamate in the extracellular space.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Ácidos Dicarboxílicos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Neurotoxinas/farmacología , Pirrolidinas/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Animales , Astrocitos/efectos de los fármacos , Recuento de Células/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
Early disruption of the serotonin neurotransmitter system may have important consequences for normal neuro-physiological development. In order to further understand the neurochemical changes which occur after early insults to the system, intracerebroventricular injections of the serotonin-selective toxin, 5,7-dihydroxytryptamine were given to Sprague-Dawley rat pups on day 3 of life. Serotonin and 5-hydroxyindoleacetic acid concentration in cortex, striatum, hippocampus, and brainstem were measured after 4, 8, and 12 weeks. In controls, serotonin concentration in the striatum and cortex increased from 4 to 12 weeks, while it reached adult levels by 4 weeks in the hippocampus. 5-Hydroxyindoleacetic acid increased in cortex and hippocampus and was unchanged after 4 weeks in the striatum. An index of serotonin turnover (5-hydroxyindoleacetic acid/serotonin) decreased in striatum and cortex, but increased in the hippocampus over time. 5,7-Dihydroxytryptamine treatment induced permanent decreases in serotonin and 5-hydroxyindoleacetic acid concentration in all three forebrain regions, but had no region- and time-specific effects on serotonin turnover. In the brainstem of controls, serotonin concentration increased from 4 to 12 weeks, while 5-hydroxyindoleacetic acid concentration was unchanged, resulting in decreasing turnover. After 5,7-dihydroxytryptamine treatment, the brainstem did not show depletions of serotonin concentration in spite of significant serotonin neuronal loss, suggesting that compensatory mechanisms in remaining neurons may permit increased serotonin production. Regional and time-dependent responses to serotonin depletion may have functional implications for the developmental regulation of serotonin transmission after early insults to this neurotransmitter system.
Asunto(s)
5,7-Dihidroxitriptamina/farmacología , Animales Recién Nacidos , Tronco Encefálico/química , Corteza Cerebral/química , Cuerpo Estriado/química , Hipocampo/química , Animales , Tronco Encefálico/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cuerpo Estriado/efectos de los fármacos , Femenino , Hipocampo/efectos de los fármacos , Ácido Hidroxiindolacético/análisis , Ácido Hidroxiindolacético/metabolismo , Inmunohistoquímica , Masculino , Embarazo , Ratas , Ratas Sprague-Dawley , Serotonina/análisis , Serotonina/metabolismoRESUMEN
Phenylketonuria (PKU) is a genetic disorder in which the hydroxylation of phenylalanine (Phe) to tyrosine is severely disrupted. If PKU is left untreated, severe mental retardation results. The accepted treatment is to restrict dietary intake of Phe. It has generally been thought that cognitive impairments are prevented if levels of Phe in plasma are maintained at or below five times the normal level. However, we recently documented that children treated early and continuously for PKU or children mildly hyperphenylalaninemic, who have levels of Phe in plasma approximately three to five times normal, still have cognitive impairments. These impairments are specific to the functions of frontal cortex (A. Diamond, W. Hurwitz, E. Lee, W. Grover, and C. Minarcik, unpublished observations). To investigate the mechanism underlying these cognitive deficits, an animal model of this condition was developed and characterized. Thirty-six rat pups were divided into three groups. The first group was treated pre- and postnatally with Phe and alpha-methylphenylalanine (a phenylalanine hydroxylase inhibitor). The second group was injected postnatally with Phe and alpha-methylphenylalanine. The third group received postnatal control injections. The mild plasma Phe elevations in the two experimental groups produced significant behavioral and neurochemical effects. Both experimental groups were impaired on a task dependent on frontal cortex, delayed alternation. Levels of dopamine, homovanillic acid (HVA), norepinephrine, and 5-hydroxyindole acetic acid (5-HIAA) were measured in medial prefrontal cortex, anterior cingulate cortex, striatum, and nucleus accumbens. The largest neurochemical reductions observed were in HVA and were in the two frontal cortical areas (medial prefrontal cortex and anterior cingulate cortex). There were modest reductions in HVA in the nucleus accumbens but no significant changes in HVA, or in any other metabolite or neurotransmitter, in the striatum. The levels of 5-HIAA were also reduced in all brain regions examined. There was no effect on norepinephrine in any of the four regions examined. Reduced levels of HVA in medial prefrontal cortex were the only neurochemical effect that significantly correlated with every measure of performance on the delayed alternation task. This study provides evidence of deleterious effects from mild elevations in the levels of Phe in plasma previously considered small enough to be safe. These effects include impaired performance on a cognitive task dependent on frontal cortex and reduced HVA levels in frontal cortex.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Fenilcetonurias/tratamiento farmacológico , Animales , Animales Recién Nacidos/metabolismo , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Quimioterapia Combinada , Feto/efectos de los fármacos , Feto/metabolismo , Ácido Homovanílico/metabolismo , Ácido Hidroxiindolacético/metabolismo , Inyecciones , Neurotransmisores/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/uso terapéutico , Fenilcetonurias/metabolismo , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
Sodium-dependent, high-affinity glutamate transport is generally assumed to limit the toxicity of glutamate in vivo and in vitro, but there is very little direct evidence to support this hypothesis. In the present study, the effects of the specific uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate on the toxicity and clearance of glutamate were examined in hippocampal neuronal cultures. At a concentration that was not toxic by itself, L-trans-pyrrolidine-2,4-dicarboxylate increased the toxicity of glutamate approximately fivefold and slowed the clearance of glutamate from the extracellular space. This toxicity was almost completely blocked by the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These studies provide direct evidence that sodium-dependent, high-affinity glutamate transport limits glutamate toxicity in vitro.
Asunto(s)
Ácidos Dicarboxílicos/farmacología , Glutamatos/metabolismo , Glutamatos/toxicidad , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Pirrolidinas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Sinergismo Farmacológico , Embrión de Mamíferos , Femenino , Ácido Glutámico , Hipocampo/metabolismo , Hipocampo/patología , Cinética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/metabolismo , Neuronas/patología , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Valina/análogos & derivados , Valina/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Levels of the excitotoxin quinolinic acid (QUIN) were measured in the cerebrospinal fluid of infants and children with congenital hyperammonemia. Twofold to tenfold elevations of QUIN were found in 4 neonates in hyperammonemic coma (QUIN range, 250-990 nM; control mean, 110 +/- 90 nM; p < 0.005). Similar elevations of neopterin were found (range, 24-75 nM; control mean, 9.0 +/- 4.9 nM; p < 0.005). In addition, significant elevations of QUIN were found in 14 older children with congenital hyperammonemia (mean, 50 +/- 20 vs 17 +/- 6 nM; p < 0.05). Neopterin levels were not elevated in these children. The QUIN may originate from an increase in tryptophan transport across the blood-brain barrier or from induction of indolamine-2,3-dioxygenase activity. These findings support a role for QUIN in the neuropathology of congenital hyperammonemia. They also suggest the potential utility of N-methyl-D-aspartate receptor-blocking agents or inhibitors of QUIN synthesis in the treatment of hyperammonemic coma.
Asunto(s)
Amoníaco/sangre , Coma/líquido cefalorraquídeo , Errores Innatos del Metabolismo/líquido cefalorraquídeo , Ácido Quinolínico/líquido cefalorraquídeo , Aminas Biogénicas/líquido cefalorraquídeo , Biopterinas/análogos & derivados , Biopterinas/líquido cefalorraquídeo , Preescolar , Coma/sangre , Humanos , Ácido Hidroxiindolacético/líquido cefalorraquídeo , Lactante , Recién Nacido , NeopterinRESUMEN
Exposure to HBO causes hypothermia, bradycardia, head weaving, resting tremor, piloerection, and straub tail in rats. These physiological and behavioral responses can also be evoked by selective activation of serotonin1A (5-HT1A) receptors. The purpose of the current study was to determine if hypothermia caused by HBO is due to increased activation of 5-HT1A receptors. The levels of brain biogenic amines were measured in brain regions of Sprague-Dawley (SD) rats exposed to HBO. Exposure to HBO caused an increase in the levels of 5-hydroxyindoleacetic acid (5-HIAA) in the striatum (92%, p < 0.05) and occipital-temporal cortex (116%, p < 0.05), but not in other brain regions. Exposure to HBO did not change the levels of tryptophan, serotonin (5-HT), other biogenic amines, or their metabolites. It is hypothesized that the Fawn Hood (FH) rat, which is reported to be resistant to hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), has an abnormality of 5-HT1A receptor activity. Although the FH rat was more resistant to hypothermia induced by HBO than the SD rat, we were not able to confirm that this rat was resistant to hypothermia induced by 8-OH-DPAT. The 5-HT receptor antagonists, 1-(1H-Indol-4-yloxy)-3-[(1-methylethyl)amino]-2-propanol (Pindolol), 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190), and methysergide, did not block hypothermia induced by HBO in SD rats. A series of control experiments were used to confirm that the antagonists blocked hypothermia induced by serotonin agonists.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Regulación de la Temperatura Corporal/efectos de los fármacos , Oxigenoterapia Hiperbárica , Receptores de Serotonina/efectos de los fármacos , 5-Hidroxitriptófano/antagonistas & inhibidores , 8-Hidroxi-2-(di-n-propilamino)tetralin/antagonistas & inhibidores , Animales , Aminas Biogénicas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Masculino , Ratas , Ratas Endogámicas , Ratas Sprague-Dawley , Antagonistas de la Serotonina/farmacología , Especificidad de la EspecieRESUMEN
Children with inborn errors of urea synthesis who survive neonatal hyperammonemic coma commonly exhibit cognitive deficits and neurologic abnormalities. Yet, there is evidence that ammonia is not the only neurotoxin. Hyperammonemia appears to induce a number of neurochemical alterations. In rodent models of hyperammonemia, uptake of L-tryptophan into brain is increased. It has been reported that in an experimental rat model of hepatic encephalopathy, in the ammonium acetate-injected rat, and in patients with hepatic failure and inborn errors of ammonia metabolism, quinolinate, a tryptophan metabolite, is increased. Elevations in quinolinate are of particular concern, as quinolinate could excessively activate the N-methyl-D-aspartate subclass of excitatory amino acid receptors, thereby causing selective neuronal necrosis. We sought to identify an animal model that would replicate the increases in quinolinate that have been associated with hyperammonemia in humans. Levels of quinolinate were measured in hyperammonemic urease-infused rats and ammonium acetate-injected rats. In the urease-infused rat, brain tryptophan was doubled, and serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly increased. Yet, despite the increase in tryptophan and evidence for increased metabolism of tryptophan to serotonin, there were no observed increases of quinolinate in brain, cerebrospinal fluid, or plasma. In the ammonium acetate-injected rat, significant increases of 5-hydroxyindoleacetic acid in cerebral cortex were also observed, but quinolinate did not change in cerebrospinal fluid or cerebral cortex. In summary, we were unable to demonstrate an increase of quinolinate in brain or cerebrospinal fluid in these rat models of hyperammonemia.