RESUMEN
Animal models are important for the investigation of mechanisms and therapeutic approaches in various human diseases, including schizophrenia. Recently, two neurodevelopmental rat models of this psychosis were developed based upon the use of subunit selective N-methyl-D-aspartate receptor agonists--quinolinic acid (QUIN) and N-acetyl-aspartyl-glutamate (NAAG). The aim of this study was to evaluate pain perception in these models. QUIN or NAAG was infused into lateral cerebral ventricles neonatally. In the adulthood, the pain perception was examined. The rats with neonatal brain lesions did not show any significant differences in acute mechanical nociception and in formalin test compared to controls. However, the neonatally lesioned rats exhibited significantly higher pain thresholds in thermal nociception. Increased levels of mechanical hyperalgesia, accompanying the sciatic nerve constriction (neuropathic pain), were also observed in lesioned rats. Although hyperalgesia was more pronounced in QUIN-treated animals, the number of c-Fos-immunoreactive neurons of the lumbar spinal cord was similar in experimental and control rats. We conclude that neonatal brain lesions attenuated the thermal perception in both nociceptive and neuropathic pain whereas mechanical pain was increased in the model of neuropathic pain only. Thus, nociceptive and neuropathic pain belongs--in addition to behavioral changes--among the parameters which are affected in described animal models of schizophrenia.
Asunto(s)
Modelos Animales de Enfermedad , Neuralgia/fisiopatología , Umbral del Dolor/fisiología , Ratas Wistar , Esquizofrenia/fisiopatología , Factores de Edad , Animales , Animales Recién Nacidos , Dipéptidos/farmacología , Femenino , Calor , Humanos , Inyecciones Intraventriculares , Masculino , Nociceptores/fisiología , Dimensión del Dolor , Estimulación Física , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ácido Quinolínico/farmacología , Ratas , Receptores de N-Metil-D-Aspartato/agonistas , Esquizofrenia/inducido químicamenteRESUMEN
Perinatal cerebral hypoxia represents a major cause of obstetric complications and the resulting transient oxygen deficiency might belong to early risk factors for schizophrenia. The aim of this study was to evaluate possible long-term behavioral changes induced by one hour of continuous bilateral common carotid artery occlusion in 12-day-old male rats. Post-ischemic behavioral disturbances were evaluated in social (play) behavior on postnatal day 22 (PND 22), open field test (PND 35 and 50) and prepulse inhibition of the acoustic startle reflex (PND 50). Transient ischemia in neonatal rats was not significantly altered in social dyadic interactions evaluated in pre-weaning pups, but resulted in enhanced locomotor activity in pubertal rats (PND 35) and impaired prepulse inhibition of the startle reflex in post-pubertal males (PND 50). These behavioral alterations suggest that perinatal hypoxic/ischemic insults may represent a risk factor for later manifestation of specific features relevant to schizophrenia in predisposed individuals.
Asunto(s)
Animales Recién Nacidos/fisiología , Conducta Animal/fisiología , Isquemia Encefálica/psicología , Psicología del Esquizofrénico , Estimulación Acústica , Animales , Análisis de los Gases de la Sangre , Arteria Carótida Común/fisiología , Modelos Animales de Enfermedad , Femenino , Ligadura , Masculino , Actividad Motora/fisiología , Ratas , Ratas Wistar , Reflejo de Sobresalto/fisiología , Conducta SocialRESUMEN
Brain infections as well as peripheral challenges to the immune system lead to an increased production of interleukin-1beta (IL-1beta), a cytokine involved in leukocyte-mediated breakdown of the blood-brain barrier. The effects of IL-1beta have been reported to depend on whether the route of administration is systemic or intracerebral. Using 50-day-old male rats, we compared the effects of IL-1beta on brain gamma-glutamyl transpeptidase (GGT; an enzymatic marker of brain capillary endothelium) at 2, 24 and 96 h after either an intravenous (i.v.) injection of 5 microg IL-1beta or an intracerebroventricular (i.c.v. - lateral ventricle) infusion of 50 ng IL-1beta. When the i.v. route was used, the GGT activity underwent small but significant changes; decreasing in the hippocampus 2 h after the i.v. injection, increasing 24 h later and returning to control levels at 96 h. No significant changes in the hippocampal GGT activity were observed at 2 and 24 h following the i.c.v. infusion. The GGT activity in the hypothalamus remained unchanged regardless of the route of IL-1beta administrations. Similar changes in GGT activity were revealed histochemically. The labeling was found mainly in the capillary bed, the changes being most evident in the hippocampal stratum radiatum and stratum lacunosum-moleculare. A transient increase in GGT activity at 24 h, together with a less sharp delineation of GGT-stained vessels, may reflect IL-1beta induced increased turnover of glutathione and/or oxidative stress, that may in turn, be related to altered permeability of the blood-brain barrier in some neurological and mental disorders, including schizophrenia.
Asunto(s)
Hipocampo/enzimología , Hipocampo/inmunología , Interleucina-1/metabolismo , gamma-Glutamiltransferasa/metabolismo , Animales , Biomarcadores/metabolismo , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Radicales Libres/metabolismo , Hipocampo/irrigación sanguínea , Hipotálamo/irrigación sanguínea , Hipotálamo/enzimología , Hipotálamo/inmunología , Interleucina-1/inmunología , Interleucina-1/farmacología , Masculino , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/inmunología , Ratas , Ratas WistarRESUMEN
The effect of lesions induced by bilateral intracerebroventricular (i.c.v.) injection of quinolinate (250 nmol of QUIN/ventricle), a selective N-methyl-D-aspartate (NMDA) receptor agonist, on [3H]glutamate ([3H]Glu) binding to the main types of both ionotropic and metabotropic glutamate receptors (iGluR and mGluR) was investigated in synaptic membrane preparations from the hippocampi of 50-day-old rats. The membranes from QUIN injured brains revealed significantly lowered binding in iGluR (by 31%) as well as in mGluR (by 22%) as compared to the controls. Using selected glutamate receptor agonists as displacers of [3H]Glu binding we found that both the NMDA-subtype of iGluR and group I of mGluR are involved in this decrease of binding. Suppression of nitric oxide (NO) production by N(G)-nitro-L-arginine (50 nmol of NARG/ventricle) or the increase of NO generation by 3-morpholinylsydnoneimine (5 nmol of SIN-1/ventricle) failed to alter [3H]Glu or [3H]CPP (3-((D)-2-carboxypiperazin-4-yl)-[1,2-(3)H]-propyl-1-phosphonic acid; NMDA-antagonist) binding declines caused by QUIN-lesions. Thus, our findings indicate that both the NMDA-subtype of iGluR and group I of mGluR are susceptible to the QUIN-induced neurodegeneration in the rat hippocampus. However, the inhibition of NO synthesis did not reveal any protective action in the QUIN-evoked, NMDA-receptor mediated decrease of [3H]Glu binding. Therefore, the additional mechanisms of QUIN action, different from direct NMDA receptor activation/NO production (e.g. lipid peroxidation induced by QUIN-Fe-complexes) cannot be excluded.
Asunto(s)
Ácido Glutámico/farmacocinética , Hipocampo/metabolismo , Molsidomina/análogos & derivados , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/patología , Ácido Kaínico/farmacología , Masculino , Molsidomina/farmacología , N-Metilaspartato/farmacología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Donantes de Óxido Nítrico/farmacología , Nitroarginina/farmacología , Ácido Quinolínico , Ácido Quiscuálico/farmacología , Ratas , Ratas Wistar , Tritio , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Autoradiographical studies revealed that 10 nM [3H]N-acetyl-aspartyl-glutamate (NAAG) labelled grey matter structures, particularly in the hippocamus, cerebral neocortex, striatum, septal nuclei and the cerebellar cortex. The binding was inhibited by (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG IV), an agonist at group II metabotropic glutamate receptors (mGluR II). (RS)-alpha-Methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-cyclopropyl-4-phosphonoglycine (CPPG) and (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE), all antagonists at mGluR II and mGluR III, also inhibited [3H]NAAG binding. Other inhibitors were (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a broad-spectrum mGluR agonist with preference for groups I and II and the mGluR I agonists/mGluR II antagonists (S)-3-carboxy-4-hydroxyphenylglycine (3,4-CHPG) and (S)-4-carboxy-3-hydroxyphenylglycine (4,3-CHPG). Neither the mGluR I specific agonist (S)-dihydroxyphenylglycine nor any of the ionotropic glutamate receptor ligands such as kainate, AMPA and MK-801 had strong effects (except for the competitive NMDA antagonist CGS 19755, which produced 20-40% inhibition at 100 microM) suggesting that, at low nM concentrations, [3H]NAAG binds predominantly to metabotropic glutamate receptors, particularly those of the mGluR II type. Several studies have indicated that NAAG can interact with mGluR II and the present study supports this notion by demonstrating that sites capable of binding NAAG at low concentrations and displaying pharmacological characteristics of mGluR II exist in the central nervous tissue. Furthermore, the results show that autoradiography of [3H]NAAG binding can be used to quantify the distribution of such sites in distinct brain regions and study their pharmacology at the same time.
Asunto(s)
Química Encefálica , Encéfalo/ultraestructura , Dipéptidos/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Alanina/análogos & derivados , Alanina/farmacología , Animales , Sitios de Unión , Carboxipeptidasas/metabolismo , Frío , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Ciclopropanos/farmacología , Dipéptidos/farmacología , Maleato de Dizocilpina/farmacología , Femenino , Glutamato Carboxipeptidasa II , Glicina/análogos & derivados , Glicina/farmacología , Ácido Kaínico/farmacología , Masculino , Proteínas del Tejido Nervioso/efectos de los fármacos , Fosfoserina/análogos & derivados , Fosfoserina/farmacología , Ácidos Pipecólicos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Tetrazoles/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Specific [3H]glutamate binding to synaptic membranes from the cerebral cortex and hippocampus of 7-, 12- and 18-day-old rats was examined, both in control animals and during seizures induced by homocysteine. In the cerebral cortex a transient peak of glutamate binding was observed in 7-day-old group, whereas in the hippocampus it occurred in 12-day-old animals. Total specific [3H]glutamate binding was not influenced by preceding seizure activity in either of the age groups and both the studied regions. NMDA- and QA-sensitive glutamate bindings represent the highest portion of the total binding. Moreover, NMDA-sensitive binding in the cerebral cortex of 7-day-old rats is significantly higher as compared to the two more mature groups. The proportion of individual receptor subtypes on total binding in each age group was not influenced by preceding seizure activity. However, NMDA-sensitive binding in the hippocampus of 12-day-old rats, sacrificed during homocysteine-induced seizures, was significantly increased as compared to corresponding controls. In contrast to the effect of NMDA, AMPA, kainate and quisqualate which displaced to a different extent [3H]glutamate binding, homocysteine had no effect when added to membrane preparations. Similarly, [3H]CPP and [3H]AMPA bindings were not affected in the presence of homocysteine. It thus seems unlikely that homocysteine is an effective agonist for conventional ionotropic glutamate receptors. Its potential activity at some of the modulatory sites at the NMDA receptor channel complex or at metabotropic receptors has to be clarified in further experiments.
Asunto(s)
Envejecimiento/metabolismo , Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Convulsiones/metabolismo , Membranas Sinápticas/metabolismo , Análisis de Varianza , Animales , Sitios de Unión , Corteza Cerebral/crecimiento & desarrollo , Hipocampo/crecimiento & desarrollo , Homocisteína , Ácido Kaínico/metabolismo , Masculino , N-Metilaspartato/metabolismo , Ácido Quiscuálico/metabolismo , Ratas , Ratas Wistar , Valores de Referencia , Convulsiones/inducido químicamente , Tritio , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
Quinolinate, an endogenous excitotoxic metabolite of tryptophan with affinity to the N-methyl-D-aspartate type of glutamate receptor, is known as a stimulator of lipid peroxidation in vitro [Neurochem. Res. (1991) 16, 1139-1143]. To analyse the mechanism of this quinolinate toxicity we used the thiobarbituric acid test to measure malondialdehyde in homogenates of rat cerebral hemispheres incubated in air at 37 degrees C for 30 min in the presence of 0.015-15.0 mM quinolinate, endogenous iron or 0.5-2.0 microM FeSO4 and with or without 250 microM ascorbate. Quinolinate in the concentrations of 0.15-2.5 mM stimulated lipid peroxidation in the homogenates in the presence of 0.5-2.0 microM Fe2+. However, quinolinate concentrations higher than 3.0 mM inhibited the lipid peroxidation at all the tested concentrations of iron. In the presence of a potent iron chelator (10 microM deferoxamine) quinolinate completely failed to induce lipid peroxidation in rat brain homogenates. Spectral analysis revealed that quinolinate is able to form a complex with Fe2+. The results suggest that quinolinate does not have a direct peroxidative effect, but that it modulates lipid peroxidation via its interaction with iron.
Asunto(s)
Ácido Ascórbico/antagonistas & inhibidores , Encéfalo/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Ácido Quinolínico/farmacología , Animales , Encéfalo/metabolismo , Deferoxamina/farmacología , Quelantes del Hierro/farmacología , Masculino , Ratas , Ratas Wistar , Sideróforos/farmacologíaRESUMEN
Quinolinic acid (QUIN) is an endogenous excitotoxic agonist of the N-methyl-D-aspartate (NMDA) type of glutamate receptor, which causes slowly progressing degeneration of vulnerable neurons in some brain regions. Using changes in the activity of membrane-bound gamma-glutamyl transpeptidase (GGT) as a marker of cell damage, we found a significant decrease of this enzyme activity, which was preferentially located in the ipsilateral hippocampal formation and entorhinal cortex, 4 d after the unilateral intracerebroventricular (icv) injection of 0.5 mumol QUIN. The dose of QUIN divided into two half-doses injected bilaterally led to a symmetrical decline of GGT activity in hippocampal areas. The lesion was characterized by a suppression of GGT activity in hippocampal and entorhinal capillaries, corresponding to 60 and 81% of their initial value, respectively, but no significant changes were ascertained in synaptosomal membranes. The changes in the activity of capillary GGT were associated with the decrease of apparent maximal velocity Vmaxapp, whereas apparent Michaelis constant K(m)app (0.69-0.79 mM) remained unaffected. In the nonlesioned brain, concanavalin A (Con A) affinity chromatography revealed five glycoforms of synaptosomal GGT in contrast to only one found in hippocampal and entorhinal capillaries. The results document that neither the saccharide moiety of GGT nor the value of enzyme K(m)app is significantly affected by the QUIN-induced lesion of the rat brain. However, the suppression of GGT activity, which is accompanied by a decrease in the value of Vmaxapp in brain microvessels, may suggest dysfunction of the blood-brain barrier (BBB) in the QUIN-injured rat brain.
Asunto(s)
Encefalopatías/enzimología , Corteza Entorrinal/enzimología , Agonistas de Aminoácidos Excitadores/farmacología , Ácido Quinolínico/farmacología , gamma-Glutamiltransferasa/metabolismo , Animales , Encefalopatías/inducido químicamente , Encefalopatías/patología , Capilares/efectos de los fármacos , Capilares/enzimología , Líquido Cefalorraquídeo/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Circulación Cerebrovascular/fisiología , Corteza Entorrinal/irrigación sanguínea , Agonistas de Aminoácidos Excitadores/administración & dosificación , Inyecciones Intraventriculares , Masculino , Membranas/efectos de los fármacos , Membranas/enzimología , Membranas/metabolismo , Ácido Quinolínico/administración & dosificación , Ratas , Ratas Wistar , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/enzimología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
The initial rate kinetics of rat kidney gamma-glutamyl transpeptidase were measured using L-gamma-glutamyl-p-nitroanilide and glycyl-glycine as the donor and the acceptor substrate, respectively. Experimental data were fitted with the initial rate equation, and the obtained results indicated that: (1) Michaelis constants for transpeptidation (Kb), autotranspeptidation (Ka), and hydrolysis (Kh) are 8.56 mmol/l, 2.02 mmol/l and 0.005 mmol/l, respectively. (2) The maximum rate of transpeptidation (Vb) exceeds that of hydrolysis (Vh) and autotranspeptidation (Va) 160 times and 5 times, respectively. (3) A comparison of the ratios of maximal rate: Michaelis constant of individual reactions shows that hydrolysis is approximately 10 times more efficient than the remaining two reactions. (4) Under routine conditions used for gamma-glutamyl transpeptidase estimation, transpeptidation is the prevalent reaction.
Asunto(s)
Riñón/enzimología , gamma-Glutamiltransferasa/metabolismo , Animales , Femenino , Glutamina/análogos & derivados , Glicilglicina , Hidrólisis , Cinética , Ratas , Ratas Wistar , Especificidad por SustratoRESUMEN
Gamma-glutamyl transpeptidase (EC 2.3.2.2; GGT) is a plasma-membrane bound glycoenzyme, the saccharide moiety of which is rather heterogeneous and organ specific. It has been stated that GGT catalyses three types of reactions, i.e., hydrolysis, transpeptidation and autotranspeptidation. The initial velocity equation, involving all these reactions, is shown in the present report. Mathematical analysis of the equation resulting in a definition of the constant of half saturation (Khs). The value of Khs was used for characterization of kinetics of GGT from rat organs differing in the structure of GGT oligosaccharide chains. No significant organ differences were found, when the Khs values of GGT from the brain, kidney and pancreas equalled 0.61 mM, 0.68 mM and 0.68, respectively. On the contrary, when two different glycoforms of GGT from the pancreas were compared, distinct values of Khs were obtained (1.43 mM and 0.67 mM, respectively). It is therefore being suggested that the saccharide chains of GGT are involved in its kinetic properties. However, this effect is masked when the enzyme, non-fractionated into glycoforms, is analysed, even though the saccharide moiety is specific for the organ studied.
Asunto(s)
gamma-Glutamiltransferasa/metabolismo , Animales , Encéfalo/enzimología , Cromatografía de Afinidad , Concanavalina A/metabolismo , Femenino , Glicoproteínas/metabolismo , Riñón/enzimología , Cinética , Oligosacáridos/química , Páncreas/enzimología , Ratas , Ratas Wistar , Bazo/enzimología , gamma-Glutamiltransferasa/químicaRESUMEN
The activity of gamma-glutamyl transpeptidase (GGT) and the sialic acid (SA) content were found to be specific in five brain regions of 7- and 50-day-old rats. While a low GGT activity was accompanied by high sialylation in the whole brain a high GGT activity and low sialylation were observed in kidney and pancreas. Similar findings were obtained for some brain regions investigated. However, the developmental differences were not characterized by such relationship. Concanavalin A affinity chromatography of GGT from brain of 7- and 50-day-old rats revealed no remarkable changes in GGT sialylation although the enzyme activity is almost 3 times higher in brain membranes of the older group. It can be concluded that in spite of an apparent relationship between GGT activity and membrane sialylation, the amount of SA linked to the GGT molecule is not related to differences in enzyme activity.
Asunto(s)
Envejecimiento/fisiología , Química Encefálica , Ácidos Siálicos/análisis , gamma-Glutamiltransferasa/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Membrana Celular/química , Membrana Celular/enzimología , Cromatografía de Afinidad/métodos , Femenino , Riñón/química , Riñón/enzimología , Riñón/crecimiento & desarrollo , Hígado/química , Hígado/enzimología , Hígado/crecimiento & desarrollo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Páncreas/química , Páncreas/enzimología , Páncreas/crecimiento & desarrollo , Ratas , Ratas Wistar , Ácidos Siálicos/metabolismo , Bazo/química , Bazo/enzimología , Bazo/crecimiento & desarrolloRESUMEN
To define the ontogeny of "excitotoxic" neurodegeneration further, bilateral intracerebroventricular injection of N-methyl-D-aspartate agonist, quinolinate (QUIN), was administered to rats at Postnatal Days 12, 30, and 50. Excitotoxic injury was quantified by means of changes in [3H]glutamate binding to membranes isolated from the entorhinal cortex, hippocampal formation, cerebellum, and medulla oblongata 4 days after the injection of QUIN. Binding was significantly decreased only in the hippocampal formation of 30- and 50-day-old rats (by 25 and 32%, respectively). In contrast, binding to cortical membranes was elevated by 29 and 56% at Postnatal Days 30 and 50, respectively. Observed changes in the binding of glutamate were due to modifications in the equilibrium binding constants rather than in the density of the receptors. In the cerebellum, which exhibited the highest developmental increase, the statistically significant decrease of the binding (by 36%) following QUIN lesion was only observable on Day 30. The effects of QUIN lesions were not statistically significant in the medulla oblongata. The results suggest that in 30- and 50-day-old rats QUIN can be implicated in neurodegeneration of the entorhinohippocampal complex.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Glutamatos/metabolismo , Ácido Quinolínico/farmacología , Animales , Animales Recién Nacidos , Ácido Glutámico , Inyecciones Intraventriculares , Masculino , Ácido Quinolínico/administración & dosificación , Ratas , Ratas Wistar , Análisis de Regresión , Distribución TisularRESUMEN
Na(+)-dependent binding of L-glutamate in cortical and hippocampal synaptic membranes from hyperammonemic rats was compared to corresponding data in the controls. In hippocampal membranes, repeated hyperammonemia resulted in a 13% and 18% decrease in binding in 20-day-old and 50-day-old rats, respectively. The decrease was statistically significant (P < 0.05) in the older animals and Scatchard analysis revealed a 19% reduction in the number of binding sites without any changes in the affinity. Within the hippocampal formation, the binding in the dentate gyrus was the most sensitive to hyperammonemia where a 21% decrease was found (P < 0.01), whilst the decline of binding in CA1 and CA3 areas of the hippocampus proper was not significant. The results support the idea that excessive accumulation of extracellular glutamate during hyperammonemia is a consequence not only of its increased release, but also of the blocking of Na(+)-dependent binding of glutamate to specific uptake sites.
Asunto(s)
Amoníaco/sangre , Corteza Cerebral/metabolismo , Glutamatos/metabolismo , Hipocampo/metabolismo , Sodio/fisiología , Membranas Sinápticas/metabolismo , Envejecimiento/metabolismo , Animales , Química Encefálica/fisiología , Corteza Cerebral/ultraestructura , Ácido Glutámico , Hipocampo/ultraestructura , Cinética , Ratas , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismoRESUMEN
gamma-Glutamyl transpeptidase (GGT) in tissue extracts from five brain regions, the kidney and pancreas of 50-day-old rats was examined for its specific activity and affinity to immobilized concanavalin A (Con A). According to their descending GGT activity, the tissue extracts were classified in the following order: kidney >> pancreas >> olfactory bulbs > medulla oblongata > hippocampus > cerebellum > frontal cortex. Using different concentrations of methyl-alpha-D-mannopyranoside for the elution of GGT from Con A-Sepharose column, the enzyme from brain regions could be separated into five fractions, two of which contained about 75% of the total GGT activity without regional differences in elution profiles. Almost complete GGT activity in kidney tissue extracts was eluted in a single peak whereas the enzyme from pancreas exhibited two peaks.
Asunto(s)
Encéfalo/enzimología , Riñón/enzimología , Páncreas/enzimología , gamma-Glutamiltransferasa/metabolismo , Animales , Fraccionamiento Químico , Cromatografía de Afinidad , Concanavalina A/química , Femenino , Riñón/química , Páncreas/química , Ratas , Ratas Wistar , Extractos de Tejidos , gamma-Glutamiltransferasa/química , gamma-Glutamiltransferasa/aislamiento & purificaciónRESUMEN
Inhibitors of gamma-glutamyl transpeptidase (mixture of serine and borate--13 mM, kainic acid--5 mM and 6-diazo-5-oxo-L-norleucine--2 mM) significantly suppressed glutamate uptake into cultured neurones and glial cells. The simultaneous application of any of these inhibitors with ouabain resulted in a further decline in glutamate uptake. It can be speculated that gamma-glutamyl transpeptidase significantly contributes to glutamate transport into nerve cells in the early period of brain development until the Na(+)-K(+)-gradient is fully constituted.
Asunto(s)
Boratos/farmacología , Encéfalo/metabolismo , Diazooxonorleucina/farmacología , Glutamatos/metabolismo , Ácido Kaínico/farmacología , Neuroglía/metabolismo , Neuronas/metabolismo , Serina/farmacología , gamma-Glutamiltransferasa/antagonistas & inhibidores , Envejecimiento/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Células Cultivadas , Pollos , Ácido Glutámico , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacosRESUMEN
Acute hyperammonemia, induced by two consecutive injections of ammonium acetate (550 and 450 mg per kg b.wt.), decreased the activity of gamma-glutamyl transpeptidase (GGT) in most brain regions of 18- and 30-day-old rats. This decrease in the brain GGT activity was more pronounced in younger than in older rats. After the addition of NH4Cl to the incubation medium, the inhibitory action of NH4+ on this enzyme activity was also demonstrated in crude synaptosomal membranes at pH 7.4, but in a range of NH4+ concentrations many-times higher than those found in the plasma or brains of young hyperammonemic rats. Because similar concentrations of NH4+ stimulated the activity of the purified enzyme from rat kidney (mainly at pH 9.0), the inhibition of GGT activity in the young rat brain is probably mediated indirectly and not by a direct interaction of ammonia with the enzyme molecules.
Asunto(s)
Amoníaco/farmacología , Encéfalo/enzimología , gamma-Glutamiltransferasa/metabolismo , Acetatos/administración & dosificación , Amoníaco/sangre , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Esquema de Medicación , Femenino , Concentración de Iones de Hidrógeno , Riñón/enzimología , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas , Sinaptosomas/enzimología , gamma-Glutamiltransferasa/antagonistas & inhibidoresRESUMEN
gamma-Glutamyl transpeptidase (GGT) is a membrane-bound sialoglycoprotein. The developmental changes in GGT activity and in sialic acid content were determined in a crude synaptosomal membrane fraction from the cerebral hemispheres of the chick embryo between days 11 and 19 of incubation. The GGT activity increased almost eightfold during the examined developmental period, while sialic acid content rose significantly only between days 11 and 15. Cortical administered on day 13 significantly increased GGT activity. On the other hand, the content of membrane bound sialic acid was not substantially affected. The value of the GGT apparent Michaelis constant (Kmapp) for gamma-glutamyl-p-nitroanilide in the presence of 20 mmol.l-1 glycylglycine was 1.5 mmol.l-1 and cortisol did not influence it. However, Vmax was increased by this hormone. The affinity of GGT to concanavalin A (ConA) did not change during development. Neither the administration of cortisol nor neuroaminidase treatment had any effect on the interaction of GGT with ConA. Desialylation of crude synaptosomal fraction did not change GGT activity. The results presented here suggest no developmental nor functional relationship between the activity of GGT and the level of sialylation in synaptosomal membranes from the cerebral hemispheres of the chick embryo.
Asunto(s)
Encéfalo/enzimología , Ácidos Siálicos/metabolismo , Sinaptosomas/enzimología , gamma-Glutamiltransferasa/metabolismo , Animales , Encéfalo/efectos de los fármacos , Embrión de Pollo , Concanavalina A/metabolismo , Glutamina/análogos & derivados , Glutamina/metabolismo , Glicilglicina/metabolismo , Hidrocortisona/farmacología , Ácido N-Acetilneuramínico , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/enzimología , Sinaptosomas/efectos de los fármacosRESUMEN
The effect of kainic acid, a structural analogue and specific agonist of glutamate, was studied on the Na(+)-dependent binding and uptake of this amino acid in cerebral cortex preparations from 7-day-old and 30-day-old mice. The specific binding of glutamate to a crude synaptic membrane fraction and uptake into cortical slices increased several fold during this period. Kainic acid (0.5 mM or 5 mM) significantly reduced glutamate binding and this effect was more pronounced in membrane fractions from older animals. In contrast to this, the inhibitory action of kainic acid on glutamate uptake was twofold more potent in 7-day-old mice. The results are discussed from the viewpoint of the relationship between the Na(+)-dependent binding of glutamate and its uptake.
Asunto(s)
Corteza Cerebral/metabolismo , Glutamatos/metabolismo , Ácido Kaínico/metabolismo , Sodio/metabolismo , Animales , Corteza Cerebral/crecimiento & desarrollo , Técnicas In Vitro , Cinética , RatonesRESUMEN
The ability of the N-methyl-D-aspartate receptor antagonists, MK-801, ketamine and alaptide [a newly synthesized cyclo(1-amino-1-cyclopentane-carbonyl-L-alanyl) with protective properties in models of hypoxia], to prevent neuronal degeneration caused by intracerebroventricular application of quinolinic acid was investigated. Neurodegenerative effects of quinolinate in the hippocampal formation were found to increase with the degree of maturity of glutamatergic target structures. A protective potency of the N-methyl-D-aspartate receptor antagonists was observed at all developmental stages studied (12- and 30-day-old and adult rats). MK-801 showed the highest efficacy, alaptide the lowest. These findings suggest a parallelism in maturity of glutamatergic transmission processes as one prerequisite of quinolinate vulnerability and postnatal increases of target fields of the protectives. Application of MK-801 or ketamine after quinolinate injection intensified their protective effects when compared to simultaneous or preadministration. This observation is interpreted as indicating that quinolinate is a prompter of a delayed neurodegenerative process rather than acting immediately as a toxicant.
Asunto(s)
Maleato de Dizocilpina/farmacología , Hipocampo/crecimiento & desarrollo , Ketamina/farmacología , Degeneración Nerviosa/efectos de los fármacos , Neuropéptidos/farmacología , Péptidos Cíclicos/farmacología , Ácidos Quinolínicos/antagonistas & inhibidores , Animales , Hipoxia de la Célula , Hipocampo/citología , Hipocampo/efectos de los fármacos , Inyecciones Intraventriculares , Masculino , Ácido Quinolínico , Ácidos Quinolínicos/farmacología , Ratas , Ratas EndogámicasRESUMEN
The effect of hyperammonemia of varying degree and duration on the gamma-glutamyl-transpeptidase (GGT) activity was studied in the homogenates and capillaries of different brain regions of the rat. "Acute" hyperammonemia (750 and 600 mg of ammonium acetate per kg b.w. were injected i.p. at 30 min interval, and the animals were decapitated immediately), in which blood ammonia was increased 14-fold, and brain ammonia six-fold above the control level, produced a 20% increase of the enzyme activity in cerebellum, and a 17% decrease in gyrus dentatus, but had no effect in the frontal cortex and the CA1 and CA3 regions of hippocampus. "Subchronic" hyperammonemia (two injections of 600 mg ammonium acetate/kg were given at 24 h intervals, and tissue samples were removed 24 h later), that was accompanied by only a 60% increase of blood or brain ammonia, increased the activity in cerebellum to 38% above control, but produced no effect in the other brain regions. "Chronic" hyperammonemia (three injections of 600 mg ammonium acetate/kg at 24 h intervals and excision of tissue samples 30 min after the last injection), in which blood and brain ammonia were, respectively, 60 and 100% higher than in control animals, elevated the GGT activity in the cerebellum by 57%, in CA1 by 15%, and in CA3 by 21%, but produced no effect in the frontal cortex or gyrus dentatus. By contrast, "chronic" hyperammonemia produced a 30% increase of GGT activity in cerebral cortical capillaries, but only a 10% increase in hippocampal capillaries, and no change in cerebellar capillaries. The results suggest that, hyperammonemia of relatively long duration may contribute to the enhancement of brain GGT activity observed in chronic forms of hepatic encephalopathy. However, ammonia does not appear to activate the enzyme directly.