[Show abstract][Hide abstract] ABSTRACT: The purpose of this paper was to examine the function of N-methyl-D-aspartate (NMDA) glutamate receptor in cortical neurons on amino acid neurotransmitters release as well as the fraction of neurons implicated in the response of this receptor. Local stimulation of these cells at different concentrations of NMDA, agonist of this ionotropic glutamate receptor, produced a dose dependent release of aspartate, glutamate, glycine and GABA. These effects were blocked by DAP5, an antagonist of the NMDA receptor. The amino acid Ca(2+) dependent release mediated by the NMDA receptor, is induced by the opening of voltage-dependent Ca(2+) channels that this receptor promotes. Ca(++) movements were explored in single cells loaded with fura-2. When single cells were stimulated with 100 microM NMDA, the calcium recording performed showed that 82% of the cells responded to this agonist increasing the intracellular calcium concentration, although the amplitude of these increments was variable. The results suggest that NMDA-elicited neurotransmitter release from cortical neurons involves Ca(2+)-dependent and Ca(2+)-independent components, as well as neuron depolarisation, and different VDCC subtypes of N, P/Q or L depending of the amino acid neurotransmitter release elicited by this receptor.
Neurochemical Research 09/2010; 35(9):1478-86. DOI:10.1007/s11064-010-0209-0 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The involvement of plasma membrane glutamate transporters (EAATs - excitatory aminoacid transporters) in the pathophysiology of ischemia has been widely studied, but little is known about the role of vesicular glutamate transporters (VGLUTs) in the ischemic process. We analyzed the expression of VGLUT1-3 in the cortex and caudate-putamen of rats subjected to transient middle cerebral artery occlusion. Western blot and immunohistochemistry revealed an increase of VGLUT1 signal in cortex and caudate-putamen until 3 days of reperfusion followed by a reduction 7 days after the ischemic insult. By contrast, VGLUT2 and 3 were drastically reduced. Confocal microscopy revealed an increase in VGLUT2 and 3 immunolabelling in the reactive astrocytes of the ischemic corpus callosum and cortex. Changes in VGLUTs and EAATs expression were differently correlated to neurological deficits. Interestingly, changes in VGLUT1 and EAAT2 expression showed a significant positive correlation in caudate-putamen. Taken together, these results suggest a contribution of VGLUTs to glutamate release in these structures, which could promote neuroblast migration and neurogenesis during ischemic recovery, and a possible interplay with EAATs in the regulation of glutamate levels, at least in the first stages of ischemic recovery.
Journal of Neurochemistry 03/2010; 113(5):1343-55. DOI:10.1111/j.1471-4159.2010.06707.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Estrogens and phytoestrogens have neuroprotective effect against neuronal damage induced by cerebral ischemia /reperfusion (I/R) injury. In preceding studies, the phytoestrogen effects have been assessed by administration previous to the ischemic period, conditions which are unusual to apply to the treatment of human stroke. Here we present a study on neuroprotection afforded by genistein on rat embryo cortical neurons subjected to oxygen and glucose deprivation (OGD) followed by re-oxigenation, when added after the stress stimulus. At 1 and 2 h of OGD times and after 24 h of reperfusion, cell viability, necrotic, apoptotic and autophagic cell death and different parameters related to oxidative stress and mitochondrial dysfunction were measured in the absence and presence of 1 µM genisteine. We found an in-creasing loss of neuronal viability after 1-5 h of OGD which was only reversed in part by 24 h of reperfusion. These changes were preceded by increases in ROS generation, caspase-3 activation, LDH release and increase in LC3B lipi-dation, indicative of autophagia. Treatment with 1 µM genistein during the 24 h reperfusion significantly attenuated neuronal necrosis and autophagia induced by 1 and 2 h of OGD exposure. Genistein also decreased ROS generation and lipid-peroxidation induced by 2 h of OGD. These results suggest an important neuroprotective effect of genistein against transient post-ischemic-like conditions.
Neuroscience & Medicine 01/2010; 01(02). DOI:10.4236/nm.2010.12008
[Show abstract][Hide abstract] ABSTRACT: Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.
Free Radical Biology and Medicine 04/2006; 40(6):940-51. DOI:10.1016/j.freeradbiomed.2005.10.062 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this paper, we present data which demonstrate that, in cortical neurons, SNAP induces loss in cell viability as evaluated by the XTT test. This cell death started at 250 microM SNAP when the treatment was performed in a serum-free medium and at 10 microM when the treatment was given in the presence of serum. This death was mediated, at least in part, by an apoptotic mechanism detected by flow cytometry and DNA fractionation. The highest SNAP concentrations induced a dual behavior on caspase-3 activity. Concentrations of 250 microM in the absence of serum and 10 microM to 300 microM in the presence of serum produced caspase-3 activation. This indicates that NO induces neuronal death by an apoptotic mechanism in which the caspase pathway is implicated. Higher SNAP concentrations (500 microM to 1 mM) diminished the caspase-3 activity to levels similar or even lower than control values. This profile was observed in the absence as well as in the presence of serum in the medium. The caspase-3 inhibition mediated by the highest SNAP concentrations did not imply NO cellular protection since the caspase-3 inhibition mediated by these SNAP concentrations neither correlated with cellular viability nor with cellular apoptosis. The possible mechanism of caspase-3 inhibition at the highest SNAP concentrations used is discussed.
Brain Research 07/2005; 1047(2):168-76. DOI:10.1016/j.brainres.2005.04.044 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study is to determine whether differences in membrane potential and/or intracellular Ca2+ increments are implicated in a programmed release of amino acid neurotransmitters (aspartate, glutamate, glycine and GABA) in cortical neurons in culture. According to our results, it is possible to assume that difference in membrane potential is not the only signal which starts the amino acid neurotransmitter release, but there are other necessary conditions at the start of this amino acid release. One of these conditions could be the increment in intracellular Ca2+, but our results indicate that, in cortical neurons in culture, the total intracellular Ca2+ increments are not important on release levels, but are the stimulating agent which produces this intracellular Ca2+ increment. From these results we may infer: (1) that in rat cortical neurons there are neurons which contain and release glutamate, aspartate, glycine and GABA, (2) that in cortical neurons the 36.6 +/- 5.8% of the neurons are GABA-ergic, (3) that the membrane potential and the total intracellular calcium are not only responsible for the release of these amino acids but also the depolarizing agent which plays an important role in this release, and (4) that glutamate and aspartate and glutamate and GABA are localized in different vesicular pools or in different cell neurons.
Neurochemistry International 04/1998; 32(3):257-64. DOI:10.1016/S0197-0186(97)00088-0 · 3.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study differences in the biochemical properties of 4-aminobutyric acid aminotransferase (GABA-T) from forebrain and cerebellum were detected. These differences may be related to: a) the characteristics of the catalytic site, b) the substrate affinities and c) their pyridoxal-phosphate requirements which suggests that PLP could be a physiological regulator of these forms of brain GABA-T.
Neurochemical Research 08/1992; 17(7):703-6. DOI:10.1007/BF00968009 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1. Differences in the GDH activity of neuronal, glial cells and synaptosomes were detected. 2. The enzyme was measured in both directions: synthesis and degradation of glutamate. 3. Synaptosomes were the region with the highest GDH activity. 4. ADP plays an important role in the regulation of the reaction sense. 5. This effector produced higher activation on the enzyme measured in the direction of glutamate synthesis than in the sense of its degradation. 6. The enhancement produced by ADP was dependent on the enzyme localization. The ADP effect is discussed.
Comparative Biochemistry and Physiology Part C Comparative Pharmacology and Toxicology 02/1990; 97(2):265-7. DOI:10.1016/0742-8413(90)90139-Z
[Show abstract][Hide abstract] ABSTRACT: The effect of several ligands and Ca2+ ions on [3H]GABA binding to bovine adrenal medulla membranes was investigated. Without any blockade, the [3H]GABA binding showed two components, one of low affinity (Kd = 139 +/- 22 nM and Bmax = 3.2 +/- 0.4 pmol/mg protein) and the other of high affinity (Kd = 41 +/- 6 nM and Bmax = 0.35 +/- 0.26 pmol/mg protein). Muscimol specifically blocked low-affinity sites, and (-)baclofen blocked high-affinity components. Ca2+ ions were strictly necessary for maximum binding to high-affinity sites, whereas they did not significantly affect sites of the lower affinity. These results show that the bovine adrenal medulla has a GABAA receptor population of low affinity together with a GABAB receptor of high affinity.
Journal of Neuroscience Research 02/1988; 20(2):241-5. DOI:10.1002/jnr.490200213 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present paper we report the presence of succinic semialdehyde dehydrogenase (SSADH) in bovine adrenal medulla and blood platelets. Both enzymes present some analogies with the brain enzyme in terms of cofactor requirements, optimal pH, mitochondrial localizaton and inhibition by AMP. However, the activity of the platelet enzyme is 100 times lower than that of the brain and affinities of both enzymes for their specific substrate succinic semialdehyde and NAD are different. The presence of SSADH in adrenal medulla and blood platelets allows us to confirm the presence of a complete GABA bypass in these tissues, where the neurotransmitter could have important regulator functions.
[Show abstract][Hide abstract] ABSTRACT: 1. The data summarized in this report reveals the existence of GABA binding in the bovine adrenal medulla membranes. 2. Since this binding was displaced not only by muscimol and bicuculline but also by baclofen, results suggest the possibility that both types of receptors (GABAA and GABAB) could be present in bovine adrenal membranes.
Comparative Biochemistry and Physiology Part C Comparative Pharmacology and Toxicology 02/1987; 88(1):155-7. DOI:10.1016/0742-8413(87)90060-0
[Show abstract][Hide abstract] ABSTRACT: Experiments carried out in the absence or presence of GABA using a synaptosomal fraction from which endogenous GABA was as far as possibly eliminated, seem to indicate that both GABA receptors are involved in the chloride channel opening. This hypothesis is supported by results obtained in the presence of GABA agonist (muscimol) or drugs which are related to the complex GABA receptor-ionophore (diazepam and phenobarbital).
Revista española de fisiología 04/1986; 42(1):1-5.
[Show abstract][Hide abstract] ABSTRACT: 4-aminobutyrate-2-oxoglutarate aminotransferase (GABA-T) has been found in adrenal medulla. The enzyme from this tissue is very similar to those found in other tissues in respect to their mitochondrial localization, optima pH and responses to cofactor. The enzyme from medulla has substrate km values similar to those for the brain enzyme, while it differs from those found for other tissues such as kidney, liver and platelets.
Revista española de fisiología 10/1983; 39(3):299-303.
[Show abstract][Hide abstract] ABSTRACT: In this paper an analytical colorimetric method is presented where 2,4-dinitrophenyl hydrazine is used to measure small amounts of succinic semialdehyde in the presence of 2-oxoglutarate. This method is applicable to the measurement of 4-aminobutyrate-2-oxoglutarate aminotransferase activity, where succinic semialdehyde formed during the enzymatic reaction has to be measured in the presence of 2-oxoglutarate.
Revista española de fisiología 07/1983; 39(2):179-82.
[Show abstract][Hide abstract] ABSTRACT: Several aspects on regulation of rat brain glutamate dehydrogenase when the enzyme catalyses the reaction of glutamate degradation have been studied. The 2-oxo-glutarate is a competitive inhibitor against glutamate and NADH competes with NAD. The enzyme seems to have three sites of binding for glutamate, two of them bind the alpha and gamma carboxylic groups and the other the NH2-group of glutamate. Between the binding of two -COO- groups, the site on the enzyme which binds the gamma -COO- group of glutamate seems to be more important than the alpha one.
Revista española de fisiología 02/1982; 38 Suppl:45-9.
[Show abstract][Hide abstract] ABSTRACT: The effect of diphenylhydantoin (DPH), granatane-3-spiro-5'-hydantoin and leptazol has been checked on both GDH activity and ammonia and urea levels in brain. Concentrations of 90 mg/kg of leptazol decreased, significantly, the ammonium and urea levels in brain with respect to normal control. DPH and G-3SH had no effect on these metabolites against normal control. The association of DPH and leptazol decreased both urea and ammonium levels in brain but the association of G-3SH and leptazol decreased, significantly, urea levels but not ammonium's. DPH, leptazol at 90 mg/kg concentration and the association of DPH or G-3SH with leptazol had no significant effect on GDH activity while G-3SH increased the enzyme activity when it was measured in the direction of glutamate degradation. DPH, G-3SH and both concentrations of leptazol decreased the GDH activity when it was checked in the direction of glutamate synthesis. The association of DPH and leptazol did not produce any effects, while the association of G-3SH-leptazol increased the enzyme activity. DPH and G-3SH and leptazol at 110 mg/kg concentration decreased the relation of GDH activity between the biosynthetic and degradative sense. The association of G-3SH-leptazol increased this relation while leptazol and its association with DPH did not affect it.
Revista española de fisiología 07/1981; 37(2):205-10.
[Show abstract][Hide abstract] ABSTRACT: This paper presents data on the elimination of hydroxylamine from Lupinus albus seeds when they were germinated in the presence of GABA and hydroxylamine. The possibility of an enzymatic reaction. ATP dependent, between GABA and hydroxylamine is discussed. Some kinetic properties from this reaction are studied.
Revista española de fisiología 04/1979; 35(1):37-40.
[Show abstract][Hide abstract] ABSTRACT: Hydroxylamine was found to stimulate germination of Lupinus albus at concentrations inferior to 10 mM and to inhibit it greatly at 20 mM concentration. This inhibition was partially restored by GABA or succinic semialdehyde. Hydroxylamine, at high concentrations, behaved as inhibitor in vivo on GABA 2-oxyglutarate amino-transferase and succinic semialdehyde dehydrogenase NAD-dependent, whereas it behaved as activator on succinic semialdehyde dehydrogenase NADP-dependent. No effects were observed on the enzymatic activities and the inhibited germination was partially restored, after GABA and succinic semialdehyde had been added to a growth medium with a 20 mM hydroxylamine concentration. A possible protection mechanism of GABA and succinic semialdehyde against hydroxylamine action is discussed.
Revista española de fisiología 07/1976; 32(2):91-4.