2-Amino-7-Phosphonoheptanoic Acid Inhibits Insulin-Induced Convulsions and Striatal Aspartate Accumulation in Rats with Frontal Cortical Ablation

Pretreatment of rats with the excitatory amino acid antagonist 2-amino-7-phosphonoheptanoic acid (2-APH; 0.5 mmol/kg, i.p.) protected against insulin-induced clonic seizures. Complete protection was observed in 38% of the rats and partial protection in an additional 50%. Lesioning of the corticostriatal pathway by frontal cortical ablation caused decreases in the striatal levels of aspartate (-28%) and glutamate (-18%), an increase in striatal glutamine level (45%), and decreased high-affinity uptake of D-[3H]aspartate (-27%) in the lesioned dorsal neostriatum. Insulin-induced hypoglycemia caused a predicted sharp increase in aspartate level (165%) and decreased glutamate (-20%) and glutamine (-38%) levels in the intact striatum. Pretreatment of rats with 2-APH significantly reversed the insulin-induced changes in striatal aspartate, glutamate, and glutamine levels, especially in the intact hemisphere. In normoglycemic control rats, the "metabolic," i.e., concentration in the lesioned hemisphere, aspartate pool constituted 72% and the "synaptic," i.e., the concentration difference between the intact and lesioned hemispheres, 28% of the total striatal aspartate pool. 2-APH had no effect on the level of "metabolic" aspartate in the striata of normoglycemic rats but caused an almost complete suppression of "synaptic" aspartate. Following insulin-induced hypoglycemia, the "metabolic" aspartate pool doubled, whereas the "synaptic" aspartate pool increased 3.5-fold in the absence of 2-APH. The insulin-induced rise in "synaptic" aspartate level was almost completely blocked by 2-APH (a 5% rise instead of a 3.5-fold rise).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Regional Neurotransmitter Amino Acid Levels in Rat Brain During Seizures Induced by l-Allylglycine, Bicuculline, and Kainic Acid

Changes in amino acid concentrations were studied in the cortex, cerebellum, and hippocampus of the rat brain, after 20 min of seizure activity induced by kainic acid, 47 mumol/kg i.v.; L-allylglycine, 2.4 mmol/kg i.v.; or bicuculline, 3.27 mumol/kg i.v. in paralysed, mechanically ventilated animals. Metabolic changes associated with kainic acid seizures predominate in the hippocampus, where there are decreases in aspartate (-26%), glutamate (-45%), taurine (-20%), and glutamine (-32%) concentrations and an increase in gamma-aminobutyric acid (GABA) concentration (+ 26%). L-Allylglycine seizures are associated with generalized decreases in GABA concentrations (-32 to -54%), increases in glutamine concentrations (+10 to +53%), and a decrease in cortical aspartate concentration (-14%). Bicuculline seizures, in fasted rats, are associated with marked increases in the levels of hippocampal GABA (+106%) and taurine (+40%). In the cerebellum, there are increases in glutamine (+50%) and taurine concentrations (+36%). These changes can be explained partially in terms of known biochemical and neurophysiological mechanisms, but uncertainties remain, particularly concerning the cerebellar changes and the effects of kainic acid on dicarboxylic amino acid metabolism.     

Excitatory Amino Acid Receptors and Depolarization-Induced Ca2+ Influx into Hippocampal Slices

Hippocampal brain slices were incubated with depolarizing agents or excitatory amino acids either alone or in the presence of excitatory amino acid antagonists [omega-phosphonic alpha-aminocarboxylic acids--2-amino-4-phosphonobutyric acid (AP4), 2-amino-5-phosphonovaleric acid (AP5), or 2-amino-7-phosphonoheptanoic acid (AP7)--or gamma-D-glutamylaminomethylsulphonic acid (GAMS)] or a calcium-channel blocker, (S)-1-(3-methoxyphenyl)-3-methylaza-7-cyano-7-(3,4-dimethoxyphenyl )-8-methyl- nonane hydrochloride [(-)-D888]. The uptake of 45Ca2+ and the efflux of glutamate or aspartate induced by veratrine or high K+ was blocked (54-76%) by AP7 (IC50 46-250 microM). AP5 and AP4 were less effective. (-)-D888 (10 microM) caused 100% block of evoked 45Ca2+ uptake. Uptake of 45Ca2+ induced by exogenous glutamate, aspartate, and N-methyl-D-aspartate (NMDA) was also inhibited by AP7, whereas GAMS completely blocked the action of kainate and partially blocked that of glutamate. The action of NMDA in stimulating 45Ca2+ uptake was Mg2+-sensitive, low Mg2+ levels in the incubation medium selectively enhancing the response. It is concluded that Ca2+ uptake evoked by excitatory amino acids is receptor-mediated, and that released excitatory amino acids are responsible for a large part of the action of veratrine and high K+ in stimulating 45Ca2+ uptake.     

Inhibition of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis in the Neonatal Rat Hippocampus by 2-Amino-3-Phosphonopropionate

The effects of excitatory amino acid agonists and alpha-amino-omega-phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7-day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, L-glutamate, L-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, L-homocysteate, and kainate. N-Methyl-D-aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by L-2-amino-4-phosphonobutyrate (10(-3) M), but this antagonist had no effect on L-glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate. At 10(-3) M, D,L-2-amino-3-phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited L-glutamate stimulation, and had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-, kainate-, or carbachol-induced [3H]inositol monophosphate formation. Concentration-effect experiments showed D,L-2-amino-3-phosphonopropionate to be five times more potent as an antagonist of ibotenate-stimulated phosphoinositide hydrolysis than L-2-amino-4-phosphonobutyrate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L-2-amino-3-phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L-2-amino-3-phosphonopropionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.     

Comparison of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis and N-[3H]Acetylaspartylglutamate Binding in Rat Brain: Selective Inhibition of Phosphoinositide Hydrolysis by 2-Amino-3-Phosphonopropionate

Abstract: The activation of phosphoinositide hydrolysis by ibotenate (IBO) in brain slices and the binding of N -[³H]acetylaspartyl-L-glutamate (NAAG) to brain membranes are biochemical parameters previously shown to be selectively inhibited by 2-amino-4-phosphonobutyrate (AP4). We have examined whether the binding of [³H]NAAG and stimulation of phosphoinositide hydrolysis by IBO are indexing the same or different populations of AP4-sensitive excitatory amino acid sites in brain. L-AP4 and D,L-2-amino-3-phosphono-propionate (D,L-AP3) were found to be about equipotent inhibitors of IBO-stimulated phosphoinositide hydrolysis. L-AP4 and D,L-AP3 did not inhibit stimulation of phosphoinositide hydrolysis by the cholinoceptor agonist carbachol. The L-isomers of serine- O -phosphate and α-aminoadipate were selective inhibitors of IBO-stimulated phosphoinositide hydrolysis, but were less potent than L-AP4 or D,L-AP3. When these compounds were examined for their ability to inhibit [³H]NAAG binding to membranes of rat forebrain, the relative order of potency was L-α-aminoadipate = D-α-aminoadipate < L-AP4 < L-serine- O -phosphate < D-AP4 < D,L-AP3. Concentrations of NAAG up to 10⁻² M did not stimulate phosphoinositide hydrolysis. Thus, although both assays are sensitive to L-AP4 inhibition, they appear to represent disparate excitatory amino acid sites in brain. Furthermore, D,L-AP3 appears to be a more selective inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis than L-AP4, and might be a more useful pharmacological tool to define the function of these receptor sites in brain.     

Stimulation of Dopamine Release from Cultured Rat Mesencephalic Cells by Naturally Occurring Excitatory Amino Acids: Involvement of Both N-Methyl-D-Aspartate (NMDA) and Non-NMDA Receptor Subtypes

In rat mesencephalic cell cultures, L-glutamate at concentrations ranging from 100 microM to 1 mM stimulated release of [3H]dopamine that was attenuated by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxalinedione, but not by the selective NMDA receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 10 microM) and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (300 microM). Even at 1 mM glutamate, this release was Ca2+ dependent. These observations suggest that the release was mediated by a non-NMDA receptor. Only release stimulated by a lower concentration (10 microM) of glutamate was inhibited by MK-801 (10 microM), indicating that glutamate at this concentration activates the NMDA receptor. By contrast, L-aspartate at concentrations of 10 microM to 1 mM evoked [3H]dopamine release that was completely inhibited by MK-801 (10 microM) and was also Ca2+ dependent (tested at 1 and 10 mM aspartate). Thus, effects of aspartate involved activation of the NMDA receptor. Sulfur-containing amino acids (L-homocysteate, L-homocysteine sulfinate, L-cysteate, L-cysteine sulfinate) also evoked [3H]dopamine release. Release evoked by submillimolar concentrations of these amino acids was attenuated by MK-801 (10 microM), indicating involvement of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Astrocyte Heterogeneity: Endogenous Amino Acid Levels and Release Evoked by Non-N-Methyl-D-Aspartate Receptor Agonists and by Potassium-Induced Swelling in Type-1 and Type-2 Astrocytes

The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.     

In Vitro and In Vivo Pharmacology of trans- and cis-(±)-1-Amino-1,3-Cyclopentanedicarboxylic Acid: Dissociation of Metabotropic and Ionotropic Excitatory Amino Acid Receptor Effects

This study explored further the function of the metabotropic excitatory amino acid receptor in the rat brain. The trans and cis isomers of (+-)-1-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were characterized for relative affinities at ionotropic and metabotropic excitatory amino acid receptors in vitro, as well as ability to produce in vivo excitatory or excitotoxic effects in rats. trans-ACPD was about 12 times more potent in vitro as an agonist for metabotropic excitatory amino acid receptors when compared to its ability to displace N-methyl-D-aspartate (NMDA) ([3H]CGS-19755) receptor binding, cis-ACPD was about 30 times more potent as a displacer of [3H]CGS-19755 binding than as a stimulant of phosphoinositide hydrolysis. When administered intraperitoneally to neonatal rats, both cis- and trans-ACPD produced convulsions that were prevented by the competitive NMDA receptor antagonists, LY233053 and LY274614. cis-ACPD was six times more potent as a convulsant when compared to trans-ACPD. Both compounds were examined for excitotoxic effects in vivo following stereotaxic injection into the mature or neonatal rat striatum. Doses of trans-ACPD of up to 5,000 or 1,200 nmol produced few signs of striatal neuronal degeneration in the mature or neonatal brain, respectively. However, cis-ACPD produced extensive dose-related neuronal degeneration at doses of 100-1,000 nmol in the mature brain and 50-200 nmol in the neonatal brain. These studies suggest that, unlike the ionotropic excitatory amino acid receptors, activation of the metabotropic excitatory amino acid receptor does not result directly in excitatory effects, such as excitotoxicity.     

Solubilization and Partial Purification of α-Amino-3- Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites from Rat Brain

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X-100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane-bound sites. Both the solubilized and the membrane-bound preparations contained high- and low-affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane-bound preparations. [3H]AMPA binding to both soluble and membrane-bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54-fold with ion-exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.     

Evidence for Metabotropic Excitatory Amino Acid Receptor Heterogeneity: Developmental and Brain Regional Studies

To examine whether multiple subtypes of the excitatory amino acid (EAA) receptor coupled to phosphoinositide (PPI) hydrolysis exist, we have pharmacologically characterized the PPI response in neonatal and adult rat brain. Activation of PPI hydrolysis was determined by the accumulation of [3H]inositol monophosphate in brain slices prelabeled with [3H]inositol. In neonatal hippocampus, D,L-2-amino-3-phosphonopropionic acid (AP3; 1 mM) inhibited the cis-1-aminocyclopentane-1,3-dicarboxylic acid (IUPAC nomenclature; ACPD; 100 microM)- and quisqualate (Quis; 100 microM)-stimulated PPI hydrolysis by 73 and 66%, respectively, but had no effect in neonatal cerebellum. In adult hippocampus, AP3 stimulated PPI hydrolysis with potency and efficacy comparable to those of Quis and ACPD and completely masked the Quis concentration-response curve. In adult cerebellum, only Quis behaved as a full agonist on the PPI response. The Quis concentration-response curve was shifted rightward with a fourfold decrease in potency in the presence of ACPD (5 mM), whereas it was nearly additive with the PPI response induced by AP3 (5 mM). Thus, our data reveal significant developmental and brain regional differences in metabotropic EAA receptor responses and support the notion that this receptor is heterogeneous, in both a regionally specific and a developmentally dependent manner.     

Amino Acid Neurotransmitters in Nucleus Tractus Solitarius: An In Vivo Microdialysis Study

Amino acid neurotransmitters in the nucleus tractus solitarius (NTS) are thought to play a key role in the mediation of visceral reflexes and glutamate has been proposed as the neurotransmitter of visceral afferent nerves projecting to this region. The present studies sought to characterize the use of in vivo microdialysis to examine extracellular fluid levels of amino acids in the NTS of anesthetized rats. Using a microdialysis probe that was 450 μm in length and a sensitive HPLC assay for amino acids, amino acids could be measured in dialysate samples collected from the NTS. Perfusion of the microdialysis probe with 60 mM K^±, to elicit depolarization of nerve terminals in the vicinity of the probe, resulted in increased dialysate fluid levels of aspartate, glutamate, glycine, taurine, and GABA. In contrast, glutamine and tyrosine were decreased and other amino acids were not significantly affected. Prior removal of the ipsilateral nodose ganglion did not alter the K^±-evoked changes in dialysate levels of any of these amino acids. Electrical stimulation of the vagus nerves, using a variety of stimulus parameters, did not significantly alter dialysate levels of glutamate or any of the other amino acids that were measured. Blockade of glutamate uptake with dihydrokainate increased dialysate levels of glutamate, aspartate, and GABA, but in the presence of dihydrokainate vagal stimulation did not alter dialysate levels of these amino acids. The results show that in vivo microdialysis can be used to examine amino acid efflux in the rat NTS and provide further evidence for amino acidergic neural transmission in the NTS. However, these studies fail to support the hypothesis that vagal afferents release glutamate or aspartate.     

Effect of Acute Stress on Hippocampal Glutamate Levels and Spectrin Proteolysis in Young and Aged Rats

Abstract: Aging in rats is associated with a loss of hippocampal neurons, which may contribute to age-related cognitive deficits. Several lines of evidence suggest that stress and glucocorticoids may contribute to age-related declines in hippocampal neuronal number. Excitatory amino acids (EAAs) have been implicated in the glucocorticoid endangerment and stress-induced morphological changes of hippocampal neurons of young rats. Previously, we have reported that acute immobilization stress can increase extracellular concentrations of the endogenous excitatory amino acid, glutamate, in the hippocampus. The present study examined the effect of an acute bout of immobilization stress on glutamate levels in the hippocampus and medial prefrontal cortex of young (3–4-month) and aged (22–24-month) Fischer 344 rats. In addition, the effect of stress on spectrin proteolysis in these two brain regions was also examined. Spectrin is a cytoskeleton protein that contributes to neuronal integrity and proteolysis of this protein has been proposed as an important component of EAA-induced neuronal death. There was no difference in basal glutamate levels between young and old rats in the hippocampus or medial prefrontal cortex. During the period of restraint stress a modest increase in glutamate levels in the hippocampus of young and aged rats was observed. After the termination of the stress procedure, hippocampal glutamate concentrations continued to rise in the aged rats, reaching a level approximately five times higher than the young rats, and remained elevated for at least 2 h after the termination of the stress. A similar pattern was also observed in the medial prefrontal cortex with an augmented post-stress-induced glutamate response observed in the aged rats. There was no increase in spectrin proteolysis in the hippocampus or medial prefrontal cortex of young or aged rats after stress or under basal nonstress conditions. The enhanced poststress glutamate response in the aged rats may contribute to the increased sensitivity of aged rats to neurotoxic insults.     

Decreases in Amino Acid and Acetylcholine Metabolism During Hypoxia

Abstract: Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucosederived amino acids [alanine, aspartate, γ-amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U-¹⁴C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO₂), hypoxic hypoxia (15 or 10% O₂), and hypoxic hypoxia plus hypercarbia (15 or 10% O₂ with 5% CO₂). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO₂) decreased the incorporation of [U-¹⁴C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9). glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO₂) of anemic hypoxia showed a parallel decrease in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r= 0.98), glutamate (r= 0.99), aspartate (r= 0.96), and glutamine (r= 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r= 0.68) and alanine (r= 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O₂) or hypoxic hypoxia with 5% CO₂ were very similar to those with the two lowest levels of anemic hypoxia. Thus, any explanation of the brain's sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.     

Solubilization and Purification of an α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Protein from Bovine Brain

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X-100 in 0.5 M phosphate buffer and 20% glycerol at 37 degrees C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high- and low-affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr approximately 500,000. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr = 110,000. Previously, we have shown that a monoclonal antibody (KAR-B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr approximately 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.     

Excitatory Amino Acid Agonist-Antagonist Interactions at 2-Amino-4-Phosphonobutyric Acid-Sensitive Quisqualate Receptors Coupled to Phosphoinositide Hydrolysis in Slices of Rat Hippocampus

Studies were carried out to define the relative affinities and intrinsic activities of excitatory amino acid agonists that activate receptor sites coupled to phosphoinositide hydrolysis in brain. Slices of rat hippocampus were prelabeled with myo-[3H]inositol, and agonist stimulation was indexed by measuring the accumulation of [3H]inositol monophosphate [( 3H]IP) in the presence of Li+. It was observed that ibotenic (IBO) and quisqualic (QUIS) acids both elicit highly significant, concentration-dependent stimulation of phosphoinositide hydrolysis. Whereas maximal stimulation by IBO (10(-3) M) was four- to fivefold over basal values, the maximal effect of QUIS (10(-4) M) was less (about twofold). Based on the relative concentrations required for 50% maximal stimulation, QUIS was 20 times more potent than IBO. Stimulation of phosphoinositide hydrolysis by either IBO or QUIS was additive to the effects of nonexcitatory amino acid agonists (carbachol and norepinephrine) in this tissue. However, the stimulatory effects of IBO plus QUIS were not additive. At greater than or equal to 10(-4) M, QUIS significantly inhibited phosphoinositide hydrolysis by a maximal stimulatory concentration of IBO (10(-3) M) to a level observed with QUIS alone. Other excitatory amino acid agonists, including kainate, N-methyl-D-aspartate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), had no stimulatory effects at concentrations as high as 10(-3) M. The D,L or L forms of 2-amino-4-phosphonobutyric acid (AP4), but not D-AP4, significantly enhanced [3H]IP levels to approximately 135% of basal values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of Excitatory Amino Acids from Cultured Hippocampal Astrocytes Induced by a Hypoxic-Hypoglycemic Stimulation

An excess release of excitatory amino acids (EAA) is an important factor for postischemic brain damage. In the present communication, we demonstrate that cultured hippocampal cells release EAA after hypoxic-hypoglycemic treatment. The amounts of EAA released from astrocytes were appreciably above those released from neurons. Furthermore, the amount of aspartate released from astrocytes was comparable to that of glutamate, although the endogenous content of aspartate was one-fifth that of glutamate. The endogenous content of aspartate in astrocytes increased even after hypoxic-hypoglycemic treatment. These results suggests that ischemic neuronal death is due, at least in part, to the excitotoxicity of aspartate and glutamate derived from surrounding astrocytes.     

Solubilization of Kainic Acid Binding Sites from Rat Brain

Kainic acid binding sites were solubilized from rat brain using a combination of Triton X-100 and digitonin. The highest percentage of solubilized binding sites (45%) was obtained by treating brain membranes with 1% Triton-X-100 and 0.2% digitonin in 0.5 M potassium phosphate containing 20% glycerol. The solubilized binding sites were stable and amenable to analysis by gel filtration and lectin affinity chromatography. Computer assisted analyses demonstrated that the solubilized sites displayed high- and low-affinity binding constants similar to the membrane-bound sites. Competition experiments further supported the pharmacological similarities of the solubilized and membrane-bound sites. Gel filtration chromatography of the solubilized binding site indicated that the detergent-bound complex had a Stokes radius of 82.7 A. The [3H]kainic acid binding site appears to be glycosylated based on its capability to bind to lectins. The lectin, wheatgerm agglutinin, proved to be a potentially useful tool for characterization because the solubilized binding sites were bound and eluted in relatively high yield.     

Administration of Vigabatrin (γ-Vinyl-γ-Aminobutyric Acid) Affects the Levels of Both Inhibitory and Excitatory Amino Acids in Rat Cerebrospinal Fluid

The effect of vigabatrin (gamma-vinyl-gamma-aminobutyric acid), a new anticonvulsant drug, on the transmitter amino acids in rat cisternal CSF was studied. CSF was collected through a permanently implanted polyethylene cannula from freely moving rats at 5, 24, 48, and 96 h after administration of 1,000 mg/kg of vigabatrin. The free gamma-aminobutyric acid (GABA) level was elevated maximally (13.5-fold; p less than 0.01) at 24 h after injection. The homocarnosine (GABA-histidine) level also was increased (123%; p less than 0.01) at 24 h after injection, and its concentration remained at the same level for the next 3 days. Glycine and taurine concentrations had increased [31% (p less than 0.05) and 63% (p less than 0.01), respectively] at 5 h after injection. It is interesting that the levels of glutamate and aspartate increased [330% (p less than 0.05) and 421% (p less than 0.01), respectively] at 96 h after injection, the time when the free GABA level had returned to the baseline concentration and the vigabatrin level was 3% of the maximal concentration. The present study indicates that a single dose of vigabatrin in rats elevates levels of both the inhibitory and excitatory amino acids in CSF. However, the temporal profile of observed changes in relation to vigabatrin injection shows that neither the long-lasting elevation of GABA content nor the increase in glutamate and aspartate levels correlates with the level of vigabatrin in CSF. These findings suggest that the excitatory mechanisms are also augmented following acute administration of vigabatrin, especially when the content of GABA had decreased to the baseline level and the level of vigabatrin was low.     

2-Amino-4-Phosphonobutyric Acid Exerts a Light-Dependent Effect on Post-Gabaculine Levels of Retinal γ-Aminobutyric Acid (GABA): Evidence that ON Synaptic Pathways Regulate Retinal GABAergic Transmission

Abstract: The effects of light, 2-amino-4-phosphonobutyric acid (APB), and kainic acid on rat retinal γ-aminobutyric acid (GABA)-ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA-transaminase. Post-gabaculine levels of retinal GABA in light-exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post-gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post-gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light-dependent increment in post-gabacu line accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post-gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either ON or ON-OFF amacrine cells.