Autoreceptor Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.     

Glutamate-Induced 45Ca2+ Uptake into Immature Cerebral Cortex Neurons Shows a Distinct Pharmacological Profile

Glutamate-induced 45Ca2+ uptake was studied in cerebral cortex neurons cultured for 4 days, i.e., at a developmental stage where the neurons are sensitive to the mixed agonist glutamate but not to the actions of N-methyl-D-aspartate or other excitatory amino acids. Using this experimental approach, allowing the investigation of effects elicited only by glutamate, it was demonstrated that the glutamate-stimulated Ca2+ influx could be completely antagonized by MK-801, phencyclidine, and cyclazocine in the nanomolar range, and by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate and D-2-amino-5-phosphonopentanoate (APV) in the low micromolar range. However, the glutamate response was unaffected by variations in the Mg2+ concentration in the exposure media. In addition, the two quinoxalinediones 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione were equipotent with APV in blocking the glutamate-stimulated Ca2+ uptake. PK 26124 blocked the response in the high micromolar concentration range. Ketamine and gamma-glutamylaminomethylsulfonate were essentially without effect at concentrations up to 10 microM and 300 microM, respectively. These results may suggest the existence of a glutamate receptor with a pharmacological profile not compatible with the existent classification of glutamate receptor subtypes.     

Release of γ-Amino[3H]butyric Acid from Cultured Amacrine-Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.     

Glutamate Release from Guinea-Pig Synaptosomes: Stimulation by Reuptake-Induced Depolarization

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.     

Effects of Ionotropic Excitatory Amino Acid Receptor Antagonists on Glutamate Transport and Transport-Mediated Changes in Extracellular Excitatory Amino Acids in the Rat Striatum

Abstract: This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High-affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, or dl -2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10–500 pmol injected). DNQX induced a dose-dependent increase in glutamate uptake rate, related to an increase in the V _max of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10⁻⁵ M ) in the dialysis probe diminished by ∼30–40% the increases in the concentrations of glutamate and aspartate elicited by l - trans -pyrrolidine-2,4-dicarboxylic acid (1 m M ). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non-NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.     

N-methyl-d-aspartate andl-aspartate activate distinct receptors in piriform cortex

The effects of ionophoretically applied N-methyl-DL-aspartate (NMDA) and aspartate on identified pyramidal neurons in rat piriform cortex were examined in isolated, submerged, and perfused brain slices. NMDA was more potent than aspartate in eliciting neuronal discharge. Perfusion of the acidic amino acid antagonists, DL-2-amino-5-phosphonovalerate (APV), 10(-6) or 10(-5) M, DL-2-amino-7-phosphonoheptanoate (APH), 10(-5) M, and gamma-D-glutamylglycine (gamma DGG), 10(-5) M, selectively blocked the response to NMDA without effect on the response to aspartate. At higher concentrations which blocked responses to both NMDA and aspartate, gamma DGG blocked kainate responses and depressed glutamate and quisqualate responses. These results suggest that in piriform neurons NMDA and aspartate act at distinct receptor sites, not a common receptor site, and that both of these sites are distinct from those that mediate responses to glutamate, quisqualate, and kainate.     

Depression by Sodium Ions of Calcium Uptake Mediated by Non-N-Methyl-d-Aspartate Receptors in Cultured Cerebellar Neurons and Correlation with Evoked d-[3H]Aspartate Release

In a previous study we noted that the release of D-[3H]aspartate evoked by non-N-methyl-D-aspartate (non-NMDA) receptor agonists in cultured rat cerebellar granule cells was enhanced in the absence of extracellular Na+. To explain this apparent paradox, we tried in the present investigation to correlate the effect of Na+ removal on the kainate (KA)- and quisqualate (QA)-induced D-[3H]aspartate release with that on KA- and QA-induced 45Ca2+ accumulation. The releasing activity of KA, which was only partially Ca2+ dependent in the presence of Na+, became totally Ca2+ dependent in its absence. Moreover, the releasing activity of QA, which was Ca2+ independent in the presence of Na+, became 50% Ca2+ dependent in the absence of the monovalent cation. The releasing action of both agonists was in all cases antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and that induced by KA was also sensitive to kynurenic acid. When glutamate was tested as an agonist in the presence of Na+, it was found that its D-[3H]aspartate releasing action was Ca2+ independent and was largely due to heteroexchange. The evoked release was Ca2+ independent, scarcely sensitive to CNQX, and insensitive to NMDA antagonists. In Na(+)-free medium, the glutamate-evoked D-[3H]aspartate release was lower (due to the abolishment of heteroexchange), but was totally Ca2+ dependent and antagonized by CNQX and kynurenate. KA (30 microM-1 mM) stimulated the accumulation of 45Ca2+ in a dose-dependent and CNQX-sensitive way, the effect being progressively higher as the Na+ concentration in the medium was decreased. Li+ affected KA-induced 45Ca2+ accumulation in a way similar to Na+, although 45Ca2+ uptake was somewhat lower in Li(+)-containing medium. The voltage-activated calcium channel antagonists La3+ and (-)-202-791 caused only a limited inhibition of the KA-induced 45Ca2+ influx both in the presence and in the absence of Na+. Under all the conditions tested [presence and absence of Na+ and of (-)-202-791], the kainate-induced 45Ca2+ uptake was scarcely sensitive to the NMDA antagonist 2-amino-5-phosphonovalerate. QA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid also stimulated 45Ca2+ influx in a CNQX-sensitive way, the effect being enhanced in Na(+)-free media. These agonists were, however, less effective than KA.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-Methyl-D-Aspartate Releases Excitatory Amino Acids in Rat Corpus Striatum In Vivo

There is a considerable amount of conflicting evidence from several studies as to the action of applied N-methyl-D-aspartate (NMDA) on the release of glutamate and aspartate in the brain. In the present study the effect of NMDA on extracellular levels of endogenous amino acids was investigated in conscious, unrestrained rats using intracerebral microdialysis. NMDA caused dose-related increases in extracellular levels of glutamate and aspartate; threonine and glutamine were unaffected. The NMDA-evoked release of glutamate and aspartate was significantly decreased by the specific NMDA receptor antagonist 3-[(+-)-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid. In addition, increasing the perfusate concentration (and therefore the extracellular concentration) of Ca2+ significantly enhanced the NMDA-evoked release of glutamate and aspartate, whereas removal of Ca2+ and addition of a high Mg2+ concentration to the perfusate caused a significant reduction in their NMDA-evoked release. Moreover, the NMDA-evoked release of glutamate and aspartate was reduced in decorticate animals. These results demonstrate that, in the striatum in vivo, NMDA causes selective release of endogenous glutamate and aspartate from neurone terminals and that this action occurs through an NMDA receptor-mediated mechanism. The ability of NMDA receptor activation to induce release of glutamate and aspartate, perhaps by a positive feedback mechanism, may be relevant to the pathologies underlying epilepsy and ischaemic and hypoglycaemic brain damage.     

Polyamines in Human Brain: Regional Distribution and Influence of Aging

Abstract: Depolarization of adult rat forebrain slices with veratrine induced the release of excitatory amino acids (glutamate and aspartate), the synthesis of nitric oxide (NO), and increases in cyclic GMP (cGMP). The NO synthase inhibitors N ^ω-monomethyl- l -arginine and N ^ω-nitro- l -arginine methyl ester decreased the release of NO and the levels of cGMP without affecting the release of excitatory amino acids. In contrast, the antiepileptic drug lamotrigine inhibited the release of excitatory amino acids and of NO, and decreased the levels of cGMP without causing a significant direct inhibition of the NO synthase. Furthermore, the synthesis of NO and the increases in cGMP induced by veratrine were partially blocked by the N -methyl- d -aspartate (NMDA) receptor antagonist MK-801 but not by 6-nitro-7-sulphamobenzo( f )quinoxaline-2,3-dione, a non-NMDA receptor antagonist. Neither of these compounds inhibited directly the NO synthase or the release of excitatory amino acids. Thus, these three types of compound act as an inhibitor of voltage-sensitive sodium channels (lamotrigine), as a receptor antagonist (MK-801), or as direct inhibitors of the NO synthase, to block the pathway leading to increased cGMP after veratrine depolarization. It is likely that some of the pharmacological and therapeutic actions shared by these three types of compound are, at least in part, a consequence of inhibition of the synthesis of NO.     

Possible role of cGMP in excitatory amino acid induced cytotoxicity in cultured cerebral cortical neurons

Using cultured cerebral cortical neurons at mature stages (9 days in culture, d.i.c.) it was demonstrated that glutamate, NMDA (N-methyl-D-aspartate) and to a lesser extent KA (kainate) increase the intracellular cGMP concentration ([cGMP]_i) whereas no such effect was observed after exposure of the cells of QA (quisqualate) and AMPA (2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate). No effect of glutamate, NMDA and KA was observed in immature neurons (2 d.i.c.). The pharmacology of these cGMP responses was investigated using the glutamate antagonists APV (2-amino-5-phosphonovalerate) with selectivity for NMDA receptors, CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) with selectivity for non-NMDA receptors and the novel KA selective antagonists AMOA (2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate) and AMNH (2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3-oxoisoxazolin-4-yl]propionate). In addition, the cytotoxicity of glutamate, NMDA and KA was studied and found to be enhanced by addition of the non-metabolizable cGMP analogue 8-Br-cGMP. On the contrary, the toxicity of QA and AMPA was not affected by 8-Br-cGMP. Pertussis toxin augmented the toxicity elicited by glutamate, NMDA, KA and QA but not that induced by AMPA. On the other hand, only glutamate and KA induced toxicity was potentiated by cholera toxin, which also enhanced the stimulatory effect of glutamate and NMDA but not that of KA on the cellular cGMP content. The toxicity as well as the effects on intracellular cGMP levels could be antagonized by the specific excitatory amino acid (EAA) antagonists. These results suggest that the mechanisms by which the various excitatory amino acids exert cytotoxicity are different, and that increased cGMP levels may participate in the mediation of glutamate, NMDA or KA induced toxicity but less likely in QA and AMPA mediated toxicity. Furthermore, G-proteins or other pertussis or cholera toxin sensitive entities seem to be involved in the cytotoxic action of all excitatory amino acids except AMPA.     

Role of Excitatory Amino Acid Receptors in K+-and Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Slices

K+ and glutamate released endogenous adenosine from superfused slices of rat parietal cortex. The absence of Ca2+ markedly diminished K+- but not glutamate-evoked adenosine release. Tetrodotoxin decreased K+- and glutamate-evoked adenosine release by 40 and 20%, respectively, indicating that release was mediated in part by propagated action potentials in the slices. Inhibition of ecto-5'-nucleotidase by alpha,beta-methylene ADP and GMP decreased basal release of adenosine by 40%, indicating that part of the adenosine was derived from the extracellular metabolism of released nucleotide. In contrast, inhibition of ecto-5'-nucleotidase did not affect release evoked by K+ or glutamate, suggesting that adenosine was released as such. Inhibition of glutamate uptake by dihydrokainate potentiated glutamate-evoked release of adenosine. Glutamate-evoked adenosine release was diminished 50 and 55% by the N-methyl-D-aspartate (NMDA) receptor antagonists, DL-2-amino-5-phosphonovaleric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), respectively. The remaining release in the presence of MK-801 was diminished a further 66% by the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione, suggesting that both NMDA and non-NMDA receptors were involved in glutamate-evoked adenosine release. Surprisingly, K+-evoked adenosine release was also diminished about 30% by NMDA antagonists, suggesting that K+-evoked adenosine release may be partly mediated indirectly through the release of an excitatory amino acid acting at NMDA receptors.     

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca(2+)-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3- dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.     

Glutamate-induced increases in intracellular Ca2+ in cultured frog tectal cells mediated by direct activation of NMDA receptor channels.

Influx of Ca2+ through NMDA channels may initiate the stabilization of coactive synapses during development of the retinotectal projection in frogs. Ca2+ imaging techniques were applied to cultured tectal cells to investigate whether excitatory amino acids cause a rise in [Ca2+]i. High [K+], NMDA, and glutamate increase [Ca2+]i in about 75% of the cells. NMDA and glutamate responses were completely blocked in the absence of extracellular Ca2+ and by the NMDA receptor or channel blockers APV and MK-801. The NMDA response was also blocked by Mg2+. Quisqualate and kainate produced little or no rise in [Ca2+]i. These studies indicate that when tectal cells are exposed to the retinal ganglion cell transmitter glutamate, the predominant means of Ca2+ entry is through NMDA channels.     

[3H]Norepinephrine Release from Hippocampal Slices Is an In Vitro Biochemical Tool for Investigating the Pharmacological Properties of Excitatory Amino Acid Receptors

[3H]Norepinephrine ([3H]NE) efflux from preloaded rat hippocampal slices was increased in a dose-dependent manner by excitatory amino acids, with the following order of potencies: N-methyl-D-aspartate (NMDA) greater than kainic acid (KA) greater than L-glutamate greater than or equal to D,L-homocysteate greater than L-aspartate greater than quinolinic acid greater than quisqualic acid. The effect of the excitatory amino acids was blocked by physiological concentrations of Mg2+, with the exception of KA. D,L-2-Amino-7-phosphonoheptanoic acid dose-dependently inhibited the NMDA effect (ID50 = 69 microM), whereas at 1 mM it was ineffective versus KA. The release of [3H]-NE induced by quinolinic acid was blocked by 0.1 mM D,L-2-amino-7-phosphonohepatanoic acid. gamma-D-Glutamylglycine dose-dependently inhibited the KA effect with an ID50 of 1.15 mM. Tetrodotoxin (2 microM) reduced by 40 and 20% the NMDA and KA effects, respectively. The data indicate that [3H]NE release from hippocampal slices can be used as a biochemical marker for pharmacological investigations of excitatory amino acid receptors and their putative agonists and antagonists.     

Magnesium-Dependent Inhibition of Agonist-Stimulated Phosphoinositide Breakdown in Rat Cortical Slices by Excitatory Amino Acids

The excitatory amino acid agonists kainate, N-methyl-D-aspartate (NMDA), and quisqualate inhibited ligand-stimulated phosphoinositide hydrolysis in rat cortical slices. The NMDA channel blocker MK-801 antagonized the inhibition by NMDA but had no effect on the inhibition due to kainate or quisqualate. The antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the effects of quisqualate and kainate but not the effect of NMDA. These data indicate that activation of the NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate types of ionotropic receptors has the same effect. In membranes prepared from cortical slices, there was no inhibition of carbachol-stimulated phosphoinositidase C activity by excitatory amino acids, suggesting that excitatory amino acids indirectly affect carbachol-stimulated phosphoinositide hydrolysis. The inhibition by excitatory amino acids of carbachol-stimulated phosphoinositide breakdown was dependent on extracellular Mg2+ and was abolished by procedures that increase intracellular Ca2+. Veratridine inhibition of carbachol-stimulated phosphoinositide hydrolysis was reversed by ouabain but not by other procedures that increase intracellular Ca2+. In contrast to excitatory amino acids, veratridine potentiated carbachol-stimulated phosphoinositide breakdown in the presence of 10 mM extracellular Mg2+. These data suggest that excitatory amino acids inhibit carbachol-stimulated phosphoinositide breakdown in rat cortex by lowering intracellular Ca2+ through a mechanism dependent on extracellular Mg2+.     

Effect of 2-Amino-7-Phosphonoheptanoic Acid on Regional Brain Amino Acid Levels in Fed and Fasted Rodents

Abstract: 2-Amino-7-phosphonoheptanoic acid, an antagonist of excitation caused by dicarboxylic amino acids with a selective action on N -methyl-d-aspartate receptors, has been administered in an anticonvulsant dose (1 mmol/kg i.p.) to fed or fasted rats and mice. The drug impaired motor activity in fasted mice. Glucose and amino acids were determined in dissected regions of brain fixed by microwave irradiation. Glucose content was low in the brains of fasted rats and mice but was restored to normal (fed) concentration 45 min after the administration of 2-amino-7-phosphonoheptanoic acid in fasted mice. In fed animals, 2-amino-7-phosphonoheptanoic acid did not change brain aspartate concentration. In fasted animals, aspartate concentration was raised in most brain regions. In fasted rats and mice, 2-amino-7-phosphonoheptanoic acid significantly increased glutamine in rat cortex and mouse striatum, decreased glutamate content in rat striatum, and decreased aspartate concentration in all regions except mouse cortex and striatum. GABA levels were significantly decreased in rat striatum and hippocampus. These changes are consistent with an increased synaptic release of glutamate and aspartate following blockage of their post-synaptic action at selected sites.     

Modulation of Glutamate and Aspartate Release from Slices of Hippocampal Area CA1 by Inhibitors of Arachidonic Acid Metabolism

Abstract: Slices of hippocampal area CA1 were used to test inhibitors of arachidonic acid metabolism for their effects on glutamate/aspartate release from the CA3-derived Schaffer collateral, commissural, and ipsilateral associational terminals. Test compounds [3 µ M nordihydroguaiaretic acid (NDGA) and 1 µ M 3-[3-(4-chlorobenzyl)-3- tert -butylthio-5-isopropylindol-2-yl]-2,2-dimethyl-propanoic acid (MK-886)] that reduced the production and release of 5-lipoxygenase metabolites also selectively reduced the K⁺-evoked release of aspartate. In contrast, the cyclooxygenase inhibitor indomethacin (100 µ M ) selectively enhanced the release of glutamate. At a concentration (100 µ M ) that nonselectively depressed the release of arachidonic acid and its metabolites, NDGA markedly depressed the release of aspartate, glutamate, and GABA. An inhibitor of the 12-lipoxygenase and an inhibitor of nitric oxide synthase did not affect the K⁺-evoked release of any transmitter amino acid. These results suggest that a 5-lipoxygenase product selectively enhances aspartate release and a cyclooxygenase product selectively depresses glutamate release. They are also consistent with previous evidence that arachidonic acid and/or platelet-activating factor enhances the release and depresses the uptake of glutamate and aspartate. The K⁺-evoked release of excitatory amino acids is much more sensitive to modulation by lipid mediators than is GABA release. Activation of NMDA receptors may enhance the K⁺-evoked release of glutamate and aspartate from CA1 slices by stimulating the production and release of lipid modulators.     

S-methyl-N,N-diethylthiocarbamate sulfoxide elicits neuroprotective effect against N-methyl-D-aspartate receptor-mediated neurotoxicity

Glutamatergic neurotransmission, particularly of the NMDA receptor type, has been implicated in the excitotoxic response to several external and internal stimuli. In the present investigation, we report that S-methyl-N,N-diethylthiocarbamate sulfoxide (DETC-MeSO) selectively and specifically blocks the NMDA receptor subtype of the glutamate receptors, and attenuates glutamate-induced neurotoxicity in rat-cultured primary neurons. Other major ionotropic glutamate receptor subtypes, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate, were insensitive to DETC-MeSO both in vitro and in vivo. Disulfiram, the parent compound of DETC-MeSO, also inhibits glutamate receptors partially in vivo; however, it fails to inhibit glutamate receptors in mice pretreated with N-butyl imidazole, a cytochrome P450 enzyme inhibitor, implicating the need for bioactivation of disulfiram to be an effective antagonist. We showed that glutamate-induced increase in (45)Ca2+ was attenuated in rat-cultured primary neurons following pretreatment with DETC-MeSO. The Ca2+ influx into primary neurons, studied by confocal microscopy of the fluorescent Ca2+ dye fura-2, demonstrated a complete attenuation of NMDA-induced Ca2+ influx. Similarly, DETC-MeSO attenuated NMDA-induced (45)Ca2+ uptake. Glutamate-induced (45)Ca2+ uptake and Ca2+ influx, however, were partially blocked by DETC-MeSO, and this is consistent with both in vitro and in vivo studies in which DETC-MeSO partially blocked mouse brain glutamate receptors. In addition, DETC-MeSO pretreatment effectively prevented seizures in mice induced either by NMDA, ammonium acetate, or ethanol-induced kindling seizures, all of which are believed to be mediated by NMDA receptors. These data demonstrate that DETC-MeSO produces the neuroprotective effect through antagonism of NMDA receptors in vivo.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

Ca2+-dependent depolarization and burst firing of rat CA1 pyramidal neurones induced by N-methyl-D-aspartic acid and quinolinic acid: antagonism by 2-amino-5-phosphonovaleric and kynurenic acids

The excitatory effects of microiontophoretically applied quisqualic (QUIS), N-methyl-D-aspartic (NMDA), and quinolinic (QUIN) acids were investigated using intracellular recording from CAl pyramidal neurones in slices of rat hippocampus. QUIS evoked only simple action potentials superimposed upon a depolarization which attained a clear plateau. When this level had been reached, increased ejecting currents did not produce further depolarization. By contrast, with low currents NMDA and QUIN elicited small membrane depolarizations which triggered bursts of action potentials superimposed upon rhythmically occurring depolarizing shifts. Larger currents caused depolarization which if sufficiently large completely blocked spike activity. Tetrodotoxin (TTX) prevented the spikes evoked by QUIS and the bursts of action potentials seen with NMDA and QUIN, and the rhythmic depolarizing shifts then appeared as broad spikes of up to 50 mV in amplitude. These and the underlying membrane depolarization were blocked by Co2+, by the NMDA antagonist D(-)-2-amino-5-phosphonovaleric acid (DAPV), and by kynurenic acid (KYNU). It thus appears that the depolarization and burst firing of rat CAl pyramidal neurones elicited by NMDA and QUIN are Ca2+ dependent while the actions of QUIS are not.