Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2–4 months) and aged (22–24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [³H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [³H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.     

Excitatory amino acids inhibit stimulated phosphoinositide hydrolysis in the rat prefrontal cortex

In rat prefrontal cortical slices, the excitatory amino acids N-methyl-D-aspartate (NMDA), ibotenate, L-aspartate, quisqualate, kainate and L-glutamate inhibit carbachol-induced phosphoinositide hydrolysis as measured by the accumulation of [3H]inositol-1-phosphate ([3H]IP1). NMDA dose-dependently inhibited the carbachol response (IC50 = 14.4 microM), and this inhibition was blocked by the NMDA receptor antagonist D,L-aminophosphonovaleric acid. Lowering medium Na+ concentration to 10 mM or exposing slices to pertussis toxin alleviated the inhibitory effect of NMDA on carbachol-induced [3H]IP1 formation. Serotonin-induced stimulation of [3H]IP1 was also inhibited by NMDA; in contrast, stimulation by norepinephrine, epinephrine or dopamine was unaffected. The results suggest that excitatory amino acids, besides their traditional role as stimulatory substances, can also act to inhibit the production of 2nd messengers activated by certain neurotransmitters in the brain.     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.     

Glutamate Inhibits Adenylate Cyclase Activity in Dispersed Rat Hippocampal Cells Directly via an N-Methyl-d-Aspartate-Like Metabotropic Receptor

Three major subtypes of glutamate receptors that are coupled to cation channels--N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors--are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 +/- 2.2 and 6.4 +/- 2.3 microM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 microM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 microM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development

The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.     

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.     

Effect of glutamate on spontaneous secretion of acetylcholine in the nerve-muscle synapse in rats]

In rats, glutamate was shown to exert no effect on the mean frequency, character of interstimuli distribution, amplitude and temporal parameters of the miniature EPPs. Glutamate suppressed nonquantal release. The glutamate effect depended on its concentration and was abolished by blockade of NMDA receptors, NO-synthase inhibitoin, and NO molecules binding by haemoglobin in extracellular medium. Glutamate seems to modulate the nonquantal acetylcholine secretion by initiation of the NO synthesis in muscle fibres via activation of the NMDA receptors.     

Generation of inositol phosphates, cytosolic Ca and secretion of noradrenaline in PC12 cells treated with glutamate

Glutamate transiently stimulated rat pheochromocytoma PC12 cells and caused an inositol trisphosphate formation and an increase in levels of Ca⁺ in the cytosol. The rank order of potency of glutamate> N-methyl-D-aspartate (NMDA) > KAINATE = quisqualate is characteristic of an interaction with NMDA receptors. The effect of glutamate on inositol trisphosphate formation disappeared in a low Mg²⁺ buffer and was not blocked by DL-2-amino-5-phosphonovalerate, an antagonist for NMDA receptors coupled to ion channels. Although glutamate failed to stimulate noradrenaline secretion, glutamate enhanced the effect of bradykinin, but not of Ca ionophore A23187, or KC1. These results suggest the existence of metabotropic glutamate receptors, different from previously reported receptors, in PC12 cells.     

Glutamate Receptor Subtypes in Cultured Cerebellar Neurons: Modulation of Glutamate and γ-Aminobutyric Acid Release

Using cerebellar, neuron-enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D-[3H]aspartate release, whereas N-methyl-D-aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20-100 microM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 microM. Kainate and dihydrokainate (20-100 microM) inhibited the initial rate of D-[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N-methyl-DL-aspartate were ineffective. D-[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D-[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50-200 microM) and, to a lesser extent, 2,3-cis-piperidine dicarboxylic acid (100-200 microM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20-100 microM) also stimulated gamma-[3H]-aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D-[3H]aspartate neuronal uptake by interfering with the acidic amino acid high-affinity transport system.     

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.     

Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons

Using neurotoxicity and inositol phosphate release as criteria for receptor expression, we report the differential expression of excitatory amino acid receptor subtypes in cerebellar granule cells grown in serum-free media containing either high (25 mM) or low (5 mM) KCl. NMDA receptors are expressed in neurons grown in high, but not low, KCl. In contrast, ionotropic quisqualate receptors are expressed in neurons grown in low KCl, but not in those grown in high KCl. Addition of NMDA to cultures containing low KCl appears to mimic high KCl conditions: NMDA receptors are expressed, but ionotropic quisqualate receptors are not. Glutamate and kainate are toxic to cells grown in either condition.     

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.     

Taurine, glutamate and GABA modulate the outgrowth from goldfish retinal explants and its concentrations are affected by the crush of the optic nerve

Summary The amino acid taurine plays an important trophic role during development and regeneration of the central nervous system. Other amino acid systems, such as those for glutamate and gamma-aminobutyric acid (GABA), are modified during the same physiological and pathological processes. After crushing the optic nerve, goldfish retinal explants were plated in the absence and in the presence of different amino acids and amino acid receptor agonists. The length and the density of the neurites were measured at 5 days in culture. Taurine increased the length and the density of neurites. Glutamate and glycine increased them at low concentration, but were inhibitors at higher concentration. The combination of N-methyl-D-aspartate (NMDA) and glycine produced a greater inhibitory effect than NMDA alone. NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) added simultaneously with taurine impaired the stimulatory effect of the latter. GABA stimulated the emission of neurites in a concentration dependent manner. Hypotaurine also elevated the length of neurites, but cysteinesulfinic acid did not produce a significant effect. The concentrations of taurine, glutamate and GABA were determined by HPLC with fluorescent detection in the retina of goldfish at various days post-crushing the optic nerve. The levels of taurine were significantly increased at 48 h after the crush, and were elevated up to 20 days. Glutamate level decreased after the lesion of the optic nerve and was still low at 20 days. GABA concentration was not significantly different from the control. The interaction of these amino acids during the regenerative period, especially the balance between taurine and glutamate, may be a determinant in restoring vision after the crush.Abbreviations AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - GABA gamma-aminobutyric acid - NMDA N-methyl-D-aspartate     

Different receptors mediate stimulation of nitric oxide-dependent cyclic GMP formation in neurons and astrocytes in culture.

The ability of various compounds to stimulate cyclic GMP accumulation was studied in neuronal and astrocyte-enriched primary cultures from rat cerebrum. Glutamate was the only agonist eliciting a response in neurons whereas several agonists had an effect in astrocytes but only those due to norepinephrine and glutamate were of considerable magnitude. The responses were markedly inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The effect of glutamate appears to be mediated predominantly by NMDA receptors in neurons and by quisqualate AMPA-insensitive receptors in astrocytes.     

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.     

Age-related changes in receptor-mediated phosphoinositide hydrolysis in various regions of rat brain.

The effects of age on cholinergic markers and receptor-stimulated phosphoinositide hydrolysis was examined in the frontal cortex and striatum of male Fischer-344 rats. Choline acetyltransferase activity was decreased 27% in the striatum of aged (24 month) rats compared to young (3 month) controls. Muscarinic receptor density as measured by [3H]-quinuclidinyl benzilate binding showed a similar 26% decrease in the striatum of aged rats. Phosphoinositide hydrolysis was measured by the release of inositol phosphate (IP) from tissue slices prelabeled with [3H]myoinositol in response to carbachol, norepinephrine, and quisqualate. In the cortex, stimulated IP release was significantly greater in slices from aged rats compared to young rats for all three agonists. In contrast, stimulated IP release was significantly decreased in striatal slices from aged rats compared to young for all three agonists. These data indicate a differential effect of age on agonist-stimulated phosphoinositide hydrolysis in the cortex and striatum. The decreased responsiveness in the latter area may result from the age-related loss of postsynaptic receptors.     

Selective blockade of NMDA-activated channel currents may be implicated in learning deficits caused by lead.

The effect of Pb2+ on glutamate receptor activity in rat hippocampal neurons was investigated with a view of explaining the cognitive and learning deficits produced by this heavy metal. Pb2+ (2.5-50 microM) selectively inhibited N-methyl-D-aspartate (NMDA)-induced whole-cell and single-channel currents in a concentration-dependent but voltage-independent manner, without significantly altering currents induced by either quisqualate or kainate. The frequency of NMDA-induced channel activation was decreased by Pb2+. Neither glycine (10-100 microM), nor Ca2+ (10 mM) reversed the effect of Pb2+. Pb2+ also inhibited the [3H]MK-801 binding to rat hippocampal membranes in vitro. The elucidation of the actions of Pb2+ on the NMDA receptor ion channel complex provides important insights into the clinical and toxic effects of this cation.     

Inhibition of Forskolin-Stimulated Cyclic AMP Formation by 1-Aminocyclopentane-trans-1,3-Dicarboxylate in Guinea-Pig Cerebral Cortical Slices

The effects of the selective metabotropic glutamate receptor agonist 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) on forskolin-stimulated cyclic AMP formation in guinea-pig cerebral cortex slices were determined. t-ACPD inhibited the accumulation of [3H]cyclic AMP by approximately 80%, with an IC50 value of 35 +/- 4 microM. The effect was reversible and stereoselective, with the 1S,3R isomer being approximately 400-fold more potent than the 1R,3S isomer. L-Glutamate (over a restricted concentration range) also partially inhibited the response to forskolin, but quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and N-methyl-D-aspartate (NMDA) were ineffective. The effect of t-ACPD was not blocked by antagonists of the phospholipase C-linked metabotropic glutamate receptor, the AMPA ionotropic glutamate receptor, or the NMDA receptor. In summary, our results indicate the presence of a glutamate receptor in guinea-pig brain that is activated selectively by t-ACPD and that is negatively linked to adenylyl cyclase.     

Effect of Kainic Acid, Glutamate, and Aspartate on CO2 Production by Goldfish Tectal Slices

For a study of the excitatory effect of kainate, glutamate, and aspartate in the goldfish optic tectum, these substances were tested on the production of CO2 from radioactive glucose in tectal slices incubated in Krebs-Ringer medium for fish. Kainate increased the rate of CO2 production for up to 30 min in a dose-related manner, the effect being maximum at 0.1 mM concentration and decreasing at higher doses. The effect was blocked by ouabain (1 mM) as well as by the substitution of choline for Na+ in the incubation medium. Glutamate and aspartate exerted a less pronounced excitatory effect on CO2 production at higher concentration than kainate. This effect was also abolished by ouabain. Glutamate, added to the medium at a concentration at least 100-fold higher than kainate, partially reversed the increase in CO2 production induced by kainic acid. No similar effect was noticed for aspartate. The supposed glutamate antagonists glutamic acid diethylester (1 mM) and proline (5 mM) did not affect the excitatory action of kainic acid or exert an antagonistic effect towards glutamate. At higher concentration (10 mM) glutamic acid diethylester increased CO2 production, an effect that was, however, ouabain insensitive. Methyltetrahydrofolic acid (1 mM), a substance reported to compete for the kainate receptor, did not inhibit the effect of kainic acid or increase CO2 production.