N-methyl-D-aspartate receptor activation in human cerebral endothelium promotes intracellular oxidant stress

Cerebral endothelial cells in the rat, pig, and, most recently, human have been shown to express several types of receptors specific for glutamate. High levels of glutamate disrupt the cerebral endothelial barrier via activation of N-methyl-d-aspartate (NMDA) receptors. We have previously suggested that this glutamate-induced barrier dysfunction was oxidant dependent. Here, we provide evidence that human cerebral endothelial cells respond to glutamate by generating an intracellular oxidant stress via NMDA receptor activation. Cerebral endothelial cells loaded with the oxidant-sensitive probe dihydrorhodamine were used to measure intracellular reactive oxygen species (ROS) formation in response to glutamate receptor agonists, antagonists, and second message blockers. Glutamate (1 mM) significantly increased ROS formation compared with sham controls (30 min). This ROS response was significantly reduced by 1) MK-801, a noncompetitive NMDA receptor antagonist; 2) 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate, an intracellular Ca(2+) antagonist; 3) LaCl(3), an extracellular Ca(2+) channel blocker; 4) diphenyleiodonium, a heme-ferryl-containing protein inhibitor; 5) itraconazole, a cytochrome P-450 3A4 inhibitor; and 6) cyclosporine A, which prevents mitochondrial membrane pore transition required for mitochondrial-dependent ROS generation. Our results suggest that the cerebral endothelial barrier dysfunction seen in response to glutamate is Ca(2+) dependent and may require several intracellular signaling events mediated by oxidants derived from reduced nicotinamide adenine dinucleotide oxidase, cytochrome P-450, and the mitochondria.     

The effect of metabotropic glutamate receptors on longitude of posttetanic reaction in spinal motoneurons of frog

Effects of metabotropic glutamate receptors of the duration of posttetanic changes in monosynaptic excitatory postsynaptic potentials (mEPSP), evoked by afferent and reticulospinal input stimulation, were investigated in lumbar motoneurons of the frog isolated spinal cord. It was found that application of MAP4 (25 microM), a selective antagonist of group III of these receptors, prolonged posttetanic potentiation and depression of synaptic transmission, whereas activation of this group of metabotropic glutamate receptors by L-AP4 (1 mM), a selective agonist of these receptors, suppressed the amplitude of synaptic responses, but did not affect the dynamics of development of posttetanic changes. The NMDA receptor antagonist AP5 (50 microM), added to the perfusing solution, blocked completely the effects produced by MAP4. Neither selective antagonist MCCG (400 microM), nor agonist tACPD (50 microM) of group II metabotropic glutamate receptors affected the terms of mEPSP posttetanic potentiation and depression, although the latter, in contrast to the antagonist, in most cases increased the synaptic potential amplitude. The data obtained permit to suggest that group III metabotropic receptors may control the duration of posttetanic changes of synaptic transmission in the frog spinal motoneurons. The long-term changes in the investigated synapses seem to be mediated by activation of postsynaptic metabotropic glutamate receptors (most likely, of group I receptors), which is normally masked with activation of group III presynaptic autoreceptors. The mechanism of such an induction essentially depends on activation of NMDA type of inotropic glutamate receptors.     

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg(2+)-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC(50) of 17.4mM, whereas EC(50) for L-glutamate was 0.17 mM. Addition of 50 microM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 microM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25 mM D,L-homocysteine with 50 microM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 microM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca(2+) to neurones.     

Ionotropic glutamate receptors are involved in malondialdehyde production in anesthetized rat brain cortex: a microdialysis study

Elevated extracellular glutamate levels can increase malondialdehyde production in the brains of anesthetized rats. Thus, we investigated whether ionotropic glutamate receptors are involved in glutamate-induced malondialdehyde production. A microdialysis probe was implanted in the brain cortex of anesthetized rats. The malondialdehyde level in microdialysates was analyzed using an HPLC system. Three different ionotropic glutamate receptor agonists were used. At a concentration of 1.5 mM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA, a selective AMPA receptor agonist) induced a dramatic increase in extracellular malondialdehyde production (as much as 14-fold relative to the basal value). N-Methyl-D-aspartic acid (NMDA, a selective NMDA receptor agonist) also induced an increase in extracellular malondialdehyde production; however, the increase was not as much as that observed in the perfusion of AMPA receptor agonist. Kainic acid (a selective kainate receptor agonist) did not significantly increase malondialdehyde production. When co-perfused with L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 31.4 mM), a glutamate uptake transport inhibitor that can increase the extracellular glutamate levels, AMPA receptor antagonist [1-(4-aminophenyl)4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride, 1.0 mM] can significantly reduce PDC-induced malondialdehyde production. Although NMDA receptor antagonist [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate, MK801] also can decrease the PDC-induced malondialdehyde production, it was not as effective as the AMPA receptor antagonist. These results suggest that ionotropic receptors are involved in the glutamate-induced increase in malondialdehdye production. Specifically, AMPA receptor seems to be predominant in the glutamate-induced malondialdehdye production in anesthetized rat brain cortex.     

Hippocampal functional hyperemia mediated by NMDA receptor/NO signaling in rats during mild exercise

Current studies have demonstrated that exercise increases regional cerebral blood flow (rCBF), an index of neuronal activity. However, neuronal regulation of the increased rCBF in the brain parenchyma is poorly understood. We developed a running model with rats for monitoring hippocampal cerebral blood flow (Hip-CBF) and found that mild treadmill running increases Hip-CBF in a tetrodotoxin-dependent manner, suggesting that functional hyperemia, an increase in rCBF in response to neuronal activation, occurs in the running rat's hippocampus (Nishijima T and Soya H. Neurosci Res 54: 186-191, 2006). To further support our hypothesis, it was important to discover the neurogenic pathways behind the increase in Hip-CBF that occurred during running. Here, we examine the possible role of N-methyl-d-aspartate (NMDA) receptor/nitric oxide (NO) signaling and group I metabotropic glutamate receptors in mediating the Hip-CBF increase. Hip-CBF during running was measured by laser-Doppler flowmetry. Intrahippocampal drug administration was performed by microdialysis. Mild treadmill running (10 m/min) increased Hip-CBF, which was remarkably attenuated by either NMDA receptor antagonists (1 mM MK-801) or NO synthase inhibitors (2 mM N(G)-nitro-l-arginine methyl ester). However, group I metabotropic glutamate receptor antagonists {1 mM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester + 1 mM 2-methyl-6-(phenylethynyl)pyridine hydrochloride} augmented the running-induced Hip-CBF increase. We also found that rCBF in the olfactory bulb was unchanged with running. These results strongly suggest that Hip-CBF during mild exercise is regulated locally under hippocampal neuronal activity, mediated mainly through NMDA receptor/NO signaling. Collectively, these results, together with our previous findings, support our hypothesis that mild exercise elicits neuronal activation, which then triggers functional hyperemia in the rat hippocampus.     

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)     

Differential roles for glutamate receptor subtypes within commissural NTS in cardiac-sympathetic reflex

Ischemic stimulation of cardiac receptors evokes excitatory sympathetic reflexes. Although the nucleus of the solitary tract (NTS) is an important site for integration of visceral afferents, its involvement in the cardiac-renal sympathetic reflex remains to be fully defined. This study examined the role of glutamate receptor subtypes in the commissural NTS in the sympathetic responses to stimulation of cardiac receptors. Renal sympathetic nerve activity (RSNA) was recorded in anesthetized rats. Cardiac receptors were stimulated by epicardial application of bradykinin (BK; 10 microg/ml). Application of BK significantly increased the mean arterial pressure from 78.2 +/- 2.2 to 97.5 +/- 2.9 mmHg and augmented RSNA by 38.5 +/- 2.5% (P < 0.05). Bilateral microinjection of 10 pmol of 6-cyano-7-nitroquinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) antagonist, into the commissural NTS eliminated the pressor and RSNA responses to BK application in 10 rats. However, microinjection of 2-amino-5-phosphonopentanoic acid (0.1 and 1 nmol, n = 8), an NMDA- receptor antagonist, or alpha-methyl-4-carboxyphenylglycine (0.1 and 1 nmol, n = 5), a glutamate metabotropic receptor antagonist, failed to attenuate significantly the pressor and RSNA responses to stimulation of cardiac receptors with BK. Thus this study suggests that non-NMDA, but not NMDA and glutamate metabotropic, receptors in the commissural NTS play an important role in the sympathoexcitatory reflex response to activation of cardiac receptors during myocardial ischemia.     

Excitatory aminoacids and epileptic seizures in immature brain

Data on convulsant and anticonvulsant action of drugs influencing excitatory amino acid receptors in developing rats are reviewed. Agonists of NMDA type of receptors NMDA and homocysteic acid, elicited an age-related seizure pattern--flexion, emprosthotonic seizures--in the first three postnatal weeks of rats. Generalized clonic-tonic seizures appeared only after a longer latency. Kainic acid administration resulted in epileptic automatisms and later in minimal, clonic seizures followed by generalized tonic-clonic seizures. A decrease of sensitivity to convulsant action with age is a general rule for all agonists tested. Different anticonvulsant action of NMDA and nonNMDA antagonists was demonstrated in a model of generalized tonic-clonic seizures induced by pentetrazol, whereas their action against epileptic afterdischarges elicited by electrical stimulation of cerebral cortex was similar. Again, higher efficacy in younger animals was a rule. As far as metabotropic glutamate receptors are concerned, agonists of groups II and III were shown to protect against convulsant action of homocysteic acid in immature rats and an antagonist of group I receptors MPEP suppressed the tonic phase of generalized tonic-clonic seizures induced by pentetrazol more efficiently in younger than in more mature rat pups. Unfortunately, a higher sensitivity to the action of antagonists of ionotropic glutamate receptors was demonstrated also for unwanted side effects (motor functions were compromized). In contrast, glutamate metabotropic receptor antagonist MPEP did not exhibit any serious side effects in rat pups.     

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

A rise in arterial Pco(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the B?tzinger/C1 region (B?tz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 ± 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Δ = +56 ± 8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the B?tz/C1 reduced PND (Δ = +43 ± 7%, +52 ± 6% or +56 ± 11%, respectively). MCPG also reduced sSND (Δ = +41 ± 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the B?tz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the B?tz/C1.     

Role of Glutamate Receptors and Glutamate Transporters in the Regulation of the Glutamate-Glutamine Cycle in the Awake Rat

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.     

Cardiovascular response to a group III mGluR agonist in NTS requires NMDA receptors

Previous studies have demonstrated that microinjection of the putative group III metabotropic glutamate receptor (mGluR) agonist, l(+)-2-amino-4-phosphonobutyric acid (L-AP4), into the nucleus tractus solitarius (NTS) produces depressor and sympathoinhibitory responses. These responses are significantly attenuated by a group III mGluR antagonist and may involve ionotropic glutamatergic transmission. Alternatively, a previous report in vitro suggests that preparations of L-AP4 may nonspecifically activate NMDA channels due to glycine contamination (Contractor A, Gereau RW, Green T, and Heinemann SF. Proc Natl Acad Sci USA 95: 8969-8974, 1998). Therefore, the present study tested whether responses to L-AP4 specifically require the N-methyl-D-aspartate (NMDA) receptor and whether they are due to actions at the glycine site on the NMDA channel. To test these possibilities in vivo, we performed unilateral microinjections of L-AP4, glycine, and selective antagonists into the NTS of urethane-anesthetized rats. L-AP4 (10 mM, 30 nl) produced sympathoinhibitory responses that were abolished by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (AP-5, 10 mM) but were unaffected by the non-NMDA antagonist 6-nitro-7-sulfamobenzoquinoxaline-2,3-dione (NBQX, 2 mM). Microinjection of glycine (0.02-20 mM) failed to mimic sympathoinhibitory responses to L-AP4, even in the presence of the inhibitory glycine antagonist, strychnine (3 mM). Strychnine blocked pressor and sympathoexcitatory actions of glycine (20 mM) but failed to reveal a sympathoinhibitory component due to presumed activation of NMDA receptors. The results of these experiments suggest that responses to L-AP4 require NMDA receptors and are independent of non-NMDA receptors. Furthermore, although it is possible that glycine contamination or other nonspecific actions are responsible for the sympathoinhibitory actions of L-AP4, our data and data in the literature argue against this possibility. Thus we conclude that responses to L-AP4 in the NTS are mediated by an interaction between group III mGluRs and NMDA receptors. Finally, we also caution that nonselective actions of L-AP4 should be considered in future studies.     

Metabotropic glutamate receptors are required for the induction of long-term potentiation.

Recent observations have led to the suggestion that the metabotropic glutamate receptor may play a role in the induction or maintenance of long-term potentiation (LTP). However, experimental evidence supporting a role for this receptor in the induction of LTP is still inconclusive and controversial. Here we report that, in rat dorsolateral septal nucleus (DLSN) neurons, which have the highest density of metabotropic receptors and show functional responses, the induction of LTP is not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate, but is blocked by two putative metabotropic glutamate receptor antagonists, L-2-amino-3-phosphonopropionic acid and L-2-amino-4-phosphonobutyrate. Furthermore, superfusion of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, a selective metabotropic glutamate agonist, resulted in a long-lasting potentiation of synaptic transmission similar to that induced by tetanic stimuli. Our results demonstrated that activation of postsynaptic metabotropic receptors is both necessary and sufficient for the induction of LTP in the DLSN, and we suggest that such a mechanism may be important at other CNS synapses.     

Ionotropic glutamate receptor activation increases intracellular calcium in prolactin-releasing cells of the adenohypophysis

Endocrine cells of the anterior pituitary are controlled by the central nervous system through hormonal interactions and are not believed to receive direct synaptic connections from the brain. Studies suggest that some pituitary cells may be modulated by the neurotransmitter glutamate. We investigated prolactin (PRL)-releasing cells of the anterior pituitary of a euryhaline fish, the tilapia (Oreochromis mossambicus), for the presence of possible glutamate receptors (GluRs). Fura-2 imaging addressed the ability of glutamate to increase intracellular calcium. We observed a dose-dependent increase in intracellular calcium with transient perfusion (1-2 min) of glutamate (10 nM to 1 mM) in two-thirds of imaged cells. This increase was attenuated by the ionotropic GluR antagonist kynurenic acid (0.5-1.0 mM). The increase was also blocked or attenuated by antagonists of L-type voltage-gated calcium channels. The GluR agonist alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA; 100 microM) produced intracellular calcium increases that were reversibly blocked by the selective AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, the selective agonist N-methyl-D-aspartate (NMDA; 100 microM to 1 mM in magnesium-free solution with 10 microM glycine) had no effect on intracellular calcium. Radioimmunoassays demonstrated that glutamate stimulated PRL release. CNQX but not the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid blocked this release. Antibodies for mammalian AMPA- and NMDA-type GluR produced a similar punctate immunoreactivity in the periphery of PRL cells. However, the NMDA antibody recognized a protein of a different molecular mass in PRL cells compared with brain cells. These results clearly indicate the presence of GluRs on tilapia PRL cells that can stimulate PRL release.     

鲫鱼脑氨基酸类神经递质受体在两栖类卵母细胞中的表达

两栖类卵母细胞表达系统经注射鲫鱼脑ｍＲＮＡ后可表达多种神经递质受体和某些离子通道。本工作利用电压箝方法结合药理学手段对ＧＡＢＡ受体和谷氨酸离子型受体作了较详细的研究。结果表明,由ＧＡＢＡ诱发的电流反应中,约９０％由ＧＡＢＡＡ受体介导,乘余约１０％的成分对ＧＡＢＡＡ受体的专一性拮抗剂Ｂｉｃｕｃｕｌｌｉｎｅ不敏感,而ＧＡＢＡＢ受体的专一性激动剂Ｂａｃｌｏｆｅｎ不能引进电流反应,因此这部分受体特性与ＧＡ     

Differential effects of chronic hyperammonemia on modulation of the glutamate-nitric oxide-cGMP pathway by metabotropic glutamate receptor 5 and low and high affinity AMPA receptors in cerebellum in vivo

Previous studies show that chronic hyperammonemia impairs learning ability of rats by impairing the glutamate-nitric oxide (NO)-cyclic guanosine mono-phosphate (cGMP) pathway in cerebellum. Three types of glutamate receptors cooperate in modulating the NO-cGMP pathway: metabotropic glutamate receptor 5 (mGluR5), (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptors. The aim of this work was to assess whether hyperammonemia alters the modulation of this pathway by mGluR5 and AMPA receptors in cerebellum in vivo. The results support that in control rats: (1) low AMPA concentrations (0.1mM) activate nearly completely Ca(2+)-permeable (glutamate receptor subunit 2 (GluR2)-lacking) AMPA receptors and the NO-cGMP pathway; (2) higher AMPA concentrations (0.3 mM) also activate Ca(2+)-impermeable (GluR2-containing) AMPA receptors, leading to activation of NMDA receptors and of NO-cGMP pathway. Moreover, the data support that chronic hyperammonemia: (1) reduces glutamate release and activation of the glutamate-NO-cGMP pathway by activation of mGluR5; (2) strongly reduces the direct activation by AMPA receptors of the NO-cGMP pathway, likely due to reduced entry of Ca(2+) through GluR2-lacking, high affinity AMPA receptors; (3) strongly increases the indirect activation of the NO-cGMP pathway by high affinity AMPA receptors, likely due to increased entry of Na(+) through GluR2-lacking AMPA receptors and NMDA receptors activation; (4) reduces the indirect activation of the NO-cGMP pathway by low affinity AMPA receptors, likely due to reduced activation of NMDA receptors.     

mGluR1 antagonist decreases tyrosine phosphorylation of NMDA receptor and attenuates infarct size after transient focal cerebral ischemia

The contribution of metabotropic glutamate receptors to brain injury after in vivo cerebral ischemia remains to be determined. We investigated the effects of the metabotropic glutamate receptor 1 (mGluR1) antagonist LY367385 on brain injury after transient (90 min) middle cerebral artery occlusion in the rat and sought to explore their mechanisms. The intravenous administration of LY367385 (10 mg/kg) reduced the infarct volume at 24 h after the start of reperfusion. As the Gq-coupled mGluR1 receptor is known to activate the PKC/Src family kinase cascade, we focused on changes in the activation and amount of these kinases. Transient focal ischemia increased the amount of activated tyrosine kinase Src and PKC in the post-synaptic density (PSD) at 4 h of reperfusion. The administration of LY367385 attenuated the increases in the amounts of PSD-associated PKCγ and Src after transient focal ischemia. We further investigated phosphorylation of the NMDA receptor, which is a major target of Src family kinases to modulate the function of the receptor. Transient focal ischemia increased the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B. Tyrosine phosphorylation of NR2A, but not that of NR2B, in the PSD at 4 h of reperfusion was inhibited by LY367385. These results suggest that the mGluR1 after transient focal ischemia is involved in the activation of Src, which may be linked to the modification of properties of the NMDA receptor and the development of cerebral infarction.     

Regulation of glutamate carboxypeptidase II hydrolysis of N-acetylaspartylglutamate (NAAG) in crayfish nervous tissue is mediated by glial glutamate and acetylcholine receptors

Glutamate carboxypeptidase II (GCPII), a glial ectoenzyme, is responsible for N-acetylaspartylglutamate (NAAG) hydrolysis. Its regulation in crayfish nervous tissue was investigated by examining uptake of [3H]glutamate derived from N-acetylaspartyl-[3H]glutamate ([3H]NAAG) to measure GCPII activity. Electrical stimulation (100 Hz, 10 min) during 30 min incubation with [3H]NAAG increased tissue [3H]glutamate tenfold. This was prevented by 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a GCPII inhibitor, suggesting that stimulation increased the hydrolysis of [3H]NAAG and metabolic recycling of [3H]glutamate. Antagonists of glial group II metabotropic glutamate receptors (mGLURII), NMDA receptors and acetylcholine (ACh) receptors that mediate axon-glia signaling in crayfish nerve fibers decreased the effect of stimulation by 58-83%, suggesting that glial receptor activation leads to stimulation of GCPII activity. In combination, they reduced [3H]NAAG hydrolysis during stimulation to unstimulated control levels. Agonist stimulation of mGLURII mimicked the effect of electrical stimulation, and was prevented by antagonists of GCPII or mGLURII. Raising extracellular K+ to three times the normal level stimulated [3H]NAAG release and GCPII activity. These effects were also blocked by antagonists of GCPII and mGLUR(II). No receptor antagonist or agonist tested or 2-PMPA affected uptake of [3H]glutamate. We conclude that NAAG released from stimulated nerve fibers activates its own hydrolysis via stimulation of GCPII activity mediated through glial mGLURII, NMDA and ACh receptors.     

Cellular Mechanisms of Protection by Metabotropic Glutamate Receptors During Anoxia and Nitric Oxide Toxicity

Abstract: Metabotropic glutamate receptors, nitric oxide (NO), and the signal transduction pathways of protein kinase C (PKC) and protein kinase A (PKA) can independently alter ischemic-induced neuronal cell death. We therefore examined whether the protective effects of metabotropic glutamate receptors during anoxia and NO toxicity were mediated through the cellular pathways of PKC or PKA in primary hippocampal neurons. Pretreatment with the metabotropic glutamate receptor agonists (±)-1-aminocyclopentane- trans -1,3-dicarboxylic acid, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD), and l (+)-2-amino-4-phosphonobutyric acid ( l -AP4) 1 h before anoxia or NO exposure increased hippocampal neuronal cell survival from ∼30 to 70%. In addition, posttreatment with 1 S ,3 R -ACPD or l -AP4 up to 6 h following an insult attenuated anoxic- or NO-induced neurodegeneration. In contrast, treatment with l -(+)-2-amino-3-phosphonopropionic acid, an antagonist of the metabotropic glutamate receptor, did not significantly alter neuronal survival during anoxia or NO exposure. Protection by the ACPD-sensitive metabotropic receptors, such as the subtypes mGluR1α, mGluR2, and mGluR5, appears to be dependent on the modulation of PKC activity. In contrast, l -AP4-sensitive metabotropic glutamate receptors, such as the subtype mGluR4, may increase neuronal survival through PKA rather than PKC. Thus, activation of specific metabotropic glutamate receptors is protective during anoxia and NO toxicity, but the signal transduction pathways mediating protection differ among the metabotropic glutamate receptor subtypes.     

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.     

Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish ( Ictalurus punctatus ) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l -glumate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.