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The enzymic formation of acetylglutamate has been studied in Chlorella vulgaris extracts. Acetyl CoA and N(2)-acetyl-l-ornithine served as substrates for glutamate acetylation whereas acetylphosphate, N(5)-acetyl-l-ornithine, and N(2)-acetyl-2,4-diamino butyrate were ineffective. Acetyl CoA-glutamate transacetylase and acetylornithine-glutamate transacetylase activities have been purified over 180-fold with no indication of any separation of activities. The acetyl CoA activity was more labile than acetylornithine activity so that preparations having acetylornithine-glutamate transacetylase activity but no acetyl CoA-glutamate transacetylase activity were obtained. The two acetylating activities appear to be properties of one enzyme with one portion more easily denatured.Both acetylating activities had pH optima between 8 and 8.5. The Km value for glutamate was 3 mm for both activities. The Km values were 0.2 mm for acetylornithine and 3.2 mm for acetyl CoA. Arginine inhibited acetyl CoA-glutamate transacetylase (Ki = 0.94 mm) and acetylglutamate phosphokinase (Ki = 0.5 mm) but had no effect on acetylornithine-glutamate transacetylase. The lack of an inhibitory effect of proline on any of the three enzymic activities indicates that acetylglutamate is not a normal intermediate in proline biosynthesis. Growth of Chlorella with arginine as a nitrogen source had no effect on enzyme levels, showing that end-product repression is not a control factor in arginine biosynthesis in Chlorella. In Chlorella, arginine controls its own biosynthesis by inhibiting acetylglutamate phosphokinase and controls the level of acetylated intermediates by inhibiting acetyl CoA-glutamate transacetylase.
Replication-defective Moloney murine leukemia virus expressing the GAD67 gene under the control of the GFAP promoter was produced using selected clones of a fibroblast-packaging cell line. A spontaneously immortalized astrocyte cell line was infected with this virus and cellular clones expressing GAD67 selected. Astrocyte and fibroblast clones expressed functional GAD (detected by glutamic acid decarboxylation), but only fibroblasts were able to also produce GABA in the extracellular medium. When exposed to 200 microM glutamate, despite an observed difference in the rates of glutamate accumulation in control and GAD67-expressing astrocytes, similar proportions of glutamate taken up were detected. In GAD67-expressing astrocytes, the glutamate was mainly converted into GABA, suggesting GAD transgene activity to be dominant over other glutamate metabolic pathways, such as glutamine synthetase and glutamate dehydrogenase. Moreover, rapid GABA release into the cell medium was also observed, suggesting the involvement of reverse GABA transporters. The use of the GFAP promoter might be able to take advantage of its activation in response to factors inducing reactive gliosis observed in pathological insults. GAD67-expressing astrocytes might therefore be used for future grafting in pathological situations in which an excess of glutamate results in neuronal dysfunction or cell death.
Previous observations indicate that spinocervical tract terminals contain relatively high levels of glutamate. To examine whether these high glutamate levels are likely to represent a neurotransmitter pool or an elevated metabolic pool, the distributions of glutamate- and glutamine-like immunoreactivities were examined in adjacent immunogold-labeled sections of the lateral cervical nucleus. Spinocervical tract terminals were identified by anterograde transport of horseradish peroxidase and wheat germ agglutinin-horseradish peroxidase conjugate from the spinal cord. Spinocervical tract terminals were found to contain significantly higher levels of glutamate-like immunoreactivity than other examined tissue compartments (large neuronal cell bodies, terminals with pleomorphic vesicles, astrocytes, and average tissue level). In contrast, the highest levels of glutamine-like immunoreactivity were detected in astrocytes. The different analyzed tissue elements formed three groups with respect to glutamate:glutamine ratios: one high ratio group including spinocervical tract terminals, a second group with intermediate ratios consisting of neuronal cell bodies and terminals containing pleomorphic synaptic vesicles, and a third low ratio group including astrocytes. Our findings indicate the presence of a compartmentation of glutamate and glutamine in the lateral cervical nucleus, similar to that postulated in biochemical studies of the central nervous system. The results also show that spinocervical tract terminals have high glutamate: glutamine ratios, similar to those previously observed in putative glutamatergic terminals in the cerebellar cortex. Thus, spinocervical tract terminals display biochemical characteristics that would be expected of glutamatergic terminals and the present findings therefore provide further evidence for glutamate as a spinocervical tract neurotransmitter.
One-week-old albino female rats were kept for 1 year in equal groups with or without monosodium glutamate in their drinking water. The concentrations of the glutamate in the brain crude extracts of the monosodium glutamate treated group were twofold higher than those of the control group. There were no significant changes in body weight, brain weight, and protein content, and total RNA and DNA content also did not vary in the two groups of rats. The activities of glutamate dehydrogenase (GDH) were decreased with a 45% reduction in V(max) values in the monosodium glutamate treated group compared to those in the control group, whereas no significant changes were observed in K(m) values between the two groups. The concentration of the enzyme on the Western blot analysis was significantly decreased in the monosodium glutamate treated group, whereas the level of GDH mRNA remained unchanged, suggesting a post-transcriptional control of the expression of GDH or an increased rate of degradation of enzyme protein. These results indicate that the prolonged monosodium glutamate feeding reduces the activity of GDH and subsequently decreases the catabolism of glutamate in rat brain.
During brain ischemia in vivo the extracellular concentration of the excitotoxic amino acid, glutamate, increases. This increase could be caused either by an enhanced formation rate of glutamate (from glutamine) or by an impaired re-uptake (or both). This re-uptake occurs to a large extent in astrocytes. In the present study we have determined glutamate uptake and the ability of the cells to maintain their glutamate content during exposure to anoxia, substrate deprivation and combined substrate deprivation and anoxia ('simulated ischemia') for a duration of up to 4 h. Isolated anoxia had no significant effect, whereas both substrate deprivation alone and 'simulated ischemia' reduced glutamate uptake and glutamate content by one-half after 2 h. Under hypothermic conditions (incubation at 32 degrees C), which in in vivo experiments exerts some protection against ischemic cell death in neurons, ischemia of intermediate duration (2 h) decreased glutamate uptake and glutamate content to a less extent than at 37 degrees C. Hypothermia did not have a similar effect during exposure to isolated substrate deprivation.
The purposes of our study were to determine the type of respiratory changes that would occur when either an excitatory amino acid receptor agonist or an uptake inhibitor was administered into the caudal subretrofacial area. This was done by microinjecting either L-glutamate or L-pyrrolidine-2,4-dicarboxylate (L-trans-2,4-PDC) into the caudal subretrofacial area while monitoring tidal volume, respiratory rate, mean arterial blood pressure and heart rate. Bilateral microinjection of 2.5 nmol of L-glutamate into the caudal subretrofacial area produced apnea in eight of eight animals tested, and the duration of apnea was 27 +/- 2 s. To determine the type of L-glutamate receptor responsible for mediating the apneic response, antagonists of the N-methyl-D-aspartate (NMDA) and non-NMDA receptor, namely, 3-[(RS)-carboxypiperazin-4-yl]-propyl-phosphonic acid (CPP), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, were tested. Neither antagonist in doses that blocked NMDA (in the case of CPP) and amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) (in the case of CNQX) blocked apnea elicited by L-glutamate. In addition, kynurenic acid, an antagonist of NMDA and non-NMDA ionotropic receptors, failed to block the effect of L-glutamate. Microinjection of the metabotropic receptor agonist drug, trans-L-1-amino-1,3-cyclopentone-dicarboxylic acid (L-trans-ACPD), into the caudal subretrofacial area failed to have any effect on respiratory activity. Because of the inability to block the effect of L-glutamate in the caudal subretrofacial area, and the lack of effect of L-trans-ACPD, the data suggest that the apneic response produced by L-glutamate is mediated by an as yet undefined receptor. Microinjection of the L-glutamate uptake inhibitor, L-trans-2,4-PDC, was found to produce apnea. Using the dose of 0.5 nmol of L-trans-2,4-PDC, we examined the type of excitatory amino acid receptor that mediated the response. Neither pretreatment with the NMDA receptor antagonist, CPP, nor the non-NMDA receptor antagonist, CNQX, affected L-trans-2,4-PDC-induced apnea. However, combined use of these two antagonists prevented L-trans-2,4-PDC-induced apnea. These data suggest that the effect of synaptically released exitatory amino acid at the caudal subretrofacial area on breathing is apnea, and that this effect is mediated by simultaneous activation of both NMDA and non-NMDA ionotropic receptors.
The effect of glutamate receptor activation on the high-affinity sodium-dependent glutamate transport expressed in chick Bergmann glia cells was examined. Pre-exposure to glutamate produced a time- and dose-dependent decrease in 3H-labeled D-aspartate uptake. This effect could not be reproduced by selective glutamate receptor agonists. Furthermore, it was insensitive to both ionotropic and metabotropic glutamate receptor antagonists. Replacement of extracellular sodium ions with choline in the preincubation media, abolished the reduction of the uptake. When the cells were pre-exposed to competitive transportable inhibitors of the transporter, such as D-aspartate, DL-threo-hydroxyaspartate (DL-THA), and aspartate-beta-hydroxamate (ABH), the glutamate effect was mimicked. From saturation experiments, it was found that the reduction on the uptake, after glutamate treatment, is related to an increase in K(m). Interestingly, the effect is blocked by staurosporine, a Ca(2+)/diacylglycerol-dependent protein kinase (PKC) inhibitor. The present findings suggest that glutamate regulates its transport in a non-receptor fashion, a phenomena that is most probably linked to changes induced by the translocation process of the substrate through the transporter.
During the formation of neuromuscular junctions in Drosophila embryos, glutamate receptors undergo a drastic change in distribution. To study the underlying mechanism of this developmental process, it is desirable to map the distribution of functional receptors with accurate spatial resolution. Since glutamate receptors desensitize within several milliseconds, the agonist must be applied rapidly. To fulfil these requirements we used laser stimulation of a caged compound to release L-glutamate at a focal spot. Since the glutamate receptor channel is permeable to Ca2+, we assayed the change in internal Ca2+ concentration using a Ca2+ indicator, fluo-3. Using this approach, we mapped the distribution of functional glutamate receptors in cultured embryonic Drosophila myotubes and myoblasts. Consistent with previous immunofluorescence studies using an antibody against a glutamate receptor subunit, a large increase of internal Ca2+ concentration was observed when laser stimulation was located close to some nuclei in the myotube. No change was detected when the laser stimulus was applied over any regions of the myoblasts. No increase of the internal Ca2+ concentration in myotubes was observed when the external solution contained either glutamate at a desensitizing concentration (1 mM) or a glutamate receptor channel blocker, argiotoxin (1 microg/ml). These results indicate that a rise in intracellular Ca2+ concentration can be used to show the distribution of the functional receptor on the muscle surface membrane.
Intrathecal (i.t.) administration of the group I metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG) to rats produces an immediate display of spontaneous nociceptive behaviors (SNBs) persisting for up to 10 h after injection (NeuroReport 7 (1996) 2743). The mechanisms underlying these behavioral effects are not entirely understood but may include enhanced release of glutamate within the dorsal horn of the spinal cord. The current experiments used microdialysis in awake moving animals to test: (1), whether i.t. (S)-3,5-DHPG increases the local release of glutamate at doses that also induce SNBs; and (2), whether the effects on glutamate release (as well as SNBs) can be blocked by pretreatment with the mGluR5 selective antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Male Sprague-Dawley rats were implanted with a microdialysis probe inserted into the i.t. space of the spinal cord (J. Neurosci. Methods 62 (1995) 43) and then tested under i.t. drug conditions (0.01, 0.1 and 1 mM (S)-3,5-DHPG) following a 2-3 day recovery period. As predicted, local application of (S)-3,5-DHPG via the microdialysis probe increased the release of glutamate in a dose-dependent manner. Significant SNBs were also noted in the 0.1 and 1 mM groups in a manner paralleling the onset and duration of the glutamate response. Pretreatment with MPEP (55 mg/kg, intraperitoneally) blocked glutamate release to the 0.1 mM dose of (S)-3,5-DHPG, and also decreased the proportion of animals displaying SNBs in this dose group. No effects of MPEP were seen against the higher dose of (S)-3,5-DHPG (1 mM). These results suggest that stimulation of spinal mGluR5 leads to glutamate release within the spinal cord, a response that may in part account for the nociceptive behaviors evoked by i.t. (S)-3,5-DHPG.
Simultaneous imposition of a pH gradient (delta pH, interior alkaline) and a sodium gradient (delta pNa, [Na+]out greater than [Na+]in) across cytoplasmic membrane vesicles from Escherichia coli B led to a several hundred fold accumulation of glutamate. Although less effective, delta pH (interior alkaline)( alone caused accumulation of glutamate in the presence of Na+. In addition, delta pNa ([Na+]out greater than [Na+]in) alone also drove the transport system, where the maximum level of glutamate accumulation was affected by the pH of the medium. A membrane potential imposed by valinomycin-induced K+ diffusion (interior negative) enhanced the accumulation, indicating that the system operation in an electrogenic manner. The Michaelis constant of glutamate transport was greatly affected by changes in the concentrations of both Na+ and H+ and could be expressed by a linear combination of the reciprocals of the Na+ and H+ concentrations in the medium. On the contrary, a membrane potential (interior negative) exerted its effect by increasing the maximum velocity. When membrane vesicles were loaded with glutamate and Na+, but not with glutamate alone, rapid efflux of glutamate with Na+ as the cocation down the concentration gradients took place upon dilution. These results indicate that both Na+ and H+ are syn-coupled ions of glutamate transport in E. coli B and that the carrier/Na+/H+/Glu- complex observed in the binding reaction is an intermediate in the transport.
We hypothesized that presynaptic glutamate regulates postsynaptic ionotropic glutamate receptor number during synaptogenesis. To test this idea, we genetically manipulated presynaptic glutamate levels at the glutamatergic Drosophila neuromuscular junction (NMJ), then microscopically and electrophysiologically measured postsynaptic glutamate receptor field size and function. Our data show that presynaptic glutamate is a strong negative regulator of postsynaptic receptor field size and function during development. Glutamate-triggered receptor downregulation was not affected by block of synaptic vesicle fusion, demonstrating that receptors are regulated by nonvesicular glutamate release. Our results reveal an elegant mechanism for receptor field regulation during synaptogenesis and reveal a nonpathological role for nonvesicular glutamate release at the synapse.
The nucleus tractus solitarii (NTS) is essential for coordinating arterial baroreflex control of blood pressure. The primary baroreceptor afferent fibres make their first excitatory synaptic contact at second-order NTS neurones with glutamate as the major neurotransmitter. Glutamate regulates its own release by activating presynaptic metabotropic glutamate autoreceptors (mGluRs) on the baroreceptor central terminals to suppress its further release in frequency-dependent manner. Gamma-aminobutyric acid (GABA) interneurones provide the major inhibitory synaptic input. It is the integration of excitatory and inhibitory inputs that shapes the NTS output of baroreceptor signals. We hypothesized that glutamate released from the primary central afferent terminals can spill over to presynaptic mGluRs on GABA interneurones to suppress GABA release at the second-order baroreceptor neurones. We assessed GABA transmission in second-order baroreceptor neurones identified by attached aortic depressor nerve (ADN) boutons. The medial NTS was stimulated to evoke GABA inhibitory postsynaptic currents (eIPSCs). Glutamate spillover, generated by brief 2 s, 25 Hz trains of stimuli applied to the tractus solitarius (TS), induced a small (10%) but significant reduction in the eIPSC amplitudes. The depression was enhanced to a 25% decrease by increasing glutamate in the cleft with a glutamate-uptake inhibitor (M-trans-pyrrolidine-2,4-dicarboxylic acid, 1 mum), blocked by a Group II mGluR antagonist (LY341495, 200 nm) and mimicked by a Group II agonist ((2S,3S,4S)-CCG/(2S,1'S,2'S)-2-carboxycyclopropyl; L-CCG-I). A presynaptic mGluR locus was established by the mGluR agonist-mediated increase in the paired-pulse ratio of two consecutive eIPSCs in conjunction with the decrease in the first eIPSC, and a decrease in the frequency (39-46% reduction at EC(50) concentration), but not amplitude, of spontaneous and miniature GABA IPSCs. The data indicate that endogenous glutamate activation of Group II presynaptic mGluRs can decrease GABA release at the first central synapses, suggesting a heterosynaptic role for the Group II mGluRs in shaping baroreceptor signal transmission.
Group I metabotropic glutamate receptors (mGluRs) form stable, disulfide-linked homodimers. Lack of a verifiably monomeric mGluR1 mutant has led to difficulty in assessing the role of dimerization in the molecular mechanism of mGluR1 activation. The related GABA(B) receptor exhibits striking intradimer cross talk (ligand binding at one subunit effectively produces G protein activation at the other), but it is unclear whether group I mGluRs exhibit analogous cross talk. Signaling of heterologously expressed mGluR1 was examined in isolated rat sympathetic neurons by measuring glutamate-mediated inhibition of native calcium currents. To examine mGluR1 activity when only one dimer subunit has access to glutamate ligand, wildtype mGluR1 was coexpressed with mGluR1 Y74A, a mutant with impaired glutamate binding, and the activity of the heterodimer (mutant/wild type) was examined. The mGluR1 Y74A mutant alone had a dose-response curve that was shifted by about 2 orders of magnitude. The half-maximal dose of glutamate shifted from 1.3 (wild-type mGluR1) to about 450 (mGluR1 Y74A) microM. However, the maximal effect was similar. Wild-type mGluR1 was expressed with excess Y74A mGluR1 to generate a receptor population consisting largely of mutant homodimers and mutant/wild-type heterodimers but without detectable wild-type homodimers. Under these conditions, no glutamate-mediated calcium current inhibition was observed below approximately 300 microM glutamate, although wild-type mGluR1 protein was detectable with immunofluorescence. These data suggest that mutant/wild-type heterodimeric receptors are inactive at ligand concentrations favoring glutamate association with receptor dimers at only one subunit.
1. Using the patch-clamp technique, we investigated whether the glutamate-elicited current in mechanically isolated cone photoreceptors from the salamander retina is generated by a Cl- channel or a glutamate transporter. 2. The current reversed near the equilibrium potential for Cl-, was decreased by three Cl- channel blockers, 5-nitro-2-(3-phenyl-propylamino) benzoic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and diphenylamine 2,2'-dicarboxylic acid, and was eliminated when gluconate was substituted for both internal and external Cl-, features consistent with the current being mediated by a Cl- channel. 3. The single-channel conductance of the Cl- channel was estimated by noise analysis of the glutamate-elicited current fluctuations to be 0.7 pS with an open time of 2 ms. 4. The magnitude of the current was dependent on both internal and external Na+ and K+, features consistent with the current being related to the activation of a glutamate transporter. Yet changes in their concentrations did not affect the reversal potential of the current. 5. Taken together with earlier reports on this current showing that it has a glutamate-transporter-like pharmacology, our results suggest that the glutamate-elicited current is carried by a Cl- channel but gated by a glutamate receptor whose pharmacology and ionic requirement resemble those previously described for glutamate transporters.
Glutamate is the primary excitatory transmitter in the suprachiasmatic nucleus (SCN). Ionotropic glutamate receptors (iGluRs) mediate transduction of light information from the retina to the SCN, an important circadian clock phase shifting pathway. Metabotropic glutamate receptors (mGluRs) may play a significant modulatory role. mGluR modulation of SCN responses to glutamate was investigated with fura-2 calcium imaging in SCN explant cultures. SCN neurons showed reproducible calcium responses to glutamate, kainate, and N-methyl-D-aspartate (NMDA). Although the type I/II mGluR agonists L-CCG-I and t-ACPD did not evoke calcium responses, they did inhibit kainate- and NMDA-evoked calcium rises. This interaction was insensitive to pertussis toxin. Protein kinase A (PKA) activation by 8-bromo-cAMP significantly reduced iGluR inhibition by mGluR agonists. The inhibitory effect of mGluRs was enhanced by activating protein kinase C (PKC) and significantly reduced in the presence of the PKC inhibitor H7. Previous reports show that L-type calcium channels can be modulated by PKC and PKA. In SCN cells, about one-half of the calcium rise evoked by kainate or NMDA was blocked by the L-type calcium channel antagonist nimodipine. Calcium rises evoked by K+ were used to test whether mGluR inhibition of iGluR calcium rises involved calcium channel modulation. These calcium rises were primarily attributable to activation of voltage-activated calcium channels. PKC activation inhibited K+-evoked calcium rises, but PKC inhibition did not affect L-CCG-I inhibition of these rises. In contrast, 8Br-cAMP had no effect alone but blocked L-CCG-I inhibition. Taken together, these results suggest that activation of mGluRs, likely type II, modulates glutamate-evoked calcium responses in SCN neurons. mGluR inhibition of iGluR calcium rises can be differentially influenced by PKC or PKA activation. Regulation of glutamate-mediated calcium influx could occur at L-type calcium channels, K+ channels, or at GluRs. It is proposed that mGluRs may be important regulators of glutamate responsivity in the circadian system.
We cloned and expressed a human metabotropic glutamate receptor 1 alpha (HmGluR1 alpha) in a novel cell line. The human mGluR1 alpha cDNA was found to be 86% identical to rat mGluR1 alpha, and the predicted protein sequence was found to be 93% identical to rat mGluR1 alpha. We expressed HmGluR1 alpha in AV12-664, an adenovirus-transformed Syrian hamster cell line. To prevent tonic activation of HmGluR1 alpha by glutamate that may be released by these cells into the extracellular medium, HmGluR1 alpha was co-expressed in AV12-664 cells with a rat glutamate/aspartate transporter (GLAST). This allowed investigation of the effect that clearance of glutamate from the extracellular space would have on HmGluR1 alpha function. A comparison of mRNA levels revealed that HmGluR1 alpha was similarly expressed in cells with or without co-expression of GLAST. However, HmGluR1 alpha-mediated phosphoinositide hydrolysis was efficiently elicited only in cells co-expressing rat GLAST. Blockade of glutamate transport by L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in an increase in glutamate levels in the media and an increase in basal HmGluR1 alpha-mediated phosphoinositide hydrolysis. Long-term pretreatment of cells with L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in media glutamate levels similar to those in cells not expressing GLAST. However, this resulted in a dramatic decrease in 1-aminocyclopentane-1S,3R-dicarboxylic acid- and glutamate-stimulated phosphoinositide hydrolysis. These studies suggest that co-expression of mGluR1 alpha with a glutamate transporter prevents desensitization of the receptor, thus achieving optimal coupling of the receptor with its effector system.