Endogenous N-acetylaspartylglutamate reduced NMDA receptor-dependent current neurotransmission in the CA1 area of the hippocampus

N-Acetylaspartylglutamate (NAAG) is a neuropeptide found in high concentrations in the brain. Using whole-cell recordings of CA1 pyramidal neurons in acute hippocampal slices, we found that either (i) the application of exogenous NAAG or (ii) an increase of endogenous extracellular NAAG, caused by the inhibition of its catabolic enzyme glutamate carboxypeptidase II (GCP II), resulted in a significant reduction in the amplitude of the isolated NMDA receptor (NMDAR) component of the evoked excitatory postsynaptic current (EPSC). Conversely, reduction of endogenous extracellular NAAG caused by either (i) perfusion with a soluble form of pure human GCP II or (ii) affinity purified antibodies against NAAG, enhanced the amplitude of the isolated NMDAR current. Bath application of GCP II inhibitor induced a progressive loss of spontaneous NMDAR miniatures. Furthermore, NAAG blocked the induction of long-term potentiation at Schaffer collateral axons-CA1 pyramidal neuron synapses. All together, these results suggest that NAAG acts as an endogenous modulator of NMDARs in the CA1 area of the hippocampus.     

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate.

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.     

Development and characterisation of a glutamate-sensitive motor neurone cell line

Modification of the growth conditions of NSC-34 mouse neuroblastoma x motor neurone cells by serum depletion promotes the expression of functional glutamate receptors as the cells mature into a form that bears the phenotypic characterisation of motor neurones. Immunocytochemical studies demonstrated the presence of the glutamate receptor proteins NMDAR1, NMDAR2A/B, GluR1, GluR2, GluR2/3, GluR4, GluR6/7, and KA2. Toxicity assays using cell counting techniques demonstrated a mild but significant cell death (approximately 30%, p < 0.01) following a 24-h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (10 microM) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 microM). As an indication of glutamate receptor functional activity a novel approach was used to detect the production of free radicals following stimulation with glutamate receptor agonists. The release of superoxide free radicals was detected using a micro-electrochemical sensor following addition of glutamate receptor agonists to the cell bathing solution. Alterations in intracellular calcium concentrations were examined using fura-2 imaging. Exposure of the differentiated NSC-34 cells to glutamate leads to an increase in intracellular calcium concentrations that is prevented by the presence of glutamate receptor antagonists. The motor neurone origin of these cells makes them particularly useful for investigating the potential role of glutamatergic toxicity in motor neurone degeneration.     

Rapid Communication Activation of Protein Kinase C Inhibits Kainate-Induced Currents in Oocytes Expressing Glutamate Receptor Subunits

Abstract: The effect of protein kinase C (PKC) activation on maximal kainate (KA)-induced currents was studied in Xenopus oocytes expressing the glutamate receptor (GluR) subunits GluR3, GluR1+3, GluR2+3, and GluR6. The PKC activator phorbol 12- myristate 13-acetate (PMA) inhibited peak KA responses in a time-dependent manner. The magnitude of inhibition was greatest in GluR6-expressing oocytes. Desensitizing KA currents characterized by a peak, transient current followed by a slower, desensitizing current were observed in oocytes expressing GluR3 and GluR 1+3 receptors. PMA inhibited the desensitization, and this effect could be observed before PMA's inhibition of peak current amplitude. PMA-mediated inhibition of both desensitization and peak current amplitude was prevented by intracellular injection of the protein kinase C (PKC) inhibitor peptide. These results suggest that the function of GluRs is regulated by PKC-dependent phosphorylation     

Fimbria-Fornix Transections Selectively Down-Regulate Subtypes of Glutamate Transporter and Glutamate Receptor Proteins in Septum and Hippocampus

Abstract: The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.     

Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.     

Neuroadaptations in Ionotropic and Metabotropic Glutamate Receptor mRNA Produced by Cocaine Treatment

Abstract : The expression of glutamate receptor/subunit mRNAs was examined 3 weeks after discontinuing 1 week of daily injections of saline or cocaine. The level of mRNA for GluR1-4, NMDAR1, and mGluR5 receptors was measured with in situ hybridization and RT-PCR. In nucleus accumbens, acute cocaine treatment significantly reduced the mRNA level for GluR3, GluR4, and NMDAR1 subunits, whereas repeated cocaine reduced the level for GluR3 mRNA. Acute cocaine treatment also reduced the NMDAR1 mRNA level in dorsolateral striatum and ventral tegmental area. In prefrontal cortex, repeated cocaine treatment significantly increased the level of GluR2 mRNA. The GluR2 mRNA level was not changed by acute or repeated cocaine in any other brain regions examined. Repeated cocaine treatment also significantly increased mGluR5 mRNA levels in nucleus accumbens shell and dorsolateral striatum. Functional properties of the ionotropic glutamate receptors are determined by subunit composition. In addition, metabotropic glutamate receptors can modulate synaptic transmission and the response to stimulation of ionotropic receptors. Thus, the observed changes in levels of AMPA and NMDA receptor subunits and the mGluR5 metabotropic receptor may alter excitatory neurotransmission in the mesocorticolimbic dopamine system, which could play a significant role in the enduring biochemical and behavioral effects of cocaine.     

A light and electron microscopic study of glutamate receptors in the monkey subthalamic nucleus

The distribution of glutamate receptors in the monkey subthalamic nucleus was studied using affinity purified polyclonal antibodies to GluR1, phosphorylated GluR1, GluR2/3, NMDAR1, mGluR1a and mGluR5. Intense staining for both the unphosphorylated and the phosphorylated forms of the AMPA receptor subunit GluR1 was observed in the cell bodies and proximal dendrites of neurons in this nucleus. In comparison to GluR1, less intense staining for GluR2/3 was observed in the cell bodies and processes. NMDAR1 immunoreactivity was present in cell bodies and large numbers of small diameter dendrites. Light staining was observed in cell bodies with mGluR1a and no staining was observed on cell bodies with mGluR5. The neuropil, however, contained many processes that were labeled for mGluR1a or mGluR5. Electron microscopy showed that label was present in cytoplasmic locations in cell bodies and dendrites, in addition to components of the synaptic region, in sections stained for GluR1, GluR2/3 and NMDAR1. In contrast, very lightly labeled or unlabeled cell bodies but labeled dendrites and axon terminals, was observed in sections stained for mGluR1a and mGluR5. In addition to neural processes, occasional astrocytic processes were also labeled for mGluR5. Of the immunogold particles that were associated with components of the synaptic region, label for ionotropic glutamate receptors was mostly present on postsynaptic densities, whilst that for metabotropic glutamate receptors was mostly present in a perisynaptic location. The ratio of GluR1/GluR2 messenger RNAs has been reported to increase in the aged hippocampus (PAGLIUSI, S. R., GERRARD, P., ABDALLAH, M., TALABOT, D. & CATSICAS, S. (1994) Neuroscience 61, 429–433.), and it is possible that a similar change in the ratio of GluR1 and GluR2 may occur in neurons of the subthalamic nucleus with age. It is postulated that this could result an increase in calcium permeability via AMPA receptors, and an enhancement of excitatory transmission in this nucleus.     

Reconstitution of an insulin signaling pathway in Xenopus laevis oocytes: coexpression of a mammalian insulin receptor and three different mammalian hexose transporters.

We report the functional expression of the mammalian muscle-adipocyte insulin-sensitive hexose transporter in Xenopus laevis oocytes. Oocytes microinjected with RNA synthesized in vitro showed enhanced hexose transport activity compared with uninjected controls. However, like the endogenous oocyte hexose transporter, activity was stimulated only twofold by 1 microM insulin. X. laevis oocytes injected with in vitro-synthesized RNA encoding the human insulin proreceptor expressed a functionally active insulin receptor that enhanced the insulin sensitivity of injected oocytes. This increase was not observed in oocytes expressing a mutant insulin receptor that lacked protein tyrosine kinase activity. In the presence of the coexpressed human insulin receptor, insulin induced a two- to threefold increase in hexose transport. The muscle-, brain-, and liver-type hexose carriers normally expressed in tissues with different responses to insulin exhibited the same insulin sensitivity when expressed in oocytes. This was observed whether or not the insulin signal was transduced through a coexpressed human insulin receptor or the endogenous oocyte insulin-like growth factor I receptor. We conclude that the expressed human insulin receptor is able to couple efficiently with preexisting postreceptor regulatory pathways in oocytes and that the regulation of hexose transport in these cells can be mediated through the combined actions of the expressed human insulin receptor and the endogenous oocyte insulin-like growth factor I receptor.     

Expression of NMDAR1, GluR1, GluR7, and KA1 Glutamate Receptor mRNAs Is Decreased in Frontal Cortex of "Neuroleptic-Free" Schizophrenics: Evidence on Reversible Up-Regulation by Typical Neuroleptics

Abstract: Schizophrenics exhibit abnormalities in many memory-associated functions mediated by the frontal cortex. Glutamate receptors play key roles in learning and memory. Hence, abnormalities in glutamate receptors within the frontal cortex may be associated with schizophrenia. In addition, emerging evidence indicates that glutamate receptors may be involved in the actions of antipsychotic drugs. To test these hypotheses, we measured mRNAs encoding the NMDAR1, GluR1, GluR7, and KA1 subunits of glutamate receptor in the left superior frontal gyrus from 21 elderly schizophrenics with varying histories of antipsychotic drug treatment and nine normal drug-free elderly controls. There were significant negative correlations between NMDAR1, GluR1, GluR7, and KA1 mRNA levels and time without neuroleptic medication before death in schizophrenics, indicating that levels of the glutamate receptor mRNAs decline rapidly after drug withdrawal. Further analysis revealed that in "neuroleptic-free" (>6 months) schizophrenics, levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs were significantly lower than in controls. By contrast, in schizophrenics who were receiving neuroleptics until death, levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs did not differ significantly from controls. These findings indicate that decreased levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs may be present in the frontal cortex of some schizophrenics and that typical neuroleptics may reversibly increase levels of these mRNAs.     

Some interactions of L-DOPA and its related compounds with glutamate receptors

L-DOPA is probably a transmitter and/or modulator in the central nervous system (1). L-DOPA methyl ester (DOPA ME) is a competitive L-DOPA antagonist. However, it remains to be clarified whether there exist L-DOPAergic receptors. In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, L-DOPA induced small inward currents with ED50 of 2.2 mM at a holding potential of -70 mV. The currents were abolished by kynurenic acid or CNQX. Similar L-DOPA-currents were seen in oocytes co-injected with AMPA receptors, GluRs1,2,3 and 4. In brain membrane preparations, L-DOPA inhibited specific binding of [3H]-AMPA with IC50 of 260 microM. This inhibition was not modified by 200 microM ascorbic acid, an antioxidant. L-DOPA did not inhibit binding of [3H]-ligands of MK-801, kainate, DCKA and CGP39653. DOPA ME and L-DOPA cyclohexyl ester, a novel, potent and competitive antagonist (2), inhibited specific binding of [3H]-MK-801 with respective IC50 of 1 and 0.68 mM, but elicited no effect on that of the other [3H]-ligands. With low affinities, L-DOPA acts on AMPA receptors, while competitive antagonists act on NMDA ion channel domain. L-DOPAergic agonist and antagonist may not interact on ionotropic glutamate receptors. DOPA ME-sensitive L-DOPA recognition sites (1) seem to differ from glutamate receptors.     

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.     

The N-methyl-d-aspartic acid receptor antagonist memantine ameliorates and delays the development of arthritis by enhancing regulatory T cells

The neuroendocrine impact on rheumatoid arthritis is not yet fully described although numerous neurotransmitters are shown to act as inflammatory modulators. One of these is the excitatory transmitter glutamate (Glu). In this study, the influence of the Glu receptor (GluR)-mediated effects on collagen-induced arthritis (CIA) was investigated. CIA was induced in DBA/1 mice by immunization with chicken collagen type II (CII). Mice were exposed to the following GluR antagonists: group 1, the N-methyl-D-aspartic acid (NMDA) receptor channel blocker memantine; group 2, the metabotropic GluR antagonist AIDA, and group 3, the excitatory amino acid receptor antagonist kynurenic acid (KA). Arthritis was evaluated clinically and histologically and compared to PBS-treated controls. The effects of treatment on T cell populations and the levels of anti-CII and anti-citrullinated peptide antibodies were evaluated. Memantine treatment significantly improved the course of CIA, reducing synovitis (p = 0.007) and the frequency of erosions (p = 0.007). Memantine treatment up-regulated the expression of Foxp3 in spleen CD4+ T cells followed by an increase in CD4+CD25+ regulatory T cells. The other GluR antagonists, AIDA and KA, had no effect on CIA. These results demonstrate that blockade of the NMDA receptor channel with memantine delays and attenuates the development of arthritis, probably by promoting the development of regulatory T lymphocytes.     

Functional analysis of Caenorhabditis elegans glutamate receptor subunits by domain transplantation

Glutamate receptors are not only abundant and important mediators of fast excitatory synaptic transmission in vertebrates, but they also serve a similar function in invertebrates such as Drosophila and the nematode Caenorhabditis elegans. In C. elegans, an animal with only 302 neurons, 10 different glutamate receptor subunits have been identified and cloned. To study the ion channel properties of these receptor subunits, we recorded glutamate-gated currents from Xenopus oocytes that expressed either C. elegans glutamate receptor subunits or chimeric rat/C. elegans glutamate receptor subunits. The chimeras were constructed between the C. elegans glutamate receptor pore domains and either the rat kainate receptor subunit GluR6, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit GluR1, or the N-methyl-d-aspartate (NMDA) receptor subunit NMDAR1-1a. Although native subunits were nonfunctional, 9 of 10 ion pores were found to conduct current upon transplantation into rat receptor subunits. A provisional classification of the C. elegans glutamate receptor subunits was attempted based on functionality of the chimeras. C. elegans glutamate receptor ion pores, at a position homologous to a highly conserved site critical for ion permeation properties in vertebrate glutamate receptor pores, contain amino acids not found in vertebrate glutamate receptors. We show that the pore-constricting Q/R site, which in vertebrate receptors determines calcium permeability and rectification properties of the ion channel, in C. elegans can be occupied by other amino acids, including, surprisingly, lysine and proline, without loss of these properties.     

Calcium influx through subunits GluR1/GluR3 of kainate/AMPA receptor channels is regulated by cAMP dependent protein kinase.

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by three major classes of glutamate receptors, namely the ionotropic NMDA (N-Methyl-D-Aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid) receptors and the metabotropic receptor type. Among the ionotropic receptors, NMDA receptors are thought to mediate their physiological response mainly through the influx of extracellular calcium, while KA/AMPA receptor channels are mainly thought to carry the influx of monovalent cations. Recently, we have challenged this view by showing that cloned KA/AMPA receptor subunits GluR1 and GluR3 form ion channels which are permeable to calcium. We now directly demonstrate large increases in intracellular calcium concentrations induced by calcium fluxes through KA/AMPA receptor channels in solutions with physiological calcium concentrations. Calcium fluxes were observed through glutamate receptor channels composed of the subunits GluR1 and GluR3, which are both abundantly present in various types of central neurones. The calcium influx was fluorometrically monitored in Xenopus oocytes injected with the calcium indicator dye fura-2. Bath application of the membrane permeable analogue of adenosine cyclic monophosphate (cAMP) potentiated the current and also the flux of calcium through open KA/AMPA receptor channels. Further pharmacological experiments suggested that this effect was mediated by the activation of protein kinase A. Our results provide a molecular interpretation for the function of calcium permeable KA/AMPA receptor channels in neurones and identify two of the subunits of the KA/AMPA receptor channel which are regulated by the cAMP dependent second messenger system.     

Changes in messenger RNAs coding for neurotransmitter receptors and voltage-operated channels in the developing rat cerebral cortex.

The ontogenetic development of poly(A)+ mRNAs coding for receptors to several neurotransmitters (kainate, glutamate, acetylcholine, and serotonin) and voltage-operated channels (sodium and calcium) was studied by isolating total poly(A)+ mRNA from the brains of rats at various developmental stages and injecting it into Xenopus oocytes. The oocytes translated the foreign mRNA and incorporated functional receptor/ion channel complexes into the cell membrane. Thus, recording of induced membrane currents in voltage-clamped oocytes gave a measure of the relative amounts of the different messengers. Responses induced by kainate, glutamate, acetylcholine, and serotonin all increased with age and reached a maximum in oocytes injected with mRNA from adult cortex. Messenger RNAs for the earliest ages examined, Embryonic Days 15 and 18, expressed little or no response to kainate, glutamate, or acetylcholine, while 50-70% of the adult response was reached by Postnatal Day 10. In contrast, the serotonin-induced response was already comparatively large (16% of the adult level) in oocytes injected with mRNA from Embryonic Day 15 brain and increased postnatally to adult levels. The expression of voltage-dependent sodium and calcium channels was small in oocytes injected with mRNA from embryonic animals and increased postnatally to reach a maximum in oocytes injected with mRNA from adult animals.     

Glutamatergic regulation of bone remodeling

L-glutamate (Glu) is the predominant neuromediator in the mammalian central nervous system (CNS). Bone is highly innervated and there is growing evidence of a neural control of bone cell metabolism. The recent discovery of Glu-containing nerve fibers in bone and Glu receptors (GluR) and transporters in bone cells suggest that this neuromediator may also act as a signaling molecule in bone and regulate bone cell function. Our previous studies have demonstrated that ionotropic N-Methyl-D-Aspartate (NMDA) GluR are highly expressed by mammalian osteoclasts. NMDA receptors (NMDAR) are heteromers associating the NR1 subunit and one of the four types of NR2 subunits (NR2A to D). We showed that osteoclasts express NR1, NR2B and NR2D subunits, suggesting a molecular diversity of NMDAR in these cells. Electrophysiological studies have confirmed that NMDAR are functional in mature osteoclasts, and features of Glu-induced current recorded in these cells indicate a major NR2D subunit composition. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDAR binding to different sites of the receptor inhibit bone resorption. In particular, the specific NMDAR channel blocker MK801 had no effect on osteoclast attachment to bone and survival while it rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. NMDAR may thus be involved in adhesion-induced formation of the sealing zone required for bone resorption. NMDAR are also expressed by osteoclast precursors isolated from mouse bone marrow. We recently confirmed the presence of NR1, NR2B and NR2D in these cells and demonstrated their expression at all differentiation stages from osteoclast precursors to mature resorbing osteoclasts. No regulation of these subunits mRNA expression levels was observed throughout the osteoclastic differentiation sequence. Activation of NMDAR may therefore represent a new mechanism for regulating osteoclast formation and activity. While the origin of Glu in bone is still unknown, the possibility of a glutamatergic neurotransmission in this tissue is suggested by the detection of Glu in nerve fibers in close contact to bone cells. Furthermore, we recently demonstrated that sciatic neurectomy in growing rats induces a bone loss associated with a reduction of nerve profiles immunostained for Glu. These results suggest that Glu may be released from glutamatergic nerve profiles present in bone and therefore contribute to the local regulation of bone cell function.     

Angiotensin II stimulates an endogenous response in Xenopus laevis ovarian follicles

While responses to angiotensin II have previously been induced in Xenopus laevis oocytes after injection of messenger RNA extracted from mammalian tissue, no endogenous responses of ovarian tissue to this hormone have been reported. Here we describe such an endogenous dose-dependent response to angiotensin II, detected by conventional electrophysiological techniques, in follicular oocytes. The ED50 of the response was estimated to be 0.15 +/- 0.07 microM (S.E.M.). Maximal depolarization, obtained at 1 microM angiotensin II, was 18.3 +/- 1.4 mV (n = 18, three experiments using oocytes from two toads, mean resting membrane potential = -42 +/- 2 mV). The response was absent from collagenase-treated oocytes or follicular oocytes treated with octanol, suggesting that the receptors are predominantly in the follicular layer surrounding the oocytes.     

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.     

Channel-opening kinetics of GluR6 kainate receptor

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.