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P L Chazot

Publications and source records attributed to P L Chazot.

At least 19 recordsLinked to original sources
PubMed2003

Modulation of [3H] TBOB binding to the rodent GABAA receptor by simple disaccharides.

We have recently reported evidence that a simple beta-linked alkylated mannose reversibly increased the magnitude of GABA(A) receptor currents evoked in cultured rat pyramidal neurons whilst concomitantly reducing the incidence of spontaneous synaptic activity. In this present study, the effects of the simple beta-linked disaccharide, lactose was investigated using a [3H] TBOB (t-[3H] butylbicycloorthobenzoate) binding assay in adult rat forebrain and cerebellum membranes. Lactose elicited a significant potentiation of [3H] TBOB binding to well-washed forebrain and cerebellar membranes (mean E(max) values=367 and 287%; mean EC(50) values=1.5 and 30 microM, respectively, N=4). The alpha-linked disaccharides, maltose and sucrose also potentiated [3H] TBOB binding, but with 100-600-fold higher EC(50) values than lactose. The lactose-mediated potentiation of [3H] TBOB in the forebrain and cerebellum was completely abolished in the presence of 0.3 microM GABA. Over the concentration range in which significant potentiation of [3H] TBOB binding was detected, lactose elicited no significant effect upon [3H] flunitrazepam binding. This study demonstrated that lactose can modulate the GABA(A) receptor channel, allosterically coupled to the agonist site, but independent of the benzodiazepine site. Furthermore, lactose displayed differential effects upon forebrain and cerebellar GABA(A) receptors indicating that it may be a novel subtype selective agent.

Animals↗
PubMed2002

Spreading depression-induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors.

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in alpha-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12-15-fold increase in alpha7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the alpha4 nAChR subunit was not altered. These data point to the alpha7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating alpha7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).

Animals↗
PubMed2001

Immunological identification of the mammalian H3 histamine receptor in the mouse brain.

Affinity-purified antibodies raised against the peptide sequence H3 (349-358) receptor specifically recognized two protein species with Mr 62,000 and 93,000 in adult mouse forebrain membranes. Both immunoreactive species were suppressed greatly by preincubation of the antibody with the respective peptide. Immunohistochemical analysis using affinity-purified anti-H3 (349-358) antibodies yielded a high degree of coincidence with ligand-autoradiographical information, with high levels detected in the CA3 and dentate gyrus of the hippocampus, laminae V of the cerebral cortex, the olfactory tubercle, Purkinje cell layer of the cerebellum, substantia nigra, globus pallidus, thalamus and striatum. This study suggests further biochemical evidence for multiple H3 receptor subtypes and the widespread distribution of the H3 receptor in the mammalian brain.

Age Factors↗
PubMed2001

Safinamide (Newron Pharmaceuticals).

Safinamide (formerly PNU-151774E), a sodium and calcium channel modulator that also inhibits monoamine oxidase B (MAOB), is under development by Newron Pharmaceuticals for the potential treatment of epilepsy, Parkinson's disease (PD), pain and stroke [345222], [348351]. Phase I trials for epilepsy and PD have been completed, and dose-finding studies for both indications had commenced in March 2001 [401685]. The compound was previously developed by Pharmacia & Upjohn (P&U) for the potential treatment of epilepsy, an indication for which it initially reached phase I trials [294891], [345007]. Newron acquired the rights to safinamide from P&U at the end of 1998. Results from two phase I trials of the compound (single ascending dose and steady state at three doses), completed in March 2000, demonstrated that the drug is well tolerated with good bioavailability and linear pharmacokinetics [359652].

Alanine↗
PubMed2000

A simple polar deacetylated caloporoside derivative is a positive modulator of the GABA(A) chloride channel complex in cortical mammalian neurones.

Synthesis of octyl-O-beta-D-mannopyranoside, a caloporoside analogue was achieved by the activation of 2,3,4,6-rerra-O-benzyl-1-O-1',3'2'-dioxaphosphacyclohexane-a lpha,beta-D-mannopyranosyl-2-oxide with TMSOTf (Trimethyl silyl triflate) and subsequent debenzylation. At 100 microM the molecule significantly and reversibly increased the magnitude of GABA(A) currents evoked in cultured rat pyramidal neurones whilst concomitantly reducing the incidence of spontaneous synaptic activity. These results contradict earlier proposals that such molecules bind to the TBPS (tert-Butylbicyclophosphorothionate) site to block the chloride channel.

Acetylation↗
PubMed2000

Immunohistochemical localization of N-methyl-D-aspartate receptor NR1, NR2A, NR2B and NR2C/D subunits in the adult mammalian cerebellum.

The distributions of the N-methyl-D-aspartate (NMDA) receptor NR1, NR2A, NR2B and NR2C/D subunits were mapped in adult mouse cerebellum using subunit-specific antibodies. Immunostaining with anti-NR1 antibodies was prominent in cell bodies and dendritic arbors of Purkinje cells, was light to moderate in cerebellar granule cells, Golgi interneurons and interneurons in the molecular layer. Anti-NR2A subunit-specific antibody staining of mouse cerebellum was moderate in the granule cells, and moderate to dense in Purkinje neurons and Bergmann glia. However, Purkinje neurons were not immunolabelled in adult rat brain. Anti-NR2B subunit-specific immunostaining was prominent in Purkinje cell bodies and dendrites but absent from the granule cell layer. Anti-NR2C/D subunit-specific immunostaining was largely restricted to cerebellar granule cells. These studies reveal that NMDA receptor subunits display distinct but overlapping expression patterns in the adult mammalian cerebellum. Furthermore, we have observed some differences between rats and mice in terms of the NMDA receptor subunits expressed in specific cerebellar cell types.

Amino Acid Sequence↗
PubMed2000

Characterization of the binding of two novel glycine site antagonists to cloned NMDA receptors: evidence for two pharmacological classes of antagonists.

The potency of two novel glycine site antagonists, GV150,526A and GV196,771A, was assessed by their ability to inhibit the binding of [(3)H]-MDL105,519 to cell homogenates prepared from mammalian cells transfected with either NR1-1a, NR1-2a, NR1-1a/NR2A, NR1-1a/NR2B, NR1-1a/NR2C or NR1-1a/NR2D NMDA receptor clones. The inhibition constants (K(i)s) for GV150,526A displacement of [(3)H]-MDL105,519 binding to either NR1-1a or NR1-2a expressed alone were not significantly different and were best fit by a one-site binding model. GV150,526A inhibition to NR1-1a/NR2 combinations was best fit by a two-site model with the NR1-1a/NR2C having an approximate 2 - 4 fold lower affinity compared to other NR1-1a/NR2 receptors. The K(i)s for GV196,771A displacement of [(3)H]-MDL105,519 binding to NR1-1a, NR1-2a and all NR1-1a/NR2 combinations was best fit by a two-site binding model. There was no significant difference between the K(i)s for the binding to NR1-1a and NR1-2a; NR1-1a/NR2A receptors had an approximate 4 fold lower affinity for GV196,771A compared to other NR1-1a/NR2 combinations. The K(i)s for both GV150, 526A and GV196,771A for the inhibition of [(3)H]-MDL105,519 binding to membranes prepared from adult rat forebrain were determined and compared to the values obtained for binding to cloned NMDA receptors. The K(i)s for a series of glycine site ligands with diverse chemical structures were also determined for the inhibition of [(3)H]-MDL105,519 binding to NR1-1a/NR2A receptors. L689,560 displayed similar binding characteristics to GV150,526A. It is suggested that glycine site antagonists may be divided into two classes based on their ability to distinguish between NR1 and NR1/NR2 receptors with respect to binding curve characteristics.

Animals↗
PubMed2000

CP-101606 Pfizer Inc.

CP-101606 is a potent, selective NMDA receptor (NR2B subunit) antagonist under development by Pfizer for its potential as a neuroprotectant in head injury and neurodegenerative disease. It is in phase II trials in the US and in phase I in Japan for the potential treatment of head injury [267139], [320737], [378812]. As of March 2000, CP-101606 had completed US phase I trials successfully and was well tolerated with a good safety profile. Results of an open-label study in patients treated with CP-101606 (24 to 72 h infusion) following severe head trauma showed that 80% of patients had good recovery at the 3-month follow-up stage [360864]. In August 2000, Merrill Lynch predicted that the phase II trials program could be complete by the end of 2000 with the possibility of a filing based on its results if positive [379892]. The drug lacks the amnesic side effects of other NMDA receptor antagonists and it has no effects on long-term potentiation (LTP) [347642]. According to presenters at the 1999 Society for Neuroscience meeting, this may result from a novel activity-dependent or NR2B-selective mechanism of action [345079]. It is thought to act by inhibiting the damaging influx of Ca2+ across neuronal cell membranes following glutamate release triggered by tissue injury, e.g., stroke [300847]. CP-101606 prevents glutamate-induced toxicity in cultured hippocampal neurons when given during or immediately after glutamate perfusion, (IC50 = 11 and 35 nM, respectively). In contrast, CP-101606 does not protect against glutamate-induced neurotoxicity in cultured cerebellar neurons, up to a dose of 10 microM. These results are consistent with CP-101606 being a potent NMDA antagonist, selective for the type of NMDA receptor associated with the hippocampus [195435]. CP-101606 is a single isomer analog of CP-98113 (Pfizer Inc) [254010]. CP-101581 (Pfizer Inc) is an isomer of CP-101606 [254010]. In December 1998, Morgan Stanley Dean Witter predicted sales of US $5 million in 2001, rising to US $80 million by 2005 [315350].

Animals↗
PubMed1999

Biochemical evidence for the co-association of three N-methyl-D-aspartate (NMDA) R2 subunits in recombinant NMDA receptors.

Functional characterization of wild-type and mutant cloned N-methyl-D-aspartate (NMDA) receptors has been used to deduce their subunit stoichiometry and quaternary structure. However, the results reported from different groups have been at variance and are thus inconclusive. This study has employed a biochemical approach to determine the number of NMDA R2 (NR2) subunits/receptor together with the NMDA R1 (NR1)/NR2 subunit ratio of both cloned and native NMDA receptors. Thus, human embryonic kidney 293 cells were transfected with the NR1-1a and NR2A NMDA receptor subunits in combination with both FLAG- and c-Myc epitope-tagged NR2B subunits. The expressed receptors were detergent-extracted and subjected to double immunoaffinity purification using anti-NR2A and anti-FLAG antibody immunoaffinity columns in series. Immunoblotting of the double immunopurified NR2A/NR2B(FLAG)-containing material demonstrated the presence of anti-NR1, anti-NR2A, anti-FLAG, and, more important, anti-c-Myc antibody immunoreactivities. The presence of anti-c-Myc antibody immunoreactivity in the double immunoaffinity-purified material showed the co-assembly of three NR2 subunits, i.e. NR2A/NR2B(FLAG)/NR2B(c-Myc), within the same NMDA receptor complex. Control experiments excluded the possibility that the co-immunopurification of the three NR2 subunits was an artifact of the solubilization procedure. These results, taken together with those previously described that showed two NR1 subunits/oligomer, suggest that the NMDA receptor is at least pentameric.

Cells, Cultured↗
PubMed1998

[3H]MDL 105,519 binds with equal high affinity to both assembled and unassembled NR1 subunits of the NMDA receptor.

[3H]MDL 105,519 (((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1[3H]-indole-2-ca rboxylic acid) is a novel radioligand which binds with high affinity, Kd = 2.5 nM, to the glycine site of adult rodent forebrain, N-methyl-D-aspartate subtype of glutamate receptors. As with other glycine site antagonists, the major determinants for high-affinity binding of [3H]MDL 105,519 resides upon the NRI subunit, and not the NR2 subunits. [3H]MDL 105,519 binds with equal affinity, Kd = 3 nM, to both NR1-1a or NR1-4b splice variants, as well as the NRI-1a/NR2A receptor expressed in human embryonic kidney (HEK) 293 cells. One percent Triton X-100/1 M NaCl solubilises with a recovery of 15+/-3%, a mixed pool of assembled and unassembled forebrain NR1 subunit polypeptides. In this preparation, the recovery of [3H]MK801 ((+)-5-[3H]methyl-10,11-dihydrodibenzo[alpha,d]cyclohepten-5 ,10-imine binding activity (7+/-1%) reflects the amount of assembled NR1 subunits whereas [3H]MDL 105,519 binds quantitatively, with a recovery of 19+/-4% and Kd = 3 nM, to both assembled and unassembled NRI subunits. Therefore, [3H]MDL 105,519 should prove a useful ligand, in conjunction with immunopurification approaches, to address the question of NMDA receptor subunit stoichiometry.

Animals↗
PubMed1997

Biochemical evidence for the existence of a pool of unassembled C2 exon-containing NR1 subunits of the mammalian forebrain NMDA receptor.

Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [3H]MK-801 radioligand binding activity. The most efficient conditions were 1% Triton X-100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X-100/1 M NaCl-solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti-NR1 911-920 antibody affinity chromatography from 1% Triton X-100/1 M NaCl solubilised extracts yielded purification of the NR1 Mr 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X-100/1 M NaCl extracts with anti-NR1 911-920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti-NR1 17-35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a co-association of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti-NR1 911-920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon-containing NR1 subunits, i.e., NR1-1a, NR1-1b, NR1-2a, and NR1-2b, selectively solubilised by 1% Triton X-100/1 M NaCl.

Age Factors↗
PubMed1997

Molecular dissection of native mammalian forebrain NMDA receptors containing the NR1 C2 exon: direct demonstration of NMDA receptors comprising NR1, NR2A, and NR2B subunits within the same complex.

The subunit compositions of the NR1 C2 exon-containing N-methyl-D-aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit-specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti-NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti-NR1 C2, anti-NR1 N1, anti-NR1 C2', anti-NR2A, and anti-NR2B NMDA receptor antibodies. The NR1 C2-receptor subpopulation was subjected to immunoprecipitation using anti-NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti-NR1 C2, anti-NR1 C2', anti-NR2A, and anti-NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2', in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2', NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.

Alternative Splicing↗
PubMed1994

Molecular characterization of N-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule.

The N-methyl-D-aspartate R1 (NMDA R1), NMDA R2A, and NMDA R2C subunits were expressed transiently in double or triple combinations in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult mouse forebrain and cerebellum. Under conditions established for maximal expression, cotransfection of the NMDA R1 and R2C subunits yielded a protein detected immunologically with a molecular size of 780,000-850,000 daltons. No cell death was observed in the transfected cells, and the KD for [3H]MK801 binding to the NMDA R1/R2C receptor was 346 +/- 158 nM. This was in contrast to a value of KD = 22 +/- 9 nM found for native cerebellar receptors. Co-transfection with NMDA R1/R2A/R2C subunits with a DNA ratio, 1:3:3, resulted in the expression of a protein with a size similar to the NMDA R1/R2C combination, but the affinity of [3H]MK801 was now 22 +/- 5 nM, and the percentage cell death post-transfection was 89 +/- 17%. Immunoprecipitation assays of detergent-solubilized transfected cells with NMDA R1 subunit-specific antibodies co-precipitated the NMDA R2A and NMDA R2C subunits in 1/2A and 1/2C transfections, respectively. Similarly, immunoprecipitations with either NMDA R1 or NMDA R2C subunit-specific antibodies co-precipitated the NMDA R2A subunit in the R1/2A/2C triple transfections. These results show that the three NMDA receptor subunit types can co-assemble following their co-expression in mammalian cells with a pharmacological profile that is similar to that found for adult cerebellar NMDA receptors.

Amino Acid Sequence↗
PubMed1994

Expression of NMDAR1-1a (N598Q)/NMDAR2A receptors results in decreased cell mortality.

Transient expression of wild-type N-methyl-D-aspartate, NMDAR1-1a/NMDAR2A heteromeric receptors, in mammalian cells yields cell death which was prevented by the inclusion of NMDA receptor antagonists in the cell culture media post-transfection. Transient expression of mutant NMDAR1-1a (N598Q)/NMDAR2A receptors resulted in a significant decrease in the percentage of cell death post-transfection. This mutation has been shown to reduce the Ca2+ permeability of cloned NMDA receptors. Thus these results provide indirect evidence for cell death via an NMDA receptor, Ca(2+)-mediated mechanism.

2-Amino-5-phosphonovalerate↗
PubMed1993

Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization.

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.

6-Cyano-7-nitroquinoxaline-2,3-dione↗