Biomedical subjects

J Bockaert

Publications and source records attributed to J Bockaert.

At least 37 records · Page 2Linked to original sources

PubMed2001

BAY36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity.

L-Glutamate (Glu) activates at least eight different G protein-coupled receptors known as metabotropic glutamate (mGlu) receptors, which mostly act as regulators of synaptic transmission. These receptors consist of two domains: an extracellular domain in which agonists bind and a transmembrane heptahelix region involved in G protein activation. Although new mGlu receptor agonists and antagonists have been described, few are selective for a single mGlu subtype. Here, we have examined the effects of a novel compound, BAY36-7620 [(3aS,6aS)- 6a-Naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on], on mGlu receptors (mGlu1-8), transiently expressed in human embryonic kidney 293 cells. BAY36-7620 is a potent (IC(50) = 0.16 microM) and selective antagonist at mGlu1 receptors and inhibits >60% of mGlu1a receptor constitutive activity (IC(50) = 0.38 microM). BAY36-7620 is therefore the first described mGlu1 receptor inverse agonist. To address the mechanism of action of BAY36-7620, Glu dose-response curves were performed in the presence of increasing concentrations of BAY36-7620. The results show that BAY36-7620 largely decreases the maximal effect of Glu. Moreover, BAY36-7620 did not displace the [(3)H]quisqualate binding from the Glu-binding pocket, further indicating that BAY36-7620 is a noncompetitive mGlu1 antagonist. Studies of chimeric receptors containing regions of mGlu1 and regions of DmGluA, mGlu2, or mGlu5, revealed that the transmembrane region of mGlu1 is necessary for activity of BAY36-7620. Transmembrane helices 4 to 7 are shown to play a critical role in the selectivity of BAY36-7620. This specific site of action of BAY36-7620 differs from that of competitive antagonists and indicates that the transmembrane region plays a pivotal role in the agonist-independent activity of this receptor. BAY36-7620 will be useful to further delineate the functional importance of the mGlu1 receptor, including its putative agonist-independent activity.

Animals↗

PubMed2000

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

Animals↗

PubMed2000

Dendritic and axonal targeting of type 5 metabotropic glutamate receptor is regulated by homer1 proteins and neuronal excitation.

The physiological actions of neurotransmitter receptors are intimately linked to their proper neuronal compartment localization. Here we studied the effect of the metabotropic glutamate receptor (mGluR)-interacting proteins, Homer1a, b, and c, in the targeting of mGluR5 in neurons. We found that mGluR5 was exclusively localized in cell bodies when transfected alone in cultured cerebellar granule cells. In contrast, mGluR5 was found also in dendrites when coexpressed with Homer1b or Homer1c, and in both dendrites and axons when cotransfected with Homer1a. In dendrites, cotransfected mGluR5 and Homer1b/c formed clusters that colocalized with the synaptic marker synaptophysin. Interestingly when transfected alone, the Homer proteins were also translocated to neurites but did not form such clusters. Depolarization of the neurons with a mixture of ionotropic glutamate receptor agonists, NMDA and kainate, or potassium channel blockers, tetraethylammonium and 4-aminopyridine, induced transient expression of endogenous Homer1a and persistent neuritic localization of transfected mGluR5 even long after degradation of Homer1a. These results suggest that Homer1a/b/c proteins are involved in the targeting of mGluR5 to dendritic synaptic sites and/or axons and that this effect can be regulated by neuronal activity. Because the activity-dependent effect of endogenous Homer1a was also long-lasting, the axonal targeting of mGluR5 by this protein is likely to play an important role in synaptic plasticity.

Animals↗

PubMed2000

Selective blockade of P/Q-type calcium channels by the metabotropic glutamate receptor type 7 involves a phospholipase C pathway in neurons.

Although presynaptic localization of mGluR7 is well established, the mechanism by which the receptor may control Ca(2+) channels in neurons is still unknown. We show here that cultured cerebellar granule cells express native metabotropic glutamate receptor type 7 (mGluR7) in neuritic processes, whereas transfected mGluR7 was also expressed in cell bodies. This allowed us to study the effect of the transfected receptor on somatic Ca(2+) channels. In transfected neurons, mGuR7 selectively inhibited P/Q-type Ca(2+) channels. The effect was mimicked by GTPgammaS and blocked by pertussis toxin (PTX) or a selective antibody raised against the G-protein alphao subunit, indicating the involvement of a G(o)-like protein. The mGuR7 effect did not display the characteristics of a direct interaction between G-protein betagamma subunits and the alpha1A Ca(2+) channel subunit, but was abolished by quenching betagamma subunits with specific intracellular peptides. Intracellular dialysis of G-protein betagamma subunits did not mimic the action of mGluR7, suggesting that both G-protein betagamma and alphao subunits were required to mediate the effect. Inhibition of phospholipase C (PLC) blocked the inhibitory action of mGluR7, suggesting that a coincident activation of PLC by the G-protein betagamma with alphao subunits was required. The Ca(2+) chelator BAPTA, as well as inhibition of either the inositol trisphosphate (IP(3)) receptor or protein kinase C (PKC) abolished the mGluR7 effect. Moreover, activation of native mGluR7 induced a PTX-dependent IP(3) formation. These results indicated that IP(3)-mediated intracellular Ca(2+) release was required for PKC-dependent inhibition of the Ca(2+) channels. Possible control of synaptic transmission by the present mechanisms is discussed.

Animals↗

PubMed2000

A novel site on the Galpha -protein that recognizes heptahelical receptors.

Specific domains of the G-protein alpha subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between alpha4 and alpha5 helices of the G-protein alpha subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Galpha(15) and its human homologue Galpha(16), whereas metabotropic glutamate receptor 8 activated Galpha(15) only. The extreme C-terminal 20 amino acid residues are identical between the Galpha(15) and Galpha(16) and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Galpha(16) that inhibit its coupling to metabotropic glutamate receptor 8. On a three-dimensional model, both regions are found in a close proximity to the extreme C terminus of Galpha(16). One module comprises alpha4 helix, alpha4-beta6 loop (L9 Loop), beta6 sheet, and alpha5 helix. The other, not described previously, is located within the loop that links the N-terminal alpha helix to the beta1 strand of the Ras-like domain of the alpha subunit. Coupling of Galpha(16) protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully.

Antibodies↗

PubMed2000

p53 and Bax implication in NMDA induced-apoptosis in mouse hippocampus.

Seven days after in vivo intrahippocampal administration of NMDA, 3'-OH DNA fragmentations and Bax protein expression were detected in hippocampal neurons of p53+/+ but not p53-/- transgenic mice. Interestingly, neurons showing pycnosis, an early apoptotic phenomena, were present in all genotypes. These results confirm that apoptotic 3'OH DNA fragmentations and Bax protein induction during NMDA-induced apoptosis in adult hippocampal neurons are p53 dependent.

Animals↗

PubMed2000

Differential modulation of the 5-HT(4) receptor agonists and antagonist on rat learning and memory.

Recent data suggest that activation of 5-HT(4) receptors may modulate cognitive processes such as learning and memory. In the present study, the effects of two potent and selective 5-HT(4) agonists, RS 17017 [1-(4-amino-5-chloro-2-methoxyphenyl)-5- (piperidin-1-yl)-1-pentanone hydrochloride] and RS 67333 [1(4-amino-5-chloro-2-methoxyphenyl)-3- (1-n-butyl-4-piperidinyl)-1-propanone], were studied in an olfactory associative discrimination task. The implication of 5-HT(4) receptors in the associative discriminative task was suggested by the following observation. Injection of a selective 5-HT(4) receptor antagonist RS 67532 [1-(4-amino-5-chloro-2-(3, 5-dimethoxybenzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone; 1 mg/kg: i.p.] before the third training session induced a consistent deficit in associative memory during the following training sessions. This deficit was absent when the antagonist was injected together with either a specific hydrophilic 5-HT(4) (RS 17017, 1 mg/kg) or a specific hydrophobic (RS 67333, 1 mg/kg) 5-HT(4) receptor agonist. RS 67333 was more potent than RS 17017. This difference in potency certainly reflects a difference in their capacity to enter into the brain. This is also likely to be the reason why, injected alone, the hydrophobic 5-HT(4) agonist (RS 67333) but not the hydrophilic 5-HT(4) agonist (RS 17017) improved learning and memory performance.

Aniline Compounds↗

PubMed2000

Conservation of the ligand recognition site of metabotropic glutamate receptors during evolution.

Mammalian metabotropic glutamate receptors (mGluRs) are classified into 3 groups based on their sequence similarity and ligand recognition selectivity. Recently, we identified a Drosophila mGluR (DmGlu(A)R) which is about equidistant, phylogenetically, from the 3 mGluR groups. However, both the G-protein coupling selectivity and the pharmacological profile of DmGlu(A)R, as analysed with mutated G-proteins and a few compounds, look similar to those of mammalian group-II mGluRs. In the present study we carefully examined the pharmacological profile of DmGlu(A)R, and compared it to those of the rat mGlu(1a), mGlu(2) and mGlu(4a) receptors, representative of group-I, II and III respectively. The pharmacological profile of DmGlu(A)R was found to be similar to that of mGlu(2)R, and only very small differences could be identified at the level of their pharmacophore models. These data strongly suggest that the binding sites of these two receptors are similar. To further document this idea, a 3D model of the mGlu(2) binding domain was constructed based on the low sequence similarity with periplasmic amino acid binding proteins, and was used to identify the residues that possibly constitute the ligand recognition pocket. Interestingly, this putative binding pocket was found to be very well conserved between DmGlu(A)R and the mammalian group-II receptors. These data indicate that there has been a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs.

Amino Acid Sequence↗

PubMed2000

Pharmacological characterization of the rat metabotropic glutamate receptor type 8a revealed strong similarities and slight differences with the type 4a receptor.

In the brain, group-III metabotropic glutamate (mGlu) receptors mGlu(4), mGlu(7) and mGlu(8) receptors play a critical role in controlling the release process at many glutamatergic synapses. The pharmacological profile of mGlu(4) receptor has been studied extensively, allowing us to propose a pharmacophore model for this receptor subtype. Surprisingly, the activity of only a few compounds have been reported on mGlu(7) and mGlu(8) receptors. In order to identify new possibilities for the design of selective compounds able to discriminate between the members of the group-III mGlu receptors, we have undertaken a complete pharmacological characterization of mGlu(8) receptor and compared it with that of mGlu(4) receptor, using the same expression system, and the same read out. The activities of 32 different molecules revealed that these two mGlu receptors subtypes share a similar pharmacological profile. Only small differences were noticed in addition to that previously reported with S-carboxyglutamate (S-Gla) being a partial agonist at mGlu(4) receptor and a full antagonist at mGlu(8) receptor. These include: a slightly higher relative potency of the agonists 1S,3R and 1S,3S-aminocyclopentane-1,3-dicarboxylic acid (ACPD), S-4-carboxyphenylglycine (S-4CPG) and S-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG), and a slightly higher potency of the antagonists 2-aminobicyclo[3.1.0]hexane-2, 6-dicarboxylic acid (LY354740) and RS-alpha-methyl-4-phosphonophenylglycine (MPPG) on mGlu(8) receptor. When superimposed on the mGlu(4) receptor pharmacophore model, these molecules revealed three regions that may be different between the ligand binding sites of mGlu(8) and mGlu(4) receptors.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Imidazoline-induced neuroprotective effects result from blockade of NMDA receptor channels in neuronal cultures.

Imidazolines have been shown to be neuroprotective in focal and global ischemia in the rat. However, their mechanism of action is still unclear. We have studied the neuroprotective effects of imidazolines against NMDA-induced neuronal death and hypoxic insult in cerebellar and striatal neuronal cultures. All of the imidazolines tested decreased the NMDA-mediated neurotoxicity in a non-competitive manner. Antazoline was the most effective (IC(50) of 5 microM, maximal neuroprotection reaching 90% at 100 microM). The neuroprotective effects were still present when the imidazolines were applied during the post-insult period. Antazoline, idazoxan and guanabenz also showed neuroprotective effects against hypoxia-induced neuronal death (neuroprotection reaching 95% for antazoline at 100 microM). Antazoline was still active if applied during the reoxygenation period (15% neuroprotection). To determine the mechanism of the neuroprotective effects, the possible interaction of imidazolines with NMDA receptors was studied. Imidazolines dose-dependently and non-competitively inhibited NMDA currents. As found for the neuroprotective effects, antazoline was the most effective imidazoline, with an IC(50) of 4 microM and a maximal inhibition of 90% at 100 microM. This blockade was rapid, reversible and voltage-dependent. We compared these effects to those of the classical non-competitive antagonist of NMDA channels, MK-801. In contrast to imidazolines, blockade of the NMDA current by MK-801 was voltage-independent and reversible only at positive potentials. When co-applied with MK-801, antazoline prevented the long lasting blockade of the NMDA current by MK-801. These results are consistent with the existence of overlapping binding sites for these drugs on the NMDA receptor channel. They indicate that imidazolines exert a strong neuroprotective effect against excitotoxicity and hypoxia in cerebellar and striatal primary neuronal cultures by inhibiting NMDA receptors. Since these effects were non-competitive, imidazolines appear to be interesting new drugs with therapeutic potential.

Animals↗

PubMed2000

Complex interactions between mGluRs, intracellular Ca2+ stores and ion channels in neurons.

Metabotropic glutamate receptors (mGluRs) can increase intracellular Ca2+ concentration via Ins(1,4,5)P3- and ryanodine-sensitive Ca2+ stores in neurons. Both types of store are coupled functionally to Ca2+-permeable channels found in the plasma membrane. The mGluR-mediated increase in intracellular Ca2+ concentration can activate Ca2+-sensitive K+ channels and Ca2+-dependent nonselective cationic channels. These mGluR-mediated effects often result from mobilization of Ca2+ from ryanodine-sensitive, rather than Ins(1,4, 5)P3-sensitive, Ca2+ stores, suggesting that close functional interactions exist between mGluRs, intracellular Ca2+ stores and Ca2+-sensitive ion channels in the membrane.

Animals↗

PubMed2000

Sequential expression of surface antigens and transcription factor NFkappaB by hippocampal cells in excitotoxicity and experimental epilepsy.

Neurodegeneration and gliosis have been extensively described after long-lasting seizures; evidence for cytokine involvement in neuron-glia interactions does exist. We have therefore studied the hippocampal expression of molecules responsible for immune and inflammatory reactions, at different time-points following either experimental status epilepticus (SE) or direct excitotoxic damage. Experiments consisting of immunohistochemical labeling of glial markers, major histocompatibility complex (MHC) and nuclear factor kappaB (NFkappaB), were performed. NFkappaB nuclear translocation was controlled and measured using the electrophoretic mobility shift assay. One day after SE, neurodegeneration was obvious in CA3 pyramidal layers; NFkappaB staining in neurons and its translocation to the nucleus enhanced. From day 4 to at least day 8 post-SE, MHC-positive microglia, NFkappaB over-expression in thickened astrocytes, and increased levels of its activated form could be observed. The excitotoxic model caused more severe lesions, but NFkappaB and MHC expression were similar in both models. These results suggest that during long-lasting seizures: (i) neuronal firing activates NFkappaB expression and translocation; (ii) microglia expresses MHC; (iii) astrocytes, probably stimulated by microglial cytokines, over-express NFkappaB, the activation of which induces a cascade of reactions, particularly the transcription of cytokines and or neuroprotective molecules. Further clarification of the toxic or protective consequences of delayed inflammatory responses may be interesting in therapy of epilepsy.

Animals↗

PubMed2000

Cytokine signals propagate through the brain.

Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) are proinflammatory cytokines that are constitutively expressed in healthy, adult brain where they mediate normal neural functions such as sleep. They are neuromodulators expressed by and acting on neurons and glia. IL-1 and TNFalpha expression is upregulated in several important diseases/disorders. Upregulation of IL-1 and/or TNFalpha expression, elicited centrally or systemically, propagates through brain parenchyma following specific spatio-temporal patterns. We propose that cytokine signals propagate along neuronal projections and extracellular diffusion pathways by molecular cascades that need to be further elucidated. This elucidation is a prerequisite for better understanding of reciprocal interactions between nervous, endocrine and immune systems.

Animals↗

PubMed2000

"Inflammatory" cytokines: neuromodulators in normal brain?

If cytokines are constitutively expressed by and act on neurons in normal adult brain, then we may have to modify our current view that they are predominantly inflammatory mediators. We critically reviewed the literature to determine whether we could find experimental basis for such a modification. We focused on two "proinflammatory" cytokines, interleukin (IL)-1 and tumor necrosis factor-alpha (TNFalpha) because they have been most thoroughly investigated in shaping our current thinking. Evidence, although equivocal, indicates that the genes coding for these cytokines and their accessory proteins are expressed by neurons, in addition to glial cells, in normal brain. Their expression is region- and cell type-specific. Furthermore, bioactive cytokines have been extracted from various regions of normal brain. The cytokines' receptors selectively are present on all neural cell types, rendering them responsive to cytokine signaling. Blocking their action modifies multiple neural "housekeeping" functions. For example, blocking IL-1 or TNFalpha by several independent means alters regulation of sleep. This indicates that these cytokines likely modulate in the brain behavior of a normal organism. In addition, these cytokines are likely involved in synaptic plasticity, neural transmission, and Ca2+ signaling. Thus, the evidence strongly suggests that these cytokines perform neural functions in normal brain. We therefore propose that they should be thought of as neuromodulators in addition to inflammatory mediators.

Animals↗

PubMed2000

Characterization of intestinal receptors for VIP and PACAP in rat and in PAC1 receptor knockout mouse.

The receptors for VIP and PACAP were characterized in vitro on rat ileal and colonic longitudinal smooth muscle with adherent myenteric ganglia. Colon strips from PAC1 receptor knockout and wildtype mice were also examined. VIP, PACAP-38 and PACAP-27 all caused concentration dependent relaxations. In rat ileum three different types of smooth muscle VIP/PACAP receptors were defined: (1) a PACAP-27 preferring receptor coupled to apamin sensitive Ca(2+)-dependent K+ channels, (2) a PAC1 receptor activated by both PACAP-27 and PACAP-38, and (3) a VIP specific receptor regulated by NPY. The receptors identified in rat colon were: (1) a PAC1 receptor localized on NO synthesizing neurones. Activation leads to increased NO production. (2) A smooth muscle PAC1 receptor. The responses elicited by both receptors were abolished by apamin. (3) A smooth muscle VIP specific receptor. PAC1 receptor knockout mice did not respond to PACAP-27 or PACAP-38, whereas VIP induced a relaxatory response indicating the presence of a VIP specific receptor. In wildtype mice all three peptides elicited relaxatory responses. Pharmacological characterization of intestinal VIP/PACAP receptors indicates the existence of receptors, such as a PACAP-27 preferring receptor and a VIP specific receptor, distinct from those that have been cloned (VPAC1, VPAC2, and PAC1).

Animals↗

PubMed2000

Nerve growth factor upregulates the PAC1 promoter by activating the MAP kinase pathway in rat PC12 cells.

We report that: (1) An increase in the transcription activity is a mechanism by which trophic peptides may regulate the expression of PAC1. (2) An activation of the PAC1 promoter does not necessarily correlate with the neurotrophin-promoted neuritogenesis. (3) Activation of the PAC1 promoter is probably an early event since the EGF response is rather weak and transient in PC12 cells. (4) MAP kinase pathway activation is necessary for the NGF effect. The mechanism of the antagonism between PACAP and NGF observed on the PAC1 promoter activity and already described in regulating chromaffin cell proliferation, remains to be explained.

Alternative Splicing↗

PubMed2000

PAC1 null females display decreased fertility.

We report here that PAC1 KO females display decreased fertility, whereas male fertility was normal. ICC on pituitary section showed that FSH, LH, and prolactin synthesis were not affected in KO mice. Moreover, the pituitary-gonadal axis responded properly to an acute fasting test in KO mice. Hence, the phenotype of PAC1 null mice provides clear evidence for the role of PAC1 receptor in reproduction process.

Animals↗

PubMed2000

Pharmacological properties of 5-Hydroxytryptamine(4) receptor antagonists on constitutively active wild-type and mutated receptors.

We studied the pharmacological properties of twenty-four 5-hydroxytryptamine (5-HT)(4) receptor ligands known to act as antagonists on 5-HT(4) receptors positively coupled to adenylyl cyclase endogenously expressed in mouse colliculi neurons. In COS-7 cells expressing human or mouse 5-HT(4(a)) receptors (100-8000 fmol/mg of protein), we found neutral antagonists, partial agonists, and inverse agonists. The majority of neutral antagonists belong to the benzodioxanyl ketone class, whereas partial agonists belong to different chemical classes. We found only two inverse agonists, GR 125487 and SB 207266, which are both indoles. Analysis of pharmacological characteristics of the constitutively active wild-type and constitutively active mutated receptors revealed that 1) the ratio between the efficiencies of the full agonist 5-HT and the partial agonist RS 23597 was invariable when the receptor density increased, but was dependent on receptor structure; 2) similarly, the efficacy of the inverse agonist SB 207266 was not dependent on receptor density but was dependent on receptor structure; 3) when the receptor concentration increased, the EC(50) values of the full agonist 5-HT were not modified and the increase in basal constitutive activity, as well as its stimulation by 5-HT, followed a parallel evolution; and 4) the stimulation of basal constitutive activity by 5-HT was not modified by the overexpression of Galphas. All these results indicate that in COS-7 cells, the coupling of the 5-HT(4) receptor to adenylyl cyclase was linear with no indication of spare receptors even at high receptor density (8 pmol/mg). These results are also in accordance with a precoupling between the activated receptor (f(R*)) and adenylyl cyclase. Such observations allowed us to use the two-state model to calculate the constant J, i.e., the equilibrium allosteric constant denoting the ratio of the receptor in the inactive versus active state (J = [R]/[R*]). We found that J was a receptor structural characteristic, independent of receptor density.

Adenylyl Cyclases↗