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J Bockaert

Publications and source records attributed to J Bockaert.

At least 181 records · Page 10Linked to original sources
PubMed1991

Cholinergic inositol phosphate formation in striatal neurons is mediated by distinct mechanisms.

In murine striatal neurons devoid of functional synapses (6 days in vitro) the cholinergic agonists carbachol and arecoline evoked dose-dependent inositol phosphate (InsP) responses with mean log EC50s of -4.1 +/- 0.5 and -4.48 +/- 0.1, respectively. Carbachol (1 mM) and arecoline (1 mM) responses were insensitive to tetrodotoxin, a voltage-sensitive Na+ channel blocker, and were blocked by pirenzepine with relatively low affinity (logIC50 = -5.9 +/- 0.3 for the carbachol response and logIC50 = -5.8 +/- 0.3 for the arecoline response). After synaptogenesis (13 days in vitro) the maximal carbachol effect doubled whereas the arecoline response remained unchanged. This additional effect was sensitive to tetrodotoxin and the voltage-dependent Ca2+ channel blocker, omega-conotoxin. The tetrodotoxin-sensitive carbachol response was blocked by lower concentrations of pirenzepine than the tetrodotoxin-insensitive carbachol response. More than 75% of the InsP response evoked by low concentrations of muscarine (1 and 10 microM) was sensitive to tetrodotoxin whereas only 38% of the InsP response stimulated by 1 mM of muscarine could be blocked by tetrodotoxin. These results suggest that there are at least two different mechanisms (depending on the stage of development), activated most probably by two different muscarinic receptors responsible for the carbachol-induced InsP formation in striatal neurons.

Animals↗
PubMed1991

Azabicycloalkyl benzimidazolone derivatives as a novel class of potent agonists at the 5-HT4 receptor positively coupled to adenylate cyclase in brain.

Recent experimental evidence indicates that central 5-HT4 receptors which are positively coupled to adenylate cyclase, are stimulated by a family of 2-methoxy-4-amino-5-chloro substituted benzamide derivatives. These compounds are also potent stimulants of the gastro-intestinal motility. In this study the ability of three azabicycloalkyl benzimidazolone derivatives, BIMU 1, BIMU 8, and DAU 6215 (structural formulas are given in the text), to stimulate cAMP formation in colliculi neurons in primary culture have been tested. Two of the compounds, BIMU 1 and BIMU 8, which show prokinetic activity in various animal models, were also good agonists at the 5-HT4 receptors, whereas DAU 6215, a drug devoid of prokinetic activity, was only a weak, partial agonist at 5-HT4 receptors. The rank order of their potencies as compared with those of 5-HT and cisapride was as follows: BIMU 8 = cisapride greater than 5-HT greater than BIMU 1 greater than DAU 6215. The efficacies of BIMU 8 and cisapride were comparable (133 +/- 9% and 124 +/- 8% of the maximal 5-HT efficacy, respectively), whereas BIMU 1 and DAU 6215 elicited, respectively, only 72 +/- 11% and 16 +/- 4% of the maximal 5-HT effect. The activities of the azabicycloalkyl benzimidazolone derivatives and 5-HT on cAMP formation were not additive and ICS 205-930 antagonized the stimulatory effect of these compounds with low potency (pKi = 6.1-6.4), further strengthening the notion of interaction with 5-HT4 receptors. In addition, cross desensitization between the effects of 5-HT and the azabicycloalkyl benzimidazolones on adenylate cyclase was noted, another argument in favor of an interaction of these drugs on 5-HT4 receptors.

Adenylyl Cyclases↗
PubMed1991

Expression of the guanine nucleotide-binding protein Go correlates with the state of neural competence in the amphibian embryo.

The nucleotide-binding protein Go is a transducing molecule closely associated with neural structures in vertebrates. Because of the potential importance of molecules of this type during the first step of neurogenesis, we have investigated the kinetics of expression of Go in the amphibian (Pleurodeles waltl) embryo, focusing our attention on the stages corresponding to the acquisition of neural competence by presumptive ectoderm and to the process of neural induction. Using affinity-purified IgGs directed against the alpha subunit of Go, Go-like immunoreaction (GoLI) is first detected at the midblastula stage in some animal cap (future ectodermal) cells just before they have attained competence to be neuralized. At the early gastrula stage, GoLI is almost exclusively expressed by neural-competent tissue as a whole, with no obvious difference between the dorsal (prospective neural) and the ventral (prospective epidermal) ectoderm. The expression of GoLI is therefore related to the state of competence of the tissue rather than to its fate. At the early neurula stage, immediately following neural induction, the expression of GoLI persists essentially in that part of ectoderm that has been diverted from epidermal differentiation towards the neural pathway; in the ventral ectoderm, as neural competence is lost GoLI disappears. Furthermore, in the neurectoderm, only approximately 70% of the cells conserve GoLI, demonstrating that immediately following neural induction the population of neurectodermal cells is not homogeneous.

Animals↗
PubMed1991

The transduction signalling protein Go during embryonic development of Drosophila melanogaster.

G proteins are heterotrimeric proteins that play a key role in signalling transduction conveying signals from cell surface receptors to intracellular effector proteins. In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacted in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the alpha subunit of the mammalian Go protein. The Drosophila Go alpha protein was present at all stages of embryonic development; however, its expression markedly increased after 10 h embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appeared to be highly labelled. The distribution of the Go alpha protein was also examined in several neurogenic mutants. The Go alpha protein expression was not altered in any of them but the pattern of labelling was disorganized as was the neuronal network. These results suggest a possible role for the Go protein during axonogenesis.

Amino Acid Sequence↗
PubMed1991

beta-N-methylamino-L-alanine is a low-affinity agonist of metabotropic glutamate receptors.

beta-N-methylamino-L-alanine (L-BMAA) is an excitotoxin whose neurodegenerative effects are associated with its agonist properties at the N-methyl-D-aspartate (NMDA) receptor. We measured the effects of L-BMAA on inositol phosphate (InsP) formation in primary cultured striatal neurons. This culture is almost devoid of glial cells and the pharmacology of glutamate receptors is well-defined. This allowed us to show that L-BMAA induced InsP formation via a direct action at the glutamate metabotropic (Qp) receptors coupled to InsP formation. We demonstrated that L-BMAA is a full-agonist of the Qp receptor, but with a low potency. Therefore, the neurotoxic properties of L-BMAA might implicate the activation of the Qp receptor in association with the NMDA receptor.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1991

[Monoclonal antibodies obtained against G-protein epitopes: comparison of immunoreactions seen in the brain, retina and striated muscular tissue].

Monoclonal antibodies have been obtained against a purified fraction of brain G proteins containing the Gi alpha, G0 alpha, G beta, and G gamma subunits. After characterization, two monoclonal antibodies have been used to detect the cellular distribution of the two epitopes in neural, retinal and muscular tissues: ELISA, cross-dot and Western blot demonstrated that F.IV.5 is an anti-G beta antibody specific for the 36 kDa beta-subunit. ELISA, cross-dot and immunocytochemical distribution of the epitopes recognized by F.VII.9 suggested that this antibody recognizes epitopes which are also detected with polyclonal anti-G0 alpha antibodies. With both monoclonal antibodies, we confirmed that G proteins demonstrated a sub-membranous distribution as well as extensively cytoplasmic, axoplasmic or sarcoplasmic distributions in different cell types.

Animals↗
PubMed1991

5-HT1A-sensitive adenylyl cyclase of rodent hippocampal neurons: effects of antidepressant treatments and chronic stimulation with agonists.

The effects of chronic treatment with desimipramine (a tricyclic antidepressant), fluoxetine [a specific 5-hydroxytryptamine (5-HT) uptake inhibitor], clorgyline (a specific monoamine oxydase inhibitor of A type), ipsapirone (a specific 5-HT1A receptor agonist) as well as electroconvulsive shock treatment were investigated on rat hippocampal 5-HT1A receptors negatively coupled to adenylyl cyclase. Drugs were injected intraperitoneally in rats for 2 or 3 weeks, and biochemical determinations were made 4 to 72 hr after the final dose. Chronic treatments with desimipramine, ipsapirone and fluoxetine did not induce any change in the 5-HT1A-induced inhibition of the adenylyl cyclase activity. In contrast, chronic treatment with clorgyline and electroconvulsive shock treatment induced a slight but significant reduction of 5-HT's ability to inhibit hippocampal adenylyl cyclase. This indicates that, at least in hippocampal neurons, the 5-HT1A receptor coupled to adenylyl cyclase is not easily desensitized. This was verified in vitro on murine hippocampal neurons in culture, by measuring the effects of intense stimulation (1 and 2 hours), with 5-HT, ipsapirone and 8-hydroxy-2-(di-n-propylamino)tetralin. Indeed, such stimulations did not significantly affect the 5-HT1A receptor-induced inhibition of cAMP production in these hippocampal neurons in culture. Our results indicate that it is not the post-synaptic 5-HT1A receptor of hippocampus that is modified during antidepressant treatments, at least at the level of its coupling to adenylyl cyclase.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Dual mechanisms of inhibition by dopamine of basal and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells. Evidence for an inhibition not mediated by voltage-dependent Ca2+ channels.

In primary cultures of anterior pituitary cells, dopamine inhibited basal and thyrotropin-releasing hormone (TRH)-stimulated inositol monophosphate, bisphosphate, and trisphosphate production. This inhibition by dopamine can be resolved into two distinct components. One of the components was rapid and already present after 10 s. The other was slower, starting after 1 min, and was mimicked by nimodipine, a dihydropyridine calcium channel antagonist. The effects of dopamine and nimodipine were not additive on both basal and TRH-stimulated inositol phosphate production. Furthermore, the dopamine inhibition in the presence of TRH was much higher than the inhibition induced by nimodipine. It is thus likely that calcium entry through voltage-dependent calcium channels triggers a positive feedback on TRH stimulation of phospholipase C. However, depolarizing concentrations of K+ or BAY-K-8644, a voltage-dependent calcium channel agonist, had no effect on inositol monophosphate and bisphosphate accumulation. Ionomycin, even at a very high concentration (10 microM), had only a slight and transient effect on inositol phosphate formation. In addition, these agents did not affect the TRH dose-dependent stimulation of inositol phosphate production. These results suggest that the intracellular calcium concentrations that we measured under basal and TRH-stimulated conditions are sufficient to allow the maximal activity of phospholipase C which can be obtained under these two experimental conditions. In contrast, any decrease in the intracellular calcium concentration by a dihydropyridine antagonist, suppression of extracellular calcium, or inactivation of a voltage-dependent calcium channel by long term depolarization with K+ decreased the phospholipase C activities measured under basal and TRH-stimulated conditions. From these data it can be concluded that dopamine inhibits inositol phosphate production by two distinct mechanisms. The slow dopamine-induced inhibition of TRH-stimulated inositol phosphate production which is mimicked by nimodipine is likely because of an inhibition of a voltage-dependent calcium channel. This is substantiated further by the fact that ionomycin (10 microM) was able to reverse the nimodipine inhibitions as well as this slow component of dopamine inhibition. The nature of the rapid inhibition of TRH-stimulated inositol phosphate production induced by dopamine, but not by nimodipine, remains to be determined. It is suppressed in the absence of extracellular Ca2+. This may suggest that this inhibition is related to blockade of non-dihydropyridine-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

On concanavalin A-treated striatal neurons quisqualate clearly behaves as a partial agonist of a receptor fully activated by kainate.

In cultured striatal neurons, maximal [3H]GABA release stimulated by quisqualate (QA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) was 10-20 times smaller than that stimulated by kainate (KA), and we have previously reported that QA or AMPA competitively inhibited KA-evoked GABA release. Since the lectin concanavalin A (Con A) has been shown to inhibit QA receptor desensitization, the interaction between QA and KA was further studied in Con A-treated neurons. Con A dose-dependently and specifically potentiated QA- or AMPA-evoked [3H]GABA release, so that maximal responses of QA or AMPA were half of that of KA. The responses of these agonists were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with similar apparent Ki values, indicating that they resulted from non-NMDA receptor activation. In Con A-treated neurons, QA and AMPA competitively inhibited the KA-induced GABA release. The apparent affinities of QA and AMPA in inhibiting the KA response were identical to their affinities in stimulating GABA release. Moreover, the maximal KA response measured in the presence of QA or AMPA was identical to that measured with KA alone. These results clearly indicate that to stimulate GABA release from Con A-treated striatal neurons, QA and AMPA behave as partial agonists of a receptor fully activated by KA. These results further support the hypothesis that QA, AMPA and KA act on a common receptor type in striatal neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1990

Arachidonic acid released from striatal neurons by joint stimulation of ionotropic and metabotropic quisqualate receptors.

Associative stimulation of N-methyl-D-aspartate (NMDA) receptors and quisqualate ionotropic receptors (Qi) induces long-term potentiation at particular glutamatergic synapses. Release of arachidonic acid as a result of stimulation of NMDA receptors has been proposed to play a part in the establishment of long-term potentiation. But long-term plasticity events at some other glutamatergic synapses do not involve activation of NMDA receptors. Here we report that in mature striatal neurons in primary cultures, quisqualate can release arachidonic acid by associatively activating both quisqualate metabotropic receptors coupled to phospholipase C (Qp) and Qi receptors. Independent activation of these two receptor types with specific agonists did not stimulate arachidonic acid release. These results support a role for the associative activation of Qp and Qi receptors in synaptic plasticity events, including long-term potentiation at particular synapses.

Arachidonic Acid↗
PubMed1990

Establishment of a long-term primary culture of striatal neurons.

A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2-5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30-50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical, pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.

Animals↗
PubMed1990

Omega-conotoxin GVIA and dihydropyridines discriminate two types of Ca2+ channels involved in GABA release from striatal neurons in culture.

We previously reported that the opening of the L-type Ca2+ channel was only partly involved in the K(+)-evoked Ca2(+)-dependent gamma-aminobutyric acid (GABA) release from striatal neurons, suggesting that probably different types of voltage-sensitive Ca2+ channels were implicated in this physiological process. Here we demonstrate that omega-conotoxin GVIA, which has been reported to block L- and N-type neuronal Ca2+ channels, also partly inhibits the Ca2(+)-dependent GABA release. The maximal effects of omega-conotoxin GVIA and nifedipine, a highly specific antagonist of the L-type channels, were additive, a total inhibition of the Ca2(+)-dependent GABA release being obtained in the presence of both drugs. We therefore propose that omega-conotoxin GVIA and nifedipine block two different types of Ca2+ channels, both involved in the GABA release process.

Calcium Channels↗
PubMed1990

Direct activation of GTP-binding regulatory proteins (G-proteins) by substance P and compound 48/80.

The neuropeptide substance P and the polyamine compound 48/80, both known to activate mast cell secretory processes, increased the rate of GTP S binding to G-proteins purified from calf brain (Go/Gi mixture). The GTPase activity of G-proteins was also increased by substance P and compound 48/80 in a dose-dependent and Mg2+-dependent way. These effects were similar to those of the wasp venom peptide mastoparan, another histamine releaser of rat peritoneal and human skin mast cells. This suggests that the secretory property of compound 48/80 and substance P is not due to a receptor-mediated process but, like mastoparan, results from a direct activation of G-proteins.

Animals↗
PubMed1990

Cellular distribution and biochemical characterization of G proteins in skeletal muscle: comparative location with voltage-dependent calcium channels.

GTP binding proteins have been proposed to play a role in excitation--contraction coupling. In a precedent study [Toutant et al., (1988), Biochem. J., 405-409], we determined that Bordetella pertussis toxin is able to catalyse ADP-ribosylation of two substrates in the detergent soluble fraction of total muscle extracts. Purified fractions of transverse tubule membranes (T-tubule membranes), a key element of the excitation--contraction coupling, were shown to exhibit a major ADP-ribosylated substrate at 40 kd and an immunoreactivity with antisera raised against purified bovine brain Go alpha or G beta. In the present study, we have investigated the cellular distribution of G protein subunits in comparison with that of the voltage-dependent Ca2+ channels by immunofluorescence on transverse and longitudinal sections of fast and slow muscles. With affinity-purified antibodies against G beta subunits, a fluorescent labelling underlined the myofibrils and sarcolemma, whereas a strong immunoreaction in a dotted pattern evoked the presence of the subunit in repetitive triadic structures. With anti-Go alpha antibodies, the immunofluorescence was more clearly focussed on a dotted pattern and the co-location with the voltage-dependent Ca2+ channel immunoreactivity indicates that both proteins were located in very close subcellular structures. Immunoblot analysis and PTX ADP-ribosylation of the purified light sarcoplasmic reticulum (LSR), heavy sarcoplasmic reticulum (HSR) and T-tubule subcellular fractions indicate the discrete presence of G proteins in LSR, an unambiguous labelling of the HSR fraction, while T-tubule membranes clearly appear very rich in a Go-like protein, confirming the observed preferential immunocytochemical distribution of G protein subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A Go-like protein in Drosophila melanogaster and its expression in memory mutants.

G proteins couple receptors for extracellular signals to several intracellular effector systems and play a key role in signalling transduction mechanisms. In particulate preparations of Drosophila melanogaster heads, only one substrate for pertussis toxin at 39-40 kd was detected. This substrate, which showed only one isoform when analysed by isoelectric focusing, was recognized by immunoblotting and immunoprecipitation techniques using a polyclonal antibody against the alpha subunit of the Go protein purified from bovine brain and can be thus considered as a Go-like protein. Antibodies obtained against a carboxy-terminal sequence of the alpha subunit of Go (but not of Gi1 or Gi2) and against an internal sequence shared by all the alpha subunits, were also able to cross-react with the alpha subunit of this protein in insects. We have also studied the Go-like protein in several D.melanogaster mutants, primarily in memory and learning mutants. In these mutants there was a sex-dependent enhancement in pertussis toxin-catalysed ADP-ribosylation with respect to the wild-type. This increase could be attributed in part to an increase in the alpha subunit of the Go-like protein, as revealed by immunoblotting with anti-Go alpha polyclonal antibody. This report constitutes the first evidence for the participation of a Go protein in learning and memory.

Adenosine Diphosphate Ribose↗
PubMed1990

Rapid, sensitive, and simple method for quantification of both neurotoxic and neurotrophic effects of NMDA on cultured cerebellar granule cells.

A simple and sensitive method adapted from the staining of living cells with fluorescein diacetate was developed to rapidly estimate the number of living cells remaining in a culture dish 24 hr after a few min of NMDA treatment of cerebellar neurons. This method consists of the measurement, after cell lysis, of the total amount of fluorescein produced from fluorescein diacetate by the living granule cells present in each culture dish. We show that this method can also be used to quantify the survival effect of chronic exposure of granule cells to either K+ or NMDA. In both cases, the fluorescence measured was found to be proportional to the number of fluorescein-labelled cells counted under a fluorescence microscope, indicating that the present method can be used to quantify both toxic and trophic effects of NMDA on cerebellar granule cells. This study confirms that these two NMDA effects occur at the same NMDA concentration, and both are inhibited by MK 801 in the same concentration range. We showed, moreover, that granule neurons developed in the presence of NMDA are much less sensitive to NMDA toxicity than neurons developed in K(+)-enriched medium.

Animals↗