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J Bockaert

Publications and source records attributed to J Bockaert.

At least 127 records · Page 7Linked to original sources
PubMed1994

Plasticity of NMDA receptor expression during mouse cerebellar granule cell development.

A period of hypersensitivity to N-methyl-D-aspartate (NMDA) has been described during the early development of different types of neuron. Since activation of NMDA receptors can also induce rapid neuron death, the hypersensitivity to NMDA may be tightly controlled. In the present study we show that mouse cerebellar granule neurons become transiently hypersensitive to NMDA between days 10 and 14 after plating in a culture medium containing 30 mM K+. The NMDA sensitivity is higher when cells are cultured in the presence of an NMDA receptor antagonist [30 mM K+ plus 100 microM 3-((+/-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP)], and no hypersensitivity is observed when cells are cultured in the continuous presence of NMDA (12.5 mM K+ plus 100 microM NMDA). The high NMDA sensitivity in control cells is associated with a higher density of NMDA receptors than that measured in NMDA-treated cells, suggesting that the sensitivity to NMDA may be partly controlled by activity-dependent NMDA receptor down-regulation. We also examined the level of NMDA-zeta 1 mRNA and found no correlation between this parameter and the transient pattern of NMDA sensitivity. Such NMDA receptor plasticity may be of importance in the central nervous system, protecting developing cells from excitotoxicity at critical developmental stages.

Animals↗
PubMed1994

Mutation of valine residue unique to alpha subunit of Gs abolishes activation.

We recently characterized a decapeptide sequence (residues 367-376) that is important for the membrane association of the activated alpha subunit of Gs. We report here that when this sequence is replaced by the cognate sequence of Gi1 alpha subunit, the chimeric protein (Gsis alpha) still interacts with the membrane but cannot be activated, regardless of the mode of activation. Construction of various chimeras demonstrates that the single replacement of valine 367 by threonine, the cognate residue of Gi1 alpha subunit, fully reproduces the loss of activation. Analysis of nucleotide interaction reveals that the mutant V367T Gs alpha protein poorly binds GDP or GTP. On the other hand, the conservative change of valine to isoleucine does not alter activation. Interestingly, members of the Gs and G12 classes have a valine and an isoleucine, respectively, at this position, whereas members of the Gi or Gq class contain a threonine residue. The evolutionary relationship between the different classes suggests that the presence of a hydrophobic or a hydrophilic residue is not fortuitous in these alpha subunits and might provide distinctive structural and/or functional properties.

Amino Acid Sequence↗
PubMed1994

5-Hydroxytryptamine1A receptor synthetic peptides. Mechanisms of adenylyl cyclase inhibition.

The 5-hydroxytryptamine1A receptor (5-HT1AR) is a G-protein-coupled receptor negatively coupled to adenylyl cyclase (AC). We have studied the functional domains of 5-HT1AR using synthetic peptides to block or mimic receptor function. The entire second intracellular loop (5-HT1AR-i2) and the carboxyl end of the third intracellular loop (5-HT1AR-i3-C) strongly inhibited forskolin-stimulated AC activity. These effects were not additive with those of 5-HT. Like 5-HT, the peptides 5-HT1AR-i3-C and -i2 weakly inhibited AIF4- and Mn2+ stimulated AC activity. 5-HT1AR binding assays indicated that peptides could interact with the same G-protein pool as the 5-HT1AR. 5-HT1AR-i3-C- and -i2-stimulated [35S]guanosine 5'-O-(thiotriphosphate) binding on Go/Gi proteins. Only 5-HT1AR-i3-C partially adopted an alpha-helical conformation in solution. These data show that different domains in the 5-HT1AR second and third intracellular loops can couple to and activate Gi proteins in order to mediate AC inhibition. Peptide-induced AC inhibition was not sensitive to pertussis toxin as opposed to the 5-HT1AR-mediated effect. Our data show that the 5-HT1AR and the 5-HT1AR peptides activate Gi proteins in a slightly different manner.

3T3 Cells↗
PubMed1994

Glutamate receptors induce a burst of superoxide via activation of nitric oxide synthase in arginine-depleted neurons.

We have previously shown in cultured cerebellar granule neurons (Lafon-Cazal, M., Pietri, S., Culcasi, M., and Bockaert, J. (1993) Nature 364, 535-537) that upon N-methyl-D-aspartate stimulation, a nitric oxide synthase (NOS)-independent, arachidonic acid-dependent generation of superoxide free radicals (O2-.) is observed after a lag time of 10-15 min. Using the electron spin resonance spin trapping technique, we show that N-methyl-D-aspartate stimulation produced a more rapid burst of O2-. in L-arginine (L-Arg)-depleted neurons. These O2-. radicals are synthesized by NOS. KCl and kainate, which also stimulated NOS in these neurons, produced this rapid burst of O2-., which was blocked as follows: (a) in the presence of L-NG-nitro-arginine (L-Narg), and (b) by L-Arg repletion. This burst of O2-. was arachidonic acid-independent, and its time course was similar to that of nitric oxide production. It was also responsible for a weak but significant cell death that was suppressed by L-Narg and L-Arg.

Amino Acid Oxidoreductases↗
PubMed1994

Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors.

G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G protein-coupled receptors. They constitute therefore a new family of receptors. Whereas mGluR1 and mGluR5 activate phospholipase C (PLC), mGluR2, mGluR3, mGluR4 and mGluR6 inhibit adenylyl cyclase (AC) activity. The third putative intracellular loop, which determines the G protein specificity in many G protein-coupled receptors, is highly conserved among mGluRs, and may therefore not be involved in the specific recognition of G proteins in this receptor family. By constructing chimeric receptors between the AC-coupled mGluR3 and the PLC-coupled mGluR1c, we report here that both the C-terminal end of the second intracellular loop and the segment located downstream of the seventh transmembrane domain are necessary for the specific activation of PLC by mGluR1c. These two segments are rich in basic residues and are likely to be amphipathic alpha-helices, two characteristics of the G protein interacting domains of all G protein-coupled receptors. This indicates that whereas no amino acid sequence homology between mGluRs and the other G protein-coupled receptors can be found, their G protein interacting domains have similar structural features.

Amino Acid Sequence↗
PubMed1994

Identification of multiple subunits of heterotrimeric G proteins on the membrane of secretory granules in rat prolactin anterior pituitary cells.

The subcellular distribution of multiple subunits of heterotrimeric GTP-binding proteins has been investigated in rat anterior pituitary cells in primary culture, and more precisely in prolactin cells, by immunocytochemistry and subcellular fractionation followed by immunoblotting or ADP ribosylation, using polyclonal affinity-purified antibodies directed against Gi3 alpha, Gs alpha, Go1 alpha, Go2 alpha, and G beta. As expected, all these subunits were detected on the plasma membrane. They were, however, also detected on the membrane of several intracellular compartments involved in the secretory pathway, particularly on the secretory granule membrane. Differences appeared between the precise subcellular distribution and the local concentration of each subunit. The main subunits present on the secretory granule membrane were Gi3 alpha and Gs alpha. Go1 alpha, Go2 alpha, and G beta were detected, to a lesser extent, on parts of the membrane of a few secretory granules located near the plasma membrane. Domains of the rough endoplasmic reticulum cisternae were immunolabeled with anti-Gs alpha and anti-Go1 alpha. In the Golgi zone, the membrane of some vesicles was stained only with anti-Gs alpha and anti-Go2 alpha. The association of this set of heterotrimeric G protein subunits on the membrane of the secretory granules suggests that these subunits could be involved in the regulation of formation, storage, targeting, and/or exocytosis of these organelles.

Animals↗
PubMed1994

The PACAP receptor: generation by alternative splicing of functional diversity among G protein-coupled receptors in nerve cells.

Recent molecular characterization of new G protein-coupled receptors (GPCR) draw attention to alternative splicing as a source of structural diversity. After a brief overview of characterized GPCR splice variants, we will describe in more detail the functional properties of the PACAP type I receptor splice variants. Some of these variants are positively coupled to both adenylate cyclase (AC) and phospholipase C (PLC) whereas others do not elicit any stimulation of the PLC or display a qualitatively intermediate phenotype. The PACAP type I receptor is therefore one of the few examples in which alternative splicing is clearly linked to functional diversity.

Alternative Splicing↗
PubMed1994

Inhibitory effects of dihydropyridines on macroscopic K+ currents and on the large-conductance Ca(2+)-activated K+ channel in cultured cerebellar granule cells.

In cultured cerebellar granule cells, we examined the effects of dihydropyridines (DHPs) on K+ currents, using the whole-cell recording configuration of the patch-clamp technique and on Ca(2+)-activated K+ channels ("maxi K+ channels") using outside-out patches. We found that micromolar concentrations of nicardipine, nifedipine, (+) and (-) BAY K 8644, nitrendipine, nisoldipine and (-) nimodipine block 10-60% of macroscopic K+ currents. The most potent of these DHPs was nicardipine and the least potent, (-) BAY K 8644. (+) Nimodipine had no effect on this current. The inhibitory effects of nifedipine and nicardipine were not additive with those of 1 mM tetraethylammonium (TEA). Outside-out recordings of "maxi K+ channels" showed a main conductance of 200 pS (in 77% of the patches) and two subconductance states (in 23% of the patches). Neither nifedipine nor nicardipine affected the main conductance, but decreased the values of the subconductance levels. In 10% of these patches, nicardipine induced a flickering activity of the channel. These findings show that both Ca2+ and K+ channels have DHP-sensitive sites, suggesting similarity in electrostatic binding properties of these channels. Furthermore, cerebellar granule cells may express different subtypes of "maxi K+ channels" having different sensitivities to DHPs. These drugs may provide new tools for the molecular study of K+ channels.

Animals↗
PubMed1994

Blockade of nitric oxide synthesis by tyrosine kinase inhibitors in neurones.

In striatal neurones in culture, N-methyl-D-aspartate-(NMDA), kainate-(Kai) and K(+)-dependent cGMP production is entirely mediated via nitric oxide (NO). Low concentrations of lavendustin-A (< or = 0.3 microM), a highly specific tyrosine kinase inhibitor, reduced irreversibly and in a time-dependent manner NMDA-stimulated cGMP production. After a preincubation period of 20 min with lavendustin-A (0.3 microM), the inhibition of NMDA-induced cGMP production was equal to 56 +/- 8% (n = 6). After the same preincubation period, the IC50 of the lavendustin-A blockade was 30 +/- 15 nM. Genistein, another tyrosine kinase inhibitor also inhibited NMDA-dependent cGMP production with high potencies (< or = 3 microM). Whatever the tyrosine kinase inhibitor tested, the basal cGMP production remained unaffected. Kai-, K(+)-, and ionomycin-induced cGMP production was also inhibited by lavendustin-A, and genistein. In contrast, tyrosine kinase inhibitors were unable to block NO donor-induced cGMP production. Using patch clamp experiments, we have also found that lavendustin-A (0.3-1 microM), the most potent tyrosine kinase inhibitor used, (a) did not reduce the NMDA receptor-mediated current, (b) only slighly affected Kai receptor-mediated current (16.4 +/- 3.4% inhibition) and (c) had a marked effect on voltage-sensitive Ca2+ channel- (VSCC) mediated currents (44.4 +/- 4.9% inhibition). A reduction in VSCC activity certainly explains the inhibition of K(+)-, Kai- and possibly part of the NMDA-induced cGMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

The metabotropic glutamate receptor types 2/3 inhibit L-type calcium channels via a pertussis toxin-sensitive G-protein in cultured cerebellar granule cells.

Modulation of Ca2+ channels by metabotropic glutamate receptors (mGluRs) was investigated in cerebellar granule cells using the cell-attached configuration of the patch-clamp technique. Experiments were performed in the absence of external Ca2+ and Ba2+ was used as charge carrier. Bath applied glutamate or (1S,3R) trans-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R t-ACPD) inhibited Ca2+ channels activated by depolarizing pulses. These channels were sensitive to dihydropyridines and displayed a 23 pS conductance. This effect was mimicked by (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), a selective agonist of mGluR2/R3 receptors, but not by quisqualate at a concentration that stimulated inositol phosphate (InsP) synthesis, showing that mGluR1 and mGluR5 did not participate to this mechanism. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), did not alter the action of the mGluR agonists and biochemical measurements showed that 1S,3R t-ACPD, in the presence of IBMX, decreased cAMP formation in such a small amount that this change could not explain the almost complete inhibition of the channel activity observed under similar experimental conditions. Moreover, whole-cell recorded L-type Ca2+ currents were inhibited by L-CCG-I, in the presence of 1 mM intracellular cAMP. These observations were consistent with the hypothesis that cyclic nucleotide second messengers were not involved in this effect. Neither the protein kinase C activator phorbol-12,13-dibutyrate (PDBU) nor the phosphatase inhibitor okadaic acid affected the action of 1S,3R t-ACPD. The inhibitory action of 1S,3R t-ACPD was abolished by pertussis toxin (PTX). These results suggest that mGluR2 or mGluR3 receptors suppress the activity of L-type Ca2+ channels by a mechanism involving Gi or G(o) proteins. A likely direct effect of G-proteins on the channels is discussed.

Animals↗
PubMed1994

Pharmacological characterization of metabotropic glutamate receptors in several types of brain cells in primary cultures.

Several cDNAs coding for metabotropic glutamate receptors (mGluR1-7) have now been isolated. mGluR1 and -5 are positively coupled to phospholipase C, whereas mGluR2, -3, -4, -6, and -7 are negatively coupled to adenylyl cyclase (AC) when they are expressed in Chinese hamster ovary or baby hamster kidney cells. However, the exact transduction mechanisms of these receptors in their natural environment remain to be determined. In a previous work, we demonstrated that striatal neurons in primary culture expressed a mGluR that is negatively coupled to AC and that has a pharmacology different from that of mGluR2. In the present study, the pharmacology of mGluRs negatively coupled to AC in several neuronal types and in glial cells was compared with the pharmacology of mGluR2, -3, and -4. Like striatal neurons, cerebral cortical neurons express a mGluR that is able to inhibit AC both in intact cells and in membrane preparations, via a pertussis toxin-sensitive G protein. This mGluR has a pharmacological profile similar to that of mGluR3, because quisqualate is active at relatively low concentrations (EC50 < 100 microM). Similar experiments revealed that cerebellar granule cells expressed mGluR2-like and mGluR4-like receptors. Striatal glial cells also expressed a mGluR negatively coupled to AC via a pertussis toxin-sensitive G protein. However, only glutamate and aspartate, and not quisqualate, 2-(carboxycyclopropyl)glycine, trans-1-aminocyclopentane-1,3-dicarboxylate, or L-2-amino-4-phosphonobutyrate, were agonists for this glial mGluR. This pharmacology is different from that of any cloned mGluR. Reverse transcription associated with polymerase chain reaction revealed that mGluR2 and mGluR3 mRNAs are present in striatal, cortical, and cerebellar neurons but not in striatal glial cells. Interestingly, mGluR4 mRNA was found at a high level in cerebellar granule cells and at a lower level in cortical neurons and glial cells. However, the mGluR4-specific agonist L-2-amino-4-phosphonobutyrate was found to inhibit AC very slightly in granule cells only. In conclusion, our data show that mGluR2- and mGluR3-like receptors can directly inhibit AC in neurons, and they raise the question of whether mGluR4 is really negatively coupled to AC in its normal environment. We also present evidence for a new mGluR subtype expressed in glial cells.

Adenylate Cyclase Toxin↗
PubMed1993

[3H]-GR113808 labels 5-HT4 receptors in the human and guinea-pig brain.

A tritiated specific and highly potent 5-HT4 antagonist: [3H]-GR113808 was used to label specific binding sites in human brain and for comparison in guinea-pig brain. [3H]-GR113808 association and dissociation were rapid, the binding was saturable and displaced by various serotoninergic agents with an affinity corresponding to their previously reported 5-HT4 receptor mediated effects. In vitro ligand binding autoradiography was used to investigate the distribution of [3H]-GR113808 recognition sites on human brain sections. The highest density was observed in the striato-nigral system. Low densities of binding sites were also found in hippocampus, neocortex and colliculus. This distribution of [3H]-GR113808 binding sites is similar to that found in the guinea-pig brain.

Aged↗
PubMed1993

Differential signal transduction by five splice variants of the PACAP receptor.

The two forms of pituitary adenylyl cyclase-activating polypeptide (PACAP-27 and -38) are neuropeptides of the secretin/glucagon/vasoactive intestinal polypeptide/growth-hormone-releasing hormone family and regulate hormone release from the pituitary and adrenal gland. They may also be involved in spermatogenesis, and PACAP-38 potently stimulates neuritogenesis and survival of cultured rat sympathetic neuroblast and promotes neurite outgrowth of PC-12 cells. The PACAP type-I receptor (found in hypothalamus, brain stem, pituitary, adrenal gland and testes), specific for PACAP, is positively coupled to adenylyl cyclase and phospholipase C. The recently cloned type II receptor does not discriminate between PACAP and vasoactive intestinal polypeptide and is coupled to only adenylyl cyclase. Here we have used a new expression cloning strategy, based on the induction of a reporter gene by cyclic AMP, to isolate a complementary DNA encoding the type-I PACAP receptor. On transfection of this cDNA, both PACAP-27 and -38 stimulate adenylyl cyclase with similar EC50 values (50% effective concentration, 0.1-0.4 nM), whereas only PACAP-38 stimulates phospholipase C with high potency (EC50 = 15 nM). Four other splice variants were isolated with insertions at the C-terminal end of the third intracellular loop. Expression of these cDNAs revealed altered patterns of adenylyl cyclase and phospholipase C stimulation, suggesting a novel mechanism for fine tuning of signal transduction.

Adenylyl Cyclases↗
PubMed1993

L-nitroarginine, an inhibitor of NO synthase, dramatically worsens limbic epilepsy in rats.

During status epilepticus provoked by an intra-amygdala injection of kainic acid, the severity of seizures and of consequent neuronal damage was considerably increased in rats treated with L-NOARG, at a dose which completely inhibited NO synthesis. We propose that the effects of L-NOARG could be related to the loss of a retrograde inhibition exerted by NO on NMDA receptors. The complete suppression of NO formation in the brain finally facilitated the development and the generalization of seizures and their neurotoxic consequences.

Amino Acid Oxidoreductases↗
PubMed1993

NMDA-dependent superoxide production and neurotoxicity.

Neuronal injury resulting from acute brain insults and some neurodegenerative diseases implicates N-methyl-D-aspartate (NMDA) glutamate receptors. The fact that antioxidants reduce some types of brain damage suggests that oxygen radicals may have a role. It has been shown that mutations in Cu/Zn-superoxide dismutase (SOD), an enzyme catalysing superoxide (O2.-) detoxification in the cell, are linked to a familial form of amyotrophic lateral sclerosis (ALS). Here we report that O2.- is produced upon NMDA receptor stimulation in cultured cerebellar granule cells. Electron paramagnetic resonance was used to assess O2.- production that was due in part to the release of arachidonic acid. Activation of kainic acid receptors, or voltage-sensitive Ca2+ channels, did not produce detectable O2.-. We also find that the nitrone DMPO (5,5-dimethyl pyrroline 1-oxide), used as a spin trap, is more efficient than the nitric oxide synthase inhibitor, L-NG-nitro-arginine, in reducing NMDA-induced neuronal death in these cultures.

Amino Acid Oxidoreductases↗
PubMed1993

Glutamate stimulates glucagon secretion via an excitatory amino acid receptor of the AMPA subtype in rat pancreas.

The effect of L-glutamate was studied on glucagon secretion from rat isolated pancreas perfused with 2.8 mM glucose. L-Glutamate (3.10(-5)-10(-4)M) induced an immediate, transient and concentration-dependent glucagon release. The three non-N-methyl-D-aspartate (NMDA) receptor agonists, kainate (3.10(-5)-3.10(-3)M), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (3.10(-5)-10(-4)M) and quisqualate (3.10(-6)-10(-5)M), all elicited a peak-shaped glucagon response. Compared to glutamate, AMPA and quisqualate exhibited a similar efficacy, whereas kainate caused a 4-fold higher maximal glucagon response. In contrast, NMDA (10(-3)M) was ineffective. The selective antagonist of non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5.10(-5)M), totally prevented the glucagon response to 10(-4) M glutamate (IC50 congruent to 0.8 +/- 0.3 10(-6)M) and 3.10(-4)M kainate. Furthermore, quisqualate at a maximal effective concentration (3.10(-4)M) inhibited the response to kainate (10(-3)M). This study showed that L-glutamate stimulates glucagon release in rat pancreas by activating a receptor of the AMPA subtype.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Transfected Go1 alpha inhibits the calcium dependence of beta-adrenergic stimulated cAMP accumulation in C6 glioma cells.

Increasing evidence indicates that heterotrimeric G proteins, and in particular Go, regulate ionic channel activities. In order to investigate the role of Go proteins in the modulation of the Ca2+ influx, C6 glioma cells were stably transfected with alpha o1 cDNA. Expression of the Go1 alpha protein was checked by Bordetella pertussis toxin-catalyzed ADP-ribosylation and Western blots using one- and two-dimensional gel analyses. Three clones were selected based on their degree of Go1 alpha expression. In alpha o1-transfected cells, cAMP accumulations, in response to isoproterenol or forskolin, were lower than in control cells. This inhibitory effect was a function of the amount of expressed Go1 alpha. In contrast, Go1 alpha expression was not followed by a significant inhibition of isoproterenol- or forskolin-stimulated adenylyl cyclase activities in particulate fractions. In C6 parental cells, 50-60% of the isoproterenol-induced cAMP accumulation was dependent on external Ca2+ concentration. This Ca(2+)-dependent cAMP accumulation was related to an induced transient Ca2+ influx. In transfected cells, expression of Go1 alpha inhibited the Ca2+ influx and the Ca(2+)-dependent component of isoproterenol-induced cAMP accumulation. In conclusion, beta-adrenergic agonists stimulate an entry of Ca2+ which exerts a positive feedback on cAMP production, and Go1 alpha blocks this positive feedback by inhibiting the Ca2+ influx.

Adenylyl Cyclases↗