Localization of the human pituitary adenylate cyclase-activating polypeptide receptor (PACAP1-R) gene to 7p15-p14 by fluorescence in situ hybridization.
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Cells, Cultured↗
Biomedical subjects
J Bockaert
Publications and source records attributed to J Bockaert.
The CaM-kinase II inhibitor KN-62 blocks NMDA but not kainate stimulation of NO synthesis.
We have studied the effect of CaM-kinase II inhibition induced by KN-62 on NO synthesis mediated by the activation of NMDA and kainate receptors in striatal neurones. KN-62 partially inhibited NMDA-mediated NO synthesis but the effect of kainate on NO production was unaffected by the specific CaM-kinase II inhibitor. Ionomycin-induced cGMP production and the NMDA-mediated superoxide generation by nNOS were also inhibited by KN-62. Since we determined the NO synthesis via its ability to increase intracellular cGMP, we checked that the kinase inhibitor was unable to block the cGMP production induced by a NO-donor like 3-morpholinosydnonimine (SIN-1). In conclusion, these results indicate that, depending on the glutamatergic receptors involved, CaM-kinase II could participate in the regulation of NO synthesis in striatal neurones.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Cloning and functional expression of a Drosophila metabotropic glutamate receptor expressed in the embryonic CNS.
The excitatory neurotransmitter glutamate plays important roles in the mammalian brain, ranging from synaptic plasticity to memory. To mediate these functions, glutamate activates two types of receptors: ligand-gated channels and metabotropic receptors coupled to G-proteins. Both families of glutamate receptors share no sequence homology and possess original structural features compared with other ligand-gated channels and G-protein-coupled receptors, respectively. Glutamate-gated receptor-channel subunits have already been characterized in invertebrates. Here we report the cloning and functional characterization of an invertebrate metabotropic glutamate receptor (DmGluRA) isolated from Drosophila melanogaster. This receptor displays 45 and 43% amino acid sequence identity with its mammalian homologs mGluR3 and mGluR2, respectively. Moreover, its pharmacology and transduction mechanisms are surprisingly similar to those of mGluR2 and mGluR3. DmGluRA is expressed in the CNS of the late embryo. These results indicate that the original structural features of both glutamate receptor types are conserved from insects to mammals and suggest that the functions of these receptors have been highly conserved during evolution.
Animals↗
Alternative splicing in the N-terminal extracellular domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor modulates receptor selectivity and relative potencies of PACAP-27 and PACAP-38 in phospholipase C activation.
Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). In addition, we demonstrated that the potencies of the two forms of PACAP are similar for adenylate cyclase stimulation, whereas PACAP-38 is more potent than PACAP-27 in phospholipase C activation. In the present work, we document the existence of a new splice variant of the PACAP receptor that was characterized by a 21-amino-acid deletion in the N-terminal extracellular domain. We demonstrate that this domain modulates receptor selectivity with respect to PACAP-27 and -38 binding and controls the relative potencies of the two agonists in phospholipase C stimulation.
Alternative Splicing↗
Functional coupling between ryanodine receptors and L-type calcium channels in neurons.
In skeletal muscle, L-type Ca2+ channels act as voltage sensors to control ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum. It has recently been demonstrated that these ryanodine receptors generate a retrograde signal that modifies L-type Ca2+ -channel activity. Here we demonstrate a tight functional coupling between ryanodine receptors and L-type Ca2+ channel in neurons. In cerebellar granule cells, activation of the type-1 metabotropic glutamate receptor (mGluR1) induced a large, oscillating increase of the L-type Ba2+ current. Activation occurred independently of inositol 1,4,5-trisphosphate and classical protein kinases, but was mimicked by caffeine and blocked by ryanodine. The kinetics of this blockade were dependent on the frequency of Ba2+ current stimulation. Both mGluR1 and caffeine-induced increase in L-type Ca2+ -channel activity persisted in inside-out membrane patches. In these excised patches, ryanodine suppressed both the mGluR1- and caffeine-activated L-type Ca2+ channels. These results demonstrate a novel mechanism for Ca2+ -channel modulation in neurons.
Animals↗
Pharmacological comparison between [3H]GR 113808 binding sites and functional 5-HT4 receptors in neurons.
5-HT4 receptors positively coupled to adenylyl cyclase and possessing unique pharmacological properties were first described in mouse colliculi neurons using functional studies. The recent introduction of a radiolabeled 5-HT4 receptor antagonist, [3H]GR 113808 [1-[2-(methylsulphonylamino)ethyl]4-piperidinyl]methyl-1-methyl-in dole-3 carboxylate] having high specificity and affinity allowed the pharmacological comparison between the specific binding sites identified with this compound and the functional 5-HT4 receptors in the same preparation, the colliculi neurons. We show here that [3H]GR 113808 binding is saturable in this preparation and reveals a homogeneous population of sites with a pKd value of 9.5 +/- 0.2 and a Bmax of 75 +/- 23 fmol/mg protein. Seventeen agonists and six antagonists with molecules structurally related either to indoles, benzamides or benzimidazolones and previously known as 5-HT4 receptor ligands, were tested for their ability to compete with [3H]GR 113808 binding sites and to stimulate or inhibit 5-HT-stimulated adenylyl cyclase activity. Highly significant correlations were obtained between the affinities of either agonists or antagonists for [3H]GR 113808 binding sites and their potencies for functional 5-HT4 receptors (r = 0.87 and 0.99, respectively). In addition, we also found good correlations between the Kd of several 5-HT4 receptor ligands determined in cell membranes of mouse colliculi neurons and their Kd determined in previous studies in guinea-pig striatum (0.95) and in human caudate (0.97). [3H]GR 113808 binding studies demonstrated that the 50% decrease in 5-HT-stimulated cAMP accumulation which followed a 5 min exposure period with 5-HT (10 microM) was not accompanied by any significant decrease in the number of binding sites. Longer exposure periods with 5-HT resulted in a decrease in [3H]GR 113808 binding sites which started to be significant after 30 min.
Animals↗
The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins.
Metabotropic glutamate receptors (mGluR) share no sequence homology with any other G-protein-coupled receptors (GPCRs). The characterization of their G-protein coupling domains will therefore help define the general rules for receptor-G-protein interaction. To this end, the intracellular domains of mGluR3 and mGluR1, receptors coupled negatively to adenylyl cyclase and positively to phospholipase C, respectively, were systematically exchanged. The ability of these chimeric receptors to induce Ca2+ signals were examined in Xenopus oocytes and HER 293 cells. The chimeric receptors that still possessed the second intracellular loop (i2) of these proteins were targeted correctly to the plasma membrane. Consistent Ca2+ signals could be recorded only with chimeric mGluR3 receptors that contains i2 and at least one other intracellular domains of mGluR3 have to be replaced by their mGluR1 equivalent to produce optimal coupling to G protein. These observations indicate that i2 of mGluR1 is a critical element in determining the transduction mechanism of this receptor. These results suggest that i2 of mGluRs may play a role similar to i3 of most other GPCRs in the specificity of coupling to the G-proteins. Moreover, as in many other GPCRs, our data revealed cooperation between the different mGluR intracellular domains to control efficient coupling to G-proteins.
Amino Acid Sequence↗
Regional distribution and ontogeny of 5-HT4 binding sites in rat brain.
The tritiated specific antagonist [3H]GR 113808 was used to study the brain regional distribution of 5-HT4 binding sites in rat during development. In adult brain, high densities of binding sites were found in the limbic system (islands of Calleja, olfactory tubercle, fundus striati, ventral pallidum, septum, hippocampus and amygdala) and in the striato-nigro-tectal pathway. Only low densities of binding sites were found in the prenatal brain, except in the brainstem. Marked density increases were observed during the second and third postnatal week in most forebrain regions. This ontogenic pattern indicates that 5-HT4 receptors probably exert a minor role in developmental processes, while, in the adult, the expression of 5-HT4 receptors in limbic areas strongly suggests a role for these receptors in emotional functions. The synchronous appearance of 5-HT4 receptors and cholinergic markers also indicates that these receptors might control acetylcholine release. This hypothesis has been confirmed by functional evidence and further supports the view that 5-HT4 drugs could improve cognitive functions in diseases with impaired cholinergic transmission (e.g., Alzheimer's disease).
Animals↗
Physiological and pharmacological profile of trans-azetidine-2,4-dicarboxylic acid: metabotropic glutamate receptor agonism and effects on long-term potentiation.
In this study, we biochemically analysed the effects of the novel metabotropic glutamate receptor agonist trans-azetidine-2,4-dicarboxylic acid and examined its role in hippocampal long-term potentiation. In cell lines expressing metabotropic receptor 1 or 5 subtypes, the compound stimulated phosphoinositide hydrolysis with EC50 values of 189.4 +/- 6.4 and 32.2 +/- 8.3 microM, respectively. In hippocampal slices, trans-azetidine-2,4-dicarboxylic acid also increased phosphoinositide hydrolysis, yet failed to show any effect on forskolin-stimulated formation of cyclic AMP, even if 1 mM azetidine was applied. Since trans-azetidine-2,4-dicarboxylic acid (20 mM in 5 microliters) injected cerebroventricularly prolongs long-term potentiation induced by weak tetanization, a possible interaction with N-methyl-D-aspartate receptors was investigated using patch-clamp techniques. Neither facilitation of N-methyl-D-aspartate (500 microM) currents nor induction of non-specific currents was observed in the presence of 50 and 500 microM azetidine. Strong tetanus-induced long-term potentiation in the dentate gyrus of freely moving rats was not influenced by azetidine. In combination with the antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (200 mM in 5 microliters), however, the potentiation was attenuated and returned to baseline within 90 min. Blockade of N-methyl-D-aspartate receptors using 2-amino-5-phosphonopentanoate (20 mM in 5 microliters) prevented the potentiation in controls, but not in the azetidine group, where normal potentiation was observed for both the population spike amplitude and the excitatory postsynaptic potential. These data suggest that (i) trans-azetidine-2,4- dicarboxylic acid is an agonist at glutamate metabotropic receptors; (ii) a facilitation of induction and maintenance of long-term potentiation via N-methyl-D-aspartate receptors seems unlikely; and (iii) pharmacological activation of metabotropic receptors prior to tetanization appears to bypass the N-methyl-D-aspartate receptor dependence of the potentiation. In conclusion, a role for metabotropic glutamate receptors in both short-term and long-term potentiation is indicated by these data.
Animals↗
Effects of nitric oxide on glutamate-gated channels and other ionic channels.
Nitric oxide is an endogenous molecule that plays a role of second messenger in the central and peripheral nervous system. A major action of this molecule is to control ionic channel activity. Because of technical difficulties to use nitric oxide as a gaseous compound, nitric oxide donors are often utilized under controlled experimental conditions. Here we will review the advantages and limitations in using these compounds. Nitric oxide can affect ionic channels through direct interactions or through the production of cGMP. We will describe an example of direct action of nitric oxide on glutamate-gated channels. We will also review indirect actions of nitric oxide on various potassium and calcium channels. Finally, we will discuss the complex physiological consequences of the action of nitric oxide on these ionic channels.
Animals↗
Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus.
The regional distribution of 5-hydroxytryptamine (5-HT4) receptors labelled with [3H]GR113808 was examined in rat basal ganglia and hippocampus after specific lesions. Lesion of serotonin neurons induced by injections of 5,7-dihydroxytryptamine into the dorsal and medial raphe nuclei resulted in increased 5-HT4 receptor binding in most regions examined, compared with controls. More precisely, there was a 78% increase in the rostral but no change in the caudal part of caudate-putamen, and 83% and 54% increases in the shell and core of the nucleus accumbens respectively. In the substantia nigra, the increase in 5-HT4 binding was larger (72%) than that in the globus pallidus (32%). In the hippocampus, 63%, 30% and 28% increases were measured in CA2, CA1 and CA3 respectively. Following lesion of dopamine neurons by intranigral injection of 6-hydroxydopamine, increased 5-HT4 receptor binding was observed in the caudal (59%), but not the rostral part of caudate-putamen, as well as in the globus pallidus (93%). Since no decreases in 5-HT4 receptor density were detected after the dopamine lesion, it was concluded that these receptors are not expressed in dopamine neurons. Kainic acid lesions of the caudate-putamen were associated with dramatic local decreases in 5-HT4 receptor binding on the injected side (-89%), which suggested that striatal neurons express 5-HT4 receptors. Corresponding decreases of 72 and 20% in receptor density were detected in globus pallidus and substantia nigra, consistent with a presumed localization of 5-HT4 receptors on striatal GABA neurons projecting to these regions. In the substantia nigra, the decrease in [3H]GR113808 binding was localized to the pars lateralis, indicating that striatal neurons belonging to the cortico-striato-nigro-tectal pathway, and containing GABA and dynorphin, express 5-HT4 receptors.
5,7-Dihydroxytryptamine↗
Comparative effects of PACAP and VIP on pancreatic endocrine secretions and vascular resistance in rat.
1. The effects of pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and secretin on pancreatic endocrine secretions and vascular resistance were investigated and compared in the isolated perfused pancreas of the rat. The PACAP/VIP receptor types involved have been characterized. 2. On insulin secretion, in the range 10(-11) to 10(-8) M, PACAP and VIP elicited a concentration-dependent biphasic response from pancreas perfused with 8.3 mM glucose; the peptides were equipotent. In contrast, secretin was ineffective in the range 10(-11) to 10(-9) M; at 10(-8) and 10(-7) M, it induced only low and transient insulin responses. On the other hand, the peptides did not modify the basal insulin release in the presence of a non stimulating glucose concentration (2.8 mM). 3. On glucagon secretion, PACAP and VIP (10(-11) to 10(-8) M) but also secretin (10(-9) to 10(-7) M) caused a concentration-dependent peak shaped response from pancreas perfused with 2.8 mM glucose; PACAP and VIP were equipotent and 20 times more potent then secretin. On the other hand, the peptides did not affect the glucagon release in the presence of 8.3 mM glucose. 4. On pancreatic vessels, in the range 10(-11) to 10(-9) M, the three peptides were equipotent in inducing a concentration-dependent sustained increase in pancreatic flow rate. On the other hand, at the high concentration of 10(-7) M PACAP but not VIP provoked a transient decrease of flow rate. 5. This study provides evidence for PACAP/VIP type II receptors mediating insulin and glucagon secretion as well as vasodilatation in rat pancreas. In addition, the different efficacies of secretin suggest that these effects are mediated by different PACAP/VIP type II receptor subtypes.
Animals↗
Changes in the carboxyl-terminal domain of metabotropic glutamate receptor 1 by alternative splicing generate receptors with differing agonist-independent activity.
The metabotropic glutamate receptors (mGluRs) share no sequence homology and show different structural features compared with most other G protein-coupled receptors (GPCRs). In particular, some isoforms of the phospholipase C (PLC)-coupled mGluRs (mGluR1a, mGluR5a, and mGluR5b) have a surprisingly long carboxyl-terminal intracellular domain of more than 350 residues, whereas the splice variants mGluR1b and mGluR1c have a much shorter carboxyl terminus. In the current study, the different splice variants of mGluR1 were expressed in porcine kidney epithelial (LLC-PK1) or the human embryonic kidney (HEK 293) cells, and their levels of expression were examined with the use of Western blot analysis. Expression of the short isoforms mGluR1b and mGluR1c did not modify the basal inositol phosphate production. In contrast, expression to similar levels of mGluR1a resulted in a 2-fold increase in the basal inositol phosphate formation. This increase in basal PLC activity was due to neither the presence of a low concentration of glutamate in the incubation medium nor a modification of the PLC pathway, resulting, for example, from the constant activation of mGluR1a++ by glutamate during the culture. Surprisingly none of the known competitive antagonists of mGluR1 inhibited the basal PLC activity, indicating that none of these molecules act as inverse agonists. Taken together, these results indicate that the long carboxyl-terminal domain confers a small agonist-independent activity to mGluR1. This indicates that, as already observed for other GPCRs, little constitutive activity of wild-type mGluRs can be detected. Our results also add to the splice variants and further suggest that the long carboxyl-terminal domain of mGluR1a confers better coupling efficiency to the G proteins.
Alternative Splicing↗
Coupling of metabotropic glutamate receptors 2 and 4 to G alpha 15, G alpha 16, and chimeric G alpha q/i proteins: characterization of new antagonists.
Together with the calcium-sensing receptor, the metabotropic glutamate receptors (mGluRs) share no sequence homology with the other G protein-coupled receptors (GPCRs) and therefore constitute a new family of receptors. Recently, it was reported that G alpha 15 and G alpha 16 subunits allow many GPCRs to activate phospholipase C (PLC). Furthermore, the exchange of a few carboxyl-terminal residues of G alpha q by those of G alpha 12 or G alpha o allows the resulting chimeric G alpha subunits (G alpha ql and G alpha qol respectively) to couple Gi-coupled receptors to PLC. We report that mGluR2 and mGluR4, two receptors negatively coupled to adenylyl cyclase, activate PLC when coexpressed with G alpha 15, G alpha ql or G alpha qo. This indicates that the carboxyl-terminal end of the G alpha subunit also plays an important role in the specific interaction between mGluRs and the G proteins. In addition, the measurement of PLC activation by Gi-coupled mGluRs coexpressed with these G alpha subunits constitutes an easy functional assay for the pharmacological characterization of these receptors. The rank order of potency of antagonists was found to be (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine approximately (R,S)- alpha-methyl-4-phosphonophenylglycine > (R,S)-alpha-methyl-4-sulfonophenylglycine > (R,S)-alpha-methyl-4-tetrazolylphenylglycine = (S)-2-amino-2-methyl-4-phosphonobutyrate for mGluR2 and to be (R,S)-alpha-methyl-4-phosphonophenylglycine > or = (S)-2-amino-2-methyl-4-phosphonobutyrate > > (R,S)-alpha-methyl-4-sulfonophenylglycine [(R,S)-alpha-methyl-4-tetrazolylphenylglycine and (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine being inactive at 1 mM] for mGluR4. Using this functional assay, (R,S)-alpha-methyl-4-phosphonophenylglycine was found to have a similar KB value for mGluR2 and mGluR4.
Adenylate Cyclase Toxin↗
[Large and small G proteins in vesicular transport].
The movement of proteins between compartments of the exocytic and endocytic pathways of eukaryotic cells is mediated by carrier vesicles. They bud from a donor compartment and are targetted to and fuse with the acceptor compartment. GTPases, proteins which bind and hydrolyze GTP, play key roles in the regulation of this vesicular protein transport. Heterotrimeric and small GTPases are involved in this vesicular traffic.
Coated Vesicles↗
Metabotropic glutamate receptors inhibiting excitatory synapses in the CA1 area of rat hippocampus.
In the CA1 region of hippocampal slices prepared from young adult rats, we studied the ability of several specific agonists of metabotropic glutamate receptors (mGluRs) to depress excitatory synaptic transmission at the CA3-CA1 pyramidal cell synapses. Three groups of mGluRs have been described: group 1 (mGluR1 and 5) receptors are positively coupled to phospholipase C whereas group 2 (mGluR2 and 3) and group 3 (mGluR4, 6, 7 and 8) receptors are negatively coupled to adenylate cyclase. We found that the broad-spectrum agonist (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate and the group 1-specific agonist (R,S)-dihydroxyphenylglycine both reversibly inhibited evoked field excitatory postsynaptic potentials, indicating the involvement of group 1 mGluRs. (R,S)-3,5-dihydroxyphenylglycine presumably inhibited transmission via a presynaptic mechanism, as whole-cell voltage-clamp recordings revealed that inhibition of the synaptic transmission was always accompanied with an increase in paired-pulse facilitation. Treatment with a specific blocker of mGluR1 receptors, the phenylglycine derivative (S)-4-carboxyphenylglycine, was without effect on the (1S,3R)-1-amino-cyclopentyl-1,3-dicarboxylate-induced depression of the field excitatory postsynaptic potentials, strongly suggesting that mGluR5 receptors are responsible for the (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate effect. Two selective agonists of group 2 mGluRs, (2S,1's,2's)-2-(2'-carboxycyclopropyl)glycine and 4-carboxy-3-hydroxyphenylglycine, were totally ineffective in blocking CA3-CA1-evoked synaptic transmission, excluding the involvement of mGluR2/3 subtypes at this developmental stage.
Animals↗
Oxygen free radicals enhance the nitric oxide-induced covalent NAD(+)-linkage to neuronal glyceraldehyde-3-phosphate dehydrogenase.
Nitric oxide (NO) induces a covalent modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from various tissues. This phenomenon, which has previously been interpreted as an auto-ADP-ribosylation, is in fact a covalent binding of NAD+ to the enzyme. In the present study, we show that 3-morpholino-sydnonimine (SIN-1) is much more efficient than sodium nitroprusside (SNP) in stimulating the covalent labelling of GAPDH from cultured striatal neurones in the presence of [adenylate-32P]NAD+ (877 +/- 110 and 266 +/- 33% increase in NAD(+)-labelling induced by maximally effective concentrations of SIN-1 and SNP respectively). The difference in the efficacy of both NO-generating compounds could be due to the additional release of superoxide by SIN-1, since superoxide dismutase and the nitrone 5,5'-dimethyl pyrroline-1-oxide markedly inhibited the SIN-1-induced covalent binding of NAD+ to GAPDH. Catalase and selective scavengers of hydroxyl radicals, mannitol and dimethyl sulphoxide, did not alter the SIN-1-induced covalent modification of GAPDH, ruling out the involvement of hydroxyl radicals in this phenomenon. Supporting further a role of oxygen free radicals in the NAD+ linkage to GAPDH, pyrogallol, a superoxide generator, which alone was ineffective, potentiated the SNP-evoked response. The NAD+ linkage to neuronal GAPDH measured in the presence of NO and superoxide probably involves sulphydryl groups, since the radiolabelling of the protein was reversed by exposure to HgCl2 and prevented by pretreatment with the alkylating agent N-ethylmaleimide. Moreover, the NO-induced inhibition of GAPDH activity was enhanced by pyrogallol, which was ineffective alone. In conclusion, the present study indicates that superoxide anions potentiate NO-induced covalent NAD(+)-linkage to GAPDH and enzyme inactivation.
Animals↗
cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.
We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.
8-Bromo Cyclic Adenosine Monophosphate↗