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Major depressive disorder (MDD) is one of the most disabling psychiatric disorders in the world. First-line treatments such as selective serotonin reuptake inhibitors (SSRIs) still have many limitations, including a resistance to treatment in 30% of patients and a delayed clinical benefit that is observed only after several weeks of treatment. Increasing clinical evidence indicates that the acute administration of psychedelic agonists of the serotonin 5-HT2Areceptor (5-HT2AR), such as psilocybin, to patients with MDD induce fast antidepressant effects, which persist up to five weeks after the treatment. However, the involvement of the 5-HT2AR in these antidepressant effects remains controversial. Furthermore, whether the hallucinogenic properties of 5-HT2AR agonists are mandatory to their antidepressant activity is still an open question. Here, we addressed these issues by investigating the effect of two psychedelics of different chemical families, DOI and psilocybin, and a non-hallucinogenic 5-HT2AR agonist, lisuride, in a chronic despair mouse model exhibiting a robust depressive-like phenotype. We show that a single injection of each drug to wild type mice induces anxiolytic- and antidepressant-like effects in the novelty-suppressed feeding, sucrose preference and forced swim tests, which last up to 15 days. DOI and lisuride administration did not produce antidepressant-like effects in 5-HT2A−/−mice, whereas psilocybin was still effective. Moreover, neither 5-HT1AR blockade nor dopamine D1or D2receptor blockade affected the antidepressant-like effects of psilocybin in 5-HT2A−/−mice. Collectively, these findings indicate that 5-HT2AR agonists can produce antidepressant-like effects independently of hallucinogenic properties through mechanisms involving or not involving the receptor.

The serotonin 5-HT2Aand glutamate mGlu2receptors continue to attract particular attention, given their implication in psychosis associated with schizophrenia and the mechanism of action of atypical antipsychotics and a new class of antipsychotics, respectively. A large body of evidence indicates a functional crosstalk between both receptors in the brain, but the underlying mechanisms are not entirely elucidated. Here, we have explored the influence of 5-HT2Areceptor upon the phosphorylation pattern of mGlu2receptor in light of the importance of specific phosphorylation events in regulating G protein-coupled receptor signaling and physiological outcomes. Among the five mGlu2receptor-phosphorylated residues identified in HEK-293 cells, the phosphorylation of Ser843was enhanced upon mGlu2receptor stimulation by the orthosteric agonist LY379268 only in cells co-expressing the 5-HT2Areceptor. Likewise, administration of LY379268 increased mGlu2receptor phosphorylation at Ser843in prefrontal cortex of wild-type mice but not 5-HT2A−/−mice. Exposure of HEK-293 cells co-expressing mGlu2and 5-HT2Areceptors to 5-HT also increased Ser843phosphorylation state to a magnitude similar to that measured in LY379268-treated cells. In both HEK-293 cells and prefrontal cortex, Ser843phosphorylation elicited by 5-HT2Areceptor stimulation was prevented by the mGlu2receptor antagonist LY341495, while the LY379268-induced effect was abolished by the 5-HT2Areceptor antagonist M100907. Mutation of Ser843into alanine strongly reduced Gi/osignaling elicited by mGlu2or 5-HT2Areceptor stimulation in cells co-expressing both receptors. Collectively, these findings identify mGlu2receptor phosphorylation at Ser843as a key molecular event that underlies the functional crosstalk between both receptors.

The serotonin 5-HT2Areceptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2Areceptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2Areceptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2Aagonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versuslisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2Areceptor, a region important for its desensitization. The specific phosphorylation of Ser280by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2Areceptors at Ser280in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT2Areceptor in response to hallucinogenic versusnonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor.

Although most antidepressants suppress serotonin (5-HT) and/or noradrenaline reuptake, blockade of 5-HT(2C) receptors and α(2)-adrenoceptors likewise enhances monoaminergic transmission. These sites are targeted by the urea derivative N- [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3-H-benzo[e]indole-3-carboxamide (S32212). S32212 was devoid of affinity for monoamine reuptake sites, yet displayed pronounced affinity (pK(i), 8.2) for constitutively active human 5-HT(2CINI) (h5-HT(2CINI)) receptors, behaving as an inverse agonist in reducing basal Gα(q) activation, [(3)H]inositol-phosphate production, and the spontaneous association of h5-HT(2CINI)-Renilla luciferase receptors with β-arrestin2-yellow fluorescent protein. Furthermore, upon 18-h pretreatment, S32212 enhanced the plasma membrane expression of h5-HT(2CINI) receptors as visualized by confocal microscopy and quantified by enzyme-linked immunosorbent assay. Its actions were prevented by the neutral antagonist 6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl]indoline-1-carboxamide (SB242,084), which also impeded the induction by long-term exposure to S32212 of otherwise absent Ca(2+) mobilization in mouse cortical neurones. In vivo, S32212 blunted the inhibitory influence of the 5-HT(2C) agonist 2-(3-chlorobenzyloxy)-6-(1-piperazinyl)pyrazine (CP809,101) on ventrotegmental dopaminergic neurones. S32212 also blocked 5-HT-induced Gα(q) and phospholipase C activation at the h5-HT(2A) and, less potently, h5-HT(2B) receptors and suppressed the discriminative stimulus properties of the 5-HT(2A) agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in rats. S32212 manifested marked affinity for human α(2A)- (pK(i) 7.2), α(2B)- (pK(i) 8.2), and α(2C)- (pK(i) 7.4) adrenoceptors, at which it abolished noradrenaline-induced recruitment of Gα(i3), Gα(o), adenylyl cyclase, and extracellular-regulated kinase1/2. Moreover, S32212 dose-dependently abolished the discriminative stimulus effects of the α(2)-adrenoceptor agonist (S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(1',2',3',4'-tetrahydronaphthalene)] (S18616). Finally, S32212 displayed negligible affinity for α(1A)-adrenoceptors, histamine H(1) receptors, and muscarinic M(1) receptors. In conclusion, S32212 behaves as an inverse agonist at h5-HT(2C) receptors and as an antagonist at human α(2)-adrenoceptors (and h5-HT(2A) receptors). Its promising profile in preclinical models potentially relevant to the treatment of depression is described in J Pharmacol Exp Ther 340:765-780, 2012.

Serotonin (5-HT)2Creceptor is a Gq-coupled receptor exhibiting a high degree of constitutive activity toward phospholipase C effector pathway, a process regulated by receptor mRNA editing. In addition to G protein-dependent signaling, 5-HT2Creceptors also activate the extracellular signal-regulated kinase (ERK) 1/2 pathway independently of receptor coupling to G proteins. Constitutive activity at ERK signaling has not yet been explored. Transient expression of unedited 5-HT2C-INIreceptors in human embryonic kidney (HEK) 293 cells resulted in a marked increase in ERK1/2 phosphorylation compared with nontransfected cells. No increase in ERK1/2 phosphorylation was measured in cells expressing fully edited (5-HT2C-VGV) receptors. Basal ERK1/2 phosphorylation in 5-HT2C-INIreceptor-expressing cells was abolished by 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole (SB206,553), a 5-HT2Cinverse agonist toward phospholipase C. This effect was prevented by the neutral antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline (SB242,084), which alone did not alter basal activity. Similar observations were made in vivo in mouse choroid plexus, a structure rich in constitutively active 5-HT2Creceptors. Reminiscent of agonist-induced ERK1/2 phosphorylation, basal activity in HEK 293 cells was unaffected by cellular depletion of Gαq/11and Gα13proteins but strongly reduced in cells expressing a dominant-negative β-arrestin or depleted of β-arrestin by RNA interference and in cells expressing a dominant-negative calmodulin or a 5-HT2C-INIreceptor mutant not capable of interacting with calmodulin. The tetracyclic antidepressants mirtazapine and mianserin likewise reduced basal ERK activation. On the other hand, the m-chlorophenylpiperazine derivative trazodone and the selective serotonin reuptake inhibitor fluoxetine were inactive alone but blocked 5-HT-induced ERK1/2 phosphorylation. Together, these data provide the first evidence of constitutive activity of a G protein-coupled receptor toward G-independent, β-arrestin-dependent, receptor signaling.

The serotonin (5-hydroxytryptamine; 5-HT)2Creceptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT2Creceptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca2+-dependent "1-10" CaM-binding motif located in the proximal region of the 5-HT2Creceptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT2Creceptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT2Creceptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT2Creceptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT2Creceptor agonists and their antidepressant-like activity.

Despite the importance of 5-hydroxytryptamine (5-HT)2C(serotonin) receptors in the control of depressive states, actions of antidepressants at these receptors remain poorly characterized. This issue was addressed both in human embryonic kidney (HEK)-293 cells coexpressing unedited human 5-HT2CINIreceptors and Gαqprotein and in cultured mouse cortical neurons. Indicative of constitutive activity, the inverse agonist SB206,553 decreased basal inositol phosphate (IP) production in HEK-293 cells. The tetracyclic antidepressants mirtazapine and mianserin likewise suppressed basal IP formation. Conversely, the tricyclics amitriptyline and clomipramine, the m-chlorophenylpiperazine derivatives trazodone and nefazodone, and the 5-HT reuptake inhibitors fluoxetine and citalopram were inactive alone, although they blocked 5-HT-induced IP production. Inverse agonist actions of 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole (SB206,553) and mirtazapine were abolished by the neutral antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline (SB242,084), which was inactive alone. As assessed by confocal microscopy and enzyme-linked immunosorbent assay, prolonged treatment of HEK-293 cells with SB206,553, mirtazapine, or mianserin, but not the other antidepressants, enhanced cell surface expression of 5-HT2Creceptors: 5-HT-induced IP production was also increased, and both these actions were blocked by SB242,084. Cortical neurons were shown by reverse transcription-polymerase chain reaction to predominantly express constitutively active 5-HT2Creceptor isoforms. Prolonged pretreatment with SB206,553 or mirtazapine triggered an otherwise absent 5-HT-induced elevation in cytosolic Ca2+concentrations. SB242,084, which was inactive alone, abolished these effects of SB206,553 and mirtazapine. In conclusion, the tetracyclic antidepressants mirtazapine and mianserin, but not other clinically established antidepressants, suppress constitutive activity at recombinant and native 5-HT2Creceptors. The clinical significance of inverse agonist versus neutral antagonist properties both during and after drug administration will be of interest to elucidate.

Glutamate is the predominant excitatory neurotransmitter in the vertebrate brain, whereas acetylcholine has been considered to play the same role in insects. Recent studies have, however, questioned the latter view by showing a rather general distribution of glutamate transporters. Here, we describe the expression pattern of the receptor DmGlu-A (DmGluRA), the unique homolog of vertebrate metabotropic glutamate receptors. Metabotropic glutamate receptors play important roles in the regulation of glutamatergic neurotransmission. Using a specific antibody, we report DmGluRA expression in most neuropile areas in both larvae and adults, but not in the lobes of the mushroom bodies. These observations suggest a key role for glutamate in the insect brain.

Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remain a challenge. In this study, we established appropriate conditions to consider spatiotemporally resolved images of such protein assemblies using bioluminescence resonance energy transfer (BRET) in mammalian living cells. Using covalently linked Renillaluciferase and yellow fluorescent proteins, we depicted the time course of dynamic changes in the interaction between the V2-vasopressin receptor and β-arrestin induced by a receptor agonist. The protein-protein interactions were resolved at the level of subcellular compartments (nucleus, plasma membrane, or endocytic vesicules) and in real time within tens-of-seconds to tens-of-minutes time frame. These studies provide a proof of principle as well as experimental parameters and controls required for high-resolution dynamic studies using BRET imaging in single cells.

The 5-hydroxytryptamine4(5-HT4) receptors have recently emerged as key modulators of learning, memory, and cognitive processes. In neurons, 5-hydroxytryptamine4receptors (5-HT4Rs) activate cAMP production and protein kinase A (PKA); however, nothing is known about their ability to activate another key signaling pathway involved in learning and memory: the extracellular signal-regulated kinase (ERK) pathway. Here, we show that 5-HT4R stimulation, in primary neurons, produced a potent but transient activation of the ERK pathway. Surprisingly, this activation was mostly PKA independent. Similarly, using pharmacological, genetic, and molecular tools, we observed that 5-HT4Rs in human embryonic kidney 293 cells, activated the ERK pathway in a Gs/cAMP/PKA-independent manner. We also demonstrated that other classical G proteins (Gq/Gi/Go) and associated downstream messengers were not implicated in the 5-HT4R–activated ERK pathway. The 5-HT4R–mediated ERK activation seemed to be dependent on Src tyrosine kinase and yet totally independent of β-arrestin. Immunocytofluorescence revealed that ERK activation by 5-HT4R was restrained to the plasma membrane, whereas p-Src colocalized with the receptor and carried on even after endocytosis. This phenomenon may result from a tight interaction between 5-HT4R and p-Src detected by coimmunoprecipitation. Finally, we confirmed that the main route by which 5-HT4Rs activate ERKs in neurons was Src dependent. Thus, in addition to classical cAMP/PKA signaling pathways, 5-HT4Rs may use ERK pathways to control memory process.

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)2Creceptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT2Creceptor-mediated Ca2+response, whereas MPP3 prevented desensitization of the Ca2+response. The effects of the PDZ proteins on the desensitization of the Ca2+response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT2Creceptor response in neurons, by using a peptidyl mimetic of the 5-HT2Creceptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT2Creceptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT2Creceptor-mediated Ca2+response. This indicates that, overall, interaction of 5-HT2Creceptors with PDZ proteins inhibits receptor desensitization in cortical neurons.

We showed recently that imidazolines exert neuroprotection against hypoxia and NMDA toxicity in cerebellar and striatal neuronal cultures, through a voltage-dependent blockade of glutamatergic NMDA receptors. Here, we report that in striatal neuronal cultures from mouse embryos the imidazoline compound, antazoline, inhibits voltage-gated Ca2channels by acting at a phencycli-dine-like site. This effect was fast, fully reversible, voltage-dependent and predominant on PQ- and N-type Ca2channels. Taken together, these results suggest that imidazolines may elicit neuroprotective effects also by decreasing the release of glutamate through inhibition of presynaptic Ca2channels.

Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT4receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D1003.32A in transmembrane domain (TMD)-III and F2756.51A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third “silent” state stabilized by inverse agonists. We also found another mutation in TMD-VI (W2726.48A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.

L‐glutamate is the excitatory neurotransmitter at neuromuscular junctions in insects. It may also be involved in neurotransmission within the central nervous system (CNS), but its function therein remains elusive. The roles of glutamatergic synapses in the Drosophila melanogasterCNS were investigated, with focus on the study of DmGluRA, a G‐protein‐coupled glutamate receptor. In a first attempt to determine the function of this receptor, we describe its distribution in the larval and adult DrosophilaCNS, using a polyclonal antibody raised against the C‐terminal sequence of the protein. DmGluRA is expressed in a reproducible pattern both in the larva and in the adult. In particular, DmGluRA can be found in the antennal lobes, the optic lobes, the central complex, and the median bundle in the adult CNS. However, DmGluRA‐containing neurons represented only a small fraction of all CNS neurons. DmGluRA immunoreactivity was not detected at the larval neuromuscular junction nor in the body wall muscles. The correlations between DmGluRA distribution and previously described glutamate‐like immunoreactivity patterns, as well as the implications of these observations concerning the possible functions of DmGluRA in the DrosophilaCNS, are discussed. J. Comp. Neurol. 438:213–225, 2001. © 2001 Wiley‐Liss, Inc.

ZAC is a recently isolated zinc finger protein that induces apoptosis and cellcycle arrest. The corresponding gene is imprinted maternally through an unknown mechanism and maps to 6q24–q25, within the minimal interval harboring the gene responsible for transient neonatal diabetes mellitus (TNDM) and a tumor suppressor gene involved in breast cancer. Because of its functional properties, imprinting status, and expression pattern in mammary cell lines and tumors,ZACis the best candidate so far for both disease conditions. In the present work, we delineated ZACgenomic organization and mapped its transcriptional start site. It is noteworthy that the ZACpromoter localized to the CpG island harboring the methylation imprint associated with TNDM and methylation of this promoter silenced its activity. These data indicate that the methylation mark may have a direct effect on the silencing of the ZACimprinted allele. Our findings further strengthen the hypothesis that ZACis the gene responsible for TNDM and suggest a novel mechanism for ZACinactivation in breast tumors.

Specific domains of the G-protein α subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between α4 and α5 helices of the G-protein α subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Gα15and its human homologue Gα16, whereas metabotropic glutamate receptor 8 activated Gα15only. The extreme C-terminal 20 amino acid residues are identical between the Gα15and Gα16and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Gα16that 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 Gα16. One module comprises α4 helix, α4−β6 loop (L9 Loop), β6 sheet, and α5 helix. The other, not described previously, is located within the loop that links the N-terminal α helix to the β1 strand of the Ras-like domain of the α subunit. Coupling of Gα16protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully.

Seven days after in vivointrahippocampal 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.

Pituitary adenylate cyclase-activating polypeptides (PACAP-27 and -38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)secretinglucagon family. PACAP receptors are expressed in different brain regions including the cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP-38 on apoptosis induced by potassium deprivation. We demonstrated that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. We showed that PACAP-38 increased survival of cerebellar neurons in a dose-dependent manner by specifically decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP-38 induced activation of the extracellular signal-regulated kinase (ERK)-type of MAP kinase through a cAMP-dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the anti-apoptotic effect of PACAP-38, suggesting that MAP kinase pathway activation is necessary for PACAP-38 effect.

Glutamate and dopamine are important neurotransmitters in the basal ganglia. Dopamine can act via D1 receptors to activate adenylyl cyclase in striatal neurons, while glutamate stimulation of NMDA receptors leads to an increase in intracellular calcium. Increases in intracellular calcium or cAMP can induce immediate early gene expression in striatal neurons. In the present study, NMDA receptor stimulation or adenylyl cyclase activation resulted in the activation of MAP kinase in striatal neurons in primary culture. The effect of cAMP appeared to involve cAMP‐dependent protein kinase, in addition to a tyrosine kinase and MEK. NMDA‐induced MAP kinase activation was also dependent on a tyrosine kinase and MEK. The EGF receptor, which has been implicated in calcium‐ and G protein‐induced MAP kinase activation, did not mediate the effects of NMDA or forskolin on MAP kinase. Furthermore, the src kinase inhibitor, herbimycin A, and the phosphoinositol‐3‐kinase inhibitor, wortmannin, did not prevent MAP kinase activation by these stimuli. However, the ability of both NMDA and forskolin to activate MAP kinase in striatal neurons was blocked by SB 203580, an inhibitor of p38 reactivating kinase. These results indicate that both NMDA receptor activation and elevations in cAMP can result in MEK‐induced MAP kinase activation in striatal neurons. However, the signal transduction pathways mediating these responses appear to be distinct from those known to mediate MAP kinase activation by other stimuli. Synapse 29:29–36, 1998.© 1998 Wiley‐Liss, Inc.

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedoelectric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetellapertussis toxin, known to catalyze the ADP-ribosylation of the α-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the G oα-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedosynaptic vesicles either by ADP-ribosylation or by immunoblotting.

Regulation of inositol phosphate synthesis was examined in a primary culture of vascular smooth muscle cells grown on collagen-coated microcarriers. In the presence of Lid (10 mM), four agonists [serotonin, angiotensin, (arginine) vasopressin and noradrenaline] were found to stimulate the formation of inositol phosphates in a dose-dependent manner. All agonists were found to have identical and additive effects on the time course of inositol phosphate formation. Therefore, our primary cell culture technique was proved to give smooth muscle cells suitable for the study of modulation of phosphoinositide metabolism in response to physiological effectors.

Alpha-2 adrenergic receptors were solubilized from calf cerebral cortex using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The soluble extract retained the ability to bind the partial α2-adrenergic agonist [3H] p-aminoclonidine. This binding was of high affinity (KD= 1.5 ± 0.3 nM) saturable (Bmax = 150 ± 24 fmol/mg) and reversible. The orders of potencies of α-adrenergic drugs and nucleotides, for inhibition of binding, paralleled those previously observed on membrane bound α2-adrenoceptors. Furthermore the very rapid decrease of [3H] p-amino-clonidine binding induced by GTP suggest that the guanyl nucleotide effect is due to an allosteric mechanism. These results indicate that in the soluble extract the interaction between the α2-adrenoceptor and the GTP-binding regulator protein is preserved thus providing an interesting model to study the molecular structure of this system.

We have previously shown that a non-classical 5-hydroxytryptamine (5-HT4) receptor mediates the stimulation of adenylate cyclase activity in mouse embryo colliculi neurons in primary culture. The pharmacological characteristics of this receptor exclude the possibility that it belongs to the known 5-HT1, 5-HT2 or 5-HT3 receptor types. Here we report that this 5-HT receptor can be stimulated by 4-amino-5-chloro-2-methoxy substituted benzamide derivatives. All these compounds have been reported to be potent stimulants of gastrointestinal motility and some of them are 5-HT3 receptor antagonists. The rank order of potency of these substituted benzamide derivatives in stimulating cAMP formation was: cisapride > BRL 24924 > 5-HT > zacopride > BRL 20627 > metoclopramide. The non-additivity of benzamide and 5-HT activities suggests that 5-HT and the substituted benzamide derivatives act on the same receptor. Only ICS 205930, a recognized 5-HT3 receptor antagonist, competitively antagonized the stimulatory effect of cisapride, zacopride and BRL 24924. However, its pKi (6–6.3) for this new receptor was very different from its pKi for 5-HT3 receptors (pKi = 8 –10). Other selective 5-HT3 receptor antagonists with an indole group (BRL 43694 and GR 38032F), with a benzoate group (cocaïne, MDL 72222) or with a piperazine group (quipazine) were ineffective in reversing the stimulatory effect of benzamide derivatives. Exposure of neuronal cells to potent agonists at this receptor such as BRL 24924 rapidly reduces its capacity to stimulate cAMP production. For example, a preincubation of 10 min with BRL 24924 (100 µmol/l) reduced by 42% the ability of 5-HT to stimulate cAMP production. Cross-desensitization occurs between the effects of 5-HT and benzamides. The unique pharmacology of these nonclassical 5-HT receptors that we propose to call 5-HT4 is very close and even identical to the pharmacology of the high affinity 5-HT receptors involved in the indirect stimulation of smooth muscle in the guinea pig ileum. These receptors are different from the 5-HT3 receptors also present in guinea pig ileum.

The effects of a nitric oxide (NO) donor, sodium nitroprusside (SNP), on N-methyl-D-aspartate (NMDA) receptors were assessed by optical measurements of intracellular calcium concentration ([Ca2+];) and patch-clamp techniques in cultured central neurons. SNP selectively blocked NMDA-mediated currents and increases in [Ca2+]iSNP inhibited the binding of [3H]-CGS 19755. The blockade of NMDA responses by SNP was prevented by CPP or APV which are selective competitive NMDA receptor antagonists. These effects were not necessarily mediated by NO, since they were mimicked by ferrocyanide ions, the NO companion photolysis product of SNP.

SERUM and potassium withdrawal-induced cell death of cerebellar granule cells is one of the most popular models used for studying neuronal apoptosis in vitro, and it is generally assumed that compounds preventing cell death in this model prevent apoptosis. In the present study we demonstrate that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. Each of these death mechanisms could be blocked by activation of different pathways, such as raising cAMP production or stimulation of the IGF-1 receptor. Studies on serum and potassium withdrawalinduced cell death of cerebellar granule cells should therefore carefully assess whether a given compound is preventing apoptosis or necrosis using appropriate techniques.

We have shown that DA receptors of the D2 subtype inhibit prolactin release by several mechanisms. DA receptors inhibit cyclic AMP production through a GTP binding protein sensitive to the Bordetella pertussis toxin. However, this mechanism cannot be involved in the blockade of the AII stimulated prolactin secretion by DA. This blockade is probably partly due to the inhibition of the AII-stimulated inositol phosphate production by DA. This inhibition is also sensitive to the Bordella pertussis toxin. The toxin is able to ADP-ribosylate three substrates in anterior pituitary cells (39, 40 and 41 kDa). In addition, we show here that AII receptors inhibit adenylate cyclase of anterior pituitary cell homogenates, but not in intact cells.

Heterotrimeric Goproteins have recently been described as regulators of vesicular traffic. The Goα gene encodes, by alternative splicing, two Goα polypeptides, Go1α and Go2α. By immunofluorescence and electron microscopy, we detected Go1α on the membrane of small intracellular vesicles in C6 glioma cells. After stable transfection of these cells, overexpression of Go1α but not Go2α was followed by a rise in the secretion of a serine protease inhibitor, protease nexin-1 (PN-1). This secretion was enhanced as a function of the amount of expressed Go1α. Metabolic cell labeling indicated that this increase in PN-1 secretion was not the result of an enhancement in PN-1 biosynthesis or a decrease in its uptake, but revealed a potential role of Go1α in the regulation of vesicular PN-1 trafficking. Furthermore, activators of Goproteins, mastoparan and a peptide derived from the amino terminus of the growth cone-associated protein GAP43, increased PN-1 secretion in parental and Go1α-overexpressing cells. Brefeldin A, an inhibitor of vesicular traffic, inhibited both basal and mastoparan-stimulated PN-1 secretions. These results indicate, that in C6 glioma cells, PN-1 secretion could be regulated by both Go1α expression and activation.

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 Gi3α, Gsα, Go1α, Go2α, and Gβ. 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 Gi3α and Gsα. Go1α, Go2α, and Gβ 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-Gsα and anti-Go1α. In the Golgi zone, the membrane of some vesicles was stained only with anti-Gsα and anti-Go2α. 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.

Since most of our knowledge on pharmacological properties of brain 5-HT 4receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT 4 Lreceptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline 75in mouse, alanine 75in the already published rat sequences was not confirmed. Therefore this proline is part of the consensus sequence present in all 5-HT receptor transmembrane domain II (LVMP). Comparing the affinities of 11 agonists and five antagonists for the cloned mouse receptor (5-HT 4 L) expressed in LLCPK1 and the corresponding receptor in mouse colliculi shows an excellent correlation. The transfected mouse 5-HT 4 Lreceptor stimulated cAMP production. When expressed at high density, it exhibited intrinsic activity. In contrast to the previously described distribution, we found that mRNA encoding for both the short (5-HT 4 S) and the long form (5-HT 4 L) of 5-HT 4receptors are expressed in all mouse and rat brain areas.

SINCE nitric oxide (NO) is supposed to mediate excitotoxicity in various brain structures, the effects of two NO synthase inhibitors were studied on rat hippocampal lesions induced by the focal injection of N-methyl-D-aspartate (NMDA). Although both drugs (NG-nitro-L-arginine methyl ester: L-NAME and L-NG-nitroarginine: L-NOARG) were given twice daily for 4 days before NMDA injection, at doses which are known to profoundly inhibit NO synthase activity, no significant decrease of NMDA-induced damage could be observed. These results do not confirm the current hypothesis of a NO involvement in NMDA toxicity at least on hippocampal neurons, in vivo.

A TRITIATED specific and highly potent 5-HT4antagonist: [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 sero-toninergic agents with an affinity corresponding to their previously reported 5-HT4receptor mediated effects. In vitroligand 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.

USING NADPH-diaphorase (NADPH-d) histochemistry, the expression of nitric oxide synthase (NOS) was studied in the rat brain 1 week after kainate-induced status epilepticus. Major changes were observed in the hippocampi of epileptic animals, especially a loss of NADPH-d-positive fibres in the periphery of degenerative pyramidal cells, the survival of NOS-containing interneurones in the dentate hilus, a different pattern of NADPH-d staining in lesioned areas, probably corresponding to the expression of inducible NOS by glial cells and an increased staining of the vasculature. These different sources of NO may exert different functions in the epileptic focus.

We investigated the mode of action of argiotoxin636on isolated N-methyl-D-aspartate (NMDA) receptor channels in cultured cerebellar granule cells. We found that the toxin blocks NMDA channels by decreasing their opening probability and by inducing a flickering activity, in a voltage-dependent manner. Our results indicate that argiotoxin636acts as an open-channel blocker and might therefore be a useful tool for studying the structure of glutamate-gated channels.

WE have isolated a cDNA encoding the 5-HT4Sreceptor by RT-PCR on poly (A)RNA from both human heart and brain. The sequence homology with the rat and mouse 5-HT4receptors was high: 93.8 of identity in the amino acid sequence. None of the 24 amino acid substitutions observed between rat and human receptors are at positions likely to modify their pharmacology. Comparing the pharmacological properties of six agonists and five antagonists on rat and human 5-HT4Sreceptors revealed no significant differences. We have analyzed the behavior of renzapride, a full and a partial agonist on mouse colliculi neurons and human heart biological responses respectively. The coupling efficiency of renzapride was two-fold lower than that of 5-HT for the stimulation of 5-HT4Sreceptors transfected in two different cell lines (LLC-PK1 and COS-7), but increasing the receptor density suppressed the partial agonist effect of renzapride.

GTP-binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussistoxin is known to catalyze ADP-ribosylation of several GTP-binding proteins. In this paper, the subcellular localization of B. pertussistoxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP-ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of G iα. Significant immunoreactivity with anti-G oα antibodies was detected at the level of the 39 kDa faster component. The association of G-proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

GTP‐binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussistoxin is known to catalyze ADP‐ribosylation of several GTP‐binding proteins. In this paper, the subcellular localization of B. pertussistoxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP‐ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Giα. Significant immunoreactivity with anti‐Goα antibodies was detected at the level of the 39 kDa faster component. The association of G‐proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

A subcellular fraction prepared from rat kidney medulla contained vasopressin-sensitive adenylate cyclase and vasopressin binding sites. Vasopressin stimulation resulted in an increase in the maximal velocity of the reaction with no change in the apparent Kmfor ATP. A maximum activation ratio (5 to 10) was obtained at low Mg2+concentration (0.75 mm) and pH 7.4. The apparent Kmfor vasopressin was (1 to 7 x10-8m). Vasopressin binding sites (0.3 pmole per mg of protein) can be identified with the hormonal receptors involved in adenylate cyclase activation on the basis of the two following criteria: (a) saturation of receptor sites and adenylate cyclase activation occurred in the same range of hormone concentrations and (b) a good correlation was observed between the relative potencies of unlabeled [8-arginine]vasopressin, [8-lysine]vasopressin, and oxytocin as inhibitors of [3H]vasopressin binding and as activators of the adenylate cyclase.

Two putative anxiolytic drugs [ipsapirone (TVXQ 7821) and buspirone], structurally unrelated to benzodiazepines, have negligible ataxic and sedative side effects. These drugs are piperazine analogs which interact at 5-HT1 binding sites. It is demonstrated here that these drugs and two other piperazine derivatives, trifluoromethylphenylpiperazine (TFMPP) and m-chlorophenylpiperazine (mCPP), are agonists at 5-HT1A receptors, a subclass of the 5-HT1 receptor, mediating inhibition of forskolin (100 µM) stimulated adenylate cyclase in particulate fractions of guinea pig hippocampus as well as inhibition of the formation of cyclic AMP promoted by vasoactive intestinal polypeptide (0.1 µM) plus forskolin (1 µM) in mouse hippocampal neurons in primary culture. This study demonstrates that these piperazine based drugs act in both brain homogenate preparations and in intact neurons in a similar manner. The biochemical models described here may aid in the development of even more active drugs in this class.

We previously showed that electrical stimulation of the nucleus locus coeruleus was followed 4 weeks later by a greatly improved performance in the acquisition of a food-reinforced operant task. To ascertain whether adrenergic receptors were involved in this long-term behavioral modification, we studied the characteristics of the a1, a2, and ß-adrenoreceptors of the cerebral cortex 4 weeks after stimulation of the locus coeruleus. This stimulation induced a slight (14%) but significant increase in the number of a1-receptor [(3H) WB 4101 binding sites] as well a rise in the number of a2-receptor [(3H) clonidine binding sites]. The later rise mainly affected high-affinity a2 sites (36%) and the number of low-affinity sites remained unchanged. No significant alteration in the number of ß-receptors [(3H)-dihydroalprenolol binding sites] was observed. To confirm this biochemical result, the effect of very small doses of clonidine (1, 2.5, 5 and 10 µg/kg) was tested on locomotor activity in the open-field. In rats stimulated 4 weeks before injection, clonidine induced a biphasic effect, comprising firstly sedation which occurred 30 min after injection, and secondly, long-term hyperactivity which began 24 h after injection. For the 5 µg/kg dose, this rebound of activity was detectable 8 days after injection. In implanted, control rats, only the sedative effect was observed. These findings are interpreted in relation to the current theories about a-adrenoreceptors.

Among phospholipase C-coupled metabotropic glutamate receptors (mGluRs), some have a surprisingly long carboxyl-terminal intracellular domain (mGluR1a, -5a, and -5b), and others have a short one (mGluR1b, -1c, and -1d). All mGluR1 sequences are identical up to 46 residues following the 7th transmembrane domain, followed by 313, 20, 11, and 26 specific residues in mGluR1a, mGluR1b, mGluR1c, and mGluR1d, respectively. Several functional differences have been described between the long isoforms (mGluR1a, -5a, and -5b) and the short ones (mGluR1b, -1c, and -1d). Compared with the long receptors, the short ones induce slower increases in intracellular Ca2+, are activated by higher concentration of agonists, and do not exhibit constitutive, agonist-independent activity. To identify the residues responsible for these functional properties, a series of truncated, chimeric, and mutated receptors were constructed. We found that the deletion of the last 19 carboxyl-terminal residues in mGluR1c changed its properties into those of mGluR1a. Moreover, the exchange of the long carboxyl-terminal domain of mGluR5a with that of mGluR1c generated a chimeric receptor that possessed functional properties similar to those of mGluR1c. Mutagenesis of specific residues within the 19 carboxyl-terminal residues of mGluR1c revealed the importance of a cluster of 4 basic residues in defining the specific properties of this receptor. Since this cluster is part of the sequence common to all mGluR1 variants, we conclude that the long carboxyl-terminal domain of mGluR1a suppresses the inhibitory action of this sequence element.

Metabotropic glutamate receptors (mGlu receptors), the Ca2+-sensing receptor, γ-aminobutyric acid type B receptors, and one group of pheromone receptors constitute a unique family (also called family 3) of heptahelical receptors. This original family shares no sequence similarity with any other G protein-coupled receptors. The identification and comparison of the molecular determinants of receptor/G protein coupling within the different receptor families may help identify general rules involved in this protein/protein interaction. In order to detect possible contact sites important for coupling selectivity between family 3 receptors and the G protein α-subunits, we examined the coupling of the cyclase-inhibiting mGlu2 and mGlu4 receptors to chimeric αq-subunits bearing the 5 extreme C-terminal amino acid residues of either Gαi, Gαo, or Gαz. Whereas mGlu4 receptor activated all three chimeric G proteins, mGlu2 receptor activated Gαqiand Gαqobut not Gαqz. The mutation of isoleucine −4 of Gαqzinto cysteine was sufficient to recover coupling of the mutant G protein to mGlu2 receptor. Moreover, the mutation of cysteine −4 of Gαqointo isoleucine was sufficient to suppress the coupling to mGlu2 receptor. Mutations at positions −5 and −1 had an effect on coupling efficiency, but not selectivity. Our results emphasize the importance of the residue −4 of the α-subunits in their specific interaction to heptahelical receptors by extending this finding on the third family of G protein-coupled receptors.

Sequential interactions between a serotonin receptor and its interaction partners underlie neurite extension and branching.