RESUMO
Neuropeptides 2001, 2nd Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference (11th Annual Meeting). 6-11 May 2001 with Satellite Symposium, Israeli-French Symposium, Israel Ministry of Science, Culture and Sport, 6 May 2001, held at Maale Hachmicha and Tel Aviv University, Israel.
Assuntos
Química Encefálica/fisiologia , Neuropeptídeos/fisiologia , AnimaisRESUMO
The hypocretins (hcrts), also known as orexins, are two recently identified excitatory neuropeptides that in rat are produced by approximately 1200 neurons whose cell bodies are located in the lateral hypothalamus. The hypocretins/orexins have been implicated in the regulation of rapid eye movement (REM) sleep and the pathophysiology of narcolepsy. In the present study, we investigated whether the locus coeruleus (LC), a structure receiving dense hcrtergic innervation, which is quiescent during REM sleep, might be a target for hcrt to regulate REM sleep. Local administration of hcrt1 but not hcrt2 in the LC suppressed REM sleep in a dose-dependent manner and increased wakefulness at the expense of deep, slow-wave sleep. These effects were blocked with an antibody that neutralizes hcrt binding to hcrt receptor 1. In situ hybridization and immunocytochemistry showed the presence of hcrt receptor 1 but not the presence of hcrt receptor 2 in the LC. Iontophoretic application of hcrt1 enhanced the firing rate of LC neurons in vivo, and local injection of hcrt1 into the LC induced the expression of c-fos in the LC area. We propose that hcrt receptor 1 in the LC is a key target for REM sleep regulation and might be involved in the pathophysiological mechanisms of narcolepsy.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Locus Cerúleo/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Sono REM/fisiologia , Animais , Anticorpos/farmacologia , Proteínas de Transporte/administração & dosagem , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Hibridização In Situ , Iontoforese , Locus Cerúleo/citologia , Locus Cerúleo/efeitos dos fármacos , Locus Cerúleo/fisiopatologia , Masculino , Microinjeções , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neuropeptídeos/administração & dosagem , Receptores de Orexina , Orexinas , Polissonografia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Receptores Acoplados a Proteínas G , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/metabolismo , Sono/efeitos dos fármacos , Sono/fisiologia , Transtornos do Sono do Ritmo Circadiano/induzido quimicamente , Transtornos do Sono do Ritmo Circadiano/fisiopatologia , Sono REM/efeitos dos fármacos , Vigília/efeitos dos fármacos , Vigília/fisiologiaRESUMO
Nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors are present on presynaptic nerve terminals in the striatum, where they have been shown to be involved in the regulation of dopamine release. Here, we explored the possibility that both receptor systems function on the same individual nerve terminals in the striatum, as assessed by confocal imaging of synaptosomes. On performing sequential stimulation, nicotine (500 nM) induced changes in [Ca(2+)](i) in most of the synaptosomes ( approximately 80%) that had previously responded to stimulation with the 5-HT(3) receptor agonist m-chlorophenylbiguanide (mCPBG; 100 nM), whereas mCPBG induced [Ca(2+)](i) responses in approximately half of the synaptosomes that showed responses on nicotinic stimulation. The 5-HT(3) receptor-specific antagonist tropisetron blocked only the mCPBG-induced responses, but not the nicotinic responses on the same synaptosomes. Immunocytochemical staining revealed extensive co-localization of the 5-HT(3) receptor with the alpha4 nicotinic receptor subunit on the same synaptosomes, but not with the alpha3 and/or alpha5 subunits. Immunoprecipitation studies indicate that the 5-HT(3) receptor and the alpha4 nicotinic receptor subunit do not interact on the nerve terminals. The presence of nicotinic and 5-HT(3) receptors on the same presynaptic striatal nerve terminal indicates a convergence of cholinergic and serotonergic systems in the striatum.
Assuntos
Neostriado/metabolismo , Terminações Nervosas/metabolismo , Receptores Nicotínicos/metabolismo , Receptores de Serotonina/metabolismo , Animais , Biguanidas/farmacologia , Western Blotting , Cálcio/metabolismo , Estimulação Elétrica , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Microscopia Confocal , Neostriado/efeitos dos fármacos , Neostriado/ultraestrutura , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/ultraestrutura , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Receptores 5-HT3 de Serotonina , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
The serotonin 5-hydroxytryptamine-3 receptor is a ligand-gated ion channel that is distributed widely in the nervous system. Within the CNS, a significant portion of the 5-hydroxytryptamine-3 receptors appears to be present on presynaptic nerve terminals and, using an imaging approach, it was shown previously that presynaptic 5-hydroxytryptamine-3 receptors on individual isolated nerve terminals (synaptosomes) from rat corpus striatum display a distinctive set of properties-slow onset, little desensitization and high apparent permeability for Ca2+-when compared to those observed for 5-hydroxytryptamine-3 receptors localized at postsynaptic sites on neuronal cell bodies. To consider whether their characteristic nature is a common feature of presynaptic 5-hydroxytryptamine-3 receptors across the brain, we used confocal microscopy to measure changes in intracellular Ca2+ concentration resulting from 5-hydroxytryptamine-3 agonist-induced responses in synaptosomes from representative rat brain regions, ranging in expression of overall levels of 5-hydroxytryptamine-3 receptors from relatively low (cerebellum) to intermediate (corpus striatum and hippocampus) to high (amygdala). Application of 100 nM m-chlorophenyl biguanide, a specific 5-hydroxytryptamine-3 receptor agonist, induced changes in relative intracellular Ca2+ concentration in subsets of synaptosomes from the corpus striatum (approximately 6% of total), hippocampus (approximately 3% of total), amygdala (approximately 30% of total) and cerebellum (approximately 32% of total). In order to assure the viability of the synaptosomes that did not respond to 5-hydroxytryptamine-3 agonist stimulation, KCl (45 mM) was subsequently added to depolarize the same population of synaptosomes, and increases in intracellular Ca2+ concentration were then seen in 80-90% of the synaptosomes from all four regions. The kinetics of the intra synaptosomal Ca2+ changes produced by K+-evoked depolarization were similar in all regions, showing a rapid rise to a peak followed by an apparent plateau phase. In contrast, the changes in intracellular Ca2+ concentration evoked by m-chlorophenyl biguanide displayed substantially slower kinetics, similar to previous findings, but which varied among responding synaptosomes from one region to another. In particular, m-chlorophenyl biguanide-induced changes were notably slower in synaptosomes from the amygdala (rise time constant, tau = 25 s), when compared to responses in synaptosomes from other regions (striatum, tau = 12 s; hippocampus, tau= 9.6 s; cerebellum, tau = 7 s). To independently demonstrate the presence of 5-hydroxytryptamine-3 receptors on nerve terminals in the various regions using a molecular approach, we double-immunostained the synaptosomes for the 5-hydroxytryptamine-3 receptor and the synaptic vesicle protein synaptophysin, using, respectively, a polyclonal antibody raised against an N-terminal peptide of the 5-hydroxytryptamine-3 receptor and a monoclonal anti-synaptophysin antibody, and observed 5-hydroxytryptamine-3 receptors in varying subsets of the synaptosomes from each region, providing direct support for the results obtained in our functional experiments. These results suggest that the distinctive properties of presynaptic 5-hydroxytryptamine-3 receptors are found throughout the brain, with evident differences in the kinetics of the responses to agonist stimulation observed across the brain regions studied. As expected, the proportion of the synaptosomal population that responded on application of 5-hydroxytryptamine-3 agonist varied in preparations from one region to another; however, the presence of a relatively high proportion of presynaptic 5-hydroxytryptamine-3 receptors in the cerebellum contrasts with previous binding studies demonstrating a relatively low overall density of 5-hydroxytryptamine-3 receptors in this region. We hypothesize that presynaptic 5-hydroxytryptamine-3 receptors present on nerve terminals regulate the
Assuntos
Encéfalo/fisiologia , Cálcio/fisiologia , Terminações Nervosas/fisiologia , Receptores Pré-Sinápticos/fisiologia , Receptores de Serotonina/fisiologia , Algoritmos , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Cinética , Masculino , Microscopia Confocal , Terminações Nervosas/efeitos dos fármacos , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Pré-Sinápticos/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Receptores 5-HT3 de Serotonina , Agonistas do Receptor de Serotonina/farmacologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/fisiologiaRESUMO
We have developed polyclonal antibodies (pAb120) against a peptide corresponding to a region within the extracellular domain of the 5-hydroxytryptamine3 (5-HT3) receptor subunit, thus permitting, for the first time, localization of 5-HT3 receptors at the cell surface in intact (non-permeabilized) systems. The antibodies are both specific and sensitive: pAb120 recognized as little as 63 ng of protein from HEK293 cells expressing recombinant 5-HT3 receptors, whilst Western blots of recombinant 5-HT3 receptors purified from Sf9 cells revealed two bands at 48 and 54 kDa, and native 5-HT3 receptors from N1E-115 cell membranes produced a broad band at 50-54 kDa with a smaller band at 35 kDa. These bands were also labelled by antibodies against the intracellular loop of the 5-HT3 receptor. Immunofluorescent labelling revealed a ring of intense fluorescence in the plasma membrane of non-permeabilized HEK293 cells expressing recombinant 5-HT3 receptors. Studies on native 5-HT3 receptors revealed that pAb120 could recognize 5-HT3 receptors on presynaptic terminals isolated from rat striatum, and immunohistochemical studies in rat brain sections revealed labelling of cell bodies, dendrites and varicose axons in hippocampus, cortex and lateral hypothalamus; all of these areas have been reported to express 5-HT3 receptors. We conclude that pAb120 is a highly specific and sensitive antiserum that will assist in clarifying fundamental questions about 5-HT3 receptor neurobiology.
Assuntos
Especificidade de Anticorpos , Corpo Estriado/química , Receptores de Serotonina/genética , Receptores de Serotonina/imunologia , Animais , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Rim/citologia , Ligantes , Masculino , Terminações Pré-Sinápticas/química , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Serotonina/química , Receptores 5-HT3 de Serotonina , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
Cortistatin (CST) is a presumptive neuropeptide that shares 11 of its 14 amino acids with somatostatin (SST). CST and SST are expressed in partially overlapping but distinct populations of cortical interneurons. In the hippocampal formation, most CST-positive cells are also positive for SST. In contrast to SST, administration of CST into the rat brain ventricles reduces locomotor activity and specifically enhances slow wave sleep. Intracerebroventricular injection of CST or SST has been shown to protect against the neurotoxic effects of kainic acid. Here, we show that CST and SST mRNAs respond differently to kainate-induced seizures. Furthermore, comparison of the upstream sequences from the CST and SST precursor genes reveal that they contain binding motifs for different transcriptional regulatory factors. Our data demonstrate that CST and SST, which are often co-expressed in the same neurons, are regulated by different stimuli.
Assuntos
Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes , Ácido Caínico/farmacologia , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Precursores de Proteínas/genética , RNA Mensageiro/biossíntese , Somatostatina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Clonagem Molecular , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hibridização In Situ , Interneurônios/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Neuropeptídeos/biossíntese , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Sequências Reguladoras de Ácido Nucleico , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , Somatostatina/biossíntese , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Cortistatin is a recently discovered neuropeptide relative of somatostatin named after its predominantly cortical expression and ability to depress cortical activity. Cortistatin-14 shares 11 of the 14 amino acids of somatostatin-14 yet their nucleotide sequences and chromosomal localization clearly indicate they are products of separate genes. Now cloned from human, mouse and rat sources, cortistatin is known to bind all five cloned somatostatin receptors and share many pharmacological and functional properties with somatostatin including the depression of neuronal activity. However, cortistatin also has many properties distinct from somatostatin including induction of slow-wave sleep, apparently by antagonism of the excitatory effects of acetylcholine on the cortex, reduction of locomotor activity, and activation of cation selective currents not responsive to somatostatin. Expression of mRNA encoding cortistatin follows a circadian rhythm and is upregulated on deprivation of sleep, suggesting cortistatin is a sleep modulatory factor. This review summarizes recent advances in our understanding of the neurobiology of cortistatin, examines the similarities and differences between cortistatin and somatostatin, and asks the question: does cortistatin bind to a cortistatin-specific receptor?
Assuntos
Neuropeptídeos/fisiologia , Somatostatina/fisiologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Neuropeptídeos/biossíntese , Somatostatina/biossínteseRESUMO
Aromatic amino acids are important components of the ligand binding site in the Cys loop family of ligand-gated ion channels. To examine the role of tryptophan residues in the ligand binding domain of the 5-hydroxytryptamine(3) (5-HT(3)) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT(3A) receptor subunit to tyrosine or serine. The mutants were expressed as homomeric 5-HT(3A) receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Trp(90), Trp(183), and Trp(195) to tyrosine resulted in functional receptors, although with increased EC(50) values (2-92-fold) to 5-HT(3) receptor agonists. Changing these residues to serine either ablated function (Trp(90) and Trp(183)) or resulted in a further increase in EC(50) (Trp(195)). Mutation of residue Trp(60) had no effect on ligand binding or receptor function, whereas mutation of Trp(95), Trp(102), Trp(121), and Trp(214) ablated ligand binding and receptor function, and all but one of the receptors containing these mutations were not expressed at the plasma membrane. We propose that Trp(90), Trp(183), and Trp(195) are intimately involved in ligand binding, whereas Trp(95), Trp(102), Trp(121), and Trp(214) have a critical role in receptor structure or assembly.
Assuntos
Receptores de Serotonina/química , Triptofano/fisiologia , Sequência de Aminoácidos , Biguanidas/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Cinética , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Serotonina/genética , Homologia de Sequência de Aminoácidos , Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
Serotonin (5-hydroxytryptamine) type 3 receptors (5-HT3R) and nicotinic acetylcholine receptors are structurally and functionally related proteins, yet distinct members of the family of ligand-gated ion channels. For most members of this family a diversity of heteromeric receptors is known at present. In contrast, known 5-HT3R subunits are all homologs of the same 5-HT3R-A subunit and form homopentameric receptors. Here we show, by heterologous expression followed by immunoprecipitation, that 5-HT3R and nicotinic acetylcholine receptor alpha4 subunits coassemble into a novel type of heteromeric ligand-gated ion channel, which is activated by 5-HT. The Ca2+ permeability of this heteromeric ion channel is enhanced as compared with that of the homomeric 5-HT3R channel. Heteromeric 5-HT3/alpha4 and homomeric 5-HT3Rs have similar pharmacological profiles, but distinct sensitivities to block by the antagonist d-tubocurarine. Coassembly of subunits beyond the boundaries of ligand-gated ion channel families may constitute an important mechanism contributing to the diverse properties and functions of native neurotransmitter receptors.