RESUMO
We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/química , Receptores de AMPA/metabolismo , Medula Espinal/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte/química , Proteínas de Transporte/genética , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Sinapses/ultraestrutura , Transcrição GênicaRESUMO
In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.
Assuntos
Trifosfato de Adenosina/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Receptores de AMPA/fisiologia , Proteínas de Transporte Vesicular , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Precipitação Química , Dendritos/metabolismo , Interações Medicamentosas , Proteínas Sensíveis a N-Etilmaleimida , Neurônios/metabolismo , Proteínas Qa-SNARE , Ratos , Ratos Sprague-Dawley , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Leveduras/genéticaRESUMO
In the present study, we examined the targeting of neuropeptide-containing vesicles in terminals of neurons that release both neuropeptides and classical transmitters. Single neurons were electrically stimulated with patterns of activity that were recorded in freely behaving animals. The amount of peptide release was measured biochemically using a radioimmunoassay, and the targeting of peptidergic vesicles was quantified using immunoelectronmicroscopy. Repeated electrical stimulation of single neurons produced a very large increase in peptide release. Peptide release is paralleled by a twofold increase in the number of peptidergic vesicles docked at the portion of the terminal membrane that is away from the target muscle. This is in stark contrast to cholinergic vesicles, which aggregate at, and are released from the conventional release sites in close apposition to the muscle. This differential targeting of cholinergic and peptidergic vesicles may play a significant role in the distinct release requirements and spatial and temporal characteristics of the actions of conventional and peptidergic transmitters.
Assuntos
Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Aplysia , Estimulação Elétrica , Técnicas In Vitro , Microscopia Imunoeletrônica , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Neuropeptídeos/análise , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Radioimunoensaio , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Many neurons contain multiple peptide cotransmitters in addition to their classical transmitters. We are using the accessory radula closer neuromuscular system of Aplysia, which participates in feeding in these animals, to define the possible consequences of multiple modulators converging on single targets. How these modulators are released onto their targets is of critical importance in understanding the outcomes of their modulatory actions and their physiological role. Here we provide direct evidence that the partially antagonistic families of modulatory peptides, the myomodulins and buccalins, synthesized by motorneuron B16 are costored and coreleased in fixed ratios. We show that this release is calcium-dependent and independent of muscle contraction. Furthermore, we show that peptide release is initiated at the low end of the physiological range of motorneuron firing frequency and that it increases with increasing motorneuron firing frequency. The coordination of peptide release with the normal operating range of a neuron may be a general phenomenon and suggests that the release of peptide cotransmitters may exhibit similar types of regulation and plasticity as have been observed for classical transmitters. Stimulation paradigms that increase muscle contraction amplitude or frequency also increase peptide release from motor neuron B16. The net effect of the modulatory peptide cotransmitters released from motorneuron B16 would be to increase relaxation rate and therefore allow more frequent and/or larger contractions to occur without increased resistance to antagonist muscles. The end result of this modulation could be to maximize the efficiency of feeding.
Assuntos
Neurônios Motores/metabolismo , Neuropeptídeos/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Aplysia , Cálcio/farmacologia , Bloqueadores Ganglionares/farmacologia , Hexametônio/farmacologia , Microscopia Eletrônica , Neurônios Motores/química , Neurônios Motores/ultraestrutura , Neuropeptídeos/análise , Radioimunoensaio , Sinapses/química , Sinapses/ultraestrutura , Transmissão Sináptica/efeitos dos fármacosRESUMO
We use a multidisciplinary approach to identify, map, and characterize the bioactivity of modulatory neuropeptides in the circuitry that generates feeding behavior in Aplysia. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the cerebral-buccal connective (CBC), a nerve containing axons of many interneurons that control feeding behavior of Aplysia, was used to identify neuropeptides that may participate in generation and shaping of feeding motor programs. Using this functionally oriented search, we identified a novel family of peptides that we call the feeding circuit-activating peptides (FCAPs). Two peptides with masses identical to those observed in the CBCs (molecular weight 1387 and 1433) were purified from buccal ganglia and partially sequenced using mass spectrometry. The amino acid sequence was then used to clone the FCAP precursor, which encodes multiple copies of eight different FCAPs. The two FCAPs present in highest copy number correspond to those observed in the CBC. The distribution of FCAP expression was mapped using Northern analysis, whole-mount in situ hybridization, and immunocytochemistry. Consistent with our initial findings, FCAP-immunopositive axons were observed in the CBC. Furthermore, we found that FCAP was present in some cerebral-buccal and buccal-cerebral interneurons. As their name suggests, FCAPs are capable of initiating rhythmic feeding motor programs and are the first neuropeptides with such activity in this circuit. The actions of FCAPs suggest that these peptides may contribute to the induction and maintenance of food-induced arousal. FCAPs were also localized to several other neuronal systems, suggesting that FCAPs may play a role in the regulation of multiple behaviors.
Assuntos
Comportamento Alimentar/fisiologia , Rede Nervosa/química , Rede Nervosa/fisiologia , Neuropeptídeos/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Animais , Aplysia , Transporte Axonal/fisiologia , Axônios/metabolismo , Northern Blotting , Clonagem Molecular , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Técnicas In Vitro , Interneurônios/química , Interneurônios/fisiologia , Dados de Sequência Molecular , Rede Nervosa/efeitos dos fármacos , Neuropeptídeos/análise , Neuropeptídeos/genética , Neuropeptídeos/farmacologia , Especificidade de Órgãos , Periodicidade , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Análise de Sequência de ProteínaRESUMO
The first Aplysia californica insulin gene is characterized and its proteolytic processing from prohormone to final peptides elucidated using a combination of biochemical and mass spectrometric methods. Aplysia insulin (AI) is one of the largest insulins found, with a molecular weight of 9146 Da, and an extended A chain compared with other invertebrate and vertebrate insulins. The AI prohormone produces a series of C peptides and also a unique N-terminally acetylated D peptide. AI-producing cells are restricted to the central region of the cerebral ganglia mostly within the F and C clusters, and AI is transported to neurohemal release sites located on the upper labial and anterior tentacular nerves. The expression of AI mRNA decreases when the animal is deprived of food, and injections of AI reduce hemolymph glucose levels, suggesting that the function of insulin-regulating metabolism has been conserved.
Assuntos
Aplysia/metabolismo , Gânglios dos Invertebrados/metabolismo , Regulação da Expressão Gênica , Insulina/genética , Neurônios/metabolismo , Proinsulina/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Aplysia/genética , Sequência de Bases , Peptídeo C/química , Peptídeo C/genética , Humanos , Imuno-Histoquímica , Lymnaea , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/imunologia , Proinsulina/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.
Assuntos
Gânglios dos Invertebrados/química , Gânglios dos Invertebrados/metabolismo , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Aplysia , Bivalves , Clonagem Molecular , Gânglios dos Invertebrados/citologia , Imuno-Histoquímica , Técnicas In Vitro , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.
Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Entérico/metabolismo , Hormônios de Invertebrado/isolamento & purificação , Hormônios de Invertebrado/metabolismo , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Aplysia , Sistema Nervoso Central/química , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/inervação , Eletrofisiologia , Sistema Nervoso Entérico/química , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Técnicas In Vitro , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/farmacologia , Dados de Sequência Molecular , Família Multigênica , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/farmacologia , Especificidade de Órgãos , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The distribution of myomodulin-like peptides in the nervous system of Aplysia californica was examined by using immunocytochemical techniques. Neurons and cell clusters containing immunoreactive material were located in each of the major central ganglia. Myomodulin-like immunoreactivity was also present in fibers in each of the connectives between the ganglia and in peripheral nerves. Varicosities containing immunoreactive material were located on specific regions of peripheral tissues associated with the feeding, digestive, cardiovascular, and reproductive systems. Double-labeling experiments were used to demonstrate myomodulin-like immunoreactivity in two identified neurons, the motor neuron B16 in the buccal ganglion and the widely acting interneuron L10 in the abdominal ganglion. Structures in the eye and cerebral ganglion that may correspond to the optic circadian pacemaker system were also stained. The central and peripheral distribution of myomodulin-like immunoreactivity indicates that this family of neuropeptides is present in specific efferent, afferent, and interneuronal elements that participate in a diversity of neural circuits in Aplysia.
Assuntos
Aplysia/química , Sistema Nervoso/química , Neuropeptídeos/análise , Sequência de Aminoácidos , Animais , Fenômenos Fisiológicos Cardiovasculares , Olho/química , Comportamento Alimentar/fisiologia , Gânglios Autônomos/química , Dados de Sequência Molecular , Neurônios/química , Neuropeptídeos/imunologia , Neuropeptídeos/fisiologia , Reprodução/fisiologia , Vísceras/químicaRESUMO
The cDNA coding for the bovine mu-opioid receptor has been cloned and sequenced. Conserved sequences from murine delta-receptor cDNA were used as primers in polymerase chain reaction (PCR) to amplify cDNA, prepared by reverse transcription of bovine brain mRNA. This cDNA was used to probe a bovine brain library. The partial sequence obtained was extended to provide the full length clone by PCR. The cDNA has an open reading frame of 1203 base pairs (bp) with a 3'-untranslated region of 1900 bp and a 5'-untranslated region of 265 bp. The protein contains 401 amino acids and has 94% amino acid identity with the human and 91% with the rat mu-opioid receptor. It has the putative seven transmembrane domains, characteristic of G protein-coupled receptors and contains 5 potential N-linked glycosylation sites near the N-terminus. Several potential phosphorylation sites and a putative palmitoylation site are also present. The receptor was stably expressed in HEK293 cells. The binding profile was found to be that of a typical mu receptor, i. e., mu agonists and antagonists, but not delta and kappa ligands, bound with high affinity. Functional assays, namely, opioid stimulation of [35S]GTPgammaS binding and inhibition of forskolin-activated adenylyl cyclase, were also found to be highly specific for mu-opioid agonists. The receptor was downregulated by chronic exposure to mu agonists but not delta or kappa agonists. Evidence is presented indicating that the cloned receptor is the same as the bovine mu receptor previously purified to homogeneity in our laboratory. No evidence was found for genes for multiple mu-type opioid receptors.
Assuntos
Benzenoacetamidas , Receptores Opioides mu/genética , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Animais , Sequência de Bases , Ligação Competitiva/efeitos dos fármacos , Bovinos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Clonagem Molecular , Colforsina/farmacologia , Corpo Estriado/química , DNA Complementar/química , DNA Complementar/genética , Diprenorfina/metabolismo , Diprenorfina/farmacologia , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , D-Penicilina (2,5)-Encefalina/metabolismo , D-Penicilina (2,5)-Encefalina/farmacologia , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Dados de Sequência Molecular , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Ensaio Radioligante , Receptores Opioides mu/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Radioisótopos de Enxofre , TrítioRESUMO
The cholinergic motor neurons for the accessory radula closer (ARC) contain several neuropeptides that affect muscle contractions. In the present study, we have purified and sequenced a sixth ARC neuropeptide, using a combination of high pressure liquid chromatography and bioassays. This neuropeptide, Gly-Leu-Asp-Arg-Tyr-Gly-Phe-Val-Gly-Gly-Leu-amide, has been named buccalin B (BUCb) because it is significantly homologous to the previously characterized neuropeptide buccalin A. BUCb was found to be two-three times more potent than buccalin A in depressing motor neuron induced contractions.
Assuntos
Aplysia/química , Neurônios Motores/química , Neuropeptídeos/química , Sequência de Aminoácidos , Animais , Nível de Alerta/fisiologia , Comportamento Alimentar/fisiologia , Marcação por Isótopo , Potenciais da Membrana , Dados de Sequência Molecular , Neurônios Motores/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Important insights into mechanisms by which neuromuscular activity can be modulated have been gained by the study of experimentally advantageous preparations such as the ARC neuromuscular system of Aplysia. Previous studies have indicated that one source of modulatory input to the ARC muscle is its own two motor neurons, B15 and B16. Both of these neurons synthesize multiple peptide cotransmitters in addition to their primary neurotransmitter acetylcholine (ACh). Peptides present in the ARC motor neurons include SCPA, SCPB, buccalin A and B, and myomodulin A. We have now purified a novel neuropeptide, myomodulin B, which is structurally similar to myomodulin A. Myomodulin B is present in two identified Aplysia neurons that contain myomodulin A; the ARC motor neuron B16 and the abdominal neuron L10. Ratios of myomodulin A to myomodulin B are approximately 6:1 in both cells. Like myomodulin A, myomodulin B potentiates ARC neuromuscular activity; it acts postsynaptically, and increases the size and relaxation rate of muscle contractions elicited either by motor neuron stimulation or by direct application of ACh to the ARC. When myomodulin A is applied to the ARC in high doses (e.g., at about 10(-7) M), it decreases the size of motor neuron-elicited muscle contractions. This inhibitory effect is never seen with myomodulin B. Thus, despite the structural similarity between the two myomodulins, there exists what may be an important difference in their bioactivity.
Assuntos
Neuropeptídeos/genética , Sequência de Aminoácidos , Animais , Aplysia , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiologia , Neurônios/metabolismo , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/metabolismo , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.
Assuntos
Neuropeptídeos/genética , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Aplysia , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The marine mollusc Aplysia, whose simple nervous system facilitates study of the neural basis of behavior, was used to investigate the role of peptidergic cotransmission in feeding behavior. Several novel modulatory neuropeptides were purified and localized to identified cholinergic motoneurons. Physiological and biochemical studies demonstrated that these peptides are released when the motoneurons fire at frequencies that occur during normal behavior, and that the peptides modify the relationship between muscle contraction amplitude and relaxation rate so as to maintain optimal motor output when the intensity and frequency of feeding behavior change.
Assuntos
Aplysia/fisiologia , Comportamento Alimentar/fisiologia , Neuropeptídeos/fisiologia , Transmissão Sináptica/fisiologia , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Junção Neuromuscular/fisiologia , Neurotransmissores/metabolismoRESUMO
The accessory radula closer (ARC) buccal muscle motor neuron B16 is buccalin-immunoreactive and it synthesizes a peptide with chromatographic properties identical to those of buccalin. Terminal varicosities in the ARC muscle are buccalin-immunoreactive, and buccalin reduces muscle contractions evoked by stimulation of neuron B16, probably by a presynaptic action.
Assuntos
Aplysia/metabolismo , Fibras Colinérgicas/metabolismo , Neurônios Motores/metabolismo , Contração Muscular/efeitos dos fármacos , Neuropeptídeos/metabolismo , Animais , Aplysia/citologia , Estimulação Elétrica , Imuno-Histoquímica , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Contração Muscular/fisiologiaRESUMO
An approximately 750-kDa member of the family of giant titin/twitchin-like myosin-associated proteins was highly purified from muscle of the marine mollusc Aplysia californica. Purified twitchin was able to autophosphorylate on threonine, which demonstrates its protein serine/threonine kinase activity. cDNA sequence analysis of the cloned kinase domain of molluscan twitchin revealed that it is most closely related with the kinase domains of Caenorhabditis elegans twitchin (62% identity) and vertebrate myosin light chain kinases (45% average identity). Analysis of the cDNA sequence further suggested the presence of a potential calmodulin-binding site in a putative autoinhibitory region. The functional activity of this site was demonstrated by the calcium-dependent binding of purified twitchin to immobilized calmodulin and the fact that this interaction could be competed with synthetic peptides deduced from the cDNA sequence. Furthermore, biotinylated calmodulin bound to immobilized twitchin in gel-overlay assays with nanomolar affinity (EC50 approximately equal to 70 nM). The potential regulation of twitchin by calcium/calmodulin indicates that titin-like molecules may serve dynamic functions during contraction-relaxation cycles in muscle in addition to their functions as cytoskeletal proteins.
Assuntos
Proteínas de Caenorhabditis elegans , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina , Calmodulina/metabolismo , Proteínas de Helminto/metabolismo , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Aplysia , Eletroforese em Gel de Poliacrilamida , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/ultraestrutura , Humanos , Dados de Sequência Molecular , Contração Muscular , Proteínas Musculares/isolamento & purificação , Proteínas Musculares/ultraestrutura , Relaxamento Muscular , Músculos/metabolismo , Músculos/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Homologia de Sequência de AminoácidosRESUMO
In many neurons more than one peptide is colocalized with a classical neurotransmitter. The functional consequence of such an arrangement has been rarely investigated. Here, within the feeding circuit of Aplysia, we investigate at a single synapse the actions of two modulatory neuropeptides that are present in a cholinergic interneuron. In combination with previous work, our study shows that the command-like neuron for feeding, CBI-2, contains two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Previous studies showed that high-frequency prestimulation or repeated stimulation of CBI-2 increases the size of CBI-2 to B61/62 excitatory postsynaptic potentials (EPSPs) and shortens the latency of firing of neuron B61/62 in response to CBI-2 stimulation. We find that both FCAP and CP2 mimic these two effects. The variance method of quantal analysis indicates that FCAP increases the calculated quantal size (q) and CP2 increases the calculated quantal content (m) of EPSPs. Since the PSP amplitude represents the product of q and m, the joint action of the two peptides is expected to be cooperative. This observation suggests a possible functional implication for multiple neuropeptides colocalized with a classical neurotransmitter in one neuron.
Assuntos
Potenciais de Ação/fisiologia , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Sinapses/fisiologia , Animais , Aplysia , Neurônios/química , Neuropeptídeos/análise , Sinapses/químicaRESUMO
Many neurons that contain a classical neurotransmitter also contain modulatory peptides, but it has been difficult to establish unequivocally that these peptides are functional cotransmitters. Here, we provide evidence for functional cotransmission in a neuromuscular system of Aplysia. Using immunocytochemical techniques, we localize members of two peptide families, the small cardioactive peptides (SCPs) and the buccalins (BUCs), to a single subset of dense-core vesicles in the terminals of the cholinergic motorneuron B15. We describe a new preparation and method for the direct detection of released peptides and show that the SCPs and BUCs are released when neuron B15 is intracellularly stimulated. Consistent with their subcellular localization, the SCPs and BUCs are released in a stoichiometric ratio that is constant across conditions that change the absolute amount of peptides released. Peptide release is calcium-dependent but does not require muscle contractions. Thus, the release cannot be attributed to a displacement of peptides that may be present in the extracellular space. In previous studies, we characterized the physiological firing patterns of neuron B15. Here, we simulate these firing patterns and show that peptide release occurs. Additionally, we find that significant quantities of material are released under behaviorally relevant conditions. We find that concentrations of released peptides in the muscle are in the concentration range in which exogenously applied peptides exert characterized modulatory actions on muscle contractions. Together, our findings provide strong support for the hypothesis that peptides contained in neuron B15 are functional cotransmitters.
Assuntos
Aplysia/fisiologia , Músculos/metabolismo , Neurotransmissores/metabolismo , Neurotransmissores/fisiologia , Animais , Cálcio/fisiologia , Contagem de Células , Eletrofisiologia , Imuno-Histoquímica/métodos , Neurônios Motores/fisiologia , Contração Muscular/fisiologia , Músculos/inervação , Neurônios/citologia , Neuropeptídeos/metabolismo , Coloração e Rotulagem , Fatores de Tempo , Distribuição TecidualRESUMO
To gain insights into the physiological role of cotransmission, we measured peptide release from cell B15, a motorneuron that utilizes ACh as its primary transmitter but also contains putative peptide cotransmitters, the small cardioactive peptides (SCPs) and the buccalins (BUCs). All stimulation parameters used were in the range in which B15 fires in freely moving animals. We stimulated neuron B15 in bursts and systematically varied the interburst interval, the intraburst frequency, and burst duration. Both peptides were preferentially released when B15 was stimulated at higher intra- or interburst frequencies or with longer burst durations. Across stimulation patterns, the amount of peptide released depended on the mean frequency of stimulation and was independent of the specific pattern of stimulation. The parameters of stimulation that produce a larger release of peptides correspond to those that evoke larger contractions. Large and frequent contractions are likely to fuse or summate, thus disrupting the rhythmic behavior mediated by the muscle innervated by motorneuron B15. Because the combined effect of the SCPs and BUCs is to accelerate the relaxation and shorten the duration of muscle contractions, these peptides reduce the probability of the disruptive fusion or summation of muscle contractions. Because these cotransmitters regulate an aspect of muscle contractions that is not controlled by acetylcholine (ACh), the primary transmitter of B15, we suggest that peptides and ACh form parallel but functionally distinct lines of transmission at the neuromuscular junction. Both types of transmission may be necessary to ensure that behavior remains efficient over a wide range of conditions.
Assuntos
Aplysia/fisiologia , Neuropeptídeos/metabolismo , Neuropeptídeos/fisiologia , Animais , Estimulação Elétrica , Neurônios Motores/metabolismo , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/farmacologia , Tempo de Reação , Serotonina/farmacologia , TemperaturaRESUMO
1. Neuromodulation by multiple related peptides with different spectra of physiological effects appears an effective way to integrate complex physiological functions. A good opportunity to examine this issue occurs in the accessory radula closer (ARC) neuromuscular circuit of Aplysia, where, extensive previous work has shown, acetylcholine-induced contractions of the muscle are variously modulated by several families of peptide cotransmitters released under appropriate behavioral circumstances from the muscle's own two motor neurons. 2. In this work we focused on the myomodulins (MMs) released from motor neuron B16. Previous work has characterized MMA (PMSMLRLamide) and MMB (GSYRMMRLamide). We now similarly purified from ARC neuromuscular material and sequenced MMC (GWSMLRLamide), MMD (GLSMLRLamide), MME (GLQMLRLamide), and MMF (SLNMLRLamide). Three additional MMs, MMG (TLSMLRLamide), MMH (GLHMLRLamide), and MMI (SLSMLRLamide), are encoded by a known MM gene. B16 probably synthesizes, and coreleases, all nine MMs. Further MMs have been found in other mollusks. All evidence indicates that the MMs are a major, widely distributed family of molluscan neuropeptides important as neuromuscular modulators and probably also central transmitters or modulators. 3. MM effects on motor neuron B16-elicited ARC muscle contractions were best analyzed as the sum of three distinct actions: potentiation, depression of the amplitude of the contractions, and acceleration of their relaxation rate. We compared the effectiveness of all nine MMs in these respects. We correlated this with their effectiveness in enhancing the L-type Ca current and activating a specific K current in voltage-clamped dissociated ARC muscle fibers, effects we previously proposed to underlie, respectively, the potentiation and the depression of contractions. 4. All nine MMs were similarly effective in enhancing the Ca current and, as far as it was possible to determine, potentiating the amplitude as well as accelerating the relaxation rate of the contractions. 5. In contrast, the MMs' ability to activate the K current and depress the contractions varied greatly. MMB and MMC, in particular, were weak, whereas the other seven MMs were considerably more effective in both respects. 6. Altogether, we were able to explain the potentiating and depressing strengths of the MMs by the magnitude of their modulation of the Ca and K currents, providing further support for our hypothesis that the effects on contraction amplitude are mediated by the effects on the two currents. 7. The net effect on contraction amplitude was determined by the balance between the potentiation and depression. Although most MM concentrations had both potentiating and depressing actions, potentiated contractions predominated at low and depressed contractions (but with accelerated relaxation rate) at high concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)