RESUMO
A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.
Assuntos
Lymnaea/química , Fatores de Crescimento Neural/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Cálcio/metabolismo , Hemolinfa/química , Humanos , Dados de Sequência Molecular , Neurônios Motores/ultraestrutura , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/isolamento & purificação , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Fator de Crescimento Neural , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Lymnaea/fisiologia , Proteínas de Membrana/fisiologia , Ocitocina/análogos & derivados , Ocitocina/fisiologia , Receptores de Vasopressinas/fisiologia , Vasopressinas/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/farmacologia , Canais de Cloreto/fisiologia , Clonagem Molecular , Condutividade Elétrica , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Oócitos/fisiologia , Especificidade de Órgãos , Ocitocina/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Ocitocina/química , Receptores de Vasopressinas/química , Receptores de Vasopressinas/genética , Alinhamento de Sequência , XenopusRESUMO
Insulin is a molecule that has played a key role in several of the most important landmarks in medical and biological research. It is one of the most extensively studied protein hormones, and its structure and function have been elucidated in many vertebrate species, ranging from man to hagfish and turkey. The structure, function as well as tissue of synthesis of vertebrate insulins are strictly conserved. The structural identification of insulin-related peptides from invertebrates has disrupted the picture of an evolutionary stable peptide hormone. Insulin-related peptides in molluscs and insects turned out to be a structurally diverse group encoded by large multi-gene families that are uniquely expressed in the brain and serve functions different from vertebrate insulin. In this review, we discuss invertebrate insulins in detail. We examine how these peptides relate to the model role that vertebrate insulin has played over the years; however, more importantly, we discuss several unique principles that can be learned from them. We show how diversity of these peptides is generated at the genetic level and how the structural diversity of the peptides is linked to the exclusive presence of a single type of neuronal insulin receptor-related receptor. We also discuss the fact that the invertebrate peptides, in addition to a hormonal role, may also act in a synaptic and/or nonsynaptic fashion as transmitters/neuromodulators on neurons in the brain. It can be expected that the use of well-defined neuronal preparations in invertebrates may lead to a further understanding of these novel functions and may act as guide preparations for a possible role of insulin and its relatives in the vertebrate brain.
Assuntos
Química Encefálica , Insulina/fisiologia , Hormônios de Invertebrado/química , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência MolecularRESUMO
We studied the regenerative properties of one of two electrically coupled molluscan neurons, the serotonergic cerebral giant cells (CGCs) of Lymnaea stagnalis, after axotomy. The CGCs play a crucial role in feeding behavior, and when both cells are disconnected from their target neurons, animals no longer feed. When one CGC was permanently disconnected from its targets and the other was reversibly damaged by a nerve crush, the latter one regenerated over a period of 2 weeks to reform functional synapses with specific target neurons. At the same time, recovery of the feeding behavior was observed. After the crush, neuropeptide gene expression in the CGC was downregulated to approximately 50%. Serotonin synthesis, on the other hand, remained unaffected, suggesting that serotonin might have an active role in regeneration. In primary neuron culture, CGCs failed to extend neurites in the presence of serotonin; in cells that extended neurites in the absence of serotonin, focally applied serotonin, but not neuropeptides, induced growth cone collapse. Using serotonin-sensitive sniffer cells, we show that CGC neurites and growth cones release serotonin in culture. Finally, both the spontaneous and stimulation-induced release of serotonin from CGCs in culture resulted in growth cone collapse responses that could be blocked by the serotonin receptor antagonist methysergide. Our data suggest that auto-released serotonin is inhibitory to CGC neurite outgrowth in vitro. During regeneration in vivo, serotonin release might fine-tune axon guidance and branching by inducing local collapse responses in extending neurites.
Assuntos
Axônios/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Neurotransmissores/biossíntese , Animais , Axônios/efeitos dos fármacos , Axotomia , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/fisiologia , Técnicas In Vitro , Lymnaea , Metisergida/farmacologia , Modelos Neurológicos , Dados de Sequência Molecular , Compressão Nervosa , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Neurotransmissores/isolamento & purificação , Neurotransmissores/farmacologia , RNA Mensageiro/biossíntese , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Serotonina/metabolismo , Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triptofano Hidroxilase/genéticaRESUMO
Immunocytochemistry and in situ hybridization were used to identify 200-250 central neurons that synthesize and contain the peptide APGWamide in the central nervous system of Lymnaea. The majority of these cells reside within the right anterior lobe of the cerebral ganglion and most appear to have projections to the ventral lobe of the ganglion. The neurites then branch to innervate the lobe and to send further projections into the penial nerve and to the PeIb cluster of the right pedal ganglion. The right ventral lobe also contains a cluster of about 30-40 somata, which apparently synthesize and contain APGWamide. Other populations of cells found in both the right and left anterior lobes project ipsilaterally to the pleural, parietal, and visceral ganglia. Prominent somata are also located in clusters in the visceral and right parietal ganglia. These cells project ipsilaterally into caudal neuropilar regions of the cerebral ganglia. Peripheral projections of central neurons form a dense plexus of varicose, APGWamide-containing fibres in superficial layers of the penis and preputium. Other peripheral projections were noted in the prostate and vas deferens. No peripherally located cell bodies appeared to contain or synthesize the peptide. The results show that APGWamide is widely present in the central nervous system and male reproductive organs and suggest that it plays a major role in control of reproduction.
Assuntos
Lymnaea/metabolismo , Sistema Nervoso/metabolismo , Neuropeptídeos/metabolismo , Animais , Feminino , Imunofluorescência , Gânglios/citologia , Gânglios/imunologia , Gânglios/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Neuritos/imunologia , Neuritos/ultraestrutura , Neurônios/imunologia , Neurônios/metabolismo , Neuropeptídeos/imunologia , Nervos Periféricos/imunologia , Nervos Periféricos/metabolismoRESUMO
The male copulation behaviour of the hermaphrodite pond snail Lymnaea stagnalis is under the control of five groups of central neurons that produce a variety of neuropeptides and a classical transmitter, 5-hydroxy tryptamine (5HT). In this article, we describe how the male sexual organs of this snail are innervated by axons from these central neurons. We carried out immunocytochemistry with antisera against the tetra peptide Ala-Pro-Gly-TRP-NH2 (APGWamide), the Lymnaea form of neuropeptide tyrosine (LNPY), conopressin, pedal peptide, the FRMFamide copeptide SEEPLY, the GDPFLRFamide co-peptide DEILSR, myomodulin, Lymnaea inhibitory peptide, and 5HT on tissue sections of the following male sexual organs that receive input from the penis nerve: the prostate gland, vas deferens, preputium, and penis. The results demonstrate that the axons of the separate muscle systems contain particular combinations of transmitters. In addition, two networks of peripheral neurons were revealed. In the tip of the everted preputium lies what appears to be a network of conopressin-containing sensory neurons, which is possibly involved in probing; probing is the part of copulation behaviour in which the male searches for the female genital pore. The other network of peripheral neurons surrounds the most proximal part of the vas deferens and is most likely involved in the pacemaker control of vas deferens motility. On the basis of the data obtained, we hypothesize how the preputium and penis are everted during copulation and which transmitters and central neurons might be involved.
Assuntos
Lymnaea/anatomia & histologia , Neurônios/ultraestrutura , Neuropeptídeos/metabolismo , Animais , Axônios/ultraestrutura , Genitália Masculina/inervação , Lymnaea/metabolismo , Masculino , Pênis/inervação , Próstata/inervação , Comportamento Sexual Animal/fisiologia , Ducto Deferente/inervaçãoRESUMO
In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.
Assuntos
Copulação/fisiologia , Lymnaea/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Neuropeptídeos/análise , Neurotransmissores/análise , Animais , Imuno-Histoquímica , Lymnaea/citologia , Masculino , Modelos NeurológicosRESUMO
The neuroendocrine caudodorsal cells play an important role in the control of reproduction in Lymnaea stagnalis. These neurons produce at least nine neuropeptides which are encoded by caudodorsal cell hormone-I and -II genes. The role of some of these peptides in the control of reproduction has been established. The present study demonstrates that the transcription and translation of the caudodorsal cell hormone genes also proceeds abundantly in the reproductive tract of this hermaphroditic animal. In the female part of the reproductive tract neurons were found to express gene I. These neurons are most likely involved in the control of transport of the eggs and egg-masses and in the regulation of secretory activity from the female accessory sex glands. In the male part of the reproductive tract exocrine secretory cells express gene I or gene II. The gene products are secreted into the male duct and transferred to the female copulant during copulation. Furthermore, putative sensory neurons in the skin were found to express gene I. The results indicate that in L. stagnalis the complex process of reproduction is regulated--at least in part--by a set of neuropeptides which are encoded by a small multigene family, viz. the caudodorsal cell gene family.
Assuntos
Glândulas Exócrinas/metabolismo , Regulação da Expressão Gênica , Hormônios de Invertebrado/genética , Lymnaea/metabolismo , Nervos Periféricos/metabolismo , Reprodução , Animais , Hibridização de Ácido NucleicoRESUMO
We report here the neuron-specific co-expression of four genes coding for neuropeptides involved in the control of male behaviour. These neurons are located in the anterior lobe of the right cerebral ganglion in the central nervous system of Lymnaea stagnalis and project via the penis nerve to the penial complex. In order to accomplish optimal assurance we applied in situ hybridization, immunocytochemistry and matrix-assisted laser desorption ionization mass spectrometry. The anterior lobe neurons express the gene encoding the amidated tetrapeptide APGWamide. Subsets of these cells are now shown to co-express the APGWamide gene exclusively with one of three other neuropeptide genes, encoding Lymnaea neuropeptide Y, conopressin or pedal peptide, respectively. All four genes are also expressed in other neurons in other centres projecting to the penial complex, but in these cells co-expression was not observed. The neuropeptides encoded by the genes could be identified in the anterior lobe cell bodies on the basis of immunocytochemistry and mass spectrometrical analysis. The neuropeptides APGWamide and Lymnaea neuropeptide Y, which are co-localized in the anterior lobe cells as well as in axons innervating the penis retractor muscle, do not induce muscle contraction but have a modulatory action by affecting the relaxation rate and amplitude of the contraction. APGWamide and conopressin had earlier been suggested to modulate peristalsis of the vas deferens. Thus, it seems that the neurons co-expressing the various combinations of neuropeptide genes in the anterior lobe represent functional units, each acting in the fine tuning of different muscles involved in specific aspects of male copulation behaviour.
Assuntos
Copulação/fisiologia , Regulação da Expressão Gênica/fisiologia , Lymnaea/fisiologia , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Comportamento Sexual Animal/fisiologia , Animais , Química Encefálica/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Hibridização In Situ , Técnicas In Vitro , Masculino , Contração Muscular/fisiologia , Músculos/efeitos dos fármacos , Músculos/inervação , Neuropeptídeos/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Specimens of the freshwater snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata show a strongly inhibited development of their reproductive tract. We hypothesised that the effects of the underdevelopment of targets are reflected at the level of the neuronal development of (i) the motor neurons innervating the male copulation organ and (ii) neuroendocrine cells regulating the gonad. We determined the state of neuronal development by measuring cell number, cell size and neuropeptide gene expression. Our results show that the neuronal development of both copulation controlling anterior lobe motor neurons of the right cerebral ganglion and neuroendocrine caudodorsal cells, which produce neuropeptides regulating ovulation, egg laying and accompanying behaviour, are affected in parasitised animals in which their respective target organs were not developed. The cell bodies were smaller and fewer cells were found to express neuropeptide genes compared to those in non-parasitised animals. These effects were not observed in the appropriate controls. Backfills and lesions of the penis nerve have shown that the inhibited development of central motor neurons in parasitised snails is target dependent; neighbouring neurons that have no connection with the male copulation organ are not affected. Our data suggest that this effect is established by target-derived neurotrophic factors that need this connection for being transported to the innervating motor neurons. We propose that the effect on the neuroendocrine caudodorsal cells is mediated by a humoral factor, since they have no known connection with their target. We have shown that the size and gene expression of motor neurons controlling copulation behaviour in the pond snail Lymnaea stagnalis are related to the size of their target, the copulation organ, and depend on the connection with this target.
Assuntos
Neurônios Motores/citologia , Sistemas Neurossecretores/citologia , Animais , Contagem de Células , Diferenciação Celular , Tamanho Celular , Feminino , Gônadas/inervação , Imuno-Histoquímica , Masculino , Moluscos/parasitologia , Neurônios Motores/metabolismo , Neuropeptídeo Y/metabolismo , Neuropeptídeos/metabolismo , SchistosomaRESUMO
A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.
Assuntos
Neuropeptídeos/genética , Peptídeos/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , Hibridização In Situ , Lymnaea , Dados de Sequência Molecular , RNA Mensageiro/metabolismoRESUMO
Neurons are highly polarized cells that contain a wealth of cytoplasmic and membrane proteins required for neurotransmission, synapse formation and various forms of neuronal plasticity. Typically, these proteins are differentially distributed over somatic, dendritic and axonal compartments. Until recently, it was believed that all proteins destined for various neuronal sites were synthesized exclusively in the somata and were subsequently targeted to appropriate extrasomal compartments. The discovery of various messenger RNA molecules in both dendrites and axons is suggestive of de novo protein synthesis in extrasomatic regions. The latter process has been demonstrated in few neuronal svrstems, but direct proof for the axonal transcription of a specific protein from a given messenger RNA is still lacking. This lack of fundamental knowledge in the field of cellular and molecular neurobiology is due primarily to both anatomical and experimental difficulties encountered in most animal preparations studied thus far. In this study we developed a neuronal experimental system comprising of individually identified neurons and their isolated axons from the mollusc Lymnaea stagnalis. We injected a foreign messenger RNA encoding a peptide precursor into the isolated axons of cultured neurons; and utilizing cellular, molecular and immunocytochemical techniques, we provide direct evidence for specific protein synthesis in isolated axons. The Lymnaea model provides us with an opportunity to examine the role and specificity of de novo protein synthesis in the extrasomal regions.
Assuntos
Axônios/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas , Animais , Técnicas In Vitro , LymnaeaRESUMO
To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.
Assuntos
Lymnaea/análise , Neuropeptídeos/genética , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , 2-Acetilaminofluoreno , 3,3'-Diaminobenzidina , Animais , Sondas de DNA , Imuno-Histoquímica , Insulina/genética , Hormônios de Invertebrado/metabolismo , Sondas de Oligonucleotídeos , Peptídeos/genética , Radioisótopos de Fósforo , Radioisótopos de EnxofreRESUMO
The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.
Assuntos
Hibridização In Situ/métodos , RNA Mensageiro/metabolismo , RNA Ribossômico 28S/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Gânglios/metabolismo , Gânglios/ultraestrutura , Imuno-Histoquímica , Lymnaea , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia Eletrônica , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/ultraestrutura , RNA Ribossômico 28S/ultraestruturaRESUMO
Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.
Assuntos
Lymnaea/citologia , Neuropeptídeos/genética , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , Animais , Sequência de Bases , Biotina , Fluoresceína-5-Isotiocianato , Fluoresceínas , Imuno-Histoquímica , Lymnaea/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Sondas RNA , RNA Mensageiro/análise , TiocianatosRESUMO
Cells immunoreactive to antisera specifically directed against Lymnaea stagnalis caudo dorsal cells egg-laying hormone (CDCH) or against alpha- and beta-peptides (CDCP), encoded on the egg-laying hormone precursor, were detected in central nervous system (CNS) of the rhynchobdellid leech Theromyzon tessulatum. A co-localization of the CDC-like hormone and CDC-like peptides was found in T. tessulatum as in L. stagnalis CNS. approximately 45 immunoreactive cells to the anti-CDCH were detected in leech brain but this number varies according to the stage of the animal life cycle, i.e. it reaches a maximum just before egg-laying while after it decreases to 2-3 cells. CDCH and alpha-CDCP epitopes recognized by anti-CDCH and anti-alpha-CDCP were contained in neurosecretory granules. Following an extensive purification, including HPGPC and reverse-phase HPLC, the CDC-like hormone contained in the T. tessulatum CNA was isolated. The sequence (GSGVSNGGTEMIQLSHIRERQRYWAQDNLRRRFLEK-amide) was established by a combination of automated Edman degradation, arginyl-endopeptidase digestion, electrospray mass spectrometry measurement and carboxypeptidase A treatment. The results demonstrate that the peptide recognized by the anti-CDCH in the leech CNS possesses 27.8, 37.2 and 47.2% sequence identity with Aplysia parvula, Lymnaea stagnalis and Aplysia californica ELH, respectively. This molecule was named the leech egg-laying-like hormone (L-ELH). The secondary structure prediction of the L-ELH and all mollusks ELH, revealed the existence of a conserved segment (segment 29-34) in a strong helicoidal bend that might be important for receptor recognition and/or activation. This finding constitutes the first biochemical characterization of an egg-laying hormone in other invertebrates than mollusks.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Hormônios de Invertebrado/química , Sanguessugas/fisiologia , Neurônios/fisiologia , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Hormônios de Invertebrado/análise , Lymnaea , Espectrometria de Massas , Dados de Sequência Molecular , Oviposição , Filogenia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The neuroendocrine light yellow cells of Lymnaea stagnalis form two clusters of cells in the visceral and right parietal ganglion, respectively. These cells are endogenously bursting neurons whose activities are modified during egg-laying and feeding. Using gel permeation chromatography and reverse phase HPLC we have purified two highly related novel peptides from the light yellow cells. These peptides differ only in length, due to truncation of the amino-terminal aspartic acid residue, which causes a major shift in the charge of the molecule. We conclude that the longer peptide is the immediate precursor of the shorter form. Using whole mount immunocytochemistry, it was confirmed that the light yellow cells produce these peptides.
Assuntos
Gânglios/química , Lymnaea/metabolismo , Neuropeptídeos/química , Sistemas Neurossecretores/química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Formação de Anticorpos/imunologia , Gânglios/citologia , Imuno-Histoquímica , Dados de Sequência Molecular , Neuropeptídeos/análise , Neuropeptídeos/isolamento & purificação , Sistemas Neurossecretores/citologiaRESUMO
We used a strategy combining immunodetection, peptide chemistry, and a novel method, direct peptide fingerprinting of neurons and small pieces of nerve by using matrix-assisted laser desorption ionization mass spectrometry, to structurally identify and localize the neuropeptide myomodulin-A in the mollusc, Lymnaea stagnalis. Lymnaea myomodulin appeared to be identical to Aplysia myomodulin-A and is produced by many central neurons, including neurons located in the ventral lobe of the right cerebral ganglion that innervate the penis complex via the penis nerve. Myomodulin-A could also be characterized from the penis complex, and physiological concentrations of the peptide enhanced the relaxation rate of electrically induced contractions of the penis retractor muscle in vitro in a dose-dependent fashion.
Assuntos
Lymnaea/metabolismo , Neurônios/metabolismo , Neuropeptídeos/fisiologia , Animais , Copulação/fisiologia , Immunoblotting , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Neuropeptídeos/análise , Neuropeptídeos/biossíntese , Oligopeptídeos/análise , Relação Estrutura-AtividadeRESUMO
A neuropeptide that strongly inhibits the spontaneous contractions of the oesophagus in Lymnaea has been characterized as GAPRFVamide. Direct mass spectrometry of nervous tissues and immunocytochemical studies show that the peptide is synthesized by neurones in the buccal ganglia and transported to the oesophagus via the dorso-buccal nerve. In accordance with the function of the peptide, immunoreactive fibres are detected within the muscle layer of the oesophagus. Finally, mass spectrometry reveals the presence of a number of unidentified peptides in the nerves that innervate the oesophagus, which suggests that oesophageal activities may be modified by multiple peptides.
Assuntos
Esôfago/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Neuropeptídeos/fisiologia , Animais , Imuno-Histoquímica , Lymnaea , Neuropeptídeos/análise , Fatores de TempoRESUMO
Monoclonal antibodies were raised against the small molecular weight fraction (less than 30 kilodaltons) of an extract from 200 central nervous systems (CNS) of the freshwater snail Lymnaea stagnalis. In a first screening step the supernatants of the 297 emerging hybridomas were immunocytochemically tested on sections of the CNS of L. stagnalis. Sixty-six appeared to produce neuron-specific antibodies, five reacted with non-neuronal elements. In a second step the 66 neuron-specific antibodies were tested on sections of the CNS of the guppy. Three reacted positively. In the third step the three antibodies were tested on the CNS of the rat. One antibody (Mab4H5) appeared to give positive results. In the snail brain Mab4H5 stains two identified giant neurons, one in the visceral ganglion (VD1), and one in the right parietal ganglion (RPD2)--these neurons form part of the network controlling the respiratory system--and a small number of cells in the cerebral ganglia (in the anterior and ventral lobes). Ultrastructural observations using immunogold labelling in VD1 showed the antigen to be localized to the secretory vesicles. In the guppy Mab4H5 stains fibres in the tectum and cell bodies in the reticular formation. In rat CNS staining was observed in Purkinje neurons of the cerebellum, in cortical pyramidal neurons and in neurons and fibres in other brain areas. Subsequent Mab4H5 staining of the CNS of the lizard, the cockroach and parts of the human CNS showed that these tissues also contain Mab4H5-positive neurons. In the human cortex and cerebellum the staining pattern appeared to be similar to that of the rat. On the basis of the results it is hypothesized that the antibody reacts with phylogenetically ancient amino acid sequences.