RESUMO
In a study of the specificity of neuronal connections in lobster abdominal ganglia, the dye Procion Yellow M4RS was electrophoretically injected into identified cell bodies. This dye spreads into fine branches of cells, survives fixation and routine histological procedures, and permits the reconstruction of cell shapes through examination of serial sections of ganglia. Certain cells were found to have an internal bilateral symmetry. Repeated injection of the same cells in ganglia from different animals showed that cells have characteristic shapes and that the neuropil is highly structured. This method of dye injection should have general applicability in studies where a knowledge of the geometry of specific cells is important.
Assuntos
Gânglios/anatomia & histologia , Neurônios/anatomia & histologia , Coloração e Rotulagem , Animais , Corantes , Crustáceos , Microscopia de Fluorescência , Microscopia de Contraste de FaseRESUMO
An FMRFamide-like neuropeptide, named AF1, was isolated from head extracts of the nematode Ascaris suum using five steps of HPLC. AF1 is a heptapeptide with the amino acid sequence Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2. Synthetic AF1 (10(-9) to 10(-7) M) rapidly and reversibly abolished slow membrane potential oscillations of identified ventral and dorsal inhibitory motoneurons and selectively reduced their input resistances. Synaptic transmission was not blocked. In intact Ascaris, AF1 inhibited locomotory movements. This study indicates a potential physiological role for an endogenous neuropeptide in nematodes.
Assuntos
Ascaris/análise , Neuropeptídeos/isolamento & purificação , Potenciais de Ação , Sequência de Aminoácidos , Animais , Ascaris/fisiologia , Cromatografia Líquida de Alta Pressão , Locomoção/fisiologia , Dados de Sequência Molecular , Neurônios Motores/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/fisiologiaRESUMO
The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d-tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic-like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.
Assuntos
Ascaris suum/fisiologia , Neurônios Motores/efeitos dos fármacos , Músculos/efeitos dos fármacos , Parassimpatomiméticos/farmacologia , Acetilcolina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Colina/análogos & derivados , Colina/farmacologia , Eletrofisiologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Microeletrodos , Neurônios Motores/enzimologia , Contração Muscular/efeitos dos fármacos , Músculos/enzimologia , Músculos/inervação , N-Metilescopolamina , Junção Neuromuscular/efeitos dos fármacos , Nicotina/farmacologia , Parassimpatolíticos/farmacologia , Pilocarpina/farmacologia , Derivados da Escopolamina/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/enzimologiaRESUMO
The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, located at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations (Guastella et al., J Comp Neurol 307:584-597, 1991). Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously (Guastella et al., J Comp Neurol 307:584-597, 1991), these results define three classes of GABA-associated neurons in Ascaris: 1) neurons that contain endogenous GABA and possess a GABA uptake system; 2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; 3) neurons that possess a GABA uptake system, but that lack endogenous GABA.
Assuntos
Ascaris/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Autorradiografia , Neurônios/metabolismo , TrítioRESUMO
An immunocytochemical method was used for localization of various peptide-like substances in the Ascaris nervous system. Out of 45 antipeptide antisera, 12 demonstrated immunoreactivity in different subsets of neurons; these 12 antisera were raised against luteinizing hormone-releasing hormone (LHRH), Aplysia peptide L11 (L11), Aplysia peptide 12B (12B), small cardioactive peptide B (SCPB), neuropeptide Y (NPY), FMRFamide, gastrin-17, cholecystokinin octapeptide (CCK-8), alpha-melanocyte stimulating hormone (alpha MSH), calcitonin gene related peptide (CGRP), corticotropin releasing factor (CRF), and vasoactive intestinal peptide (VIP). Several peptide-like substances were colocalized to the same neuron. Our results suggest that Ascaris, like other organisms, contains multiple peptidergic systems.
Assuntos
Ascaris/metabolismo , Sistema Nervoso/metabolismo , Neuropeptídeo Y/metabolismo , Neuropeptídeos/metabolismo , Animais , FMRFamida , Feminino , Imuno-Histoquímica , Masculino , Sistema Nervoso/citologiaRESUMO
gamma-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses. In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the RME-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.
Assuntos
Ascaris/citologia , Neurônios/química , Ácido gama-Aminobutírico/análise , Animais , Ascaris/análise , Gânglios/química , Imuno-Histoquímica , Masculino , Sistema Nervoso/química , Sistema Nervoso/citologiaRESUMO
A wholemount immunocytochemical method was used for the localization of cholecystokinin (CCK8)-like and gastrin-like immunoreactivity in Ascaris. The patterns of specific neuronal staining given by two antisera and four monoclonal antibodies made against CCK8, and one antiserum made against gastrin were investigated. Preabsorption of these antibodies with CCK8 or gastrin 17 resulted in complete loss of immunoreactivity in almost all of the neurons (two antisera also contained nonspecific antibodies), suggesting that all of the antibodies recognize epitopes, in Ascaris neurons, that include some or all of the C-terminal five amino acids that are identical in CCK8 and gastrin 17. However, the seven different antibodies showed immunoreactivity in different subpopulations of neurons, implying that there are at least seven different species of CCK-like molecules in Ascaris. Fractionation of Ascaris peptide extracts by high performance liquid chromatography (HPLC), monitoring fractions with a CCK8 radioimmunoassay (RIA), also shows heterogeneity of molecules immunologically related to CCK8.
Assuntos
Ascaris suum/metabolismo , Colecistocinina/imunologia , Gastrinas/imunologia , Sistema Nervoso/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Neurônios/metabolismo , SuínosRESUMO
By immunocytochemical and immunohistochemical methods, FMRFamide-like immunoreactivity (FLI) was localized to many neurons and processes in the Ascaris nervous system, including the head, tail, and lateral lines. Some of these cells were identified; they included sensory neurons, interneurons, and motor neurons. FLI was also present in the pharyngeal neurons and in their varicosities near the surface of the pharynx. By HPLC analysis of extracts, only a subset of the FMRFamide-like peptides (FLPs) expressed in Ascaris heads, and heads from which the pharynx had been removed, were expressed in the pharynx. Furthermore, FLPs appeared to be differentially expressed in female heads and tails and male heads and tails. Acetone and acid methanol differentially extracted subforms of FLI from Ascaris heads and from C. elegans.
Assuntos
Ascaris suum/metabolismo , Neuropeptídeos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , FMRFamida , Feminino , Imuno-Histoquímica , Hormônios de Invertebrado/metabolismo , Masculino , Neurônios Motores/metabolismo , Neurônios/metabolismo , Faringe/inervação , Radioimunoensaio , Distribuição TecidualRESUMO
The nematode nervous system is distinguished by the small number and morphological simplicity of its neurons. Recently, the shapes and synaptic interactions of each of the 302 neurons in the small free-living nematode, Caenorhabditis elegans, have been determined from reconstructions of serial sections by electron microscopy. Comparable anatomical studies of the large parasitic nematode Ascaris have concentrated on the dorsal and ventral nerve cords where reconstructions of motor neurons by light microscopy led to the identification of seven distinct types of motor neurons, each corresponding to a homologous cell type in C. elegans. In this study the shapes of the 13 neurons with cell bodies in the retrovesicular ganglion (RVG) of Ascaris suum were reconstructed from light micrographs of serial sections. In other preparations the morphology of RVG neurons was observed in whole mounts after the cells were impaled with microelectrodes and injected with the fluorescent dye Lucifer yellow. The intracellular electrodes also permitted electrical recordings and revealed that one type of cell, the AVF-like interneuron, expresses spontaneous repetitive plateau potentials. Comparisons of neuronal morphologies in the retrovesicular ganglia of Ascaris and C. elegans suggest that each neuron in Ascaris can be assigned a corresponding homolog in C. elegans. These data provide further evidence for a remarkable conservation of neuronal morphology in nematodes despite large differences in size and habitat.
Assuntos
Ascaris/anatomia & histologia , Gânglios/citologia , Interneurônios/citologia , Neurônios Motores/citologia , Potenciais de Ação , Animais , Feminino , Corantes Fluorescentes , Gânglios/fisiologia , Interneurônios/classificação , Interneurônios/fisiologia , Neurônios Motores/classificação , Neurônios Motores/fisiologia , Neurônios/classificação , Neurônios/citologiaRESUMO
Monoclonal antibodies that cross-react with Ascaris neural antigens were generated in mice immunized with a conjugate made with keyhole limpet hemocyanin (KLH) linked to a crude peptide extract from Caenorhabditis elegans. The response to KLH was suppressed by injection of cyclophosphamide 3 days after immunization with a gamma-aminobutyric acid (GABA)-KLH conjugate. Screening of hybridomas was carried out by enzyme-linked immunosorbent assay and whole mount immunocytochemistry. Two similar clones produced antibodies that recognized a small subset of Ascaris neurons. This result suggests that the monoclonal antibody technique might be useful for identifying new neuropeptides since the antibodies can be used for localization of the neuropeptidelike substances and, potentially, for immunoaffinity chromatography. As a by-product of this experiment, monoclonal antibodies that recognize GABA-like immunoreactivity in whole mounts and plastic sections were also obtained.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Ascaris/metabolismo , Neurônios/metabolismo , Neuropeptídeos/imunologia , Ácido gama-Aminobutírico/imunologia , Animais , Imuno-Histoquímica , Extratos de Tecidos/imunologiaRESUMO
Previous electrophysiological and anatomical studies of Ascaris suum motor neurons demonstrated a strong correlation between functional interactions and the presence of anatomically defined synapses. However, one example of a physiologically robust synaptic connection was encountered for which no anatomical evidence of direct chemical synapses was found. This involved synaptic transmission from an identified excitatory motor neuron to its inhibitory partner. In this study, pressure injection of horseradish peroxidase or nickel lysine into inhibitory motor neurons revealed numerous spines projecting from the main neuronal process toward the neuromuscular surface that then branched and extended fine, longitudinal processes up to 130 microm in length. Subsequent examination of nickel lysine-injected spines by electron microscopy revealed numerous chemical synapses, including for the first time direct inputs from the excitatory neuron. However, the numbers of synapses from this motor neuron were very small relative to inputs from other identified cells. Thus, direct synapses are unlikely to explain the robust nature of this physiological interaction.
Assuntos
Ascaris suum/anatomia & histologia , Ascaris suum/fisiologia , Dendritos/ultraestrutura , Neurônios Motores/ultraestrutura , Sinapses/ultraestrutura , Animais , Estruturas da Membrana Celular/ultraestrutura , Antagonistas Colinérgicos/farmacologia , Cobalto/farmacologia , Dendritos/fisiologia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Técnicas In Vitro , Mecamilamina/farmacologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Vias Neurais/citologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
We have used immunocytochemical techniques to investigate the distribution of serotonin-like immunoreactivity in the nematode Ascaris suum. Antisera raised against serotonin (5-hydroxytryptamine, 5-HT) conjugated to bovine serum albumin (BSA) labelled a pair of neurons in the pharynx of both sexes and five cells in the ventral cord of the male tail. The labelling was blocked by 5-HT or by 5-HT conjugated to BSA. The 5-HT-immunoreactive cells in the pharynx resemble neurosecretory cells and are probably homologous to the neurosecretory motor neurons (NSM) in Caenorhabditis elegans; the cells in the male tail appear to be motor neurons that are homologous to CP neurons in C. elegans. Other cells that stain with 5-HT antisera have been observed in C. elegans but are not seen in Ascaris.
Assuntos
Ascaris suum/química , Caenorhabditis elegans/química , Neurônios/química , Faringe/química , Serotonina/análise , Animais , Ascaris suum/citologia , Caenorhabditis elegans/citologia , Imuno-Histoquímica , Masculino , Faringe/citologiaRESUMO
Several protocols for conjugating peptides in situ to a protein carrier on paper, nitrocellulose, or nylon membranes were explored for their usefulness in dot-ELISA detection of the peptides. The most sensitive method in which peptide diluted in bovine serum albumin is applied to nitrocellulose, then fixed with glutaraldehyde, can detect several peptides, ranging from 4 to 38 amino acids in length, at the level of 2-10 fmol. Both immunohistochemical grade antisera and monoclonal antibodies have been used successfully. The method may be a useful alternative to radioimmunoassay since there is no requirement for radiolabelled peptide, or (for quantitation) for known quantities of unlabelled peptide. The method has been used to monitor, semiquantitatively, the fractionation of FMRFamide-like or CCK-like peptides from the nematode Ascaris, and to detect peptide-like immunoreactivities in tissue extracts.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Peptídeos/análise , Animais , Anticorpos Monoclonais , Ascaris/análise , Colecistocinina/análise , Colecistocinina/imunologia , Cromatografia Líquida de Alta Pressão , FMRFamida , Hormônios de Invertebrado/análise , Neuropeptídeos/análise , Neuropeptídeos/imunologia , Coelhos , Sensibilidade e EspecificidadeRESUMO
We have examined some aspects of the neuropharmacology of the nematode Ascaris suum using a divided chamber and selective stimulation technique to localize the sites of action of drugs. These techniques enabled us to investigate separately excitatory neuromuscular transmission, inhibitory neuromuscular transmission, and transmission from interneurons to excitatory motorneurons. We find that a curare-sensitive mechanism is involved in the excitation of the excitatory motorneuron via interneurons. The anthelmintic avermectin Bla (AVM) also blocks interneuronal stimulation of excitatory motorneurons. This action of AVM can be reversed by picrotoxin. AVM has no effect on excitatory neuromuscular transmission. Two GABAergic agonists in nematodes, muscimol and piperazine, mimic the effects of AVM when applied ventrally. This suggests that the action of AVM is related to a GABAergic mechanism. Ventral inhibitory neuromuscular transmission is also blocked by AVM, but this action is not reversed by picrotoxin. Thus AVM has two distinct sites of action in A. suum.
Assuntos
Acetilcolina/farmacologia , Anti-Helmínticos/farmacologia , Ascaris/efeitos dos fármacos , Ivermectina/análogos & derivados , Lactonas/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Animais , Ascaris/fisiologia , Curare/farmacologia , Estimulação Elétrica , Técnicas In Vitro , Neurônios Motores/efeitos dos fármacos , Muscimol/farmacologia , Piperazina , Piperazinas/farmacologiaRESUMO
Injection of Ascaris FMRFamide-like (AF) peptides and peptides encoded by genes in Caenorhabditis elegans were analyzed for effects on locomotion, body waveforms, and cAMP concentrations in adult female Ascaris suum. Injection of AF1 (KNEFIRFamide) or AF2 (KHEYLRFamide) inhibited the propagation of locomotory waves and reduced the number of waveforms, decreased the body length, and caused a large, long-lasting increase in cAMP. Muscle tissue was identified as a major source of the cAMP response induced by AF1. The AF1 analog AF1R6A did not affect cAMP levels by itself, but inhibited the cAMP response produced by AF1. AF8 (KSAYMRFamide) produced ventral coiling in the behavioral assay, and AF10 (GFGDEMSMPGVLRFamide) decreased the body length and increased the number of body waveforms. In dorsal muscle strips, AF10 produced a long-lasting contraction. Neither AF8 nor AF10 changed cAMP concentrations. AF17 (FDRDFMHFamide) increased body length and decreased cAMP. The neuropeptides encoded by C. elegans genes flp-4, flp-7, flp-9, and flp-13 produced paralysis and loss of waveforms, increased body length and, like AF17, decreased cAMP. Three new predicted peptides from C. elegans genome sequences were synthesized and tested. One produced ventral coiling but no change in cAMP; the other two gave no detectable responses. The fact that C. elegans neuropeptides produce behavioral and physiological effects in A. suum suggests that structurally related peptides may exist in A. suum. The profound changes in cAMP produced by some neuropeptides has important implications for understanding cAMP signaling and shows that neuropeptide-mediated signal transduction pathways are potential targets for anthelmintic drug development.
Assuntos
Ascaris suum/fisiologia , Caenorhabditis elegans/química , AMP Cíclico/metabolismo , Neuropeptídeos/farmacologia , Oligopeptídeos/farmacologia , Animais , Ascaris suum/efeitos dos fármacos , Ascaris suum/metabolismo , Comportamento Animal/efeitos dos fármacos , Feminino , Cinética , Movimento/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/metabolismo , Radioimunoensaio , Transdução de SinaisRESUMO
A FMRFamide-like neuropeptide, KHEYLRFamide (Lys-His-Glu-Tyr-Leu-Arg-Phe-amide; AF2) was isolated from a head extract of the nematode Ascaris suum by using a three-step HPLC separation. In a dorsal muscle strip preparation, synthetic AF2 produced multiple effects on muscle tension: a slow relaxation was followed by contraction and rhythmic activity. Sulfated AF2 was no more potent than AF2. The effects on muscle tension were correlated with electrical activity recorded intracellularly from muscle cells. AF2 markedly increased the tension change associated with change in muscle membrane potential.
Assuntos
Ascaris suum/química , Neuropeptídeos/isolamento & purificação , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Animais , Ascaris suum/fisiologia , Técnicas In Vitro , Dados de Sequência Molecular , Contração Muscular/fisiologia , Relaxamento Muscular/fisiologia , Tono Muscular/fisiologia , Neuropeptídeos/química , Neuropeptídeos/fisiologiaRESUMO
Neuropeptides play an important role in all nervous systems and structure-activity studies of related peptides is one approach to understanding this role. This study of the motor nervous system of the parasitic nematode Ascaris suum describes the physiological effects of a family of 18 endogenous Ascaris FMRFamide-like peptides (AF peptides) on the membrane potential and input resistance of the dorsal excitatory type 2 (DE2) and dorsal inhibitory (DI) motor neurons. These motor neurons are part of the final common output pathway from the motor nervous system to the somatic muscle cells responsible for locomotion. AF peptide effects on the frequency of excitatory postsynaptic potentials (EPSPs) in DE2 motor neurons were also measured to infer peptide effects on central presynaptic spiking neurons. AF peptide injections into intact worms were made to assess their qualitative effects on behavior, providing a context for interpreting motor neuron data. One category of AF peptides, N-terminally extended -FIRFa peptides (AF5, AF7 and AF1), has pronounced behavioral effects and qualitatively similar, but quantitatively different effects on DE2 and DI motor neurons. A second category of AF peptides (AF2, AF9, and AF8) also produces dramatic behavioral effects and strong electrophysiological effects on DE2 and/or DI motor neurons. A third category of AF peptides, consisting of six members of the -PGVLRFa group (which are encoded by the same gene and have closely related sequences) and peptide AF11, have pronounced behavioral effects, but relatively weak or negligible effects on DE2 and DI motor neurons. A fourth category of AF peptides, also consisting of structurally unrelated members, has pronounced behavioral effects and, as individual peptides, similar effects on both DE2 and DI motor neurons; AF15 is excitatory, while AF17 and AF19 are inhibitory, on both motor neuron types. Finally, two AF peptides (AF6, AF16) are relatively weak or inactive in producing behavioral or motor neuronal effects. Based on comparisons of the effects of AF peptides on DE2 and DI motor neurons, a tentative list of 5 major response-types is proposed as a working hypothesis to guide the search for AF peptide receptors. The findings attest to the potential complexity of neurosignaling in this comparatively simple nervous system.
Assuntos
Ascaris suum/metabolismo , Neuropeptídeos , Animais , Potenciais da Membrana , Neurônios Motores/fisiologia , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Eight FMRFamide-like neuropeptides were isolated from an extract of heads and tails from the nematode Ascaris suum using seven steps of HPLC. The peptides ranged in size from 8 to 14 amino acid residues: AF3 (Ala-Val-Pro-Gly-Val-Leu-Arg-Phe-amide), AF4 (Gly-Asp-Val-Pro-Gly-Val-Leu-Arg-Phe-amide), AF5 (Ser-Gly-Lys-Pro-Thr-Phe-Ile-Arg-Phe-amide), AF7 (Ala-Gly-Pro-Arg-Phe-Ile-Arg-Phe-amide), AF9 (Gly-Leu-Gly-Pro-Arg-Pro-Leu-Arg-Phe-amide), AF10 (Gly-Phe-Gly-Asp-Glu-Met-Ser-Met-Pro-Gly-Val-Leu-Arg-Phe-amide), AF11 (Ser-Asp-Ile-Gly-Ile-Ser-Glu-Pro-Asn-Phe-Leu-Arg-Phe-amide), and AF12 (Phe-Gly-Asp-Glu-Met-Ser-Met-Pro-Gly-Val-Leu-Arg-Phe-amide). The effect of synthetic AF4 on muscle tension in a dorsal muscle strip preparation was a strong, long-lasting contraction. PF1, a peptide present in Panagrellus redivivus and Caenorhabditis elegans, relaxed the AF4-induced contraction.
Assuntos
Ascaris suum/química , Hormônios de Invertebrado/isolamento & purificação , Neuropeptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , FMRFamida , Feminino , Masculino , Dados de Sequência MolecularRESUMO
We have identified a gene, afp-1, that encodes a new subfamily of six FMRFamide-like neuropeptides in the nematode Ascaris suum. The predicted peptides share the C-terminal sequence PGVLRF-NH2 but have different N-terminal extensions. We discuss possible functional roles of these different peptides based upon experiments with Ascaris as well as results from other organisms. Three of the peptides were previously isolated from extracts of A. suum (4) and three other are novel sequences. The translated product of afp-1 is a precursor protein containing two main halves: a C-terminal region containing a series of putative peptides separated by characteristic processing sites and a relatively hydrophobic N-terminal region with no obvious peptides. Although the overall structure of the translated product of afp-1 is similar to flp-1 from C. elegans (18), there is little evidence for homology between the two nematode neuropeptide genes. At least four different transcripts of afp-1 have been identified. These transcripts differ in their 3' and 5' untranslated regions, and one of the transcripts predicts a truncated precursor protein which contains only the C-terminal peptide-containing region.