RESUMO
In amphibians, there is a close interaction between the interrenal and the thyroidal axes. Hypothalamic corticotropin-releasing hormone or related peptides stimulate thyroidal activity by increasing thyrotropin synthesis and release, while corticosterone accelerates both spontaneous and thyroid hormone-induced metamorphosis. One of the mechanisms that is thought to contribute to this acceleration is a corticosterone-induced change in peripheral deiodinating activity. The present experiments were designed to investigate further the effects of glucocorticoid treatment on amphibian deiodinase activities and to explore the possible role of these effects in metamorphosis. Neotenic axolotls (Ambystoma mexicanum) were treated either acutely or chronically with dexamethasone (DEX) and changes in type II and type III iodothyronine deiodinase (D2 and D3) activities were studied in liver, kidney, and brain. In addition, gill length, tail height, and body weight were measured at regular intervals in the chronically treated animals in search of metamorphosis-related changes. A single injection of 50 microg DEX decreased hepatic D3 activity (6-48 h) while it increased D2 activity in brain (6-48 h) and to a lesser extent in kidney (24 h). These changes were accompanied by an increase in plasma T(3) levels (48 h). Samples taken during chronic treatment with 20 or 100 microg DEX showed that both hepatic D2 and D3 activities were decreased on day 26, while renal D3 activity was decreased but only in the 20 microg dose group. All other deiodinase activities were not different from those in control animals. At 25 days, all DEX-treated axolotls showed a clear reduction in gill length, tail height, and body weight, changes typical of metamorphosis. Prolongation of the treatment up to 48 days resulted in complete gill resorption by days 44-60. Although probably several mechanisms contribute to these DEX-induced metamorphic changes, the interaction with thyroid function via a sustained downregulation of hepatic D3 may be one of them.
Assuntos
Ambystoma/metabolismo , Dexametasona/farmacologia , Iodeto Peroxidase/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Química Encefálica/fisiologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Brânquias/efeitos dos fármacos , Brânquias/crescimento & desenvolvimento , Técnicas In Vitro , Isoenzimas/metabolismo , Rim/metabolismo , Fígado/enzimologia , Masculino , Microssomos/enzimologia , Microssomos/metabolismo , Tamanho do Órgão/fisiologia , Cauda/crescimento & desenvolvimento , Tiroxina/sangue , Tiroxina/farmacologia , Tri-Iodotironina/sangueRESUMO
The ontogeny of the neurohormonal peptides vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), and somatostatin (SOM) as well as serotonin (SER) and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, using immunohistochemical techniques. The first regulatory substances to appear were SP, SOM, and SER that could be immunohistochemically detected up from stage 1. At early stage 2, VIP immunoreactivity was observed infrequently in enteric nerve fibers. With the onset of external feeding at late stage 2, SP-immunoreactive (IR) and SER-IR endocrine cells and VIP-IR nerve fibers were present throughout the gastrointestinal tract. Furthermore, in the small intestine NT-IR and GAS/CCK-IR endocrine cells appeared. At stage 3, SER immunoreactivity was observed not only in endocrine cells but also in nerve fibers. CGRP-IR and SP-IR nerve fibers were detectable at stage 4 and stage 5, respectively. From stage 5 on, a minority of the CGRP immunoreactivity occurred in SP-IR nerve fibers. NOS immunoreactivity did not appear before stage 6 when it was found infrequently in nerve fibers. Thus, several phases of development can be distinguished: (1) at the yolk sac stages only few regulatory substances are present. (2) At the onset of external feeding, all endocrine cell types investigated were readily detectable. Thus, the onset of external feeding seems to trigger the development of the gastrointestinal endocrine system. (3) The endocrine cells are first found in the proximal part of the gastrointestinal tract and later in higher numbers in the distal parts. (4) The dually distributed neurohormonal peptides and SER first appear in endocrine cells and later additionally in nerve fibers. Thus, the nerve fibers likely set up the fine regulation of gastrointestinal blood flow and motility.
Assuntos
Ambystoma/crescimento & desenvolvimento , Sistema Digestório/crescimento & desenvolvimento , Neuropeptídeos/análise , Sistemas Neurossecretores/crescimento & desenvolvimento , Óxido Nítrico Sintase/análise , Serotonina/análise , Ambystoma/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/análise , Colecistocinina/análise , Sistema Digestório/química , Gastrinas/análise , Imuno-Histoquímica , Larva/crescimento & desenvolvimento , Larva/metabolismo , Sistemas Neurossecretores/química , Neurotensina/análise , Somatostatina/análise , Substância P/análise , Peptídeo Intestinal Vasoativo/análiseRESUMO
Amphibians occupy a central position in phylogeny between aquatic and terrestrial vertebrates and are widely used as model systems for studying vertebrate development. We have undertaken a comprehensive molecular approach to understand the early events related to embryonic development in the Mexican axolotl, Ambystoma mexicanum, which is an exquisite animal model for such explorations. Axolotl RBP is a RNA-binding protein which was isolated from the embryonic Mexican axolotl by subtraction hybridization and was found to show highest similarity with human, mouse, and Xenopus cold-inducible RNA-binding protein (CIRP). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis suggests that it is expressed in most of the axolotl tissues except liver; the expression level appears to be highest in adult brain. We have also determined the temporal and spatial pattern of its expression at various stages of development. RT-PCR and in situ hybridization analyses indicate that expression of the AxRBP gene starts at stage 10-12 (gastrula), reaches a maxima around stage 15-20 (early tailbud), and then gradually declines through stage 40 (hatching). In situ hybridization suggests that the expression is at a maximum in neural plate and neural fold at stage 15 (neurula) of embryonic development.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Ligação a RNA/genética , Ambystoma/embriologia , Ambystoma/metabolismo , Animais , Northern Blotting , Desenvolvimento Embrionário , Humanos , Hibridização In Situ , Camundongos , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , XenopusRESUMO
To improve basic knowledge about the neurochemical organization of the urodele brain, and to study discrepancies in the localization of monoaminergic markers, we immunohistochemically charted the distribution of four such markers (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine, and serotonin) in the axolotl (Ambystoma mexicanum) forebrain. Catecholaminergic and serotoninergic systems were found in similar locations to those seen in other Urodela. As seen in other vertebrates, the localization of the different monoaminergic markers reveals some inconsistencies. Cells that are exclusively tyrosine hydroxylase-immunoreactive are observed in the olfactory bulb, anterior olfactory nucleus/nucleus accumbens region, the epichiasmatic portion of the preoptic nucleus, and in the pars intercalaris thalami, whereas cells that are only labelled by aromatic L-amino acid decarboxylase are seen in the anterior olfactory nucleus/nucleus accumbens region, the bed nuclei of the anterior commissure, the posterior portion of the preoptic nucleus, the ventral hypothalamus, and the pars intercalaris thalami. The presence of cells solely serotonin (5-HT)-immunoreactive is suggested for the nucleus infundibularis dorsalis. Conversely, there were no areas that appeared to be exclusively immunoreactive for dopamine. Double-labelling for aromatic L-amino acid decarboxylase/tyrosine hydroxylase and aromatic L-amino acid decarboxylase/serotonin, together with cell counting, confirmed the existence of neurons that express only one monoaminergic marker in amphibian, supporting the hypothesis that these cells are universally present in the central nervous system of vertebrates.
Assuntos
Ambystoma/metabolismo , Descarboxilases de Aminoácido-L-Aromático/análise , Prosencéfalo/química , Serotonina/análise , Tirosina 3-Mono-Oxigenase/análise , Animais , Mapeamento Encefálico/métodos , Diencéfalo/química , Feminino , Hipotálamo/química , Imuno-Histoquímica , Masculino , Telencéfalo/químicaRESUMO
Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.
Assuntos
Ambystoma/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Tropomiosina/biossíntese , Tropomiosina/genética , Ambystoma/genética , Ambystoma/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Complementar/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum) inner ear. Hair cells showed an intense NADPH-d reaction; afferent neurones also stained but less intensely than hair cells. Effects of NG-nitro-L-arginine (L-NOARG) on the basal discharge and mechanical responses of semicircular canal afferent neurones recorded extracellularly were also studied. L-NOARG (1 mu M) diminished the basal discharge and the response of afferent neurones to sinusoidal mechanical stimuli to 45 +/- 6.4% and 65 +/- 5.3% (mean +/- SEM) of control value, respectively. These findings suggest that production of nitric oxide (NO) by hair cells and probably also by afferent neurones contributes to the basal discharge and the response of afferent neurones to mechanical stimuli.
Assuntos
Ambystoma/metabolismo , Orelha Interna/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Orelha Interna/citologia , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Potenciais Evocados/efeitos dos fármacos , Células Ciliadas Vestibulares/efeitos dos fármacos , Células Ciliadas Vestibulares/enzimologia , Histocitoquímica , NADPH Desidrogenase/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/enzimologia , Neurônios Eferentes/efeitos dos fármacos , Neurônios Eferentes/enzimologia , Nitroarginina/farmacologiaRESUMO
1. The myelin protein profiles in the CNS and PNS of three species of amphibians were analyzed by biochemical and immunohistochemical methods. 2. The CNS myelin of the African clawed frog (Xenopus) and the Mexican salamander (axolotl) contained, in addition to proteolipid protein, a unique protein zero (P0)-like protein, whereas the adult bullfrog did not. 3. A strong expression of the P0-like protein in the bullfrog CNS myelin was found transiently at ontogenetically early phases including at the time of metamorphosis. 4. The CNS P0-like protein and the PNS P0 protein showed a difference in reactivity with lectins and anti-L2/HNK-1 antibodies, suggesting that the two proteins differ in some aspects of their carbohydrate structures.
Assuntos
Ambystoma/metabolismo , Sistema Nervoso Central/metabolismo , Proteínas da Mielina/biossíntese , Rana catesbeiana/metabolismo , Xenopus laevis/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Técnicas Imunoenzimáticas , Lectinas/metabolismo , Camundongos , Peso Molecular , Proteína P0 da Mielina , Proteínas da Mielina/química , Proteínas da Mielina/metabolismo , Reação do Ácido Periódico de SchiffRESUMO
The Leydig cells in the lingual epithelium of the axolotl were investigated by immunohistochemistry using serotonin antiserum. Serotonin-immunoreactivity was found in their secretory granules. The physiological role of serotonin in the Leydig cell, a type of exocrine cell, is unknown.
Assuntos
Ambystoma/metabolismo , Mucosa Bucal/metabolismo , Serotonina/metabolismo , Língua/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Células Epiteliais , Epitélio/metabolismo , Imuno-Histoquímica , Mucosa Bucal/citologia , Língua/citologiaRESUMO
1. Lactose-inhibitable hemagglutination activity was identified in extracts of axolotl (Ambystoma mexicanum) larvae. 2. Two types of lectin were isolated from extracts by affinity chromatography on lactose-Sepharose. 3. A thiol-independent lectin of subunit mol. wt 15 kDa and a thiol-dependent lectin of subunit mol. wt 18 kDa were identified. 4. The 15 kDa and a 18 kDa polypeptides were weakly reactive with polyclonal anti-human galaptin serum.
Assuntos
Ambystoma/metabolismo , Hemaglutininas/isolamento & purificação , Lectinas/isolamento & purificação , Animais , Galectina 4 , Galectinas , Hemaglutininas/química , Hemaglutininas/imunologia , Imunoquímica , Lectinas/química , Lectinas/imunologia , Peso Molecular , Conformação ProteicaRESUMO
Six major neutral and acidic oligosaccharide-alditols were prepared from the jelly coat of Mexican axolotl eggs. These compounds were demonstrated to contain 3-deoxy-D-glycero-D-galacto-nonulosonic acid (dNloA) and L-fucose (Fuc). The structures of the six major oligosaccharides were established as follows: [sequence: see text]
Assuntos
Ambystoma/metabolismo , Oligossacarídeos/química , Álcoois Açúcares/química , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Óvulo/químicaRESUMO
The presence of kainic (KA) and quisqualic acid (QA) receptors on inner retinal neurones of the axolotl Ambystoma mexicanum has been studied using intracellular recording techniques. In the presence of CoCl2, which blocks neurotransmitter release, KA and QA depolarized the membrane. The minimum concentration of KA that induced a response was 1 microM and a maximum response was obtained with 10 microM (EC50: 3 microM). The operating range of QA was between 0.5 and 5 microM with an EC50 of 1 microM. These data show that inner retinal cells of the axolotl are sensitive to KA and QA. Cis-2,3-piperidine dicarboxylic acid (PDA, 3 mM) completely blocked responses to 5 microM KA, but not those induced by 2 microM QA. This suggests that the KA- and QA-sensitive receptors on inner retinal cells of the salamander are pharmacologically different and that PDA can be a valuable tool in distinguishing KA- and QA-sensitive receptors on these neurones.
Assuntos
Ambystoma/metabolismo , Receptores de Neurotransmissores/metabolismo , Retina/metabolismo , Animais , Cobalto/farmacologia , Eletrodos , Técnicas In Vitro , Ácido Caínico/farmacologia , Neurotransmissores/metabolismo , Estimulação Luminosa , Ácidos Pipecólicos/farmacologia , Ácido Quisquálico/farmacologia , Receptores de AMPA , Receptores de Ácido Caínico , Retina/citologiaRESUMO
Plasma total calcium and calcium influx, measured during 4-h 45Ca influx experiments, were lower in control axolotls, Ambystoma mexicanum, during August when compared to those in July. A single intraperitoneal injection of 10 micrograms of synthetic eel calcitonin significantly reduced the calcium influx rates during both months but the effect was greater in July (P less than 0.001) than in August (P less than 0.05). Plasma levels of calcium, sodium, potassium, and magnesium were not affected. In neotonous wholly aquatic amphibians, eel calcitonin may work in the same way as it does in fish by reducing the uptake of dissolved calcium from the ambient medium.
Assuntos
Ambystoma mexicanum/metabolismo , Ambystoma/metabolismo , Calcitonina/farmacologia , Cálcio/metabolismo , Animais , Cálcio/sangue , Feminino , Masculino , Estações do AnoRESUMO
The pancreas of the axolotl, Ambystoma mexicanum, was investigated by immunocytochemical methods for the presence of immunoreactivity to a number of antisera raised against mammalian insulins. All anti-insulin antisera tested revealed substantial amounts of reaction products confined solely to the aldehyde-fuchsinophilic B cells of the endocrine pancreas. The reactive cell population was detected by use of one polyclonal antiserum against bovine insulin and eight different monoclonal antibodies against insulins from various mammalian species. Six of these antibody clones have known specificity to sub-regions of the insulin molecule. Additionally, fractions of an ethanol-HCl extract of pancreatic tissue from Ambystoma was studied in both conventional dot-blot tests by means of the same panel of antibodies and a two-site sandwich time-resolved immunofluorometric assay for human insulin involving two of the monoclonal antibodies. These experiments support the immunocytochemical observations by demonstrating the existence of an insulin-related peptide with a great deal of structural resemblance to mammalian insulins and displaying antigenic determinants in common at least with the amino acid residues A8-10 and B26-30. In conclusion, we interpret the findings as indicating that the immunocytochemically revealed tissue bound antigen in the Ambystoma pancreatic B-cells may be a peptide related to human insulin.
Assuntos
Ambystoma/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Feminino , Imuno-Histoquímica , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Larva , MasculinoRESUMO
The beta-adrenergic ligand iodocyanopindolol (ICP) bound specifically to hepatic plasma membrane preparations from the axolotl, Ambystoma mexicanum (Bmax, 40 fmol/mg protein (P) at free concentration above 140 pM; KD, 42 pM); the toad, Xenopus laevis (Bmax, 200 fmol/mg P at 1 nM; KD, 300 pM); and the Australian lungfish, Neoceratodus forsteri (Bmax, 100 fmol/mg P at 5 nM). For the lungfish, the Scatchard plot was curved showing two classes of binding site with KD's of 20 and 500 pM. Neither the alpha 1-adrenergic ligand prazosin nor the alpha 2-adrenergic ligand yohimbine bound specifically to hepatic membrane preparations from any of the three species. Several adrenergic ligands displaced ICP from hepatic membrane preparations of all three species with KD's of Axolotl--propranolol, 50 nM; isoprenaline, 600 nM; adrenaline, 10 microM; phenylephrine, 20 microM; noradrenaline, 40 microM; and phentolamine, greater than 100 microM; X. laevis--propranolol, 30 nM; isoprenaline, 100 microM; adrenaline, 200 microM; noradrenaline, 300 microM; phenylephrine, 1 mM; and phentolamine, greater than 1 mM; N. forsteri,--propranolol, 25 nM; isoprenaline, 1 microM; adrenaline, 20 microM; phenylephrine, 35 microM; noradrenaline, 600 microM; and phentolamine, 400 microM. These findings suggest that alpha-adrenergic receptors are not present in hepatic plasma membrane preparations from these three species and that the hepatic actions of catecholamines are mediated via beta-adrenergic receptors. The order of binding of the beta-adrenergic ligands suggests that the receptors are of the beta 2 type.
Assuntos
Ambystoma mexicanum/metabolismo , Ambystoma/metabolismo , Peixes/metabolismo , Fígado/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , Xenopus laevis/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Iodocianopindolol , Ligantes/metabolismo , Pindolol/análogos & derivados , Pindolol/metabolismo , Prazosina/metabolismo , Ensaio Radioligante , Receptores Adrenérgicos alfa/fisiologiaRESUMO
Glucagon increases the rate of glycogenolysis in in vitro cultures of hepatic tissue from the axolotl Ambystoma mexicanum. The hormone causes an increase in the concentration of cyclic AMP in the tissue which is followed by activation of glycogen phosphorylase and subsequent breakdown of glycogen and release of glucose from the tissue. Insulin counteracts the glycogenolytic effect of glucagon by inhibiting the increase in tissue cyclic AMP concentration brought about by glucagon. This inhibitory effect of insulin is not seen in the presence of the phosphodiesterase inhibitor IBMX and so it appears that the initial action of insulin is a stimulation of cyclic AMP phosphodiesterase activity which lowers the tissue concentration of cyclic AMP and so counters the actions of hormones that act by raising the tissue concentration of cyclic AMP. This model for the mode of action of insulin is supported by the finding that insulin also interferes with the glycogenolytic actions of adrenaline, a second hormone which acts by raising tissue cyclic AMP concentrations.
Assuntos
Ambystoma/metabolismo , AMP Cíclico/metabolismo , Glucagon/fisiologia , Insulina/fisiologia , Glicogênio Hepático/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , AMP Cíclico/fisiologia , Ativação Enzimática/efeitos dos fármacos , Epinefrina/antagonistas & inibidores , Epinefrina/farmacologia , Glucose/metabolismo , Técnicas In Vitro , Fosforilase a/metabolismoRESUMO
The cellular binding sites of anti-oPRL IgG and anti-bSTH IgG were demonstrated in the pituitary glands of Lepidosiren paradoxa, Rana temporaria and Ambystoma mexicanum by means of the unlabeled antibody enzyme method by light and electron microscopy (the latter only in Lepidosiren). With the light microscope PRL or PRL-like substances and STH or STH-like substances were revealed in two different cell types in the distal lobe corresponding to the acidophils. However, as a result of the insufficient differentiation of the acidophils in Lepidosiren after staining with Brookes' procedure it was not possible to distinguish the two types of acidophils in this animal. Treatment with low dilutions of both anti-oPRL and anti-bSTH IgG revealed simultaneous immunocytochemical staining in both types of acidophils in Lepidosiren and in Rana. These results, indicating that there is antigenic cross-reaction between anti-oPRL and anti-bSTH IgG and both PRL and STH in these animals, are discussed. The electron microscopic investigations of Lepidosiren revealed that the specific anti-oPRL IgG reactive cells contain granules ranging in size from 200 to 300 nm, while the specific anti-bSTH IgG reactive cells contain smaller immunoreactive granules ranging from 80 to 160 nm.