RESUMO
We have studied the distribution of the CD15 epitope in the developing brain of an Australian diprotodontid metatherian mammal, the tammar wallaby ( Macropus eugenii), using immunohistochemistry in conjunction with hematoxylin and eosin staining. At the time of birth (28 days after conception), CD15 immunoreactivity labeled somata in the primordial plexiform layer of the parietal cortex in a similar position to that seen in the early fetal eutherian brain. CD15 immunoreactivity in the brain of the developing pouch-young wallaby was found to be localized on the surface of radial glia at boundaries between developmentally significant forebrain compartments in a similar distribution to that seen in developing eutherian brain. These were best seen in the developing diencephalon, delineating epithalamus, ventral and dorsal thalamus and hypothalamic anlage, and in the striatum. Immunoreactivity for CD15 identified radial glia marking the lateral migratory stream at the striatopallial boundary, peaking in intensity at P19 to P25. From P37 to P54, CD15 immunoreactivity also demarcated patch compartments in the developing striatum. In contrast, CD15 immunoreactivity in hindbrain structures showed some differences from the temporospatial pattern seen in eutherian brain. These may reflect the relatively early brainstem maturation required for the newborn wallaby to be able to traverse the distance from the maternal genital tract to the pouch. The wallaby provides a convenient model for testing hypotheses concerning the role of CD15 in forebrain development because all events in which CD15 may play a critical role in forebrain morphogenesis occur during pouch life, when the young wallaby is accessible to experimental manipulation.
Assuntos
Encéfalo/crescimento & desenvolvimento , Antígenos CD15/metabolismo , Macropodidae/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Imuno-Histoquímica , Macropodidae/metabolismo , Modelos Animais , Modelos Biológicos , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismoRESUMO
In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca(2+) -dependent and Ca(2+) -independent NOS isoforms.
Assuntos
Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/enzimologia , Isoenzimas/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Feminino , Idade Gestacional , Isoenzimas/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , GravidezRESUMO
For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.
Assuntos
Células Epiteliais/transplante , Células Epiteliais/ultraestrutura , Iris/citologia , Epitélio Pigmentado Ocular/transplante , Epitélio Pigmentado Ocular/ultraestrutura , Animais , Diferenciação Celular , Movimento Celular , Separação Celular , Células Epiteliais/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Sobrevivência de Enxerto/fisiologia , Humanos , Ferro/metabolismo , Neostriado/citologia , Neostriado/cirurgia , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/terapia , Epitélio Pigmentado Ocular/metabolismo , Ratos , Ratos Long-Evans , Técnicas EstereotáxicasRESUMO
Conventional therapeutical approaches such as surgery, radiotherapy, or chemotherapy have been shown to be rather unsuccessful in the treatment of infiltrative growing tumors such as the malignant glioblastoma multiforme. Thus, new therapeutical strategies have to be developed that are suitable for inducing cell death also in migrating tumor cells. These new therapeutical stategies include cell and/or gene therapeutical approaches. We demonstrate that glial-restricted progenitor cells as well as embryonic stem cell-derived neural stem cells belong to cell populations applicable to such therapeutical concepts. Both cell types can be efficiently transduced using a third-generation high-capacity "gutless" adenoviral vector, and show a tropism for the F98 glioma cells by migrating towards a spheroid of F98 glioma cells with a tendency to form a barrier around the tumor spheroid in an in vitro tumor confrontation model. Moreover, in a migration assay, secretion products of glial-restricted precursor cells have shown a potency to inhibit the migratory activity of glioma cells in vitro. In vivo, F98 glioma cell-derived tumor formation in the right striatum resulted in migration of glial as well as neural precursor cells towards the tumor area when cotransplanted in the corpus callosum of the contralateral hemisphere. After arrival, both cell types surround the tumor mass and even invade the experimentally induced tumor. These data indicate that glial-restricted as well as embryonic stem cell-derived neural precursor cells are good candidates as carriers for an ex vivo gene therapeutical approach in tumor therapy.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , Neoplasias/terapia , Neuroglia/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Transplante de Células , Células Cultivadas , Quimiotaxia/fisiologia , Embrião de Mamíferos/fisiologia , Masculino , Transplante de Neoplasias , Neoplasias/metabolismo , Neoplasias/patologia , Neuroglia/citologia , Neurônios/citologia , Ratos , Ratos Endogâmicos F344 , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células-Tronco/citologiaRESUMO
Nitric oxide (NO), a cell-derived highly diffusible and unstable gas is regarded to be involved in inter- and intracellular communication in the nervous system. Based on findings about the expression of the inducible NO synthase (NOS) isoform during development of early mouse olfactory as well as vestibulocochlear receptor neurons, we intended to prove a general role of this isoform for neuronal differentiation. Using immunohistochemical techniques, an exclusive expression of the inducible NOS-II isoform in early post-mitotic neurons of the developing mouse cortex and retina can be detected. In a pharmacological approach using cultures of the mouse cortex as well as embryonic stem cell-derived neural precursor cells, we investigated the functional role of NO on initial neuronal differentiation. Effects of NOS inhibitors and NO donors on the morphological differentiation were correlated with developmentally regulated calcium current densities, focusing on the effects of the specific NOS-II inhibitor GW 274150. Furthermore, involvement of the soluble guanylate cyclase (sGC)/cGMP signaling cascade was pharmacologically investigated. Our data indicate that while a specific block of NOS-II provokes a clear inhibition of neurite outgrowth formation as well as a decrease of calcium current densities, the inverse is true for exogenous NO donation. In line with lacking immunoreactivity for the sGC and cGMP there are only minor effects of compounds manipulating the sGC/cGMP pathway, suggesting the downstream sGC/cGMP pathway not to be essential in these early differentiation steps.
Assuntos
Córtex Cerebral/enzimologia , Córtex Cerebral/crescimento & desenvolvimento , Neurônios/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Retina/enzimologia , Retina/crescimento & desenvolvimento , Animais , Cálcio/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , GMP Cíclico/metabolismo , Eletrofisiologia , Embrião de Mamíferos , Imuno-Histoquímica , Camundongos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfetos/farmacologiaRESUMO
To investigate the ability of genetically modified astrocytes to integrate into adult rat brain, two spontaneously immortalized cell lines and the allogenic nontumorigenic glioma cell line F98 were transduced with a high-capacity adenoviral vector (HC-Adv) expressing the EGFP gene from the hCMV promoter. In organotypic slice cultures the transduced astrocytes were shown to integrate into the brain tissue. Following transplantation of the transduced astrocytes into the striatum of adult rats, the transplanted cells survived at least for 6 weeks, continuously expressed the EGFP transgene, in close neighborhood with cells of the recipient tissue executing their differentiation capacity along the glial lineage. Thus, HC-Adv transduced astrocytes are promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases.
Assuntos
Adenoviridae/genética , Astrócitos/transplante , Linhagem Celular Transformada/transplante , Transplante de Células/métodos , Vetores Genéticos/uso terapêutico , Sobrevivência de Enxerto/genética , Células Tumorais Cultivadas/transplante , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Diferenciação Celular/genética , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/metabolismo , Linhagem da Célula/genética , Tamanho Celular/genética , Sobrevivência Celular/genética , Transformação Celular Viral/genética , Transplante de Células/tendências , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/transplante , Feto , Regulação Viral da Expressão Gênica/genética , Vetores Genéticos/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Regiões Promotoras Genéticas/genética , Ratos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismoRESUMO
Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.
Assuntos
Embrião de Mamíferos/citologia , Transplante de Células-Tronco Hematopoéticas , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas do Tecido Nervoso , Animais , Diferenciação Celular , Linhagem Celular , Separação Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Nestina , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Wistar , Fatores de Tempo , TransgenesRESUMO
The distribution of the CD15 antigen (CD15, 3-fucosyl-N-acetyl-lactosamine, Lewis x) has been studied immunohistochemically in the fetal human thalamus. Its changing patterns could be related to three successive, but overlapping, periods primarily due to its association with radial glial cells, neuropil, and neural cell bodies, respectively. From 9 weeks of gestation (wg), a subset of CD15-positive radial glial cells distinguished the neuroepithelium of the ventral thalamus, a characteristic also seen in the developing mouse. Distal processes of the radial glial cells converged at the root of the forebrain choroid tenia, which was also CD15 positive. From 13 wg until approximately 20 wg, CD15-positive neuropil labeling marked the differentiation areas of prospective nuclei within the dorsal thalamus and progressively outlined their territories in a time sequence, which appeared specific for each nucleus. CD15 labeling of differentiating nuclei of the ventral, medial, anterior, and intralaminar thalamic divisions showed a transient topographic relationship with restricted areas of the ventricular wall. After 26 wg, CD15 immunoreactivity was observed in subpopulations of glial cells and neurons. Transient CD15 immunoreactivity was also found in delimited compartments within the subventricular region. The time of CD15 expression, its location, and cellular association suggest that CD15 is involved in segmentation of diencephalon, in the specification of differentiating nuclear areas and initial processes regarding the formation of intercellular contacts and cellular maturation.
Assuntos
Antígenos CD15/análise , Proteínas do Tecido Nervoso/análise , Tálamo/anatomia & histologia , Biomarcadores , Calbindina 2 , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Humanos , Recém-Nascido , Antígenos CD15/biossíntese , Antígenos CD15/genética , Morfogênese , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neuroglia/química , Neurônios/química , Neurópilo/química , Proteína G de Ligação ao Cálcio S100/análise , Núcleos Talâmicos/anatomia & histologia , Núcleos Talâmicos/embriologia , Núcleos Talâmicos/crescimento & desenvolvimento , Tálamo/embriologia , Tálamo/crescimento & desenvolvimentoRESUMO
In the present study the localization of heme oxygenase-2 (HO-2) in developing and mature olfactory epithelium of the bovine is investigated using immunohistochemistry and post embedding immunogold labelling. HO-2 immunoreactivity is first seen in epithelial cells localized along the luminal surface of the olfactory pit. Up to midgestation the number of HO-2 immunoreactive cells increases throughout all layers of the developing olfactory epithelium. From midgestation through adulthood immunostaining is restricted to perinuclear cytoplasm and axons of mature olfactory receptor neurons localized in intermediate epithelial regions. The temporal and spatial expression patterns of HO-2 immunohistochemistry support the notion that CO plays a role in neuronal differentiation while its presence in mature neurons might be functionally related to olfactory transduction.
Assuntos
Heme Oxigenase (Desciclizante)/análise , Mucosa Olfatória/embriologia , Mucosa Olfatória/enzimologia , Fatores Etários , Animais , Anticorpos , Antígenos CD57/análise , Antígenos CD57/imunologia , Bovinos , Feminino , Feto/enzimologia , Heme Oxigenase (Desciclizante)/imunologia , Masculino , Microscopia Imunoeletrônica , Mucosa Olfatória/ultraestrutura , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/imunologiaRESUMO
According to the view of differentiation-related alterations in the glycosylation pattern of neurones, recent studies have shown development dependent expression patterns of lactoseries carbohydrate epitopes, CD15 and HNK-1, on olfactory receptor neurones in rats and chicks. In order to evaluate a general role for these epitopes during development of vertebrate olfactory receptor neurones, this investigation focuses on the situation in the mouse, bovine and Xenopus olfactory epithelium. In all three species CD15 expression was found on a subpopulation of morphologically mature receptor cells starting at the time of initial synaptogenesis. Whereas for bovine and Xenopus the timetable of HNK-1 expression is similar to that described for the chick, suggesting involvement in pathfinding, in the mouse HNK-1 is found on immature cells when mature CD15 positive receptor cells could already be discerned. By our results a role for CD15 during establishment of synaptic contacts and for HNK-1 during their formation is suggested.
Assuntos
Antígenos CD57/análise , Epitopos/análise , Antígenos CD15/análise , Neurônios Receptores Olfatórios/química , Neurônios Receptores Olfatórios/crescimento & desenvolvimento , Animais , Antígenos CD57/genética , Bovinos , Epitopos/genética , Glicosilação , Imuno-Histoquímica , Antígenos CD15/genética , Metamorfose Biológica , Camundongos , Neurônios Receptores Olfatórios/embriologia , Xenopus laevisRESUMO
The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.
Assuntos
Blastocisto/citologia , Proteínas do Tecido Nervoso , Células Fotorreceptoras de Vertebrados/citologia , Retina , Degeneração Retiniana/prevenção & controle , Transplante de Células-Tronco , Animais , Núcleo Celular/ultraestrutura , Células Cultivadas , Células Clonais , Proteínas de Fluorescência Verde , Indicadores e Reagentes/análise , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Luminescentes/análise , Camundongos , Nestina , Células Fotorreceptoras de Vertebrados/ultraestrutura , Epitélio Pigmentado Ocular/patologia , Ratos , Ratos Endogâmicos , Retina/patologia , Retina/ultraestrutura , Degeneração Retiniana/patologiaRESUMO
Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.
Assuntos
Axônios/metabolismo , Proteínas Fúngicas/genética , Junção Neuromuscular/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae , Traumatismos do Sistema Nervoso , Degeneração Walleriana/genética , Degeneração Walleriana/metabolismo , Potenciais de Ação/genética , Animais , Axônios/ultraestrutura , Sequência de Bases/fisiologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Sobrevivência Celular/genética , Proteínas Fúngicas/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Mutação/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Sistema Nervoso/patologia , Junção Neuromuscular/ultraestrutura , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Nervo Isquiático/ultraestrutura , Transmissão Sináptica/genética , Vesículas Sinápticas/metabolismo , Enzimas de Conjugação de Ubiquitina , Degeneração Walleriana/fisiopatologiaRESUMO
OBJECT: The aim of this investigation was to assess new information concerning the capacity of transplanted embryonic stem cell (ESC)-derived neuronal cells to migrate into host brain and to evaluate these cells as a possible source for cell replacement therapy in neurodegenerative disorders such as Parkinson's disease (PD). METHODS: The authors investigated the ability of ESC-derived neural precursor cells to migrate and differentiate in a host striatum by using a D3-derived ESC clone that was transfected stably with a chicken beta-actin cytomegalovirus enhancer-driven green fluorescent protein (GFP)-labeled construct. This procedure allowed easy monitoring of all transplanted cells because of the green fluorescent labeling of donor cells. This approach also afforded easy estimation of cell integration and simultaneous observation of the entire transplanted cell population in relation to immunocytochemically identified neuronal and glial differentiation. After selection of nestin-positive neural precursor cells in a synthetic medium, they were implanted into the striatum of male adult Wistar rats. Their integration was analyzed on morphological studies performed 3 days to 4 weeks posttransplantation. CONCLUSIONS: The investigators found that after transplantation, a subpopulation of GFP-labeled cells differentiated into various neural morphological types that were positive for the mouse-specific Thy-1 antigen, which is known be expressed on neurons, as well as being positive for the astroglial marker glial fibrillary acidic protein. Moreover, GFP-expressing cells that were negative for either of these markers remained close to the injection site, presumably representing other derivatives of the neural lineage. Together, these findings contribute to basic research regarding future transplantation strategies in neurodegenerative diseases such as PD.
Assuntos
Diferenciação Celular/fisiologia , Corpo Estriado/cirurgia , Transplante de Tecido Fetal , Neuroglia/transplante , Neurônios/transplante , Doença de Parkinson/cirurgia , Transplante de Células-Tronco , Antígenos Thy-1/análise , Animais , Movimento Celular/fisiologia , Galinhas , Corpo Estriado/patologia , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes , Masculino , Neuroglia/patologia , Neurônios/patologia , Doença de Parkinson/patologia , Ratos , Ratos Wistar , Células-Tronco/patologiaRESUMO
It has recently been suggested that, in addition to nitric oxide (NO), carbon monoxide (CO) is an important gaseous messenger which might be involved in vertebrate olfactory transduction because its effects include activation of guanylyl cyclase and the formation of cGMP. As there is no information regarding the presence of heme oxygenase-2 -- the constitutive isoform of the heme oxygenase system -- in olfactory neurons of non-rodent species, we have investigated the distribution pattern of heme oxygenase-2 in the olfactory epithelium of the bovine, a representative of macrosmatics. Localization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity of the olfactory epithelium was compared with heme oxygenase-2 and NO synthase (NOS) immunoreactivities in order to obtain possible hints at functional significance. NADPH-d activity was particularly intense in apical dendrites of receptor neurons. It was also found in Bowman glands and intraepithelial duct cells. Less intense, discrete NADPH-d activity was present also at intermediate and basal levels of the olfactory epithelium, corresponding to the layer of receptor neuron somata and basal cells. While heme oxygenase-2 activity mainly occurred in neuronal perikarya, a very intense NOS immunoreactivity, exclusively for the inducible isoform, was detected in the apical dendrites. Ultrastructurally, NADPH-d histochemistry showed distinct labelling of membranes, in particular of endoplasmic reticulum, mitochondria and nucleus. The coincident localization of the moderate NADPH-d activity and heme oxygenase-2 immunoreactivity in receptor cell perikarya suggest a functional association between NADPH-cytochrome P450 reductase and heme oxygenase-2. In contrast, dendritic localization of NADPH-d activity is topically and possibly functionally related to the presence of the inducible isoform of NOS. The results suggest that both CO and NO may be generated in bovine receptor neurons and thus involved in odorant stimulation. Based on immunocytochemical localization of synthesizing enzymes, NO might be regarded as a direct regulator of transduction related processes while CO might act as a modulator of the initial signal.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase/metabolismo , Neurônios Receptores Olfatórios/enzimologia , Animais , Bovinos , Imuno-Histoquímica/métodos , Septo Nasal/enzimologia , Septo Nasal/patologia , Septo Nasal/ultraestrutura , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo IIIRESUMO
There are remarkable changes of calcium binding proteins and voltage dependent Ca(2+) channel subtypes during in vitro differentiation of embryonic stem cell derived neurons. To observe these maturation dependent changes neurones were studied using combined immunohistochemical, patch clamp and videomicroscopic time lapse techniques. Embryonic stem cell derived neuronal maturation proceeds from apolar to bi- and multipolar neurones, expressing all Ca(2+) channel subtypes. There is, however, a clear shift in channel contribution to whole cell current from apolar neurones with mainly N- and L-type channel contribution in favour of P/Q- and R-type participation in bi- and multipolar cells. Expression of the calcium binding protein parvalbumin could be detected in bipolar, while calretinin and calbindin was preferentially found in multipolar neurones. Our data provides new insights into fundamental neurodevelopmental mechanisms related to Ca(2+) homeostasis, and clarifies contradictory reports on the development of Ca(2+) channel expression using primary cultures of neurones already committed to certain brain compartments.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Neurônios/química , Células-Tronco/química , Animais , Cloreto de Cádmio/farmacologia , Calbindina 2 , Calbindinas , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/biossíntese , Canais de Cálcio Tipo L/biossíntese , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo N/biossíntese , Canais de Cálcio Tipo N/fisiologia , Canais de Cálcio Tipo R/biossíntese , Canais de Cálcio Tipo R/fisiologia , Proteínas de Ligação ao Cálcio/análise , Diferenciação Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Glicina/farmacologia , Isradipino/farmacologia , Ácido Caínico/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microscopia de Vídeo , Neurônios/citologia , Neurônios/metabolismo , Parvalbuminas/análise , Parvalbuminas/metabolismo , Proteína G de Ligação ao Cálcio S100/análise , Proteína G de Ligação ao Cálcio S100/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Ácido gama-Aminobutírico/farmacologia , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologiaRESUMO
Based on the idea of differentiation-related changes in the glycosylation pattern of neurons, the expression of two cell surface oligosaccharide epitopes, N-acetyl-lactosamine (NALA), and its sulpho-glucuronyl derivative (HNK-1), was studied, by immunohistochemistry and Western blot experiments, in the developing chick retina beginning on day 2 of incubation (E2) until day 18 post-hatching. NALA was detectable on neuroepithelial cells as soon as the primary optic vesicles formed, and this pattern continued until E3. During subsequent retinal development NALA expression became progressively restricted in concert with the appearance of postmitotic neurons as revealed by neurite outgrowth, and with the formation of synaptic contacts until it disappeared at the end of the incubation period. The pattern of NALA expression was the inverse of HNK-1 which was detected for the first time at E3 on postmitotic ganglion cells accumulating at the vitreal surface. The number of HNK-1+ cells steadily increased until around E10, when the entire neural epithelium was labelled. Synchronously to synaptogenesis, most neurons lost their HNK-1 immunoreactivity. At the time of hatching the adult-like pattern was found, characterised by subpopulations of labelled horizontal, bipolar, amacrine, and ganglion cells. Immunoblot experiments demonstrated transient NALA glycosylation of protein bands, partially identical in their apparent molecular weight to those proteins with HNK-1 glycosylation. The observed temporospatial changes in the glycosylation patterns of distinct proteins during retinal development suggest NALA as a suitable marker for neuronal proliferation, and HNK-1 for differentiation and establishment of final synaptic configuration.
Assuntos
Amino Açúcares/análise , Glicoproteínas de Membrana/análise , Retina/química , Retina/crescimento & desenvolvimento , Fatores Etários , Amino Açúcares/imunologia , Animais , Western Blotting , Diferenciação Celular , Galinhas , Epitopos/análise , Regulação da Expressão Gênica , Glicosilação , Imunoglobulina M/imunologia , Imuno-Histoquímica , Antígenos CD15/análise , Proteínas de Membrana/análise , Neurônios/química , Epitélio Pigmentado Ocular/química , Retina/citologia , Fatores de TempoRESUMO
This study examined the occurrence of endothelial nitric oxide (NO)-synthase (NOS-III) in terminal mesenteric vessels and the involvement of NO in microvascular permeability. Possible effects were studied in bradykinin (BK)-induced and basal conditions. NOS expression was investigated by using NOS-III immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry on the light- and electron-microscopic levels. Permeability was examined in dissected mesenteries of male rats weighing 250-300 g. Tissue treatment was performed with BK (100 nM), sodium nitroprusside (SNP, 1 and 10 microM), L-nitroarginine (L-NA, 300 microM), BK and L-NA, BK and SNP, L-NA and SNP, as well as with BK, SNP (10 microM), and the guanylylcyclase inhibitor ODQ (10 microM), and BK and ODQ alone. Pharmacologically induced permeability changes were studied with fluorescein isothiocyanate (FITC)-dextran 70 kDa as a tracer for macromolecular transport. Video images were analyzed with computer determination of integrated optical density (IOI). Results were statistically verified by analysis of variance and t test. Microvascular permeability was increased by 168% after BK treatment and was enhanced by NO-synthesis inhibition with L-NA by 607%. However, the NO donor SNP led to a reduced tracer extravasation to 105 and 58%, respectively, an effect blocked by ODQ. Under basal conditions without prior BK induction, L-NA also causes an increase of IOI by 25%, whereas coapplication with SNP resulted in only a 10% increase of permeability. These results point out that NO has a modulatory role for microvascular permeability by supporting the barrier function of the endothelial lining in stimulated and nonstimulated conditions.
Assuntos
Bradicinina/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/farmacologia , Animais , Interações Medicamentosas , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Mesentério/efeitos dos fármacos , Mesentério/patologia , Microscopia Eletrônica , NADPH Desidrogenase/análise , Óxido Nítrico/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.
Assuntos
Cóclea/embriologia , Cóclea/crescimento & desenvolvimento , Óxido Nítrico Sintase/metabolismo , Células Receptoras Sensoriais/citologia , Vestíbulo do Labirinto/embriologia , Vestíbulo do Labirinto/crescimento & desenvolvimento , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Divisão Celular/fisiologia , Cóclea/inervação , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Imuno-Histoquímica , Camundongos/embriologia , Óxido Nítrico Sintase Tipo II , Vestíbulo do Labirinto/inervaçãoRESUMO
In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the beta-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltage-dependent L-type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 +/-3% (n=34) in early stage cardiomyocytes lacking functional beta-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized beta-adrenoceptor-stimulated ICa by 43 +/-4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.
Assuntos
Inibidores de Adenilil Ciclases , Canais de Cálcio/fisiologia , Inibidores Enzimáticos/farmacologia , Coração/embriologia , Coração/fisiologia , Ativação do Canal Iônico , Óxido Nítrico/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Eletrofisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Células-Tronco/fisiologia , ômega-N-Metilarginina/farmacologiaRESUMO
OBJECTIVE: The aim of the present study was the investigation of the expression of NOS during cardiomyogenesis and its functional role. DESIGN: The qualitative and quantitative expression of NOS isoforms during different stages of cardiac development was evaluated using immunocytochemistry and dot blots, respectively. The functional relevance of NOS expression during cardiomyogenesis was investigated using the in vitro ES cell-differentiation model and selective pharmacological agents. RESULTS: On day 7.5 of embryonic development (E7.5) none of the NOS isoforms were expressed in the embryo, whereas the inducible (iNOS), as well as the endothelial (eNOS) isoforms were detected in the extraembryonic parts. In contrast, starting from E9.5 rat and murine embryos displayed prominent iNOS and eNOS expression. This was correlated with high expression of soluble guanylylcyclase (sGC) as well as high cyclic GMP (cGMP) content. During further development after E14.5 both, iNOS as well as eNOS, started to be downregulated and shortly prior to birth reduced staining for eNOS was found, whereas iNOS was hardly detectable. We further investigated whether NO plays a role for cardiomyogenesis, using in vitro ES cell-derived cardiomyocytes differentiating within embryoid bodies (EBs). The NOS expression pattern in these cells paralleled the one detected in vivo. We demonstrate that continuous incubation of EBs with the NOS inhibitors L-NMMA (2-10 mM) or L-NA (2-10 mM) for 4 to 9 days after plating resulted in a pronounced differentiation arrest of cardiomyocytes, whereas this effect could be reversed by coapplication of the NO-donor spermine-NONOate (10 microM). CONCLUSIONS: Both, iNOS and eNOS isoforms are prominently expressed during early stages of cardiomyogenesis. Around E14.5 NOS expression starts to decline. Moreover, the NO-generation is required for cardiomyogenesis since NOS inhibitors prevent the maturation of terminally differentiated cardiomyocytes using the ES cell system.