RESUMEN
In spite of keen clinical and neuroscientific interest, the aetiology and immunopathology of multiple sclerosis (MS) remain to be elucidated. The present work seeks to give insight into the important, but thus far underestimated contribution of B cells to the disease. Emphasis will be placed on the role of B cells as producers of autoantibodies and as antigen presenting cells. In addition, the development of ectopic B cell follicles in the CNS and their potential correlation with the course of the disease and MS severity will be discussed. Finally, regulatory functions of a B cell-dependent immunopathology should be mentioned. A better understanding of the complex pathomechanisms of MS will allow for therapeutic options that are causative. Potential targets of a B cell-oriented therapy will be delineated in the following review. We hereby aim at triggering a critical re-evaluation of traditional paradigms assigned to MS, appreciating the importance of B cells in the disease.
Asunto(s)
Linfocitos B/inmunología , Esclerosis Múltiple/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos B/patología , Agregación Celular , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Humanos , Sistema Inmunológico/patología , Inmunoterapia , Esclerosis Múltiple/patología , Esclerosis Múltiple/terapiaRESUMEN
In spite of tremendous scientific effort, the mechanisms underlying multiple sclerosis (MS) still remain to be elucidated. The prevalent pathogenetic concept adheres to the assumption of a strict hierarchical sequence of the triad inflammation, demyelination and axonal damage. However, recent studies have provided evidence that axonal pathology can occur independently of inflammation and demyelination. The present article critically re-evaluates the traditional paradigm of MS pathology. Potential cellular, humoral and metabolic mechanisms of axonal pathology are delineated and the development of isolated axonal damage is assessed. A better understanding of the pathological processes underlying MS is likely to result in an improvement of current therapeutic strategies. These should not only target the inflammatory, but also the neurodegenerative component of the disease.
Asunto(s)
Axones/patología , Esclerosis Múltiple/patología , Degeneración Nerviosa/patología , Transporte Axonal/fisiología , Axones/ultraestructura , Enfermedades Desmielinizantes/patología , Humanos , Inflamación/patología , Mitocondrias/metabolismo , Esclerosis Múltiple/complicaciones , Degeneración Nerviosa/etiología , Fármacos Neuroprotectores/uso terapéutico , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
The pleiotrophic but overlapping functions of the cytokine family that includes interleukin (IL)-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1 are mediated by the cytokine receptor subunit gp130 as the common signal transducer. Although mice lacking individual members of this family display only mild phenotypes, animals lacking gp130 are not viable. To assess the collective role of this cytokine family, we inducibly inactivated gp130 via Cre-loxP-mediated recombination in vivo. Such conditional mutant mice exhibited neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects, demonstrating the widespread importance of gp130-dependent cytokines.
Asunto(s)
Antígenos CD/metabolismo , Citocinas/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Proteínas Virales , Animales , Anticuerpos/sangre , Antígenos CD/genética , Linfocitos T CD8-positivos/inmunología , Receptor gp130 de Citocinas , Marcación de Gen/métodos , Sistema Hematopoyético/patología , Histocitoquímica , Integrasas/genética , Lipopolisacáridos/farmacología , Listeria monocytogenes/patogenicidad , Hígado/patología , Pulmón/patología , Glicoproteínas de Membrana/genética , Ratones , Microscopía Electrónica , Nervios Periféricos/patología , Nervios Periféricos/ultraestructura , Recombinación Genética/genética , Virus Vaccinia/patogenicidad , Virus de la Estomatitis Vesicular Indiana/patogenicidadRESUMEN
Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of beta 1 integrin during teratoma formation, we compared teratomas induced by normal and beta1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, beta 1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of beta 1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in beta1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface. Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell-derived endothelial cells. In contrast, beta 1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although beta 1- deficient endothelial cells were absent in teratomas, beta 1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in beta1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in beta 1-null embryoid bodies.
Asunto(s)
Integrina beta1/fisiología , Neovascularización Patológica , Teratoma/irrigación sanguínea , Teratoma/patología , Animales , Antígenos CD/biosíntesis , Apoptosis/genética , Diferenciación Celular/genética , División Celular/genética , Línea Celular , Embrión de Mamíferos , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Endotelio Vascular/fisiología , Integrina alfaV , Integrina beta1/biosíntesis , Integrina beta1/genética , Linfocinas/fisiología , Masculino , Ratones , Neovascularización Patológica/genética , Células Madre/patología , Teratoma/genética , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.
Asunto(s)
Cartílago/fisiología , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/fisiología , Proteoglicanos/fisiología , Animales , Membrana Basal/anomalías , Membrana Basal/crecimiento & desarrollo , Membrana Basal/fisiología , Calcificación Fisiológica/genética , Cartílago/anomalías , Cartílago/crecimiento & desarrollo , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Exostosis Múltiple Hereditaria/genética , Marcación de Gen , Genes Letales , Cardiopatías Congénitas/genética , Heparitina Sulfato/deficiencia , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Homocigoto , Ratones , Ratones Congénicos , Ratones Mutantes , Mutagénesis Insercional , Defectos del Tubo Neural/genética , Osificación Heterotópica/genética , Proteoglicanos/deficiencia , Proteoglicanos/genética , Proteoglicanos/metabolismo , Mapeo RestrictivoRESUMEN
beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.
Asunto(s)
Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Integrina beta1/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Factor Natriurético Atrial/farmacología , Canales de Calcio Tipo L/metabolismo , Compartimento Celular , Células Cultivadas , Citocalasina D/farmacología , Adhesiones Focales , Integrina beta1/genética , Isoproterenol/farmacología , Ratones , Antagonistas Muscarínicos/farmacología , Miocardio/citología , Óxido Nítrico/farmacología , Canales de Potasio/metabolismo , Transducción de SeñalRESUMEN
In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.
Asunto(s)
Actinas/biosíntesis , Canales de Calcio/biosíntesis , Señalización del Calcio , Corazón Fetal/citología , Proteínas Fetales/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas Luminiscentes/biosíntesis , Proteínas Musculares/biosíntesis , Canal Liberador de Calcio Receptor de Rianodina/biosíntesis , Células Madre/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Actinas/genética , Animales , Cafeína/farmacología , Calcio/fisiología , Canales de Calcio/genética , Canales de Calcio/fisiología , Canales de Calcio Tipo L , ATPasas Transportadoras de Calcio/fisiología , Diferenciación Celular , Colforsina/farmacología , Proteínas Fetales/genética , Genes Reporteros , Proteínas Fluorescentes Verdes , Receptores de Inositol 1,4,5-Trifosfato , Proteínas Luminiscentes/genética , Ratones , Proteínas Musculares/genética , Contracción Miocárdica , Especificidad de Órganos , Técnicas de Placa-Clamp , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
Efficient transformation of primary human amniocytes by E1 gene functions of human adenovirus serotype 5 (Ad5) yield in stable cell lines, which exhibit morphological features of epithelial like cells. A thorough investigation using immunocytochemistry confirmed the expression of epithelial cell markers. The analysis also revealed the expression of neuronal and glial marker proteins, such as nestin, vimentin, A2B5 and GFAP. Using RT-PCR, transcripts of the neurotrophic factors nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), and neurotrophin 3 (NT-3) could be detected. Neurotrophic factors could also be detected in the cell culture supernatants of transformed amniocytes. In line with previous experimental data on a human Ad5 E1-transformed embryonal kidney cell line (HEK-293), the results suggest a co-expression of epithelial and neuronal marker proteins in E1-transformed human amniotic fluid derived cells and thus a preferential transformation into neuronal-like cells.
Asunto(s)
Adenoviridae/genética , Amnios/citología , Células Epiteliales/citología , Neuronas/citología , Transformación Genética , Proteínas E1 de Adenovirus/genética , Amnios/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Diferenciación Celular/genética , Línea Celular Transformada , Células Cultivadas , Células Epiteliales/metabolismo , Vectores Genéticos/genética , Células HeLa , Humanos , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
Axones/metabolismo , Proteínas Fúngicas/genética , Unión Neuromuscular/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/genética , Proteínas Recombinantes de Fusión/genética , Proteínas de Saccharomyces cerevisiae , Traumatismos del Sistema Nervioso , Degeneración Walleriana/genética , Degeneración Walleriana/metabolismo , Potenciales de Acción/genética , Animales , Axones/ultraestructura , Secuencia de Bases/fisiología , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Supervivencia Celular/genética , Proteínas Fúngicas/metabolismo , Inmunohistoquímica , Ratones , Ratones Mutantes , Ratones Transgénicos , Microscopía Electrónica , Datos de Secuencia Molecular , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Mutación/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/metabolismo , Sistema Nervioso/patología , Unión Neuromuscular/ultraestructura , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Nervio Ciático/ultraestructura , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismo , Enzimas Ubiquitina-Conjugadoras , Degeneración Walleriana/fisiopatologíaRESUMEN
The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Capilares/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Animales , Aorta/embriología , Aorta/patología , Northern Blotting , Southern Blotting , Embrión de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Vectores Genéticos , Homocigoto , Inmunohistoquímica , Riñón/embriología , Riñón/metabolismo , Riñón/patología , Pulmón/embriología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Transgénicos , Microscopía Electrónica , Microscopía Fluorescente , Modelos Genéticos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10(-7) M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10(-6) M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.
Asunto(s)
Bradiquinina/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Odontoblastos/metabolismo , Animales , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Antagonistas de los Receptores de Bradiquinina , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Microscopía Confocal , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Serina/metabolismo , Treonina/metabolismo , Regulación hacia ArribaRESUMEN
By the formation of cyclic guanosine 3',5'-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC alpha(2)-subunit/beta(1)-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC alpha(2), and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10(-5) M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10(-4) M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.
Asunto(s)
GMP Cíclico/metabolismo , Pulpa Dental/enzimología , Dentina/enzimología , Óxido Nítrico/metabolismo , Odontoblastos/enzimología , Animales , Pulpa Dental/ultraestructura , Dentina/ultraestructura , Guanilato Ciclasa/metabolismo , Inmunohistoquímica , Masculino , Diente Molar/citología , Diente Molar/enzimología , Diente Molar/ultraestructura , Óxido Nítrico Sintasa/metabolismo , Odontoblastos/ultraestructura , Ratas , Ratas Wistar , Transducción de Señal/fisiología , Distribución TisularRESUMEN
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.
Asunto(s)
Corazón/embriología , Óxido Nítrico Sintasa/fisiología , Transducción de Señal/fisiología , Células Madre/enzimología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Edad Gestacional , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Nitroarginina/farmacología , Óxidos de Nitrógeno , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Espermina/análogos & derivados , Espermina/farmacología , Células Madre/efectos de los fármacos , omega-N-Metilarginina/farmacologíaRESUMEN
We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac alpha-actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac alpha-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.
Asunto(s)
Corazón/embriología , Proteínas Luminiscentes/genética , Miocardio/metabolismo , Actinas/genética , Animales , Femenino , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , Corazón/fisiología , Humanos , Proteínas Luminiscentes/biosíntesis , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Regiones Promotoras GenéticasRESUMEN
Immunogold labeling on samples of isolated perfused rat hearts embedded by an innovative low-temperature LR White procedure provided detailed insight into the interaction of caveolin-1 and endothelial NOS in myocardial capillary endothelium at the subcellular level. Separately, the localization of caveolin-1 and eNOS at caveolae under steady state conditions was visualized. A double-labeling experiment supported their close co-localization. Short-term bradykinin stimulation caused a detectable dissociation of eNOS from caveolin and its redistribution to different cell compartments, whereas caveolin itself remained stationary at caveolae. Morphometric analysis revealed that more than 80% of detectable eNOS was co-localized with caveolin-1 at caveolae under control conditions. After brief stimulation for 2 min with 10(-7) M bradykinin, only 26% of the eNOS signals were associated with caveolin-1 and randomly distributed over the endothelial cells. After stimulation, eNOS was found at the plasmalemmal and intracellular membranes, freely in the cytoplasm, and at outer mitochondrial membranes.
Asunto(s)
Caveolinas/metabolismo , Vasos Coronarios/metabolismo , Endotelio Vascular/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Capilares/metabolismo , Caveolina 1 , Masculino , Microscopía Inmunoelectrónica , Óxido Nítrico Sintasa de Tipo III , Ratas , Ratas WistarRESUMEN
PURPOSE: Because earlier studies indicate that the choroid close to the ora serrata may have unique anatomic features such as wandering cells, blood vessels in Bruch's membrane, and accumulated pigment in the retinal pigment epithelium (RPE), the morphology of the normal human eye at the ora serrata region was investigated. METHODS: Specimens from the ora serrata region of two normal human eyes (male donors, 48 and 52 years old) were investigated by light and electron microscope. Specimens from all quadrants were studied in one eye. RESULTS: The elastic layer of Bruch's membrane extended as far as 15 microm into the peripheral choroid; capillaries were included between the elastin layer and the RPE. Nasally, from the anterior end to 2 mm posterior of the ora serrata, the RPE cells contained more melanin than did those in the adjacent posterior region. Melanin granules in the RPE cells close to the ora either formed large clusters or appeared unusually small because of fragmentation. A unique, fine lamellar, membranous material with a fingerprint-like structure was found between the basal folds of the RPE. This material is also found within the extracellular matrix of the choroid and in association with red blood cells. CONCLUSIONS: The morphology of Bruch's membrane is varied near the ora serrata because capillaries and wandering cells are present in its outer collagenous layer. Unique, fine lamellar, fingerprint-like structures are extruded from the RPE and are removed from the eye together with red blood cells. Capillaries within the inner collagenous region of Bruch's membrane at the ora serrata may not necessarily represent a pathologic response but may be a normal characteristic of thick regions of Bruch's membrane.
Asunto(s)
Lámina Basal de la Coroides/irrigación sanguínea , Capilares/ultraestructura , Lámina Basal de la Coroides/ultraestructura , Ojo/irrigación sanguínea , Humanos , Masculino , Melaninas/metabolismo , Melanocitos/metabolismo , Melanocitos/ultraestructura , Persona de Mediana Edad , Epitelio Pigmentado Ocular/ultraestructuraRESUMEN
The effects of morphine (10 mg kg-1 i.p.) on haemodynamics, arrhythmias and plasma and myocardial catecholamines (CA) were studied after coronary artery occlusion in anaesthetized rats. Myocardial intraneuronal CA were assessed histofluorimetrically and CA concentrations measured by high performance liquid chromatography. Morphine increased blood pressure, presumably due to higher plasma noradrenaline (NA) concentrations found in morphine-treated rats. Morphine increased the area of catecholamine-containing fluorescing neurones in the myocardium (as a percentage of total field area) 60 min after sham-operation (0.87 +/- 0.07%) or occlusion (0.57 +/- 0.05%) compared to untreated animals (0.67 +/- 0.06 and 0.38 +/- 0.03% respectively). Tissue NA content was not significantly affected by coronary occlusion and/or morphine within the first 60 min. Morphine had no effect on ischaemia-induced arrhythmias. Whether the higher intraneuronal NA content following morphine resulted from reduced central sympathetic outflow to the heart, presynaptic inhibition of NA release, or increased uptake due to higher plasma concentrations is unclear. Ischaemia-induced local NA release appears independent of these mechanisms, as it was unaffected by morphine.
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Arritmias Cardíacas/fisiopatología , Catecolaminas/metabolismo , Enfermedad Coronaria/metabolismo , Morfina/farmacología , Miocardio/metabolismo , Animales , Arritmias Cardíacas/etiología , Enfermedad Coronaria/complicaciones , Fluorescencia , Hemodinámica/efectos de los fármacos , Masculino , Ratas , Ratas EndogámicasRESUMEN
Selective removal of sialic acid from isolated guinea pig left atrial strips and rabbit thoracic aortic ring segments was performed by neuraminidase prepared from Clostridium perfringens and was controlled electron microscopically. Preincubation of these organs (2 units/mL; 2 hr) resulted in enzyme mediated hydrolysis of total tissue sialic acid; 55.2% for atria and 60.9% for aorta. Contractile force of atria and arterial diameter of thoracic aorta were measured isometrically and isotonically by means of a force displacement transducer. Pretreatment of both organs with neuraminidase (2 units/mL; 2 hr) in a carbogen saturated organ bath caused a moderate left-hand shift of the cumulative concentration response curves for the dihydropyridine type calcium antagonist nisoldipine, the phenylalkylamine derivative gallopamil and the benzothiazepine diltiazem. EC50 values were significantly lower (P less than 0.05), particularly in the atrial muscle, when compared to untreated preparations. There was no effect of neuraminidase on the negative inotropic and vasodilator potency of the calcium channel modulator fendiline. Conversely, neuraminidase induced a right-hand shift in the concentration response curves shown by the pure calcium agonist (-)-S-Bay K 8644 leading to significantly higher EC50 values in both organs. Similarly, the contractile potency of calcium chloride (atria) and potassium chloride (aorta) was attenuated upon neuraminidase treatment. From the results obtained it is concluded that sialic acid removal may modulate the action of calcium channel ligands through an inhibitory effect on transmembrane calcium fluxes and/or by decreasing the external calcium availability. Whether the present results suggest a functional role for sialic acid in the regulation of calcium channels warrants further investigation.
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Canales de Calcio/metabolismo , Músculo Liso Vascular/metabolismo , Miocardio/metabolismo , Ácidos Siálicos/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Aorta Torácica , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Cricetinae , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Fendilina/farmacología , Galopamilo/farmacología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/ultraestructura , Miocardio/ultraestructura , Ácido N-Acetilneuramínico , Neuraminidasa , Nisoldipino/farmacología , Conejos , Ácidos Siálicos/aislamiento & purificaciónRESUMEN
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.
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Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/enzimología , Isoenzimas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Femenino , Edad Gestacional , Isoenzimas/genética , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , EmbarazoRESUMEN
In spite of accumulating evidence for a modulation of sympathetic neurotransmission by endogenously produced nitric oxide (NO), it remains unclear in which parts of the vascular system and at what level this interaction takes place. The aim of the present study was to investigate the distribution of endothelial and neuronal NO synthase (NOS) along the vascular tree of the heart at the light and electron microscopic level using NADPH-diaphorase (NADPH-d) staining as a marker for NOS. In addition, the functional effects of exogenous NO on coronary vascular resistance and cardiac adrenergic nerves was studied using the isolated perfused rat heart as a model. The intraaxonal catecholamine content of adrenergic nerve fibers was visualised and morphometrically assessed by applying glyoxylic acid-induced histofluorescence. The expression of endothelial NOS in the heart was found to depend on the diameter of the blood vessel. Arteries > 100 microns always showed intense staining, whereas staining in smaller arteries and veins was considerably weaker. Smooth-muscle free vessels were essentially devoid of NADPH-d activity. In atrial and ventricular myocardium, neuronal NOS localised in autonomic nerve fibers along the entire vascular tree. Ultrastructurally, NADPH-d staining revealed adjacent localisation of NOS-positive and -negative axons, suggesting and interaxonal modulation of adjacent autonomic nerve fibers by NO. In isolated perfused rat hearts, the intracoronary application of 10(-8) M NO produced a marked decrease of coronary perfusion pressure, which was accompanied by a distinct increase in intraaxonal catecholamine levels of intramural adrenergic nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)