RESUMO
BACKGROUND: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. RESULTS: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. CONCLUSIONS: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.
Assuntos
Cálcio/análise , Imagem Molecular/métodos , Neurônios/metabolismo , Proteínas Recombinantes/metabolismo , Troponina C/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Fluorescência , Fluorometria/métodos , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/instrumentação , Técnicas de Patch-Clamp , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Imagem com Lapso de Tempo , Troponina C/genéticaRESUMO
New neurons are continuously produced from neural stem cells in specific regions of the adult brain of animals and humans. In the hippocampus, a region crucial for cognitive function, neurogenesis responds to a multitude of extrinsic stimuli; emerging evidence indicates that it may be important for behavior, pathophysiology, brain repair, and response to drugs. We have developed an approach to identify and quantify the cellular targets of pro- and anti-neurogenic stimuli, based on reporter transgenic mouse lines in which neural stem and progenitor cells or their progeny are marked by fluorescent proteins. Here, we demonstrate the feasibility of using MIMS for studying adult neurogenesis.
RESUMO
The localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion ofthe same components. ROS-generating complexes have been suggested to follow this principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model ofredox signaling compartmentalization. Nevertheless, evidences of local ROS production and restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH-oxidase isoforms, their adapter proteins and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant system which prevents uncontrolled ROS diffusion from the sites of generation to the adjacent subcellular compartments; the current data of the specific localization NADPH-oxidases activity and its influence on intracellular processes; the recent evidences of the ROS diffusion restriction.
Assuntos
Compartimento Celular/genética , Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Apoptose/genética , Movimento Celular/genética , Proliferação de Células , Retículo Endoplasmático/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo/genética , Proteínas/metabolismoRESUMO
Finding biomarkers of human neurological diseases is one of the most pressing goals of modern medicine. Most neurological disorders are recognized too late because of the lack of biomarkers that can identify early pathological processes in the living brain. Late diagnosis leads to late therapy and poor prognosis. Therefore, during the past decade, a major endeavor of clinical investigations in neurology has been the search for diagnostic and prognostic biomarkers of brain disease. Recently, a new field of metabolomics has emerged, aiming to investigate metabolites within the cell/tissue/ organism as possible biomarkers. Similarly to other "omics" fields, metabolomics offers substantial information about the status of the organism at a given time point. However, metabolomics also provides functional insight into the biochemical status of a tissue, which results from the environmental effects on its genome background. Recently, we have adopted metabolomics techniques to develop an approach that combines both in vitro analysis of cellular samples and in vivo analysis of the mammalian brain. Using proton magnetic resonance spectroscopy, we have discovered a metabolic biomarker of neural stem/progenitor cells (NPCs) that allows the analysis of these cells in the live human brain. We have developed signal-processing algorithms that can detect metabolites present at very low concentration in the live human brain and can indicate possible pathways impaired in specific diseases. Herein, we present our strategy for both cellular and systems metabolomics, based on an integrative processing of the spectroscopy data that uses analytical tools from both metabolomic and spectroscopy fields. As an example of biomarker discovery using our approach, we present new data and discuss our previous findings on the NPC biomarker. Our studies link systems and cellular neuroscience through the functions of specific metabolites. Therefore, they provide a functional insight into the brain, which might eventually lead to discoveries of clinically useful biomarkers of the disease.
Assuntos
Biomarcadores/metabolismo , Metabolômica/métodos , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Encefalopatias/diagnóstico , Encefalopatias/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Metabolômica/estatística & dados numéricos , Processamento de Sinais Assistido por Computador , Biologia de SistemasRESUMO
Nestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 microm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration.
Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Isquemia/metabolismo , Rim/irrigação sanguínea , Rim/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Movimento Celular , Citometria de Fluxo , Expressão Gênica , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Vídeo , Neovascularização Fisiológica , NestinaRESUMO
Nitric oxide (NO) has a profound role in the generation, differentiation, survival, and physiology of neurons. We have created a novel transgenic model to study the action of NO in the adult brain, in which the neuronal isoform of NO synthase (nNOS) is expressed under control of the promoter of the calcium-calmodulin multifunctional kinase IIalpha (CaMKIIalpha) gene. We show that the transgenic nNOS RNA and protein are expressed in the cortex, the hippocampus and the striatum of the transgenic mice. We also show that expression of several genes involved in the protection of neurons from oxidative stress and cell death is not affected in neurons of the transgenic mice. Furthermore, generation of new cells is depressed in the neurogenic brain areas in transgenics. In addition, we analyze gene expression in the hippocampus of the transgenic animals using microarray RNA profiling and Q-PCR. Our experiments describe specific changes in cell division and gene activity in the CaMKII-nNOS transgenic model and demonstrate its utility for studying the action of NO in the adult brain.
Assuntos
Regulação Enzimológica da Expressão Gênica , Sistema Nervoso/metabolismo , Óxido Nítrico Sintase Tipo I/biossíntese , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/genética , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Neurônios/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA/biossíntese , RNA/genética , TransgenesRESUMO
Mechanisms controlling the transition of a neural precursor cell from proliferation to differentiation during brain development determine the distinct anatomical features of the brain. Nitric oxide (NO) may mediate such a transition, because it can suppress DNA synthesis and cell proliferation. We cloned the gene encoding the neuronal isoform of Xenopus NO synthase (XNOS) and found that in the developing brain of Xenopus tadpoles, a zone of XNOS-expressing cells lies adjacent to the zone of dividing neuronal precursors. Exogenous NO, supplied to the tadpole brain in vivo, decreased the number of proliferating cells and the total number of cells in the optic tectum. Conversely, inhibition of NOS activity in vivo increased the number of proliferating cells and the total number of cells in the optic tectum. NOS inhibition yielded larger brains with grossly perturbed organization. Our results indicate that NO is an essential negative regulator of neuronal precursor proliferation during vertebrate brain development.
Assuntos
Encéfalo/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Bromodesoxiuridina , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Implantes de Medicamento , Inibidores Enzimáticos/farmacologia , Hibridização In Situ , Larva , Dados de Sequência Molecular , Morfogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Especificidade de Órgãos , XenopusRESUMO
Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia in Drosophila. We cloned and analyzed the only gene in the fly genome that encodes Drosophila nitric-oxide synthase (dNOS). It consists of 19 exons and is dispersed over 34 kilobases of genomic DNA. Alternative transcription start sites and alternative splice sites are used to generate a remarkable variety of mRNAs from the dNOS gene. We identified eight new transcripts that are widely expressed throughout Drosophila development and encode a family of DNOS-related proteins. Alternative splicing affects both the 5'-untranslated region and the coding region of the dNOS primary transcript. Most of the splicing alterations in the coding region of the gene lead to premature termination of the open reading frame. As a result, none of the alternative transcripts encode an enzymatically active protein. However, some of these shorter DNOS protein products can effectively inhibit enzymatic activity of the full-length DNOS1 protein when co-expressed in mammalian cells, thus acting as dominant negative regulators of NO synthesis. Using immunoprecipitation, we demonstrate that these short DNOS protein isoforms can form heterodimers with DNOS1, pointing to a physical basis for the dominant negative effect. Our results suggest a novel regulatory function for the family of proteins encoded by the Drosophila NOS gene.
Assuntos
Drosophila/genética , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Dimerização , Éxons , Dados de Sequência Molecular , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/química , Testes de PrecipitinaRESUMO
Nefiracetam, a pyrrolidone derivative developed as an anti-dementia drug, persistently potentiated currents through neuronal nicotinic acetylcholine (ACh) receptors (alpha7, alpha4beta2) expressed in Xenopus oocytes, and the potentiation was blocked by either the selective protein kinase C (PKC) inhibitors, GF109203X and staurosporine, or co-expressed active PKC inhibitor peptide. In primary cultures of rat hippocampal neurons, nefiracetam increased the rate of nicotine-sensitive miniature excitatory postsynaptic currents, without affecting the amplitude, and the increase was inhibited by GF109203X. In addition, the drug caused a marked increase in the glutamate release from electrically stimulated guinea pig hippocampal slices, and the effect was abolished by the nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. Nefiracetam induced a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by alpha-bungarotoxin and mecamylamine. Such facilitatory action was still found in the hippocampus with selective cholinergic denervation. The results of the present study, thus, suggest that nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission. This may represent a cellular mechanism underlying the cognition-enhancing action of nefiracetam. The results also provide the possibility that nefiracetam could be developed as a promising therapeutic drug for senile dementia or Alzheimer's disease.
Assuntos
Fármacos do Sistema Nervoso Central/farmacologia , Hipocampo/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Pirrolidinonas/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Células Cultivadas , Denervação , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/metabolismo , Indóis/farmacologia , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , RatosRESUMO
Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development [1] [2]. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway [3] [4], which integrates signals from multiple effectors ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger [5], can reversibly suppress DNA synthesis and cell division [6] [7]. In developing flies, the antiproliferative action of NO is essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly [8] [9] [10]. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however. We used transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors to address this issue. We found that manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. Our data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Óxido Nítrico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transativadores , Animais , Animais Geneticamente Modificados , Drosophila/genética , Fatores de Transcrição E2F , Olho/efeitos dos fármacos , Olho/ultraestrutura , Genes do Retinoblastoma/efeitos dos fármacos , Microscopia Eletrônica , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Proteína do Retinoblastoma , Proteína 1 de Ligação ao Retinoblastoma , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologiaRESUMO
We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neuritos/ultraestrutura , Neurônios/citologia , Neurônios/ultraestrutura , Células PC12 , Mutação Puntual , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Sinaptotagminas , TransfecçãoRESUMO
Nitric oxide (NO) is an efficient inhibitor of cell proliferation. Here we show that part of the antiproliferative activity of NO in fibroblasts is mediated through p53 signaling pathway. Cells from p53-/- knockout mice are compromised in their ability to stop dividing in the presence of NO. NO strongly induces expression of genes which are transcriptional targets of p53, and p53 is necessary for some, but not all, of the transcription activation effects of NO. Furthermore, NO strongly increases the cellular level of p53 protein. Since phosphorylation of particular residues of the p53 molecule has been correlated with its functional activity, we determined the phosphorylation pattern of p53 molecule after exposure to NO and compared it with the phosphorylation patterns that develop upon treatment with gamma-irradiation, UV light, and adriamycin. We found that NO induces a specific signature pattern of p53 phosphorylation, distinct from the patterns evoked by other inducers. This study suggests that NO activates specific signaling pathways that may partially overlap, but that do not coincide, with signaling pathways activated by other known inducers of p53 activity.
Assuntos
Perfilação da Expressão Gênica , Óxido Nítrico/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Células 3T3 , Animais , Ciclo Celular , Divisão Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Camundongos , Camundongos Knockout , Fosforilação , Transdução de Sinais , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Raios UltravioletaRESUMO
Mechanisms controlling the transition of precursor cells from proliferation to differentiation during organism development determine the distinct anatomical features of tissues and organs. NO may mediate such a transition since it can suppress DNA synthesis and cell proliferation. Inhibition of NOS activity in the imaginal discs of Drosophila larvae results in hypertrophy of tissues and organs of the adult fly, whereas ectopic overexpression of NOS has the reciprocal, hypotrophic, effect. Furthermore, NO production is crucial for the establishment of ordered neuronal connections in the visual system of the fly, indicating that NO affects the acquisition of the differentiated phenotype by the neural tissue. Increasing evidence points to a broad role that NO may play in animal development by acting as an essential negative regulator of precursor cell proliferation during tissue and organ morphogenesis.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Óxido Nítrico/química , Óxido Nítrico/fisiologia , Animais , Drosophila melanogaster/químicaRESUMO
The present study was conducted to understand the effect of arachidonic acid on nicotinic acetylcholine (ACh) receptor-mediated synaptic plasticity. Arachidonic acid persistently (>/=1 h) potentiated currents through neuronal nicotinic ACh receptors (alpha7 and alpha4beta2) expressed in Xenopus oocytes, and the effect was blocked by the selective protein kinase C (PKC) inhibitors, such as GF109203X, PKCI, and co-expressed active PKC inhibitor peptide. This free fatty acid markedly increased nicotine-sensitive glutamate release from hippocampal slices and enhanced the rate of nicotine-sensitive miniature excitatory postsynaptic currents without affecting the amplitude in cultured hippocampal CA1 neurons under the influence of PKC. Furthermore, arachidonic acid induced a long-lasting (>/=3 h) facilitation of hippocampal CA1 synaptic transmission in slices, and the effect was blocked by nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. The facilitation, whereas independent of N-methyl-D-aspartate (NMDA) receptors, shares a common mechanism with long-term potentiation (LTP) induced by tetanic stimulation. The results of the present study thus suggest that arachidonic acid sustains enhanced activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to an 'LTP-like' facilitation of hippocampal synaptic transmission.
Assuntos
Ácido Araquidônico/farmacologia , Hipocampo/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Técnicas de Patch-Clamp , Ratos , Fatores de Tempo , XenopusRESUMO
The diffusible factors, nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) are both suggested to be intercellular messengers that have similar synaptic activities and developmental influences in the brain. In the present study, we have analysed their mutual regulation with respect to their production in rodent neocortical neurons. Some of the cultured rat neocortical neurons exhibited immunoreactivity for both neuronal NO synthase (NOS) and the BDNF receptor trkB. Neuronal NOS appeared to be activated autonomously and produced NO in culture as monitored by nitrite accumulation. Inhibition of the endogenous NO production in culture by a NOS inhibitor, NG-monomethyl-L-arginine (NMMA), enhanced basal expression of BDNF mRNA and protein. Similarly, cerebroventricular administration of another NOS inhibitor, N-omega-nitro-L-arginine methylester (L-NAME), but not D-NAME or saline, increased BDNF content in the neocortex. In the opposite direction, however, BDNF appeared to function as a positive regulator for NO synthesis. Addition of BDNF upregulated the neuronal NOS expression as well as NO production in neocortical culture. In agreement, BDNF knock-out mice exhibited significant impairment of neuronal NOS expression in the neocortex. Taken together, these observations suggest that the trans-synaptic signalling molecules, NO and BDNF, influence the production of each other and mutually regulate the strength of their intercellular communications.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Comunicação Celular/fisiologia , Neurônios/citologia , Neurônios/enzimologia , Óxido Nítrico/fisiologia , Animais , Células Cultivadas , Sondas de DNA , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Neocórtex/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Fenótipo , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/fisiologia , Receptor do Fator Neutrófico Ciliar , Receptores de Fator de Crescimento Neural/fisiologia , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/fisiologia , ômega-N-Metilarginina/farmacologiaRESUMO
The present study investigated the effect of arachidonic acid on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, presumably heteromeric receptors formed of GluR1, GluR2, and GluR3, expressed in Xenopus oocytes. Arachidonic acid (10 microM) potentiated currents through receptors expressing GluR1 and 3 (GluR1,3) to 170% of basal level during initial 20 min following application, being still evident at 60-min washing-out of the drug, while it never or little enhanced currents through receptors expressing GluR1 and 2 (GluR1,2) or GluR1, 2, and 3 (GluR1,2,3) (110% 30 min after treatment). The effect of arachidonic acid on GluR1,3 currents was not observed in Ca2+-free extracellular solution, and the potentiation was blocked by either KN-93, a selective Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, or NP217, an active CaMKII inhibitor peptide, when co-expressed with the receptors. In contrast, the protein synthesis inhibitor, cycloheximide, the selective inhibitor of cAMP-dependent protein kinase (PKA), H-89, the selective inhibitors of protein kinase C (PKC), PKCI and GF109203X, the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059, or the inactive CaMKII inhibitors, KN-92 and NP218, had no effect on the currents. In the assay of intracellular calcium mobilizations, Ca2+ influx in response to receptor activation was greatest with receptors formed in oocytes expressing GluR1,3. The results of the present study indicate that arachidonic acid induces a long-lasting potentiation of GluR1,3 currents, possibly as a result of the interaction with a CaMKII pathway.
Assuntos
Ácido Araquidônico/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Receptores de AMPA/metabolismo , Animais , Benzilaminas/farmacologia , Cálcio/análise , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Flavonoides/farmacologia , Indóis/farmacologia , Ativação do Canal Iônico/fisiologia , Isoquinolinas/farmacologia , Ácido Caínico/farmacologia , Maleimidas/farmacologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , Receptores de AMPA/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , XenopusRESUMO
We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Melanóforos/fisiologia , Organelas/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Proteína Quinase C/fisiologia , Células 3T3 , Animais , Agregação Celular , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Melanócitos/metabolismo , Melanócitos/fisiologia , Camundongos , Microtúbulos/fisiologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Pigmentos Biológicos/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Transfecção , XenopusRESUMO
Cellular NO synthase was assayed in bone marrow, spleen, thymus, lymph nodes, epidermis, hypodermic connective tissue, small gut, peritoneal exudate, liver, kidney, and heart by cytochemical and immunofluorescent methods. As a rule the enzymatic activity was found in differentiated cells that finished proliferation (mature hemopoietic cells, epithelium of gut villi, etc.). The enzyme activity was undetectable in actively reproducing low-differentiated cells (basal layer of the epidermis, crypt cells of the small gut, or blast hemopoietic forms). NO is proposed to participate in regulation of tissue-specific terminal differentiation (maturation) of the cells.
Assuntos
Óxido Nítrico Sintase/metabolismo , Animais , Diferenciação Celular/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Histocitoquímica , Isoenzimas/metabolismo , CamundongosRESUMO
Cell division and subsequent programmed cell death in imaginal discs of Drosophila larvae determine the final size of organs and structures of the adult fly. We show here that nitric oxide (NO) is involved in controlling the size of body structures during Drosophila development. We have found that NO synthase (NOS) is expressed at high levels in developing imaginal discs. Inhibition of NOS in larvae causes hypertrophy of organs and their segments in adult flies, whereas ectopic expression of NOS in larvae has the opposite effect. Blocking apoptosis in eye imaginal discs unmasks surplus cell proliferation and results in an increase in the number of ommatidia and component cells of individual ommatidia. These results argue that NO acts as an antiproliferative agent during Drosophila development, controlling the balance between cell proliferation and cell differentiation.
Assuntos
Divisão Celular/efeitos dos fármacos , Drosophila melanogaster/crescimento & desenvolvimento , Óxido Nítrico/farmacologia , Animais , Apoptose/efeitos dos fármacos , DNA/biossíntese , Di-Hidrolipoamida Desidrogenase/isolamento & purificação , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Inibidores Enzimáticos/farmacologia , Extremidades/crescimento & desenvolvimento , Extremidades/patologia , Olho/crescimento & desenvolvimento , Olho/ultraestrutura , Histocitoquímica , Hipertrofia , Proteínas Inibidoras de Apoptose , Larva/enzimologia , Larva/crescimento & desenvolvimento , Camundongos , Microinjeções , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Transgenes , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection leads to a general exhaustion of the immune system. Prior to this widespread decline of immune functions, however, there is an evident hyperactivation of the monocyte/macrophage arm. Increased levels of cytokines and other biologically active molecules produced by activated monocytes may contribute to the pathogenesis of HIV disease both by activating expression of HIV-1 provirus and by direct effects on cytokine-sensitive tissues, such as lung or brain. In this article, we investigate mechanisms of hyperresponsiveness of HIV-infected monocytes. MATERIALS AND METHODS: The study was performed on monocyte cultures infected in vitro with a monocytetropic strain HIV-1ADA. Cytokine production was induced by stimulation of cultures with lipopolysaccharides (LPS) and measured by ELISA. To study involvement of nitric oxide (NO) in the regulation of cytokine expression, inhibitors of nitric oxide synthase (NOS) or chemical donors of NO were used. RESULTS: We demonstrate that infection with HIV-1 in vitro primes human monocytes for subsequent activation with LPS, resulting in increased production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). This priming effect can be blocked by Ca(2+)-chelating agents and by the NOS inhibitor L-NMMA, but not by hemoglobin. It could be reproduced on uninfected monocyte cultures by using donors of NO, but not cGMP, together with LPS. CONCLUSIONS: NO, which is expressed in HIV-1-infected monocyte cultures, induces hyperresponsiveness of monocytes by synergizing with calcium signals activated in response to LPS stimulation. This activation is cGMP independent. Our findings demonstrate the critical role of NO in HIV-1-specific hyperactivation of monocytes.