RESUMO
The microscopic assessment of the colocalization of fluorescent signals has been widely used in cell biology. Although imaging techniques have drastically improved over the past decades, the quantification of colocalization by measures such as the Pearson correlation coefficient or Manders overlap coefficient, has not changed. Here, we report the development of an R-based application that allows to (i) automatically segment cells and subcellular compartments, (ii) measure morphology and texture features, and (iii) calculate the degree of colocalization within each cell. Colocalization can thus be studied on a cell-by-cell basis, permitting to perform statistical analyses of cellular populations and subpopulations. ColocalizR has been designed to parallelize tasks, making it applicable to the analysis of large data sets. Its graphical user interface makes it suitable for researchers without specific knowledge in image analysis. Moreover, results can be exported into a wide range of formats rendering post-analysis adaptable to statistical requirements. This application and its source code are freely available at https://github.com/kroemerlab/ColocalizR.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Espaço Intracelular , Microscopia de Fluorescência/métodos , Software , Linhagem Celular Tumoral , Humanos , Espaço Intracelular/diagnóstico por imagem , Espaço Intracelular/fisiologia , Biologia de Sistemas , Interface Usuário-ComputadorRESUMO
Single, isolated epithelial cells move randomly; however, during wound healing, organism development, cancer metastasis, and many other multicellular phenomena, motile cells group into a collective and migrate persistently in a directed manner. Recent work has examined the physics and biochemistry that coordinates the motions of these groups of cells. Of late, two mechanisms have been touted as being crucial to the physics of these systems: leader cells and jamming. However, the actual importance of these to collective migration remains circumstantial. Fundamentally, collective behavior must arise from the actions of individual cells. Here, we show how biophysical activity of an isolated cell impacts collective dynamics in epithelial layers. Although many reports suggest that wound closure rates depend on isolated cell speed and/or leader cells, we find that these correlations are not universally true, nor do collective dynamics follow the trends suggested by models for jamming. Instead, our experimental data, when coupled with a mathematical model for collective migration, shows that intracellular contractile stress, isolated cell speed, and adhesion all play a substantial role in influencing epithelial dynamics, and that alterations in contraction and/or substrate adhesion can cause confluent epithelial monolayers to exhibit an increase in motility, a feature reminiscent of cancer metastasis. These results directly question the validity of wound-healing assays as a general means for measuring cell migration, and provide further insight into the salient physics of collective migration.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Animais , Fenômenos Biomecânicos , Adesão Celular , Simulação por Computador , Cães , Células Epiteliais/citologia , Espaço Intracelular/fisiologia , Células Madin Darby de Rim Canino , Microscopia , Modelos Biológicos , Cicatrização/fisiologiaRESUMO
The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Espaço Intracelular/fisiologia , Microscopia de Contraste de Fase/métodos , Processamento de Sinais Assistido por Computador , Análise de Célula Única/métodos , Algoritmos , Linhagem Celular , Eritrócitos/citologia , HumanosRESUMO
Several growth factors (GFs) have been implicated in long-term memory (LTM), but no single GF can support all of the plastic changes that occur during memory formation. Because GFs engage highly convergent signaling cascades that often mediate similar functional outcomes, the relative contribution of any particular GF to LTM is difficult to ascertain. To explore this question, we determined the unique contribution of distinct GF families (signaling via TrkB and TGF-ßr-II) to LTM formation in Aplysia. We demonstrate that TrkB and TGF-ßr-II signaling are differentially recruited during two-trial training in both time (by trial 1 or 2, respectively) and space (in distinct subcellular compartments). These GFs independently regulate MAPK activation and synergistically regulate gene expression. We also show that trial 1 TrkB and trial 2 TGF-ßr-II signaling are required for LTM formation. These data support the view that GFs engaged in LTM formation are interactive components of a complex molecular network.
Assuntos
Aplysia/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Espaço Intracelular/fisiologia , Memória de Longo Prazo/fisiologia , Animais , Glicoproteínas de Membrana/fisiologia , Técnicas de Cultura de Órgãos , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptor trkB , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais/fisiologia , Fatores de Tempo , Fator de Crescimento Transformador beta2/fisiologiaRESUMO
We developed new cationic fluorescent polymeric thermometers containing both benzothiadiazole and BODIPY units as an environment-sensitive fluorophore and as a reference fluorophore, respectively. The temperature-dependent fluorescence spectra of the thermometers enabled us to perform highly sensitive and practical ratiometric temperature sensing inside living mammalian cells. Intracellular temperatures of non-adherent MOLT-4 (human acute lymphoblastic leukaemia) and adherent HEK293T (human embryonic kidney) cells could be monitored with high temperature resolutions (0.01-1.0 °C) using the new cationic fluorescent polymeric thermometer.
Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Espaço Intracelular/química , Polímeros/química , Termômetros , Linhagem Celular Tumoral , Células HEK293 , Humanos , Espaço Intracelular/fisiologia , Espectrofotometria Ultravioleta/métodos , TemperaturaRESUMO
There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension). The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg(2+), the approximate Mg(2+) concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg(2+) concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg(2+) alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations.
Assuntos
Nucleotídeos de Adenina/química , Espaço Intracelular/fisiologia , Conformação de Ácido Nucleico , Estabilidade de RNA/fisiologia , RNA Bacteriano/química , Riboswitch/fisiologia , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Espaço Intracelular/química , Magnésio/química , Magnésio/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Concentração Osmolar , Estabilidade de RNA/efeitos dos fármacos , Riboswitch/efeitos dos fármacosRESUMO
Mitochondrial density in skeletal muscle fibers is governed by the demand for aerobic ATP production, but the heterogeneous distribution of these mitochondria appears to be governed by constraints associated with oxygen diffusion. We propose that each muscle fiber has an optimal mitochondrial distribution at which it attains a near maximal rate of ATP consumption (RATPase ) while mitochondria are exposed to a minimal oxygen concentration, thus minimizing reactive oxygen species (ROS) production. We developed a coupled reaction-diffusion/cellular automata (CA) mathematical model of mitochondrial function and considered four fiber types in mouse extensor digitorum longus (EDL) and soleus (SOL) muscle. The developed mathematical model uses a reaction-diffusion analysis of metabolites including oxygen, ATP, ADP, phosphate, and phosphocreatine (PCr) involved in energy metabolism and mitochondrial function. A CA approach governing mitochondrial life cycles in response to the metabolic state of the fiber was superimposed and coupled to the reaction-diffusion approach. The model results show the sensitivity of important model outputs such as the RATPase , effectiveness factor (η) and average oxygen concentration available at each mitochondrion to local oxygen concentration in the fibers through variation in the CA model parameter θdet , which defines the sensitivity of mitochondrial death to the oxygen concentration. The predicted optimal mitochondrial distributions matched previous experimental findings. Deviations from this optimal distribution corresponding to higher CA model parameter values (a more uniform mitochondrial distribution) lead to lower aerobic rates. In contrast, distributions corresponding to lower CA model parameter values (a more asymmetric distribution) lead to an increased exposure of mitochondria to oxygen, usually without substantial increases in aerobic rates, which would presumably result in increased ROS production and thus increased risks of cytotoxicity.
Assuntos
Espaço Intracelular/fisiologia , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Oxigênio/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Aerobiose , Animais , Difusão , Metabolismo Energético , Camundongos , Mitocôndrias Musculares/química , Modelos Biológicos , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/citologia , Fosfocreatina/metabolismoRESUMO
Chaotic rhythms in deterministic models can arise as a consequence of changes in model parameters. We carried out experimental studies in which we induced a variety of complex rhythms in aggregates of embryonic chick cardiac cells using E-4031 (1.0-2.5 µM), a drug that blocks the hERG potassium channel. Following the addition of the drug, the regular rhythm evolved to display a spectrum of complex dynamics: irregular rhythms, bursting oscillations, doublets, and accelerated rhythms. The interbeat intervals of the irregular rhythms can be described by one-dimensional return maps consistent with chaotic dynamics. A Hodgkin-Huxley-style cardiac ionic model captured the different types of complex dynamics following blockage of the hERG mediated potassium current.
Assuntos
Ventrículos do Coração/citologia , Dinâmica não Linear , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Função Ventricular/efeitos dos fármacos , Animais , Agregação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Ventrículos do Coração/efeitos dos fármacos , Espaço Intracelular/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Modelos Cardiovasculares , Piperidinas/farmacologia , Piridinas/farmacologiaRESUMO
In cardiovascular computational physiology the importance of understanding cardiac contraction as a multi-scale process is of paramount importance to understand causality across different scales. Within this study, a multi-scale and multi-physics model of the left ventricle that connects the process of cardiac excitation and contraction from the protein to the organ level is presented in a novel way. The model presented here includes the functional description of a cardiomyocyte (cellular scale), which explains the dynamic behaviour of the calcium concentration within the cell whilst an action potential develops. The cell domain is coupled to a domain that determines the kinetics of the sliding filament mechanism (protein level), which is at the basis of cardiac contraction. These processes are then linked to the generation of muscular force and from there to the generation of pressure inside the ventricle. This multi-scale model presents a coherent and unified way to describe cardiac contraction from the protein to the organ level.
Assuntos
Coração/fisiologia , Proteínas dos Microfilamentos/fisiologia , Modelos Cardiovasculares , Contração Miocárdica/fisiologia , Potenciais de Ação/fisiologia , Fenômenos Biomecânicos/fisiologia , Cálcio/fisiologia , Simulação por Computador , Ventrículos do Coração/metabolismo , Humanos , Espaço Intracelular/fisiologia , Miosinas/fisiologia , Pressão , Proteoma , Troponina C/fisiologia , Função Ventricular/fisiologiaRESUMO
Intracellular pathogens such as Mycobacterium tuberculosis have evolved strategies for coping with the pressures encountered inside host cells. The ability to coordinate global gene expression in response to environmental and internal cues is one key to their success. Prolonged survival and replication within macrophages, a key virulence trait of M. tuberculosis, requires dynamic adaptation to diverse and changing conditions within its phagosomal niche. However, the physiological adaptations during the different phases of this infection process remain poorly understood. To address this knowledge gap, we have developed a multi-tiered approach to define the temporal patterns of gene expression in M. tuberculosis in a macrophage infection model that extends from infection, through intracellular adaptation, to the establishment of a productive infection. Using a clock plasmid to measure intracellular replication and death rates over a 14-day infection and electron microscopy to define bacterial integrity, we observed an initial period of rapid replication coupled with a high death rate. This was followed by period of slowed growth and enhanced intracellular survival, leading finally to an extended period of net growth. The transcriptional profiles of M. tuberculosis reflect these physiological transitions as the bacterium adapts to conditions within its host cell. Finally, analysis with a Transcriptional Regulatory Network model revealed linked genetic networks whereby M. tuberculosis coordinates global gene expression during intracellular survival. The integration of molecular and cellular biology together with transcriptional profiling and systems analysis offers unique insights into the host-driven responses of intracellular pathogens such as M. tuberculosis.
Assuntos
Adaptação Fisiológica/fisiologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose , Animais , Espaço Intracelular/fisiologia , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Microscopia Eletrônica de Transmissão , Mycobacterium tuberculosis/ultraestrutura , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that â¼ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca(2+) release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca(2+) influx also persisted in the presence of nitric oxide, delivered from spermine NONOate, or increased ectonucleotidase levels (apyrase). Surprisingly, apyrase was more effective than prostacyclin and NO at limiting secondary P2X1 activation. Dilution of platelets reduced the average extracellular ATP level without affecting the percentage contribution of P2X1 receptors to collagen-evoked Ca(2+) responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca(2+) mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores Toll-Like/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Bovinos , Células Cultivadas , Colágeno Tipo I/farmacologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Epoprostenol/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Agonistas do Receptor Purinérgico P2/metabolismo , Agonistas do Receptor Purinérgico P2/farmacologiaRESUMO
Maintaining cardiac preload throughout the perioperative period is a generally accepted target. As perioperative fasting is believed to cause intravascular hypovolaemia it traditionally triggers aggressive preemptive intravenous fluid infusion. Physiology suggests that extracellular losses via urinary output and evaporation decrease the extracellular compartment. Representing a relevant part of the latter, the intravascular space is also affected, even without blood loss. Measurements in humans, however, have revealed that even a prolonged fasting period does not decrease absolute blood volume. Beyond that, modern fasting guidelines recommend to refrain from clear liquids only two hours prior to surgery. Nevertheless, an intravenous colloid challenge can increase stroke volume after induction of anaesthesia in the majority of surgical patients. While perioperative stroke volume maximisation in high-risk surgery probably improves outcome, the implication of this observation for the routine patient remains unclear. It appears as though there are two important targets to preserve cardiac preload: normovolaemia and vasotension.
Assuntos
Volume Sanguíneo/fisiologia , Jejum/fisiologia , Hemodinâmica/fisiologia , Assistência Perioperatória/métodos , Espaço Extracelular/fisiologia , Jejum/efeitos adversos , Humanos , Hipovolemia/fisiopatologia , Hipovolemia/prevenção & controle , Espaço Intracelular/fisiologia , Resultado do TratamentoRESUMO
The transient receptor potential channels family (TRP channels) is a relatively new group of cation channels that modulate a large range of physiological mechanisms. In the nervous system, the functions of TRP channels have been associated with thermosensation, pain transduction, neurotransmitter release, and redox signaling, among others. However, they have also been extensively correlated with the pathogenesis of several innate and acquired diseases. On the other hand, the omega-3 polyunsaturated fatty acids (n-3 fatty acids) have also been associated with several processes that seem to counterbalance or to contribute to the function of several TRPs. In this short review, we discuss some of the remarkable new findings in this field. We also review the possible roles played by n-3 fatty acids in cell signaling that can both control or be controlled by TRP channels in neurodegenerative processes, as well as both the direct and indirect actions of n-3 fatty acids on TRP channels.
Assuntos
Animais , Humanos , /fisiologia , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Cálcio/metabolismo , Morte Celular/fisiologia , Espaço Intracelular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Over the past 3 decades it has become clear that a large number of extracellular signaling proteins/peptides also act in the intracellular space. These factors are termed intracrines and, although diverse in structure, they share a variety of functional features. In recent years, attention has increasingly turned to identifying the intracellular mechanisms of intracrine action and their implications for human disorders, such as cancer and cardiovascular disease. Perhaps not surprisingly, some intracrines have been shown to bind to and activate their cognate receptors located on intracellular membranes, such as the nuclear envelope. Here we discuss known intracrine actions and argue that mechanisms distinct from membrane receptor activation (that is, "noncanonical" actions) are often operative and physiologically relevant. These actions, we argue, expand our understanding of peptide signaling in important ways. Moreover, an appreciation of noncanonical intracrine functionality informs our understanding of the major effector protein of the renin-angiotensin system, angiotensin II, as well as other hormones operative in cardiovascular biology.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Angiotensina II/fisiologia , Comunicação Celular/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Glicoproteínas/fisiologia , Proteínas de Homeodomínio/fisiologia , Humanos , Espaço Intracelular/fisiologia , Proteína Relacionada ao Hormônio Paratireóideo/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Any mutations in the human neural cell adhesion molecule L1 (hL1CAM) gene might cause various types of serious neurological syndromes in humans, characterized by increased mortality, mental retardation, and various malformations of the nervous system. Such missense mutations often cause severe abnormalities or even fatalities, and the reason for this may be a disruption of the adhesive function of L1CAM resulting from a misdirection of the degradative pathway. Transfection studies using neuroblastoma N2a cells demonstrated that hL1CAM carrying the missense mutations in the fibronectin-like type III (FnIII) domains most likely is located within the endoplasmic reticulum (ER), but it is less well expressed on the cell surface. One mutant, L935P, in the fourth FnIII domain, was chosen from six mutants (K655 and G698 at Fn1, L935P and P941 at Fn4, W1036 and Y1070 at Fn5) in the FnIII domains to study in detail the functions of hL1CAM(200 kDa) , such as the intracellular traffic and degradation, because only a single band at 200 kDa was detected in the hL1CAM(L935P) -transfected cells. hL1CAM(200 kDa) is expressed predominantly in the ER but not on the cell surface. In addition, this missense mutated hL1CAM(200 kDa) is polyubiquitylated at some sites in the extracellular domain and thus becomes degraded by proteasomes via the ER-associated degradation pathway. These observations demonstrate that the missense mutations of hL1CAM in the FnIII domain may cause the resultant pathogenesis because of a loss of expression on the cell surface resulting from misrouting to the degradative pathway.
Assuntos
Retículo Endoplasmático/metabolismo , Fibronectinas/genética , Mutação de Sentido Incorreto/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Ubiquitinação/genética , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/patologia , Fibronectinas/química , Fibronectinas/metabolismo , Fibronectinas/fisiologia , Humanos , Espaço Intracelular/genética , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Camundongos , Mutagênese Sítio-Dirigida/métodos , Molécula L1 de Adesão de Célula Nervosa/química , Molécula L1 de Adesão de Célula Nervosa/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/patologia , Estrutura Terciária de Proteína/genética , Transporte Proteico/genéticaRESUMO
Here, we demonstrate that coupling to N-hydroxypropyl methacrylamide (HPMA) copolymer greatly enhances the activity of apoptosis-inducing peptides inside cells. Peptides corresponding to the BH3 domain of Bid were coupled to a thioester-activated HPMA (28.5 kDa) via native chemical ligation in a simple one-pot synthesis. Peptides and polymer conjugates were introduced into cells either by electroporation or by conjugation to the cell-penetrating peptide nona-arginine. The molecular basis of the increased activity is elucidated in detail. Loading efficiency and intracellular residence time were assessed by confocal microscopy. Fluorescence correlation spectroscopy was used as a separation-free analytical technique to determine proteolytic degradation in crude cell lysates. HPMA conjugation strongly increased the half-life of the peptides in crude cell lysates and inside cells, revealing proteolytic protection as the basis for higher activity.
Assuntos
Espaço Intracelular/metabolismo , Metacrilatos/metabolismo , Peptídeos/metabolismo , Apoptose/fisiologia , Células HeLa , Humanos , Espaço Intracelular/química , Espaço Intracelular/fisiologia , Células Jurkat , Metacrilatos/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/fisiologia , Peptídeos/química , Peptídeos/fisiologia , Polímeros/química , Polímeros/metabolismo , Ligação Proteica/fisiologia , Estabilidade Proteica , Estrutura Terciária de Proteína/fisiologiaRESUMO
The medulla contains central chemosensitive cells important for the maintenance of blood gas and pH homeostasis. To identify the intrinsic chemosensitive cells, we measured responses of intracellular Ca(2+) ([Ca(2+)](i)) and H(+) ([H(+)](i)), and membrane potential of rat primary-cultured medullary cells to 6-s exposure to acidosis. The cells showed transient [Ca(2+)](i) increases to extracellular pH 6.8, which was inhibited by the specific ASIC1a blocker (psalmotoxin-1), but did not respond to pH 7.1 in the HEPES-buffered solution. Isocapnic acidosis induced no changes in [Ca(2+)](i), whereas hypercapnic acidosis induced a remarkable Ca(2+) response and an increase in membrane potential in the HCO(3)(-)-buffered solution (pH 7.1). In glia-rich cultures, intracellular acidification preceded the hypercapnic acidosis-induced Ca(2+) response, and acetazolamide, a carbonic anhydrase inhibitor suppressed these responses. Transient receptor potential (TRP) channel broad-spectrum blockers Ni(2+) and ruthenium red, and a TRPV1- and TRPM8-specific blocker N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-carbox-amide attenuated the hypercapnic acidosis-induced Ca(2+) response. Subpopulations of cells that exhibited the hypercapnic acidosis-induced Ca(2+) response also responded to the application of capsaicin (TRPV1 agonist) and menthol (TRPM8 agonist). These results suggest that the TRP channel family partially mediates the fast hypercapnic acidosis-induced Ca(2+) response via changes in [H(+)](i) and is a candidate of central chemosensing proteins.
Assuntos
Sinalização do Cálcio/fisiologia , Espaço Extracelular/fisiologia , Hipercapnia/metabolismo , Hipercapnia/patologia , Neuroglia/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Acidose/metabolismo , Acidose/patologia , Animais , Células Cultivadas , Técnicas de Cocultura , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Líquido Extracelular/metabolismo , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Bulbo/embriologia , Bulbo/metabolismo , Bulbo/patologia , Neuroglia/patologia , Neuroglia/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Regulação para Cima/fisiologiaRESUMO
Dynamic speckle from 3-D coherence-gated optical sections provides a sensitive label-free measure of cellular activity up to 1 mm deep in living tissue. However, specificity to cellular functionality has not previously been demonstrated. In this work, we perform fluctuation spectroscopy on dynamic light scattering captured using coherence-domain digital holography to obtain the spectral response of tissue that is perturbed by temperature, osmolarity, and antimitotic cytoskeletal drugs. Different perturbations induce specific spectrogram response signatures that can show simultaneous enhancement and suppression in different spectral ranges.
Assuntos
Corrente Citoplasmática/fisiologia , Holografia/métodos , Espaço Intracelular/fisiologia , Análise Espectral/métodos , Algoritmos , Animais , Corrente Citoplasmática/efeitos dos fármacos , Processamento de Imagem Assistida por Computador , Luz , Nocodazol/farmacologia , Óptica e Fotônica , Ratos , Espalhamento de Radiação , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Secretory phospholipase A2 (sPLA2) activity is present in the CNS and the sPLA2-IIA isoform has been shown to induce exocytosis in cultured hippocampal neurons. However, little is known about possible contributions of various lysophospholipid species to exocytosis in neuroendocrine cells. This study was therefore carried out to examine the effects of several lysophospholipid species on exocytosis on rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion, indicating exocytosis, was observed in PC12 cells after external infusion of lysophosphatidylinositol (LPI), but not lysophosphatidylcholine or lysophosphatidylserine by total internal reflection microscopy. Similarly, external infusion of LPI induced significant increases in capacitance, or number of spikes detected at amperometry, indicating exocytosis. Depletion of cholesterol by pre-incubation of cells with methyl beta cyclodextrin and depletion of Ca2+ by thapsigargin and incubation in zero external Ca2+ resulted in attenuation of LPI induced exocytosis, indicating that exocytosis was dependent on the integrity of lipid rafts and intracellular Ca2+. Moreover, LPI induced a rise in intracellular Ca2+ suggesting that this could be the trigger for exocytosis. It is postulated that LPI may be an active participant in sPLA2-mediated exocytosis in the CNS.
Assuntos
Exocitose/fisiologia , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos/metabolismo , Animais , Cálcio/metabolismo , Colesterol/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/fisiologia , Capacitância Elétrica , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Hipolipemiantes/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/fisiologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/fisiologia , Células PC12 , Ratos , Tapsigargina/farmacologia , beta-Ciclodextrinas/farmacologiaRESUMO
The main properties of Na+ /K(+)-ATPase as a natural receptor for cardiotonic steroids have been discusses. Primary attention is focused on structural and functional differences between the alpha-subunit isoforms of Na+/K(+)-ATPase in different tissues. General information on the role of the Na pump in signaling cascades in kidney epithelial cells, cardiomyocytes and neurons is presented. The data obtained indicate that, in neurons, several alpha-isoforms of Na+/K(+)-ATPase possessing different sensitivity to ouabain may have different signaling functions.