RESUMEN
Osteosarcoma (OS) is a highly metastatic bone cancer that usually affects children. Rhizoma Paridis saponins (RPS) have been identified to show a broad-spectrum anti-tumor activity. Our previous study has identified vasculogenic mimicry (VM) as an indicator of poor prognosis for OS. Rhizoma Paridis ethanol extract exhibits potent anti-OS property. However, the anti-metastatic effect of RPS on OS and the detailed mechanisms remain unknown. RPS was characterized by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) analysis. The anti-OS, anti-metastasis and anti-VM activities of RPS were investigated using in vitro biological assays and a xenograft mouse model. Western blot, qRT-PCR, ELISA, Phalloidin staining and immunohistochemistry assays were conducted to investigate the molecular mechanism of RPS. A total of 34 phytochemicals from RPS were identified by LC/Q-TOF/MS. RPS dose-dependently suppressed the OS cell proliferation, metastasis and VM formation in vitro and in vivo. Mechanically, we found that RPS downregulated migration-inducing gene 7 (MIG-7) expression, resulting in inhibition of the PI3K/MMPs/Ln-5γ2 pathway and cell protrusion formation. Additionally, we confirmed that RPS downregulated MIG-7 by upregulating miR-520d-3p expression. Our results suggests that RPS inhibits the VM formation and metastasis of OS by modulating the miR-520d-3p/MIG-7 signaling axis.
Asunto(s)
Neoplasias Óseas , MicroARNs , Osteosarcoma , Saponinas , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Línea Celular Tumoral , Proliferación Celular/genética , Etanol , Humanos , Ratones , MicroARNs/genética , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Osteosarcoma/patología , Faloidina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Extractos Vegetales/farmacología , Saponinas/farmacología , Saponinas/uso terapéuticoRESUMEN
AIMS: Recently, the European Association of Urology recommended hexane-extracted fruit of Serenoa repens (HESr) in their guidelines on management of non-neurogenic male lower urinary tracts symptoms (LUTS). Despite previously lacking recommendations, Permixon® is the most investigated HESr in clinical trials, where it proved effective for male LUTS. In contrast, underlying mechanisms were rarely addressed and are only marginally understood. We therefore investigated effects of Permixon® on human prostate and detrusor smooth muscle contraction and on growth-related functions in prostate stromal cells. MAIN METHODS: Permixon® capsules were dissolved using n-hexane. Contractions of human prostate and detrusor tissues were induced in organ bath. Proliferation (EdU assay), growth (colony formation), apoptosis and cell death (flow cytometry), viability (CCK-8) and actin organization (phalloidin staining) were studied in cultured human prostate stromal cells (WPMY-1). KEY FINDINGS: Permixon® inhibited α1-adrenergic and thromboxane-induced contractions in prostate tissues, and methacholine-and thromboxane-induced contractions in detrusor tissues. Endothelin-1-induced contractions were not inhibited. Neurogenic contractions were inhibited in both tissues in a concentration-dependent manner. In WPMY-1 cells, Permixon® caused concentration-dependent breakdown of actin polymerization, inhibited colony formation, reduced cell viability, and proliferation, without showing cytotoxic or pro-apoptotic effects. SIGNIFICANCE: Our results provide a novel basis that allows, for the first time, to fully explain the ubiquitous beneficial effects of HESr in clinical trials. HESr may inhibit at least neurogenic, α1-adrenergic and thromboxane-induced smooth muscle contraction in the prostate and detrusor, and in parallel, prostate stromal cell growth. Together, this may explain symptom improvements by Permixon® in previous clinical trials.
Asunto(s)
Hiperplasia Prostática , Serenoa , Actinas/metabolismo , Adrenérgicos/farmacología , Endotelina-1/metabolismo , Hexanos/metabolismo , Hexanos/farmacología , Hexanos/uso terapéutico , Humanos , Masculino , Cloruro de Metacolina/metabolismo , Contracción Muscular , Músculo Liso , Faloidina/metabolismo , Faloidina/farmacología , Faloidina/uso terapéutico , Extractos Vegetales/uso terapéutico , Próstata/metabolismo , Hiperplasia Prostática/tratamiento farmacológico , Hiperplasia Prostática/metabolismo , Sincalida/metabolismo , Células del Estroma/metabolismo , Tromboxanos/metabolismo , Vejiga Urinaria/metabolismoRESUMEN
The neurovascular structures in the cranial dura mater have been studied with various histological techniques in the past years. In order to obtain a proper approach to reveal the detailed structures, different labeling methods for the cranial vessels and nerve fibers were tested in this study. Firstly, the labeling characteristics of phalloidin, alpha smooth muscle actin (α-SMA), and CD31 were compared in rat whole-mount cranial dura mater by using fluorescent immunohistochemistry or histochemistry. Secondly, according to their properties, phalloidin and α-SMA were selected to combine with calcitonin gene-related peptide (CGRP) to further demonstrate the cranial neurovascular structure. By these approaches, a three-dimensional map of blood vessels and nerve fibers within the whole-mount rat cranial dura mater was obtained. The results showed that phalloidin, α-SMA, and CD31 were preferably expressed in the wall of cranial vessels, corresponding to the arteriors, venules, and capillaries, respectively. Additionally, CGRP + nerve fibers were clearly demonstrated together with phalloidin + or α-SMA + vessels, forming a delicate neurovascular network in the cranial dura mater. The thick nerve bundles ran closely to the phalloidin + or α-SMA + vessels in parallel pattern, while the thin nerve fibers branched off from the bundles tending to surround the phalloidin + arterioles rather than α-SMA + venules. These findings suggest that phalloidin could be an appropriate biochemical maker to be effectively used together with CGRP for experiments examining the detailed spatial correlation of cranial blood vessels and nerve fibers in a three-dimensional view, which may provide clues for understanding the underlying mechanisms of cranial neurovascular disorders.
Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Péptido Relacionado con Gen de Calcitonina/química , Duramadre/irrigación sanguínea , Duramadre/efectos de los fármacos , Faloidina/farmacología , Animales , Biomarcadores , Péptido Relacionado con Gen de Calcitonina/farmacología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Fibras Nerviosas/metabolismo , RatasRESUMEN
Actin undergoes structural transitions during polymerization, ATP hydrolysis, and subsequent release of inorganic phosphate. Several actin-binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here, we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin-binding proteins. Furthermore, high-resolution cryoelectron microscopy structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.
Asunto(s)
Citoesqueleto de Actina/química , Antifúngicos/química , Depsipéptidos/química , Proteínas Fúngicas/química , Micotoxinas/química , Faloidina/química , Citoesqueleto de Actina/metabolismo , Antifúngicos/farmacología , Sitios de Unión , Microscopía por Crioelectrón , Depsipéptidos/farmacología , Proteínas Fúngicas/farmacología , Micotoxinas/farmacología , Faloidina/farmacología , Unión ProteicaRESUMEN
We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, and three live-cell actin probes in five fixation conditions across three imaging platforms as a basis for the design of optimized protocols. Of the fluorescent phalloidin-dye conjugates tested, Alexa Fluor-488 Phalloidin ranked best in overall labeling of the actin cytoskeleton and maintenance of the fluorescence signal over time. Use of actin monoclonal antibodies revealed significant limitations under a variety of fixation-permeabilization conditions. Evaluation of commonly used live-cell probes provides evidence for actin filament bias, with TagRFP-Lifeact excluded from lamellipodia, but not mEGFP-Lifeact or F-tractin-EGFP.
Asunto(s)
Citoesqueleto de Actina/genética , Actinas/aislamiento & purificación , Colorantes Fluorescentes/química , Citoesqueleto de Actina/química , Actinas/química , Anticuerpos/química , Anticuerpos/farmacología , Colorantes Fluorescentes/farmacología , Maleimidas/química , Maleimidas/farmacología , Faloidina/química , Faloidina/farmacología , Seudópodos/química , Seudópodos/genéticaRESUMEN
Sodium influx is tightly regulated in the cells of blood origin. Amiloride-insensitive sodium channels were identified as one of the main sodium-transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics. Here, to search for physiological mechanisms controlling epithelial Na+ channel (ENaC)-like channels, we utilized leukemia K562 cells as a unique model to examine single channel behavior in a whole-cell patch-clamp experiments. We have shown for the first time that extracellular serine protease trypsin directly activates sodium channels in plasma membrane of K562 cells. The whole-cell single current recordings clearly demonstrate no inhibition of trypsin-activated channels by amiloride or benzamil. Involvement of proteolytic cleavage in channel opening was confirmed in experiments with soybean trypsin inhibitor. More importantly, stabilization of F-actin with intracellular phalloidin did not prevent trypsin-induced channel activation indicating no implication of cytoskeleton rearrangements in stimulatory effect of extracellular protease. Our data reveals a novel mechanism modulating amiloride-insensitive ENaC-like channel activity and integral sodium permeability in leukemia cells.
Asunto(s)
Amilorida/farmacología , Canales Epiteliales de Sodio/metabolismo , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Tripsina/farmacología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Amilorida/análogos & derivados , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Citocalasina D/farmacología , Bloqueadores del Canal de Sodio Epitelial/farmacología , Humanos , Células K562 , Potenciales de la Membrana/efectos de los fármacos , Microscopía Fluorescente , Modelos Biológicos , Técnicas de Placa-Clamp , Faloidina/farmacología , Sodio/metabolismo , Tripsina/metabolismo , Inhibidores de Tripsina/farmacologíaRESUMEN
Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.
Asunto(s)
Apoptosis , Oryza/citología , Oryza/metabolismo , Vacuolas/metabolismo , Citoesqueleto de Actina/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Catepsina B/antagonistas & inhibidores , Catepsina B/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Forma del Núcleo Celular/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Citocalasina B/farmacología , Fusión de Membrana/efectos de los fármacos , Oryza/efectos de los fármacos , Permeabilidad , Faloidina/farmacología , Protoplastos/efectos de los fármacos , Protoplastos/metabolismo , Semillas/citología , Semillas/metabolismo , Vacuolas/efectos de los fármacosRESUMEN
This chapter gives an overview of the most common F-actin-perturbing substances that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement, or when apoptosis has to be induced. These substances can be divided into two major subclasses: F-actin-stabilizing and -polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and -polymerizing agent, which may even have anticancer activities. Cytochalasins, derived from fungi, show an F-actin-severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given.
Asunto(s)
Actinas/metabolismo , Citocalasinas/farmacología , Depsipéptidos/farmacología , Faloidina/farmacología , Células Vegetales/efectos de los fármacos , Tiazolidinas/farmacología , Actinas/química , Microscopía Inmunoelectrónica , Células Vegetales/metabolismo , Células Vegetales/ultraestructura , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de ProteínaRESUMEN
To investigate plasmodesmata (PD) function, a useful technique is to monitor the effect on cell-to-cell transport of applying an inhibitor of a physiological process, protein, or other cell component of interest. Changes in PD transport can then be monitored in one of several ways, most commonly by measuring the cell-to-cell movement of fluorescent tracer dyes or of free fluorescent proteins. Effects on PD structure can be detected in thin sections of embedded tissue observed using an electron microscope, most commonly a Transmission Electron Microscope (TEM). This chapter outlines commonly used inhibitors, methods for treating different tissues, how to detect altered cell-to-cell transport and PD structure, and important caveats.
Asunto(s)
Arabidopsis/fisiología , Citotoxinas/farmacología , Procesamiento de Imagen Asistido por Computador/métodos , Raíces de Plantas/fisiología , Plasmodesmos/fisiología , Tradescantia/fisiología , Actinas/antagonistas & inhibidores , Actinas/genética , Actinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/ultraestructura , Transporte Biológico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citocalasina B/farmacología , Depsipéptidos/farmacología , Fijadores/química , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Microinyecciones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microtomía , Faloidina/farmacología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/ultraestructura , Plasmodesmos/efectos de los fármacos , Plasmodesmos/ultraestructura , Profilinas/farmacología , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Tiazolidinas/farmacología , Fijación del Tejido , Tradescantia/efectos de los fármacos , Tradescantia/ultraestructuraRESUMEN
A diminution in the order of membrane lipids, which occurs during apoptosis, has been shown to correlate with increased membrane susceptibility to hydrolysis by secretory phospholipase A2. Studies with artificial membranes, however, have demonstrated that the relationship between membrane order and hydrolysis is more complex than suggested thus far by cell studies. To better resolve this relationship, this study focused on comparisons between increasing temperature and calcium ionophore as means of decreasing membrane order in S49 cells. Although these two treatments caused comparable changes in apparent membrane order as detected by steady-state fluorescence measurements, only ionophore treatment enhanced phospholipase activity. Experiments with exogenously-added phosphatidylserine indicated that the difference was not due to the presence of that anionic phospholipid in the outer membrane leaflet. Instead, analysis of the equilibration kinetics of various cationic membrane probes revealed that the difference could relate to the spacing of membrane lipids. Specifically, ionophore treatment increased that spacing while temperature only affected overall membrane order and fluidity. To consider the possibility that the distinction with ionophore might relate to the actin cytoskeleton, cells were stained with phalloidin and imaged via confocal microscopy. Ionophore caused disruption of actin fibers while increased temperature did not. This apparent connection between membrane hydrolysis and the cytoskeleton was further corroborated by examining the relationship among these events during apoptosis stimulated by thapsigargin.
Asunto(s)
Ionóforos de Calcio/farmacología , Membrana Celular/enzimología , Calor , Ionomicina/farmacología , Fluidez de la Membrana/efectos de los fármacos , Fosfolipasas A2 Secretoras/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Línea Celular Tumoral , Ratones , Faloidina/farmacología , Fosfolípidos/metabolismo , Venenos/farmacologíaRESUMEN
We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.
Asunto(s)
Citoesqueleto de Actina/fisiología , Fertilización/fisiología , Oocitos/fisiología , Paracentrotus/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Señalización del Calcio/efectos de los fármacos , Citocalasina B/farmacología , Depsipéptidos/farmacología , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Femenino , Fertilización/efectos de los fármacos , Masculino , Proteínas de Microfilamentos/fisiología , Oocitos/efectos de los fármacos , Faloidina/farmacología , Tiazolidinas/farmacología , Cigoto/efectos de los fármacos , Cigoto/fisiologíaAsunto(s)
Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Encéfalo , Colesterol/química , Citoesqueleto/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacología , Humanos , Faloidina/farmacología , Seudópodos/efectos de los fármacos , Fibras de Estrés/efectos de los fármacosRESUMEN
Cigarette smoke extracts (CSE) may play a significant role in diseases of the upper airway including chronic rhinosinusitis. Even short term exposure of cigarette smoke has adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking. Cigarette smoke alters toll-like receptor 4 (TLR4) expression and activation in bronchial epithelial cells. Carbocysteine is an anti-oxidant and mucolytic agent. The effects of carbocysteine on CSE induced oxidative stress and on associated innate immune and inflammatory responses in nasal epithelial cells are largely unknown. The present study was aimed to assess in CSE stimulated nasal epithelial cells (RPMI 2650) the effects of carbocysteine (10(-4)M) on: cell survival, intracellular reactive oxygen species (ROS) production, TLR4 expression, LPS binding and neutrophil chemotaxis (actin reorganization). We found that CSE increased ROS production, TLR4 expression, LPS binding and neutrophil chemotaxis and all these events were counteracted by pre-incubating CSE stimulated RPMI 2650 cells with carbocysteine. In conclusion, the present study provides compelling evidence that carbocysteine may be considered a promising therapeutic strategy in chronic inflammatory nasal diseases.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Mucosa Nasal/efectos de los fármacos , Nicotiana , Estrés Oxidativo/efectos de los fármacos , Humo/efectos adversos , Productos de Tabaco , Actinas/metabolismo , Apoptosis/efectos de los fármacos , Carbocisteína/farmacología , Línea Celular , Separación Celular , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Expectorantes/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Lipopolisacáridos/metabolismo , Mucosa Nasal/citología , Mucosa Nasal/inmunología , Necrosis , Neutrófilos/efectos de los fármacos , Faloidina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/biosíntesisRESUMEN
The objective of this study was to investigate the mechanisms of increase in the efficacy of ATP-sensitive K(+) channel (KATP) openings by hypo-osmotic stress. The whole-cell KATP currents (IK,ATP) stimulated by 100 µmol/L pinacidil, a K(+) channel opening drug, were significantly augmented during hypo-osmotic stress (189 mOsmol/L) compared with normal conditions (303 mOsmol/L). The EC50 and Emax value for pinacidil-activated IK,ATP (measured at 0 mV) was 154 µmol/L and 844 pA, respectively, in normal solution and 16.6 µmol/L and 1266 pA, respectively, in hypo-osmotic solution. Augmentation of IK,ATP during hypo-osmotic stress was attenuated by wortmannin (50 µmol/L), an inhibitor of phosphatidylinositol 3- and 4-kinases, but not by (i) phalloidin (30 µmol/L), an actin filament stabilizer, (ii) the absence of Ca(2+) from the internal and external solutions, and (iii) the presence of creatine phosphate (3 mmol/L), which affects creatine kinase regulation of the KATP channels. In the single-channel recordings, an inside-out patch was made after approximately 5 min exposure of the myocyte to hypo-osmotic solution. However, the IC50 value for ATP under such conditions was not different from that obtained in normal osmotic solution. In conclusion, hypo-osmotic stress could augment cardiac IK,ATP through intracellular mechanisms involving the phosphatidylinositol kinase pathway.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Activación del Canal Iónico , Canales KATP/metabolismo , Miocitos Cardíacos/enzimología , Potasio/metabolismo , 1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Androstadienos/farmacología , Animales , Calcio/metabolismo , Tamaño de la Célula , Creatina Quinasa/metabolismo , Relación Dosis-Respuesta a Droga , Activación del Canal Iónico/efectos de los fármacos , Canales KATP/efectos de los fármacos , Masculino , Potenciales de la Membrana , Moduladores del Transporte de Membrana/farmacología , Miocitos Cardíacos/efectos de los fármacos , Presión Osmótica , Faloidina/farmacología , Fosfocreatina/metabolismo , Pinacidilo/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Endogámicas WKY , Transducción de Señal , Factores de Tiempo , WortmaninaRESUMEN
In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Glucólisis , Metaboloma , Complejos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Actinas/agonistas , Actinas/antagonistas & inhibidores , Actinas/química , Anticuerpos Antifúngicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citocalasina D/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/metabolismo , Estabilidad de Enzimas/efectos de los fármacos , Fermentación/efectos de los fármacos , Glucólisis/efectos de los fármacos , Cinética , Metaboloma/efectos de los fármacos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Faloidina/farmacología , Polimerizacion/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/agonistas , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Tiazolidinas/farmacología , Trehalosa/farmacología , Moduladores de Tubulina/farmacología , ViscosidadRESUMEN
PURPOSE: Recent evidence suggests that the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli and signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder. This led us to establish a method of co-culturing dorsal root ganglion sensory neurons and bladder urothelial cells. MATERIALS AND METHODS: Sensory neurons and urothelial cells obtained from dorsal root ganglia and bladders dissected from adult female Sprague-Dawley® rats were isolated by enzyme treatment and mechanical dissociation. They were plated together or separately on collagen coated substrate and cultured in keratinocyte medium for 48 to 72 hours. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing. RESULTS: Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to that in neuronal monocultures. The growth promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor K252a but upstream inhibition of nerve growth factor signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to adenosine triphosphate (100 µM) and activation of TRPV4 (4α-PDD, 10 µM) but not TRPV1 (capsaicin, 1 µM), TRPV3 (farnesyl pyrophosphate, 1 µM) or TRPA1 (mustard oil, 100 µM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate. CONCLUSIONS: Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Results suggest that neuronal properties are maintained in the presence of urothelium and neurite growth is potentiated by a nerve growth factor independent mechanism.
Asunto(s)
Ganglios Espinales/metabolismo , Células Receptoras Sensoriales/metabolismo , Urotelio/citología , Adenosina Trifosfato/farmacología , Análisis de Varianza , Animales , Capsaicina/farmacología , Carbazoles/farmacología , Técnicas de Cocultivo , Dronabinol/farmacología , Femenino , Fura-2/farmacología , Inmunohistoquímica , Alcaloides Indólicos/farmacología , Indoles/farmacología , Mentol/farmacología , Modelos Animales , Factor de Crecimiento Nervioso/farmacología , Faloidina/farmacología , Fosfatos de Poliisoprenilo/farmacología , Ratas , Ratas Sprague-Dawley , Sesquiterpenos/farmacología , Canales Catiónicos TRPV/biosíntesisRESUMEN
The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Arabidopsis/citología , Arabidopsis/fisiología , Fusión de Membrana , Estomas de Plantas/citología , Estomas de Plantas/fisiología , Vacuolas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/efectos de la radiación , Proteína 2 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Actinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Citocalasina D/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Luz , Fusión de Membrana/efectos de los fármacos , Fusión de Membrana/efectos de la radiación , Mutación/genética , Faloidina/farmacología , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/efectos de la radiación , Polimerizacion/efectos de los fármacos , Polimerizacion/efectos de la radiación , Imagen de Lapso de Tiempo , Vacuolas/efectos de los fármacos , Vacuolas/efectos de la radiaciónRESUMEN
Mutations in cardiac actin (ACTC) have been associated with different cardiac abnormalities in humans, including dilated cardiomyopathy and septal defects. However, it is still poorly understood how altered ACTC structure affects cardiovascular physiology and results in the development of distinct congenital disorders. A zebrafish mutant (s434 mutation) was identified that displays blood regurgitation in a dilated heart and lacks endocardial cushion (EC) formation. We identified the mutation as a single nucleotide change in the alpha-cardiac actin 1a gene (actc1a), resulting in a Y169S amino acid substitution. This mutation is located at the W-loop of actin, which has been implicated in nucleotide sensing. Consequently, s434 mutants show loss of polymerized cardiac actin. An analogous mutation in yeast actin results in rapid depolymerization of F-actin into fragments that cannot reanneal. This polymerization defect can be partially rescued by phalloidin treatment, which stabilizes F-actin. In addition, actc1a mutants show defects in cardiac contractility and altered blood flow within the heart tube. This leads to downregulation or mislocalization of EC-specific gene expression and results in the absence of EC development. Our study underscores the importance of the W-loop for actin functionality and will help us to understand the structural and physiological consequences of ACTC mutations in human congenital disorders.
Asunto(s)
Actinas/química , Actinas/genética , Endocardio/embriología , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Mutación , Pez Cebra/embriología , Actinas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Embrión no Mamífero/anomalías , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Endocardio/anomalías , Endocardio/metabolismo , Corazón/efectos de los fármacos , Corazón/embriología , Corazón/fisiología , Corazón/fisiopatología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/fisiopatología , Modelos Moleculares , Datos de Secuencia Molecular , Morfogénesis , Miocardio/metabolismo , Miofibrillas/genética , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Faloidina/farmacología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Toxins that impair the function of actin microfilaments in cytoskeleton, cytochalasin B (disrupts microfilaments by inhibiting actin polymerization) and phalloidin (binds polymeric F-actin, stabilizing it and interfering with the function of actin-rich structures) reduce the depression of acetylcholine-induced inward current in Helix lucorum command neurons of defensive behavior during rhythmical local acetylcholine applications to soma (cellular analogue of habituation). These results and mathematical simulation allow us to suggest that the depression of cholinosensitivity of extrasynaptic membrane zones in command neurons on the cellular analogue of habituation is associated with the involvement of actin microfilaments in reduction of the number of membrane cholinoreceptors.
Asunto(s)
Acetilcolina/metabolismo , Actinas/metabolismo , Habituación Psicofisiológica , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Neuronas/metabolismo , Acetilcolina/farmacología , Actinas/antagonistas & inhibidores , Animales , Simulación por Computador , Citocalasina B/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Caracoles Helix , Modelos Neurológicos , Faloidina/farmacologíaRESUMEN
Actin, which is normally depleted in the nuclei of somatic cells, accumulates in high amounts in giant nuclei of amphibian oocytes. The supramolecular organization and functions of this nuclear pool of actin in growing vertebrate oocyte are controversial. Here, we investigated the role of nuclear actin in the maintenance of the spatial architecture of intranuclear structures in avian and amphibian growing oocytes. A meshwork of filamentous actin was not detected in freshly isolated or fixed oocyte nuclei of Xenopus, chicken or quail. We found that the actin meshwork inside the oocyte nucleus could be induced by phalloidin treatment. Actin polymerization is demonstrated to be required to stabilize the specific spatial organization of nuclear structures in avian and amphibian growing oocytes. In experiments with the actin depolymerizing drugs cytochalasin D and latrunculin A, we showed that disassembly of nuclear actin polymers led to chromosome condensation and their transportation to a limited space within the oocyte nucleus. Experimentally induced "collapsing" of chromosomes and nuclear bodies, together with global inhibition of transcription, strongly resembled the process of karyosphere formation during oocyte growth.