RESUMEN
Atrial myocytes in a number of species lack transverse tubules. As a consequence the intracellular calcium signals occurring during each heartbeat exhibit complex spatio-temporal dynamics. These calcium patterns arise from saltatory calcium waves that propagate via successive rounds of diffusion and calcium-induced calcium release. The many parameters that impinge on calcium-induced calcium release and calcium signal propagation make it difficult to know a priori whether calcium waves will successfully travel, or be extinguished. In this study, we describe in detail a mathematical model of calcium signalling that allows the effect of such parameters to be independently assessed. A key aspect of the model is to follow the triggering and evolution of calcium signals within a realistic three-dimensional cellular volume of an atrial myocyte, but with low computational costs. This is achieved by solving the linear transport equation for calcium analytically between calcium release events and by expressing the onset of calcium liberation as a threshold process. The model makes non-intuitive predictions about calcium signal propagation. For example, our modelling illustrates that the boundary of a cell produces a wave-guiding effect that enables calcium ions to propagate further and for longer, and can subtly alter the pattern of calcium wave movement. The high spatial resolution of the modelling framework allows the study of any arrangement of calcium release sites. We demonstrate that even small variations in randomly positioned release sites cause highly heterogeneous cellular responses. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Asunto(s)
Señalización del Calcio/fisiología , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Humanos , Miocitos Cardíacos/citologíaRESUMEN
Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.
Asunto(s)
Axones/metabolismo , Axotomía , Calcio/metabolismo , Neuropatía Ciática/etiología , Degeneración Walleriana/metabolismo , Degeneración Walleriana/patología , Animales , Axones/patología , Axones/ultraestructura , Benzofuranos , Células Cultivadas , Ganglios Espinales/citología , Regulación de la Expresión Génica/genética , Imidazoles , Microscopía Electrónica de Transmisión , Mutación/genética , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Proteínas del Tejido Nervioso/genética , Neuritas/metabolismo , Neuritas/ultraestructura , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Mutantes , Neuropatía Ciática/complicaciones , Factores de Tiempo , Degeneración Walleriana/etiologíaRESUMEN
Ca(2+) increases in the heart control both contraction and transcription. To accommodate a short-term increased cardiovascular demand, neurohormonal modulators acting on the cardiac pacemaker and individual myocytes induce an increase in frequency and magnitude of myocyte contraction respectively. Prolonged, enhanced function results in hypertrophic growth of the heart, which is initially also associated with greater Ca(2+) signals and cardiac contraction. As a result of disease, however, hypertrophy progresses to a decompensated state and Ca(2+) signalling capacity and cardiac output are reduced. Here, the role that Ca(2+) plays in the induction of hypertrophy as well as the impact that cardiac hypertrophy and failure has on Ca(2+) fluxes will be discussed.
Asunto(s)
Calcio/metabolismo , Miocardio/metabolismo , Señalización del Calcio , Cardiomegalia/metabolismo , Humanos , Contracción MiocárdicaRESUMEN
The S100 family member S100A9 and its heterodimeric partner, S100A8, are cytosolic Ca2+ binding proteins abundantly expressed in neutrophils. To understand the role of this EF-hand-containing complex in Ca2+ signalling, neutrophils from S100A9 null mice were investigated. There was no role for the complex in buffering acute cytosolic Ca2+ elevations. However, Ca2+ responses to inflammatory agents such as chemokines MIP-2 and KC and other agonists are altered. For S100A9 null neutrophils, signalling at the level of G proteins is normal, as is release of Ca2+ from the IP(3) receptor-gated intracellular stores. However MIP-2 and FMLP signalling in S100A9 null neutrophils was less susceptible than wildtype to PLCbeta inhibition, revealing dis-regulation of the signalling pathway at this level. Downstream of PLCbeta, there was reduced intracellular Ca2+ release induced by sub-maximal levels of chemokines. Conversely the response to FMLP was uncompromised, demonstrating different regulation compared to MIP-2 stimulation. Study of the activity of PLC product DAG revealed that chemokine-induced signalling was susceptible to inhibition by elevated DAG with S100A9 null cells showing enhanced inhibition by DAG. This study defines a lesion in S100A9 null neutrophils associated with inflammatory agonist-induced IP3-mediated Ca2+ release that is manifested at the level of PLCbeta.
Asunto(s)
Señalización del Calcio , Calgranulina B/metabolismo , Factores Quimiotácticos/farmacología , Neutrófilos/metabolismo , Animales , Calgranulina B/genética , Células Cultivadas , Quimiocina CXCL2 , Quimiocinas/farmacología , Diglicéridos/metabolismo , Estrenos/farmacología , Homeostasis , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Ratones , Ratones Noqueados , Modelos Biológicos , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Pirrolidinonas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a novel mechanism for treating a variety of CNS disorders. The aim of this study was to investigate pathways controlling neurite outgrowth in human neural precursor cells, in particular in response to platelet-derived growth factor (PDGF). PDGF-AA, -AB and -BB were found to initiate calcium signalling and produce robust increases in neurite outgrowth. PDGF-induced outgrowth of Tuj1-positive precursors was abolished by the addition of EGTA, suggesting that calcium entry is a critical part of the signalling pathway. Wortmannin and PD098059 failed to inhibit PDGF-induced outgrowth. Clostridium Toxin B increased the amount of PDGF-induced neurite branching but had no effect on basal levels. In contrast, WHI-P154, an inhibitor of Janus protein tyrosine kinase (JAK3), Hck and Syk, prevented PDGF-induced neurite outgrowth. PDGF activates multiple signalling pathways with considerable potential for cross-talk. This study has highlighted the complexity of the pathways leading to neurite outgrowth in human neural precursors, and provided initial evidence to suggest that calcium entry is critical in producing the morphological changes observed.
Asunto(s)
Señalización del Calcio/fisiología , Diferenciación Celular/fisiología , Neuritas/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinas/farmacología , Células Madre/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Janus Quinasa 3 , Neuritas/metabolismo , Neuritas/ultraestructura , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Trasplante de Células Madre/métodos , Células Madre/citología , Células Madre/metabolismo , Toxina Tetánica/farmacología , Tubulina (Proteína)/metabolismoRESUMEN
Calcium is a pleiotropic messenger controlling a diverse array of intracellular events from fertilization to cell death. One of the main mechanisms by which intracellular calcium is elevated is through InsP(3) [Ins(1,4,5)P(3)]-induced mobilization of calcium from its receptor on the endoplasmic reticulum calcium store. The activity of the InsP(3)R (InsP(3) receptor) is subject to regulation by many factors other than InsP(3), most notably calcium itself, which regulates the channel in a bell-shaped dependent manner. InsP(3)R sensitivity is also regulated by post-translational modifications such as phosphorylation and by binding of accessory proteins. Taken together it appears that the InsP(3)R can be regarded as a cellular sensor for many signalling pathways, qualitatively and quantitatively regulating intracellular calcium signals with consequences for downstream cellular physiology.
Asunto(s)
Canales de Calcio/química , Calcio/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Transducción de Señal , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Modelos Biológicos , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Elementary Ca(2+) signals, such as "Ca(2+) puffs", which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca(2+) signalling. We characterized Ca(2+) puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca(2+) puffs in these cell types suggests that they are a generic elementary Ca(2+) signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca(2+) signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca(2+) puffs. The amplitude and duration of Ca(2+) puffs were substantially reduced. In addition, the number of Ca(2+) puff sites active during the onset of a Ca(2+) wave declined. The consequence of the changes in Ca(2+) puff properties was that cells displayed a lower propensity to trigger regenerative Ca(2+) waves. Therefore, Ca(2+) puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Adenosina Trifosfato/farmacología , Animales , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Línea Celular , Núcleo Celular/metabolismo , Regulación hacia Abajo , Histamina/farmacología , Humanos , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line
Asunto(s)
Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas/metabolismo , Androstadienos/farmacología , Animales , Brefeldino A/farmacología , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/genética , Endosomas/química , Endosomas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Aparato de Golgi/efectos de los fármacos , Proteínas de la Matriz de Golgi , Proteínas Fluorescentes Verdes , Humanos , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de Superficie , Wortmanina , Dedos de ZincRESUMEN
Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca(2+) to play a pivotal role in cell biology results from the facility that cells have to shape Ca(2+) signals in space, time and amplitude. To generate and interpret the variety of observed Ca(2+) signals, different cell types employ components selected from a Ca(2+) signalling 'toolkit', which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca(2+) signals that suit their physiology. Recent studies have demonstrated the importance of local Ca(2+) signals in defining the specificity of the interaction of Ca(2+) with its targets. Furthermore, local Ca(2+) signals are the triggers and building blocks for larger global signals that propagate throughout cells.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Potenciales de Acción , Animales , Canales de Calcio Tipo T/metabolismo , Humanos , Transporte Iónico , Mitocondrias/metabolismo , Músculos/citología , Músculos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neurotransmisores/metabolismo , Páncreas/citología , Páncreas/metabolismoRESUMEN
Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.
Asunto(s)
Calcio/metabolismo , Mitocondrias/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Transporte IónicoRESUMEN
Calcium (Ca2+) is an almost universal intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca2+ to play a pivotal role in cell biology results from the facility that cells have to shape Ca2+ signals in the dimensions of space, time and amplitude. To generate the variety of observed Ca2+ signals, different cell types employ components selected from a Ca2+ signalling 'toolkit', which comprizes an array of signalling, homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca2+ signals that suit their physiology.
Asunto(s)
Señalización del Calcio/fisiología , Animales , Calcio/metabolismo , Calcio/fisiología , Canales de Calcio/fisiología , Homeostasis/fisiología , HumanosRESUMEN
Excitation-contraction coupling (E-C coupling) was studied in isolated fluo-3-loaded rat atrial myocytes at 22 and 37 degrees C using rapid confocal microscopy. Within a few milliseconds of electrical excitation, spatially discrete subsarcolemmal Ca2+ signals were initiated. Twenty to forty milliseconds after stimulation the spatial overlap of these Ca2+ signals gave a 'ring' of elevated Ca2+ around the periphery of the cells. However, this ring was not continuous and substantial Ca2+ gradients were observed. The discrete subsarcolemmal Ca2+-release sites, which responded in a reproducible sequence to repetitive depolarisations and displayed the highest frequencies of spontaneous Ca2+ sparks in resting cells, were denoted 'eager sites'. Immunostaining atrial myocytes for type II ryanodine receptors (RyRs) revealed both subsarcolemmal 'junctional' RyRs, and also 'non-junctional' RyRs in the central bulk of the cells. A subset of the junctional RyRs comprises the eager sites. For cells paced in the presence of 1 mM extracellular Ca2+, the response was largely restricted to a subsarcolemmal 'ring', while the central bulk of the cell displayed a approximately 5-fold lower Ca2+ signal. Under these conditions the non-junctional RyRs were only weakly activated during E-C coupling. However, these channels are functional and the Ca2+ stores were at least partially loaded, since substantial homogeneous Ca2+ signals could be stimulated in the central regions of atrial myocytes by application of 2.5 mM caffeine. Neither the location nor activation order of the eager sites was affected by increasing the trigger Ca2+ current (by increasing extracellular Ca2+ to 10 mM) or the sarcoplasmic reticulum (SR) Ca2+ load (following 1 min incubation in 10 mM extracellular Ca2+), although with increased SR Ca2+ load, but not greater Ca2+ influx, the delay between the sequential activation of eager sites was reduced. In addition, increasing the trigger Ca2+ current or the SR Ca2+ load changed the spatial pattern of the Ca2+ response, in that the Ca2+ signal propagated more reliably from the subsarcolemmal initiation sites into the centre of the cell. Due to the greater spatial spread of the Ca2+ signals, the averaged global Ca2+ transients increased by approximately 500 %. We conclude that rat atrial myocytes display a predetermined spatiotemporal pattern of Ca2+ signalling during early E-C coupling. A consistent set of eager Ca2+ release sites with a fixed location and activation order on the junctional SR serve to initiate the cellular response. The short latency for activation of these eager sites suggests that they reflect clusters of RyRs closely coupled to voltage-operated Ca2+ channels in the sarcolemma. Furthermore, their propensity to show spontaneous Ca2+ sparks is consistent with an intrinsically higher sensitivity to Ca2+-induced Ca2+ release. While the subsarcolemmal Ca2+ response can be considered as stereotypic, the central bulk of the cell grades its response in direct proportion to cellular Ca2+ load and Ca2+ influx.
Asunto(s)
Calcio/metabolismo , Corazón/fisiología , Contracción Miocárdica/fisiología , Animales , Cafeína/farmacología , Señalización del Calcio/fisiología , Adhesión Celular , Células Cultivadas , Atrios Cardíacos , Cinética , Masculino , Miocardio/citología , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Sarcolema/fisiología , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/fisiología , Factores de TiempoRESUMEN
BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Endocitosis/fisiología , Ácidos Grasos/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Sitios de Unión , Transporte Biológico , Proteínas Sanguíneas/genética , Proteínas Portadoras/genética , Línea Celular , Membrana Celular/metabolismo , Pollos , Activación Enzimática , Ácidos Grasos/genética , Lipoproteínas/genética , Proteínas de la Membrana/genética , Fosfoproteínas/metabolismo , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Porcinos , Vesículas Transportadoras/metabolismo , Tirosina/metabolismoRESUMEN
Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon several considerations including Ca2+ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high-amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower in-situ Ca2+ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca2+ binding affinities between nuclear and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptible to photobleaching than many of the other Ca2+ indicators.
Asunto(s)
Señalización del Calcio , Colorantes Fluorescentes , Compuestos de Anilina , Benzofuranos , Calcio/metabolismo , Calibración , Compartimento Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Imidazoles , Compuestos Orgánicos , XantenosRESUMEN
The Ca(2+)-modulating cyclophilin ligand (CAML) protein causes stimulation of transcription factors via activation of a store-operated Ca(2+) entry pathway. Since CAML is widely expressed in mammalian tissues, it may be an important regulator of Ca(2+) store function. In the present study, we investigated the consequence of CAML overexpression on Ca(2+) signaling using rapid confocal imaging of Fluo3-loaded NIH3T3 fibroblasts. Control and CAML-expressing cells gave concentration-dependent responses to the Ca(2+) mobilizing agonist ATP. CAML expression reduced the sensitivity of the cells so that higher concentrations of ATP were needed to achieve global Ca(2+) waves. The amplitudes of Ca(2+) waves were significantly reduced in CAML expressing cells, consistent with earlier suggestions that CAML causes depletion of internal Ca(2+) stores. With low ATP concentrations, only local Ca(2+) release events were observed. CAML did not affect the characteristics of these local Ca(2+) signals, suggesting that it does not directly affect Ca(2+) release channels.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfato/farmacología , Calcio/fisiología , Proteínas Portadoras/fisiología , Transducción de Señal , Células 3T3 , Animales , Transporte Biológico , Relación Dosis-Respuesta a Droga , Ratones , Transducción de Señal/efectos de los fármacosRESUMEN
Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Lisofosfolípidos/farmacología , Receptores de Superficie Celular/fisiología , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Cafeína/farmacología , Canales de Calcio/fisiología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Neuroblastoma , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores del Ácido Lisofosfatídico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/metabolismo , Esfingosina/farmacología , Células Tumorales CultivadasRESUMEN
The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.
Asunto(s)
Canales de Calcio/metabolismo , Corazón/fisiología , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Western Blotting , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato , Miocardio/citología , Reacción en Cadena de la Polimerasa , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/fisiologíaRESUMEN
The topic of nuclear Ca2+ signalling is beset by discrepant observations of substantial nuclear/cytoplasmic gradients. The reasons why some labs have recorded such gradients, whilst other workers see equilibration of Ca2+(cyt) and Ca2+(nuc) using the same cells and techniques, is unexplained. Furthermore, how such gradients could arise across the NE that possesses many highly-conductive NPCs is a mystery. Although nuclei may have the capacity to be autonomous signalling entities, with functional Ca2+ release channels and an inositide cycle, the balance of evidence suggests that Ca2+ release on the inner NE does not occur during physiological stimulation. Our work suggests that elementary Ca2+ release events originating in the cytoplasm can give rise to Ca2+ signals without causing elevation of the bulk cytoplasm. Clearly, the many Ca2+ signalling mechanisms that may impinge on Ca2+(nuc) will remain a topic of controversy and debate for some time.
Asunto(s)
Calcio/fisiología , Núcleo Celular/fisiología , Transducción de Señal , Animales , HumanosRESUMEN
We investigated the consequences of depolarizing the mitochondrial membrane potential (Deltapsi(mit)) on Ca(2+) signals arising via inositol 1,4,5-trisphosphate receptors (InsP(3)R) in hormone-stimulated HeLa cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or a mixture of antimycin A+oligomycin were found to rapidly depolarize Deltapsi(mit). Mitochondrial depolarization enhanced the number of cells responding to a brief application of a Ca(2+)-mobilizing hormone and prolonged the recovery of cytosolic Ca(2+) after washout of the hormone; effects consistent with the removal of a passive Ca(2+) buffer. However, with repeated application of the same hormone concentration both the number of responsive cells and peak Ca(2+) changes were observed to progressively decline. The inhibition of Ca(2+) signalling was observed using different Ca(2+)-mobilizing hormones and also with a membrane-permeant Ins(1,4,5)P(3) ester. Upon washout of FCCP, the Ca(2+) signals recovered with a time course similar to the re-establishment of Deltapsi(mit). Global measurements indicated that none of the obvious factors such as changes in pH, ATP concentration, cellular redox state, permeability transition pore activation or reduction in Ca(2+)-store loading appeared to underlie the inhibition of Ca(2+) signalling. We therefore suggest that local changes in one or more of these factors, as a consequence of depolarizing Deltapsi(mit), prevents InsP(3)R activation.