Cadmium and/or copper excess induce interdependent metal accumulation, DNA methylation, induction of metal chelators and antioxidant defences in the seagrass Zostera marina

Publisher’s embargo period: Embargo set on 01.03.2019 by SR (TIS)

ROS Production via P2Y(1)-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

Artículo de publicación ISIDuring exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7-dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y(1) receptor agonist, induced a large signal while UTPyS (P2Y(2) agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y(1). Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y(1) receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.CONICYT-PIA ACT111 FONDECYT 111046

Electrical stimulation induced ROS production via ATP release and purinergic receptor.

<p>Muscle fibers were isolated, load with DCF fluorescence (30 min) under control or stimulated, in the absence or presence of different inhibitors (30 min of incubation). A, representative traces of DCF fluorescence under control or stimulated, in absence or presence of CBX (10μM). B, slope of fluorescence from muscle cells stimulated with ES (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>), in the absence or presence of CBX. C, representative traces of DCF fluorescence under control or stimulated, in the absence or presence of suramin (10μM). D, slope of fluorescence from muscle cells stimulated with ES, in the absence or presence of suramin. (n = 6), **p<0.01, ***p<0.001.</p

Exogenous ATP increases H₂O₂ production via P2Y₁ purinergic receptor.

<p>Muscle fibers were isolated and transfected with HyPer plasmid, 24h post transfection the cells were stimulated. A, H₂O₂ generation was measured before and after ATP (10μM) addition. Left panel shows a representative image of Hyper transfection, right panel image fluorescence in pseudo-color. The scale bar represents 50μm. B, kinetics of extracellular ATP-induced H₂O_2. C, muscle fibers were transfected with with HyPer plasmid and stimulated with ES in absence or presence of apyrase (2U/ml). Maximal fluorescence was plotted D, C. Effect of Mrs2365, Mrs2179, UTPγS or exogenous ATP, maximal fluorescence was plotted (n = 4), *p<0.05, **p<0.01.</p

Working model.

<p>Electrical stimulation in adult FDB fibers activates Cav1.1 with each depolarizing event. This activation in turn induces ATP release via PnX1 channel. These events will trigger in turn a signaling cascade where, through activation of P2Y₁ receptors, PI3K and PLC and consequent PKC activation induces NOX2 activation and ROS production. Dotted lines show signaling pathways already described. Solid lines show our observations.</p

Electrical stimulation and exogenous ATP increase ROS production via NOX2.

<p>Muscle fibers were isolated, loaded with DCF (30min) and stimulated with electrical stimulation (ES) or exogenous ATP A, representative traces of DCF fluorescence under control conditions or stimulated with ES in the absence or presence of apocynin. B, Muscle cells were stimulated with ES and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">Materials and Methods</a></i>). C, Representative traces of DCF fluorescence under control conditions or stimulated with ES in the absence or presence of gp91ds-TAT or scrambled peptide. D, muscle cells were stimulated with ES and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>). E, representative traces of DCF fluorescence under control or stimulated with exogenous ATP in the absence or presence of gp91ds-TAT or scrambled peptide. F, muscle cells were stimulated with ES and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>)(n = 5, *p<0.05, **p<0.01, ***p<0.001).</p

Extracellular ATP induces NOX2 activation via PKC.

<p>Muscle fibers were isolated, loaded with DCF (30min) and stimulated with exogenous ATP. A, representative traces of DCF fluorescence under control or stimulated with ATP in the absence or presence of BIM (5μM). B, muscle cells were stimulated with ATP and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>) (n = 3, *p<0.05). C, muscle fibers were isolated and transfected with HyPer plasmid, 24h post transfection the cells were stimulated in the presence of PMA, BIM or Rotterin (Rotl) as indicated in the graph, maximal fluorescence was plotted (n = 5), *p<0.05, **p<0.01.</p

Anabolic Androgenic Steroids and Intracellular Calcium Signaling: A Mini Review on Mechanisms and Physiological Implications

Artículo de publicación ISIIncreasing evidence suggests that nongenomic effects of testosterone and anabolic androgenic steroids (AAS) operate concertedly with genomic effects. Classically, these responses have been viewed as separate and independent processes, primarily because nongenomic responses are faster and appear to be mediated by membrane androgen receptors, whereas long-term genomic effects are mediated through cytosolic androgen receptors regulating transcriptional activity. Numerous studies have demonstrated increases in intracellular Ca(2+) in response to AAS. These Ca(2+) mediated responses have been seen in a diversity of cell types, including osteoblasts, platelets, skeletal muscle cells, cardiac myocytes and neurons. The versatility of Ca(2+) as a second messenger provides these responses with a vast number of pathophysiological implications. In cardiac cells, testosterone elicits voltage-dependent Ca(2+) oscillations and IP(3)R-mediated Ca(2+) release from internal stores, leading to activation of MAPK and mTOR signaling that promotes cardiac hypertrophy. In neurons, depending upon concentration, testosterone can provoke either physiological Ca(2+) oscillations, essential for synaptic plasticity, or sustained, pathological Ca(2+) transients that lead to neuronal apoptosis. We propose therefore, that Ca(2+) acts as an important point of crosstalk between nongenomic and genomic AAS signaling, representing a central regulator that bridges these previously thought to be divergent responses

Electrical stimulation and exogenous ATP induced increase of ROS production.

<p>Muscle fibers were isolated, load with DCF (30min) and stimulated with electrical stimulation (ES) (20Hz) or exogenous ATP (10μM). A. Representative image in pseudo-color of a cell loaded with DCF in control condition (upper panel) and stimulated with ES (lower panel). B, representative traces of DCF fluorescence under control or stimulated with ES or exogenous ATP. C, muscle cells were stimulated with ES or ATP and the slope of fluorescence is shows (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>). D, muscle cells were stimulated with ES at 20Hz or 90Hz and the slope of fluorescence is show (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">Materials and Methods</a></i>) (n = 4, *p<0.05, **p<0.01).</p