Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

Submitted by Sandra Infurna ([email protected]) on 2016-12-13T15:06:03Z No. of bitstreams: 1 leonardo_ferreira_etal_IOC_2016.pdf: 3073404 bytes, checksum: bcdeb147a5807f576266e60f4c1d2e0e (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-13T15:23:31Z (GMT) No. of bitstreams: 1 leonardo_ferreira_etal_IOC_2016.pdf: 3073404 bytes, checksum: bcdeb147a5807f576266e60f4c1d2e0e (MD5)Made available in DSpace on 2016-12-13T15:23:31Z (GMT). No. of bitstreams: 1 leonardo_ferreira_etal_IOC_2016.pdf: 3073404 bytes, checksum: bcdeb147a5807f576266e60f4c1d2e0e (MD5) Previous issue date: 2016Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Ciências Biológicas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neurogeneses. Rio de Janeiro, RJ, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inflamação. Rio de Janeiro, RJ. Brasil.Universidade Federal Fluminense. Departamento de Fisiologia e Farmacologia. Laboratório de Neurofarmacologia. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxoplasmose. Rio de Janeiro, RJ, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil.Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as βIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 μM and MK-801 20 μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit

Immunocytochemistry of avian mixed neuron-Müller glial cells in culture and calcium responses by selective agents.

<p>Mixed neuron-glia retina cells in culture. A. Neurons were immunostained with the Tuj-1 antibody (β_III Tubulin, red) while glia cells were labeled with 2M6 (green). Nuclei were labeled with DAPI (blue). B. Cell calcium imaging traces of emission fluorescence (ratio 340/380 nm excitation) of a representative β_III tubulin labeled neuron (red) activated by 50mM KCl. C. A representative 2M6 labeled glial cell (green) activated by 5mM GSH; D. Quantification of the functional responses of 2M6+ cells (glia) show that 92% were responsive to 5mM GSH and none responded to 50 mM KCl; Alternatively, 64% of Tuj-1 β_III Tubulin+ cells were activated by 50mM KCl; at least 280 cells were analyzed (n = 5). A. 50μm scale bar. *Calculated significance was at least p < 0.05.</p

GSH does not induce death in retinal cells in culture.

<p>GSH does not induce cell death in Müller glia cells in culture A-C. A 60 seconds 5mM GSH does not induce cell death, but 30nM thapsigargin induce apoptosis. A and B. No TUNEL positive cells are found in the control or in the GSH-treated (5mM) cells in cultures. C. Retinal cells in culture treated with thapsigargin (30nM) show innumerous TUNEL-labeled regions. 50μm scale bar. N = 3, performed in triplicate. D-G. Live and Dead viability assay for a 60s GSH 5mM stimulus. D. Control or E. GSH 5mM treated cultures show few or none labeled cells (red as propidium iodine positive cells), while F. H₂O₂ 0.1%. treated cultures were essentially labelled with propidium iodine (red). G. GSH 5mM treated cultures for 60s is sufficient to protect partialy retinal cells in culture. D. 50μm scale bar. Total of three individual experiments (n = 3).</p

Analysis of ROS activity based on a DCF assay in Müller cells.

<p>(A and B.) Live/Dead cell viability in the presence of 0.1% H₂O₂ (C to F) ROS activity (DCFH oxidation) in the presence of 5mM GSH or compared to control levels. (C and D.) Cells incubated with hydrogen peroxide (H₂O₂ 0.1%) did not survive or were extremely stressed. (E and F.) Cells were able to survive when pre-incubated for 60 seconds with 5mM GSH followed by H₂O₂-induced (0.1%) oxidative stress. G. Even through intense ROS activity, cells incubated with H₂O₂ 0.1% + GSH 5mM were protected and able to survive in H₂O₂-induced toxicity condition. At least five (n = 5) individual experiments were performed for each condition. B and F. 100μm and 50μm scale bars, respectively. *Calculated significance was at least <i>p</i> < 0.05.</p

GSH-induced calcium shifts in enriched glial retinal culture cells.

<p>A. Mixed neuron-glia cells in culture in bright field or in fura-2 loaded fluorescence. Neurons are identified by brighter soma compared to glia. B. Cells were chosen (colored circles) and a dose response curve for GSH-induced calcium responses is seen for each cell. As shown, only glia cells respond to GSH, indicated in the arrows below (5–10mM). C. Panel C shows that the majority of glial cells respond to GSH. At least 350 cells were analyzed (n = 6). A. 50μm scale bar. *Calculated significance was at least <i>p</i> < 0.05.</p

Quantification of GSH in Müller glial cells after a 60 seconds incubation.

<p>Intracellular GSH concentrations after a 60s incubation with either Krebs solution or with 5mM GSH. Incubation time aimed to mimic single cell calcium imaging experiments, where cellular response to antioxidants were evaluated (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153677#pone.0153677.g001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153677#pone.0153677.g006" target="_blank">6</a>). *Calculated significance was at least <i>p</i> < 0.05.</p

Effect of ionotropic NMDA (MK-801) and non-NMDA (DNQX) or P2X7 (BBG) receptor antagonists on calcium responses induced by GSH in glial cells.

<p>A. Cultured Müller cells in bright field or in fura-2 loaded fluorescence. B. A 30 minutes pre-incubation with 70μM DNQX + 25μM MK-801 followed by a single GSH (5mM) stimulus with or without antagonists. C. After quantification, both GSH (5mM) stimuli were able to evoke calcium responses in Müller glial cells. Total of 117 cells were analyzed for GSH 5mM without inhibitors (n = 3), while 115 cells were evaluated for GSH 5mM in the presence of inhibitors (n = 3). D. Data quantification of calcium responses shows that no cells are activated by 5mM GSH in the presence of 100 nM BBG. E. Calcium transient is recovered when 100nM BBG is washed and only 5mM GSH is incubated. No statistical differences were found between samples in C. *Calculated significance was at least <i>p</i> < 0.05. A. 100μm scale bar.</p

GSH induced PI uptake is inhibited by P2X7 receptor antagonists in Müller glia cell culture.

<p>Permeabilization in Müller glia cells depends on P2X7 receptor. (A-H) Left panels are phase contrast, while right are fluorescence images showing PI uptake. No drugs were added (A), or 1 mM ATP (B), and in the presence of 1 μM A74003 (C) or 5 μM BAPTA-AM in avian Muller glia (D). Alternatively, 5mM GSH was added (E), plus 500 nM BBG (F), or with 1 μM A74003 (C) or 5 μM BAPTA-AM (H). P2X7 receptor antagonists or BAPTA-AM were added 10 minutes before ATP or GSH for 20 min. Quantification of the percentage of fluorescente cells is shown in I. N = 3 plates done in triplicate. (J) GSH induced PI uptake is inhibited by P2X7R antagonists. Permeabilization assays with 1 (in black), 5 (in yellow) and 10 mM (in brown) GSH measured in intervals of 2.5 minutes until 60 minutes. A740003 was added for 10 minutes before 5 mM GSH treatment (orange) or 10 mM GSH (in green). Negative control (saline) is in blue while cells stimulated with 1 mM ATP are in red. These signals are representative of duplicate measurements performed in 2–3 independent days.</p