Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model
<p>(<b>a</b>) Example of SEM images of Croc and FAV173 fibers. (<b>b</b>) Vitelline membrane of non-treated (Ctrl), Croc-treated, and ground FAV173-treated oocytes. Note, in (<b>b</b>), a Croc fiber partially inserted into the pore of the vitelline membrane. Croc = 15 μg/mL, FAV173 = 200 μg/mL.</p> "> Figure 2
<p>Dose–response effect of FAV173 fiber exposure (from 15 to 400 μg/mL) on resting potential (RP) in (<b>a</b>) and membrane resistance (R<sub>m</sub>) in (<b>b</b>). In (<b>a</b>,<b>b</b>), the green bars show the effect of 15 μg/mL of Croc for comparison. The effect of FAV173 and Croc is expressed as % of decrease with respect to untreated cells (Ctrl) from the same donor. * <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001 fibers vs. Ctrl (unpaired <span class="html-italic">t</span>-test). In (<b>c</b>), the time course effect of Croc and FAV treatments on RP (<span class="html-italic">n</span> ≥ 3 donors). <sup>§§§</sup> <span class="html-italic">p</span> < 0.001 FAV173 vs. Croc, <sup>##</sup> <span class="html-italic">p</span> < 0.01 Croc (15–20) vs. Croc (5–10), (unpaired <span class="html-italic">t</span>-test). In (<b>d</b>), the effect on RP was irreversible only at higher fiber concentrations in Croc-treated cells (120 min, Croc: 45 μg/mL; FAV173: 600 μg/mL). *** <span class="html-italic">p</span> < 0.001 Croc vs. Ctrl (one-way ANOVA with Tukey’s post hoc).</p> "> Figure 3
<p>In (<b>a</b>), examples of current traces recorded by stepping the membrane potential from −80 to +40 mV (10 mV steps, 3 s, V<sub>h</sub> = −40 mV) in non-treated (Ctrl, black), Croc-treated (green), and FAV173-treated (red) cells. (<b>b</b>) I–V relationships from Ctrl, Croc-treated, and FAV173-treated cells (same batch). Note the outward rectification in treated cells. (<b>c</b>) Percentage increase in the evoked current amplitude measured at −80 mV and +40 mV and normalized to their respective Ctrl (of the same donor). *** <span class="html-italic">p</span> < 0.001, Fiber-treatments vs. Ctrl; <sup>§§</sup> <span class="html-italic">p</span> < 0.001, Croc vs. FAV173 (unpaired <span class="html-italic">t</span>-test). Croc: 15 μg/mL; FAV173: 200 μg/mL.</p> "> Figure 4
<p>(<b>a</b>, <b>left</b>) Examples of recording traces from Ctrl cells in normal bathing solution, in presence of [Ca<sup>2+</sup>]<sub>e</sub> = 11 mM (Ca11) and Ca11 + Ani9 (1 μM). (<b>a</b>, <b>right</b>) I–V relationships in Ctrl (normal bathing solution), Ca11, and Ca11 + Ani9 (1 μM) conditions. Note that, in Ca11, the currents at negative potentials increased with respect to those recorded in normal bathing solution, and the evident blocking effect of Ani9. (<b>b</b>, <b>left</b>) Evoked currents from FAV173-treated (200 μg/mL) cells in presence of Ca11 and Ca11 + Ani9 (1 μM). (<b>b</b>, <b>right</b>) The I–V relationships revealed a significant increase in the evoked currents at positive and negative potentials in presence of Ca11 and a partial blocking effect induced by Ani9. Ctrl: <span class="html-italic">n</span> = 5; FAV173: <span class="html-italic">n</span> = 7, same donor. * <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001, Fibers vs. Ctrl (unpaired <span class="html-italic">t</span>-test); <sup>§</sup> <span class="html-italic">p</span> < 0.05, <sup>§§</sup> <span class="html-italic">p</span> < 0.01, <sup>§§§</sup> <span class="html-italic">p</span> < 0.001, Ca11 vs. Ca11 + Ani9 (paired <span class="html-italic">t</span>-test).</p> "> Figure 5
<p>Example of SEM images of microvilli in (<b>a</b>) Ctrl oocyte, and oocytes in Ca11 bath condition in the absence and in the presence of FAV173 (200 μg/mL). In the inset of Ctrl, the vitelline membrane (VM) above the microvilli (MV) is shown. The FAV173 effect on the diameter (<b>b</b>) and density (<b>c</b>) of the microvilli (<b>c</b>), *** <span class="html-italic">p</span> < 0.001 vs. Ca11 (unpaired <span class="html-italic">t</span>-test). In (<b>d</b>) microvilli of Croc and (Fe<sup>3+</sup> + H<sub>2</sub>O<sub>2</sub>)-treated oocytes. Comparison of microvilli diameter (<b>e</b>) and density (<b>f</b>) values measured under the 3 test conditions (* <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001 vs. Ctrl, <sup>§</sup> <span class="html-italic">p</span> < 0.05, <sup>§§§</sup> <span class="html-italic">p</span> < 0.001 (Fe<sup>3+</sup> + H<sub>2</sub>O<sub>2</sub>) vs. Croc, One-Way ANOVA with Tukey’s post hoc). Cells were treated for 60 min. (values are in the text). Scale bar 1 μm.</p> "> Figure 6
<p>(<b>a</b>) Comparison of RP values in oocytes in the presence of CyTD alone (<span class="html-italic">n</span> = 5), or with FAV173 (<span class="html-italic">n</span> = 13) and Ani9 (<span class="html-italic">n</span> = 13), Croc (<span class="html-italic">n</span> = 12) and Ani9 (<span class="html-italic">n</span> = 15), (Fe<sup>3+</sup> + H<sub>2</sub>O<sub>2</sub>) (<span class="html-italic">n</span> = 12), and Ani9 (<span class="html-italic">n</span> = 14). * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001 vs. CyTD, <sup>§§§</sup> <span class="html-italic">p</span> < 0.001 CyTD + Croc vs. CyTD + Croc + Ani9 (ANOVA with Tukey’s post hoc). (<b>b</b>) Examples of SEM images of the plasmalemma from CyTD, CyTD + Croc, and (Fe<sup>3+</sup> + H<sub>2</sub>O<sub>2</sub>) + CyTD-treated oocytes. In both cases, damage on the plasmalemma are visible (red asterisks). In (<b>c</b>), the presence of Ani9 fully recovered the membrane damage in the Croc-treated cells, while it is still visible in (Fe<sup>3+</sup> + H<sub>2</sub>O<sub>2</sub>)-treated cells (white asterisk). Scale bar: 1 μm.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Effects of FAV173 Fiber Exposure on the Passive Membrane Properties of Xenopus Oocytes
2.2. FAV173 Fiber Exposure Stimulates TMEM16A Channel Activity
2.3. Effects of FAV173 Fiber Exposure on Cell Morphology
2.4. The Role of Actin in the TMEM16A-Mediated Effect Induced by FAV173 Fiber Exposure
3. Discussion
4. Materials and Methods
4.1. Isolation of Xenopus Oocytes
4.2. Electrophysiological Recordings
4.3. Asbestos and FAV173 Fibers
4.4. Scanning and Transmission Electron Microscopy
4.5. Chemicals
4.6. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Zangari, M.; Zabucchi, G.; Conti, M.; Lorenzon, P.; Borelli, V.; Constanti, A.; Dellisanti, F.; Leone, S.; Vaccari, L.; Bernareggi, A. Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model. Int. J. Mol. Sci. 2024, 25, 8661. https://doi.org/10.3390/ijms25168661
Zangari M, Zabucchi G, Conti M, Lorenzon P, Borelli V, Constanti A, Dellisanti F, Leone S, Vaccari L, Bernareggi A. Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model. International Journal of Molecular Sciences. 2024; 25(16):8661. https://doi.org/10.3390/ijms25168661
Chicago/Turabian StyleZangari, Martina, Giuliano Zabucchi, Martina Conti, Paola Lorenzon, Violetta Borelli, Andrew Constanti, Francesco Dellisanti, Sara Leone, Lisa Vaccari, and Annalisa Bernareggi. 2024. "Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model" International Journal of Molecular Sciences 25, no. 16: 8661. https://doi.org/10.3390/ijms25168661