Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation

Gilbert, D, Stebbing, M, Kuenzel, K, Murphy, R, Zacharewicz, E, Buttgereit, A, Stokes, L, Adams, D and Friedrich, O 2016, 'Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation', Frontiers in Molecular Neuroscience, vol. 9, no. OCT2016, 111, pp. 1-15.


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Title Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation
Author(s) Gilbert, D
Stebbing, M
Kuenzel, K
Murphy, R
Zacharewicz, E
Buttgereit, A
Stokes, L
Adams, D
Friedrich, O
Year 2016
Journal name Frontiers in Molecular Neuroscience
Volume number 9
Issue number OCT2016
Article Number 111
Start page 1
End page 15
Total pages 15
Publisher Frontiers Research Foundation
Abstract Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca 2+ -mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca 2+ entry (ROCE). Although store-operated Ca 2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca 2+ imaging. After depleting internal Ca 2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca 2+ ]i whereas lower concentrations (≤100 µM) also induced Ca 2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca 2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca 2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca 2+ oscillations (P2X4) and sustained [Ca 2+ ]i increases. We demonstrate specific drug effects on purinergic Ca 2+ pathways and provide new pharmacologi
Subject Clinical Sciences not elsewhere classified
Neurosciences not elsewhere classified
Keyword(s) ATP
BV2 microglia
Calcium
High-content screening
Minocycline
Multiphoton imaging
Store-operated calcium entry
DOI - identifier 10.3389/fnmol.2016.00111
Copyright notice © 2016 Gilbert, Stebbing, Kuenzel, Murphy, Zacharewicz, Buttgereit, Stokes, Adams and Friedrich. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
ISSN 1662-5099
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