Ca(2+) spiking activity caused by the activation of store-operated Ca(2+) channels mediates TNF-α release from microglial cells under chronic purinergic stimulation

• Published in: Biochimica et biophysica acta Vol. 1833; no. 12; pp. 2573 - 2585
• Main Authors: Ikeda, Masayuki, Tsuno, Saki, Sugiyama, Takashi, Hashimoto, Ayami, Yamoto, Kurumi, Takeuchi, Kouhei, Kishi, Hiroyuki, Mizuguchi, Hiroyuki, Kohsaka, Shin-Ichi, Yoshioka, Tohru
• Format: Journal Article
• Language: English
• Published: Netherlands 01.12.2013
• Subjects: Adenosine Triphosphate - pharmacology; Adenoviridae - drug effects; Adenoviridae - metabolism; Animals; Calcium - metabolism; Calcium Channels - metabolism; Calcium Signaling - drug effects; Cell Line; Cell Survival - drug effects; Cytosol - drug effects; Cytosol - metabolism; Dendrites - drug effects; Dendrites - metabolism; Gene Expression Profiling; Intracellular Space - drug effects; Intracellular Space - metabolism; Mice; Mice, Inbred C57BL; Microglia - drug effects; Microglia - secretion; Models, Biological; Purinergic P2X Receptor Antagonists - pharmacology; Pyridines - pharmacology; Receptors, Purinergic P2X7 - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Tetrazoles - pharmacology; Transfection; Tumor Necrosis Factor-alpha - secretion; tumor necrosis factor α; Calcium oscillation; phosphate-buffered saline; buffered salt solution; 2-APB; DMEM; analysis of variance; cytomegalovirus; ANOVA; pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid; uridine diphosphate; pore-forming subunit of the Ca(2+) release-activated Ca(2+) channel; PPADS; macrophage scavenger receptor with collagenous structure; BzATP; PBS; UDP; small interference ribonucleic acid; 2-aminoethoxy diphenyl borate; BSS; stromal interacting molecules; SOC; 4′,6-diamidino-2-phenylindole; DAPI; siRNA; store-operated channel; CMV; Dulbecco's Modified Eagle Medium; ORAI; EGTA; Microglia; MARCO; 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphoric acid; Yellow Cameleon; TNF-α; STIMs; Cytokine release; ethylene glycol tetraacetic acid; GAPDH; Macrophage; glyceraldehyde 3-phosphate dehydrogenase
• ISSN: 0006-3002; 1878-2434
• DOI: 10.1016/j.bbamcr.2013.06.022

Cytokines released from microglia mediate defensive responses in the brain, but the underlying mechanisms are obscure. One proposed process is that nucleotide leakage or release from surrounding cells is sensed by metabotropic (P2Y) and ionotropic (P2X) purinergic receptors, which may trigger long-term intracellular Ca(2+) flux and tumor necrosis factor α (TNF-α) release. Indeed, 3h of exposure to ATP was required to evoke TNF-α release from a murine microglial cell line (MG5). A Ca(2+) chelator, ethylene glycol tetraacetic acid (EGTA), reduced ATP-induced TNF-α release, suggesting that intracellular Ca(2+) is important in this response. Therefore, Ca(2+) sensor genes (YC3.6) were transfected into MG5 cells to investigate the Ca(2+) dynamics underlying ATP-induced TNF-α release. The results demonstrated ATP-induced biphasic Ca(2+) mobilization mediated by P2Y (~5min) and P2X7 receptors (5-30min). Moreover, Ca(2+) spiking activity in cell processes progressively increased with a reduction in P2X7 receptor-mediated Ca(2+) elevation during 3-h ATP stimulation. Increased Ca(2+) spiking activity paralleled the reduction in thapsigargin-sensitive internal Ca(2+) stores, dendrite extension, and expression of macrophage scavenger receptors with collagenous structure. The Ca(2+) spiking activity was enhanced by a P2X7 receptor antagonist (A438079), but inhibited by a store-operated channel antagonist (SKF96365) or by co-transfection of small interference ribonucleic acid (siRNA) targeted on the channel component (Orai1). Furthermore, ATP-induced TNF-α release was enhanced by A438079 but was inhibited by SKF96365. Because store-operated channels (Stim1/Orai1) were expressed both in MG5 and primary microglial cultures, we suggest that P2X7 receptor signaling inhibits store-operated channels during ATP stimulation, and disinhibition of this process gates TNF-α release from microglial cells.