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Extracellular phosphates enhance activities of voltage-gated proton channels and production of reactive oxygen species in murine osteoclast-like cells

  • Ion channels, receptors and transporters
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Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Osteoclasts are highly differentiated bone-resorbing cells and play a significant role in bone remodelling. In the resorption pit, inorganic phosphate (Pi) concentrations increase because of degradation of hydroxyapatite. We studied effects of extracellular Pi on voltage-gated H+ channels in osteoclast-like cells derived from a macrophage cell line (RAW264). Extracellular Pi (1.25–20 mM) increased the H+ channel currents dose dependently and reversibly. The Pi-induced increases were attenuated by removal of extracellular Na+ and by phosphonoformic acid, a blocker of Na+-dependent Pi transporters. Pi increased the maximal conductance, decreased activation time constant, increased deactivation time constant, and shifted the conductance-voltage relationship to more negative voltages. The most marked change was enhanced gating which was mainly caused by elevation of intracellular Pi levels. The Pi-induced enhanced gating was partially inhibited by protein kinase C (PKC) inhibitors, GF109203X and staurosporine, indicating that PKC-mediated phosphorylation was involved in part. The increase in the maximal conductance was mainly due to accompanying decrease in intracellular pH. These effects of Pi were not affected by intracellular Mg2+, bafilomycin A1 (V-ATPase inhibitor) and removal of intracellular ATP. Extracellular Pi also upregulated reactive oxygen species (ROS). Diphenyleneiodonium chloride, an inhibitor of NADPH oxidases, decreased ROS production and partially attenuated the enhanced gating. In the cells during later passages where osteoclastogenesis declined, H+ channel activities and ROS production were both modest. These results suggest that, in osteoclasts, ambient Pi is a common enhancer for H+ channels and ROS production and that potentiation of H+ channels may help ROS production.

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Acknowledgments

We thank Y. Hino, Y. Moriura and J. Kawawaki for technical assistance. This work was supported by JSPS KAKENHI Grant Number JP15K08184.

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Authors and Affiliations

  1. Department of Physiology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan

    Guangshuai Li & Miyuki Kuno

  2. Department of Applied Pharmacology and Therapeutics, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka, 545-8585, Japan

    Guangshuai Li & Katsuyuki Miura

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Correspondence to Miyuki Kuno.

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Fig. S1

PMA-induced ROS production in RAW-derived osteoclasts. The time courses of ROS production in control (squares) and after addition of PMA (phorbol 12-myristate 13-acetate) (circles). Triangles are data obtained in the presence of PMA and ZnCl2. The cells (0.3 × 105 cells) were incubated with L012 for 8 min before the recordings. The data represent the average of triplicate measurements of chemiluminescence intensities, expressed as relative to the values at the onset of recordings in a batch. PMA; 200 nM. ZnCl2; 200 μM. *p < 0.05; the PMA-induced ROS production with and without ZnCl2 are compared. (PDF 14 kb)

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Li, G., Miura, K. & Kuno, M. Extracellular phosphates enhance activities of voltage-gated proton channels and production of reactive oxygen species in murine osteoclast-like cells. Pflugers Arch - Eur J Physiol 469, 279–292 (2017). https://doi.org/10.1007/s00424-016-1931-5

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