Zinc-Induced Effects on Osteoclastogenesis Involves Activation of Hyperpolarization-Activated Cyclic Nucleotide Modulated Channels via Changes in Membrane Potential

Key points The contribution of HCN4 pacemaker channels in the autonomic regulation of the sino‐atrial node (SAN) has been a matter of debate.The transgenic overexpression of HCN4 did not induce tachycardia, but reduced heart rate variability, while the conditional knockdown of HCN4 gave rise to sinus arrhythmia.The response of the SAN to β‐adrenergic stimulation was not affected by overexpression or knockdown of HCN4 channels.When HCN4 channels were knocked down, the parasympathetic response examined by cervical vagus nerve stimulation (CVNS) was enhanced; the CVNS induced complete sinus pause. The overexpression of HCN4 attenuated bradycardia induced by CVNS only during β‐adrenergic stimulation.We concluded that HCN4 pacemaker channels stabilize the spontaneous firing by attenuating the parasympathetic response of the SAN. AbstractThe heart rate is dynamically controlled by the sympathetic and parasympathetic nervous systems that regulate the sinoatrial node (SAN). HCN4 pacemaker channels are the well‐known causative molecule of congenital sick sinus syndrome. Although HCN4 channels are activated by cAMP, the sympathetic response of the SAN was preserved in patients carrying loss‐of‐function mutations of the HCN4 gene. In order to clarify the contribution of HCN4 channels in the autonomic regulation of the SAN, we developed novel gain‐of‐function mutant mice in which the expression level of HCN4 channels could be reversibly changed from zero to ∼3 times that in wild‐type mice, using tetracycline transactivator and the tetracycline responsive element. We recorded telemetric ECGs in freely moving conscious mice and analysed the heart rate variability. We also evaluated the response of the SAN to cervical vagus nerve stimulation (CVNS). The conditional overexpression of HCN4 did not induce tachycardia, but reduced heart rate variability. The HCN4 overexpression also attenuated bradycardia induced by the CVNS only during the β‐adrenergic stimulation. In contrast, the knockdown of HCN4 gave rise to sinus arrhythmia, and enhanced the parasympathetic response; complete sinus pause was induced by the CVNS. In vitro, we compared the effects of acetylcholine on the spontaneous action potentials of single pacemaker cells, and found that similar phenotypic changes were induced by genetic manipulation of HCN4 expression both in the presence and absence of β‐adrenergic stimulation. Our study suggests that HCN4 channels attenuate the vagal response of the SAN, and thereby stabilize the spontaneous firing of the SAN.

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“…; Notomi et al . ). However, GFP fluorescence was too weak and indistinguishable from the autofluorescence signal.…”

Section: Resultsmentioning

confidence: 97%

HCN4 pacemaker channels attenuate the parasympathetic response and stabilize the spontaneous firing of the sinoatrial node

Kozasa

Nakashima

Itô

et al. 2018

The Journal of Physiology

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“…PI(3,5)P 2 inhibited osteoclastogenesis and depolarized the membrane potential. Previously, we have confirmed that membrane hyperpolarization promotes osteoclast differentiation (2). Thus, we tested our additional hypothesis, suggesting that membrane depolarization would affect osteoclastogenesis.…”

Section: Membrane Depolarization Inhibits Osteoclast Differentiationmentioning

confidence: 73%

“…(sRANKL, 50 ng/ml; Oriental Yeast, Tokyo, Japan). TRAP staining was performed as previously described (2,10). Ned19 (1 M; Tocris Bioscience, Bristol, UK), a TPC2 inhibitor, and YM201636 (100 nM; Tocris Bioscience), a PIKfyve inhibitor, were added to culture media in some experiments.…”

Section: Role Of Tpc2 In Osteoclastogenesis Under Low-mg 2؉ Conditionsmentioning

confidence: 99%

“…Mouse bone marrow cells were obtained from the tibia and femur (C57BL/6 mice, 4-week-old males; Charles River Japan, Kanagawa, Japan). Bone marrow stromal cells were depleted from the mouse bone marrow by using a Sephadex G10 (GE Healthcare) column as previously described (2,10). The obtained cells were cultured in Mg 2ϩ -free DMEM with/without 1 mM MgCl 2 in the presence of macrophage colony-stimulating factor (50 ng/ml; R&D Systems, Minneapolis, MN) and sRANKL (50 ng/ml).…”

Section: Bone Marrow-derived Osteoclastsmentioning

confidence: 99%

“…Ca 2ϩ is recognized as a necessary factor for bone health, and the calcium signaling pathway has been extensively studied in the bone. However, other minerals, including zinc, copper, and magnesium, are also known to be critical for bone health (1,2). In particular, bones are known to store Ͼ50% of the total Mg 2ϩ in the body (3,4), and Mg 2ϩ deficiency causes bone loss, coupled with an increase in osteoclastogenesis (5,6).…”

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confidence: 99%

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Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions

Notomi

Kuno

Hiyama

et al. 2017

Journal of Biological Chemistry

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The bone is the main storage site for Ca and Mg ions in the mammalian body. Although investigations into Ca signaling have progressed rapidly and led to better understanding of bone biology, the Mg signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) signaling pathway played a role in the TPC2 activation under low-Mg conditions. Furthermore, PI(3,5)P depolarized the membrane potential by increasing the intracellular Na levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of -inositol supplementation, which increased the PI(3,5)P levels in mice fed a low-Mg diet. The -inositol supplementation rescued the low-Mg diet-induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg-induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P pathway. Our findings also suggest that -inositol consumption might provide beneficial effects in Mg deficiency-induced skeletal diseases.

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Acid-inducible proton influx currents in the plasma membrane of murine osteoclast-like cells

Kuno

Moriura

et al. 2016

Pflugers Arch - Eur J Physiol

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Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H(+)-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to <5.5 induced non-ohmic inward currents. The reversal potentials depended on the pH gradients across the membrane and were independent of concentrations of Na(+), Cl(-), and HCO3 (-), suggesting that they were carried largely by protons. The acid-inducible proton influx currents were not inhibited by amiloride, a widely used blocker for cation channels/transporters, or by 4,4'-diisothiocyanato-2,2'-stilbenesulfonate(DIDS) which blocks anion channels/transporters. Additionally, the currents were not significantly affected by V-ATPase inhibitors, bafilomycin A1 and N,N'-dicyclohexylcarbodiimide. Extracellular Ca(2+) (10 mM) did not affect the currents, but 1 mM ZnCl2 decreased the currents partially. The intracellular pH in the vicinity of the plasma membrane was dropped by the acid-inducible H(+) influx currents, which caused overshoot of the voltage-gated H(+) channels after removal of acids. The H(+) influx currents were smaller in undifferentiated, mononuclear RAW cells and were negligible in COS7 cells. These data suggest that the acid-inducible H(+) influx (H(+) leak) pathway may be an additional mechanism modifying the pH environments of osteoclasts upon exposure to strong acids.

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Zinc-Induced Effects on Osteoclastogenesis Involves Activation of Hyperpolarization-Activated Cyclic Nucleotide Modulated Channels via Changes in Membrane Potential

Cited by 14 publications

References 39 publications

HCN4 pacemaker channels attenuate the parasympathetic response and stabilize the spontaneous firing of the sinoatrial node

HCN4 pacemaker channels attenuate the parasympathetic response and stabilize the spontaneous firing of the sinoatrial node

Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions

Acid-inducible proton influx currents in the plasma membrane of murine osteoclast-like cells

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