Phospholipase C-dependent Ca2+-sensing pathways leading to endocytosis and inhibition of the plasma membrane vacuolar H+-ATPase in osteoclasts.
Phospholipase C-dependent Ca2+-sensing pathways leading to endocytosis and inhibition of the plasma membrane vacuolar H+-ATPase in osteoclasts.
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DOI：
10.1152/ajpcell.00486.2009
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发表时间：
2010-06
期刊：
影响因子：
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通讯作者：
H. Sakai;Yoshie Moriura;T. Notomi;J. Kawawaki;K. Ohnishi;M. Kuno
中科院分区：
文献类型：
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作者：
H. Sakai;Yoshie Moriura;T. Notomi;J. Kawawaki;K. Ohnishi;M. Kuno
在破骨细胞中，细胞外 Ca2+ 升高是抑制骨吸收的内源性信号。我们最近发现细胞外 Ca2+ 的升高会迅速减少通过质膜液泡 H+-ATP 酶 (V-ATP 酶) 的质子排出。在这项研究中，我们研究了这种早期 Ca2+ 感应反应的机制，特别是质膜 V-ATP 酶的活性和膜修复。全细胞钳记录使我们能够同时测量 V-ATP 酶电流和细胞电容 (C(m))。 C(m) 是细胞表面的量度。细胞外 Ca2+ (2.5-40 mM) 同时降低 C(m) 和 V-ATPase 电流。 C(m) 降低，加上亲脂性染料 (FM1-43) 的摄取增强，表明 Ca2+ 促进内吞作用。内吞作用被动力抑制剂（dynasore 和动力抑制肽）、针对 dynanmin-2 的小干扰 RNA (siRNA) 以及 V-ATP 酶阻滞剂巴弗洛霉素 A(1) 阻断。磷脂酶 C 抑制剂 (U73122) 和针对磷脂酶 C gamma2 亚基的 siRNA 减弱了细胞外 Ca2+ 诱导的内吞作用和 V-ATPase 电流的抑制。将胞质 Ca2+ 保持在高水平 (0.5-5 µM) 或低水平，或通过抑制剂 (W7) 或抗体 (抗 CaM) 抑制钙调蛋白，可减少刺激的内吞作用和 V-ATP 酶电流的抑制。这些数据表明，细胞外 Ca2+ 促进动力蛋白和 V-ATP 酶依赖性内吞作用，并与质膜 V-ATP 酶的抑制相关。磷脂酶 C、胞质 Ca2+ 和钙调蛋白参与信号传导途径。膜修复和质膜 V-ATP 酶活性可能在破骨细胞 Ca2+ 感应反应的早期阶段协同作用。
In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance (C(m)) simultaneously. C(m) is a measure of cell surface. Extracellular Ca2+ (2.5-40 mM) decreased C(m) and the V-ATPase current simultaneously. The decreased C(m), together with the enhanced uptake of a lipophilic dye (FM1-43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A(1), a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C gamma2 subunit. Holding the cytosolic Ca2+ at either high (0.5-5 microM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.