Berger C E, Rathod H, Gillespie J I, Horrocks B R, Datta H K
Department of Biosciences, University of Kent, Canterbury, United Kingdom.
J Bone Miner Res. 2001 Nov;16(11):2092-102. doi: 10.1359/jbmr.2001.16.11.2092.
Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by-product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a "sensor" for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion (O2.-) generation by bone-resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of O2.- by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone-resorbing osteoclasts at the surface of a bone slice revealed a continuous steady-state release of Ca2+. Even after prolonged incubation lasting 3 h the near-surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real-time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast-bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on O2.- generation, bone-matrix, or bone resorption. Similarly, osteoclast adhesion and bone-resorbing activity was affected by [Ca2+]e' but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.
尽管破骨细胞半液泡内存在极高水平的游离钙（[Ca2+]），破骨细胞的吸收活性仍会发生，这种高浓度的[Ca2+]是其吸收活性的副产物。以往的体外观察表明，周围培养基中细胞外[Ca2+]（[Ca2+]e）的增加可抑制破骨细胞的吸收活性。因此，有人提出破骨细胞可作为[Ca2+]e的“传感器”，高[Ca2+]e会导致细胞内[Ca2+]（[Ca2+]i）增加，从而以负反馈方式抑制破骨细胞。在本报告中，我们对破骨细胞活动期间的钙处理进行了实验和理论分析，以评估体外模型与体内破骨细胞活动的关系，在体内，高[Ca2+]e可能存在于半液泡中，但不存在于细胞的非吸收表面。将扫描电化学显微镜（SECM）对牛皮质骨切片表面吸收骨的破骨细胞产生的[Ca2+]和超氧阴离子（O2.-）的研究，与单破骨细胞中[Ca2+]i水平的显微分光荧光测定以及[Ca2+]i对破骨细胞功能各个方面的影响进行了比较。破骨细胞产生O2.-已被证明与破骨细胞吸收功能呈正相关，因此可作为破骨细胞活性急性变化的指标。骨切片表面吸收骨的破骨细胞的SECM显示Ca2+持续稳定释放。即使经过长达3小时的长时间孵育，细胞上方溶液中的近表面[Ca2+]e仍<2 mM。SECM实时测量数据与破骨细胞作为从破骨细胞 - 骨界面持续排出Ca2+的通道一致。我们得出结论，破骨细胞能够区分半液泡中和细胞上方细胞外液中的[Ca2+]e。我们发现[Ca2+]i的增加可能与激活、抑制有关，或者对O2.-产生、骨基质或骨吸收没有影响。同样，破骨细胞的黏附及骨吸收活性受[Ca2+]e影响，但与[Ca2+]i无关。数据表明破骨细胞内[Ca2+]i存在功能区室化，其中升高的钙可能对破骨细胞的整体活性具有抑制、兴奋或无作用，同时对不同的功能模式发挥选择性作用。这些观察结果得出的结论是，破骨细胞远未受到所产生的Ca2+的抑制，由于观察到的功能区室化，即使吸收腔隙中的[Ca2+]水平升高，破骨细胞也能高度适应进行其活动。
