Miyauchi A, Hruska K A, Greenfield E M, Duncan R, Alvarez J, Barattolo R, Colucci S, Zambonin-Zallone A, Teitelbaum S L, Teti A
Department of Medicine, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri 63110.
J Cell Biol. 1990 Dec;111(6 Pt 1):2543-52. doi: 10.1083/jcb.111.6.2543.
The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.
在培养的鸡破骨细胞以及由单核细胞前体融合产生的假定破骨细胞中，研究了钙离子（Ca2+）进入细胞的机制及其对细胞功能的影响。通过钾离子（K+）、BAY K 8644和二氢吡啶拮抗剂引起的膜去极化效应来检测电压门控钙离子通道（VGCC）。K+可使玻璃盖玻片上的破骨细胞内的胞质钙浓度（[Ca2+]i）呈剂量依赖性增加。在70 mM K+时达到半数最大效应。K+的作用被二氢吡啶衍生物钙离子通道阻滞剂完全抑制。VGCC激动剂BAY K 8644（5×10−6 M）刺激钙离子进入细胞，这种作用被尼卡地平抑制。破骨细胞附着于骨时，VGCC会失活，这表明与破骨细胞与其吸收底物的黏附相关的钙离子进入机制发生了快速的表型变化。细胞外钙离子浓度（[Ca2+]e）升高会诱导细胞内钙库释放钙离子以及钙离子内流。丹曲林（10−5 M）可阻断钙离子释放，镧离子（La3+）可阻断钙离子内流。[Ca2+]e对[Ca2+]i的影响表明破骨细胞膜上存在一种钙离子受体，该受体可能与调节细胞功能的机制相关联。无论有无骨基质底物，[Ca2+]e对[Ca2+]i的影响表现相似。每种导致[Ca2+]i升高的机制（膜去极化、BAY K 8644和[Ca2+]e）都会降低破骨细胞特异性黏附结构——足体的表达。足体表达的减少与骨吸收活性降低50%相对应。因此，破骨细胞[Ca2+]i的刺激升高会导致细胞骨架变化，影响细胞黏附并降低骨吸收活性。
