We have shown that superoxide anion (O2−) production by the osteoclast can be used as an index of the osteoclast activity since the agents that inhibit and stimulate the osteoclast also diminish and stimulate O2− production respectively. Therefore, we have investigated the mechanism of parathyroid hormone (PTH)-mediated stimulation of osteoclast function in terms of its effect on O2− generation. The determination of O2− generation was carried out by employing cytochrome c immobilised on a surface-modified gold electrode. The basal level of free radical production by the osteoblast-like cells (ROS 17/2·8) was 104-fold lower than by osteoclasts cultured on bone. PTH had no acute effect on free radical production by the osteoblasts. The exposure of the osteoclasts cultured on bone to PTH led to a dramatic and immediate stimulation of O2− generation which was unaffected by the presence of ROS 17/2·8 cells. The osteoclasts cocultured with ROS 17/2·8 cells and exposed to PTH for 3 h were also found to produce greater stimulation of O2− than the osteoclasts exposed to PTH alone. A competitive leukotriene D4 antagonist REV 5901, which also inhibits 5-lipoxygenase, did not block O2− generation by osteoclasts cultured alone or in the presence of osteoblasts. Therefore, we conclude that PTH directly stimulates osteoclasts to produce O2−; this may be the main mode of activation of the osteoclasts, although an osteoblast-mediated effect of the hormone cannot be ruled out.
Journal of Endocrinology (1996) 149, 269–275
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