ELECTROPHYSIOLOGICAL DETECTION OF H^+ SECRETION IN OSTEOCLAST PROTON-PUMP

实验采用1周龄以内的Wister大鼠幼鼠。取4只经Nembutal麻醉的幼鼠股骨和胫骨，纵向切开。从骨头中取出软组织，然后通过巴斯德移液管反复通过使其分散。将细胞悬液（0.004ml）置于涂有纤维连接蛋白的玻璃罩上，在30℃下孵育60 min。我发现在这项研究中观察到的破骨细胞有两种形态类别。一种是由扩散良好的扁平细胞（“扩散的”破骨细胞）组成。第二类细胞由圆顶状(“圆形”破骨细胞组成。“扩散”和“圆形”破骨细胞均呈酒石酸抗性酸性磷酸酶（TRAP）阳性。采用膜片钳技术的全细胞模式检测大鼠破骨细胞的膜特性。“扩张性”破骨细胞的静息电位为-10 ~ - 80mv，输入电阻为1 ~ 10gomega，膜容量为10 ~ 55pf . ...更多的扩张性细胞在超极化电压阶下显示向内电流，在去极化电压阶下显示向外电流。向内电流对活性的电压依赖性以及对细胞外Ba^<2+>和Cs^+阻断的敏感性与向内整流K^+电流相似。在少数情况下，质子泵的抑制剂DCCD对电流有一定的抑制作用。“圆形”破骨细胞的静息电位为-30 ~ -60 mV，输入电阻为2 ~ 8 GOMEGA，膜容量为10 ~ 100 pf。圆形细胞仅在比静息电位更负的电压下显示漏电流成分，而在比静息电位更正的电压下显示大的外向电流成分。外部Ba^<2+>和DCCD. 13ea03抑制了向外电流：虽然本研究发现质子泵抑制剂DCCD降低了电流成分，但DCCD的抑制成分可能是K^+电流。在没有K^+电流的情况下可以检测到H^+电流的进一步实验必须进行。少

Wister rat pups (up to 1 week old) were used for the experiments. Femora and tibiae were removed from four pups anesthetized with Nembutal and were split longitudinally. Soft tissue was removed from bones, which were then dispersed by repeated passage through a Pasteur pipette. Cell suspension (0.004ml) was placed on a glass coverslip coated with fibronectin and were incubated at 30ﾟC for 60 min. I found the two morphological categories of osteoclasts observed during this study. One was composed of well-spread, flattened cells ( "spread" osteoclast). The second category of the cells consisted of dome-shaped ( "rounded" osteoclasts). Both "spread" and "rounded" osteoclasts stained positive for tartrate-resistant acid phosphatase (TRAP).Membrane properties of isolated rat osteoclasts were examined using whole-cell mode of the patch-clamp technique. "Spread" osteoclast had resting potentials from -10 to -80 mV,input resistances from 1 to 10 GOMEGA and membrane capacities from 10 to 55 pF. … More The spread cells displayd an inward current in response to hyperpolarizing voltage steps and little outward current in response to depolarizing voltage steps.The inward current was similar in its voltage dependence of activity and its sensitivity to blockade by extracellular Ba^<2+> and Cs^+ to the inward rectifying K^+ current. In a few cases, The current was a little inhibited by DCCD which is the inhibitor for the proton-pump. The "rounded" osteoclast had resting potentials from -30 to -60 mV,input resistances from 2 to 8 GOMEGA and membrane capacities from 10 to 100 pF.The rounded cells displayd only leak components of current in the voltages more negative than the resting potential, while the cells exhibited a large outward current in the voltages more positive than the resting potential. The outward current was inhibited by external Ba^<2+> and DCCD.13EA03 : Although the present study identified the current components decreased by DCCD,an inhibitor for the proton pump, the DCCD-inhibitable component may be K^+ currents. Further experiments in which H^+ current without K^+ current can be detectable must be carried on. Less