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

实验选用Wistar大鼠幼鼠(最大1周龄)。取4只用Nembutal麻醉的幼鼠的股骨和胫骨，纵向切开。从骨骼中取出软组织，然后通过巴斯德吸管反复进行分散。将细胞悬液(0.004ml)放置在包被纤维连接蛋白的玻璃片上，在30ﾟC下孵育60min。我发现了在这项研究中观察到的两种形态的破骨细胞。一种是由分布良好的扁平细胞(“散布的”破骨细胞)组成。第二类细胞由圆顶状(圆形破骨细胞)组成。抗酒石酸酸性磷酸酶(TRAP)染色阳性的“散布”和“圆形”破骨细胞用膜片钳全细胞模式检测分离的大鼠破骨细胞的膜特性。“扩张型”破骨细胞的静息电位为-10~-80 mV，输入电阻为1~10GoMega，膜容量为10~55pF。…内向电流的活性与电压依赖性相似，对胞外K~(2+)和Cs~(++)的阻断敏感性相似。在少数情况下，质子泵的抑制剂DCCD对电流有轻微的抑制作用。“圆形”破骨细胞的静息电位为-30~-60 mV，输入电阻为2~8GoMega，膜容量为10~100pF。圆形破骨细胞在比静息电位更负的电压中只显示漏电流分量，而在比静息电位更正的电压中表现出较大的外向电流。外源性Ba2+和DCCD.13EA03抑制外向电流：虽然本研究证实质子泵抑制剂DCCD2+和DCCD13EA03使电流成分减少，但DCCD2+和DCCD13EA03抑制的电流成分可能是K+电流。必须进行进一步的实验，以便在没有K^+电流的情况下能够检测到H^+电流。较少

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