The regulatory mechanisms of mechanical stress-induced ionic channels in osteocytes

骨细胞力学敏感的机制和信号转导机制尚不清楚。在本研究中，我们专注于跨质膜的离子转运，并研究了骨细胞膜拉伸与低渗细胞肿胀对离子通道活性的调节。采用全细胞电压钳技术，对分离自新生大鼠颅骨的骨细胞进行了研究。在生理盐溶液（290 mOsm）中，外向电流较小。暴露于低渗溶液（210 mOsm）强烈激活了与细胞肿胀相关的外向整流电流。高正电压（>=+70mV）的膜去极化引起外向电流的时间依赖性衰减。这种激活是可逆的和低渗依赖性的。即使在细胞内Cs^+抑制K^+通道的情况下，也能观察到激活电流，并依赖于细胞外Cl^-浓度，表明为外向整流Cl^-电流。溶胀激活Cl^-电流的阴离子渗透性顺序为I^->Br^-> Cl ^-> glucoate ^-。此外，经典的Cl^-通道阻断剂，如DIDS、NPPB和尼氟灭酸，可逆地抑制肿胀激活的Cl^-电流。Cl^-电流的这些电生理学、药理学特性和阴离子选择性与在其他细胞类型中显示为体积调节阴离子通道的CLC-3 Cl^-通道非常相似。在免疫细胞化学方法中，大鼠骨细胞中不仅存在CLC-3，而且还存在CLC-2。RT-PCR结果显示，大鼠骨细胞中CLC-2和CLC-3的mRNA均有表达。竞争性RT-PCR结果显示，CLC-3 mRNA在总RNA中的表达量明显高于CLC-2。这些结果表明，大鼠骨细胞CLC-3氯通道主要被机械应力激活，并在骨中作为机械感受器发挥作用。

The mechanisms and signal-transduction of mechanosensing in osteocytes are not well understood. In the present study, we focused on ionic transport across the plasma membrane, and have investigated the modulation of ionic channel activities by membrane stretch with hypo-osmotic cell swelling in osteocytes. Osteocytes, isolated from neonatal rat calvaria, were studied by the whole-cell voltage-clamp technique with cell image recording through a CCD camera. In physiological salt solution (290mOsm), small outward currents. Exposure to hypotonic solution (210mOsm) strongly activated an outwardly rectifying current associated with cell swelling in few minutes. Membrane depolarization of high positive voltage (>=+70mV) caused a time-dependent decay in outward currents. This activation was reversible and hypotonicity-dependent. The activated current was observed even in condition of inhibiting K^+ channel by intracellular Cs^+, and dependent on extracelluar Cl^- concentration, indicating the outwardly rectifying Cl^- current. The anion permeability sequence the swelling-activated Cl^- current was I^->Br^->C1^->gluconate^-. Moreover, classical Cl^- channel blockers, such as DIDS, NPPB and niflumic acid, reversibly inhibited the swelling-activated Cl^- current. These electro-physiological, pharmacological properties and anion selectivity of the Cl^- current were well similar to the CLC-3 Cl^- channels shown in other cell types as volume-regulated anion channels. In the immunocytochemistry method, not only CLC-3 but also CLC-2 existed in rat osteocytes. Moreover, both mRNA of CLC-2 and CLC-3 expressed in rat osteocytes by RT-PCR. However, the expression level of CLC-3 mRNA in total RNA was predominantly higher than that of CLC-2 using competitive RT-PCR. These results suggested that CLC-3 chloride channels in rat osteocytes are mainly activated by mechanical stress, and act as mechanoreceptor in bone.