| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
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.
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