A novel method for assessing effects of hydrostatic fluid pressure on intracellular calcium: a study with bovine articular chondrocytes
A novel method for assessing effects of hydrostatic fluid pressure on intracellular calcium: a study with bovine articular chondrocytes
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DOI：
10.1152/ajpcell.00131.2004
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发表时间：
2005-02-01
影响因子：
5.5
通讯作者：
Mizuno, S
中科院分区：
文献类型：
--
作者：
Mizuno, S
关节软骨中的软骨细胞在承重和关节载荷过程中受到静水压力和变形应力的影响。由于这些应力同时发生在关节软骨中，因此仅在软骨细胞中由静水压力引起的机械信号转导机制尚不清楚。在这项研究中，我们试图通过使用荧光指示剂(X-Rhod-1AM)、定制的耐压光学小室和激光共聚焦显微镜来表征静水压力(HFP)对从限定表面(SZ)和中间带(MZ)分离的牛关节软骨细胞施加静液压力(HFP)后细胞内钙离子浓度([Ca+](I))的变化。在这项实验中实施的关键方法包括将高水平的HFP应用于细胞，并使用一种新的成像设备来测量单个细胞中的峰值[Ca~(2+)](I)。培养5d的MZ细胞在恒定0.5 Mpa的HFP作用5min后，[Ca~(2+)](I)峰值显著增加一倍。SZ细胞的[Ca~(2+)](I)峰值低于MZ细胞(43%)。丹曲烯、Gd的抑制剂或无钙缓冲液可抑制该峰，但维拉帕米不能抑制该峰。这项研究表明，软骨细胞内[Ca~(2+)](I)对HFP的升高与带状起源有关。HFP刺激钙动员和拉伸激活的通道。
Chondrocytes in articular cartilage are exposed to hydrostatic pressure and distortional stress during weight bearing and joint loading. Because these stresses occur simultaneously in articular cartilage, the mechanism of mechanosignal transduction due to hydrostatic pressure alone in chondrocytes is not clear. In this study, we attempted to characterize the change in intracellular calcium concentration ([Ca2+](i)) in response to the application of hydrostatic fluid pressure (HFP) to cultured bovine articular chondrocytes isolated from defined surface (SZ) and middle zones (MZ) by using a fluorescent indicator (X-rhod-1 AM), a novel custom-made pressure-proof optical chamber, and laser confocal microscopy. Critical methodology implemented in this experiment involved application of high levels of HFP to the cells and the use of a novel imaging apparatus to measure the peak [Ca2+](i) in individual cells. The peak [Ca2+](i) in MZ cells cultured for 5 days showed a significant twofold increase after the application of HFP at constant 0.5 MPa for 5 min. The peak [Ca2+](i) in SZ cells was lower (43%) than that of MZ cells. The peak was suppressed with an inhibitor of dantrolene, gadolinium, or a calcium ion-free buffer, but not with verapamil. This study indicated that the increase in [Ca2+](i) in chondrocytes to HFP is dependent on the zonal origin. HFP stimulates calcium mobilization and stretch-activated channels.