Kerrigan Mark J P, Hall Andrew C
School of Biomedical and Clinical Laboratory Science, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.
Biorheology. 2005;42(4):283-93.
Articular chondrocytes are exposed to significant changes in extracellular osmolarity during normal joint activity, which can lead to changes in cell volume and metabolism of the extracellular matrix (ECM). Chondrocytes can respond to cell swelling/shrinking by volume regulatory pathways, but the signalling pathways are poorly understood although a role for the cytoskeleton is frequently implicated. Here, we have investigated the effects of disruption of the chondrocyte F-actin cytoskeleton on the recovery of cell volume by RVD. The cytoskeleton was perturbed using the relatively specific agent latrunculin B (5 microM; 30 min) and loss of F-actin integrity quantified using fluorescent phalloidin-labelling and confocal laser scanning microscopy (CLSM). Imaging of isolated chondrocytes labelled with Fura-2 to measure the fluorescence associated with cell volume changes, showed that the extent of hypo-osmotic swelling was unaffected by latrunculin B treatment. Two categories of the chondrocyte RVD response were observed: 'fast' RVD where at 3 min post-osmotic challenge there was a recovery in cell fluorescence of >or=80%, whereas other cells exhibited 'slow' RVD. Latrunculin B increased the proportion of chondrocytes demonstrating 'fast' RVD by approximately 10 fold and reduced those cells showing 'slow' RVD. An inhibitor of chondrocyte RVD (REV 5901) had no significant effect on the integrity of the cytoskeleton showing that the RVD response could be inhibited independent of the state of the F-actin cytoskeleton. These results suggest that the intact cortical F-actin cytoskeleton has a restraining effect on the RVD response of isolated bovine articular chondrocytes.
在正常关节活动过程中，关节软骨细胞会暴露于细胞外渗透压的显著变化中，这可能导致细胞体积和细胞外基质（ECM）代谢的改变。软骨细胞可通过体积调节途径对细胞肿胀/收缩做出反应，但尽管细胞骨架的作用经常被提及，其信号通路却知之甚少。在此，我们研究了软骨细胞F-肌动蛋白细胞骨架的破坏对调节性容积减小（RVD）介导的细胞体积恢复的影响。使用相对特异的试剂Latrunculin B（5微摩尔；30分钟）扰乱细胞骨架，并使用荧光鬼笔环肽标记和共聚焦激光扫描显微镜（CLSM）对F-肌动蛋白完整性的丧失进行定量。用Fura-2标记分离的软骨细胞以测量与细胞体积变化相关的荧光，成像结果显示低渗肿胀程度不受Latrunculin B处理的影响。观察到软骨细胞的RVD反应分为两类：“快速”RVD，即在渗透刺激后3分钟，细胞荧光恢复≥80%，而其他细胞表现为“缓慢”RVD。Latrunculin B使表现出“快速”RVD的软骨细胞比例增加了约10倍，并减少了表现出“缓慢”RVD的细胞。软骨细胞RVD的抑制剂（REV 5901）对细胞骨架的完整性没有显著影响，表明RVD反应可以独立于F-肌动蛋白细胞骨架的状态而被抑制。这些结果表明，完整的皮质F-肌动蛋白细胞骨架对分离的牛关节软骨细胞的RVD反应具有抑制作用。
