Sánchez Julio C, Wilkins Robert J
University Laboratory of Physiology, University of Oxford, Parks Road, Oxford, UK.
Comp Biochem Physiol A Mol Integr Physiol. 2004 Jan;137(1):173-82. doi: 10.1016/j.cbpb.2003.09.025.
Intracellular calcium concentration ([Ca2+]i) in articular chondrocytes changes during mechanical challenges associated with joint movements, because of the fluctuation of the extracellular osmotic environment during joint loading. Matrix synthesis by chondrocytes is modulated by loading patterns, possibly mediated by variations in intracellular composition, including [Ca2+]i. The present study has employed the Ca(2+)-sensitive fluoroprobe Fura-2 to determine the effects of hypertonic shock on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a hypertonic shock, [Ca2+]i rapidly increased by approximately 300%, reaching a maximal value within 50 s following the hypertonic shock with a recovery of more than 90% towards the initial [Ca2+]i within 5 min. The effect was inhibited by removal of extracellular Ca2+ ions, but not by thapsigargin, indicating that the rise in [Ca2+]i is only a result of influx from the extracellular medium. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels, TRPV channels or stretch-activated cation channels. Non-specific inhibitors of Ca2+ channels like CdCl2, NiCl2, LaCl3 and ZnCl2 significantly attenuated the response, although the extent in which CdCl2 and NiCl2 (both of them inhibitors of annexin-mediated Ca2+ fluxes) inhibited the response was significantly greater. The rise was also sensitive to KBR7943, inhibitor of NCE reverse mode and trifluoperazine, inhibitor of the activity of annexins. Hypertonic shock also produced also hyperpolarisation of chondrocytes (Em measured by means of Di-BA-C4(3), a membrane potential sensitive dye), which was inhibited by TEA-Cl and BaCl, but was not affected by changing the extracellular solution to Ca(2+)-free HBS. Inhibition of hyperpolarisation completely abolished the [Ca2+]i rise following hypertonic shock. Treatment with retinoic acid, which can increase the activity of annexins as Ca2+ transport pathways caused a significant increase in [Ca2+]i. The recovery of [Ca2+] was inhibited by benzamil and was dependent on extracellular Na+, but was unaffected by Na-orthovanadate, an inhibitor of plasma Ca(2+)-ATPase. We conclude that in response to hypertonic shock, NCE reverse mode and annexins are the pathways responsible for the [Ca2+]i increase, while forward mode operation of NCE is responsible for the subsequent extrusion of Ca2+ and recovery of [Ca2+]i towards initial values.
在与关节运动相关的机械刺激过程中，由于关节负荷期间细胞外渗透环境的波动，关节软骨细胞内的钙浓度（[Ca2+]i）会发生变化。软骨细胞的基质合成受负荷模式调节，可能由包括[Ca2+]i在内的细胞内成分变化介导。本研究采用钙敏感荧光探针Fura-2来确定高渗休克对细胞内钙浓度（[Ca2+]i）的影响，并表征分离的牛关节软骨细胞反应所涉及的机制。在遭受高渗休克的细胞中，[Ca2+]i迅速增加约300%，在高渗休克后50秒内达到最大值，5分钟内恢复到初始[Ca2+]i的90%以上。去除细胞外钙离子可抑制该效应，但毒胡萝卜素不能抑制，这表明[Ca2+]i的升高仅是细胞外介质内流的结果。该升高对L型电压激活钙通道、TRPV通道或牵张激活阳离子通道的抑制剂不敏感。Ca2+通道的非特异性抑制剂如CdCl2、NiCl2、LaCl3和ZnCl2显著减弱了该反应，尽管CdCl2和NiCl2（两者均为膜联蛋白介导的Ca2+通量抑制剂）抑制反应的程度明显更大。该升高对NCE反向模式抑制剂KBR7943和膜联蛋白活性抑制剂三氟拉嗪也敏感。高渗休克还使软骨细胞发生超极化（通过膜电位敏感染料Di-BA-C4(3)测量Em），该超极化被TEA-Cl和BaCl抑制，但将细胞外溶液换成无Ca(2+)的HBS对其无影响。超极化的抑制完全消除了高渗休克后[Ca2+]i的升高。用视黄酸处理可增加作为Ca2+转运途径的膜联蛋白的活性，导致[Ca2+]i显著增加。[Ca2+]的恢复被苯甲酰米抑制且依赖于细胞外Na+，但不受血浆Ca(2+)-ATP酶抑制剂原钒酸钠的影响。我们得出结论，在对高渗休克的反应中，NCE反向模式和膜联蛋白是[Ca2+]i增加的途径，而NCE的正向模式操作负责随后Ca2+的排出以及[Ca2+]i恢复到初始值。
