Sun Junqing, Liu Xifang, Tong Jie, Sun Lijun, Xu Hao, Shi Liang, Zhang Jianbao
The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi׳an Jiaotong University, Xi׳an 710049, China.
The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi׳an Jiaotong University, Xi׳an 710049, China; Hong-hui Hospital, College of Medicine, Xi׳an Jiaotong University, Xi׳an 710054, China.
J Biomech. 2014 Dec 18;47(16):3903-8. doi: 10.1016/j.jbiomech.2014.10.003. Epub 2014 Oct 16.
Intracellular calcium transient ([Ca(2+)]i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca(2+)]i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca(2+)]i transient in osteoblasts. Fluorescent dyes DiBAC4(3) and fura-2AM were respectively used to detect membrane potential and [Ca(2+)]i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca(2+)]i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca(2+)]i transients. Replacing extracellular Na(+) with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca(2+)]i transients. However, voltage-activated K(+) channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca(2+) or blocking of L-type voltage-sensitive Ca(2+) channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca(2+)]i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was -43.3mV and the depolarization induced by FSS was about 44mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca(2+) mobilization wherein FSS activated SACs to promote Na(+) entry to depolarize membrane that, in turn, activated L-VSCCs and Ca(2+) influx though L-VSCCs switched on [Ca(2+)]i response in osteoblasts.
流体剪切力（FSS）诱导的细胞内钙瞬变（[Ca(2+)]i瞬变）在成骨细胞机械转导中起重要作用。膜电位的变化通常会影响[Ca(2+)]i水平。在此，我们试图确定成骨细胞中膜电位与FSS诱导的[Ca(2+)]i瞬变之间是否存在关系。分别使用荧光染料DiBAC4(3)和fura-2AM来检测膜电位和[Ca(2+)]i。我们的结果表明，FSS首先诱导成骨细胞膜电位去极化，然后[Ca(2+)]i出现短暂升高。FSS诱导膜电位去极化和[Ca(2+)]i瞬变具有相同的阈值。用四乙铵替代细胞外Na(+)或用钆阻断牵张激活通道（SACs）均能有效抑制FSS诱导的膜去极化和[Ca(2+)]i瞬变。然而，电压激活的K(+)通道抑制剂4-氨基吡啶并不影响这些反应。去除细胞外Ca(2+)或用硝苯地平阻断L型电压敏感Ca(2+)通道（L-VSCCs）也能抑制FSS诱导的成骨细胞[Ca(2+)]i瞬变。用膜片钳对膜电位进行量化显示，成骨细胞的静息电位为-43.3mV，FSS诱导的去极化约为44mV。电压钳表明这种去极化足以激活成骨细胞中的L-VSCCs。这些结果提示了Ca(2+)动员的时间线，即FSS激活SACs促进Na(+)内流使膜去极化，进而激活L-VSCCs，并且通过L-VSCCs的Ca(2+)内流开启了成骨细胞中的[Ca(2+)]i反应。
