Department of Anatomy and Graduate School of Oral Biology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand.
J Periodontal Res. 2011 Oct;46(5):607-15. doi: 10.1111/j.1600-0765.2011.01379.x. Epub 2011 May 25.
Our previous studies showed that mechanical stress could induce ATP release in human periodontal ligament (HPDL) cells. By signaling through P2 purinergic receptors, ATP increased the expression and the synthesis of osteopontin and RANKL. In this study, the mechanism of stress-induced ATP release was investigated.
Continuous compressive forces were applied on cultured HPDL cells. The ATP released was measured using luciferin-luciferase bioluminescence. The expression of gap-junction proteins was examined using RT-PCR and western blot analysis. The opening of hemichannels was demonstrated by cellular uptake of a fluorescent dye, 5(6)-carboxyfluorescein, which is known to penetrate hemichannels. Intracellular signal transduction was investigated using inhibitors and antagonists.
Mechanical stress induced the release of ATP into the culture medium, which was attenuated by carbenoxolone, a nonspecific gap-junction inhibitor. Addition of meclofenamic acid sodium salt, a connexin43 inhibitor, inhibited ATP release by mechanical stress. Knockdown of connexin43 expression by small interfering RNA reduced the amount of ATP released by mechanical stress, suggesting the role of connexin43 hemichannels. In addition, intracellular Ca(2+) blockers could also inhibit mechanical stress-induced ATP release and the opening of the gap junction.
Our study demonstrated the involvement of gap-junction hemichannels, especially connexin43, in the stress-induced ATP-release mechanism. Furthermore, this mechanism may be regulated by the intracellular Ca(2+) signaling pathway. These results suggest an important role of gap-junction hemichannels in the function and behavior of HPDL cells.
我们之前的研究表明，机械应力可诱导人牙周韧带（HPDL）细胞释放 ATP。通过 P2 嘌呤能受体信号，ATP 可增加骨桥蛋白和 RANKL 的表达和合成。在本研究中，我们研究了应激诱导 ATP 释放的机制。
对培养的 HPDL 细胞施加连续的压缩力。使用荧光素-荧光素酶生物发光法测量释放的 ATP。使用 RT-PCR 和 Western blot 分析检测缝隙连接蛋白的表达。通过荧光染料 5(6)-羧基荧光素的细胞摄取来证明半通道的开放，该染料已知可穿透半通道。使用抑制剂和拮抗剂研究细胞内信号转导。
机械应力诱导 ATP 释放到培养基中，该过程被非特异性缝隙连接抑制剂 carbenoxolone 减弱。添加 connexin43 抑制剂 meclofenamic acid sodium salt 可抑制机械应力诱导的 ATP 释放。小干扰 RNA 敲低 connexin43 表达可减少机械应力释放的 ATP 量，表明 connexin43 半通道的作用。此外，细胞内 Ca(2+) 阻滞剂也可抑制机械应力诱导的 ATP 释放和缝隙连接的开放。
本研究表明缝隙连接半通道，特别是 connexin43，参与了应激诱导的 ATP 释放机制。此外，该机制可能受细胞内 Ca(2+) 信号通路的调节。这些结果表明缝隙连接半通道在 HPDL 细胞的功能和行为中起重要作用。
