Li Duo-ling, Ma Zhi-yong, Fu Zhi-jie, Ling Ming-ying, Yan Chuan-zhu, Zhang Yun
Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, China ; Department of Neurology, Qilu Hospital, Shandong University, Jinan, China.
Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, China.
PLoS One. 2014 Feb 18;9(2):e89083. doi: 10.1371/journal.pone.0089083. eCollection 2014.
Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca(2+)]i) handling in Raw 264.7 macrophages. In the present study, [Ca(2+)]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+) channel blockers had no effect on the resting [Ca(2+)]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca(2+)]i transient elevation independent of extracellular Ca(2+). The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca(2+)]i transient elevation, but pinacidil and other unselective K(+) channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+)]i transient induced by extracellular thapsigargin (Tg, 1 µM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+)]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+)]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.
越来越多的证据表明，格列本脲具有广泛的抗炎作用。然而，尚不清楚格列本脲是否会影响Raw 264.7巨噬细胞静息状态下以及三磷酸腺苷（ATP）诱导的细胞内钙（[Ca(2+)]i）处理。在本研究中，分别使用Fura 2-am、DCFDA和罗丹明-123作为指示剂，通过高速TILLvisION数字成像系统测量[Ca(2+)]i瞬变、活性氧（ROS）和线粒体活性。我们发现，格列本脲、吡那地尔和其他非选择性钾（K(+)）通道阻滞剂对Raw 264.7细胞的静息[Ca(2+)]i没有影响。细胞外ATP（100 µM）诱导[Ca(2+)]i瞬态升高，且不依赖于细胞外钙。ROS清除剂（钛铁试剂）和线粒体抑制剂（鱼藤酮）可抑制这种瞬态升高。格列本脲和5-羟基癸酸（5-HD）也可降低ATP诱导的[Ca(2+)]i瞬态升高，但吡那地尔和其他非选择性钾（K(+)）通道阻滞剂则无此作用。格列本脲还可降低细胞外毒胡萝卜素（Tg，1 µM）诱导的[Ca(2+)]i瞬变峰值。此外，格列本脲可降低细胞内ROS和线粒体活性。当用钛铁试剂和鱼藤酮预处理后，格列本脲无法进一步降低ATP和Tg诱导的最大[Ca(2+)]i瞬变。我们得出结论，格列本脲可能通过阻断线粒体ATP敏感性钾通道来抑制ATP诱导的[Ca(2+)]i瞬态升高，从而导致Raw 264.7巨噬细胞中ROS生成和线粒体活性降低。
