Department of Clinical Pharmacology, Ruhr-University Bochum, Universitätsstrasse 150, D-44801 Bochum, Germany.
Cardiovasc Res. 2011 Feb 1;89(2):392-400. doi: 10.1093/cvr/cvq330. Epub 2010 Oct 20.
Bicarbonate transport has been shown to participate in apoptosis under ischaemic stress. However, the precise transporting mechanisms involved in ischaemic apoptosis are unknown and were thus the aim of the present study.
Rat coronary endothelial cells (EC) were exposed to simulated in vitro ischaemia for 2 h, and apoptosis was subsequently determined by chromatin staining and caspase-3 activity analysis. By examining the expression of bicarbonate transporters (BT) in EC by reverse transcriptase polymerase chain reaction and western blotting, a marked expression of the electroneutral sodium bicarbonate co-transporter (SLC4A7) was defined. To analyse the potential role of this transporter during apoptosis, a selective inhibitor (S0859, Sanofi-Aventis) was applied. Treatment with S0859 significantly increased caspase-3 activity and elevated the number of apoptotic EC. These results were comparable with an unselective inhibition of all BT due to withdrawal of bicarbonate in the anoxic medium. Knockdown of SLC4A7 in EC by transfecting appropriate siRNA similarly increased apoptosis of EC under simulated ischaemia. The initial characterization of the participating mechanisms of SLC4A7-dependent apoptosis revealed an activation of the mitochondrial pathway of apoptosis, i.e. cleavage of caspase-9 and binding of Bax to mitochondria. In contrast, no activation of the endoplasmic reticulum-dependent pathway (caspase-12 cleavage) or the extrinsic apoptotic pathway (caspase-8 cleavage) was found. Finally, a mitochondrial localization of SLC4A7 was demonstrated.
The electroneutral sodium bicarbonate co-transporter SLC4A7 localizes in mitochondria and suppresses the ischaemia-induced activation of the mitochondrial pathway of apoptosis in coronary EC.
已有研究表明，在缺血应激下，碳酸氢盐转运参与了细胞凋亡。然而，缺血性凋亡中涉及的确切转运机制尚不清楚，因此本研究旨在探讨这一问题。
将大鼠冠状动脉内皮细胞（EC）暴露于模拟的体外缺血 2 小时，随后通过染色质染色和 caspase-3 活性分析来确定细胞凋亡情况。通过逆转录聚合酶链反应和 Western blot 检测 EC 中碳酸氢盐转运体（BT）的表达，发现电中性的钠离子碳酸氢盐共转运体（SLC4A7）表达明显上调。为了分析该转运体在凋亡过程中的潜在作用，应用了一种选择性抑制剂（S0859，赛诺菲-安万特）。S0859 处理显著增加了 caspase-3 的活性，并增加了凋亡的 EC 数量。这些结果与由于缺氧培养基中碳酸氢盐的缺失而对所有 BT 进行非选择性抑制的结果相当。通过转染适当的 siRNA 使 EC 中的 SLC4A7 沉默，也可在模拟缺血条件下增加 EC 的凋亡。SLC4A7 依赖性凋亡参与机制的初步表征揭示了线粒体凋亡途径的激活，即 caspase-9 的切割和 Bax 与线粒体的结合。相反，未发现内质网依赖性途径（caspase-12 的切割）或外源性凋亡途径（caspase-8 的切割）的激活。最后，还证明了 SLC4A7 的线粒体定位。
电中性的钠离子碳酸氢盐共转运体 SLC4A7 定位于线粒体，并抑制冠状动脉内皮细胞中缺血诱导的线粒体凋亡途径的激活。
