Andersen Ann-Dorit, Poulsen Kristian Arild, Lambert Ian H, Pedersen Stine Falsig
Department of Biology, Univesity of Copenhagen, DK-2100 Copenhagen, Denmark.
Am J Physiol Cell Physiol. 2009 May;296(5):C1227-42. doi: 10.1152/ajpcell.00370.2008. Epub 2009 Mar 4.
The Ca(2+)-independent phospholipase A(2) VI (iPLA(2)-VI) and the Na(+)/H(+) exchanger isoform 1 (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH (pH(o)) in ischemia-reperfusion injury and death and in regulation and function of iPLA(2)-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% O(2), 8 mM K(+), and 20 mM lactate) at pH(o) 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA(2)-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content. iPLA(2)-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pH(o)-dependent manner during SI/R. This also affected the subcellular localization of iPLA(2)-VIA. Thus, the mode of cell death and the roles and regulation of iPLA(2)-VI and NHE1 are at least in part determined by the pH(o) during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of iPLA(2)-VIA and NHE1 during cardiac I/R.
钙离子非依赖性磷脂酶A2 VI（iPLA2-VI）和钠/氢交换体同工型1（NHE1）是高度pH敏感的蛋白质，在心脏缺血再灌注中发挥着保护和有害的双重作用。在此，我们研究了细胞外pH（pHo）在缺血再灌注损伤和死亡中以及在这些条件下iPLA2-VI和NHE1的调节及功能中的作用。HL-1心肌细胞在pHo 6.0和7.4条件下暴露于模拟缺血（SI；0.5% O2、8 mM K+和20 mM乳酸），伴有或不伴有4或8小时的再灌注（SI/R）。与中性SI相比，酸性SI后细胞色素c释放和半胱天冬酶-3激活减少，而以葡萄糖-6-磷酸脱氢酶释放估算的坏死性死亡在两种条件下相似。在常氧期间以及酸性SI后（而非中性SI后），溴烯醇内酯（BEL）抑制iPLA2-VI活性会引发心肌细胞坏死。异构体选择性对映体R-BEL和S-BEL在酸性SI后均模拟了消旋BEL的作用。相反，EIPA抑制NHE活性对SI后的坏死无显著影响。中性和酸性SI均与F-肌动蛋白和皮层肌动蛋白完整性的可逆丧失相关。抑制iPLA2-VI会破坏F-肌动蛋白、皮层肌动蛋白和线粒体完整性，而抑制NHE会轻微降低应力纤维含量。iPLA2-VIA和NHE1 mRNA水平在SI期间降低，并在SI/R期间以pHo依赖性方式上调。这也影响了iPLA2-VIA的亚细胞定位。因此，细胞死亡模式以及iPLA2-VI和NHE1的作用及调节至少部分由SI期间的pHo决定。除了具有临床相关意义外，这些发现还可以部分解释先前关于心脏缺血/再灌注期间iPLA2-VIA和NHE1的研究中获得的矛盾结果。
