Wang H, Harrison-Shostak D C, Lemasters J J, Herman B
Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina at Chapel Hill 27599-7090, USA.
FASEB J. 1996 Sep;10(11):1319-25. doi: 10.1096/fasebj.10.11.8836046.
Numerous studies have suggested that enhanced membrane phospholipid degradation contributes to hypoxic and ischemic injury. Recently, acidosis has been found to potently protect against hypoxic and ischemic injury. To investigate the interrelationships of these two events in hypoxic injury, we studied the role of a pH-dependent group II phospholipase A2 (PLA2, E.C. 3.1.1.4) in chemical hypoxic injury in rat hepatocytes. Northern blot analysis of RNA extracted from normoxic and hypoxic rat hepatocytes with group II rat liver PLA2-specific oligonucleotide cDNA probes revealed a 0.9 kb transcript whose abundance was significantly increased in rat hepatocytes within 15 min after initiation of chemical hypoxia and remained high until cells lost viability. Immunofluorescence staining of hepatocytes with polyclonal antibodies that recognize group II PLA2 demonstrated a substantial increase in the level of PLA2 protein in hypoxic rat hepatocytes within 30 min of initiation of hypoxic injury. Treatment of hepatocytes with group II PLA2-specific anti-sense DNA oligonucleotides: 1) abolished accumulation of PLA2 protein in hypoxic rat hepatocytes as assessed by immunofluorescence staining with anti-PLA2 antibodies; 2) decreased the enzymatic activity of PLA2 manifested as decreased arachidonic acid release in hypoxic hepatocytes; and 3) significantly delayed cell death evoked by chemical hypoxia as indicated by a decrease in propidium iodide uptake. These findings suggest that pH-dependent group II PLA2 plays an important role in chemical hypoxic injury of rat hepatocytes.
众多研究表明，增强的膜磷脂降解会导致缺氧和缺血性损伤。最近，已发现酸中毒能有效预防缺氧和缺血性损伤。为了研究这两个事件在缺氧损伤中的相互关系，我们研究了pH依赖性的Ⅱ型磷脂酶A2（PLA2，E.C. 3.1.1.4）在大鼠肝细胞化学性缺氧损伤中的作用。用Ⅱ型大鼠肝脏PLA2特异性寡核苷酸cDNA探针，对从正常和缺氧大鼠肝细胞中提取的RNA进行Northern印迹分析，结果显示有一个0.9 kb的转录本，在化学性缺氧开始后的15分钟内，大鼠肝细胞中该转录本的丰度显著增加，并一直保持高水平，直到细胞失去活力。用识别Ⅱ型PLA2的多克隆抗体对肝细胞进行免疫荧光染色，结果表明在缺氧损伤开始后的30分钟内，缺氧大鼠肝细胞中PLA2蛋白水平大幅增加。用Ⅱ型PLA2特异性反义DNA寡核苷酸处理肝细胞：1）通过用抗PLA2抗体进行免疫荧光染色评估，消除了缺氧大鼠肝细胞中PLA2蛋白的积累；2）降低了PLA2的酶活性，表现为缺氧肝细胞中花生四烯酸释放减少；3）如碘化丙啶摄取减少所示，显著延迟了化学性缺氧引起的细胞死亡。这些发现表明，pH依赖性的Ⅱ型PLA2在大鼠肝细胞化学性缺氧损伤中起重要作用。
