Turman M A, Bates C M, Mathews A, Haun S E
Wexner Institute for Pediatric Research, Department of Pediatrics, Children's Hospital, Ohio State University, Columbus 43205, USA.
Ren Fail. 1995 Jul;17(4):421-35. doi: 10.3109/08860229509037606.
Removal of extracellular calcium has been demonstrated to improve membrane integrity of rodent myocytes, astrocytes, and renal tubular cells injured by hypoxia. In this study, the effect of extracellular calcium on long-term survival of cultured human proximal tubular epithelial cells (PTEC) subjected to hypoxia was evaluated. In addition, the effect of extracellular calcium on release of arachidonic acid metabolites (AAM) was assessed during and after hypoxia. To induce hypoxic injury, PTEC were incubated in an anaerobic chamber in glucose-free buffer (combined oxygen/glucose deprivation, COGD). Long-term survival was assessed by measuring lactate dehydrogenase (LDH) efflux during COGD and after an additional 24-h "recovery" period (in routine culture medium in 95% air/5% CO2). To determine if extracellular calcium influenced AAM release from membrane phospholipids, cells were preincubated with [3H]arachidonic acid and the release of AAM was measured during COGD and recovery. With this model system, PTEC exhibited minimal LDH efflux during < or = 12 h COGD, but LDH efflux increased to 73.9 +/- 4.7% by 24 h COGD. With 12-18 h of COGD, the extent of LDH efflux was greater during recovery than during COGD, indicating that, for human PTEC, the extent of membrane damage does not become fully evident by LDH efflux for hours after hypoxia. PTEC exposed to 24 h of COGD in the absence of extracellular calcium exhibited strikingly less LDH efflux during COGD than cells incubated in the presence of extracellular calcium, suggesting that extracellular calcium contributes to membrane damage during COGD. However, upon reexposure of PTEC to extracellular calcium, LDH efflux rapidly increased to control levels. Furthermore, despite allowing cells to recover in oxygen or oxygen and glucose before exposure to calcium-containing medium, a rapid increase in LDH efflux could not be avoided. These results suggest that COGD induces an irreversible injury that ultimately leads to loss of membrane integrity whether or not extracellular calcium is present; however, extracellular calcium accelerates the loss of membrane integrity caused by hypoxia. Extracellular calcium did not alter AAM release, indicating that the effect of extracellular calcium on membrane damage (as indicated by LDH efflux) was not mediated by an increased activity of phospholipases (such as phospholipase A2) that are involved in the release of AAM.
业已证明，去除细胞外钙可改善受缺氧损伤的啮齿类动物心肌细胞、星形胶质细胞和肾小管细胞的膜完整性。在本研究中，评估了细胞外钙对缺氧培养的人近端肾小管上皮细胞（PTEC）长期存活的影响。此外，还评估了缺氧期间及之后细胞外钙对花生四烯酸代谢产物（AAM）释放的影响。为诱导缺氧损伤，将PTEC置于无氧培养箱中无糖缓冲液中孵育（联合氧/葡萄糖剥夺，COGD）。通过测量COGD期间及额外24小时“恢复”期（在95%空气/5%二氧化碳的常规培养基中）的乳酸脱氢酶（LDH）流出量来评估长期存活情况。为确定细胞外钙是否影响AAM从膜磷脂中的释放，细胞先用[3H]花生四烯酸预孵育，并在COGD和恢复期间测量AAM的释放。在这个模型系统中，PTEC在COGD≤12小时期间LDH流出量最小，但到COGD 24小时时，LDH流出量增加到73.9±4.7%。在COGD 12 - 18小时时，恢复期间的LDH流出程度比COGD期间更大，这表明对于人PTEC，缺氧数小时后膜损伤程度通过LDH流出量并未完全显现。在无细胞外钙的情况下暴露于24小时COGD的PTEC在COGD期间的LDH流出量明显低于在有细胞外钙存在下孵育的细胞，这表明细胞外钙在COGD期间促成了膜损伤。然而，当PTEC重新暴露于细胞外钙时，LDH流出量迅速增加至对照水平。此外，尽管在暴露于含钙培养基之前让细胞在有氧或有氧和葡萄糖条件下恢复，但仍无法避免LDH流出量的快速增加。这些结果表明，无论是否存在细胞外钙，COGD都会诱导不可逆损伤，最终导致膜完整性丧失；然而，细胞外钙会加速缺氧引起的膜完整性丧失。细胞外钙并未改变AAM的释放，这表明细胞外钙对膜损伤的影响（如通过LDH流出量所示）不是由参与AAM释放的磷脂酶（如磷脂酶A2）活性增加介导的。
