Venkatachalam M A, Weinberg J M, Patel Y, Hussong U, Davis J A
Department of Pathology, University of Texas Health Science Center at San Antonio, USA.
Kidney Int. 1995 Jul;48(1):118-28. doi: 10.1038/ki.1995.275.
The relationships between cytosolic free calcium (Caf), cell associated glycine, phospholipid hydrolysis and cell death were investigated in Madin-Darby canine kidney (MDCK) cells injured by depletion of adenosine triphosphate (ATP). Glucose free incubation for three hours with a mitochondrial uncoupler resulted in progressive loss of glycine from cells. However, they were not lethally injured unless a perturbation of Ca++ homeostasis was also induced. Exposure to a Ca++ ionophore and uncoupler in 1.25 mM Ca++ medium (+Ca) resulted in accelerated cell death. ATP depleted cells with ionophore in 100 nM Ca++ medium (-Ca) were also lethally injured, but after a significant delay. Depletion of glycine preceded death in both groups of cells. Exogenous glycine (5 mM) protected +Ca cells against lethal membrane damage, but the beneficial effects were lost over a period of time. In contrast, -Ca cells were completely protected throughout. Phospholipid mass and radioactive label in lipid fractions of cells prelabeled with 3H-oleic acid were measured. Accelerated death of +Ca cells was accompanied by large decreases of phospholipid mass, loss of phospholipid label, and accumulation of unesterified labeled fatty acid. These changes were greatly decreased by incubation in -Ca medium. On the other hand, protection by glycine could not be attributed to modifications of either the massive breakdown of phospholipids that occurred in +Ca cells, or the modest changes seen in -Ca cells. In +Ca cells, the deleterious effects of increased Caf and phospholipid breakdown ultimately prevailed over protection by the amino acid. Thus, separate pathways of cell death associated with increased Caf and decreased glycine were defined in ATP depleted, Ca(+)- permeabilized MDCK cells. Calcium excess and massive phospholipid loss are features of a damage process that occurs independently of whether cells are protected by glycine or not. Conversely, the glycine sensitive component of injury is expressed regardless of whether intracellular Ca++ is increased, or large phospholipid losses occur. ATP depletion in -Ca medium provides a system to study mechanisms of glycine cytoprotection uncomplicated by Ca++ toxicity.
在因三磷酸腺苷（ATP）耗竭而受损的犬肾上皮细胞（MDCK）中，研究了胞质游离钙（Caf）、细胞相关甘氨酸、磷脂水解与细胞死亡之间的关系。用线粒体解偶联剂在无葡萄糖条件下孵育三小时，导致细胞内甘氨酸逐渐丧失。然而，除非同时诱导Ca++稳态的扰动，否则它们不会受到致命损伤。在1.25 mM Ca++培养基（+Ca）中暴露于Ca++离子载体和解偶联剂会导致细胞死亡加速。在100 nM Ca++培养基（-Ca）中用离子载体处理的ATP耗竭细胞也会受到致命损伤，但会有显著延迟。两组细胞在死亡前均出现甘氨酸耗竭。外源性甘氨酸（5 mM）可保护+Ca细胞免受致命的膜损伤，但一段时间后这种有益作用会消失。相比之下，-Ca细胞在整个过程中都得到了完全保护。测量了用3H-油酸预标记的细胞脂质部分中的磷脂质量和放射性标记。+Ca细胞的加速死亡伴随着磷脂质量的大幅下降、磷脂标记的丧失以及未酯化标记脂肪酸的积累。在-Ca培养基中孵育可大大减少这些变化。另一方面，甘氨酸的保护作用不能归因于+Ca细胞中发生的磷脂大量分解或-Ca细胞中观察到的适度变化的改变。在+Ca细胞中，Caf增加和磷脂分解的有害作用最终超过了氨基酸的保护作用。因此，在ATP耗竭、Ca(+)-通透的MDCK细胞中定义了与Caf增加和甘氨酸减少相关的细胞死亡的不同途径。钙过量和大量磷脂损失是一个损伤过程的特征，该过程的发生与细胞是否受到甘氨酸保护无关。相反，无论细胞内Ca++是否增加或是否发生大量磷脂损失，损伤的甘氨酸敏感成分都会表达。在-Ca培养基中ATP耗竭提供了一个系统，用于研究不受Ca++毒性影响的甘氨酸细胞保护机制。
