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 mw 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 occured 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.
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