EFFECTS OF CA-DEPLETED MDCK CELLS(+ AND GLYCINE ON LIPID BREAKDOWN AND DEATH OF ATP)

The relationships between cytosolic free calcium (Caf), cell associate d glycine, phospholipid hydrolysis and cell death were investigated in Madin-Darby canine kidney (MDCK) cells injured by depletion of adenos ine 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 o f Ca++ homeostasis was also induced. Exposure to a Ca++ ionophore and uncoupler in 1.25 mM Ca++ medium (+Ca) resulted in accelerated cell de ath. ATP depleted cells with ionophore in 100 nM Ca++ medium (-Ca) wer e also lethally injured, but after a significant delay. Depletion of g lycine preceded death in both groups of cells. Exogenous glycine (5 mM ) protected +Ca cells against lethal membrane damage, but the benefici al effects were lost over a period of time. In contrast, -Ca cells wer e completely protected throughout. Phospholipid mass and radioactive l abel in lipid fractions of cells prelabeled with H-3-oleic acid were m easured. Accelerated death of +Ca cells was accompanied by large decre ases of phospholipid mass, loss of phospholipid label, and accumulatio n of unesterified labeled fatty acid. These changes were greatly decre ased by incubation in -Ca medium. On the other hand, protection by gly cine could not be attributed to modifications of either the massive br eakdown of phospholipids that occured in +Ca cells, or the modest chan ges seen in -Ca cells. In +Ca cells, the deleterious effects of increa sed Caf and phospholipid breakdown ultimately prevailed over protectio n 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 phospholipi d loss are features of a damage process that occurs independently of w hether cells are protected by glycine or not. Conversely, the glycine sensitive component of injury is expressed regardless of whether intra cellular Ca++ is increased, or large phospholipid losses occur. ATP de pletion in -Ca medium provides a system to study mechanisms of glycine cytoprotection uncomplicated by Ca++ toxicity.