PROCEEDINGS |
Inhibition of cellular ATP synthesis induced by complete ischemia or chemical inhibitors rapidly initiates cell death via the oncosis pathway (Majno, G. and Joris, I. Am. J. Pathol. 146:3-15, 1995), whereas hypoxia often induces cell death through the apoptosis pathway. Additionally, other types of injury may induce apoptosis in some situations and oncosis in others. In order to determine the cellular "switching" involved in the decision to follow the apoptotic vs the oncotic pathway, we tested the hypothesis that ATP concentration was an important determining factor. Apoptosis was induced in NRK-52E (normal rat kidney) cells by microinjecting cytochrome c. NRK-52E monolayers were grown on grid slides to permit precise mapping of injected cells for later studies by light and electron microscopy. Injection of cytochrome c into untreated NRK cells resulted in apoptosis of 35% of cells by 30 min and 94% by 1 hr as characterized by phase, DAPI staining, and TEM. In order to test the hypothesis that ATP was a critical determining factor, we depleted ATP prior to cytochrome c microinjection by pretreatment of the cells with 0.25 mM KCN + 0.25 mM IAA. Such pretreatment resulted in marked decrease of the normal concentration of 24 femtomoles of ATP/cell to 5 femtomoles/cell after 1 hr. If cytochrome c was injected following 30 min, only 5% underwent apoptosis by 30 min and 23% by 1 hr. Instead, the ATP-depleted cells proceeded to show oncosis only after cytochrome c injection. These results therefore clearly support the hypothesis that ATP concentration (with a threshold of ~5 femtomoles/cell or ~5% of normal ATP concentration) represents one critical determinant in the cells' decision to choose apoptosis rather than oncosis on its pathway to cell death following a lethal injury. (Supported by NIH DK15440).