Mechanisms of Hydroxyl Free Radical-induced Cellular Injury and Calcium Overloading in Alveolar Macrophages

Excessive production of reactive oxygen radicals by alveolar macrophages is proposed to play an important role in oxidative lung injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical (•OH) generated via a biologic Fenton reaction. In addition to its known ability to induce lipid peroxidation, recent studies have suggested that the •OH may exert its cytotoxic effect through the alteration of [Ca2+], homeostasis. To test this potential mechanism as well as to investigate the relationship between •OH and Ca" overloading in cytotoxic injury, isolated rat alveolar macrophages were exposed to externally generated radical system, H2O2 (0.01 to 1 mM) and Fe (1 mM) and their [Ca2+] levels and cell injury were monitored using quantitative fluorescence microscopy with the aid of the specific Ca" indicator, Fura-2, and membrane integrity indicator, propidium iodide. Electron spin resonance measurements using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) confirmed the production of the .OH radical by this system. Upon the addition of the radicals, the macrophages displayed a rapid initial rise in [Ca2+] which was followed by a slower but more pronounced [Ca2+], elevation that reached a level 3 to 5 times higher than the basal level. This process preceded cell death as evident by nuclear propidium iodide fluorescence. Depletion of extracellular Ca" inhibited both the [Ca2+], response and cell injury. Preincubation of the cells with the Ca" channel blocker verapamil or .0H radical scavenger mannitol similarly inhibited the [Ca2+], rise and loss of viability. Firefly luciferase assay of cellular ATP content demonstrated that the alterations in [Ca2+], following .0H treatment preceded the depletion of ATP. Changes in membrane potential caused by Na* substitution with K* had no significant effect on either the unstimulated or stimulated [Ca"], levels. Taken together, these measurements indicate that .OH-induced injury in alveolar macrophages is associated with the alteration in [Ca2+], homeostasis. The mechanism of •OH-induced [Ca2+] loading is not due to nonspecific membrane damage or energy depletion, but mainly due to an increase in Ca2+ influx through Ca2+ selective channels. This Ca2+ over-load state as well as energy depletion caused by the oxidant may activate certain cellular degradative processes leading to cell injury and death.