MECHANISMS OF HYDROXYL FREE-RADICAL INDUCED CELLULAR INJURY AND CALCIUM OVERLOADING IN ALVEOLAR MACROPHAGES

Excessive production of reactive oxygen radicals by alveolar macrophag es is proposed to play an important role in oxidative lung injury. A m ajor product of oxygen radical formation is the highly reactive hydrox yl radical (.OH) generated via a biologic Fenton reaction. In addition to its known ability to induce lipid peroxidation, recent studies hav e suggested that the .OH may exert its cytotoxic effect through the al teration of [Ca2+]i homeostasis. To test this potential mechanism as w ell as to investigate the relationship between .OH and Ca2+ overloadin g in cytotoxic injury, isolated rat alveolar macrophages were exposed to externally generated radical system, H2O2 (0.01 to 1 mM) and Fe2+ ( 1 mM) and their [Ca2+]i levels and cell injury were monitored using qu antitative fluorescence microscopy with the aid of the specific Ca2+ i ndicator, 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 radica l by this system. Upon the addition of the radicals, the macrophages d isplayed a rapid initial rise in [Ca2+]i which was followed by a slowe r but more pronounced [Ca2+]i elevation that reached a level 3 to 5 ti mes higher than the basal level. This process preceded cell death as e vident by nuclear propidium iodide fluorescence. Depletion of extracel lular Ca2+ inhibited both the [Ca2+]i response and cell injury. Preinc ubation of the cells with the Ca2+ channel blocker verapamil or .OH ra dical scavenger mannitol similarly inhibited the [Ca2+]i rise and loss of viability. Firefly luciferase assay of cellular ATP content demons trated that the alterations in [Ca2+]i following .OH treatment precede d the depletion of ATP. Changes in membrane potential caused by Na+ su bstitution with K+ had no significant effect on either the unstimulate d or stimulated [Ca2+]i levels. Taken together, these measurements ind icate that .OH-induced injury in alveolar macrophages is associated wi th the alteration in [Ca2+]i homeostasis. The mechanism of .OH-induced [Ca2+]i loading is not due to nonspecific membrane damage or energy d epletion, but mainly due to an increase in Ca2+ influx through Ca2+ se lective channels. This Ca2+ over-load state as well as energy depletio n caused by the oxidant may activate certain cellular degradative proc esses leading to cell injury and death.