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 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+]i homeostasis. To test this potential mechanism as well as to investigate the relationship between .OH and Ca2+ overloading 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 quantitative fluorescence microscopy with the aid of the specific Ca2+ 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+]i which was followed by a slower but more pronounced [Ca2+]i 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 Ca2+ inhibited both the [Ca2+]i response and cell injury. Preincubation of the cells with the Ca2+ channel blocker verapamil or .OH radical scavenger mannitol similarly inhibited the [Ca2+]i rise and loss of viability. Firefly luciferase assay of cellular ATP content demonstrated that the alterations in [Ca2+]i following .OH treatment preceded the depletion of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
West Virginia University, School of Pharmacy, Morgantown 26506.
Y
Y
Rojanasakul
L
L
Wang
A H
AH
Hoffman
X
X
Shi
N S
NS
Dalal
D E
DE
Banks
J K
JK
Ma
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006878:Hydroxides / Q000378:metabolism*; D017665:Hydroxyl Radical; D007700:Kinetics; D016676:Macrophages, Alveolar / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D005609:Free Radicals; D006878:Hydroxides; D017665:Hydroxyl Radical; D002118:Calcium