Mechanisms of hydroxyl free radical-induced cellular injury and calcium overloading in alveolar macrophages.

Y. Rojanasakul, L. Wang, A. H. Hoffman, X. Shi, N. S. Dalal, D. E. Banks, J. K. Ma

Research output: Contribution to journalArticlepeer-review

33 Scopus citations


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)

Original languageEnglish
Pages (from-to)377-383
Number of pages7
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Issue number4
StatePublished - Apr 1993

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology


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