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

Abstract

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)

Hydrogen Peroxide Induces Intracellular Calcium Overload by Activation of a Non-selective Cation Channel in an Insulin-secreting Cell Line

“…Such a change in NAD combined with the initial rise in [Ca 2ϩ ] i could conspire to create an environment conducive to NS NAD channel activation. DISCUSSION Previous studies have demonstrated that H 2 O 2 , most probably through the generation of reactive oxygen species, causes an increase in intracellular calcium levels that precedes, if not causes, cell death in a wide variety of cell types, including cardiac (27) and smooth muscle cells (26), pancreatic acinar cells (34), alveolar macrophages (35), and central nervous system neurons (36). Here, we describe the effects of H 2 O 2 on [Ca 2ϩ ] i in the insulin-secreting cell line CRI-G1 and clearly demonstrate that H 2 O 2 also disrupts calcium homeostasis in these cells.…”

“…Here, we describe the effects of H 2 O 2 on [Ca 2ϩ ] i in the insulin-secreting cell line CRI-G1 and clearly demonstrate that H 2 O 2 also disrupts calcium homeostasis in these cells. There is no clear consensus in the literature indicating the likely mechanism by which H 2 O 2 causes an increase in [Ca 2ϩ ] i ; suggestions that have been promulgated include influx through voltage-dependent calcium channels (25,35), nonspecific changes in membrane calcium permeability (37,38), alteration in Na ϩ -Ca 2ϩ exchange (39), or changes in calcium release from intracellular stores (40,41).…”

“…Activation of voltage-gated calcium channels has been implicated in the autoimmune-associated destruction of pancreatic beta cells (44) and has been suggested to contribute to cellular response to oxidative stress (25,35,45). Therefore, a possible contribution to the observed effects of H 2 O 2 from this permeation pathway was investigated in CRI-G1 cells using the inorganic calcium channel blocker Cd 2ϩ .…”

Fluoromicroscopic Studies of Bleomycin-Induced Intracellular Oxidation in Alveolar Macrophages and Its Inhibition by Taurine

“…For the measurement of intracellular free calcium (16), AM cultures were incubated with 5 pM Fura-2 AM in HEPES buffer with 2% FBS for 1 hr at 37°C and washed with the same buffer to remove free dye and nonadherent cells. (Stock solution of Fura-2 AM was made by dispersing 50 pg Fura-2 AM in 100 pl of 20% w/v of Pluronic F-127 in dimethyl sulfoxide.)…”

“…The most significant and deleterious consequences of oxidation, such as DNA strand breakage (5), damage to the cytoskeleton (14,15), and a rise in intracellular calcium (16), occur intracellularly and can lead to a cascade of cellular events resulting in cell death. Detection of intracellular oxygen radicals is difficult as they are highly reactive and extracellular modes of detection such as luminol-derived chemiluEnvironmental Health Perspectives 91 minescence or electron spin resonance signals rely on detection of extracellularly generated radicals (17) or leakage from a compromised cell membrane.…”