Abstract
Peroxynitrite toxicity has been implicated in the pathogenesis of white matter injury. The mechanisms of peroxynitrite toxicity to oligodendrocytes (OLs), the major cell type of the white matter, are unknown. Using primary cultures of mature OLs that express myelin basic protein, we found that 3-morpholinosydnonimine, a peroxynitrite generator, caused toxicity to OLs. N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a zinc chelator, completely blocked peroxynitrite-induced toxicity. Use of FluoZin-3, a specific fluorescence zinc indicator, demonstrated the liberation of zinc from intracellular stores by peroxynitrite. Peroxynitrite caused the sequential activation of extracellular signal-regulated kinase 42/44 (ERK42/44), 12-lipoxygenase, and generation of reactive oxygen species, which were all dependent upon the intracellular release of zinc. The same cell death pathway was also activated when exogenous zinc was used. These results suggest that in addition to preventing the formation of peroxynitrite, useful strategies in preventing disease progression in pathologies in which peroxynitrite toxicity plays a critical role might include maintaining intracellular zinc homeostasis, blocking phosphorylation of ERK42/44, inhibiting activation of 12-lipoxygenase, and eliminating the accumulation of reactive oxygen species.
Highlights
Peroxynitrite, the reaction product of nitric oxide and superoxide [1], is a potent oxidant capable of inducing DNA damage [2], lipid peroxidation [3], and protein nitration [4, 5]
Using primary cultures of rat brain OLs that express myelin basic protein, we found that peroxynitrite toxicity to OLs is mediated via a zinc-12-LOX-ROS pathway, and that ERK activation is upstream of 12-LOX activation and ROS generation
Similar to our observations in neuronal cultures [38], the toxicity of SIN-1 (500 M) to OLs was blocked by carboxyl-PTIO (50 M), a nitric oxide scavenger, or superoxide dismutase (100 units/ml) plus catalase (100 units/ml), which block the accumulation of superoxide (Fig. 1B)
Summary
Materials—3-Morpholinosydnonimine (SIN-1), peroxynitrite, carboxyl-PTIO, and FeTMPyP were obtained from Cayman Chemical Co. (Ann Arbor, MI). OLs were treated with SIN-1 or peroxynitrite for 2 h or zinc chloride (ZnCl2) for 90 min, washed twice with Hank’s balanced salt solution containing 0.1% BSA, and placed in BDM with T3 and ciliary neurotrophic factor. Western Blot Analysis of ERK and p38 Phosphorylation—At various times after SIN-1 or zinc treatment, OLs were placed on ice. After medium aspiration, cells were washed once with ice-cold phosphatebuffered saline, and lysed with lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerolphosphate, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride.
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