Abstract
We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 microM ONOO-) disappearance at pH 7. 4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ss-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 microM ONOO-). However, a lack of effect of ss-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that (ONOO-)-induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 microM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.
Highlights
Peroxynitrite anion (ONOO-), a highly reactive and biologically important species, is produced under physiological conditions and in vivo by the reaction of superoxide anion radical (O2·-) with nitric oxide (·NO),(·NO + O2·- ® ONOO-) [1]
It has been shown that ONOO- promotes lipoperoxidation, protein nitration and a decrease of intracellular reduced glutathione in human erythrocyte [15]
Red blood cell (RBC) shape and deformability are regulated by a submembrane cytoskeleton whose major proteins responsible for regulating the membrane topography are: spectrin, actin, ankyrin, band 4.1, band 4.9, and tropomyosin
Summary
Peroxynitrite anion (ONOO-), a highly reactive and biologically important species, is produced under physiological conditions and in vivo by the reaction of superoxide anion radical (O2·-) with nitric oxide (·NO),. Peroxynitrites are able to react with lipids, DNA, proteins and small antioxidant molecules such as glutathione [13,14] These rapid and specific reactions are likely to inactivate important cellular targets. Red blood cell (RBC) shape and deformability are regulated by a submembrane cytoskeleton whose major proteins responsible for regulating the membrane topography are: spectrin, actin, ankyrin, band 4.1, band 4.9, and tropomyosin These proteins are arranged in a network connected to integral membrane proteins along the bilayer membrane through associations between ankyrin and band 3 and between band 4.1 and glycophorin [16]. Red blood cell membrane ghosts offer a good model for studying protein damage induced by ONOO- because their protein composition is well known and they lack organelles, which makes them a simple and suitable biological system
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