Original Contribution
Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes

https://doi.org/10.1016/j.freeradbiomed.2006.10.035Get rights and content

Abstract

CO2 changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO3radical dot / radical dotNO2 radical pair, and (ii) peroxynitrite diffusion distance is significantly reduced. For peroxynitrite generated extracellularly this last effect is particularly dramatic at low cell density because CO3radical dot and radical dotNO2 are short-lived and decay mostly in the extracellular space or at the cell surface/membrane. This study was aimed to distinguish between peroxynitrite-induced extra- and intracellular modifications of red blood cells (RBC). Our results show that at low cell density and in the presence of CO2 peroxynitrite induced the oxidation of surface thiols, the formation of 3-nitrotyrosine and DMPO-RBC adducts, and the down-regulation of glycophorins A and C (biomarkers of senescence). Reactivation of glycolysis reversed only the oxidation of surface thiols. Without CO2 peroxynitrite also induced the oxidation of hemoglobin and glutathione, the accumulation of lactate, a decrease in ATP, the clustering of band 3, the externalization of phosphatidylserine, and the activation of caspases 8 and 3 (biomarkers of apoptosis). The latter biomarkers were all reversed by reactivation of glycolysis. We hypothesize that cell senescence could (generally) be derived by irreversible radical-mediated oxidation of membrane targets, while the appearance of apoptotic biomarkers could be bolstered by oxidation of intracellular targets. These results suggest that, depending on extracellular homolysis or diffusion to the intracellular space, peroxynitrite prompts RBCs toward either senescence or apoptosis through different oxidation mechanisms.

Introduction

Peroxynitrite2 is a strong oxidant and reacts directly with several cellular targets including thiols [1], [2] and hemoproteins [3], [4], [5], [6]. These direct reactions are biologically relevant especially with substrates present in tissues at high concentrations. The O–O bond of peroxynitrite is exceptionally weak so that its conjugated acid peroxynitrous acid (ONOOH, pKa = 6.5–6.8) homolyzes to yield 20–30% of a geminate pair of radicals, radical dotOH and radical dotNO2, and 70–80% NO3 [7].

The free radical chemistry based on the radical dotOH/radical dotNO2 pair is of minor relevance in vivo due to the presence of targets which can react with peroxynitrite before homolysis [8]. In apparent contradiction, several recent reports (see, for example, [9], [10], [11], [12], [13]) have shown that under biologically relevant conditions peroxynitrite still behaves in a way that suggests free radical chemistry. This discrepancy is explained by the presence in tissues of (bi)carbonate (i.e., CO2, H2CO3, HCO3, and CO3–2) at 20–30 mM. Indeed, CO2 is the species that reacts with peroxynitrite to form about 35% of a geminate pair of radicals, CO3radical dot and radical dotNO2, and ∼ 65% NO3 [14]. Due to the relatively high concentration of CO2 in tissues (1.3 mM at pH 7.4), the CO2-catalyzed homolysis of peroxynitrite outcompetes many other reactions and suggests that under physiological conditions the formation of CO3radical dot and radical dotNO2 radicals is a relevant indirect peroxynitrite oxidation pathway [15].

A second relevant effect of CO2 is the reduction in the half-life of peroxynitrite from about 1–2 to 0.01–0.02 s [16]. CO2 thus drastically limits the diffusion and cell killing activity of peroxynitrite [17], [18], [19]. This finding can easily be rationalized if it is considered that the half-life of CO3radical dot and radical dotNO2 is shorter than that of peroxynitrite and these radicals can therefore cover a much shorter diffusion distance. Even though radical dotNO2 is significantly more stable and hydrophobic than CO3radical dot [19], its diffusion distance is shorter than that of peroxynitrite (e.g., 0.8–0.2 μm [20]). In experiments with diluted cell suspensions the addition of (bi)carbonate seems to be a means of “detoxifying” extracellular peroxynitrite, but this cannot be extrapolated to the in vivo situation for at least two reasons. First, the cell density of tissues is generally high, so that a fraction of extracellular peroxynitrite reacts with CO2 and a fraction enters neighboring cells. Secondly, CO3radical dot and radical dotNO2 are reactive radicals able to cause the oxidation not only of extracellular components but also of the cell surface/membrane. It is worth noting that these two oxidation mechanisms of peroxynitrite, also referred to as direct and indirect pathways [16], usually coexist and that their effects on cells have not been analyzed separately.

These considerations are particularly appropriate in the case of red blood cells (RBCs). These cells are, in fact, the major scavengers of peroxynitrite in blood and it has been calculated that at 45% hematocrit about 40–45% of peroxynitrite crosses the cell membrane and quickly reacts with hemoglobin (Hb), while the remainder reacts extracellularly with CO2 [21], [22]. These authors developed a theoretical model to estimate the percentage of peroxynitrite that would reach a target located at a certain distance from its site of production. For example, at room temperature and in the presence of 1.3 mM CO2 about 99% of peroxynitrite decays within a radius of approx 20 μm from the site of addition whereas without CO2 99% of this decay is predicted to occur within a radius of approx 250 μm.

In a previous work [23] we reported that, without (bi)carbonate and at low cell density, the addition of peroxynitrite to RBCs caused the formation of methemoglobin (metHb) as well as the appearance of several biomarkers of cell aging (senescence) and cell death (apoptosis). The formation of metHb clearly indicates that peroxynitrite diffuses into the cell, although it was unclear if the appearance of the other biomarkers arises from extra- or intracellular reactions. Although devoid of those organelles that play a key role in apoptosis (mitochondria, nucleus), RBCs can still display phosphatidylserine (PS) externalization (annexin V binding) and the activation of caspases [23], [24], [25], [26]. Another marker of RBC apoptosis is band 3 clustering, which generates a cell surface epitope identified by autologous IgG antibodies and may act as a signal for the removal of RBCs from circulation [27], [28]. It is also widely accepted that RBC senescence (or cell aging) is accompanied by a loss of the surface negative charge which is associated with the sialic acid of glycophorins [29]. The down-regulation of these glycoproteins, and in particular of glycophorin A, is considered a biomarker (indicative) of cell senescence [29], [30], [31].

The aim of this study was to distinguish between extra-and intracellular RBC modifications induced by peroxynitrite. Therefore, we treated RBCs with peroxynitrite in the presence or in the absence of (bi)carbonate at low cell density (0.25% hematocrit).

Section snippets

Chemicals and peroxynitrite preparation

ONOO was synthesized from sodium nitrite and hydrogen peroxide and stabilized by alkali [1] with minor modifications [23]. Unless otherwise indicated, all chemicals were from Sigma-Aldrich (Milan, Italy).

Erythrocyte isolation and treatments

Heparinized fresh human blood was obtained from healthy donors following informed consent. Plasma and buffy coat were removed by centrifugation (10 min, 1000g) and RBCs were washed three times with isotonic phosphate-buffered saline (PBS), pH 7.4. Washed RBCs were suspended in PBS at 0.25%

Kinetics and reversibility of peroxynitrite-induced Hb oxidation

Without (bi)carbonate the metHb induced by 50 μM peroxynitrite at 0.25% hematocrit (1:1 Hb:peroxynitrite molar ratio) was 11.2 μM (Fig. 1A). At this hematocrit the mean distance between two RBCs is about 900 μm, so that peroxynitrite might be expected partly to cross some cells (those nearer than 250 μm) and in part to decay in the extracellular space by proton-catalyzed decomposition [21], [22]. Instead, in the presence of CO2 the metHb induced by peroxynitrite was only 1.2 μM because most of

Discussion

The present results show that the erythrocyte is a suitable cell model to study the modifications induced by peroxynitrite. Besides, it has been demonstrated that peroxynitrite is able to induce distinct cellular biomarkers when it acts extra-or intracellularly. Intracellular oxidations are due mostly to direct reactions of peroxynitrite, whereas surface/membrane oxidations are due principally to indirect radical reactions generated by CO2-catalyzed homolysis. It is worth noting that both

Acknowledgments

This work was supported in part by ISS-NIH collaborative project “Peripheral blood determinants of redox changes in human respiratory diseases: biochemical and pathophysiological evaluations” Rif. 0F14. We are grateful to Prof. G. Girelli, Centro Trasfusionale, Università La Sapienza, Roma, for providing blood samples.

References (51)

  • N.S. Cohen et al.

    Biochemical characterization of density-separated human erythrocytes

    Biochim. Biophys. Acta

    (1976)
  • M.E. Reid et al.

    Red blood cell blood group antigens: structure and function

    Semin. Hematol.

    (2004)
  • H.U. Lutz et al.

    Total sialic acid content of glycophorins during senescence of human red blood cells

    J. Biol. Chem.

    (1979)
  • C.C. Winterbourn

    Oxidative reactions of hemoglobin

    Methods Enzymol.

    (1990)
  • D. Pietraforte et al.

    Scavenging of reactive nitrogen species by oxygenated hemoglobin: globin radicals and nitrotyrosines distinguish nitrite from nitric oxide reaction

    Free Radic. Biol. Med.

    (2004)
  • P.F. Devaux et al.

    Maintenance and consequences of membrane phospholipid asymmetry

    Chem. Phys. Lipids

    (1994)
  • H. Ischiropoulos

    Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species

    Arch. Biochem. Biophys.

    (1998)
  • R.P. Mason

    Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping

    Free Radic. Biol. Med.

    (2004)
  • M.R. Gunther et al.

    Self-peroxidation of metmyoglobin results in formation of an oxygen-reactive tryptophan-centered radical

    J. Biol. Chem.

    (1995)
  • L.J. Bruce et al.

    A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane

    Blood

    (2003)
  • W. Nunomura et al.

    Regulation of protein 4.1R, p55, and glycophorin C ternary complex in human erythrocyte membrane

    J. Biol. Chem.

    (2000)
  • C. Quijano et al.

    Pathways of peroxynitrite oxidation of thiol groups

    Biochem. J.

    (1997)
  • R. Floris et al.

    Interaction of myeloperoxidase with peroxynitrite. A comparison with lactoperoxidase, horseradish peroxidase and catalase

    Eur. J. Biochem.

    (1993)
  • M. Minetti et al.

    Scavenging of peroxynitrite by oxyhemoglobin and identification of modified globin residues

    Biochemistry

    (2000)
  • M. Exner et al.

    Kinetic and mechanistic studies of the peroxynitrite-mediated oxidation of oxymyoglobin and oxyhemoglobin

    Chem. Res. Toxicol.

    (2000)
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