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Nitric oxide induced oxidative changes in erythrocyte membrane components
Cell Biology International 32 (2008) 114e120 www.elsevier.com/locate/cellbi
Nitric oxide induced oxidative changes in erythrocyte membrane components Joanna Brzeszczynska a,*, Krzysztof Gwozdzinski b a
School of Life Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK b Department of Biophysics, University of Lodz, 90-237 Lodz, Poland Received 20 March 2007; revised 7 June 2007; accepted 27 August 2007
Abstract The effects of NO in its environment may vary considerably depending on various factors. This study shows oxidative mechanism of cellular membrane alterations, which is not associated with triggering of ONOOH generation but is induced by pure NO. Our investigation examined the influence of low concentration of NO (0.1; 0.2 mmol/l) on the qualitative changes of structure and dynamics of erythrocyte membrane. NO causes a statistically significant increase in membrane fluidity on different depths of lipid bilayer that is correlated with increase of lipids peroxidation. Statistically significant changes in the conformational state of cytoskeleton proteins were also detected. NO can be considered as a molecule responsible for determining rheological properties of erythrocytes membrane. Therefore, we propose that NO acts as pro-oxidant molecule at concentrations for which membrane appeared to be the first target before it entered the cytosol. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Oxidative stress; Spin-labeling; Erythrocyte membrane; RBC
1. Introduction Nitric oxide (NO) is a small, hydrophobic molecule with a high diffusion coefficient and short life-span. Similarly to reactive oxygen species (ROS), it is an unstable molecule that can quickly react with for example hemoglobin, myoglobin, oxygen, superoxide anions, and various cellular components. The molecular targets of NO in vivo belong to the following 3 categories (Kaminski and Andrade, 2001; Reid, 1998). First, there are reactions with molecular oxygen and superoxide anions. Under biological conditions the autooxidation reaction of nitric oxide takes place with the rate constant k ¼ 106 M1 s1 (Wink et al., 1996). Subsequently formed nitrogen dioxide and/ or peroxynitrite can mediate oxidative injury of cellular components. Second, there are reactions with transition metals, such as heme iron or iron-sulphur clusters, which lead to metal-NO
* Corresponding author. Tel.: þ44 131 451 4679; fax þ44 131 451 3009. E-mail address: [email protected] (J. Brzeszczynska).
products. Third, there are reactions with reduced protein thiol groups (RSH, RS-), which form RS-NO groups. Gladwin et al. (2004) suggest that the major vascular pool of NO in vivo is nitrite, which is present at concentrations of 0.5e10 mM in plasma, erythrocytes and tissues. Depending on the concentration of NO in its biological environment, the effect of this molecule may vary considerably. At high concentrations, NO has antimicrobial, antitumor and cytotoxic effects, while at low concentrations it stimulates guanylate cyclase and influences blood pressure regulation, blood flow, neuronal transmission and neuroendocrine activity (Bergendi et al., 1999). It is also known to be a modulator of several aspects of skeletal muscle function (Stamler and Meissner, 2001). Thus, NO is considered to be an endogenous modulator of numerous cellular functions in a variety of tissues, with the properties of a cell signalling molecule (Beckman, 1996). Beside this, NO participates in the regulation of erythrocyte membrane properties and in the regulation of the rheological behavior of erythrocytes (Tsuda et al., 2003; McCord and Fridovich, 1969). Numerous studies show that cell membrane
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.08.020
J. Brzeszczynska, K. Gwozdzinski / Cell Biology International 32 (2008) 114e120
Nomenclature EPR Hb MetHb HbO2 MSL ISL 5-,12-,16-DS RBC RNS NO NO2 ONOOH Fe2þ SOD NOS PBS TBARS TBA MDA NaCl
electron paramagnetic resonance hemoglobin methemoglobin oxygenated hemoglobin 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl 4-iodoacetamide-2,2,6,6-tetramethylpiperidine-1-oxyl 5-,12-,16-doxylstearic acid red blood cell reactive nitrogen species nitric oxide nitrogen dioxide peroxynitrite iron ions superoxide dismutase nitric oxide synthase phosphate buffered saline thiobarbituric acid reactive substances thiobarbituric acid malondialdehyde sodium chloride
abnormalities of erythrocyte and also structural changes of the erythrocyte membrane are an etiological factor in many diseases of the circulatory system, e.g. hypertension (Tsuda et al., 2000, 2003). Therefore, membrane fluidity is an important physiochemical characteristic of biomembranes and an important factor in regulating membrane micro viscosity, cell functions and its rheological behavior. Membrane proteins are also very sensitive to the attack of NO. Particularly, the thiol groups are modified, which influences the regulating process of protein functions. Nitric oxide can be involved in the process of distinct protein modification, nitration of aromatic residues, nitrosylation of metal, and generation of S-nitrosothiols (Ischiropoulos and Gow, 2005). If these modifications are not prevented, they may lead to initiation of pathological responses. On the other hand, they can transduce physiological responses and signalling. Tyrosine nitration influences alteration of protein functions (Ischiropoulos and Gow, 2005). It can also result in the changes of proteinprotein interactions as well as other aspects of the protein cycle, such as the protein half-life (Ischiropoulos, 2003). Proteins peroxidation induces changes in the conformational state of membrane proteins that may contribute to the inhibition of erythrocyte membrane enzymes and transporting proteins. Alteration in the membrane transporting system can induce many processes dangerous to cells and finally can even lead to the cell death (Rohn et al., 1993). In our investigation we have introduced NO, which creates the conditions more similar to the physiological ones. The concentrations, which have been chosen in this study, are relatively low as for the examinations in vitro. Furthermore, this
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investigation shows oxidative mechanism of cellular membrane alterations, which are not associated with triggering of ONOOH generation but are induced by pure NO. Moreover, introduction of pure NO, as a factor that initiates oxidative damages in the cell, makes this investigation novel. 2. Materials and methods 2.1. Synthesis of NO NO was generated under anaerobic conditions through the reaction of nitric acid (10%) and copper, purified in alkaline solution and dried over solid potassium hydroxide and dissolved in water at 20 C (Archer, 1993). Stock solution (2 mM NO) was added to the samples and the final concentration of NO was 0.1 or 0.2 mM. 2.2. Erythrocytes and erythrocyte ghosts Human blood was collected from healthy donors of between 18 and 30 years attending the local blood bank. Donors were examined by the doctor and were recruited according to the established criteria. Packed cells were suspended in PBS to a haematocrit of 50%. Erythrocytes (isolated and in the whole blood) were treated with 0.1 or 0.2 mM NO in the presence or absence of 0.1 mM FeSO4 at 22 C 1 for 1 h, washed twice with 0.9% NaCl and once with PBS. Erythrocyte ghosts were prepared by the method of Dodge et al. (1963) with the modification of including hypertonic lysis using 20 mM sodium phosphate buffer (pH 7.4) at 4 C. The ghosts were successively washed with 20 mM, 10 mM and 5 mM phosphate buffer (pH 7.4) and finally suspended in 5 mM phosphate buffer (pH 7.4) at 4 C. 2.3. Spin labeling of erythrocytes and erythrocyte ghosts Erythrocytes were labeled with 5-DS, 12-DS and 16-DS by introduction of the doxyl derivatives in ethanol into the erythrocyte suspension and incubation for 30 min at 22 C 1. Temperature was stable during measurements. The final ethanol concentration in the erythrocyte suspension was less than 0.05% (v/v). From the spectra the ratio hþ1/h0 was calculated, with hþ1 representing the height of the low-field line and h0 representing the height of the middle-field line of the spectrum. This ratio is correlated with lipid bilayer fluidity and serves as a semi-quantitative measure of acyl side chain flexibility (Morrisett et al., 1975). To investigate conformational changes of membrane proteins, MSL and ISL were used, which bind covalently to the -SH groups of membrane proteins. Erythrocyte membranes were labeled with MSL or ISL using 2 ml of ethanol solution with 100 mM MSL or ISL per 1 ml of membrane suspension (w3 mg$ml1 of protein), and incubated for 1 h at 4 C. Unbound spin label was removed through several washings with cold phosphate buffer. The effect of physiological oxidative stress on the ratio of weakly to strongly immobilized
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residues of erythrocyte membrane-bound MSL spin labels (hw/hs) was studied (Fung, 1983). In the case of ISL the mobility of attached spin label was estimated by calculating the rotational correlation time (tc) according to the Kivelson formula (Kivelson, 1960). EPR spectra were obtained at 22 C 1 using a Bruker ESP 300 E spectrometer, operating at a microwave frequency of 9.73 GHz. The instrumental settings were: center field 3480G; scan range 80G; modulation frequency 100 kHz; modulation amplitude 1G. 2.4. Lipid peroxidation Lipid peroxidation was measured with thiobarbituric acid (TBA) using the method described by Lapage et al. (1991). The absorbance of the butanol layer of the supernatant was measured at l ¼ 535 nm to estimate the level of lipid peroxidation. 2.5. Osmotic fragility Osmotic fragility was estimated according to the spectrophotometric method described by Marimoto et al. (1995). Changes in osmotic fragility were presented as a concentration of NaCl, for which 50% of erythrocytes in the investigated groups underwent hemolysis C(50%). 2.6. Statistical analysis Normality of distributions was tested using the Shapiroe Wilk test. The significance of the difference between couples of means (control and investigated probes as well as the differences among the probes) was assessed by the Tukey test at a < 0.05 significance level. 3. Results The spin-labeling technique allows for an improved understanding of the molecular mechanisms of membrane changes and was used to determine alterations in the physical state of erythrocyte membrane components caused by treatment with NO. Spin labels were used to investigate both lipid fluidity and the conformational state of proteins. Through binding to macromolecules these substances can act as reporting groups, providing information about the changes in the microenvironment in the nearest region. Spectral analysis of samples labeled with free doxyl derivatives provides information about structural characteristics and dynamics of cell membranes. The spectral parameter hþ1/h0 was measured and served as a semi-quantitative measure of acyl chain flexibility corresponding to lipid bilayer fluidity. The changes in hþ1/h0 ratio of applied doxyl spin labels generally indicated statistically significant increases in membrane lipid fluidity at different depths of lipid bilayer after the incubation of erythrocytes with 0.2 mmol/l of NO. A statistically significant increase in lipid membrane fluidity was observed for 5-DS in both groups of erythrocytes. There were significant
changes found for the isolated erythrocytes: mC sm0:2NO and mC sm0:2NOþFe at p < 0:05 as well as for the erythrocytes in the whole blood: mC sm0:2NOþFe at p < 0:05 (Fig. 1)1. For 12-DS non-significant changes in hþ1/h0 ratio were observed. However, for 16-DS statistically significant changes were observed for the following couples of means for the erythrocytes in the whole blood: m0:1NO sm0:2NOþFe , m0:2NO sm0:1NOþFe at p < 0:05 and m0:2NO sm0:2NOþFe at p < 0:01 (Fig. 2). On the other hand, a slight tendency to an increase of lipids fluidity both for 12-DS and for 16-DS was seen in all model groups. In order to investigate conformational changes of membrane proteins we used MSL and ISL. Effect of NO on membrane proteins, in both investigated erythrocytes groups, resulted in an increase in the ratio hw/hs, indicating an increase in spin label mobility. For the isolated erythrocytes, statistically significant changes were observed for the following couples of means: mC sm0:2NO , mC sm0:2NOþFe at p < 0:005 and for the erythrocytes in the whole blood for: mC sm0:2NO and mC sm0:2NOþFe at p < 0:005 (Fig. 3). This suggests that NO induces the loss of interaction between the spectrin-actin complex and other proteins, e.g. band 4.1, which links the spectrin network to glycophorin, or ankyrin, which links it to band 3 (Dodge et al., 1963; Caprari et al., 1995). No differences were observed between isolated erythrocytes and erythrocytes suspended in the whole blood, nor was the increase in the ratio hw/hs affected by the addition of Fe2þ. In contrast to MSL, ISL attached to erythrocyte ghosts yields a triplet spectrum, from which it is possible to calculate rotational correlation time (tc), providing a measure for the mobility of this spin label. Values for tc tend to be negatively correlated with the ratio hw/hs. Thus, the observed decrease in tc in the presence of 0.2 mM NO for the isolated erythrocytes: mC sm0:2NO at p < 0:001 was in line with results obtained for MSL and for the erythrocytes in the whole blood for: mC sm0:2NO at p < 0:001. However, addition of Fe2þ appeared to attenuate this decrease: m0:2NO sm0:2NOþFe at p < 0.05 (Fig. 4). Estimation of lipids peroxidation indicated changes in the amount of releasing substances reacting with thiobarbituric acid. Fig. 5 shows the influence of NO and Fe2þ on isolated erythrocytes and erythrocytes suspended in the whole blood. For the isolated erythrocytes we observed statistically significant changes for the following couples of means: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe and mC sm0:2NOþFe at p < 0:005. For the erythrocytes in the whole blood the changes were statistically significant in the case of the following couples of means: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe , mC sm0:2NOþFe and m0:1NO sm0:2NOþFe at p < 0:005 and m0:1NO sm0:2NO and m0:1NOþFe sm0:2NOþFe at p < 0:05. Under the investigated conditions NO appears to have prooxidant properties. This effect seems to be more pronounced in isolated erythrocytes compared to erythrocytes suspended in the whole blood and is enhanced in the presence of Fe2þ.
1
For this and all the subsequent figures, n stands for the number of subjects.
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Fig. 1. Influence of different concentrations of NO in the presence or absence of Fe2þ on the changes of the parameter hþ1/h0 for 5-DS (n ¼ 7). For the isolated erythrocytes: mC sm0:2NO and mC sm0:2NOþFe at p < 0:05. For erythrocytes in the whole blood: mC sm0:2NOþFe at p < 0:05.
NO did not induce significant changes in osmotic fragility although a trend ( p ¼ 0.067) towards increased osmotic fragility was observed for isolated erythrocytes in the presence of Fe2þ. However, particularly important is the fact that oxidative changes were observed before cell lysis.
4. Discussion The aim of this study was investigation of the role of NO in the qualitative changes of structure and function of human erythrocyte membrane in vitro using an EPR and spin-labeling method. The effects of 2 concentrations of NO (0.1 and 0.2 mmol/l) on the membrane components structure were investigated in isolated erythrocytes as well as in erythrocytes suspended in the whole blood, both in the presence or absence of Fe2þ. Another purpose of the study was to measure lipid fluidity at different depths of erythrocyte membrane and to
detect oxidative changes in the structure of membrane proteins by estimating the conformational state of membrane proteins. Our results reveal changes in erythrocytes membrane integrity after NO treatment using the concentrations of NO, which are relatively low as for the examinations in vitro. We emphasise that we introduced NO, which is generated in vivo, while other investigators usually use synthetic peroxynitrite. However, the chosen concentrations have also commonly been used by other authors (Soszyn´ski and Bartosz, 1996, 1997a,b). Moreover, peroxynitrite is generated at the same time nitric oxide and superoxide anion are generated, but we introduced NO which creates the conditions more similar to the physiological ones. By introducing pure NO, we induced oxidative changes that have not previously been investigated. We presented the modulator action of exogenous NO on erythrocyte membrane and we observed the oxidative deterioration of membrane lipids and changes in conformational state of membrane proteins. Low concentrations of NO regulate
Fig. 2. Influence of different concentrations of NO in the presence or absence of Fe2þ on the changes of the parameter hþ1/h0 for 16-DS (n ¼ 7). For the erythrocytes in the whole blood: m0:1NO sm0:2NOþFe , m0:2NO sm0:1NOþFe at p < 0:05 and m0:2NO sm0:2NOþFe at p < 0:01.
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Fig. 3. Changes of hw/hs for MSL bound with erythrocytes membrane proteins in control and in RBC and RBC in the whole blood after treatment with 0.2 mmol/l NO in the presence or absence of Fe2þ (n ¼ 6). For the isolated erythrocytes: mC sm0:2NO , mC sm0:2NOþFe at p < 0:005. For the erythrocytes in the whole blood: mC sm0:2NO and mC sm0:2NOþFe at p < 0:005.
membrane fluidity; however, higher concentrations induced oxidative stress, increased lipids fluidity, lipid peroxidation and protein degradation. A slight protective antioxidant effect of the plasma on the oxidative damage of erythrocytes was found, and it was observed that Fe2þ ions had little effect on changes of erythrocyte membrane structure induced by nitric oxide. We have shown that NO introduces statistically significant increases in membrane lipid fluidity at different depths of lipid bilayer after the incubation of erythrocytes with 0.2 mmol/l of NO and a slight tendency to an increase of lipids fluidity both for 12-DS and 16-DS in all model groups. Thus NO can have a modulatory action on erythrocyte membrane fluidity and can play a key role as a molecule responsible for determining rheological properties of erythrocyte membrane in vivo. Similar results were described in patients with essential hypertension (Tsuda et al., 2000). Tsuda et al. (2000) have also shown that NO donor S-nitroso-N-acetylpenicillamine used
in concentrations from 5 104 mol/l and higher also improved membrane fluidity, which indicated that NO can have an effect on rheological behavior of erythrocyte. These results were confirmed by changes in the conformational state of membrane proteins. In both groups of erythrocytes statistically significant changes were observed in the conformational state of membrane proteins labeled with MSL as well as with ISL. We interpret our results as the effect of the dissociation of spectrin-actin complex, in view of the fact that 75e90% of the total amount of attached spin labels is bound to this complex (Fung, 1983; Bellary et al., 1995). The effect of NO on the ratio of weakly to strongly immobilized residues of membrane-bound MSL (hw/hs) was studied, indicating alterations in the conformational states and interactions of cytoskeletal proteins, according to the hypothesis of Trad and Butterfield (1994) and Hensley et al. (1994). This hypothesis says that the hw/hs ratio generally increases during protein denaturation and degradation (e.g. by proteinases),
Fig. 4. Changes of tc for ISL bound with erythrocytes membrane proteins in control and in RBC and RBC in the whole blood after treatment with 0.2 mmol/l NO in the presence or absence of Fe2þ (n ¼ 6). For the isolated erythrocytes: mC sm0:2NO at p < 0:001. For the erythrocytes in the whole blood: mC sm0:2NO at p < 0:001 and m0:2NO sm0:2NOþFe at p < 0.05.
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Fig. 5. The level of lipid peroxidation in control and in RBC and in RBC in the whole blood after treatment with different concentrations of NO in the presence or absence of Fe2þ (n ¼ 8). For the isolated erythrocytes: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe and mC sm0:2NOþFe at p < 0:005. For the erythrocytes in the whole blood: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe , mC sm0:2NOþFe and m0:1NO sm0:2NOþFe at p < 0:005 and m0:1NO sm0:2NO and m0:1NOþFe sm0:2NOþFe at p < 0:05.
which is connected with the increase of segmental motion of spin-labeled proteins. The effect of exogenous NO on membrane proteins resulted in the increase of the ratio hw/hs, indicating the increase of spin label mobility, which was bound with membrane proteins after incubation with nitric oxide. In contrast, an increase in protein-protein interactions and the oxidant-induced aggregation of membrane proteins are associated with a decrease in segmental motion of spin-labeled proteins and a decrease in the ratio hw/hs. Therefore, the ratio hw/hs is a sensitive measure of the physical state of membrane proteins (Fung, 1983). Significant changes in the MSL spectra can be induced by even small alterations in the nearest proximity of the label bound to label proteins. Obtained changes were comparable in both groups of erythrocytes. Moreover, those changes found confirmation in the values of rotational correlation time for iodoacetamide spin label. This suggests that added NO is a potent modulator of the physical state of cytoskeletal proteins. However, greater decrease of this parameter was seen in the group of erythrocytes without iron ions. Pietraforte et al. (1995) postulated that the process of peroxidation in erythrocyte membrane protein thiols is conducted by nitrogen dioxide or generated thiol radicals trapped by DMPO. Moreover, protein thiols can be oxidized to disulfides and further products and also thiol conjugates can be formed (Thomas and Park, 1998; Radi et al., 1991). These modifications may lead to initiation of pathological response and explain nitric oxide role in health and disease (Augusto et al., 2004). However, lipid peroxidation causes depolarization of lipids bilayer and yields changes in the structural organization of membrane lipids that is reflected in membrane fluidity and, in consequence, can be important for physiological functions of erythrocyte. It has been shown that added NO induced statistically significant changes among both groups of treated erythrocyte and NO proved to be a pro-oxidant molecule in investigated model reaction, which induced the process of lipids peroxidation. Obtained changes are more
intensive in the group of isolated erythrocytes and the increase of lipid peroxidation was dependent on the used concentration of nitric oxide in comparison with control group. Iron ions also influence the increase of the level of lipids membrane peroxidation. Additional iron ions deepen the process of lipids peroxidation, suggesting that they play a role in catalyzing the reaction between RNS and oxygen. Radi et al. (1991) suggested that peroxynitrite can be protonated to the ‘‘hydroxyl-like’’ form, which also can be involved in the initiation of lipid peroxidation. We suspect that this process was initiated by nitrogen dioxide or peroxynitrite. Traylor and Mayeux (1997) showed that induced generation of NO in the rat proximal tubule initiated oxidant injury and intensive lipid peroxidation before cell death. Moreover, the added Fe2þ reduced LOOH to alcoxyl radical LO, which initiated a new chain reaction (McCord, 1998). Our study confirms the oxidizing processes within erythrocyte membrane in the assay of osmotic fragility. Although, the changes were not statistically significant, the increasing tendency in osmotic fragility was remarkable, which suggests that NO induced strong oxidative processes in erythrocyte membrane, but did not cause erythrocyte hemolysis. Higher concentrations of NO lead to lysis of erythrocytes.2 Particularly important is the fact that intensive lipid peroxidation can be observed before cell lysis (Deuticke et al., 1986). It indicates and confirms our previous observations that in vitro the membrane was the first target for pro-oxidant activity of nitric oxide (Brzeszczyn´ska and Gwoz´dzin´ski, 2006). Vaughn et al. (2001) showed that erythrocytes slow the nitric oxide uptake. It is also possible that much of the NO reacts in the erythrocyte membrane and never enters erythrocyte cytosol, which can be dictated by the elevated reaction rate between NO and oxygen in lipid membrane (Liu et al., 1998). This NO bioavailability explains our results. Therefore,
2
Data not shown, but available upon request.
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we suggest that membrane is the first target for NO reactions before it enters the cytosol, as well as NO uptake is restricted by the movement of the diffusion layer linked with erythrocyte membrane. Moreover, NO consumption can be regulated by physiological or pathophysiological conditions (Han and Liao, 2005). In summary, the reactivity of nitric oxide is not only a direct function of its chemistry but it additionally is associated with NO and RNS proximity as well as local environment of the target. Iron ions have an insignificant impact on changes induced by the investigated amounts of NO. Intensive peroxidation processes can cause cell lysis and in consequence even cell death. The results obtained in our work seem to confirm the contradictory mode of actions of nitric oxide dictated by its diverse chemical activities. Therefore, we propose that nitric oxide acts as pro-oxidant molecule at the investigated concentrations for which membrane appeared to be the first target before it entered cytosol. Acknowledgements We are very grateful to Dr Zbigniew Gasyna (The University of Chicago), Dr Niels Vollaard (Heriot-Watt University, Edinburgh), and anonymous referees for helpful comments. References Archer S. Measurement of nitric oxide in biological models. FASEB J 1993;7:349e60. Augusto O, Bonini MG, Trindade DF. Spin trapping of glutathiyl and protein radicals produced from nitric oxide-derived oxidants. Free Radic Biol Med 2004;36:1224e32. Beckman JS. Physiological and pathological chemistry of nitric oxide. In: Lancaster J, editor. Nitric oxide principles and action. San Diego: Academic Press; 1996. p. 1e82. Bellary SS, Anderson KW, Arden WA, Butterfield DA. Effect of lipopolysaccharide on the physical conformation of erythrocyte cytoskeletal proteins. Life Sci 1995;56:91e8. Bergendi L, Benes L, Durackova Z, Ferencik M. Chemistry, physiology and pathology of free radicals. Life Sci 1999;65:1865e74. Brzeszczyn´ska J, Gwoz´dzin´ski K., Effect of nitric oxide on the internal components of erythrocytes. Proceedings of the XIII Biennial Meeting of the International Society for Free Radical Research (Davos, Switzerland); 2006. p. 67e70. Caprari P, Bozzi A, Malorni W, Bottini A, Josi F, Santini MT, et al. Junctional sites of erythrocyte skeletal proteins are specific targets of ter-butylhydroperoxide oxidative damage. Chem Biol Interact 1995;94:243e58. Deuticke B, Heller K, Haest CWM. Leak formation in human erythrocytes by the radical-forming oxidant t-butylhydroperoxide. Biochim Biophys Acta 1986;854:159e83. Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemical characteristics of hemoglobin e free ghosts of human erythrocytes. Arch Biochem Biophys 1963;100:119e30. Fung LW. Analysis of spin labeled erythrocyte membrane. Ann NY Acad Sci 1983;414:162e7. Gladwin MT, Crawford JH, Patel RP. The biochemistry of nitric oxide, nitrite and hemoglobin: role in blood flow regulation. Free Radic Biol Med 2004;36:707e17. Han TH, Liao JC. Erythrocyte nitric oxide transport reduced by a submembrane cytoskeletal barrier. Biochim Biophys Acta 2005;1723:135e42.
Hensley K, Carney J, Hall N, Show W, Butterfield DA. Electron paramagnetic resonance investigations of free radical-induced alterations in neocortical synaptosomal membrane protein infrastructure. Free Radic Biol Med 1994;17:321e31. Ischiropoulos H. Biological selectivity and functional aspects of protein tyrosine nitration. Biochem Biophys Res Commun 2003;305:776e83. Ischiropoulos H, Gow A. Pathophysiological functions of nitric oxidemediated protein modifications. Toxicology 2005;208:299e303. Kaminski HJ, Andrade FH. Nitric oxide: biologic effects on muscle and role in muscle diseases. Neuromuscul Disord 2001;11:517e24. Kivelson D. Theory of ESR linewidth of free radicals. J Chem Phys 1960;33: 1094e106. Lapage G, Munoz G, Champagne J, Roy CC. Preparative steps necessary for the accurate measurement of malondialdehyde by high-performance liquid chromatography. Anal Biochem 1991;197:277e83. Liu X, Miller MJS, Joshi MS, Thomas DD, Lancaster J. Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes. Proc Natl Acad Sci USA 1998;95:2175e9. Marimoto Y, Tanaka K, Iwakiri Y. Protective effects of some neutral amino acids against hypotonic hemolysis. Biol Pharm Bull 1995;18:1417e22. McCord JM. Iron, free radicals, and oxidative injury. Semin Hematol 1998; 35:5e12. McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969;244:6049e55. Morrisett JD, Pownall HJ, Plumlee RT, Smith LC, Zahner ZE, Esfahani M, et al. Multiple thermotropic phase transition in Escherichia coli membranes and membrane lipids. J Biol Chem 1975;250:6969e76. Pietraforte D, Mallozzi C, Scorza G, Minetti M. Role of thiols in the targeting of S-nitroso thiols to red blood cells. Biochem 1995;34:7177e85. Radi R, Beckman JS, Bush K, Freeman BA. Peroxynitrite induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 1991;288:481e7. Reid MB. Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance. Acta Physiol Scand 1998;162:401e9. Rohn TT, Hinds TR, Vincenzi FF. Inhibition of the Ca pump of intact red blood cells by t-butylhydroperoxide: importance of glutathione peroxidase. Biochim Biophys Acta 1993;46:525e34. Soszyn´ski M, Bartosz G. Effect of peroxynitrite on erythrocytes. Biochim Biophys Acta 1996;1291:107e14. Soszyn´ski M, Bartosz G. Decrease in accessible thiols as an index of oxidative damage to membrane proteins. Free Radic Biol Med 1997a;23:463e9. Soszyn´ski M, Bartosz G. Peroxynitrite inhibits glutathione S-conjugate transport. Biochim Biophys Acta 1997b;1325:135e41. Stamler JS, Meissner G. Physiology of nitric oxide in skeletal muscle. Physiol Rev 2001;81:1e82. Thomas JA, Park EM. Oxy radical-initiated protein S-thiolation and enzymic dethiolation. In: Simic MG, Taylor KA, Ward JF, Von Sonntag C, editors. Oxygen radicals in Biol Med. New York: Plenum Press; 1998. p. 365e73. Trad CH, Butterfield DA. Menadione-induced cytotoxity effects on human erythrocyte membranes studied by electron paramagnetic resonance. Toxicol Lett 1994;73:145e55. Traylor LA, Mayeux PR. Nitric oxide generation mediates lipid A-induced oxidant injury in renal proximal tubules. Arch Biochem Biophys 1997;338: 129e35. Tsuda K, Kimura K, Nishio I, Masuyama Y. Nitric oxide improves membrane fluidity of erythrocytes in essential hypertension: an electron paramagnetic resonance investigation. Biochem Biophys Res Comm 2000;275: 946e54. Tsuda K, Shimamoto YK, Kimura K, Nishio I. Nitric oxide is a determinant of membrane fluidity of erythrocytes in postmenopausal woman: an electron paramagnetic resonance investigation. Am J Hyper 2003;16:244e8. Vaughn MW, Huang K, Kuo L, Liao JC. Erythrocyte consumption of nitric oxide: competition experiment and model analysis. Nitric Oxide 2001;5:18e31. Wink DA, Hanbauer I, Grisham MB, Laval F, Nims RW, Laval J, et al. Chemical biology of nitric oxide: regulation and protective and toxic mechanisms. Curr Top Cell Regul 1996;34:159e87.
Nitric oxide induced oxidative changes in erythrocyte membrane components Joanna Brzeszczynska a,*, Krzysztof Gwozdzinski b a
School of Life Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK b Department of Biophysics, University of Lodz, 90-237 Lodz, Poland Received 20 March 2007; revised 7 June 2007; accepted 27 August 2007
Abstract The effects of NO in its environment may vary considerably depending on various factors. This study shows oxidative mechanism of cellular membrane alterations, which is not associated with triggering of ONOOH generation but is induced by pure NO. Our investigation examined the influence of low concentration of NO (0.1; 0.2 mmol/l) on the qualitative changes of structure and dynamics of erythrocyte membrane. NO causes a statistically significant increase in membrane fluidity on different depths of lipid bilayer that is correlated with increase of lipids peroxidation. Statistically significant changes in the conformational state of cytoskeleton proteins were also detected. NO can be considered as a molecule responsible for determining rheological properties of erythrocytes membrane. Therefore, we propose that NO acts as pro-oxidant molecule at concentrations for which membrane appeared to be the first target before it entered the cytosol. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Oxidative stress; Spin-labeling; Erythrocyte membrane; RBC
1. Introduction Nitric oxide (NO) is a small, hydrophobic molecule with a high diffusion coefficient and short life-span. Similarly to reactive oxygen species (ROS), it is an unstable molecule that can quickly react with for example hemoglobin, myoglobin, oxygen, superoxide anions, and various cellular components. The molecular targets of NO in vivo belong to the following 3 categories (Kaminski and Andrade, 2001; Reid, 1998). First, there are reactions with molecular oxygen and superoxide anions. Under biological conditions the autooxidation reaction of nitric oxide takes place with the rate constant k ¼ 106 M1 s1 (Wink et al., 1996). Subsequently formed nitrogen dioxide and/ or peroxynitrite can mediate oxidative injury of cellular components. Second, there are reactions with transition metals, such as heme iron or iron-sulphur clusters, which lead to metal-NO
* Corresponding author. Tel.: þ44 131 451 4679; fax þ44 131 451 3009. E-mail address: [email protected] (J. Brzeszczynska).
products. Third, there are reactions with reduced protein thiol groups (RSH, RS-), which form RS-NO groups. Gladwin et al. (2004) suggest that the major vascular pool of NO in vivo is nitrite, which is present at concentrations of 0.5e10 mM in plasma, erythrocytes and tissues. Depending on the concentration of NO in its biological environment, the effect of this molecule may vary considerably. At high concentrations, NO has antimicrobial, antitumor and cytotoxic effects, while at low concentrations it stimulates guanylate cyclase and influences blood pressure regulation, blood flow, neuronal transmission and neuroendocrine activity (Bergendi et al., 1999). It is also known to be a modulator of several aspects of skeletal muscle function (Stamler and Meissner, 2001). Thus, NO is considered to be an endogenous modulator of numerous cellular functions in a variety of tissues, with the properties of a cell signalling molecule (Beckman, 1996). Beside this, NO participates in the regulation of erythrocyte membrane properties and in the regulation of the rheological behavior of erythrocytes (Tsuda et al., 2003; McCord and Fridovich, 1969). Numerous studies show that cell membrane
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.08.020
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Nomenclature EPR Hb MetHb HbO2 MSL ISL 5-,12-,16-DS RBC RNS NO NO2 ONOOH Fe2þ SOD NOS PBS TBARS TBA MDA NaCl
electron paramagnetic resonance hemoglobin methemoglobin oxygenated hemoglobin 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl 4-iodoacetamide-2,2,6,6-tetramethylpiperidine-1-oxyl 5-,12-,16-doxylstearic acid red blood cell reactive nitrogen species nitric oxide nitrogen dioxide peroxynitrite iron ions superoxide dismutase nitric oxide synthase phosphate buffered saline thiobarbituric acid reactive substances thiobarbituric acid malondialdehyde sodium chloride
abnormalities of erythrocyte and also structural changes of the erythrocyte membrane are an etiological factor in many diseases of the circulatory system, e.g. hypertension (Tsuda et al., 2000, 2003). Therefore, membrane fluidity is an important physiochemical characteristic of biomembranes and an important factor in regulating membrane micro viscosity, cell functions and its rheological behavior. Membrane proteins are also very sensitive to the attack of NO. Particularly, the thiol groups are modified, which influences the regulating process of protein functions. Nitric oxide can be involved in the process of distinct protein modification, nitration of aromatic residues, nitrosylation of metal, and generation of S-nitrosothiols (Ischiropoulos and Gow, 2005). If these modifications are not prevented, they may lead to initiation of pathological responses. On the other hand, they can transduce physiological responses and signalling. Tyrosine nitration influences alteration of protein functions (Ischiropoulos and Gow, 2005). It can also result in the changes of proteinprotein interactions as well as other aspects of the protein cycle, such as the protein half-life (Ischiropoulos, 2003). Proteins peroxidation induces changes in the conformational state of membrane proteins that may contribute to the inhibition of erythrocyte membrane enzymes and transporting proteins. Alteration in the membrane transporting system can induce many processes dangerous to cells and finally can even lead to the cell death (Rohn et al., 1993). In our investigation we have introduced NO, which creates the conditions more similar to the physiological ones. The concentrations, which have been chosen in this study, are relatively low as for the examinations in vitro. Furthermore, this
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investigation shows oxidative mechanism of cellular membrane alterations, which are not associated with triggering of ONOOH generation but are induced by pure NO. Moreover, introduction of pure NO, as a factor that initiates oxidative damages in the cell, makes this investigation novel. 2. Materials and methods 2.1. Synthesis of NO NO was generated under anaerobic conditions through the reaction of nitric acid (10%) and copper, purified in alkaline solution and dried over solid potassium hydroxide and dissolved in water at 20 C (Archer, 1993). Stock solution (2 mM NO) was added to the samples and the final concentration of NO was 0.1 or 0.2 mM. 2.2. Erythrocytes and erythrocyte ghosts Human blood was collected from healthy donors of between 18 and 30 years attending the local blood bank. Donors were examined by the doctor and were recruited according to the established criteria. Packed cells were suspended in PBS to a haematocrit of 50%. Erythrocytes (isolated and in the whole blood) were treated with 0.1 or 0.2 mM NO in the presence or absence of 0.1 mM FeSO4 at 22 C 1 for 1 h, washed twice with 0.9% NaCl and once with PBS. Erythrocyte ghosts were prepared by the method of Dodge et al. (1963) with the modification of including hypertonic lysis using 20 mM sodium phosphate buffer (pH 7.4) at 4 C. The ghosts were successively washed with 20 mM, 10 mM and 5 mM phosphate buffer (pH 7.4) and finally suspended in 5 mM phosphate buffer (pH 7.4) at 4 C. 2.3. Spin labeling of erythrocytes and erythrocyte ghosts Erythrocytes were labeled with 5-DS, 12-DS and 16-DS by introduction of the doxyl derivatives in ethanol into the erythrocyte suspension and incubation for 30 min at 22 C 1. Temperature was stable during measurements. The final ethanol concentration in the erythrocyte suspension was less than 0.05% (v/v). From the spectra the ratio hþ1/h0 was calculated, with hþ1 representing the height of the low-field line and h0 representing the height of the middle-field line of the spectrum. This ratio is correlated with lipid bilayer fluidity and serves as a semi-quantitative measure of acyl side chain flexibility (Morrisett et al., 1975). To investigate conformational changes of membrane proteins, MSL and ISL were used, which bind covalently to the -SH groups of membrane proteins. Erythrocyte membranes were labeled with MSL or ISL using 2 ml of ethanol solution with 100 mM MSL or ISL per 1 ml of membrane suspension (w3 mg$ml1 of protein), and incubated for 1 h at 4 C. Unbound spin label was removed through several washings with cold phosphate buffer. The effect of physiological oxidative stress on the ratio of weakly to strongly immobilized
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residues of erythrocyte membrane-bound MSL spin labels (hw/hs) was studied (Fung, 1983). In the case of ISL the mobility of attached spin label was estimated by calculating the rotational correlation time (tc) according to the Kivelson formula (Kivelson, 1960). EPR spectra were obtained at 22 C 1 using a Bruker ESP 300 E spectrometer, operating at a microwave frequency of 9.73 GHz. The instrumental settings were: center field 3480G; scan range 80G; modulation frequency 100 kHz; modulation amplitude 1G. 2.4. Lipid peroxidation Lipid peroxidation was measured with thiobarbituric acid (TBA) using the method described by Lapage et al. (1991). The absorbance of the butanol layer of the supernatant was measured at l ¼ 535 nm to estimate the level of lipid peroxidation. 2.5. Osmotic fragility Osmotic fragility was estimated according to the spectrophotometric method described by Marimoto et al. (1995). Changes in osmotic fragility were presented as a concentration of NaCl, for which 50% of erythrocytes in the investigated groups underwent hemolysis C(50%). 2.6. Statistical analysis Normality of distributions was tested using the Shapiroe Wilk test. The significance of the difference between couples of means (control and investigated probes as well as the differences among the probes) was assessed by the Tukey test at a < 0.05 significance level. 3. Results The spin-labeling technique allows for an improved understanding of the molecular mechanisms of membrane changes and was used to determine alterations in the physical state of erythrocyte membrane components caused by treatment with NO. Spin labels were used to investigate both lipid fluidity and the conformational state of proteins. Through binding to macromolecules these substances can act as reporting groups, providing information about the changes in the microenvironment in the nearest region. Spectral analysis of samples labeled with free doxyl derivatives provides information about structural characteristics and dynamics of cell membranes. The spectral parameter hþ1/h0 was measured and served as a semi-quantitative measure of acyl chain flexibility corresponding to lipid bilayer fluidity. The changes in hþ1/h0 ratio of applied doxyl spin labels generally indicated statistically significant increases in membrane lipid fluidity at different depths of lipid bilayer after the incubation of erythrocytes with 0.2 mmol/l of NO. A statistically significant increase in lipid membrane fluidity was observed for 5-DS in both groups of erythrocytes. There were significant
changes found for the isolated erythrocytes: mC sm0:2NO and mC sm0:2NOþFe at p < 0:05 as well as for the erythrocytes in the whole blood: mC sm0:2NOþFe at p < 0:05 (Fig. 1)1. For 12-DS non-significant changes in hþ1/h0 ratio were observed. However, for 16-DS statistically significant changes were observed for the following couples of means for the erythrocytes in the whole blood: m0:1NO sm0:2NOþFe , m0:2NO sm0:1NOþFe at p < 0:05 and m0:2NO sm0:2NOþFe at p < 0:01 (Fig. 2). On the other hand, a slight tendency to an increase of lipids fluidity both for 12-DS and for 16-DS was seen in all model groups. In order to investigate conformational changes of membrane proteins we used MSL and ISL. Effect of NO on membrane proteins, in both investigated erythrocytes groups, resulted in an increase in the ratio hw/hs, indicating an increase in spin label mobility. For the isolated erythrocytes, statistically significant changes were observed for the following couples of means: mC sm0:2NO , mC sm0:2NOþFe at p < 0:005 and for the erythrocytes in the whole blood for: mC sm0:2NO and mC sm0:2NOþFe at p < 0:005 (Fig. 3). This suggests that NO induces the loss of interaction between the spectrin-actin complex and other proteins, e.g. band 4.1, which links the spectrin network to glycophorin, or ankyrin, which links it to band 3 (Dodge et al., 1963; Caprari et al., 1995). No differences were observed between isolated erythrocytes and erythrocytes suspended in the whole blood, nor was the increase in the ratio hw/hs affected by the addition of Fe2þ. In contrast to MSL, ISL attached to erythrocyte ghosts yields a triplet spectrum, from which it is possible to calculate rotational correlation time (tc), providing a measure for the mobility of this spin label. Values for tc tend to be negatively correlated with the ratio hw/hs. Thus, the observed decrease in tc in the presence of 0.2 mM NO for the isolated erythrocytes: mC sm0:2NO at p < 0:001 was in line with results obtained for MSL and for the erythrocytes in the whole blood for: mC sm0:2NO at p < 0:001. However, addition of Fe2þ appeared to attenuate this decrease: m0:2NO sm0:2NOþFe at p < 0.05 (Fig. 4). Estimation of lipids peroxidation indicated changes in the amount of releasing substances reacting with thiobarbituric acid. Fig. 5 shows the influence of NO and Fe2þ on isolated erythrocytes and erythrocytes suspended in the whole blood. For the isolated erythrocytes we observed statistically significant changes for the following couples of means: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe and mC sm0:2NOþFe at p < 0:005. For the erythrocytes in the whole blood the changes were statistically significant in the case of the following couples of means: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe , mC sm0:2NOþFe and m0:1NO sm0:2NOþFe at p < 0:005 and m0:1NO sm0:2NO and m0:1NOþFe sm0:2NOþFe at p < 0:05. Under the investigated conditions NO appears to have prooxidant properties. This effect seems to be more pronounced in isolated erythrocytes compared to erythrocytes suspended in the whole blood and is enhanced in the presence of Fe2þ.
1
For this and all the subsequent figures, n stands for the number of subjects.
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Fig. 1. Influence of different concentrations of NO in the presence or absence of Fe2þ on the changes of the parameter hþ1/h0 for 5-DS (n ¼ 7). For the isolated erythrocytes: mC sm0:2NO and mC sm0:2NOþFe at p < 0:05. For erythrocytes in the whole blood: mC sm0:2NOþFe at p < 0:05.
NO did not induce significant changes in osmotic fragility although a trend ( p ¼ 0.067) towards increased osmotic fragility was observed for isolated erythrocytes in the presence of Fe2þ. However, particularly important is the fact that oxidative changes were observed before cell lysis.
4. Discussion The aim of this study was investigation of the role of NO in the qualitative changes of structure and function of human erythrocyte membrane in vitro using an EPR and spin-labeling method. The effects of 2 concentrations of NO (0.1 and 0.2 mmol/l) on the membrane components structure were investigated in isolated erythrocytes as well as in erythrocytes suspended in the whole blood, both in the presence or absence of Fe2þ. Another purpose of the study was to measure lipid fluidity at different depths of erythrocyte membrane and to
detect oxidative changes in the structure of membrane proteins by estimating the conformational state of membrane proteins. Our results reveal changes in erythrocytes membrane integrity after NO treatment using the concentrations of NO, which are relatively low as for the examinations in vitro. We emphasise that we introduced NO, which is generated in vivo, while other investigators usually use synthetic peroxynitrite. However, the chosen concentrations have also commonly been used by other authors (Soszyn´ski and Bartosz, 1996, 1997a,b). Moreover, peroxynitrite is generated at the same time nitric oxide and superoxide anion are generated, but we introduced NO which creates the conditions more similar to the physiological ones. By introducing pure NO, we induced oxidative changes that have not previously been investigated. We presented the modulator action of exogenous NO on erythrocyte membrane and we observed the oxidative deterioration of membrane lipids and changes in conformational state of membrane proteins. Low concentrations of NO regulate
Fig. 2. Influence of different concentrations of NO in the presence or absence of Fe2þ on the changes of the parameter hþ1/h0 for 16-DS (n ¼ 7). For the erythrocytes in the whole blood: m0:1NO sm0:2NOþFe , m0:2NO sm0:1NOþFe at p < 0:05 and m0:2NO sm0:2NOþFe at p < 0:01.
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Fig. 3. Changes of hw/hs for MSL bound with erythrocytes membrane proteins in control and in RBC and RBC in the whole blood after treatment with 0.2 mmol/l NO in the presence or absence of Fe2þ (n ¼ 6). For the isolated erythrocytes: mC sm0:2NO , mC sm0:2NOþFe at p < 0:005. For the erythrocytes in the whole blood: mC sm0:2NO and mC sm0:2NOþFe at p < 0:005.
membrane fluidity; however, higher concentrations induced oxidative stress, increased lipids fluidity, lipid peroxidation and protein degradation. A slight protective antioxidant effect of the plasma on the oxidative damage of erythrocytes was found, and it was observed that Fe2þ ions had little effect on changes of erythrocyte membrane structure induced by nitric oxide. We have shown that NO introduces statistically significant increases in membrane lipid fluidity at different depths of lipid bilayer after the incubation of erythrocytes with 0.2 mmol/l of NO and a slight tendency to an increase of lipids fluidity both for 12-DS and 16-DS in all model groups. Thus NO can have a modulatory action on erythrocyte membrane fluidity and can play a key role as a molecule responsible for determining rheological properties of erythrocyte membrane in vivo. Similar results were described in patients with essential hypertension (Tsuda et al., 2000). Tsuda et al. (2000) have also shown that NO donor S-nitroso-N-acetylpenicillamine used
in concentrations from 5 104 mol/l and higher also improved membrane fluidity, which indicated that NO can have an effect on rheological behavior of erythrocyte. These results were confirmed by changes in the conformational state of membrane proteins. In both groups of erythrocytes statistically significant changes were observed in the conformational state of membrane proteins labeled with MSL as well as with ISL. We interpret our results as the effect of the dissociation of spectrin-actin complex, in view of the fact that 75e90% of the total amount of attached spin labels is bound to this complex (Fung, 1983; Bellary et al., 1995). The effect of NO on the ratio of weakly to strongly immobilized residues of membrane-bound MSL (hw/hs) was studied, indicating alterations in the conformational states and interactions of cytoskeletal proteins, according to the hypothesis of Trad and Butterfield (1994) and Hensley et al. (1994). This hypothesis says that the hw/hs ratio generally increases during protein denaturation and degradation (e.g. by proteinases),
Fig. 4. Changes of tc for ISL bound with erythrocytes membrane proteins in control and in RBC and RBC in the whole blood after treatment with 0.2 mmol/l NO in the presence or absence of Fe2þ (n ¼ 6). For the isolated erythrocytes: mC sm0:2NO at p < 0:001. For the erythrocytes in the whole blood: mC sm0:2NO at p < 0:001 and m0:2NO sm0:2NOþFe at p < 0.05.
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Fig. 5. The level of lipid peroxidation in control and in RBC and in RBC in the whole blood after treatment with different concentrations of NO in the presence or absence of Fe2þ (n ¼ 8). For the isolated erythrocytes: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe and mC sm0:2NOþFe at p < 0:005. For the erythrocytes in the whole blood: mC sm0:1NO , mC sm0:2NO , mC sm0:1NOþFe , mC sm0:2NOþFe and m0:1NO sm0:2NOþFe at p < 0:005 and m0:1NO sm0:2NO and m0:1NOþFe sm0:2NOþFe at p < 0:05.
which is connected with the increase of segmental motion of spin-labeled proteins. The effect of exogenous NO on membrane proteins resulted in the increase of the ratio hw/hs, indicating the increase of spin label mobility, which was bound with membrane proteins after incubation with nitric oxide. In contrast, an increase in protein-protein interactions and the oxidant-induced aggregation of membrane proteins are associated with a decrease in segmental motion of spin-labeled proteins and a decrease in the ratio hw/hs. Therefore, the ratio hw/hs is a sensitive measure of the physical state of membrane proteins (Fung, 1983). Significant changes in the MSL spectra can be induced by even small alterations in the nearest proximity of the label bound to label proteins. Obtained changes were comparable in both groups of erythrocytes. Moreover, those changes found confirmation in the values of rotational correlation time for iodoacetamide spin label. This suggests that added NO is a potent modulator of the physical state of cytoskeletal proteins. However, greater decrease of this parameter was seen in the group of erythrocytes without iron ions. Pietraforte et al. (1995) postulated that the process of peroxidation in erythrocyte membrane protein thiols is conducted by nitrogen dioxide or generated thiol radicals trapped by DMPO. Moreover, protein thiols can be oxidized to disulfides and further products and also thiol conjugates can be formed (Thomas and Park, 1998; Radi et al., 1991). These modifications may lead to initiation of pathological response and explain nitric oxide role in health and disease (Augusto et al., 2004). However, lipid peroxidation causes depolarization of lipids bilayer and yields changes in the structural organization of membrane lipids that is reflected in membrane fluidity and, in consequence, can be important for physiological functions of erythrocyte. It has been shown that added NO induced statistically significant changes among both groups of treated erythrocyte and NO proved to be a pro-oxidant molecule in investigated model reaction, which induced the process of lipids peroxidation. Obtained changes are more
intensive in the group of isolated erythrocytes and the increase of lipid peroxidation was dependent on the used concentration of nitric oxide in comparison with control group. Iron ions also influence the increase of the level of lipids membrane peroxidation. Additional iron ions deepen the process of lipids peroxidation, suggesting that they play a role in catalyzing the reaction between RNS and oxygen. Radi et al. (1991) suggested that peroxynitrite can be protonated to the ‘‘hydroxyl-like’’ form, which also can be involved in the initiation of lipid peroxidation. We suspect that this process was initiated by nitrogen dioxide or peroxynitrite. Traylor and Mayeux (1997) showed that induced generation of NO in the rat proximal tubule initiated oxidant injury and intensive lipid peroxidation before cell death. Moreover, the added Fe2þ reduced LOOH to alcoxyl radical LO, which initiated a new chain reaction (McCord, 1998). Our study confirms the oxidizing processes within erythrocyte membrane in the assay of osmotic fragility. Although, the changes were not statistically significant, the increasing tendency in osmotic fragility was remarkable, which suggests that NO induced strong oxidative processes in erythrocyte membrane, but did not cause erythrocyte hemolysis. Higher concentrations of NO lead to lysis of erythrocytes.2 Particularly important is the fact that intensive lipid peroxidation can be observed before cell lysis (Deuticke et al., 1986). It indicates and confirms our previous observations that in vitro the membrane was the first target for pro-oxidant activity of nitric oxide (Brzeszczyn´ska and Gwoz´dzin´ski, 2006). Vaughn et al. (2001) showed that erythrocytes slow the nitric oxide uptake. It is also possible that much of the NO reacts in the erythrocyte membrane and never enters erythrocyte cytosol, which can be dictated by the elevated reaction rate between NO and oxygen in lipid membrane (Liu et al., 1998). This NO bioavailability explains our results. Therefore,
2
Data not shown, but available upon request.
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we suggest that membrane is the first target for NO reactions before it enters the cytosol, as well as NO uptake is restricted by the movement of the diffusion layer linked with erythrocyte membrane. Moreover, NO consumption can be regulated by physiological or pathophysiological conditions (Han and Liao, 2005). In summary, the reactivity of nitric oxide is not only a direct function of its chemistry but it additionally is associated with NO and RNS proximity as well as local environment of the target. Iron ions have an insignificant impact on changes induced by the investigated amounts of NO. Intensive peroxidation processes can cause cell lysis and in consequence even cell death. The results obtained in our work seem to confirm the contradictory mode of actions of nitric oxide dictated by its diverse chemical activities. Therefore, we propose that nitric oxide acts as pro-oxidant molecule at the investigated concentrations for which membrane appeared to be the first target before it entered cytosol. Acknowledgements We are very grateful to Dr Zbigniew Gasyna (The University of Chicago), Dr Niels Vollaard (Heriot-Watt University, Edinburgh), and anonymous referees for helpful comments. References Archer S. Measurement of nitric oxide in biological models. FASEB J 1993;7:349e60. Augusto O, Bonini MG, Trindade DF. Spin trapping of glutathiyl and protein radicals produced from nitric oxide-derived oxidants. Free Radic Biol Med 2004;36:1224e32. Beckman JS. Physiological and pathological chemistry of nitric oxide. In: Lancaster J, editor. Nitric oxide principles and action. San Diego: Academic Press; 1996. p. 1e82. Bellary SS, Anderson KW, Arden WA, Butterfield DA. Effect of lipopolysaccharide on the physical conformation of erythrocyte cytoskeletal proteins. Life Sci 1995;56:91e8. Bergendi L, Benes L, Durackova Z, Ferencik M. Chemistry, physiology and pathology of free radicals. Life Sci 1999;65:1865e74. Brzeszczyn´ska J, Gwoz´dzin´ski K., Effect of nitric oxide on the internal components of erythrocytes. Proceedings of the XIII Biennial Meeting of the International Society for Free Radical Research (Davos, Switzerland); 2006. p. 67e70. Caprari P, Bozzi A, Malorni W, Bottini A, Josi F, Santini MT, et al. Junctional sites of erythrocyte skeletal proteins are specific targets of ter-butylhydroperoxide oxidative damage. Chem Biol Interact 1995;94:243e58. Deuticke B, Heller K, Haest CWM. Leak formation in human erythrocytes by the radical-forming oxidant t-butylhydroperoxide. Biochim Biophys Acta 1986;854:159e83. Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemical characteristics of hemoglobin e free ghosts of human erythrocytes. Arch Biochem Biophys 1963;100:119e30. Fung LW. Analysis of spin labeled erythrocyte membrane. Ann NY Acad Sci 1983;414:162e7. Gladwin MT, Crawford JH, Patel RP. The biochemistry of nitric oxide, nitrite and hemoglobin: role in blood flow regulation. Free Radic Biol Med 2004;36:707e17. Han TH, Liao JC. Erythrocyte nitric oxide transport reduced by a submembrane cytoskeletal barrier. Biochim Biophys Acta 2005;1723:135e42.
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