Erythrocyte nitric oxide availability and oxidative stress following exercise

Abstract

Growing evidence has shown that acute exercise impairs erythrocyte membrane structure and function as a consequence of increased physical and chemical stress. Erythrocyte-synthesized nitric oxide (NO) is known to modulate membrane fluidity, and its bioavailability depends on the balance between its production and scavenging by reactive oxygen species. Here, we investigated whether a maximal exercise test could affect erythrocyte NO bioavailability and oxidative stress. Twelve men (26±4 years old, $\dot{V}$ O_2peak 44.1±4.3 mL·kg^–1·min^–1) performed a treadmill maximal cardiopulmonary exercise test. Blood was collected at rest and immediately after exercise for erythrocytes isolation. Maximal exercise caused an increase in erythrocytes count, haemoglobin and haematocrit levels. There was no change in L-arginine influx into erythrocytes after exercise. Yet, nitric oxide synthase activity, and thus, NO production, was increased after maximal test, as well cyclic GMP levels. In relation to biomarkers of oxidative stress, maximal test resulted in increased levels of lipid peroxidation, and diminished superoxide dismutase activity. Neither glutathione peroxidase nor catalase activity was affected by maximal test. Our findings demonstrate that the increased erythrocyte membrane rigidity caused by an acute bout of exercise may be caused, in part, by an increased lipid oxidative damage caused by ROS produced exogenously.

Keywords

Reactive oxygen species red blood cells arginine nitric oxide synthase

1 Introduction

Erythrocytes play a major role during physical exercise due to its oxygen transportation and buffer abilities. The importance of these blood cells during exercise is highlighted by the widespread use of physiological and pharmacological tools to increase their number, such as high altitude training [13] and non-authorized blood doping [21]; as well as a marked decreased exercise capacity in those with anaemia on the other side [12]. Recent evidences, however, have shown that an acute bout of intense [4 , 34] or prolonged [8 , 33] physical exercise leads to changes in erythrocyte membrane structure, increasing its rigidity, and accelerating its senescence and removal from the circulation.

Structural and functional erythrocyte changes during exercise are suggested to be caused, in part, by increased oxidative stress. Senturk et al. [31] observed the presence of oxidative damage in erythrocytes after a maximum test, demonstrated by increased lipid and protein oxidation. Indeed, lipid peroxidation affects cell membranes, and it has been shown to be associated with increased erythrocyte osmotic fragility and reduced deformability [30]. It is well known that exercise causes an increase in reactive oxygen production (ROS), particularly in active skeletal muscle, but also on other metabolic active tissues such as the heart. In skeletal muscle, ROS may be generated from the increased electron flux through the electron transport chain in the mitochondria, as well as by the “superoxide producers” NADPH oxidases located in sarcoplasmic reticulum, transverse tubules and sarcolemma [for a detailed review, see Powers et al. [27]]. Erythrocytes have their own ROS generating systems, albeit they do not possess mitochondria. In these cells, ROS are produced mainly by NADPH oxidases, and secondarily by haemoglobin auto-oxidation and xanthine oxidases. It is not known, however, whether erythrocytes are affected by endogenous or by exogenous synthesized ROS as they pass through skeletal muscle microcirculation. On the other side, erythrocytes are equipped with an efficient antioxidant defence system, making them a “mobile carrier system of free radicals” which provides a protection not only for themselves, but also for tissues and organs in the body [3, 6]. This system comprises the enzymes superoxide dismutase that performs the dismutation of the superoxide anion (O₂^–.) to hydrogen peroxide (H₂O₂) and molecular oxygen (O₂), and the second line of defence, catalase and glutathione peroxidase (GPx) that decompose H₂O₂ to water and O₂, with catalase possessing a more efficient kinetics than GPx in the removal of large amounts of H₂O₂ [23]. Acute exercise has been shown to increase, decrease or cause no changes in enzymatic antioxidant defence system [2 , 36]. These divergent findings are probably due to the various types of exercises and experimental conditions adopted in the aforementioned studies.

Equally important for erythrocyte functionality is nitric oxide (NO). Nitric oxide is a molecule synthesized by all blood cells as well as endothelial cells, that plays a major role in vasodilation, modulation of platelet function and inflammation [15, 20]. Therefore, NO can be considered a central regulator of vascular homeostasis, and its low bioavailability can be involved in the pathogenesis of many vascular diseases, such as peripheral artery disease [15] and systemic arterial hypertension [22]. Kleinbongard and collaborators [16] demonstrated that erythrocytes possess a functional endothelial nitric oxide synthase (eNOS). Previous work from our group evidenced that the NO precursor, L-arginine, is transported into human erythrocytes by the classical cationic amino acid transport systems y⁺ and y⁺L [14], and that L-arginine influx is a rate-limiting step for NO production. Erythrocyte-derived NO is proposed to have an autocrine function, by means of a cyclic guanosine monophosphate (cGMP) dependent signaling, affecting membrane fluidity and deformability, which is important for its passage through the microcirculation and delivery of oxygen to exercising muscles [16]. In this regard, one study showed a reduced expression of phosphorylated eNOS at Ser¹¹⁷⁷ after two hockey training sessions, suggesting a diminished NO production from erythrocytes [34]. It is worth noting, however, that this study was conducted in field, which increases its external validity, but limits its internal validity as it is not possible to control the independent and confounding variables. Nitric oxide bioavailability depends both on its synthesis and on its inactivation by ROS, especially O₂^–., leading to peroxynitrite production. Thus, increased ROS production might affect erythrocytes both by direct (attacking lipid and protein structures) and indirect means (reducing NO bioavailability).

In this study, we aimed to investigate whether a maximal exercise test would affect oxidative stress and NO bioavailability in erythrocytes from healthy subjects.

2 Methods

2.1 Subjects

Twelve young healthy male subjects (26±4 years old; 68.5±4.1 kg; 1.76±0.07 m; 9.9±4.5% body fat; $\dot{V}$ O_2max 44.1±4.3 mL·kg^–1·min^–1) volunteered to participate in this study. The subjects were sedentary or recreationally physically active, who were not involved in regular endurance training at the time of the study. The presence of any orthopaedic injury, anaemia, use of prescription or non-prescription medication and/or dietary supplements that might affect erythrocyte number or function were considered exclusion criteria for study participation. The experimental protocol was carried out in accordance with the Declaration of Helsinki (2008) of the World Medical Association and it was approved by the institutional Ethics Committee (#20047). All subjects signed a written informed consent prior to participation.

2.2 Procedures

Subjects were instructed to avoid performing strenuous physical exercise for at least 72 h before the test. After arriving to the laboratory, anthropometric measurements were recorded, and after a 30 min rest in a supine position, blood was collected for baseline data. In sequence, subjects underwent a maximal cardiopulmonary exercise test on a treadmill (Super ATL^®, Inbramed, Porto Alegre, Brasil), according to an individualized ramp protocol [10], and with analysis of expired gases breath-by-breath (Ultima CardiO₂^®, Medical Graphics Corporation, USA.) The tests were conducted with controlled temperature and conditions for emergency care.

2.3 Blood collection and erythrocytes isolation

Blood samples were collected at two different times: (i) at baseline (after 30 min rest), and(ii) immediately after maximal exercise test. Blood was collected by venipuncture into tubes containing the anticoagulant heparin (15 IU·mL^–1) or EDTA (for blood cell count). Erythrocytes were isolated by differential centrifugation (1000 g, 4°C), and subsequently washed three times with a saline solution.

2.4 Red blood cell count, plasma osmolality and Na⁺ levels

Red blood cell count was performed using an automated haematological analyser (ABX Pentra 60, Horiba, Japan), and plasma osmolality and Na⁺ levels were measured at the DLE Laboratory (Niteroi, Brazil).

2.5 Osmotic fragility

Osmotic fragility was determined by the addition of erythrocytes suspension (∼50% haematocrit) to tubes with increasing concentrations of a buffered NaCl solution (0–0.9% NaCl, pH 7.4). After 30 min of incubation at room temperature, samples were centrifuged (1200 g, 10 min), and the optical density of the supernatant was measured by spectrophotometry at 540 nm (Fluostar Omega, BMG Labtech, Ortenberg, Germany) [5].

2.6 L-arginine transport

Erythrocytes suspension was incubated at 37°C for 5 minutes with increasing concentration ofL-[³H]-arginine (5–500 μM). The influx was stopped by rapid cooling and centrifugation, followed by lysis of cells with Triton X-100 and 5% trichloroacetic acid for β-scintillation counting (LS 6500 Liquid Scintillation Counter, Beckman Counter Inc., CA, USA). Michaelis-Menten constant (K_m) and maximum velocity (V_max) of L-arginine transport in erythrocytes were calculated using GraphPad Prism 6.0 software.

2.7 Nitric oxide synthase activity

The activity of nitric oxide synthase was quantified by the conversion of L-[³H]-arginine toL-[³H]-citrulline. The erythrocytes suspension was incubated at 37°C with L-[³H]-arginine (500 mM) for one hour. The reactions were stopped by rapid centrifugation and two washes with MgCl₂. The erythrocytes were lysed with Triton X-100, followed by haemoglobin precipitation with 5% TCA. The lysate was transferred to a cation exchange resin column (Dowex^® 50WX8). Radioactivity was measured by liquid-scintillation counting (LS 6500 Liquid Scintillation Counter, Beckman Counter Inc., California, USA).

2.8 Measurement of cGMP levels

Samples were prepared by incubation of the erythrocytes suspension with 3-isobutyl-1-methylxanthine and perchloric acid, followed by cell lysis with one freeze/thaw cycle and sonication (MaxiClean 1400, Unique, Sao Paulo, Brazil) for 15 minutes. The erythrocytes lysate was centrifuged at 24000 g for 20 minutes and the supernatant was collected and stored at –80 °C until analysis. cGMP levels were detected using an enzyme-linked immunoassay kit (cGMP EIA kit, Cayman Chemical Company, MI, USA).

2.9 Oxidative stress

2.9.1 Lipid peroxidation

The damage to the structure in lipid membrane was quantified by formation of byproducts of lipid peroxidation (malondialdehyde, MDA), which are thiobarbituric acid reactive substances (TBARS). MDA reacts with thiobarbituric acid, resulting in a pinkish substance that was subsequently measured by spectrophotometry at 532 nm [11]. TBARS concentration was expressed as pmol/g of haemoglobin.

2.9.2 Protein carbonylation

Oxidative damage to proteins was assessed by determination of carbonyl groups based on the reaction with dinitrophenylhydrazine (Sigma, St. Louis, MO) using the method of Levine et al. [38]. Carbonyl contents were determined from the absorbance at 370 nm. Total protein concentration was assayed using the BCA assay kit (Bioagency, Sao Paulo, Brazil).

2.9.3 Activity of antioxidant enzymes

SOD activity was assessed by a colorimetric assay kit (Cayman Chemical Company, USA). Catalase activity was assessed according to Aebi [1]. Briefly, 50 μL of erythrocytes lysate were added to 1 mL of a solution containing phosphate buffered saline and hydrogen peroxide. Spectrophotometric reading was done at 0, 30 and 60 seconds at 240 nm. GPx activity was assayed according to the protocol of Paglia and Valentine [25]. Drabkins reagent was used to convert haemoglobin to a stable form, cyanomethaemoglobin. GPx activity was assessed as the rate of NADPH disappearance, measured by spectrophotometry at 340 nm during 5 minutes. All the results were expressed as U·g^–1 of haemoglobin.

2.9.4 Assessment of 3-nitrotyrosine levels

3-nitrotyrosine was measured by Western Blot to assess protein nitration. Forty micrograms of protein from erythrocyte lysate were loaded into a 12% stain-free gel (TGX Stain-Free™ FastCast™, Bio-Rad Inc., CA, USA), and subsequently transferred to PVDF membranes using a semi-dry apparatus (Trans-Blot^TM SD, Bio-Rad Inc., CA, USA). Gels were activated by ultraviolet exposure for 1 min using a Bio-Rad ChemiDocMP imager, and total protein was quantified using Image Lab 5.2 software (Bio-Rad Inc., CA, USA). After transfer, membrane was blocked with 3% BSA in TBS-T for 1 h, followed by overnight incubation with primary antibodies against 3-nitrotyrosine (#SC-32757, 1 : 1000, SantaCruz Biotechnology, CA, USA). Membrane was then washed and incubated with goat anti-mouse HRP-conjugated secondary antibody for 1 h (1 : 10,000), and after washing, proceeded with ECL detection (ECL Prime^TM, GE Healthcare, Uppsala, Sweden) in ChemiDoc MP. The bands were detected and analyzed with ImageLab 5.2 (Bio-Rad Inc., CA, USA), and were normalized against total protein (Stain-Free^TM gels, Bio-Rad Inc., CA, USA).

2.10 Statistical analysis

Data are presented as mean±SD. Differences between pre- and post-test were assessed by paired t-test, after testing the assumption that the differences between pairs are normally distributed. Significance level was set at 5%. Statistical analysis was done using the software GraphPad Prism 6 (GraphPad Inc., CA, USA).

3 Results

3.1 Maximal exercise test

Maximal exercise test lasted around ten minutes (10.1±2.1 min), which is in accordance to testing recommendations to last between eight and 12 minutes. At the end of exercise, maximal velocity reached was 14.8±1.6 km·h^–1, and maximal heart rate, 194±2 bpm. There was a body mass reduction of 0.5±0.3% in relation to pre-exercise.

3.2 Erythrogram

Maximal exercise test induced a significant increase of approximately 7% in erythrocytes count, haemoglobin concentration, mean corpuscular volume, and haematrocrit (Table 1).

3.3 Dehydration markers

Plasma osmolality and Na⁺ levels were measured in order to assess dehydration. The results are presented in Table 1. No significant differences in both Na⁺ levels and osmolality were observed after maximal exercise test.

3.4 L-arginine-NO-cGMP pathway

It was performed a kinetic analysis of L-arginine influx, and no significant changes in V_max was noted after maximal exercise (417.0±62.8 μmol·L cells^–1·h^–1) compared to baseline (353.2±45.5 μmol·L cells^–1·h^–1), or in K_m (pre, 123.6±21.2; postexercise, 158.3±32.6 μM) (Fig. 1a).

Despite no changes in L-arginine influx, erythrocyte NOS activity, assessed by the production of L-[³H]-citrulline from L-[³H]-arginine, was increased after maximal test compared to baseline (Fig. 1b). It was also observed significant higher levels of intraerythrocyte cGMP (pre, 14.1±0.4; postexercise, 39.5±15.2 fmol·10⁸ cells; p < 0.01).

3.5 Biomarkers of oxidative stress in erythrocytes

Maximal exercise induced an increase in lipid peroxidation when compared to baseline, as assessed by TBARS assay. On the other side, no changes were observed in neither protein carbonylation (Table 2) nor protein nitration (Fig. 2) after maximal test.

After exercise, SOD activity was significantly reduced, which may have contributed to the increased lipid peroxidation. No significant change was observed in GPx, and catalase activity (Table 2).

3.6 Osmotic fragility

Haemolysis was expressed as a percentage, considering as 100% the absorbance of distilled water (0% NaCl). The NaCl concentration that elicited 50% of haemolysis was calculated using GraphPad Prism 6 (GraphPad Inc., CA, USA) for further comparisons among baseline and maximal test. Osmotic fragility was increased after maximal test (pre, 0.45±0.03%; postexercise, 0.47±0.03%; p = 0.0096).

4 Discussion

In this study, we aimed to investigate whether maximal exercise test would affect oxidative stress and NO bioavailability in erythrocytes from healthy subjects. Exercise led to an increase in erythrocyte fragility to an osmotic challenge and lipid peroxidation. This might be partially caused by a reduction in erythrocyte SOD activity, the first line of defence against ROS as it catalyzes the dismutation of O₂^–. into H₂O₂ and O₂; and by increased ROS produced by erythrocytes itself and surrounding cells. As erythrocytes pass through the microcirculation, they are susceptible to ROS produced by other cell types, particularly from the exercising skeletal muscle. It is well recognized that exercise increases skeletal muscle ROS production by the following [27]: (i) increased oxygen flux via electron transport chain in the mitochondria; and (ii) activation of NADPH oxidase located in sarcoplasmic reticulum, transverse tubules and sarcolemma. These findings may have implications for the impairment of erythrocyte fragility secondary to acute vigorous exercise [35], because the oxidation of lipids, particularly polyunsaturated fatty acids, may destabilize the lipid membrane bilayer and the cytoskeleton. As a consequence, the membrane may become more rigid given that its fluidity is affected by any change in polyunsaturated/saturated fatty acid ratio. Both structural and functional membrane alterations may compromise cell survival, as erythrocytes have poor repair mechanism. Accordingly, exercise resulted in an increase in erythrocyte osmotic fragility. Hence, any chemical or physical stress can lead to haemolysis, trigger an accelerated senescence or premature removal from thecirculation [8].

The other hypothesis tested in the present investigation was that the reduced membrane fluidity after strenuous exercise [4] could be due to a diminished NO bioavailability. Our results, however, do not support this hypothesis because there was an increase in intraerythrocytic cGMP levels after maximal test, as well NO production, despite an absence of increase in L-arginine influx into erythrocytes. In addition, NO was not inactivated by O₂^–., leading to peroxynitrite formation, as it was not observed any increase in 3-nitrotyrosine. Therefore, exercise might have led to increased NO bioavailability. Similar findings were recently reported by Suhr et al. [33]. They demonstrated that exercise resulted in an increase in phosphorylated eNOS at Ser¹¹⁷⁷ by means of Akt activation, and also elevated NO production assessed with diaminobenzidine fluorescence and cGMP levels in erythrocytes [33]. We believe that this increased synthesis of NO may be an acute response to the increased levels of oxidants and also secondary to the increased vascular shear stress caused by exercise, which is known to activate eNOS through PI3K/Akt pathway [17, 37]. It is important to highlight that NO is not the unique factor known to affect membrane fluidity. Adenosine triphosphate (ATP) plays a dual role in facilitating erythrocyte passage through the microcirculation. This molecule diffuses out of erythrocytes, leading to increased NO production from endothelial cells leading to vasodilation [24]; and phosphorylates proteins from erythrocyte cytoskeleton, resulting in a decrease in interconnections between proteins and, therefore, reduced membrane rigidity [32]. We did not, however, measure erythrocyte ATP levels in this study.

The increase in red blood cell count, haemoglobin content, haematocrit, and mean corpuscular volume after maximal exercise was an unexpected finding, since it lasted only around ten minutes. This acute haematocrit increase induced by exercise has been commonly referred as “haemoconcentration”, but based on our findings and as deeply discussed by others [7], it is an incomplete picture of the complex blood rheology changes that follow exercise. We believe that loss of water by sweat in the model of exercise adopted in our study was minimal, as body mass reduction, an indicator of dehydration and reduction in plasma volume, was of only 0.5%. In addition, other dehydration markers, such as plasma osmolality and Na⁺, were not altered by exercise. So, those values do not seem to be masked by haemoconcentration secondary to exercise-induced water loss. As mentioned before, exercise-induced increase in haematocrit may have different and independent mechanisms beyond dehydration. First, there may be a fluid shift between plasma and active muscle [9], leading to an entrapment of water into muscle cells. Second, another plausible hypothesis, yet not tested, is that exercise could have caused a sympathetic-mediated spleen contraction and consequent release of erythrocytes intocirculation [18].

This study presents a few limitations. Dehydration could have also been assessed by other not blood related measurements, such as urine specific gravity. Another limitation was the absence of erythrocyte deformability measurement, but it has been demonstrated by others that the exercise model employed in our study leads to a reduction in erythrocyte membrane fluidity [4].

In conclusion, the increased erythrocyte membrane fragility caused by an acute bout of exercise may be caused, in part, by an increased lipid oxidative damage caused by ROS produced exogenously. Nitric oxide synthesis and cGMP levels might be acutely increased in order to try to maintain membrane fluidity during exercise, and thus provide adequate O₂ supply to metabolically active cells.

4.1 Perspective

Results from this study have shown that a short bout of exercise acutely affects erythrocytes ability to resist to osmotic pressure variations, indicating that they are more susceptible to lyse. This might be secondary to changes in erythrocyte membrane structure [4, 34], partially caused by increased lipid oxidative damage. It was also observed an increase in erythrocyte NO synthesis and increased levels of its second messenger, cGMP, which contributes to maintain membrane fluidity. The clinical relevance of our findings is that, despite increased NO levels, erythrocytes membrane structure and function seems to be affected by exercise. This may result in increased rigidity, impaired deformability, which may, ultimately, impair its passage through the microcirculation and adequate oxygen delivery, and thus exercise limitation.

Footnotes

Acknowledgments

We thank Carolina M.O. Chamma and Nathalia Almeida Brigido de Souza for technical assistance and FAPERJ (E-26/110.387/2014) for funding.

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