Blood cell NO synthesis in response to exercise

Nitric Oxide 15 (2006) 5–12 www.elsevier.com/locate/yniox Blood cell NO synthesis in response to exercise Antoni Sureda a, Pedro Tauler a, Antoni Aguiló a, Emilia Fuentespina b, Alfredo Córdova c, Josep A. Tur a, Antoni Pons a,¤ a Laboratori de Ciències de l’Activitat Física, Departament de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes Balears, Crtra. Valldemossa Km 7.5. E-07122-Palma de Mallorca, Illes Balears, Spain b Laboratori del Carme, Hospital Son Dureta, INSALUD, Palma de Mallorca, Illes Balears, Spain c Escuela de Fisioterapia de Soria, Universidad de Valladolid, Soria, Spain Received 7 July 2005; revised 10 October 2005 Available online 22 December 2005 Abstract Nitric oxide (NO) is important for the maintenance of cardiovascular homeostasis and is also involved in immunity and inXammation. The aim of our work was to determine the eVects of intense exercise on plasma and blood cell NO handling. Nine voluntary male professional cyclists participated in the study. Blood samples were taken in basal conditions and 3 h after Wnishing a mountain cycling stage. Exercise-induced neutrophilia, lymphopenia, and hemolysis. Plasma and erythrocytes maintained basal nitrite levels, whereas neutrophils and lymphocytes decreased nitrite concentration after intense exercise. Basal iNOS levels and SOD activity were similar in neutrophils and lymphocytes. iNOS levels and SOD activity dropped in neutrophils and rose in lymphocytes after exercise. Arginase activity rose only in lymphocytes. Neutrophil nitrite was correlated with SOD activity and iNOS levels, but not in lymphocytes. iNOS levels were correlated with SOD in both neutrophils and lymphocytes. Intense exercise maintained plasma basal arginine and ornithine concentration, and decreased citrulline concentration. Intense exercise induced important changes in NO handling in neutrophils and lymphocytes, yet the basal picture was maintained in erythrocytes. © 2005 Elsevier Inc. All rights reserved. Keywords: Exercise; Oxidative stress; Nitric oxide synthase; Nitrite; SOD NO is a free radical gas synthesized from L-arginine by a family of isoenzymes called nitric oxide synthases (EC 1.14.13.39) (NOSs). Three major isoforms of NOS have been described. There are two constitutively expressed isoforms: vascular endothelial (eNOS), neuronal (nNOS), and one inducible (iNOS) isoform. Constitutive endothelial production of NO is important for the maintenance of cardiovascular homeostasis and the basal vasodilator tone [1–3]. In addition, NO tonically inhibits platelet aggregation, leukocyte adhesion to endothelial cells and smooth muscle proliferation; modulates respiration; and exerts antioxidant and anti-inXammatory activity [4–7]. iNOS is present in many cells involved in immunity and inXammation, which produce * Corresponding author. Fax: +34 971 173184. E-mail address: [email protected] (A. Pons). 1089-8603/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.11.004 high-levels of sustained NO synthesis when cells are activated. iNOS generates greater amounts of NO compared to the constitutive isoforms of NOS. Generation of NO is intertwined with synthesis, catabolism, and availability of arginine [8]. NO is important as a toxic defense molecule against infectious organisms and also regulates the functional activity, growth and death of many immune cells [9]. NO does not act through a receptor; its speciWcity on target cells depends on its concentration, the chemical reactivity, the vicinity of target cells and the way target cells are programmed to respond. When NO is generated at high concentrations it is rapidly oxidized to reactive nitrogen oxide species (RNOS) which mediate most of the immunological eVects. These important regulatory functions, together with its short halflife, involve a very tight control of NO synthesis. NO synthesized by endothelial cells diVuses to the lumen where it is either oxidized by oxygen, resulting in the 6 A. Sureda et al. / Nitric Oxide 15 (2006) 5–12 formation of nitrite, or it is taken up by erythrocytes. Nitrite is relatively stable under intracellular reducing conditions, and has recently been pointed out as a storage pool for NO synthesis in erythrocytes [10]. Hemoglobin presents both nitrite oxidase and reductase activity, depending on its oxygenation state [10]. Under normoxic or high oxygen conditions, nitrite is oxidized by oxyhemoglobin, producing nitrate and methemoglobin and preventing NO vasodilatory eVects. In the arterioles when the partial pressure of oxygen decreases nitrite is reduced by the deoxygenated hemoglobin formed, thus regenerating vasoactive NO, and producing vasodilatation [11]. The potential routes of blood NO decomposition comprise the reaction with molecular oxygen to form nitrite or with superoxide anion to form peroxynitrite, which subsequently decomposes to yield nitrate [12]. Alternatively, NO can react with aromatic compounds, amines, alcohols, and thiols to form C-, N-, O-, and S-nitroso species [13]. Exercise enhances or reduces the immune function depending on its frequency, duration, and intensity [14]. Exhaustive exercise decreases the functional capacity of neutrophils and lymphocytes and increases susceptibility to infections. Strenuous exercise induces oxidative stress [15] and also produces tissue hypoxia. The high oxygen demands of muscles during exercise could produce low oxygen availability for other tissues. This observation suggests that exercise increases the use of nitrite as NO precursor to produce vasodilation. Blood nitrite handling during exhaustive exercise is worth studying, as well as the relationship between NO synthesis and oxidative stress. The aim of this study was to determine the inXuence of exhaustive exercise on plasma and blood cell nitrite levels, on blood cellular capabilities to synthesize NO and arginine metabolization, and on blood cellular capabilities of anion superoxide detoxiWcation. We also determined the eVects of exhaustive exercise on the plasma arginine concentration and its metabolic derivates, ornithine and citrulline. Materials and methods Subjects Nine voluntary male subjects participated in this study. They were all professional cyclists participating in the “Challenge Volta a Mallorca 2002,” a Wve-day competition for professional cyclists. Subjects were informed of the purpose of this study and the possible risks involved before giving their written consent to participate. The study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of “University Hospital Son Dureta” (Balearic Islands, Spain). The exercise was a mountain stage (171.8 Km). The mean age of the sportsmen (§SEM) was 25.2 § 2.3 years, height 177 § 5 cm; weight 69 § 5.4 kg, and body max index 22.1 § 1.1 § g/m. Their VO2 max was 78.4 § 4.9 ml/kg/min. The cyclists took a mean of 283 § 12 min to complete this stage. Experimental procedure Venous blood samples were taken from the antecubital vein with suitable vacutainers with EDTA as anticoagulant. Samples were taken the morning of the cycling stage, after overnight fasting, and 3 h after the stage. Blood samples were used to purify neutrophils, lymphocytes, erythrocytes, and to obtain plasma. Blood cells and hematological parameters were quantiWed in an automatic Xow cytometer analyser Technicon H*2 (Bayer) VCS system. Nitrite levels were determined in all cell types and plasma. SOD and arginase enzyme activities were determined in neutrophils and lymphocytes. We also determined the iNOS protein levels in neutrophils and lymphocytes. Amino acids levels were measured in plasma. Neutrophil and lymphocyte puriWcation The neutrophil fraction was puriWed following an adaptation of the method described by Boyum [16]. Blood was carefully introduced on Ficoll in a proportion of 1.5:1 and centrifuged at 900g, at 4 °C for 30 min. The lymphocyte layer was carefully removed. The precipitate containing the erythrocytes and neutrophils was incubated at 4 °C with 0.15 M ammonium chloride to hemolyse the erythrocytes. The suspension was centrifuged at 750g, 4 °C for 15 min and the supernatant was then discarded. The neutrophil phase at the bottom was washed Wrst with ammonium chloride and then with phosphate-buVered saline, pH 7.4. Finally, the neutrophils were lysed with distilled water. The lymphocyte slurry was then washed twice with PBS and centrifuged for 10 min at 1000g, 4 °C. The cellular precipitate of lymphocytes was lysed with distilled water. Erythrocyte and plasma puriWcation Blood samples were centrifuged at 900g, 18 °C for 30 min. The plasma was recovered, and the erythrocyte phase at the bottom was washed with PBS, pH 7.4. Erythrocytes were reconstituted and hemolysed with distilled water in the same volume as plasma. Nitrite determination Nitrite levels were determined in all cell types and plasma by the acidic Griess reaction using a spectrophotometric method. Lysed cells and plasma were deproteinised with acetone and kept overnight at ¡20 °C. Samples were centrifuged for 10 min at 15,000g at 4 °C, and supernatants were recovered. A 96-well plate was loaded with the samples or nitrite standard solutions (100 l) in duplicate. Fifty microliter sulfanilamide (2% w/v) in 5% HCl was added to each well, and 50 l N-(1-napthyl)-ethylenediamine (0.1% w/v) in water was then added. The absorbance at 540 nm was measured following an incubation of 30 min. 7 A. Sureda et al. / Nitric Oxide 15 (2006) 5–12 iNOS protein levels iNOS protein levels were determined in neutrophils and lymphocytes by ELISA using polyclonal antibody Anti human iNOS (Stressgen). We followed an adaptation of the previously described method [17]. Suitable dilutions of the neutrophil or lymphocyte suspensions and of the iNOS standard were placed in each well of the plate per duplicate (Polystyrene Assay Plate, Costar). The plate was then incubated at 37 °C for 3 h. A solution of 1% bovine albumin was added into each well and the plate was incubated (37 °C for 3 h) to saturate all binding protein sites of the plate. The plate was then washed 4 times with NaCl 0.9%–Tween 20. The commercial antibody (diluted 1000-fold) was placed into each well and the plate was newly incubated for 3 h at 37 °C. The plate was then washed as above. The secondary antibody against the IgG chain, conjugated to alkaline phosphatase (diluted 500-fold), was placed into each well and the plate was incubated in the same conditions as above. The wells were newly washed and the phosphatase substrate solution was added. Finally, absorbance was measured at 405 nm. Enzyme assays SOD and arginase activities were determined with a Shimadzu UV-2100 spectrophotometer at 37 °C. SOD activity was measured by an adaptation of the method of McCord and Fridovich [18]. The xanthine/xanthine oxidase system was used to generate the superoxide anion. This anion produced the reduction of cytochrome c, which was monitored at 550 nm. The SOD in the sample removed the superoxide anion and produced an inhibition of the cytochrome c reduction. Arginase activity was determined by measuring an increase in the concentrations of ornithine [19]. In the presence of acetic acid, ornithine reacts with ninhydrin with a maximal absorbance at 515 nm. Amino acid levels In a parallel experiment, we studied the changes in blood arginine, citrulline, and ornithine concentrations during maximal exercise. The test was performed on an electromagnetic cycle ergometer (Ergometrics 900) equipped with a counter to measure the exact number of revolutions. The test ended when the subjects manifested their subjective fatigue status and when increased work did not increase or decrease oxygen consumption; this value was the maximal oxygen volume (VO2 max). The subjects exercised at a pedaling rate of 60 rpm. The duration of the test was 25.3 § 0.9 min and they explained their lack of leg muscular fatigue. The sportsmen warmed up on the cycle ergometer for 3 min at 30 W prior to starting the test. The test started at 50 W and the subjects’ work rate was increased by 30 W every 3 min until fatigue. Expired oxygen was continuously monitored with a Cardiopulmonary Exercise System CPX (MedGraphics) and VO2 maximal determined automatically. The levels of arginine, citrulline, and ornithine were determined in plasma by HPLC following the previously described procedure [20]. Plasma was diluted with an equal volume of working internal-standard solution (L-methionine sulfone 0.4 mM), was deproteinized with cold acetone (1:1.5, v/v) and the protein-free supernatant fraction was used for individual amino acid measurements. Amino acids were assessed by HPLC, using the PICO.TAG method [21] developed by Waters Associates, by derivatization of amino acids using phenylisothiocyanate. Sample amino acid levels were calculated from the peak area using the Maxima 820 programme. Statistical analysis Statistical analysis was carried out using a statistical package for social sciences (SPSS 11.0 for Windows). Results are expressed as means § SEM and P < 0.05 was considered statistically signiWcant. The statistical signiWcance of nitrite, iNOS levels, and enzymatic activities were assessed by two-way analysis of variance (ANOVA). The statistical factors analyzed were cell type (Cll) and exercise (E). When signiWcant eVects were found, a Student’s t test for unpaired data was used to determine the diVerences between the groups involved. One-way ANOVA was also used to determine diVerences in the cell number, hematological parameters, and amino acid levels. We also determined the correlations between SOD, arginase, iNOS, and nitrite in neutrophils and lymphocytes. Results Exhaustive exercise, as is the mountain stage, produced neutrophilia—circulating neutrophils increased about 4 times after stage—and lymphopenia—lymphocytes decreased about 38%—(Table 1). The cycling stage produced signiWcant decreases in hematocrit (about 12%), erythrocyte number (about 8%), and hemoglobin concentration (about 10%) but the reticulocyte number was maintained. The basal characteristic parameters of erythrocytes, Table 1 Hematological parameters Neutrophils (103/l) Lymphocytes (103/l) LUC (103/l) Erythrocytes (106/l) Reticulocytes (103/l) Hemoglobin (gr/dl) Hematocrit (%) VCM (Fl) HCM (Pg) CHCM (g/dl) RDW (%) Before After 2.89 § 0.28 2.43 § 0.12 111 § 8 5.14 § 0.11 0.51 § 0.07 16.3 § 0.23 46.6 § 0.6 89.9 § 1.3 31.5 § 0.4 35.0 § 0.2 13.6 § 0.1 12.3 § 1.6¤ 1.50 § 0.13¤ 75.5 § 10¤ 4.70 § 0.13¤ 0.59 § 0.06 14.6 § 0.24¤ 41.0 § 0.6¤ 87.5 § 1.5 31.2 § 0.4 35.7 § 0.3 13.3 § 0.1 Circulating blood cells and hematological parameters before and after the cycling stage. ¤ Indicate signiWcant diVerent values (one-way ANOVA test, P < 0.05). 8 A. Sureda et al. / Nitric Oxide 15 (2006) 5–12 such as mean corpuscular volume (VCM), mean corpuscular hemoglobin (HCM), and mean corpuscular hemoglobin concentration (MCHC) maintained their values after exhaustive exercise. Overall parameters in erythrocytes and reticulocytes indicate the existence of hemolysis induced by the cycling stage. The decreased hematocrit was non indicative of hemodilution. Fig. 1 shows the plasma and blood cell nitrite concentrations before and after the mountain cycling stage. Plasma nitrite levels after the stage were not corrected because the apparent hemodilution was due to hemolysis. Basal plasma and blood cell nitrite levels were maintained after the cycling stage. Nitrite concentration in the blood cell compartment was about 7- to 10-fold that in plasma. The basal blood cell/plasma ratio was maintained after the stage. The contribution of the diVerent blood cell types to the total nitrite blood cell compartment was calculated by taking into account the nitrite concentration of each cell type and the number of each cell type in blood. 93.8–95% of all nitrite concentration in the blood cell compartment was attributable to erythrocytes, 2.6–3.1% to neutrophils, and 3.6–1.9% to lymphocytes (before–after exercise). Nitrite concentration in the diVerent blood cell types before and after exhaustive exercise is shown in Table 2. Nitrite levels were signiWcantly diVerent between diVerent blood cell types, both expressed per cellular number or per cellular volume. Erythrocytes presented the lowest and lymphocytes the highest basal nitrite concentration. The mountain cycling stage signiWcantly changed blood cell nitrite concentration depending on blood cell type. Neutrophils and lymphocytes signiWcantly decreased (67 and 43%) the nitrite concentration after the cycling stage, while erythrocytes maintained the low basal nitrite concentration. The ratio between nitrite concentrations in each blood cell type and in plasma followed the same pattern. Lymphocytes concentrated about 15 times more nitrite than erythrocytes but only 1.6 times more than neutrophils at baseline. The cycling stage decreased the ratio about 3-fold in neutrophils and about 1.9-fold in lymphocytes, but it was maintained in erythrocytes. Table 2 Neutrophil, lymphocyte, and erythrocyte nitrite levels Nitrite Before After nmol/109 cells Neutrophils Lymphocytes Erythrocytes 56.5 § 5.8a 97.2 § 12.8c 1.67 § 0.09d 18.7 § 3.5b 55.6 § 12.1a 1.75 § 0.10d pmol/l Neutrophils Lymphocytes Erythrocytes 188 § 19a 324 § 42c 18.7 § 1.0d 62.6 § 11.8b 185 § 40a 19.9 § 1.2d Ratio Neutrophils/plasma Lymphocytes/plasma Erythrocytes/plasma 146 § 19a 235 § 20c 15.0 § 2.0d b 49.1 § 12.3b 125 § 26a 15.9 § 2.4d b T £ Cll x x x x x x x x The changes in nitrite concentration in lymphocytes and neutrophils resulting from exhaustive exercise could be related either to the changes in iNOS levels or to the capability to metabolise arginine via ornithine by arginase. We determined iNOS levels and arginase activity in lymphocytes and neutrophils (Table 3). The basal iNOS levels were similar in lymphocytes and neutrophils when expressed on a cellular basis. However, the iNOS levels signiWcantly decreased about 67% in neutrophils and increased 1.5 times in lymphocytes after exhaustive exercise. Lymphocyte arginase activity was signiWcantly lower in lymphocytes with respect to neutrophils. The mountain cycling stage signiWcantly increased lymphocyte arginase activity about 5.8-fold, whereas this activity was maintained in neutrophils. Lymphocyte and neutrophil basal SOD activities were similar, but exhaustive exercise induced an opposite pattern of change in these cell activities: the mountain cycling stage lowered SOD Before After 15 µmol/L Cll x Nitrite levels expressed per nmol/109 cells, per cell volume (approximately 30 l/108 neutrophils and lymphocytes, and using the VCM for erythrocytes), and the cell type/plasma ratio. (x) SigniWcant eVects of time (T) or cell type (Cll) or interaction of supplementation and time (T£Cll) (P < 0.05, two-way ANOVA). DiVerent letters indicate signiWcant diVerent values (one-way ANOVA test, P < 0.05) when a signiWcant T£Cll interaction is observed. 20 10 5 0 Plasma T Blood cells Blood cells / plasma Fig. 1. Nitrite levels in plasma (mol/L plasma), in blood cells (mol/L blood), and the blood cells/plasma ratio. 9 A. Sureda et al. / Nitric Oxide 15 (2006) 5–12 Table 3 iNOS protein levels, arginase, and SOD activities in neutrophils and lymphocytes iNOS Neutrophils (ng/106 cells) Lymphocytes (ng/106 cells) Before After 409 § 52a 319 § 37c,a 134 § 29b 493 § 50d,a T Cll T £ Cll x x Arginase x Neutrophils (nKat/106 cells) 2.26 § 0.24a 3.65 § 0.64a Lymphocytes (nKat/109 cells) 57.2 § 9.1b 335 § 76c x x SOD x Neutrophils (pKat/109 cells) 12.4 § 1.3a 6.00 § 0.86b Lymphocytes (pKat/109 cells) 13.1 § 2.1c,a 25.0 § 2.6d x x 6 iNOS protein levels expressed as ng protein/10 cells, arginase activity (nKat/109 cells) and SOD activity (pKat/109 cells). (x) SigniWcant eVects of time (T) or cell type (Cll) or interaction of supplementation and time (T £ Cll) (P < 0.05, two-way ANOVA). DiVerent letters indicate signiWcant diVerent values (one-way ANOVA test, P < 0.05) when a signiWcant T £ Cll interaction is observed. activity about 50% in neutrophils and raised it about 50% in lymphocytes. Plasma nitrite concentration was independent of nitrite concentration in neutrophils, lymphocytes, erythrocytes or whole blood cells (results not shown). However, neutrophil nitrite concentration was strongly correlated with neutrophil SOD activity and iNOS levels, but was independent of neutrophil arginase activity (Table 5). Lymphocyte nitrite concentration was independent of lymphocyte SOD and arginase activities as well as iNOS levels. iNOS levels were correlated with SOD activity in both neutrophils and in lymphocytes. Arginine availability is another control point for cellular NO production. We determined the levels of arginine, citrulline, and ornithine in plasma. Exhaustive exercise as a maximal test maintained basal plasma concentrations of arginine and ornithine, whereas citrulline levels decreased (Table 4). We tried to determine the amino acid content in cells by subtracting plasma levels from whole blood levels (data not shown). However, amino acid levels in blood cells were lower than the values found in plasma due to their fast Table 4 Correlations in neutrophil and lymphocyte parameters Neutrophils SOD iNOS Arginase Nitrite Lymphocytes SOD iNOS Arginase Nitrite SOD iNOS Arginase Nitrite 1 0.931 1 — — 1 0.915 0.878 — 1 1 0.594¤ 1 — — 1 — — — 1 Bivariate correlations signiWcant at P < 0.01 level. ¤ Correlation is signiWcant at P < 0.05 level. Table 5 Amino acid levels in plasma and hematocrit Before After Arginine Ornithine Citrulline 52.4 § 5.3 96.5 § 14.6 30.4 § 0.1 54.7 § 6.5 61.8 § 12.5 17.0 § 1.2¤ Hematocrit (%) 44.8 § 0.8 48.6 § 1.2 Levels of L-arginine, L-ornithine, and L-citrulline in plasma (mol/L) before and after a maximal test. ¤ Indicate signiWcant diVerent values (one-way ANOVA test, P < 0.05). metabolization during blood processing, resulting in a negative value. Discussion Exhaustive exercise such as a mountain cycling stage produces a redistribution in the circulating blood cells [17]. Lymphocyte number decreased after the mountain stage, as has been evidenced after intense exercise [22]. Exerciseinduced lymphopenia is related to apoptosis induced by oxidative stress [22,23], and to increased probability to suVering upper respiratory tract infections [24]. The mountain cycling stage also induced the characteristic neutrophilia evidenced after intense exercise, and an acute phase immune response similar to infection [25]. The drop in the circulating number of erythrocytes after the cycling stage could be attributed to hemolysis, since reticulocyte number remained unchanged after exercise. The hemolysis induced by exhaustive exercise is also reXected by an important decrease in the hematocrit value and in hemoglobin concentration. Hemolysis is more evident during recovery than during, or immediately after, exhaustive exercise, as a result of the eVects of oxidative stress on plasma erythrocyte membrane [26]. Intravascular hemolysis releases hemoglobin from the erythrocytes to the plasma, altering NO availability for endothelial cells and for smooth muscle cells [27]. The erythrocyte membrane creates diVusional barriers between NO and erythrocytic hemoglobin, decreasing the rate of NO scavenging by hemoglobin [10]. However, free hemoglobin reacts quickly with NO resulting in the formation of nitrate and methemoglobin and, thereby, preventing the diVusion of NO from plasma to smooth muscle [28]. The hemolysis evidenced after the mountain cycling stage could increase the rate of NO scavenging and could limit NO bioactivity. Exercise has been evidenced to raise plasma nitrite levels by increasing NO synthesis in endothelial cells [29,30]. However, the nitrite levels in venous plasma and blood cells after 3 h of intense exercise are fairly similar to the basal ones. Apparently, nitrite plasma concentration is regulated to maintain constant plasma levels. The metabolic pathways that tightly regulate circulating nitrite are not well elucidated but were operative during exhaustive exercise. NO is mainly oxidized in human plasma to nitrite [31]. Plasma nitrite is gradually oxidized to nitrate, a process 10 A. Sureda et al. / Nitric Oxide 15 (2006) 5–12 which is greatly accelerated by the presence of heme proteins [32]. Nitrate is stable in plasma until excreted in the urine. Circulating nitrite rather than nitrate reXects endothelial-dependent NO synthesis in humans and animals [33]. It has been evidenced that urinary nitrate excretion is increased immediately after a submaximal exercise test [34]. However, in our study plasma nitrite levels remained unchanged 3 h after the exercise. The increased production of NO during exercise is probably controlled by increasing nitrate excretion, as a possible mechanism to control plasmatic homeostasis [34]. Nitrite is transported in blood mainly in the cells. The main contributors are the erythrocytes, which account for about 95% of blood cell nitrite, whereas leukocyte contribution is about 5%. However, lymphocytes are the blood cells that present the highest nitrite concentration gradient versus plasma, whereas erythrocytes are the blood cells with the lowest nitrite concentration gradient. The nitrite in erythrocytes might be the result of plasma nitrite uptake or could also be the end product of the oxidation of the NO synthesized by erythrocytic NOS from arginine. Erythrocytes contain an un-active nitric oxide synthase which is only activated under speciWc stimulus [35]. The great diVerences between erythrocytes, lymphocytes, and neutrophils in the cellular nitrite content argue in favor of a low or inexistent synthesis of NO from arginine in erythrocytes. However, erythrocytes take up plasma nitrite, concentrating it about 15-fold with respect to plasma. The mechanisms of nitrite intake by erythrocytes are under evaluation [36,37]. When oxygen tension decreases, the reduction of nitrite by deoxyhemoglobin produces NO. Erythrocyte NO generation and output, along with the oxygen concentration gradient, could be related with a role of nitrite-bound to erythrocytes in the vasodilatation processes in response to hypoxia. The cycling stage maintained the same basal picture with respect to nitrite concentration in plasma and erythrocytes. The cellular response of immune cells to exerciseinduced oxidative stress on the synthesis of NO, degradation of superoxide anion and the limitation of L-arginine availability for iNOS can be useful to understand NO handling in ‘in vivo’ situations. Although neutrophils and lymphocytes decreased the intracellular concentrations of nitrite after exhaustive exercise, both cells showed a diVerent enzymatic response. Exhaustive exercise increased arginase activity in lymphocytes nearly 6-fold, limiting L-arginine availability for lymphocyte iNOS activity. It has been pointed out that iNOS and nNOS can produce superoxide anion at low arginine concentrations [38,39]. The limitation of arginine availability for iNOS, evidenced by a decreased nitrite concentration, produces superoxide anion and NO, with the consequent peroxynitrite production [38]; then, peroxynitrite could be related to post-exercise lymphopenia by activating lymphocyte apoptosis [40]. SOD is important in NO handling strategy in lymphocytes because it avoids ROS damaging eVects, as is evidenced by the high increase in its activity after exercise. SOD protects from oxidation by superoxide anion and, therefore, confers NO with a longer half-life by sparing it from superoxide attack and, thus, allows NO to carry out its physiological functions. The eVects of exhaustive exercise on ‘in vivo’ arginine availability in immune cells are unknown. Exhaustive exercise maintained the basal L-arginine and L-ornithine levels in plasma; however it decreased plasma citrulline levels. This picture is compatible with the existence of L-arginine recycling from citrulline; the high L-arginine demands for NO synthesis during exercise is reXected by the drop in citrulline levels to maintain plasma arginine concentration. Plasma L-arginine levels are a limiting factor for NO synthesis [41]. Upon stimulation of NO production, over 80% of the L-citrulline is recycled to arginine in endothelial cells [42]. The limited L-arginine availability for the immune cells during exhaustive exercise argues in favor of peroxynitrite formation and lymphopenia induction by the mountain cyclist stage. Exhaustive exercise also induced an acute phase immune response that primed neutrophils to oxidative stress [43]. This primed situation coexists with diminished antioxidant enzyme activity [17]. This picture is evidenced again in this work because the mountain cycling stage induced neutrophilia and decreased neutrophil SOD activity. Exhaustive exercise reduced neutrophil capability to synthesize NO, as evidenced by the decrease in iNOS levels and nitrite concentration—in accordance with previous Wndings [44,45]— but maintained arginase activity. One hour of exercise at 85% VO2 max induced no changes in neutrophil immune functions but decreased nitric oxide production by decreasing iNOS expression [44]. The quick decrease in the nitrite content could indicate a fast metabolization of nitrite in primed neutrophils [46]. NO production in neutrophils can be modulated by superoxide, which, in turn, can form peroxynitrite and, subsequently, nitrate [44]. Selective inhibitors of iNOS have been observed to increase neutrophil adhesion to endothelial cells [45]. This response agrees with a previous study, where we evidenced an increase in the expression of adhesion molecules in neutrophils after intense exercise [47]. Simultaneous generation of superoxide and NO in SOD deWciency can lead to the production of peroxynitrite anion and initial lipid peroxidation [48,49]. The basal iNOS levels and SOD activity were similar in neutrophils and lymphocytes, iNOS levels and SOD activity dropped in neutrophils and rose in lymphocytes after exercise. This dual picture could diVerentially aVect the redox status and the oxidative damage in both cell types. We have described that lymphocytes increased the markers of oxidative damage after intense exercise, whereas the neutrophils maintain the basal levels [50]. In summary, NO handling in blood is speciWc for each blood compartment. Exhaustive exercise causes important changes in the strategies of NO handling in lymphocytes and neutrophils but maintains the basal picture in erythrocytes. Plasma nitrite levels are tightly regulated and activated by exercise. Exhaustive exercise induces a situation of A. 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