Erythrocyte nitric oxide availability and oxidative stress following exercise.

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, V̇O2peak 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.

Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6 kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity.

Major depression induces oxidative stress and platelet hyperaggregability.

We have previously demonstrated an impairment of intraplatelet L-arginine-nitric oxide-cGMP pathway in major depression (MD) associated to platelet dysfunction. Here, we evaluated arginase pathway and phosphodiesterase 5 (PDE5) expression in platelets, systemic and intraplatelet oxidative status in untreated MD patients, and their effects on platelet aggregation. Blood samples were collected from 22 treatment naive MD patients (31 ± 2 yr) and 27 healthy subjects (33 ± 2 yr). MD patients presented with an activation of platelet arginase II, which competes with L-arginine for the production of nitric oxide (NO). An increase in protein carbonylation, overexpression of NADPH oxidase and PDE5, an enzyme that inactivates cGMP, was observed in platelets from MD patients compared to controls. In this context, platelet hyperaggregability was found in MD patients. On the other hand, antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase activities in serum and in platelets did not differ between groups. The increased activation of intraplatelet arginase and platelet aggregability, in addition to an overexpression of PDE5 and oxidative stress may contribute to alterations in L-arginine-NO-cGMP pathway and in platelet function, and consequently to the increased thrombotic risk in MD.

Diminished nitric oxide generation from neutrophils suppresses platelet activation in chronic renal failure.

Chronic renal failure (CRF) is a complex clinical condition associated with accelerated atherosclerosis and thrombosis leading to cardiovascular events. The aim of this study was to investigate in detail the NO pathway in neutrophils obtained from hemodialysis patients and its association with platelet function and oxidative status. Fifteen CRF patients on hemodialysis and fifteen controls were included in this study. Laboratory and experimental evaluations were performed after hemodialysis in CRF patients. We evaluated L-[³H] arginine transport, NO synthase (NOS) activity, amino acid concentration in neutrophils, and expressions of NOS isoforms and p47(phox) by western blotting. Platelet aggregation was analyzed in the presence or absence of neutrophils. Oxidative status was measured through glutathione peroxidase, catalase activities, protein oxidation, lipid peroxidation, and DNA/RNA oxidation in serum. Basal NOS activity (pmol/106 cells/min) was impaired in CRF patients on hemodialysis (0.33 ± 0.17) compared to controls (0.65 ± 0.12), whereas the expression of NOS isoforms remained unaltered. L-Arginine transport into neutrophils was similar in CRF patients on hemodialysis and controls. In addition, intracellular concentration of L-arginine was increased fourfold in the patient group. Systemic oxidative stress markers were not affected by CRF. On the other hand, NADPH oxidase subunit p47(phox) in neutrophils was overexpressed in CRF. In the presence of neutrophils, there was a reduction time-dependent in platelet aggregation in both groups with no difference between them. This data suggest that reduced basal generation of NO by neutrophils in CRF patients on hemodialysis occurs independently of L-arginine bioavailability and is able to suppress platelet activation.

Role of dietary fish oil on nitric oxide synthase activity and oxidative status in mice red blood cells.

The consumption of n-3 polyunsaturated fatty acids (PUFAs) derived from fish oil concomitant with a reduced intake of saturated fats is associated with cardiovascular benefits, which may result from the participation of nitric oxide (NO). In contrast, PUFAs are vulnerable to peroxidation, which could affect the oxidative stability of the cell and reduce NO bioavailability. Therefore, we investigated the effects of high fat diets with increasing amounts of fish oil (0-40% of energy) in place of lard on the l-arginine-NO pathway, the arginase pathway and oxidative status in mice red blood cells (RBC). We found that l-arginine transport, as well as NO synthase (NOS) expression and activity, was enhanced by the highest doses of fish oil (30 and 40%). In contrast, diets rich in lard led to NOS expression and activity impairment. Arginase expression was not significantly affected by any of the dietary regimens. No significant difference in protein and lipid oxidative markers was observed among any of the fish-oil fed mice; only lard feeding induced protein damage in addition to a decreased superoxide dismutase activity. These data suggest that a substantial dose of fish oil, but not low doses, activates the RBC l-arginine-NO pathway without resulting in oxidative damage.

Effects of non-surgical periodontal treatment on the L-arginine-nitric oxide pathway and oxidative status in platelets.

Several studies have suggested an increase of cardiovascular disease (CVD) risk on periodontitis patients. An enhancement has been demonstrated on both platelet activation and oxidative stress on periodontitis patients, which may contribute for this association. Therefore, the aim of this study was to evaluate the effects of non-surgical periodontal treatment on the l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway and oxidative status in platelets. A total of eight periodontitis patients and eight controls were included in this study. Clinical, laboratory and experimental evaluations were performed on baseline and 90 days after periodontal treatment (except for western blot analysis). The clinical periodontal evaluation included measurements of probing pocket depth (PPD), clinical attachment loss (CAL), % of sites with plaque and % of sites with bleeding on probing. We evaluated: l-[(3)H]arginine influx; nitric oxide synthase (NOS) and arginase enzymes activity and expression; expression of guanylate cyclase and phosphodiesterase-5 enzymes; cGMP levels; platelet aggregation; oxidative status through superoxide dismutase (SOD) and catalase activities, and measurement of reactive oxygen species (ROS) levels and C-reactive protein (CRP) levels. The initial results showed an activation of both l-arginine influx and via system y (+ )L associated with reduced intraplatelet cGMP levels in periodontitis patients and increased systemic levels of CRP. After periodontal treatment, there was a significant reduction of the % of sites with PPD 4-5mm, % of sites with CAL 4-5 mm, and an enhancement in cGMP levels and SOD activity. Moreover, CRP levels were reduced after treatment. Therefore, alterations in the intraplatelet l-arginine-NO-cGMP pathway and oxidant-antioxidant balance associated with a systemic inflammatory response may lead to platelet dysfunction, which may contribute to a higher risk of CVD in periodontitis.

L-arginine-nitric oxide pathway and oxidative stress in plasma and platelets of patients with pre-eclampsia.

Pre-eclampsia (PE), a syndrome of pregnancy-induced hypertension, continues to be a leading cause of maternal and fetal morbidity and mortality. The aim of this study was to investigate whether changes in oxidative status are correlated with alterations in the L-arginine-nitric oxide pathway and platelet aggregation in PE. Plasma and platelets from women with PE (n=24) or normotensive pregnancy (NP, n=27) recruited in the third trimester of gestation were used to measure oxidative damage assessed by protein carbonyl content, antioxidant activities of superoxide dismutase (SOD), catalase (CAT) and nitrite levels. Transport of L-[(3)H]-arginine, as well as the activities of the nitric oxide (NO) synthase (eNOS and inducible NO synthase (iNOS)) and platelet aggregation, were also evaluated. Plasma nitrite levels and the activities of SOD and CAT were reduced in PE (5.2±2.7, 3.4±0.8, 0.3±0.4, respectively, P<0.05) compared with NP (8.7±2.3, 6.7±3.1, 1.0±0.5, respectively), whereas protein carbonyl content and L-arginine levels were not significantly different between PE and NP groups. In platelets, L-arginine transport was reduced in PE (19.2±10.5, P<0.05) compared with NP (62.0±31.1), whereas the NOS activity, eNOS and iNOS expression, nitrite levels and platelet aggregation were unaffected. Protein carbonyl content was increased, and CAT activity was reduced in platelets from PE (0.03±0.02, 0.55±0.30, respectively, P<0.05), compared with NP (0.005±0.005, 1.01±0.36, respectively). The data suggest that a systemic impairment of antioxidant defense mechanisms is associated with decreased plasma nitrite levels, which may contribute to hypertension in PE. Oxidative stress may contribute to the reduced influx of L-arginine in platelets. Compensatory mechanisms may contribute to the maintenance of NO production and its modulatory role on platelet function.

Defective nitric oxide-cyclic guanosine monophosphate signaling in patients with bipolar disorder: a potential role for platelet dysfunction.

OBJECTIVE: Bipolar disorder (BD) is associated with elevated cardiovascular mortality rates. We investigated the modulation of l-arginine-nitric oxide (NO) signaling in platelets from patients with BD at different phases. METHODS: Platelets obtained from 28 patients with BD and 10 healthy volunteers were analyzed for l-arginine transport, NO synthase (NOS) activity, cyclic guanosine monophosphate content, and biomarkers of oxidative stress. Expressions of NOS isoforms, soluble guanylyl cyclase, and arginase were also measured in platelets. Amino acid and C-reactive protein levels in plasma were assessed. RESULTS: Plasma concentrations of l-arginine (mean [M] ± standard error of the mean [SEM] = 97 ± 10 versus 121 ± 10 µM) and its transport into platelets (median [interquartile range] = 26.0 [28.6] versus 26.5 [43.9] pmol/10(9) cells per minute) did not differ between patients with BD and controls (p > .05). Patients with BD showed reduced NOS activity (M ± SEM = 0.037 ± 0.003 versus 0.135 ± 0.022 pmol/10(8) cells, p < .001), but not endothelial NOS, inducible NOS, and arginase expression, compared with controls (p > .05). Cyclic guanosine monophosphate content was reduced (M ± SEM = 0.022 ± 0.003 versus 0.086 ± 0.020 pmol/10(8) cells, p < .05) despite the absence of changes in soluble guanylyl cyclase expression (median [interquartile range] = 21.6 [15.5] versus 9.5 [9.4] arbitrary units, p > .05) in patients with BD. Superoxide dismutase activity, but not catalase activity, was increased in patients with BD in the manic phase (M ± SEM = 2094 ± 335 versus 172 ± 17 U/mg protein, p < .001). C-reactive protein was elevated only in manic episodes (M ± SEM = 0.8 ± 0.2 versus 0.1 ± 0.02 mg/L, p < .001). CONCLUSIONS: Impaired NO generation from platelets, inflammation, and oxidative stress may play pivotal roles in the multifaceted process of cardiovascular events in BD.

Low plasma levels of L-arginine, impaired intraplatelet nitric oxide and platelet hyperaggregability: implications for cardiovascular disease in depressive patients.

BACKGROUND: Major depression (MD) is an independent cardiovascular risk factor, but the exact mechanisms are not clear. In this study we have investigated the intraplatelet L-arginine-nitric oxide (NO) pathway and platelet function in depressive patients. METHODS: Nineteen unmedicated patients with MD (34±4years) and 19 control subjects (CS, 34±3years) were included. L-[(3)H]-arginine influx, NO synthase (NOS) activity and intracellular cGMP levels were evaluated in platelets, as well as the expression of eNOS, iNOS, arginase and soluble guanylate cyclase (sGC), platelet aggregation and the systemic amino acid profile in MD patients and CS. RESULTS: L-arginine influx (pmol/10(9)cells/min) in platelets was reduced from 46.2±9.5 to 20.02±2.12 in depression. NOS activity (pmol/10(8) cells) was diminished in MD patients (0.09±0.01) compared to CS (0.17±0.01). Intracellular cGMP levels were also impaired in MD patients associated with hyperaggregability. Moreover, the concentration of plasma L-arginine was reduced by 20% in MD patients. The expression of eNOS, iNOS, arginase II and sGC in platelet lysates was not affected by MD. LIMITATIONS: Small number of patients in the study. CONCLUSIONS: This study has demonstrated an impairment of L-arginine-NO signaling in platelets from MD patients, suggesting a role in platelet activation and cardiovascular events.

Upregulation of platelet (L)-arginine: nitric oxide pathway after exercise training in hypertension.

We investigated whether physical exercise can affect platelet L-arginine - nitric oxide pathway in spontaneously hypertensive rats (SHR). Sixteen male SHR and 16 Wistar Kyoto rats (WKY) were divided among exercise (EX) and sedentary (SED) groups. After 20 weeks of treadmill training, systolic blood pressure (mm Hg) was significantly lower in exercised spontaneously hypertensive rats (SHR/EX; 138 ± 8) than in sedentary spontaneously hypertensive rats (SHR/SED; 214 ± 9). Exercise significantly increased platelet L-arginine transport (pmol L-arginine·(10(9) cells)(-1)·min(-1)), assessed by incubation with L-[(3)H]-arginine, in both WKY (SED, 0.196 ± 0.054 compared with EX, 0.531 ± 0.052) and SHR (SED, 0.346 ± 0.076 compared with EX, 0.600 ± 0.049). Nitric oxide synthase (NOS) activity (pmol L-citrulline·(10(8) cells)(-1)), measured by the conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, was significantly increased in SHR/EX (0.072 ± 0.007) compared with SHR/SED (0.038 ± 0.007), but no changes were observed in WKY. The iNOS and eNOS protein levels assessed by Western blot were not affected by exercise. This upregulation of the platelet L-arginine-NO pathway may attenuate the risk of thromboembolic events, supporting the role of exercise in hypertension management.

Nitric oxide activity in platelets of dengue haemorrhagic fever patients: the apparent paradoxical role of ADMA and l-NMMA.

Dengue haemorrhagic fever (DHF) is a prevalent acute disease that occurs in patients infected by an arbovirus in tropical and subtropical regions. We have previously shown increased intraplatelet nitric oxide (NO) production in patients with dengue fever associated with reduced platelet aggregation. In this study, l-arginine transport as well as expression and activity of nitric oxide synthase (NOS) isoforms in the presence or absence of l-arginine analogues were examined in 23 DHF patients. l-arginine transport and NOS activity in platelets were increased in patients with DHF compared with controls. However, platelet endothelial NOS (eNOS) and inducible (iNOS) protein levels did not differ between healthy controls and DHF patients. Endogenous or exogenous analogues did not inhibit platelet NOS activity from DHF patients. In contrast, endogenous l-arginine analogues [N(G)-monomethyl-l-arginine (l-NMMA) and asymmetric dimethylarginine (ADMA)] inhibited NOS activity in platelets from healthy subjects. These results show the first evidence that the intraplatelet l-arginine-NO pathway is activated in DHF patients. The lack of inhibition of NO formation in vitro by all l-arginine analogues tested in DHF platelets may suggest another mechanism by which NOS activity can be regulated.

Modulation of cytokines, resistin, and distribution of adipose tissue in C57BL/6 mice by different high-fat diets.

OBJECTIVE: To investigate whether changing the lipid source induces metabolic changes and/or modulates the adipose tissue distribution in mice fed with a high-fat (HF) diet. METHODS: C57BL/6 mice were subjected to a 10-wk control diet (10% fat) or an HF diet (60% fat) containing lard (HF-L), olive oil (HF-O), sunflower oil, or canola oil. Food intake and body weight were measured. At euthanasia, blood was collected and adipose tissue was dissected. Serum hormones and cytokines were determined. RESULTS: The plasma insulin levels were higher in the HF-L and HF-O groups than in the other three groups (P < 0.0001). The levels of resistin were highest in the HF-L and HF-O groups (P < 0.0001). Leptin expression was also highest in these two groups (P < 0.0001). Of the four groups, interleukin-6 was expressed at the highest level in the HF-L group (P < 0.0005), whereas adiponectin was expressed at the lowest level (P < 0.0001). The accumulation of subcutaneous and visceral adipose tissues was higher in the HF-L group compared with the other groups. This group was hypertrophic because of excess subcutaneous fat and epididymal fat in the adipocytes. However, the ratio of subcutaneous to visceral fat was significantly lower in the HF-L and HF-O groups compared with the other groups. CONCLUSION: In mice fed fat-rich diets, the level of adipokines, the distribution of adipose tissue, and the metabolism of carbohydrates are more significantly influenced by the lipid content rather than the absolute amount of lipid.

Characterization of the L-arginine-NO-cGMP pathway in spontaneously hypertensive rat platelets: the effects of pregnancy.

Nitric oxide (NO) is a short-lived intercellular messenger that provides an efficient vascular regulatory mechanism to support homeostasis and prevent thrombosis. Endothelial dysfunction and reduced NO bioavailability have a central role in hypertension associated with pregnancy. The purpose of this study was to investigate the impact of pregnancy on the L-arginine-NO-cGMP pathway in platelets and its correlation to platelet function and blood pressure in normotensive rats and spontaneously hypertensive rats (SHRs). Platelets were obtained from blood on the 20th day of pregnancy from female SHRs (SHR-P) and normotensive controls (P) or age-matched nonpregnant rats (SHR-NP and NP). Intraplatelet NO synthase (NOS) activity was reduced in P compared to NP, despite unchanged L-arginine influx. The expression levels of endothelial NOS (eNOS) and inducible NOS (iNOS) were diminished during pregnancy in normotensive rats. Paradoxically, cyclic guanosine monophosphate (cGMP) levels were similar between NP and P, as were phosphodiesterase type 5 (PDE5) expression and platelet aggregation induced by adenosine diphosphate. In SHRs, L-arginine influx was reduced in SHR-P compared to SHR-NP. SHR-P exhibited impaired NOS activity and reduced iNOS expression compared with SHR-NP. Soluble guanylyl cyclase and PDE5 expression in platelets were lower in SHR-P than in SHR-NP, whereas no differences were noted between groups with respect to cGMP levels. However, increased levels of cGMP were observed in SHR-P compared to normotensive groups and platelet aggregability remained unaltered. In conclusion, these observations prompted the hypothesis that normal platelet aggregation in pregnant SHRs may be related to a reduction in PDE5 expression and consequently the maintenance of cGMP levels, independently of reduced platelet NO bioavailability.

Depression and cardiovascular disease: role of nitric oxide.

Both depression and cardiovascular disease are major public health problems. Growing evidence shows that depression is a risk factor for the development of coronary artery disease (CAD). However, the exact mechanisms underlying the interplay between depression and CAD remain to be elucidated. Depression adversely affects autonomic and hormonal homeostasis, resulting in metabolic abnormalities, inflammation, increased platelet aggregation and endothelial dysfunction. All of these pathological features lead to atherothrombosis and cardiovascular events. However, there is no clear evidence that anti-depressant drugs or psychotherapy will reduce the risk or improve the outcome of CAD. Recent studies suggest that the L-arginine-nitric oxide (NO) pathway is involved in the genesis of depression. NO has many physiological functions, including vasodilatation, neurotransmission and platelet aggregation inhibition. It is synthesised from the cationic amino acid L-arginine by a family of enzymes: NO synthases (NOS). There are three NOS isoforms: inducible NOS (iNOS), endothelial NOS and neuronal NOS (nNOS). The availability and transport of L-arginine modulate rates of NO biosynthesis in circulating blood cells and vasculature, which provides a protective effect against cardiovascular disease. In depressive patients, the L-arginine-nitric oxide pathway seems to be impaired. The present review seeks a better understanding of the mechanisms that could identify depression as a cardiovascular risk factor and introduce new possible therapeutic interventions.

L-Arginine transport in disease.

The importance of membrane transport in normal physiological cell function is unquestionable. However, to what extent alterations in the transport of amino acids are the cause and/or consequence of pathological changes observed in disease states is a question not yet completely clarified. Kinetic experiments with blood cells provide a simple and useful model for researching alterations in amino acid transport. The cationic amino acid L-arginine is the precursor of nitric oxide (NO), a key second messenger involved in functions such as endothelium-dependent vascular relaxation, immune defence and platelet activation. The transport of L-arginine, being rate-limiting for nitric oxide production, is extremely relevant to pathological conditions where NO synthesis and/or actions are affected. The current review provides an overview of L-arginine transport in disease, specifically in uraemia, heart failure, hypertension, diabetes mellitus, septic shock and sickle cell disease.

Role of the NO-cGMP pathway in the systemic antinociceptive effect of clonidine in rats and mice.

The mechanism underlying the analgesic effect of clonidine, an alpha(2)-adrenoceptor agonist, remains uncertain. Activation of alpha(2)-adrenoceptor induces the release of nitric oxide (NO) from endothelial cells, which has led us to test the hypothesis that the observed antinociceptive effect induced by the systemic administration of clonidine depends on the NO-cGMP pathway. The possible involvement of an opioid link in the antinociceptive effect of clonidine was also evaluated. The antinociceptive effect induced by systemic administration (intravenous or intraperitoneal) of clonidine was evaluated using the rat paw formalin, mice tail-flick and writhing tests. Clonidine (3-120 microg/kg) induces a dose-dependent antinociceptive effect in the formalin, tail-flick and writhing tests. The antinociceptive effect of clonidine in a dose that had no sedative effect assessed by rota rod test, was significantly reduced by NO-synthase and guanylyl cyclase inhibition. The antinociceptive effect of morphine, but not clonidine, was inhibited by naloxone. Our current results suggest that the antinociceptive effect of systemic clonidine does not involve the opioid receptor and is modulated by the NO-cGMP pathway.

Time interval between pairs of arterial blood pressure measurements--does it matter?

Guidelines on arterial blood pressure (BP) measurements recommend a time interval between readings. There has been little evidence supporting this recommendation. Therefore, we measured the variation in arterial pressure between the pairs of measurements without an interval (n = 269) or with venous congestion interposed (n = 79). The variation in BP readings was not significantly greater whether a 60-sec interval or venous congestion were present. These overall results do not support the hypothesis that the absence of the time interval or of presence of venous congestion, as may occur in clinical practice, significantly affects BP measurements.

Insuficiência cardíaca crônica e óxido nítrico / Chronic heart failure and nitric oxide

O presente tabalho tem por objetivo realizar um levantamento do papel do óxido nítrico (NO) e de seu precursor L-arginina no contexto da síndrome de insuficiência cardíaca crônica