Tatiana M.C. Brunini

Diminished L-arginine bioavailability in hypertension

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175+/-19 micromol/l) compared with control subjects (137+/-8 micromol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60+/-7 micromol.l(-1).cells(-1).h(-1); n=16) compared with controls (90+/-17 micromol.l(-1).cells(-1).h(-1); n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86+/-17 pmol.10(9) cells(-1).min(-1) in controls compared with 36+/-9 pmol.10(9) cells(-1).min(-1) in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12+/-0.02 pmol/10(8) cells; n=8) compared with controls (0.22+/-0.01 pmol/10(8) cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.

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.

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-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-[3H]-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.

Oxidative stress, l-arginine-nitric oxide and arginase pathways in platelets from adolescents with anorexia nervosa.

Anorexia nervosa (AN) is associated with high cardiovascular mortality. Nitric oxide (NO) inhibits platelet function and regulates the cardiovascular homeostasis. The aim of this study was to investigate the l-arginine-NO-GMPc and arginase pathways and oxidative stress in platelets from patients with AN. Intraplatelet l-arginine transport, NOS expression and activity, cGMP levels, platelet aggregation, arginase expression and oxidative stress were measured in adolescent patients with AN (n=11) and healthy controls (n=12). Plasma l-arginine levels were significantly reduced in AN. l-arginine transport, NOS activity and cGMP basal levels were reduced in platelets associated with unchanged platelet aggregability. The expression of NOS isoforms was not affected. TBARS production was diminished, while the activity of superoxide dismutase was elevated in AN patients. There was an overexpression of arginase II in AN. Alterations of l-arginine-NO-GMPc and arginase pathways in platelets can be early predictors of the incidence of cardiovascular disease into adult life in AN.

INHIBITORY EFFECTS OF ENDOGENOUS l-ARGININE ANALOGUES ON NITRIC OXIDE SYNTHESIS IN PLATELETS: ROLE IN PLATELET HYPERAGGREGABILITY IN HYPERTENSION

1 An increase in plasma concentrations of endogenous l‐arginine analogues, which are inhibitors of nitric oxide (NO) synthesis, may be involved in platelet activation and the increased risk of thrombosis in essential hypertension. Nitric oxide is synthesised in platelets from the amino acid l‐arginine by inducible and constitutive isoforms of NO synthase (NOS), which leads to increased levels of cGMP. 2 In the present study, we investigated basal intraplatelet cGMP levels, platelet aggregation and pro‐inflammatory biomarkers in hypertensive patients. The effects of endogenous (NG‐monomethyl‐l‐arginine (l‐NMMA) and asymmetric dimethylarginine (ADMA); both at 1 mmol/L) and exogenous (aminoguanidine and NG‐nitro‐l‐arginine; both at 1 mmol/L) l‐arginine analogues and the neutral amino acid l‐leucine (1 mmol/L) in inhibiting NOS activity in platelets were also investigated. 3 Twelve healthy controls and 18 hypertensive patients participated in the study. Platelet aggregation induced by collagen was increased in hypertensive patients (95 ± 5%) compared with controls (72 ± 5%). Basal NOS activity and intraplatelet cGMP levels were reduced in hypertensive platelets. Moreover, ADMA, l‐NMMA and l‐leucine were effective inhibitors of NO synthesis in both hypertensive and control platelets. Essential hypertension led to an inflammatory response, with increased plasma concentrations of fibrinogen, C‐reactive protein and cytokines. 4 These findings provide evidence that, in essential arterial hypertension, the enhanced plasma levels of endogenous l‐arginine analogues ADMA and l‐NMMA, potent inhibitors of l‐arginine transport and NO synthesis in platelets, may play a role in increased platelet aggregation via a cGMP‐dependent mechanism.

DENGUE FEVER ACTIVATES THE l-ARGININE–NITRIC OXIDE PATHWAY: AN EXPLANATION FOR REDUCED AGGREGATION OF HUMAN PLATELETS

1 In patients with Dengue fever, a viral inflammatory syndrome, haemorrhage is a significant pathological feature, yet the underlying mechanisms remain unclear. Nitric oxide (NO) is an important regulator of platelet function, inhibiting aggregation, recruitment and adhesion to the vascular endothelium. 2 We have investigated whether changes in the activity of the l‐arginine–NO pathway in human platelets may account for increased bleeding in patients with Dengue fever. A total of 16 patients with Dengue fever and 18 age‐matched healthy volunteers participated in the study. 3 Collagen induced platelet aggregation in a dose‐dependent manner in both Dengue patients and controls, but the degree of platelet aggregation was significantly reduced in the patient group. Elevated rates of l‐arginine transport in Dengue fever patients were associated with enhanced NO synthase activity and elevated plasma fibrinogen levels. 4 The present study provides the first evidence that Dengue fever is associated with increased l‐arginine transport and NO generation and reduced platelet aggregation.

Nitric Oxide, Malnutrition and Chronic Renal Failure

The conditionally essential amino acid L-arginine is the substrate for nitric oxide (NO) synthesis, a key second messenger involved in physiological functions including endothelium-dependent vascular relaxation and inhibition of platelet adhesion and aggregation. Extracellular L-arginine transport seems to be essential for the production of NO by the action of NO synthases (NOS), even when the intracellular levels of L-arginine are available in excess (L-arginine paradox). Chronic renal failure (CRF) is a complex clinical condition associated with accelerated atherosclerosis and thrombosis leading to cardiovascular events. Various studies document that markers of malnutrition and inflammation, such as low body mass index (BMI), C-reactive protein (CRP) and interleukin-6 (IL-6), are strong independent predictors of cardiovascular mortality in patients with end-stage renal disease (ESRD). There is considerable literature demonstrating that a disturbance in the nitric oxide control mechanism plays a role in mediating the haemodynamic and haemostatic disorders present in CRF. Endogenous analogues of L-arginine, ADMA and L-NMMA, which can inhibit NO synthesis and L-arginine transport, are increased whilst L-arginine is reduced in plasma from all stages of CRF patients. In this context, the uptake of L-arginine in blood cells is increased in undialysed CRF patients and in patients treated by CAPD and haemodialysis. In platelets obtained from haemodialysis patients, the activation of L-arginine transport and NO production was limited to well-nourished patients. Impairment in nitric oxide bioactivity, coupled with malnutrition and inflammation, may contribute to increased incidence of atherothrombotic events in CRF. This article summarizes the current knowledge of L-arginine-nitric oxide pathway and malnutrition in CRF and briefly describes possible therapeutic interventions.

Uremia, Atherothrombosis and Malnutrition: The Role of L-arginine- Nitric Oxide Pathway

The uraemic syndrome is a complex condition that results from an accumulation of multiple waste compounds, combined with failure of the endocrine and homeostatic functions of the kidney in end-stage chronic renal failure (CRF) patients. Recently it has become clear that uraemia is a microinflammatory condition with a significant increase in inflammation markers. Malnutrition is a common pathological condition which exacerbates cardiovascular mortality in uraemic patients. Inadequate diet and a state of persistent catabolism play major roles in uraemic malnutrition, yet the underlying mechanisms have not been completely clarified. Malnourished patients present elevated levels of circulating cytokines, further aggravating the oxidative and inflammatory characteristics of uraemia. It has been suggested that abnormalities in nitric oxide bioactivity, coupled with malnutrition and inflammation, may contribute to increased incidence of atherothrombotic events in uraemia. Amongst the earliest indications of nutritional deficiency are low concentrations of plasma amino acids, including L-arginine, the precursor for nitric oxide (NO) synthesis. Atherosclerosis is an inflammatory disorder and NO is an important mediator of inflammation. There is a close association between thrombosis and platelet aggregation, and NO is involved in all stages of platelet activation. L-arginine inhibits platelet aggregation both in vitro and in vivo, while L-NMMA (NG-monomethyl-L-arginine), an endogenous L-arginine analogue and inhibitor of NO synthase (NOS), increases platelet activation and adhesion. The majority of studies in animal models and human patients indicate that the systemic production of NO is increased in uraemia. CRF patients show reduced plasma concentration of L-arginine, and the enhancement of L-arginine transport is essential to maintain increased NO synthesis in platelets taken from these patients. The present review provides an overview of recent advances in the understanding of the association among malnutrition, chronic inflammation and the L-arginine-nitric oxide pathway in uraemic patients, and related potential interventions that could improve clinical outcome in chronic renal failure.

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.