RESUMO
OBJECTIVE: Cardiovascular mortality increases after menopause in women. Nitric oxide is essential for proper platelet function inhibiting its aggregation and maintaining vascular haemostasis. Here, we investigated whether platelet function and intraplatelet l-arginine-nitric oxide pathway are impaired in postmenopausal women. STUDY DESIGN: Cross-sectional. MAIN OUTCOMES MEASURES: Blood was collected from 16 premenopausal and 12 postmenopausal women without any additional risk factor for cardiovascular disease. Platelet reactivity was measured by light transmission aggregometry. l-Arginine-nitric oxide pathway was assessed measuring transmembrane l-[(3)H]-arginine transport, nitric oxide synthase activity by the citrulline assay, and arginase activity by the conversion of l-[(14)C]arginine to l-[(14)C]-urea. The activity of antioxidant enzymes was measured by spectrophotometric assays. Protein expression was determined by Western blotting. RESULTS: Platelet aggregation was increased in postmenopausal compared to premenopausal women. Postmenopausal women demonstrated reduced plasma levels of l-arginine, a lower nitric oxide synthase activity, similar endothelial and inducible nitric oxide synthase expression, and a compensatory increase in l-arginine transmembrane transport. Arginase expression and activity did not differ between groups. In regard to oxidative stress, no differences between groups were observed NAPDH oxidase subunits expression and protein carbonylation. However, the activity of the antioxidant enzyme superoxide dismutase and catalase protein levels in platelets were higher in postmenopausal women. CONCLUSION: Postmenopausal women present increased platelet reactivity, which may be due to a reduction in intraplatelet nitric oxide synthesis. Platelet hyperaggregability is known to be associated with arterial and venous thromboembolic event; therefore, it may contribute to the heightened risk of cardiovascular adverse events in this population.
Assuntos
Plaquetas/metabolismo , Pós-Menopausa , Adulto , Arginase/metabolismo , Arginina/metabolismo , Brasil , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Pessoa de Meia-Idade , Óxido Nítrico Sintase/metabolismo , Agregação Plaquetária , Saúde da Mulher , Adulto JovemRESUMO
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.
Assuntos
Falência Renal Crônica/sangue , Neutrófilos/metabolismo , Óxido Nítrico/sangue , Ativação Plaquetária , Adulto , Arginina/sangue , Feminino , Regulação da Expressão Gênica , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/metabolismo , Agregação Plaquetária , Diálise RenalRESUMO
BACKGROUND: Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO generated by the thermolysis of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course. METHODS: Chromatographic, mass spectroscopy, and UV-visible spectrometric techniques were used to study the oxidation of melatonin by ROO or horseradish peroxidase (HRP)/H2O2. Our focus was the characterization of products and the study of features of the reaction. RESULTS: We found that N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H2O2 as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG. CONCLUSIONS: These results show, for the first time, that melatonin radical is able to oxidize GSH. GENERAL SIGNIFICANCE: We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.