Platelet redox balance in diabetic patients with hypertension improved by n-3 fatty acids.

OBJECTIVE: Patients with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease, largely as a result of defective production of cardioprotective nitric oxide and a concomitant rise in oxidative stress. Dietary interventions that could reverse this trend would be extremely beneficial. Here we investigated whether dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation positively affected platelet nitroso-redox imbalance. RESEARCH DESIGN AND METHODS: We randomized hypertensive T2DM patients (T2DM HT; n = 22) and age-and-sex matched hypertensive study participants without diabetes (HT alone; n = 23) in a double-blind, crossover fashion to receive 8 weeks of n-3 PUFAs (1.8 g eicosapentaenoic acid and 1.5 g docosahexaenoic acid) or identical olive oil capsules (placebo), with an intervening 8-week washout period. Platelet nitrite and superoxide were measured and compared before and after treatment; 8-isoprostane was determined by ELISA and subcellular compartmentalization of the NAD(P)H oxidase subunit p47-phox examined by Western blotting. RESULTS: The n-3 PUFA supplementation reduced 8-isoprostane and superoxide levels in platelets from T2DM HT, but not HT alone, participants, without effect on nitrite production. This coincided with a significant decrease in p47-phox membrane localization and a similar reduction in superoxide to that achieved with apocynin. At baseline, a subcohort of T2DM HT and HT alone participants showed evidence of nitric oxide synthase (NOS)-derived superoxide production, indicating defective enzymatic activity. This was reversed significantly in T2DM HT participants after treatment, demonstrating improved NOS function. CONCLUSIONS: Our finding that n-3 PUFAs diminish platelet superoxide production in T2DM HT patients in vivo suggests a therapeutic role for these agents in reducing the vascular-derived oxidative stress associated with diabetes.

Docosahexaenoic acid improves the nitroso-redox balance and reduces VEGF-mediated angiogenic signaling in microvascular endothelial cells.

PURPOSE: Disturbances to the cellular production of nitric oxide (NO) and superoxide (O(2)(-)) can have deleterious effects on retinal vascular integrity and angiogenic signaling. Dietary agents that could modulate the production of these signaling molecules from their likely enzymatic sources, endothelial nitric oxide synthase (eNOS) and NADPH oxidase, would therefore have a major beneficial effect on retinal vascular disease. The effect of ω-3 polyunsaturated fatty acids (PUFAs) on angiogenic signaling and NO/superoxide production in retinal microvascular endothelial cells (RMECs) was investigated. METHODS: Primary RMECs were treated with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 48 hours. RMEC migration was determined by scratch-wound assay, proliferation by the incorporation of BrdU, and angiogenic sprouting using a three-dimensional model of in vitro angiogenesis. NO production was quantified by Griess assay, and phospho-eNOS accumulation and superoxide were measured using the fluorescent probe dihydroethidine. eNOS localization to caveolin-rich microdomains was determined by Western blot analysis after subfractionation on a linear sucrose gradient. RESULTS: DHA treatment increased nitrite and decreased superoxide production, which correlated with the displacement of eNOS from caveolar subdomains and colocalization with the negative regulator caveolin-1. In addition, both ω-3 PUFAs demonstrated reduced responsiveness to VEGF-stimulated superoxide and nitrite release and significantly impaired endothelial wound healing, proliferation, and angiogenic sprout formation. CONCLUSIONS: DHA improves NO bioavailability, decreases O(2)(-) production, and blunts VEGF-mediated angiogenic signaling. These findings suggest a role for ω-3 PUFAs, particularly DHA, in maintaining vascular integrity while reducing pathologic retinal neovascularization.

Reduced nitro-oxidative stress and neural cell death suggests a protective role for microglial cells in TNFalpha-/- mice in ischemic retinopathy.

PURPOSE: Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFalpha and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect. METHODS: Wild-type C57/BL6 (WT) and TNFalpha(-/-) mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7-12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining. RESULTS: The TNFalpha(-/-) animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity. CONCLUSIONS: Collectively, the results suggest that improved vascular recovery in the absence of TNFalpha is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Statins have beneficial effects on platelet free radical activity and intracellular distribution of GTPases in hyperlipidaemia.

In addition to lowering cholesterol, statins may alter endothelial release of the vasodilator NO and harmful superoxide free radicals. Statins also reduce cholesterol intermediates including isoprenoids. These are important for post-translational modification of substances including the GTPases Rho and Rac. By altering the membrane association of these molecules, statins affect intracellular positioning and hence activity of a multitude of substances. These include eNOS(endothelial NO synthase), which produces NO (inhibited by Rho), and NADPH oxidase, which produces superoxide (dependent on Rac). Statins may improve endothelial function by enhancing production of NO while decreasing superoxide production. A total of 40 hypercholesterolaemic patients were randomized to treatment with either atorvastatin or placebo; 20 normolipidaemic patients were also studied. Platelet nitrite, NO and superoxide were examined as was the cellular distribution of the GTPases Rho and Rac at baseline and after 8 weeks of treatment.Following atorvastatin therapy, platelet NO was increased (3.2 pmol/10(8) platelets) and superoxide output was attenuated [-3.4 pmol min(-1) (10(8) platelets)(-1)] when compared with placebo. The detection of both Rho and Rac was significantly reduced in the membranes of platelets, implying reduced activity. In conclusion, the results of the present study show altered NO/superoxide production following statin therapy. A potential mechanism for this is the change in the distribution of intracellular GTPases, which was considered to be secondary to decreases in isoprenoid intermediates, suggesting that the activity of the former had been affected by atorvastatin.

The pleiotropic effects of simvastatin on retinal microvascular endothelium has important implications for ischaemic retinopathies.

BACKGROUND: Current guidelines encourage the use of statins to reduce the risk of cardiovascular disease in diabetic patients; however the impact of these drugs on diabetic retinopathy is not well defined. Moreover, pleiotropic effects of statins on the highly specialised retinal microvascular endothelium remain largely unknown. The objective of this study was to investigate the effects of clinically relevant concentrations of simvastatin on retinal endothelium in vitro and in vivo. METHODS AND FINDINGS: Retinal microvascular endothelial cells (RMECs) were treated with 0.01-10 microM simvastatin and a biphasic dose-related response was observed. Low concentrations enhanced microvascular repair with 0.1 microM simvastatin significantly increasing proliferation (p<0.05), and 0.01 microM simvastatin significantly promoting migration (p<0.05), sprouting (p<0.001), and tubulogenesis (p<0.001). High concentration of simvastatin (10 microM) had the opposite effect, significantly inhibiting proliferation (p<0.01), migration (p<0.01), sprouting (p<0.001), and tubulogenesis (p<0.05). Furthermore, simvastatin concentrations higher than 1 microM induced cell death. The mouse model of oxygen-induced retinopathy was used to investigate the possible effects of simvastatin treatment on ischaemic retinopathy. Low dose simvastatin (0.2 mg/Kg) promoted retinal microvascular repair in response to ischaemia by promoting intra-retinal re-vascularisation (p<0.01). By contrast, high dose simvastatin(20 mg/Kg) significantly prevented re-vascularisation (p<0.01) and concomitantly increased pathological neovascularisation (p<0.01). We also demonstrated that the pro-vascular repair mechanism of simvastatin involves VEGF stimulation, Akt phosphorylation, and nitric oxide production; and the anti-vascular repair mechanism is driven by marked intracellular cholesterol depletion and related disorganisation of key intracellular structures. CONCLUSIONS: A beneficial effect of low-dose simvastatin on ischaemic retinopathy is linked to angiogenic repair reducing ischaemia, thereby preventing pathological neovascularisation. High-dose simvastatin may be harmful by inhibiting reparative processes and inducing premature death of retinal microvascular endothelium which increases ischaemia-induced neovascular pathology. Statin dosage should be judiciously monitored in patients who are diabetic or are at risk of developing other forms of proliferative retinopathy.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

BACKGROUND: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD's actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). AIM/METHOD: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. RESULTS: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O(2-) anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. CONCLUSION: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.

Influence of atenolol and nifedipine on nitric-oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency.

BACKGROUND: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. AIMS: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. METHODS: NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). RESULTS: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-alpha-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. CONCLUSION: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Microcirculatory hemodynamics and endothelial dysfunction in systemic lupus erythematosus.

OBJECTIVE: Impaired flow-mediated dilation (FMD) occurs in disease states associated with atherosclerosis, including SLE. The primary hemodynamic determinant of FMD is wall shear stress, which is critically dependent on the forearm microcirculation. We explored the relationship between FMD, diastolic shear stress (DSS), and the forearm microcirculation in 32 patients with SLE and 19 controls. METHODS AND RESULTS: DSS was calculated using (mean diastolic velocity x 8 x blood viscosity)/baseline brachial artery diameter. Doppler velocity envelopes from the first 15 seconds of reactive hyperemia were analyzed for resistive index (RI), and interrogated in the frequency domain to assess forearm microvascular hemodynamics. FMD was significantly impaired in SLE patients (median, 2.4%; range, -2.1% to 10.7% versus median 5.8%; range, 1.9% to 14%; P<0.001). DSS (dyne/cm2) was significantly reduced in SLE patients (median, 18.5; range, 3.9 to 34.0 versus median 21.8; range, 14.1 to 58.7; P=0.037). A strong correlation between FMD and DSS, r(s)=0.65, P=0.01 was found. Postischemic RI was not significantly different between the 2 groups; however, there were significant differences in the power-frequency spectrums of the Doppler velocity envelopes (P<0.05). CONCLUSIONS: These data suggest that in SLE, altered structure and function of the forearm microcirculation contributes to impaired FMD through a reduction in shear stress stimulus.

Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension.

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

NAD(P)H-dependent superoxide production in platelets: the role of angiotensin II and protein kinase C.

OBJECTIVES: Vascular NAD(P)H oxidase represents a major source for excessive superoxide production in hypertension. Angiotensin II (AngII) can activate NAD(P)H oxidase via the angiotensin II type 1 (AT1) receptor and protein kinase C (PKC). Platelets possess AT1 receptors and all the components of the NAD(P)H oxidase system. We employed this tissue model to explore mechanisms involved in AngII-mediated superoxide production. DESIGN AND METHODS: Platelet suspensions from hypertensive patients' blood were activated with AngII or phorbol 12-myristate 13-acetate (PMA). Inhibitors of NAD(P)H oxidase, PKC, and the AT1 receptor were employed to study their effects on superoxide production. RESULTS: Superoxide production was stimulated by AngII and PMA and attenuated by AT1 receptor antagonists (mean percentage reduction 80.2%, P<0.01) and inhibitors of PKC (mean reduction 94.8%, P<0.001) and NAD(P)H oxidase (mean reduction 100%, P< 0.001). CONCLUSIONS: AngII stimulates platelet superoxide production through activation of vascular NAD(P)H oxidase via the AT1 receptor and PKC.

Increased superoxide production in hypertensive patients with diabetes mellitus: role of nitric oxide synthase.

BACKGROUND: Hypertension and diabetes are important independent risk factors for increased oxidative stress and increased cardiovascular risk. The combination of hypertension and diabetes results in a dramatic increase in cardiovascular risk. Enhanced oxidative stress in hypertension and diabetes is linked to decreased nitric oxide (NO) bioavailability because of its interaction with vascular superoxide (O(2)(*-)), derived predominantly from NAD(P)H-dependent oxidases. When uncoupled from essential cofactors, NO synthase III (NOS III) can also produce O(2)(*-). We studied platelet superoxide production in patients with hypertension alone and in patients with coexistent diabetes mellitus, investigating the contribution of NOS III uncoupling to platelet superoxide production. METHODS AND RESULTS: Gel-filtered platelets were obtained and were stimulated with Phorbol 12-myristate 13-acetate, and O(2)(*-) production was detected using lucigenin-enhanced chemiluminescence. Superoxide production was significantly higher in patients with diabetes and hypertension (6.4 +/- 1.6 pmol/min/10(8) platelets) than in patients with hypertension (1.6 +/- 0.6 pmol/min/10(8) platelets) (P < .04). After incorporation of N(omega)-nitro-l-arginine methyl ester (L-NAME, 1 mmol/L), O(2)(*-) detection increased in 40% of patients with diabetes and hypertension and in 87% of patients with hypertension. This expected response results from L-NAME inhibition of NO production preventing NO scavenging of O(2)(*-). A reduction in O(2)(*-) production in response to L-NAME occurred in the remaining patients and indicates O(2)(*-) production by the uncoupled NOS III enzyme. CONCLUSIONS: This study provides first published evidence that NOS III can reside in the uncoupled state in patients with hypertension and, to a greater extent, in patients with coexisting hypertension and diabetes, and that it contributes significantly to increased superoxide production in these disease states.

Functional consequences of endothelial nitric oxide synthase uncoupling in congestive cardiac failure.

BACKGROUND: Impaired endothelium-mediated vasodilatation (EMVD) in congestive cardiac failure (CCF) has been linked to decreased nitric oxide (NO) bioavailability because of its interaction with vascular superoxide (O2*-), derived predominantly from NAD(P)H-dependent oxidases. When uncoupled from essential cofactors, endothelial nitric oxide synthase (eNOS) produces O2*-. We studied the functional consequences of eNOS uncoupling in relation to EMVD in patients with CCF. METHODS AND RESULTS: We employed the platelet as a compartmentalized ex-vivo model to examine O2*- and NO production. When eNOS is functioning normally, incorporation of Nomega-Nitro-L-Arginine methyl ester (L-NAME, 1 mmol/L), results in increased O2*- detection, as inhibition of NO production prevents NO scavenging of O2*-. This was observed in controls and 9 of the CCF patients, in whom O2*- detection increased by 63% and 101%, respectively. In the remaining 9 CCF patients, incorporation of L-NAME reduced O2*- production by 39%, indicating O2*- production by eNOS uncoupling. Detection of platelet-derived NO was significantly greater in eNOS-coupled platelets compared with the uncoupled group (2.8+/-1.4 versus 0.9+/-0.4 pmol/108 platelets, P=0.04). Endothelium-dependent and -independent vasodilator responses to acetylcholine and sodium nitroprusside recorded using venous occlusion plethysmography were significantly impaired in patients exhibiting eNOS uncoupling. CONCLUSIONS: This study provides first evidence that platelet eNOS can become uncoupled in human CCF. Impaired endothelium-dependent and -independent vasodilator responses and diminished platelet-derived NO production occurred in association with enzyme uncoupling.

Platelet nitric oxide and superoxide release during the development of nitrate tolerance: effect of supplemental ascorbate.

BACKGROUND: The therapeutic benefits that accompany the continuous administration of organic nitrates are attenuated by the development of tolerance to the compounds. Altered superoxide production and NO bioavailability have been implicated in contributing to the development of tolerance, an effect that may be ameliorated by the administration of antioxidants. METHODS AND RESULTS: We studied the effect of 3 days of continuous transdermal administration of nitroglycerin (NTG) (10 mg/24 hours) on platelet free radical (NO and superoxide anion [O2*-] activity) with and without coadministration of supplemental ascorbate (2.4 g/24 hours). NAD(P)H oxidase activity, nitric oxide synthase (NOS) activity, and cyclic guanosine monophosphate (cGMP) content were also assessed. Radial artery pressure pulse waveforms were used to track the hemodynamic actions of NTG. Three days of NTG/placebo was associated with a significant increase in platelet NO and O2*- production from 1.0+/-1.17 to 2.52+/-0.88 pmol/10(8) platelets and 13.2+/-4.8 to 72.5+/-34.4 pmol/10(8) platelets, respectively (P<0.01 for both). These changes were accompanied by increased platelet NADH oxidase activity from 47.9+/-11.0 to 65.3+/-13.6 pmol O2*- min/mg protein and cGMP content from 0.60+/-0.10 to 0.89+/-0.16 pmol/10(9) platelets (P<0.05 for both). Administration of NTG/ascorbate attenuated both NO and O2*- release in platelets. CONCLUSIONS: Three days of continuous transdermal administration of NTG was accompanied by increased platelet NO and O2*- production and NADH oxidase activity that was suppressed by coadministration of oral ascorbate. Although a significant degree of tolerance would be expected during continuous nitrate administration, a residual hemodynamic action could be identified by arterial pulse contour analysis.