Preliminary study of hypoxia-related cardiovascular mediator-markers in patients with end-stage renal disease with and without diabetes and the effects of haemodialysis.

BACKGROUND: Evidence points to activation of pro-inflammatory and pro-thrombotic stimuli during the haemodialysis process in end-stage renal disease (ESRD) with potential to predispose to cardiovascular events. Diabetes is associated with a higher incidence of cardiovascular disease in haemodialysis patients. We tested the hypothesis that a range of mediators and markers that modulate cardiovascular risk are elevated in haemodialysis patients with diabetes compared to those without. METHODS: Men and women with diabetes (n = 6) and without diabetes (n = 6) aged 18-90 years receiving haemodialysis were recruited. Blood samples were collected and analysed pre- and post-haemodialysis sessions for (platelet-monocyte conjugates (PMC), oxidised LDL (Ox-LDL), endothelin 1 (ET-1) and vascular endothelial growth factor (VEGF-A). RESULTS: PMC levels significantly increased after haemodialysis in both groups (diabetes p = 0.047; non-diabetes p = 0.005). Baseline VEGF-A was significantly higher in people with diabetes (p = 0.009) and post-dialysis levels were significantly reduced in both groups (P = 0.002). Ox-LDL and CRP concentrations were not significantly different between groups nor affected in either group post-dialysis. Similarly, ET-1 concentrations were comparable in all patients at baseline, with no change post-dialysis in either group. CONCLUSIONS: In this pilot study, we have confirmed that circulating PMCs are increased following dialysis irrespective of diabetes status. This is likely to be a mechanistic process and offers a potential explanation for high rates of vascular events associated with haemodialysis. The higher VEGF-A concentrations between patients with and without diabetes is a previously unreported finding in diabetic ESRD. Further research is merited to establish whether VEGF-A is a marker or mediator (or both) of cardiovascular risk in haemodialysis.

A randomized crossover study to assess the effect of an oat-rich diet on glycaemic control, plasma lipids and postprandial glycaemia, inflammation and oxidative stress in Type 2 diabetes.

AIMS: In the UK, lifestyle intervention is first-line management in Type 2 diabetes. It is unclear what type of diet is most efficacious for improving glycaemic control. This study investigated the effects of an oat-enriched diet on glycaemic control, postprandial glycaemia, inflammation and oxidative stress compared with standard dietary advice. METHODS: In a randomized crossover design, 27 volunteers with Type 2 diabetes, managed on diet and lifestyle only, were observed for two consecutive 8-week periods following either the oat-enriched diet or re-enforced standard dietary advice. Volunteers attended at baseline (habitual intake) and 8 and 16 weeks. Measurements included basic clinical measurements and fasted and postprandial (3-h) glucose and insulin in response to a healthy test meal. Markers of inflammation and oxidative stress, including high-sensitivity C-reactive protein, interleukin 6, interleukin 18, tumour necrosis factor-alpha, adiponectin, thiobarbituric acid reactive substances, oxygen radical antioxidant capacity, oxidized LDL and urinary isoprostanes, were also measured at fasting and in the postprandial period. RESULTS: There were no diet-related effects on glycaemic control or glycaemic or insulinaemic responses to the test meal. Total cholesterol (5.1 ± 1.0 vs. 4.9 ± 0.8 mmol/l, P = 0.019) concentrations declined following the oat-enriched diet compared with standard dietary advice. There was a postprandial decline in adiponectin concentration (P = 0.009), but no effect of dietary intervention. None of the measures of oxidative stress or inflammation were altered by the oat-enriched diet compared with standard dietary advice. CONCLUSION: The oat-enriched diet had a modest impact on lipid lowering, but did not impact on oxidative stress or inflammation in these volunteers with Type 2 diabetes.

N-Acetylcysteine inhibits platelet-monocyte conjugation in patients with type 2 diabetes with depleted intraplatelet glutathione: a randomised controlled trial.

AIMS/HYPOTHESIS: The aim of this study was to determine whether oral dosing with N-acetylcysteine (NAC) increases intraplatelet levels of the antioxidant, glutathione (GSH), and reduces platelet-monocyte conjugation in blood from patients with type 2 diabetes. METHODS: In this placebo-controlled randomised crossover study, the effect of oral NAC dosing on platelet-monocyte conjugation and intraplatelet GSH was investigated in patients with type 2 diabetes (eligibility criteria: men or post-menopausal women with well-controlled diabetes (HbA(1c) < 10%), not on aspirin or statins). Patients (n = 14; age range 43-79 years, HbA(1c) = 6.9 ± 0.9% [52.3 ± 10.3 mmol/mol]) visited the Highland Clinical Research Facility, Inverness, UK on day 0 and day 7 for each arm of the study. Blood was sampled before and 2 h after oral administration of placebo or NAC (1,200 mg) on day 0 and day 7. Patients received placebo or NAC capsules for once-daily dosing on the intervening days. The order of administration of NAC and placebo was allocated by a central office and all patients and research staff involved in the study were blinded to the allocation until after the study was complete and the data fully analysed. The primary outcome for the study was platelet-monocyte conjugation. RESULTS: Oral NAC reduced platelet-monocyte conjugation (from 53.1 ± 4.5% to 42.5 ± 3.9%) at 2 h after administration and the effect was maintained after 7 days of dosing. Intraplatelet GSH was raised in individuals with depleted GSH and there was a negative correlation between baseline intraplatelet GSH and platelet-monocyte conjugation. There were no adverse events. CONCLUSIONS/INTERPRETATION: The NAC-induced normalisation of intraplatelet GSH, coupled with a reduction in platelet-monocyte conjugation, suggests that NAC might help to reduce atherothrombotic risk in type 2 diabetes. FUNDING: Chief Scientist Office (CZB/4/622), Scottish Funding Council, Highlands & Islands Enterprise and European Regional Development Fund. TRIAL REGISTRATION: isrctn.org ISRCTN89304265.

Diabetic fatty liver disease is associated with specific changes in blood-borne markers.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) can lead to cirrhosis and hepatocellular carcinoma and is strongly associated with obesity and insulin resistance. The aim of this study was to assess if plasma markers associated with NAFLD are increased in people with concomitant diabetes compared with those without. METHODS: A total of 68 participants were recruited from diabetes and liver clinics. Fatty liver disease was indicated by routine blood tests and ultrasonography. Forty-seven participants had type 2 diabetes; of them, 18 had no fatty liver disease as defined previously (DNoFLD) and 29 had fatty liver disease (DFLD); the remaining 21 had fatty liver disease but no diabetes (NonDFLD). Serum samples were analyzed for adiponectin (APN), alanine and aspartate aminotransferases and plasma for cholesterol, triglyceride, hyaluronic acid (HA), procollagen peptide III, alkaline phosphatase and fibrinogen. RESULTS: Hyaluronic acid and procollagen peptide III were significantly higher and adiponectin significantly lower in DFLD than NonDFLD and DNoFLD, the difference being particularly marked for hyaluronic acid and APN. There was no difference in these markers between NonDFLD and DNoFLD and no association between any plasma or serum marker and ultrasound grade of steatosis. CONCLUSION: We have identified markers of hepatic steatosis that appear to be specific for people with type 2 diabetes. A further longitudinal study is merited to assess the role of these markers in understanding the progression of hepatic steatosis and fibrosis in people with and without diabetes.

NiO and Co3O4 nanoparticles induce lung DTH-like responses and alveolar lipoproteinosis.

Lung exposure to metal oxide nanoparticles (NPs) comprising soluble metal haptens may produce T-helper cell type 1 (Th1)- and Th17-associated delayed-type hypersensitivity (DTH) responses and pulmonary alveolar proteinosis (PAP). In order to study this, haptenic metal oxide NPs (NiO, Co(3)O(4), Cr(2)O(3) and CuO) were instilled into the lungs of female Wistar rats, and the immunoinflammatory responses were assessed at 24 h and 4 weeks post-instillation. Primary culture of alveolar macrophages from Wistar rats was used to evaluate the effect of the NPs on the ability to clear surfactant. NiO NPs induced chronic interstitial inflammation and pro-inflammatory Th1 and Th17 immune responses characterised by increases in the cytokines monocyte chemotactic protein (MCP)-1/CCL2, interleukin (IL)-12 p40, interferon-γ and IL-17A, whilst similar pathological responses induced by Co(3)O(4) NPs were associated with increases in MCP-1/CCL2 and IL-12 p40. However, neither Cr(2)O(3) nor CuO NPs elicited immunoinflammatory reactions. PAP was induced by both NiO and Co(3)O(4) NPs during the chronic phase. PAP was associated with over-production of surfactant by proliferation of type II cells and impaired clearance of surfactant by macrophages. These findings have implications for the risk management of occupational NP exposure and provide evidence that haptenic metal oxide NPs can induce chronic progressive lung immune responses via a DTH-like mechanism.

Acute cardiovascular effects of apelin in humans: potential role in patients with chronic heart failure.

BACKGROUND: Apelin, the endogenous ligand for the novel G protein-coupled receptor APJ, has major cardiovascular effects in preclinical models. The study objectives were to establish the effects of acute apelin administration on peripheral, cardiac, and systemic hemodynamic variables in healthy volunteers and patients with heart failure. METHODS AND RESULTS: Eighteen patients with New York Heart Association class II to III chronic heart failure, 6 patients undergoing diagnostic coronary angiography, and 26 healthy volunteers participated in a series of randomized, double-blind, placebo-controlled studies. Measurements of forearm blood flow, coronary blood flow, left ventricular pressure, and cardiac output were made by venous occlusion plethysmography, Doppler flow wire and quantitative coronary angiography, pressure wire, and thoracic bioimpedance, respectively. Intrabrachial infusions of (Pyr(1))apelin-13, acetylcholine, and sodium nitroprusside caused forearm vasodilatation in patients and control subjects (all P<0.0001). Vasodilatation to acetylcholine (P=0.01) but not apelin (P=0.3) or sodium nitroprusside (P=0.9) was attenuated in patients with heart failure. Intracoronary bolus of apelin-36 increased coronary blood flow and the maximum rate of rise in left ventricular pressure and reduced peak and end-diastolic left ventricular pressures (all P<0.05). Systemic infusions of (Pyr(1))apelin-13 (30 to 300 nmol/min) increased cardiac index and lowered mean arterial pressure and peripheral vascular resistance in patients and healthy control subjects (all P<0.01) but increased heart rate only in control subjects (P<0.01). CONCLUSIONS: Acute apelin administration in humans causes peripheral and coronary vasodilatation and increases cardiac output. APJ agonism represents a novel potential therapeutic target for patients with heart failure.

Glutathione supplementation to University of Wisconsin solution causes endothelial dysfunction.

INTRODUCTION: Glutathione (GSH) is added to University of Wisconsin (UW) organ preservation solution to protect against oxidative stress. This study assesses the effect of GSH-supplementation on endothelial function in tissues subjected to cold ischaemia and compares its effects to a mono-ethyl ester equivalent (GSH-MEE) and S-nitrosated GSH (GSNO). METHODS: Rat aortic rings were stored for 1 h or 48 h in cold, hypoxic UW solution with or without GSH (3 mM), GSH-MEE (3 mM) or GSNO (100 mciroM) supplementation. Aortic rings were reoxygenated in warm Krebs solution; smooth muscle function was assessed by responses to phenylephrine (PE), and endothelial function by vasodilatation to the endothelium-dependent dilator, acetylcholine (ACh). The protective effects against oxidant-induced endothelial cell death were assessed in cultured human umbilical vein endothelial cells (HUVEC). RESULTS: Supplementation of UW with either GSH or GSH-MEE had no effect on vascular responses to PE, but smooth muscle contraction was significantly attenuated in rings incubated for 48 h with GSNO. Endothelium-dependent relaxation was significantly impaired in tissues stored under hypoxic conditions in GSH, GSH-MEE and GSNO supplemented UW solution for 1 h. However, impairment at 48 h was significantly more pronounced in GSH-treated vessels. Cultured HUVEC death was exacerbated by GSH and GSH-MEE in unstressed cells and in those stressed with a superoxide anion generator. CONCLUSIONS: GSH supplementation of UW solution exacerbates cold-ischaemia induced endothelial dysfunction. GSNO did not share the detrimental effects of GSH and promoted NO-mediated vasodilatation.

Topical application of acidified nitrite to the nail renders it antifungal and causes nitrosation of cysteine groups in the nail plate.

BACKGROUND: Topical treatment of nail diseases is hampered by the nail plate barrier, consisting of dense cross-linked keratin fibres held together by cysteine-rich proteins and disulphide bonds, which prevents penetration of antifungal agents to the focus of fungal infection. Acidified nitrite is an effective treatment for tinea pedis. It releases nitric oxide (NO) and other NO-related species. NO can react with thiol (-SH) groups to form nitrosothiols (-SNO). OBJECTIVES: To determine whether acidified nitrite can penetrate the nail barrier and cure onychomycosis, and to determine whether nitrosospecies can bind to the nail plate. METHODS: Nails were treated with a mixture of citric acid and sodium nitrite in a molar ratio of 0.54 at either low dose (0.75%/0.5%) or high dose (13.5%/9%). Immunohistochemistry, ultraviolet-visible absorbance spectroscopy and serial chemical reduction of nitrosospecies followed by chemiluminescent detection of NO were used to measure nitrosospecies. Acidified nitrite-treated nails and the nitrosothiols S-nitrosopenicillamine (SNAP) and S-nitrosoglutathione (GSNO) were added to Trichophyton rubrum and T. mentagrophytes cultures in liquid Sabouraud medium and growth measured 3 days later. Thirteen patients with positive mycological cultures for Trichophyton or Fusarium species were treated with topical acidified nitrite for 16 weeks. Repeat mycological examination was performed during this treatment time. RESULTS: S-nitrothiols were formed in the nail following a single treatment of low- or high-dose sodium nitrite and citric acid. Repeated exposure to high-dose acidified nitrite led to additional formation of N-nitrosated species. S-nitrosothiol formation caused the nail to become antifungal to T. rubrum and T. mentagrophytes. Antifungal activity was Cu(2+) sensitive. The nitrosothiols SNAP and GSNO were also found to be antifungal. Topical acidified nitrite treatment of patients with onychomycosis resulted in > 90% becoming culture negative for T. rubrum. CONCLUSIONS: Acidified nitrite cream results in the formation of S-nitrosocysteine throughout the treated nail. Acidified nitrite treatment makes a nail antifungal. S-nitrosothiols, formed by nitrosation of nail sulphur residues, are the active component. Acidified nitrite exploits the nature of the nail barrier and utilizes it as a means of delivery of NO/nitrosothiol-mediated antifungal activity. Thus the principal obstacle to therapy in the nail becomes an effective delivery mechanism.

Recent developments in nitric oxide donor drugs.

During the 1980s, the free radical, nitric oxide (NO), was discovered to be a crucial signalling molecule, with wide-ranging functions in the cardiovascular, nervous and immune systems. Aside from providing a credible explanation for the actions of organic nitrates and sodium nitroprusside that have long been used in the treatment of angina and hypertensive crises respectively, the discovery generated great hopes for new NO-based treatments for a wide variety of ailments. Decades later, however, we are still awaiting novel licensed agents in this arena, despite an enormous research effort to this end. This review explores some of the most promising recent advances in NO donor drug development and addresses the challenges associated with NO as a therapeutic agent.

Extracellular nitric oxide release mediates soluble guanylate cyclase-independent vasodilator action of spermine NONOate: comparison with other nitric oxide donors in isolated rat femoral arteries.

Nitric oxide (NO) and NO donors exhibit actions that are not entirely mediated by soluble guanylate cyclase (sGC). The site of NO release may influence the involvement of sGC-independent effects. Here we use spermine NONOate (SPER/NO) to release NO extracellularly, compared with other NO donors. Isolated rat femoral arteries were perfused luminally and perfusion pressure monitored. Vessels were contracted with phenylephrine (2-14 microM) in the presence of an NO synthase inhibitor (N(omega)-nitro-L-arginine methyl ester; 20 microM). Vasodilator responses to NO donors were assessed before and after perfusion of an sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ; 20 microM), NO scavengers (hemoglobin; Hb & hydroquinone; HQ), and a superoxide generator (duroquinone; DQ). ODQ (20 microM) abolished the vasodilator responses to glyceryl trinitrate (10(-8) - 10(-3) M), and sodium nitroprusside (10(-8) - 10(-4) M), which release NO intracellularly. ODQ (20 microM) attenuated, but failed to abolish, the vasodilator responses to SPER/NO (10(-6) - 10(-3) M). ODQ abolished responses to S-nitrosoglutathione and S-nitroso-N-valeryl-D-penicillamine (10(-8) - 10(-4) M), but a small residual vasodilatation remained in response to 10(-3) M. In the presence of ODQ, the remaining vasodilatation to SPER/NO was all but abolished by scavengers of extracellular NO (Hb; 10 microM, HQ; 100 microM). Superoxide generation (DQ; 100 microM) also attenuated ODQ-resistant vasodilatation. The data suggest that, in rat femoral arteries, NO donors that are capable of releasing extracellular NO cause vasodilatation that is only partially mediated by sGC. Lack of augmentation of sGC-independent effects by superoxide suggests that they are not mediated by peroxynitrite.

Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis.

Apoptosis of inflammatory cells is a critical event in the resolution of inflammation, as failure to undergo this form of cell death leads to increased tissue damage and exacerbation of the inflammatory response. Many factors are able to influence the rate of apoptosis in neutrophils, eosinophils, monocytes and macrophages. Among these is the signalling molecule nitric oxide (NO), which possesses both anti- and proapoptotic properties, depending on the concentration and flux of NO, and also the source from which NO is derived. This review summarises the differential effects of NO on inflammatory cell apoptosis and outlines potential mechanisms that have been proposed to explain such actions.

Dissociation of DNA fragmentation from other hallmarks of apoptosis in nitric oxide-treated neutrophils: differences between individual nitric oxide donor drugs.

The events of apoptotic cell death can be experimentally dissociated from each other in certain cell types. Here we demonstrate the ability of structurally diverse nitric oxide (NO) donating compounds to delay or enhance neutrophil apoptosis and to differentially influence distinct parameters of programmed cell death. We provide evidence that high concentrations of the NO donors GEA 3162, SPER/NO, and DEA/NO induce morphological and biochemical markers of neutrophil apoptosis, but that only DEA/NO causes a concomitant increase in DNA fragmentation as evidenced by nuclear propidium iodide intercalation and the classical laddering pattern of electrophoresed DNA. In contrast, both GEA 3162 and SPER/NO inhibit DNA cleavage in a time- and concentration-dependent manner. We are the first to show that DNA fragmentation can be dissociated from other changes of apoptosis in NO-treated neutrophils and that it may therefore be inappropriate to assess NO-induced apoptosis solely by measuring DNA fragmentation in this cell type.

Effects of acute methionine loading and vitamin C on endogenous fibrinolysis, endothelium-dependent vasomotion and platelet aggregation.

We assessed forearm blood flow and plasma fibrinolytic factors in eight healthy males who received unilateral brachial artery infusions of the endothelium-dependent vasodilator, substance P, and the endothelium-independent vasodilator, sodium nitroprusside. These measurements, together with platelet aggregation studies, were performed on four occasions after double-blind randomized ingestion of placebo, methionine (0.1 mg/kg), vitamin C (2 g) and methionine plus vitamin C. Blood flow and platelet aggregation responses were unaffected by methionine loading. Substance P caused dose-dependent increases in plasma tissue plasminogen activator (t-PA) antigen (from 3.0+/-0.1 to 4.7+/-0.4 ng/ml; P<0.001) and activity (from 1.2+/-0.2 to 4.2+/-0.4 i.u./ml; P<0.001), which were augmented during acute methionine loading (4.7+/-0.4 to 5.6+/-0.5 ng/ml and 4.2+/-0.4 to 5.5+/-0.9 i.u./ml respectively; P

Acute methionine loading does not alter arterial stiffness in humans.

Hyperhomocystinemia is a risk factor for cardiovascular disease, and acute elevation of plasma homocysteine after methionine loading impairs endothelial function in healthy subjects. Interestingly, pretreatment with vitamin C can ameliorate this effect. We have already shown that acute oral vitamin C administration reduces arterial stiffness in healthy subjects, and the aim of the present study was to investigate the effect of methionine loading on arterial stiffness with and without concomitant vitamin C using the noninvasive technique of pulse wave analysis. Eight healthy male subjects (mean age, 29 years; range, 20-42 years) were studied on three occasions at weekly intervals. In a double-blind, double-dummy, randomized order they received orally either 100 mg/kg methionine, 100 mg/kg methionine plus 2 g of vitamin C, or matching placebos. Peripheral and central blood pressure, heart rate, cardiac index, arterial stiffness, and plasma homocysteine levels were assessed at baseline and 6 hours after dosing. Compared with placebo, there was no significant change in any of the hemodynamic parameters, including arterial stiffness, after oral methionine, although plasma homocysteine did increase from 11.5 +/- 1.6 to 28.7 +/- 4.4 microM (mean +/- SEM; p < 0.001). Combined methionine and vitamin C led to a similar increase in plasma homocysteine but significantly reduced augmentation index by 10.5 +/- 3.2% (p = 0.02). Acute hyperhomocystinemia does not significantly alter arterial stiffness, as assessed by pulse wave analysis, whereas a combination of methionine and vitamin C leads to a similar reduction in augmentation index to that previously described after vitamin C alone. These data reinforce evidence that vitamin C reduces arterial stiffness but do not indicate any important interaction with oral methionine.

Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms.

Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.

Inhibition of human platelet aggregation by a novel S-nitrosothiol is abolished by haemoglobin and red blood cells in vitro: implications for anti-thrombotic therapy.

1. S-Nitrosothiols are nitric oxide (NO) donor drugs that have been shown to inhibit platelet aggregation in platelet rich plasma (PRP) in vitro and to inhibit platelet activation in vivo. The aim of this study was to compare the platelet effects of a novel S-nitrosated glyco-amino acid, RIG200, with an established S-nitrosothiol, S-nitrosoglutathione (GSNO) in PRP, and to investigate the effects of cell-free haemoglobin and red blood cells on S-nitrosothiol-mediated inhibition of platelet aggregation. 2. The effects of GSNO and RIG200 in collagen (2.5 microg ml(-1))-induced platelet aggregation in PRP and whole blood were investigated in vitro. Both compounds were found to be powerful inhibitors of aggregation in PRP, and RIG200 was significantly more potent (IC(50)=2.0 microM for GSNO and 0.8 microM for RIG200; P=0.04). 3. Neither compound inhibited aggregation in whole blood, even at concentrations of 100 microM. Red blood cell concentrations as low as 1% of the haematocrit, and cell-free haemoglobin (> or = 2.5 microM), significantly reduced their inhibitory effects on platelets. 4. Experiments involving measurement of cyclic GMP levels, electrochemical detection of NO and electron paramagnetic resonance of haemoglobin in red blood cells, indicated that scavenging of NO generated from S-nitrosothiols by haemoglobin was responsible for the lack of effect of S-nitrosothiols on platelets in whole blood. 5. These studies suggest that scavenging of NO by haemoglobin in blood might limit the therapeutic application of S-nitrosothiols as anti-platelet agents.

Novel S-nitrosothiols do not engender vascular tolerance and remain effective in glyceryltrinitrate-tolerant rat femoral arteries.

Organic nitrates, such as glyceryltrinitrate, are nitric oxide (NO) donor drugs that engender tolerance with long-term use. Here, we tested the hypothesis that our novel S-nitrosothiols, N-(S-nitroso-N-acetylpenicillamine)-2-amino-2-deoxy-1,3,4,6, tetra-O-acetyl-beta-D-glucopyranose (RIG200) and S-nitroso-N-valeryl-D-penicillamine (D-SNVP), do not induce vascular tolerance ex vivo. Femoral arteries from adult male Wistar rats were preconstricted with phenylephrine and perfused with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Perfusion pressure was measured during 20 h treatment with supramaximal concentrations of NO donor (10 microM). Perfusion with glyceryltrinitrate caused a vasodilatation, which recovered over 2-20 h. In contrast, the S-nitrosothiols caused vasodilatations that were maintained throughout the 20 h perfusion period. Responses to S-nitrosothiols were partially reversed by the NO scavenger ferrohaemoglobin and fully reversed by the soluble guanylate cyclase inhibitor [1H-[1,2,4] oxadiazole [4,3-a]quinoxaline-1-one (ODQ). Glyceryltrinitrate-tolerant vessels were fully responsive to bolus injections of S-nitrosothiols. Resistance to tolerance is an attractive property of our novel compounds, particularly in view of their sustained activity in arteries with damaged endothelium.

S-nitrosothiols cause prolonged, nitric oxide-mediated relaxation in human saphenous vein and internal mammary artery: therapeutic potential in bypass surgery.

1. Reduced endothelial nitric oxide (NO) production in conduit vessels for coronary artery bypass grafting (CABG) has been implicated in post-operative complications, including spasm. 2. The brief effects of existing NO donors limits their applicability to improving patency of graft vessels. RIG200 is a novel S-nitrosothiol that might have advantages over conventional drugs because it has sustained effects in areas of endothelial damage. 3. Here we tested the hypothesis that RIG200 and S-nitrosoglutathione (GSNO) have prolonged, NO-mediated effects in human saphenous vein (SV) and internal mammary artery (IMA), compared with glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). 4. 84 SV and 80 IMA rings from 64 patients undergoing CABG were studied in vitro. Rings were precontracted with phenylephrine (EC(80) concentration) and the functional integrity of the endothelium tested with acetylcholine (10 microM). 5. Relaxation of precontracted SV and IMA rings to GTN and SNP (0.01 - 10 microM) generally recovered fully on washout. In contrast, responses to RIG200 and GSNO were sustained during washout (30 min). Sustained relaxation was reversed by the NO scavenger, ferrohaemoglobin (10 microM) but not by the NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 and 250 microM in SV and IMA respectively). 6. Pretreatment (30 min) of SV with both S-nitrosothiols (10 microM) inhibited phenylephrine-induced contraction for >180 min, compared with <90 min for GTN. In IMA, contractility was suppressed to 49+/-4% (GSNO) and 26+/-4% (RIG200) of baseline after 240 min washout. 7. Pretreatment of bypass conduits with S-nitrosothiols might improve their patency in the early post-operative period.

Novel S-nitrosothiols do not engender vascular tolerance and remain effective in glyceryl trinitrate-tolerant rat femoral arteries.

Organic nitrates, such as glyceryl trinitrate, are nitric oxide (NO) donor drugs that engender tolerance with long-term use. Here, we tested the hypothesis that our novel S-nitrosothiols, N-(S-nitroso-N-acetylpenicillamine)-2-amino-2-deoxy-1,3,4,6, tetra-O-acetyl-beta-D-glucopyranose (RIG200) and S-nitroso-N-valeryl-D-penicillamine (D-SNVP), do not induce vascular tolerance ex vivo. Femoral arteries from adult male Wistar rats were preconstricted with phenylephrine and perfused with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Perfusion pressure was measured during 20-h treatment with supramaximal concentrations of NO donor (10 microM). Perfusion with glyceryltrinitrate caused a vasodilatation, which recovered over 2-20 h. In contrast, the S-nitrosothiols caused vasodilatations that were maintained throughout the 20-h perfusion period. Responses to S-nitrosothiols were partially reversed by the NO scavenger ferrohaemoglobin and fully reversed by the soluble guanylate cyclase inhibitor [1H-[1,2,4] oxadiazole [4,3-a]quinoxaline-1-one (ODQ). Glyceryltrinitrate-tolerant vessels were fully responsive to bolus injections of S-nitrosothiols. Resistance to tolerance is an attractive property of our novel compounds, particularly in view of their sustained activity in arteries with damaged endothelium.