assessed using Western blot analysis. The role of NAD[P]H oxidase and cGMP was further studied by using specific inhibitors of each. RESULTSSuperoxide formation was significantly greater in cells incubated with U46619 after 1 and 16 h incubation than in controls, an effect blocked by NADP(H) oxidase inhibitors. These effects of U46619 were inhibited by sildenafil (1 and 10 nmol/L), which in turn were negated by the guanylyl cyclase inhibitor, ODQ; 10 nmol/L sildenafil inhibited p47phox expression induced by U46619. CONCLUSIONSSildenafil is a potent inhibitor of superoxide formation in CVSMCs. This effect is mediated through the inhibition of PDE-5 which in turn augments the inhibitory action of the NOcGMP axis on NAD[P]H oxidase expression and activity. This mechanism constitutes a new pharmacological action of sildenafil, consolidates the potential role of superoxide in ED, and indicates that thromboxane A 2 may be an important mediator of intrapenile oxidative stress.

Background and purpose: Superoxide (O 2 K À ), derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is associated with acute respiratory distress syndrome (ARDS). NADPH oxidase activity and expression are blocked by nitric oxide (NO) and sildenafil. As another gas, hydrogen sulphide (H 2 S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H 2 S-donating derivative of sildenafil, ACS6, on O 2 KÀ formation and the expression of gp91 phox (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated. Experimental approach: PAECs were incubated with 10 ng mL À1 tumour necrosis factor-a (TNFa) ( ± NaHS or ACS6), both of which released H 2 S, for 2 h or 16 h. O 2 K À was measured. Expression of gp91 phox was measured by western blotting and the role of cyclic AMP (cAMP) and/or cyclic GMP was assessed using protein kinase inhibitors. Key results: After either 2-or 16-h incubations, O 2K À formation by PAECs was inhibited by NaHS or ACS6, with IC 50 values of about 10 nM and less than 1 nM, respectively. Both 100 nM NaHS and 1 nM ACS6 completely inhibited gp91 phox expression induced by TNFa. The effects of NaHS were blocked by the inhibition of protein kinase A (PKA), but not PKG, and not by the inhibition of guanylyl cyclase. Effects of ACS6 were blocked by inhibition of both PKA and PKG. Both NaHS and ACS6 augmented cAMP formation. Conclusion and implications: H 2 S inhibited O 2K À formation and upregulation of NADPH oxidase in PAECs through the adenylyl cyclase-PKA pathway. ACS6 may be effective in treating ARDS through both elevation of cAMP and inhibition of phosphodiesterase type 5 activity.

The activity of NADPH oxidase (NOX) is blocked by nitric oxide (NO). Hydrogen sulfide (H₂S) is also produced by blood vessels. It is reasonable to suggest that H₂S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O₂^– formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac₁ activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A₂ analogue U46619 ± NaHS for 1 or 16 h, and O₂^– formation, NOX-1 expression and Rac₁ activity were assessed. The possible interaction between H₂S and NO was also studied by using an NO synthase inhibitor, L-NAME, and an NO donor, DETA-NONOate. The role of K_ATP channels was studied by using glibenclamide. NaHS inhibited O₂^– formation following incubation of 1 h (IC₅₀, 30 nM) and 16 h (IC₅₀, 20 nM), blocked NOX-1 expression and inhibited Rac₁ activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H₂S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac₁ and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H₂S.

. À in PA segments, PAVSMCs and PAECs, an effect inhibited by diphenyleneiodonium and apocynin (both NADPH oxidase inhibitors) and upregulated the expression of gp91 phox in PAECs and PAVSMCs. These effects were augmented by LPS, TNF-a and IL-1a but inhibited by iloprost. Under identical incubation conditions, IL-1a, LPS and TNF-a all induced an increase in the formation of TXA 2 , PGF 2a and 8-isoprostane F 2a but reduced the concomitant formation of PGI 2 . 4 These data demonstrate that LPS and cytokines influence the relative balance of TXA 2 , PGI 2 , PGF 2a and 8-isoprostane F 2a in pig PA, which in turn alter NADPH oxidase expression and O 2 . À formation. These novel findings have implications in devising effective strategies for treating ARDS.

Background: The interactive roles of cytokines, endotoxins, superoxide (O 2 v -) and nitric oxide (NO) in the pathogenesis of adult respiratory distress syndrome (ARDS) have not been fully elucidated. The effects of tumour necrosis factor-α (TNF-α), interleukin 1α (IL-1α), and lipopolysaccharide (LPS) and the role of NO and the endothelium in mediating O 2 v -formation were therefore investigated in intact porcine pulmonary arteries in vitro. Methods: Intrapulmonary artery (PA) segments were obtained from White Landrace pigs (25-35 kg) and incubated with LPS, IL-1α, and TNF-α and O 2 v -release was measured by the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. The source of O 2 v -formation was determined using a number of enzyme inhibitors. The role of NO was explored using NO synthase (NOS) inhibitors and the distribution of NOS isoforms and peroxynitrite (ONOO

1 Acute respiratory distress syndrome (ARDS) is associated with increased superoxide (O 2 K À ) formation in the pulmonary vasculature and negation of the bioavailability of nitric oxide (NO). Since NO inhibits NADPH oxidase expression through a cyclic GMP-mediated mechanism, sildenafil, a type V phosphodiesterase inhibitor, may be therapeutically effective in ARDS through an augmentation of NO-mediated inhibition of NADPH oxidase. Therefore, the effect of sildenafil citrate and NO-donating sildenafil (NCX 911) on O 2 K À formation and gp91 phox (active catalytic subunit of NADPH oxidase) expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs). 2 PAECs were incubated with 10 nM TXA 2 analogue, 9,11-dideoxy-9a,11a-methanoepoxy-prostaglandin F 2a (U46619) (7sildenafil or NCX 911), for 16 h and O 2 K À formation measured spectrophometrically and gp91 phox using Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride (SIN-1), the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ), and the protein kinase G inhibitor, 8-bromoguanosine-3 0 ,5 0 -cyclic monophosphorothioate, Rp-isomer (Rp-8-BrcGMPS). NO release was studied using a fluorescence assay and O 2 K À -NO interactions by measuring nitrites.3 After a 16-h incubation with 10 nM U46619, both NCX 911 and sildenafil elicited a concentrationdependent inhibition of O 2 K À formation and gp91 phox expression, NCX 911 being more potent (IC 50 ; 0.26 nM) than sildenafil citrate (IC 50 ; 1.85 nM). These inhibitory effects were reversed by 1 mM ODQ and 10 mM Rp-8-Br-cGMPS. NCX 911 stimulated the formation of cGMP in PAECs and generated NO in a cell-free system to a greater degree than sildenafil citrate. The inhibitory effect of sildenafil was augmented by 1 mM SIN-1 and blocked partially by the eNOS inhibitor 10 mM N 5 -(1-iminoethyl)-ornithine (L-NIO). Acutely, sildenafil and NCX 911 also inhibited O 2 K À formation, again blocked by 1 mM ODQ. NCX 911 reacted with O 2 K À generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500 U ml À1 ). 4 Since O 2 K À formation plays contributory role in ARDS, both sildenafil citrate and NCX 911 may be indicated for treating ARDS through suppression of NADPH oxidase expression and therefore of O 2 K À formation and preservation of NO bioavailability.

Background-Although

Oxidant stress [ OS ] is a condition in which cells are exposed to excessive levels of either molecular oxygen or chemical derivatives of oxygen called reactive oxygen species [ROS], principal amongst which is superoxide [O2-]. It is becoming increasingly apparent that O2- is a key risk factor for cardiovascular disease [CVD], including atherogenesis, reperfusion injury, angina, restenosis following balloon angioplasty and vein graft failure. When one considers the multiplicity of effects of O2-, this is perhaps not surprising, as it promotes vascular smooth muscle cell proliferation, damages the endothelium, promotes lipid oxidation and activates blood cells. However, perhaps the key reaction of O2- is that with nitric oxide [NO] to form peroxynitrite [ONOO] resulting in a depletion of endogenous vascular NO. Reduced NO formation is also now firmly associated with the aetiology of CVD and as such NO donors may become a major class of drugs. Furthermore, risk factors for CVD, in particular diabetes mellitus [DM], dyslipidaemia, and hyperhomocysteinaemia are all associated with OS. As such, it is becoming increasingly apparent that novel antioxidant therapies, including the gene transfer of antioxidant enzymes, are potentially valuable in the treatment of CVD. In this review, the aetiology of OS and CVD is discussed with particular emphasis on NO. The interactions of risk factors and how this pathophysiology relates to the design of effective novel strategies to treat CVD is also considered. Particular emphasis is also placed on OS and cardiovascular surgery.