Lifestyle and metabolic approaches to maximizing erectile and vascular health.

Oxidative stress and inflammation, which disrupt nitric oxide (NO) production directly or by causing resistance to insulin, are central determinants of vascular diseases including ED. Decreased vascular NO has been linked to abdominal obesity, smoking and high intakes of fat and sugar, which all cause oxidative stress. Men with ED have decreased vascular NO and circulating and cellular antioxidants. Oxidative stress and inflammatory markers are increased in men with ED, and all increase with age. Exercise increases vascular NO, and more frequent erections are correlated with decreased ED, both in part due to stimulation of endothelial NO production by shear stress. Exercise and weight loss increase insulin sensitivity and endothelial NO production. Potent antioxidants or high doses of weaker antioxidants increase vascular NO and improve vascular and erectile function. Antioxidants may be particularly important in men with ED who smoke, are obese or have diabetes. Omega-3 fatty acids reduce inflammatory markers, decrease cardiac death and increase endothelial NO production, and are therefore critical for men with ED who are under age 60 years, and/or have diabetes, hypertension or coronary artery disease, who are at increased risk of serious or even fatal cardiac events. Phosphodiesterase inhibitors have recently been shown to improve antioxidant status and NO production and allow more frequent and sustained penile exercise. Some angiotensin II receptor blockers decrease oxidative stress and improve vascular and erectile function and are therefore preferred choices for lowering blood pressure in men with ED. Lifestyle modifications, including physical and penile-specific exercise, weight loss, omega-3 and folic acid supplements, reduced intakes of fat and sugar, and improved antioxidant status through diet and/or supplements should be integrated into any comprehensive approach to maximizing erectile function, resulting in greater overall success and patient satisfaction, as well as improved vascular health and longevity.

Microcirculation and oxidative stress.

The microcirculation is a complex and integrated system, transporting oxygen and nutrients to the cells. The key component of this system is the endothelium, contributing to the local balance between pro and anti-inflammatory mediators, hemostatic balance, as well as vascular permeability and cell proliferation. A constant shear stress maintains vascular endothelium homeostasis while perturbed shear stress leads to changes in secretion of vasodilator and vasoconstrictor agents. Increased oxidative stress is a major pathogenetic mechanism of endothelial dysfunction by decreasing NO bioavailability, promoting inflammation and participating in activation of intracellular signals cascade, so influencing ion channels activation, signal transduction pathways, cytoskeleton remodelling, intercellular communication and ultimately gene expression. Targeting the microvascular inflammation and oxidative stress is a fascinating approach for novel therapies in order to decrease morbidity and mortality of chronic and acute diseases.

Effect of gestational hypercholesterolaemia on omental vasoreactivity, placental enzyme activity and transplacental passage of normal and oxidised fatty acids.

OBJECTIVE: Maternal hypercholesterolaemia during pregnancy increases lipid peroxidation in mothers and fetuses and programs increased susceptibility to atherosclerosis later in life. The objective of this study was to elucidate the role of the placenta in mediating oxidative stress from mother to offspring. DESIGN: Comparison between normo- and hypercholesterolaemic mothers (n = 36 each) and their children. SETTING: Obstetric wards, hospitals of the University of Naples and Regione Campania. POPULATION: Healthy primiparas delivering by caesarean section. METHODS: Biochemical measurements of oxidative stress and serum leptin in cord plasma and placenta, immunochemistry of placenta microvessels, and vasoreactivity studies were performed. MAIN OUTCOME MEASURES: Oxidative status (i.e. lipid composition and content of oxidised fatty acids, activity of pro- and antioxidant enzymes, immunohistochemical presence of oxidation-specific epitopes) in maternal and cord blood and in placental tissue, as well as vascular reactivity in omental arteries. RESULTS: Hypercholesterolaemia during pregnancy was associated with extensive changes in fatty acid composition of both maternal and cord blood lipids, sufficient to alter vasoreactivity of omental vessels. Results also indicated that the placenta is not only subject to substantial oxidative stress, but that it may further increase fetal oxidative stress through changes of pro- and antioxidant enzyme activities. CONCLUSIONS: The placenta plays an important role in both transmitting and enhancing pathogenic effects of gestational hypercholesterolaemia.

Influence of human menopausal gonadotrophin treatment on testicular blood flow and on seminal plasma nitric oxide levels in infertile males.

Gonadotrophin treatment is capable of increasing intratesticular blood flow and improving sperm fertilization potential. A nitric oxide (NO)-mediated mechanism may play a role. This study aimed at evaluating whether the increase in intratesticular blood flow, in response to human menopausal gonadotrophin (hMG) treatment, is accompanied by an increase in seminal plasma NO levels in 20 normogonadotropic males affected by severe oligoasthenoteratozoospermia. Patients received 150 IU of hMG, three times a week for 3 months. Before starting treatment and at month 3 of therapy, levels of NO in the seminal plasma were determined, followed by ultrasound scanning of testis, Doppler evaluation of intratesticular vascular resistance, serum hormone and conventional semen evaluation. Statistical analysis was performed by using Wilcoxon test; the limit of statistical significance was p<0.05. At the end of treatment, seminal plasma concentration of NO, conventional semen parameters and testicular volume did not increase significantly. Follicle-stimulating hormone serum level significantly increased (p=0.014) after treatment, whereas luteinizing hormone, testosterone and oestradiol values did not change. The resistance index of both intratesticular arteries significantly decreased (p=0.012 and 0.005 for right and left testes respectively). The results of this study confirmed the positive effect of exogenous hMG on testicular blood flow, but failed to demonstrate any effect of hMG treatment on NO levels in the seminal plasma in infertile males.

Sulfhydryl angiotensin-converting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension.

BACKGROUND: Essential hypertension is associated with enhanced LDL oxidation and impaired endothelium-dependent vasodilation. The antioxidant status is linked to the nitric oxide (NO) pathway. Sulfhydryl angiotensin-converting enzyme (ACE) inhibitors inhibit oxidative stress and atherogenesis in experimental models; therefore we tested whether this beneficial antioxidant activity could be also clinically relevant in patients with essential hypertension. METHODS: Plasma LDL oxidizability was investigated initially in untreated normocholesterolemic patients with moderate essential hypertension without clinically evident target organ damage (n = 96) and in control normotensive subjects (n = 46). Patients were then randomly assigned into two age- and sex-matched groups to receive the new sulfhydryl ACE inhibitor zofenopril (15 to 30 mg/d; n = 48) or enalapril (20 mg/d, n = 48). LDL oxidizability was evaluated (generation of malondialdehyde, MDA) and systemic oxidative stress was evaluated by isoprostanes (8-isoPGF2alpha). Asymmetrical dimethyl-L-arginine (ADMA), a competitive inhibitor of endothelial NO synthase, and plasma nitrite and nitrates (NOx) were also measured. RESULTS: LDL from hypertensive subjects had enhanced susceptibility to oxidation in vitro compared with that in control subjects (P <.05). Similarly, isoprostanes were significantly increased (P <.01) in hypertensive subjects versus control subjects. After 12-week treatment, MDA levels were significantly reduced by zofenopril (P <.05) but not enalapril treatment (P = not significant). Isoprostanes were normalized after zofenopril treatment (P <.03), whereas enalapril was ineffective. After treatment with both ACE inhibitors, plasma NOx concentrations were significantly reduced (P <.05). Similarly, hypertension increased ADMA concentration compared with the normotensive state, whereas ACE inihibition elicited a significant decrease. However, the reduction of ADMA concentration was significantly higher in patients receiving sulfhydryl ACE inhibition (P <.05 vs enalapril). CONCLUSIONS: The sulfhydryl ACE inhibitor zofenopril reduces oxidative stress and improves the NO pathway in patients with essential hypertension. If confirmed in a large multicenter clinical trial, our data suggest a possible vasculoprotective effect of the compound in retarding vascular dysfunction and atherogenesis that often develops rapidly in hypertensive patients.

Nitric oxide as a unique signaling molecule in the vascular system: a historical overview.

In retrospect, basic research in the fields of NO and cyclic GMP during the past two decades appears to have followed a logical course beginning with the findings that NO and cyclic GMP are vascular smooth muscle relaxants, that nitroglycerin relaxes smooth muscle by metabolism to NO, progressing to the discovery that mammalian cells synthesize NO, and finally the revelation that NO is a neurotransmitter mediating vasodilation in specialized vascular beds. A great deal of basic and clinical research on the physiological and pathophysiological roles of NO in cardiovascular function has been conducted since the discovery that EDRF is NO. The new knowledge on NO should enable investigators in this field to develop novel and more effective therapeutic strategies for the prevention, diagnosis and treatment of numerous cardiovascular disorders. Since NO elicits a protective and beneficial action in many disease states, novel NO donor drugs for clinical use should prove to be very effective drugs for the treatment of essential hypertension, stroke, coronary artery disease, vascular complications of diabetes, impotency and other disorders involving the vascular system.

Role of p42/p44 mitogen-activated-protein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide.

The purpose of this study was to determine the involvement of the p42/p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21(waf1/cip1) in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like alpha-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 microM, and DFMO elicited effects at concentrations of 100 microM or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1/2 inhibitors PD 098,059 or U0126. This finding suggests that the p42/p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42/p44 MAPK and induction of p21(waf1/cip1). This effect was prevented by MAPK kinase 1/2 inhibitors, suggesting that induction of p21(waf1/cip1) depended on activation of p42/p44. Moreover, activation of p42/p44 and induction of p21(waf1/cip1) were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42/p44 MAPK and induction of p21(waf1/cip1) were cGMP-independent. Neither 1H-(1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21(waf1/cip1) and consequent inhibition of cell proliferation.

Lipopolysaccharide-induced expression of interferon-beta mediates the timing of inducible nitric-oxide synthase induction in RAW 264.7 macrophages.

The production of nitric oxide by macrophages has been implicated as a host defense mechanism against microbial pathogens and tumor cells. Recent reports have implicated interferon-alpha/beta (IFN-alpha/beta) as an autocrine/paracrine signal critical for the induction of murine iNOS. In this report we have systematically investigated the role of IFN-beta in the induction of iNOS in the murine macrophage cell line, RAW 264.7. First, we demonstrate that IFN-beta expression is highly up-regulated, and is secreted in response to lipopolysaccharide (LPS). Treatment of RAW macrophages with LPS results in a time-dependent phosphorylation of STAT-1 on both tyrosine residue 701 (Tyr-701) and serine residue 727 (Ser-727) that is consistent with the timing of endogenous IFN-beta expression. LPS also induces interferon regulatory factor-1 expression with similar kinetics. We further demonstrate that exogenous IFN-beta accelerates the induction of iNOS by LPS. The acceleration of iNOS induction is observed at the levels of transcription, protein expression, and NO formation. Accordingly, we propose that the cytokine environment of macrophages may determine the rate and magnitude of nitric oxide production, thereby regulating the cytotoxic response to pathogen challenge.

Acute arterial thrombosis causes endothelial dysfunction: a new paradigm for thrombolytic therapy.

PURPOSE: The goals of this study were to delineate the time course of endothelial dysfunction after arterial thrombosis, to determine the cause of endothelial dysfunction in this setting, and to determine whether modulating standard thrombolytic therapy would ameliorate the thrombosis-mediated endothelial dysfunction. METHODS: Male adult rats underwent infrarenal aortic occlusion by means of clip ligature to induce arterial thrombosis. After 30 minutes, 1, 2, and 3 hours, ring segments from the infrarenal aorta were harvested and placed into physiologic buffer baths. With the use of a force transducer, both endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR) were measured. Endothelial function and presence were determined by means of factor VIII immunohistochemical staining. Endothelial morphology was evaluated with scanning electron microscopy (SEM). Nitric oxide (NO) levels were determined with a chemiluminescent assay of its nitrite/nitrate metabolites (NO(x)). Standard thrombolytic therapy with urokinase (UK) was infused into thrombosed aortic ring segments and compared with UK supplemented with both low-dose L -arginine (2 mmol) and high-dose L -arginine (20 mmol). RESULTS: Arterial thrombosis decreases EDR. The nadir of EDR occurs 1 hour after thrombosis (mean +/- SE, 13% +/- 6.4% vs 94% +/- 2.6% for controls, P <.005), with persistent lowering of EDR as long as 3 hours after thrombosis. EIR is preserved, and vasoconstriction with norepinephrine or potassium buffer is unaltered. Both endothelial function and presence (n = 6 per group) were documented by means of factor VIII immunohistochemistry. An intact monolayer of endothelium at all time intervals after thrombosis was revealed by means of SEM analysis. No differences between control and thrombosed specimens were revealed by means of the grading of SEM images. Local NO(x) levels were lower after 1 hour of thrombosis, with an increase higher than baseline values at 3 hours. The addition of low-dose L -arginine resulted in a minor increase in EDR. However, high-dose L -arginine resulted in a significant increase in EDR versus controls receiving UK alone (64% +/- 6.3% vs 38% +/- 4.4%, P <.05). Correspondingly, local NO(x) levels were 20-fold higher after the high-dose L -arginine supplementation when compared with UK thrombolysis alone (2.8 +/- 0.52 micromol/L vs 0.133 +/- 0.02 micromol/L, n = 6 samples/group, P <.005). CONCLUSION: Acute arterial thrombosis causes endothelial dysfunction, without causing endothelial cell loss. Endothelial function reaches a nadir after 1 hour of thrombosis. EIR and vasoconstriction remain unaffected, indicating normal smooth muscle cell function. NO(x) levels suggest that NO levels are decreased acutely after thrombosis. Supplementing standard thrombolytic therapy with the NO precursor, l-arginine, ameliorates the endothelial dysfunction seen after acute thrombosis by increasing local NO production.

Nitric oxide inhibits ornithine decarboxylase via S-nitrosylation of cysteine 360 in the active site of the enzyme.

Ornithine decarboxylase is the initial and rate-limiting enzyme in the polyamine biosynthetic pathway. Polyamines are found in all mammalian cells and are required for cell growth. We previously demonstrated that N-hydroxyarginine and nitric oxide inhibit tumor cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively, and, therefore, polyamine synthesis. In addition, we showed that nitric oxide inhibits purified ornithine decarboxylase by S-nitrosylation. Herein we provide evidence for the chemical mechanism by which nitric oxide and S-nitrosothiols react with cysteine residues in ornithine decarboxylase to form an S-nitrosothiol(s) on the protein. The diazeniumdiolate nitric oxide donor agent 1-diethyl-2-hydroxy-2-nitroso-hydrazine acts through an oxygen-dependent mechanism leading to formation of the nitrosating agents N(2)O(3) and/or N(2)O(4). S-Nitrosoglutathione inhibits ornithine decarboxylase by an oxygen-independent mechanism likely by S-transnitrosation. In addition, we provide evidence for the S-nitrosylation of 4 cysteine residues per ornithine decarboxylase monomer including cysteine 360, which is critical for enzyme activity. Finally S-nitrosylated ornithine decarboxylase was isolated from intact cells treated with nitric oxide, suggesting that nitric oxide may regulate ornithine decarboxylase activity by S-nitrosylation in vivo.

Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation.

Arginase, which exists as the isoforms arginase I and II, catalyzes the hydrolysis of arginine to ornithine and urea. Ornithine is the principal precursor for production of polyamines, which are required for cell proliferation. Rat aortic smooth muscle cells (RASMC) contain constitutive arginase I, and arginase inhibitors cause inhibition of cell proliferation. The objective of this study was to determine whether the elevated expression of arginase I in RASMC causes increased cell proliferation. RASMC were stably transfected with either rat arginase I cDNA or a beta-galactosidase control expression plasmid. Western blots and arginase enzymatic assays revealed high-level expression of cytosolic arginase I in arginase I-transfected RASMC. Moreover, this observation was associated with the increased production of urea and polyamines and higher rates of RASMC proliferation. The two selective inhibitors of arginase, N(G)-hydroxy-l-arginine and S-(2-boronoethyl)-l-cysteine, inhibited arginase and decreased the production of urea and polyamines in arginase I-transfected RASMC, all of which were associated with the inhibition of cell proliferation. This study demonstrates that elevated arginase I expression increases RASMC proliferation by mechanisms involving increased production of polyamines. These observations suggest that arginase I plays a potentially important role in controlling RASMC proliferation.

Platelets modulate gastric ulcer healing: role of endostatin and vascular endothelial growth factor release.

Bleeding and delayed healing of ulcers are well recognized clinical problems associated with the use of aspirin and other nonsteroidal antiinflammatory drugs, which have been attributed to their antiaggregatory effects on platelets. We hypothesized that antiplatelet drugs might interfere with gastric ulcer healing by suppressing the release of growth factors, such as vascular endothelial growth factor (VEGF), from platelets. Gastric ulcers were induced in rats by serosal application of acetic acid. Daily oral treatment with vehicle, aspirin, or ticlopidine (an ADP receptor antagonist) was started 3 days later and continued for 1 week. Ulcer induction resulted in a significant increase in serum levels of VEGF and a significant decrease in serum levels of endostatin (an antiangiogenic factor). Although both aspirin and ticlopidine markedly suppressed platelet aggregation, only ticlopidine impaired gastric ulcer healing and angiogenesis as well as reversing the ulcer-associated changes in serum levels of VEGF and endostatin. The effects of ticlopidine on ulcer healing and angiogenesis were mimicked by immunodepletion of circulating platelets, and ticlopidine did not influence ulcer healing when given to thrombocytopenic rats. Incubation of human umbilical vein endothelial cells with serum from ticlopidine-treated rats significantly reduced proliferation and increased apoptosis, effects reversed by an antibody directed against endostatin. Ticlopidine treatment resulted in increased platelet endostatin content and release. These results demonstrate a previously unrecognized contribution of platelets to the regulation of gastric ulcer healing. Such effects likely are mediated through the release from platelets of endostatin and possibly VEGF. As shown with ticlopidine, drugs that influence gastric ulcer healing may do so in part through altering the ability of platelets to release growth factors.

Nitric oxide and atherosclerosis.

Endothelial dysfunction has been shown in a wide range of vascular disorders including atherosclerosis and related diseases. Here, we examine and address the complex relationship among nitric oxide (NO)-mediated pathways and atherogenesis. In view of the numerous pathophysiological actions of NO, abnormalities could potentially occur at many sites: (a) impairment of membrane receptors in the arterial wall that interact with agonists or physiological stimuli capable of generating NO; (b) reduced concentrations or impaired utilization of l-arginine; (c) reduction in concentration or activity both of inducible and endothelial NO synthase; (d) impaired release of NO from the atherosclerotic damaged endothelium; (e) impaired NO diffusion from endothelium to vascular smooth muscle cells followed by decreased sensitivity to its vasodilator action; (f) local enhanced degradation of NO by increased generation of free radicals and/or oxidation-sensitive mechanisms; and (g) impaired interaction of NO with guanylate cyclase and consequent limitation of cyclic GMP production. Therefore, one target for new drugs should be the preservation or restoration of NO-mediated signaling pathways in arteries. Such novel therapeutic strategies may include administration of l-arginine/antioxidants and gene-transfer approaches.

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation.

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. N(G)-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1-3 microM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as alpha-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.

An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity.

Caspases are key mediators in liver inflammation and apoptosis. In the present study we provide evidence that a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA), NCX-1000 ([2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]), protects against liver damage in murine models of autoimmune hepatitis induced by i.v. injection of Con A or a Fas agonistic antibody, Jo2. Con A administration causes CD4(+) T lymphocytes to accumulate in the liver and up-regulates FasL expression, resulting in FasL-mediated cytotoxicity. Cotreating mice with NCX-1000, but not with UDCA, protected against liver damage induced by Con A and Jo2, inhibited IL-1beta, IL-18, and IFN-gamma release and caspase 3, 8, and 9 activation. Studies on HepG2 cells demonstrated that NCX-1000, but not UDCA, directly prevented multiple caspase activation induced by Jo2. Incubating HepG2 cells with NCX-1000 resulted in intracellular NO formation and a DTT-reversible inhibition of proapoptotic caspases, suggesting that cysteine S-nitrosylation was the main mechanism responsible for caspase inhibition. Collectively, these data suggest that NCX-1000 protects against T helper 1-mediated liver injury by inhibiting both the proapoptotic and the proinflammatory branches of the caspase superfamily.

Effects of nitric oxide-releasing aspirin versus aspirin on restenosis in hypercholesterolemic mice.

Restenosis is due to neointimal hyperplasia, which occurs in the coronary artery after percutaneous transluminal coronary angioplasty (PTCA). During restenosis, an impairment of nitric oxide (NO)-dependent pathways may occur. Concomitant hypercholesterolemia may exacerbate restenosis in patients undergoing PTCA. Here, we show that a NO-releasing aspirin derivative (NCX-4016) reduces the degree of restenosis after balloon angioplasty in low-density lipoprotein receptor-deficient mice and this effect is associated with reduced vascular smooth muscle cell (VSMC) proliferation and macrophage deposition at the site of injury. Drugs were administered following both therapeutic or preventive protocols. We demonstrate that NCX-4016 is effective both in prevention and treatment of restenosis in the presence of hypercholesterolemia. These data indicate that impairment of NO-dependent mechanisms may be involved in the development of restenosis in hypercholesterolemic mice. Although experimental models of restenosis may not reflect restenosis in humans in all details, we suggest that a NO-releasing aspirin derivative could be an effective drug in reducing restenosis following PTCA, especially in the presence of hypercholesterolemia and/or gastrointestinal damage.

Up-regulation of endothelial nitric oxide synthase through beta(2)-adrenergic receptor--the role of a beta-blocker with NO-releasing action.

We determined the existence and role of beta(2)-adrenergic receptor in cultured BAECs through the effect of a beta-blocker having NO releasing action; 3,4-dihydro-8(2-hydroxy-3-isopropylamino)-propoxy-3-nitroxy-2H-1-benzopyran; nipradilol on eNOS and eNOS regulatory protein caveolin-1. beta(2) receptor exists in BAECs. eNOS mRNA and protein were up-regulated by its treatment whereas those of caveolin were not altered considerably. This eNOS up-regulatory action was abolished by beta(2) receptor antagonist, ICI-118551. Increase of NO metabolites, protein and mRNA of eNOS was also partially inhibited by co-treatment of NOS inhibitor, L-NA with nipradilol. This is the first investigation of the action of non-selective beta blocker on eNOS through beta(2) receptor. The drug increases NO on incubation with BAECs about 50% as a NO donor and about 50% as results of eNOS up-regulation.

Nitric oxide improves cisplatin cytotoxicity in head and neck squamous cell carcinoma.

OBJECTIVE: To test whether nitric oxide (NO) enhances the cytotoxicity of cisplatin in a head and neck squamous cell carcinoma (HNSCC) cell line. BACKGROUND: Cisplatin is one of the most frequently used chemotherapeutic agents in the treatment of HNSCC. NO has been shown to play an important role in regulating tumor growth. Previous studies demonstrate that NO can enhance the cytotoxicity of cisplatin in Chinese hamster lung fibroblasts. In this report, we examined the in vitro interaction of NO and cisplatin in a HNSCC cell line. MATERIALS AND METHODS: CCL23 cells were pretreated with three different NO donors: PAPA/NO (t 1/2 = 15 min), DPTA/NO (t 1/2 = 3 h), and DETA/NO (t 1/2 = 20 h). The cells were rinsed and exposed for 6 hours to a culture medium containing cisplatin. Cell survival and LD50 of cisplatin were calculated with and without NO pretreatment. RESULTS: PAPA/NO and DPTA/NO did not show any cytotoxic activity and did not change the LD50 of cisplatin. DETA/NO when used alone resulted in 25.6% cell death at its peak dose (100 microM). Pretreatment with DETA/NO resulted in almost a threefold reduction of the LD50 of cisplatin (6.8 vs. 2.4 microg/mL). Pretreatment with DETA/NO sensitized the HNSCC cells to subsequent cisplatin activity (two-sided P =.00016). CONCLUSION: Pretreatment of HNSCC cells with long-acting NO donors enhances cisplatin activity. Short- and medium-acting NO donors do not exert a toxic effect and do not augment the activity of cisplatin. NO agonists should be considered in the future as a possible adjunct to cisplatin in the treatment of HNSCC. Further studies with animal models are necessary to further clarify this relationship.

Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat.

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by a variety of nitric oxide synthases (NOS). It has been demonstrated that a decrease in NOS activity, as observed in aging, is associated with a diminished erectile response. The objective of this study was to determine if adenoviral-mediated gene transfer of eNOS could reverse age-related erectile dysfunction in the rat. Two groups of animals were transfected with adenoviruses: (1) aged rats (60 weeks) with AdRSVbetagal; and (2) aged rats (60 weeks) with AdRSVeNOS. Five days after transfection, these study animals underwent cavernosal nerve stimulation (CNS) to assess erectile function and their responses were compared with young (20 weeks) control rats. Cross-sections of the rat penises transfected with AdRSVeNOS were examined after trichrome staining. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by a galacto-light chemiluminescent reporter gene assay in cavernosal tissues of rats administered AdRSVbetagal. The transgene expression of eNOS was examined by RT-PCR in rats transfected with AdRSVbetagal and AdRSVeNOS. eNOS and iNOS protein levels were measured by Western blot analysis, and cGMP levels were assessed in cavernosal tissue by enzyme immunoassay. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue for up to 30 days, with peak expression registered at 5 days after intracavernosal administration of AdRSVbetagal. Cross-sections of the rat penises transfected with the AdRSVeNOS revealed no pathological (morphological or histological) changes. Five days after administration of AdRSVeNOS, eNOS protein, mRNA and cGMP levels in the corpora cavernosa were significantly increased (P<0. 05), while iNOS protein levels remained unchanged (P>0.05). In conclusion, enhanced expression of eNOS employing an adenoviral vector significantly increased the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the aged rat.