cyclic-gmp and 3-nitrotyrosine

Endothelial dysfunction associated with decreased nitric oxide (NO) bioactivity is a major feature of vascular diseases such as atherosclerosis or diabetes. Sodium nitroprusside (SNP)-induced relaxation is entirely dependent on cyclic guanosine monophosphate (cGMP) and preserved in atherosclerosis, suggesting that smooth muscle response to NO donor is intact. However, NO gas activates both cGMP-dependent and -independent signal pathways in vascular smooth muscle cells, and oxidative stress associated with vascular diseases selectively impairs cGMP-independent relaxation to NO. Sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA), which regulates intracellular Ca(2+) levels by pumping Ca(2+) into store, is a major cGMP-independent target for NO. Physiological levels of reactive nitrogen species (RNS) S-glutathiolate SERCA at Cys674 to increase its activity, and the augmentation of RNS in vascular diseases irreversibly oxidizes Cys674 or nitrates tyrosine residues at Tyr296-Tyr297, which are associated with loss of function. S-glutathiolation of various proteins by NO can explain redox-sensitive cGMP-independent actions, and oxidative inactivation of target proteins for NO can be associated with the pathogenesis of cardiovascular diseases. Oxidative inactivation of SERCA is also implicated with dysregulation of smooth muscle migration, promotion of platelet aggregation, and impairment of cardiac function, which can be implicated with restenosis, pathological angiogenesis, thrombosis, as well as heart failure. Analysis of posttranslational oxidative modifications of SERCA and the preservation of SERCA function can be novel strategies against cardiovascular diseases associated with oxidative stress.

Topics: Animals; Antioxidants; Atherosclerosis; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Diabetic Angiopathies; Endothelium, Vascular; Glutathione; Heart Failure; Humans; Mice; Models, Animal; Muscle, Smooth, Vascular; Nitric Oxide; Oxidative Stress; Protein Processing, Post-Translational; Rabbits; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tyrosine; Vasodilation

Endothelial dysfunction accompanied by an increase in oxidative stress is a key event leading to hypertension. As dietary nitrite has been reported to exert antihypertensive effect, the present study investigated whether chronic oral administration of sodium nitrite improves vascular function in conduit and resistance arteries of hypertensive animals with elevated oxidative stress.. Sodium nitrite (50mg/L) was given to angiotensin II-infused hypertensive C57BL/6J (eight to ten weeks old) mice for two weeks in the drinking water. Arterial systolic blood pressure was measured using the tail-cuff method. Vascular responsiveness of isolated aortae and renal arteries was studied in wire myographs. The level of nitrite in the plasma and the cyclic guanosine monophosphate (cGMP) content in the arterial wall were determined using commercially available kits. The production of reactive oxygen species (ROS) and the presence of proteins (nitrotyrosine, NOx-2 and NOx-4) involved in ROS generation were evaluated with dihydroethidium (DHE) fluorescence and by Western blotting, respectively.. Chronic administration of sodium nitrite for two weeks to mice with angiotensin II-induced hypertension decreased systolic arterial blood pressure, reversed endothelial dysfunction, increased plasma nitrite level as well as vascular cGMP content. In addition, sodium nitrite treatment also decreased the elevated nitrotyrosine and NOx-4 protein level in angiotensin II-infused hypertensive mice.. The present study demonstrates that chronic treatment of hypertensive mice with sodium nitrite improves impaired endothelium function in conduit and resistance vessels in addition to its antihypertensive effect, partly through inhibition of ROS production.

Topics: Administration, Oral; Angiotensin II; Animals; Antihypertensive Agents; Antioxidants; Aorta, Thoracic; Arterial Pressure; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Hypertension; Male; Mice, Inbred C57BL; NADPH Oxidase 2; NADPH Oxidase 4; Nitric Oxide; Oxidative Stress; Renal Artery; Sodium Nitrite; Tyrosine; Vasodilation

Ischaemia reperfusion (IR) injury occurs during vascular graft harvesting and implantation during vascular/cardiac surgery. Elevated intracellular cyclic guanosine monophosphate (cGMP) levels contribute to an effective endothelial protection in different pathophysiological conditions. The hypothesis that the phosphodiesterase-5 inhibitor vardenafil would protect vascular grafts against IR injury by upregulating the nitric oxide-cGMP pathway in the vessel wall of the bypass graft was investigated.. Lewis rats (n = 6-7/group) were divided into Group 1, control; Group 2, donor rats received intravenous saline; Group 3, received intravenous vardenafil (30 μg/kg) 2 h before explantation. Whereas aortic arches of Group 1 were immediately mounted in an organ bath, aortic segments of Groups 2 and 3 were stored for 2 h in saline and transplanted into the abdominal aorta of the recipient. Two hours after transplantation, the implanted grafts were harvested. Endothelium dependent and independent vasorelaxations were investigated. TUNEL, CD-31, ICAM-1, VCAM-1, α-SMA, nitrotyrosine, dihydroethidium and cGMP immunochemistry were also performed.. The results support the view that impairment of intracellular cGMP signalling plays a role in the pathogenesis of the endothelial dysfunction of an arterial graft after bypass surgery, which can effectively be prevented by vardenafil. Its clinical use as preconditioning drug could be a novel approach in vascular/cardiac surgery.

Topics: Actins; Animals; Aorta, Thoracic; Cold Ischemia; Cyclic GMP; Cytoprotection; DNA Damage; Intercellular Adhesion Molecule-1; Male; Nitrosative Stress; Phosphodiesterase 5 Inhibitors; Platelet Endothelial Cell Adhesion Molecule-1; Rats, Inbred Lew; Reperfusion Injury; Signal Transduction; Tissue and Organ Harvesting; Tyrosine; Vardenafil Dihydrochloride; Vascular Cell Adhesion Molecule-1; Vascular System Injuries; Vasodilator Agents; Warm Ischemia

One of the medicinal plants widely used by the population in the treatment of hypertension, atherosclerosis and circulatory disorders is Cuphea carthagenensis (Jacq.) J.F. Macbr. (Lythraceae), popularly known as 'sete sangrias', being found in Brazil, Hawaii and in South Pacific Islands. Despite the widespread use of this species by the population, its long-term antihypertensive and cardioprotective activities have not yet been scientifically evaluated.. To evaluate the possible cardioprotective effects of an ethanol-soluble fraction obtained from C. carthagenensis (ESCC) using ovariectomized hypertensive rats to simulate a broad part of the female population over 50 years of age affected by hypertension. In addition, the molecular mechanism that may be responsible for its cardiorenal protective effects was also explored.. Female Wistar rats were submitted to surgical procedures of bilateral ovariectomy and induction of renovascular hypertension (two-kidneys, one-clip model). The sham-operated group was used as negative control. ESCC was obtained and a detailed phytochemical investigation about its main secondary metabolites was performed. ESCC was orally administered at doses of 30, 100 and 300  mg/kg, daily, for 28 days, 5 weeks after surgery. Enalapril (15  mg/kg) was used as standard antihypertensive drug. Renal function was evaluated on days 1, 7, 14, 21 and 28. At the end of the experimental period, systolic, diastolic, mean arterial pressure and heart rate were recorded. The activity of the tissue enzymatic antioxidant system, thiobarbituric acid reactive substances, nitrotyrosine, nitrite, aldosterone and vasopressin levels, in addition to the activity of the angiotensin-converting enzyme were also evaluated. Additionally, vascular reactivity to acetylcholine, sodium nitroprusside, and phenylephrine, and the role of nitric oxide, prostaglandins, and K. ESCC-treatment induced an important cardiorenal protective response, preserving renal function and preventing elevation of blood pressure and heart rate in ovariectomized hypertensive rats. In addition, prolonged treatment with ESCC recovered mesenteric vascular reactivity at all doses used. This effect was associated with an important modulation of the antioxidant defense system with a possible increase in NO bioavailability. Additionally, NO/cGMP activation and K. A 28-days ESCC treatment reduces the progression of the cardiorenal disease in ovariectomized hypertensive rats. These effects seem to be involved with an attenuation of oxidative and nitrosative stress, affecting endothelial nitric oxide production and K

Topics: Aldosterone; Animals; Antihypertensive Agents; Blood Pressure; Cuphea; Cyclic GMP; Endothelium, Vascular; Female; Hypertension, Renovascular; Nitric Oxide; Nitrites; Nitrosative Stress; Oxidation-Reduction; Oxidative Stress; Peptidyl-Dipeptidase A; Phytochemicals; Plant Extracts; Plants, Medicinal; Potassium Channels; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Tyrosine; Vasodilator Agents; Vasopressins

Nitric oxide is a multifunctional molecule that can modify proteins via nitrosylation; it can also initiate signaling cascades through the activation of soluble guanylate cyclase. Diabetic retinopathy is the leading cause of blindness, but its pathogenesis is unknown. Multiple mechanisms including oxidative-nitrosative stress have been implicated. Our main goal was to find significant changes in nitric oxide (NO) levels and determine their association with nitrosative stress in the rat retina at the onset of diabetes.. Diabetes was induced by a single intraperitoneal administration of streptozotocin. The possible nitric oxide effects on the rat retina were evaluated by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a specific marker for NO-producing neurons, detected by histochemistry performed on whole retinas and retina sections. Immunohistochemistry was also performed on retina sections for iNOS, 3-nitrotyrosine (3-NT) and glial fibrillary acidic protein (GFAP). Retinal nitric oxide levels were assessed by measuring total nitrate/nitrite concentrations. Retinal cGMP levels were determined by radioimmunoassay. Western blots for nitrotyrosine (3-NT) and oxidized proteins were performed.. In the present study, we found increased activity of NADPH-diaphorase and iNOS immunoreactivity in the rat retina at the onset of diabetes; this increase correlated with a remarkable increase in NO levels as early as 7 days after the onset of diabetes. However, cGMP levels were not modified by diabetes, suggesting that NO did not activate its signaling cascade. Even so, Western blots revealed a progressive increase in nitrated proteins at 7 days after diabetes induction. Likewise, positive nitrotyrosine immunolabeling was observed in the photoreceptor layer, ganglion cell layer, inner nuclear layer and some Müller cell processes in the retinas of diabetic rats. In addition, levels of oxidized proteins were increased in the retina early after diabetes induction; these levels were reduced by the administration of L-NAME. In addition, stress in Müller cells was determined by immunoreactivity to the glial fibrillary acidic protein.. Our findings indicated the occurrence of nitrosative stress at the onset of diabetes in the rat retina and emphasized the role of NO in retinal function and the pathogenesis of retinopathy.

Topics: Animals; Cyclic GMP; Diabetes Mellitus, Experimental; Glial Fibrillary Acidic Protein; Immunohistochemistry; Microscopy, Fluorescence; NADPH Dehydrogenase; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Protein Carbonylation; Rats; Rats, Long-Evans; Retina; Streptozocin; Tyrosine

Heart failure and related cardiac complications remains a great health challenge. We investigated the effects of upregulating heme-oxygenase (HO) on myocardial histo-pathological lesions, proinflammatory cytokines/chemokines, oxidative mediators and important markers of heart failure such as osteopontin and osteoprotergerin in N(ω)-nitro-l-arginine methyl ester (L-NAME)-induced hypertension. Treatment with the HO-inducer, heme-arginate improved myocardial morphology in L-NAME hypertensive rats by attenuating subendocardial injury, interstitial fibrosis, mononuclear-cell infiltration and cardiomyocyte hypertrophy. These were associated with the reduction of several inflammatory/oxidative mediators including chemokines/cytokines such as macrophage inflammatory protein-1 alpha (MIP-1α), macrophage chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, endothelin-1, 8-isoprostane, nitrotyrosine, and aldosterone. Similarly, heme-arginate abated the elevated levels of extracellular matrix/remodeling proteins including transforming-growth factor beta (TGF-β1) and collagen-IV in the myocardium. These were accompanied by significant reduction of proteins of heart failure such as osteopontin and osteoprotegerin. Interestingly, the cardio-protective effects of heme-arginate were associated with the potentiation of adiponectin, atrial-natriuretic peptide (ANP), HO-1, HO-activity, cyclic gnanosine monophosphate (cGMP) and the total-anti-oxidant capacity, whereas the HO-inhibitor, chromium-mesoporphyrin nullified the effects of heme-arginate, exacerbating inflammatory injury and oxidative insults. We conclude that heme-arginate therapy protects myocardial damage by potentiating the HO-adiponectin-ANP axis, which in turn suppressed the elevated levels of aldosterone, pro-inflammatory chemokines/cytokines, mononuclear-cell infiltration and oxidative stress, with concomitant reduction of extracellular matrix/remodeling proteins and heart failure proteins. These data suggest a cardio-protective role of the HO system against L-NAME-induced hypertension that could be explored in the design of novel strategies against cardiomyopathy.

Topics: Adiponectin; Aldosterone; Animals; Antioxidants; Arginine; Atrial Natriuretic Factor; Biomarkers; Blood Pressure; Cardiomyopathies; Cardiotonic Agents; Chemokine CCL2; Chemokine CCL3; Cyclic GMP; Dinoprost; Endothelin-1; Enzyme Induction; Extracellular Matrix Proteins; Heart Failure; Heme; Heme Oxygenase (Decyclizing); Hypertension; Interleukin-1beta; Interleukin-6; Male; NG-Nitroarginine Methyl Ester; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Tyrosine

Bioconversion of glyceryl trinitrate (GTN) into nitric oxide (NO) by aldehyde dehydrogenase-2 (ALDH-2) is a crucial mechanism which drives vasodilatory and antiplatelet effect of organic nitrates in vitro and in vivo. Oxidative stress generated by overproduction of free radical species, mostly superoxide anions and NO-derived peroxynitrite, has been suggested to play a pivotal role in the development of nitrate tolerance, though the mechanism still remains unclear. Here we studied the free radical-dependent impairment of ALDH-2 in platelets as well as vascular tissues undergoing organic nitrate ester tolerance and potential benefit when using the selective peroxynitrite decomposition catalyst Mn(III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP). Washed human platelets were made tolerant to nitrates via incubation with GTN for 4h. This was expressed by attenuation of platelet aggregation induced by thrombin (40U/mL), an effect accompanied by GTN-related induction of cGMP levels in platelets undergoing thrombin-induced aggregation. Both effects were associated to attenuated GTN-induced nitrite formation in platelets supernatants and to prominent nitration of ALDH-2, the GTN to NO metabolizing enzyme, suggesting that GTN tolerance was associated to reduced NO formation via impairment of ALDH-2. These effects were all antagonized by co-incubation of platelets with MnTBAP, which restored GTN-induced responses in tolerant platelets. Comparable effect was found under in in vivo settings. Indeed, MnTBAP (10mg/kg, i.p.) significantly restored the hypotensive effect of bolus injection of GTN in rats made tolerants to organic nitrates via chronic administration of isosorbide-5-mononitrate (IS-5-MN), thus confirming the role of peroxynitrite overproduction in the development of tolerance to vascular responses induced by organic nitrates. In conclusion, oxidative stress subsequent to prolonged use of organic nitrates, which occurs via nitration of ALDH-2, represents a key event in GTN tolerance, an effect counteracted both in vitro and in vivo by novel peroxynitrite decomposition catalyst.

Topics: Aldehyde Dehydrogenase; Animals; Aorta; Blood Platelets; Cyclic GMP; Drug Tolerance; Endothelium, Vascular; Humans; Hypotension; Isosorbide Dinitrate; Male; Metalloporphyrins; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitrites; Nitroglycerin; Platelet Aggregation; Rats; Thrombin; Tyrosine; Vasodilator Agents

The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress-induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion.. Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and inhibition of PKG (KT5823) or NOS (l-NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2(-)) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS.. These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2(-) production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling.

Topics: Animals; Biopterins; Cell Death; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Ischemic Postconditioning; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitrates; Nitric Oxide Synthase Type III; Nitrites; Oxidative Stress; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Superoxides; Time Factors; Tyrosine

The role of nitric oxide (NO) in the human pancreas and in pancreatitis still remains controversial. Furthermore, conflicting conclusions have been reached by different laboratories about the localization of the NO-generating enzyme (NO synthase, NOS) in the pancreas. Here, we investigated the co-expression of NOS with enzymes involved in regulation of NO signalling in the normal human pancreas and in pancreatitis. We found that the whole NO signalling machinery was up-regulated in pancreatitis, especially within the exocrine compartment. Furthermore, the exocrine parenchymal cells revealed higher levels of oxidative stress markers, nitrotyrosine and 8-hydroxyguanosine, in pancreatitis, which reflects the exceptional susceptibility of the exocrine parenchyma to oxidative stress. This study provides a direct link between oxidative stress and the enzymatic control of the NO bioavailability at the cellular level and endows with further insight into fundamental mechanisms underlying pancreatic disorders associated with disruptions in the L-arginine-NO-cGMP signalling enzyme cascade.

Topics: Aged; Arginine; Biomarkers; Case-Control Studies; Cyclic GMP; Female; Fluorescent Antibody Technique; Guanosine; Humans; Immunoenzyme Techniques; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Pancreas; Pancreatitis; Signal Transduction; Tyrosine

Cirrhotic patients with minimal hepatic encephalopathy (MHE) show impaired driving ability and increased vehicle accidents. The neurological deficits contributing to impair driving and the underlying mechanisms are poorly understood. Early detection of driving impairment would help to reduce traffic accidents in MHE patients. It would be therefore useful to have psychometric or biochemical parameters reflecting driving impairment. The aims of this work were as follows: (i) to shed light on the neurological deficits contributing to impair driving; (ii) to assess whether some psychometric test or biochemical parameter is a good indicator of driving impairment.. We assessed in 22 controls, 36 cirrhotic patients without and 15 with MHE, driving performance using a driving simulator (SIMUVEG) and Driver Test. MHE was diagnosed using the psychometric hepatic encephalopathy score (PHES). Psychometric tests assessing different neurological functions (mental processing speed, attention, visuo-spatial and bimanual coordination) were performed. Blood ammonia and parameters related with nitric oxide-cGMP metabolism, IL-6, IL-18 and 3-nitrotyrosine were measured.. Patients with MHE showed impaired driving ability correlating with MHE grade, with impaired vehicle lateral control in spite of reduced driving speed. Patients with MHE show psychomotor slowing, longer reaction times, impaired bimanual and visuo-spatial coordination and concentrated attention and slowed speed of anticipation and increased blood ammonia, cGMP, IL-6, IL-18 and 3-nitrotyrosine.. Impaired mental processing speed, attention and alterations in visuo-spatial and motor coordination seem main contributors to impaired driving ability in patients with MHE. Increased serum 3-nitrotyrosine is associated with impaired driving ability.

Topics: Adult; Aged; Analysis of Variance; Automobile Driving; Biomarkers; Chemokines; Cyclic GMP; Flicker Fusion; Hepatic Encephalopathy; Humans; Liver Cirrhosis; Middle Aged; Nitric Oxide; Psychometrics; Tyrosine

Nitric oxide (NO) seems to play an important role during renal ischemia/reperfusion (I/R) injury. We investigated whether rutin inhibits inducible nitric oxide synthase (iNOS) and reduces 3-nitrotyrosine (3-NT) formation in the kidneys of rats during I/R.. Wistar albino rats were nephrectomized unilaterally and, 2 weeks later, subjected to 45 minutes of left renal pedicle occlusion followed by 3 hours of reperfusion. We intraperitoneally administered L-N6-(1-iminoethyl)lysine (L-NIL; 3 mg/kg) for 30 minutes or rutin (1 g/kg) for 60 minutes before I/R. After reperfusion, kidney samples were taken for immunohistochemical analysis of iNOS and 3-NT. We measured plasma nitrite/nitrate and cyclic guanosine monophosphate (cGMP) to evaluate NO levels.. Ischemia/reperfusion caused plasma cGMP to increase significantly. Similarly, plasma nitrite/nitrate was elevated in the I/R group compared with the control group. Histochemical staining was positive for iNOS and 3-NT in the I/R group. Pretreatment with L-NIL or rutin significantly mitigated the elevation of plasma cGMP and nitrite/nitrate. These changes in biochemical parameters were also associated with changes in immunohistochemical appearance. Pretreatment with L-NIL or rutin significantly decreased the incidence and severity of iNOS and 3-NT formation in the kidney tissues.. Our findings suggest that high activity of iNOS causes renal I/R injury, and that rutin exerts protective effects, probably by inhibiting iNOS.

Topics: Animals; Biomarkers; Cyclic GMP; Enzyme Inhibitors; Immunohistochemistry; Infusions, Parenteral; Kidney; Lysine; Male; Nephrectomy; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Protective Agents; Rats; Rats, Wistar; Reperfusion Injury; Rutin; Tyrosine

Cardiovascular disease (CVD) is associated with vascular oxidative imbalance and inflammation. Increased reactive oxygen species (ROS) generation is associated with a functional inactivation of nitric oxide (NO) due to the reaction with O₂⁻, leading to peroxynitrite (ONOO⁻) formation and subsequent reduction in the beneficial effect of vascular NO bioavailability. Carotenoids'-rich diets have been associated with decreased risk of CVD, but the underlying mechanism is still unknown.. In human umbilical vein endothelial cells (HUVECs), both β-carotene (BC) or lycopene (Lyc) significantly affected tumor necrosis factor-α (TNF-α)-induced inflammation, being associated with a significant decrease in the generation of ROS (spectrofluorometry) and nitrotyrosine (an index of ONOO⁻ formation, cytofluorimetry), an increased NO/cGMP (cyclic guanosine monophosphate) levels (EIA), and a down-regulation of NF-κB-dependent adhesion molecule expression (Western blot and EMSA) and monocyte-HUVEC interaction (adhesion assay). Our results indicate that BC or Lyc treatment reduce the inflammatory response in TNF-α-treated HUVECs. This is due to the redox balance protection and to the maintenance of NO bioavailability.. Our observations provide background for a novel mechanism for carotenoids' anti-inflammatory activity in the vasculature and may contribute to a better understanding of the protective effects of carotenoid-rich diets against CVD risk.

Topics: Anti-Inflammatory Agents, Non-Steroidal; beta Carotene; Biological Availability; Carotenoids; Cell Adhesion; Cell Adhesion Molecules; Cyclic GMP; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Lycopene; Monocytes; NF-kappa B; Nitric Oxide; Oxidative Stress; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Tyrosine

NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O(2) for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-l-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-l-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22(phox) and p47(phox) and decreased the expression of Nox2 and p22(phox) in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity.

Topics: Acetophenones; Animals; Animals, Newborn; Biopterins; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelium, Vascular; Hypertension, Pulmonary; Lung; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Norepinephrine; Pulmonary Artery; Reactive Oxygen Species; Sheep; Superoxides; Tyrosine; Vasoconstriction; Vasodilation

Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (F(passive)) and cardiomyocyte hypertrophy. In experimental models, both reacted favorably to raised protein kinase G (PKG) activity. The present study assessed myocardial PKG activity, its downstream effects on cardiomyocyte F(passive) and cardiomyocyte diameter, and its upstream control by cyclic guanosine monophosphate (cGMP), nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF).. Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. F(passive) was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Lower PKG activity in HFPEF than in aortic stenosis (P<0.01) or HFREF (P<0.001) was associated with higher cardiomyocyte F(passive) (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/oxidative stress (P<0.05). Higher F(passive) in HFPEF was corrected by in vitro PKG administration.. Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte F(passive) and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

Topics: Aortic Valve Stenosis; Biopsy; Cohort Studies; Comorbidity; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Diabetes Mellitus; Female; Heart; Heart Failure; Humans; Male; Middle Aged; Myocardium; Natriuretic Peptide, Brain; Obesity; Oxidative Stress; Stroke Volume; Tyrosine

Oxidative stress has been implicated in the development of vascular disease and in the promotion of endothelial dysfunction via the reduction in bioavailable nitric oxide (NO()). Glutathione (GSH) is a tripeptide thiol antioxidant that is utilized by glutathione peroxidase (GPx) to scavenge reactive oxygen species such as hydrogen peroxide and phospholipid hydroperoxides. Relatively frequent single-nucleotide polymorphisms (SNPs) within the 5' promoters of the GSH synthesis genes GCLC and GCLM are associated with impaired vasomotor function, as measured by decreased acetylcholine-stimulated coronary artery dilation, and with increased risk of myocardial infarction. Although the influence of genetic knockdown of GPx on vascular function has been investigated in mice, no work to date has been published on the role of genetic knockdown of GSH synthesis genes on vascular reactivity. We therefore investigated the effects of targeted disruption of Gclm in mice and the subsequent depletion of GSH on vascular reactivity, NO() production, aortic nitrotyrosine protein modification, and whole-genome transcriptional responses as measured by DNA microarray. Gclm(-/+) and Gclm(-/-) mice had 72 and 12%, respectively, of wild-type (WT) aortic GSH content. Gclm(-/+) mice had a significant impairment in acetylcholine (ACh)-induced relaxation in aortic rings as well as increased aortic nitrotyrosine protein modification. Surprisingly, Gclm(-/-) aortas showed enhanced relaxation compared to Gclm(-/+) aortas, as well as increased NO() production. Although aortic rings from Gclm(-/-) mice had enhanced ACh relaxation, they had a significantly increased sensitivity to phenylephrine (PE)-induced contraction. Alternatively, the PE response of Gclm(-/+) aortas was nearly identical to that of their WT littermates. To examine the role of NO() or other potential endothelium-derived factors in differentially regulating vasomotor activity, we incubated aortic rings with the NO() synthase inhibitor L-NAME or physically removed the endothelium before PE treatment. L-NAME treatment and endothelium removal enhanced PE-induced contraction in WT and Gclm(-/+) mice, but this effect was severely diminished in Gclm(-/-) mice, indicating a potentially unique role for GSH in mediating vessel contraction. Whole-genome assessment of aortic mRNA in Gclm(-/-) and WT mice revealed altered expression of genes within the canonical Ca(2+) signaling pathway, which may have a role in mediating these observe

Topics: Acetylcholine; Adrenergic alpha-1 Receptor Agonists; Animals; Aorta; Cholinergic Agonists; Cyclic GMP; Glutamate-Cysteine Ligase; Glutathione; In Vitro Techniques; Male; Metabolic Networks and Pathways; Mice; Mice, Inbred C57BL; Mice, Knockout; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oligonucleotide Array Sequence Analysis; Phenylephrine; Reactive Oxygen Species; Transcriptome; Tyrosine; Vasoconstriction; Vasodilation

Methamphetamine (METH) is a potent stimulant that induces both acute and long-lasting neurochemical changes in the brain including neuronal cell loss. Our laboratory demonstrated that the neuropeptide substance P enhances the striatal METH-induced production of nitric oxide (NO). In order to better understand the role of the striatal neuropeptides on the METH-induced production of NO, we used agonists and antagonists of the NPY (Y1R and Y2R) receptors infused via intrastriatal microinjection followed by a bolus of METH (30 mg/kg, ip) and measured 3-NT immunofluorescence, an indirect index of NO production. One striatum received pharmacological agent while the contralateral striatum received aCSF and served as control. NPY receptor agonists dose dependently attenuated the METH-induced production of striatal 3-NT. Conversely, NPY receptor antagonists had the opposite effect. Moreover, METH induced the accumulation of cyclic GMP and activated caspase-3 in approximately 18% of striatal neurons, a phenomenon that was attenuated by pre-treatment with NPY2 receptor agonist. Lastly, METH increased the levels of striatal preproneuropeptide Y mRNA nearly five-fold 16 h after injection as determined by RT-PCR, suggesting increased utilization of the neuropeptide. In conclusion, NPY inhibits the METH-induced production of NO in striatal tissue. Consequently, production of this second messenger induces the accumulation of cyclic GMP and activated caspase-3 in some striatal neurons, an event that may precede the apoptosis of some striatal neurons.

Topics: Analysis of Variance; Animals; Arginine; Caspase 3; Corpus Striatum; Cyclic GMP; Drug Interactions; Male; Methamphetamine; Mice; Mice, Inbred ICR; Microinjections; Neuropeptide Y; Nitric Oxide Synthase Type II; Peptides; Protein Precursors; Receptors, Neuropeptide Y; RNA, Messenger; Tyrosine

The thromboxane receptor (TPr) and multiple TPr ligands, including thromboxane A(2) (TxA(2)) and prostaglandin H(2), are elevated during vascular and atherothrombotic diseases. How TPr stimulation causes vascular injury remains poorly defined. This study was conducted to investigate the mechanism by which TPr stimulation leads to vascular injury.. Exposure of bovine aortic endothelial cells to either [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(d'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1] heptan-2-yl]-5'-heptenoic acid (IBOP) or U46619, 2 structurally related TxA(2) mimetics, for 24 hours markedly increased the release of superoxide anions (O(2)(·-)) and peroxynitrite (ONOO(-)) but reduced cyclic GMP, an index of nitric oxide bioactivity. IBOP also significantly suppressed activity of endothelial nitric oxide synthase (eNOS), increased enzyme-inactive eNOS monomers, and reduced levels of tetrahydrobiopterin, an essential eNOS cofactor. IBOP- and U46619-induced increases in O(2)(·-) were accompanied by the membrane translocation of the p67(phox) subunit of NAD(P)H oxidase. Pharmacological or genetic inhibition of either NAD(P)H oxidase or TPr abolished IBOP-induced O(2)(·-) formation. Furthermore, TPr activation significantly increased protein kinase C-ζ (PKC-ζ) in membrane fractions and PKC-ζ phosphorylation at Thr410. Consistently, PKC-ζ inhibition abolished TPr activation-induced membrane translocation of p67(phox) and O(2)(·-) production. Finally, exposure of isolated mouse aortae to IBOP markedly increased O(2)(·-) in wild-type but not in those from gp91(phox) knockout mice.. We conclude that TPr activation via PKC-ζ-mediated NAD(P)H oxidase activation increases both O(2)(·-) and ONOO(-), resulting in eNOS uncoupling in endothelial cells.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Apoptosis; Biopterins; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cell Survival; Cells, Cultured; Cyclic GMP; Cytochrome P-450 Enzyme System; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxynitrous Acid; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Transport; Receptors, Immunologic; Receptors, Thromboxane A2, Prostaglandin H2; RNA Interference; Signal Transduction; Superoxides; Time Factors; Tyrosine

The purposes of these studies were to quantify the concentrations of total nitrate and nitrite (NO(x)(-)) cyclic guanosine monophosphate (cGMP), and nitrotyrosine over skin surface in normal weight healthy volunteers (n = 64) compared to overweight/obese subjects (n = 54). A semi-circular plastic tube was taped to the skin along acupuncture points (acupoints), meridian line without acupoint (MWOP), and nonmeridian control and filled with a 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution for 20 min. The concentrations of NO(x)(-), cGMP, and nitrotyrosine in the samples were quantified in a blinded fashion using chemiluminescence and enzyme-linked immunosorbent assay, respectively. In normal weight healthy volunteers, NO(x)(-) and cGMP concentrations were consistently increased over the pericardium meridian (PC) 4-7 compared with nonmeridian areas. NO(x)(-) concentration is enhanced over the bladder meridian (BL) 56-57, but cGMP level is similar between the regions. In overweight/obese subjects, NO(x)(-) contents were increased or tended to be elevated over PC and BL regions. cGMP is paradoxically decreased over PC acupoints and nonmeridian control on the forearm but the decreases were blunted along BL regions on the leg. Nitrotyrosine concentrations are markedly elevated (five- to sixfold) over both PC and BL in all areas of overweight/obese subjects. This is the first evidence showing that nitrotyrosine level is tremendously elevated over skin accompanied by paradoxical changes in nitric oxide (NO)-cGMP concentrations over PC skin region in overweight/obese subject. The results suggest that NO-related oxidant inflammation is systemically enhanced while cGMP generation is impaired over PC skin region but not over BL region in obesity.

Topics: Acupuncture Points; Adult; Cyclic GMP; Cyclic N-Oxides; Female; Humans; Imidazoles; Male; Nitrates; Nitric Oxide; Nitrites; Obesity; Skin; Tyrosine

The diabetic heart is known to be susceptible to ischemia/reperfusion (I/R) injury by increased oxidative stress. Although oxidative stress upregulates inducible nitric oxide (iNOS), the role of iNOS in I/R injury in the diabetic heart has been poorly understood. Because iNOS-derived nitric oxide (NO) plays a crucial role in cardioprotection against I/R injury, we hypothesized that inhibition of iNOS uncoupling would restore tolerance to I/R injury in the diabetic heart. The present study demonstrated that iNOS-derived superoxide generation was reduced, and that the NO bioavailability was increased, by treatment with the NOS-cofactor, tetrahydrobiopterin (BH4), before I/R in the hearts isolated from diabetic rats. This was associated with a reduction of infarct size and improvement of left ventricular (LV) function after I/R. The cardioprotective effect of BH4 was abrogated by treatment with a thiol reducing agent dithiothreitol (DTT), but not a NO-sensitive guanylyl cyclase inhibitor ODQ, suggesting that iNOS-derived NO-mediated cardioprotection occurs through protein S-nitrosylation but not cGMP-dependent signaling in the diabetic heart. Indeed, protein S-nitrosylation was increased by treatment with BH4 in the diabetic heart and was inhibited by DTT. These results suggest that the inhibition of iNOS uncoupling unmasks tolerance to I/R injury through enhanced protein S-nitrosylation in the diabetic rat heart.

Topics: Animals; Biopterins; Cyclic GMP; Diabetes Complications; Diabetes Mellitus, Experimental; Dithiothreitol; Imines; Male; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Synthase Type II; Oxadiazoles; Oxidative Stress; Quinoxalines; Rats; Rats, Sprague-Dawley; Superoxides; Tiopronin; Tyrosine; Up-Regulation; Ventricular Function, Left

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO) and atrial natriuretic peptide (ANP) systems in the kidney in association with the angiotensin (Ang) II-induced hypertension. Male Sprague-Dawley rats were used. Ang II (100 ng·min⁻¹·kg⁻¹) was infused through entire time course. Thirteenth day after beginning the regimen, kidneys were taken. The protein expression of NO synthase (NOS) and nitrotyrosine was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed hypertension and decreased creatinine clearance in the experimental group. The protein expression of eNOS, nNOS and nitrotyrosine was increased in the cortex, while that of iNOS remained unaltered. The urinary excretion of NO increased in Ang II-induced hypertensive rats. The catalytic activity of soluble guanylyl cyclase was blunted in the glomerulus in Ang II-induced hypertensive rats. The mRNA expression of ANP was increased in Ang II-induced hypertensive rats. Neither the expression of NPR-A nor that of NPR-C was changed. The protein expression of neutral endopeptidase was decreased and the activity of particulate guanylyl cyclase was blunted in the glomerulus and papilla in Ang II-induced hypertensive rats. In conclusion, the synthesis of NO and ANP was increased in the kidney of Ang II-induced hypertension, while stimulated cGMP response was blunted. These results suggest desensitization of guanylyl cyclase in the kidney of Ang II-induced hypertensive rats, which may contribute to the associated renal vasoconstriction and hypertension.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Blood Pressure; Cyclic GMP; Hypertension; Kidney; Kidney Function Tests; Male; Neprilysin; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Transcription, Genetic; Tyrosine

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension. IH causes oxidative stress that may limit bioavailability of the endothelium-derived vasodilator nitric oxide (NO) and thus contribute to this hypertensive response. We therefore hypothesized that increased vascular superoxide anion (O(2)(-)) generation reduces NO-dependent pulmonary vasodilation following IH. To test this hypothesis, we examined effects of the O(2)(-) scavenger tiron on vasodilatory responses to the endothelium-dependent vasodilator ionomycin and the NO donor S-nitroso-N-acetylpenicillamine in isolated lungs from hypocapnic-IH (H-IH; 3 min cycles of 5% O(2)/air flush, 7 h/day, 4 wk), eucapnic-IH (E-IH; cycles of 5% O(2), 5% CO(2)/air flush), and sham-treated (air/air cycled) rats. Next, we assessed effects of endogenous O(2)(-) on NO- and cGMP-dependent vasoreactivity and measured O(2)(-) levels using the fluorescent indicator dihydroethidium (DHE) in isolated, endothelium-disrupted small pulmonary arteries from each group. Both E-IH and H-IH augmented NO-dependent vasodilation; however, enhanced vascular smooth muscle (VSM) reactivity to NO following H-IH was masked by an effect of endogenous O(2)(-). Furthermore, H-IH and E-IH similarly increased VSM sensitivity to cGMP, but this response was independent of either O(2)(-) generation or altered arterial protein kinase G expression. Finally, both H-IH and E-IH increased arterial O(2)(-) levels, although this response was more pronounced following H-IH, and H-IH exposure resulted in greater protein tyrosine nitration indicative of increased NO scavenging by O(2)(-). We conclude that IH increases pulmonary VSM sensitivity to NO and cGMP. Furthermore, endogenous O(2)(-) limits NO-dependent vasodilation following H-IH through an apparent reduction in bioavailable NO.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium-Dependent Relaxing Factors; Free Radical Scavengers; Hypertrophy, Right Ventricular; Hypocapnia; Hypoxia; Ionomycin; Lung; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Polycythemia; Pulmonary Artery; Rats; Rats, Wistar; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Superoxides; Tyrosine; Vasodilation

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.

Topics: Animals; Antioxidants; Aorta; Biopterins; Catalase; Cyclic GMP; GTP Cyclohydrolase; Hydrogen Peroxide; Male; Mesenteric Arteries; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphorylation; Serine; Superoxide Dismutase; Superoxides; Tyrosine

Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of extracellular matrix and loss of pulmonary function. No cure exists for this pathologic condition, and current treatments often fail to slow its progression or relieve its symptoms. Relaxin was previously shown to induce a matrix-degrading phenotype in human lung fibroblasts in vitro and to inhibit pulmonary fibrosis in vivo. A novel peptide that targets the relaxin RXFP1/LGR7 receptor was recently identified using our computational platform designed to predict novel G protein-coupled receptor peptide agonists. In this study, we examined the antifibrotic properties of this novel peptide, designated CGEN25009, in human cell-based assays and in a murine model of bleomycin-induced pulmonary fibrosis. Similar to relaxin, CGEN25009 was found to have an inhibitory effect on transforming growth factor-β1-induced collagen deposition in human dermal fibroblasts and to enhance MMP-2 expression. The peptide's biological activity was also similar to relaxin in generating cellular stimulation of cAMP, cGMP, and NO in the THP-1 human cell line. In vivo, 2-week administration of CGEN25009 in a preventive or therapeutic mode (i.e., concurrently with or 7 days after bleomycin treatment, respectively) caused a significant reduction in lung inflammation and injury and ameliorated adverse airway remodeling and peribronchial fibrosis. The results of this study indicate that CGEN25009 displays antifibrotic and anti-inflammatory properties and may offer a new therapeutic option for the treatment of pulmonary fibrosis.

Topics: Animals; Bleomycin; Bronchi; Cell Line, Tumor; Collagen; Cyclic AMP; Cyclic GMP; Fibroblasts; Goblet Cells; Humans; Lung; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Monocytes; Muscle, Smooth; Nitric Oxide; Peptides; Peroxidase; Pulmonary Fibrosis; Receptors, G-Protein-Coupled; Receptors, Peptide; Relaxin; Signal Transduction; Thiobarbituric Acid Reactive Substances; Transforming Growth Factor beta1; Tyrosine

Hyperhomocysteinaemia has been associated with increased risk of thrombosis and atherosclerosis. Homocysteine produces endothelial injury and stimulates platelet aggregation. Several molecular mechanisms related to these effects have been elucidated. The study aimed to deeply investigate the homocysteine effect on nitric oxide formation in human platelets. The homocysteine-induced changes on nitric oxide, cGMP, superoxide anion levels and nitrotyrosine formation were evaluated. The enzymatic activity and the phosphorylation status of endothelial nitric oxide synthase (eNOS) at thr495 and ser1177 residues were measured. The protein kinase C (PKC), assayed by immunofluorescence confocal microscopy technique and by phosphorylation of p47pleckstrin, and NADPH oxidase activation, tested by the translocation to membrane of the two cytosolic subunits p47(phox) and p67(phox), were assayed. Results show that homocysteine reduces platelet nitric oxide and cGMP levels. The inhibition of eNOS activity and the stimulation of NADPH oxidase primed by PKC appear to be involved. PKC stimulates the eNOS phosphorylation of the negative regulatory residue thr495 and the dephosphorylation of the positive regulatory site ser1177. GF109203X and U73122, PKC and phospholipase Cgamma2 pathway inhibitors, respectively, reverse this effect. Moreover, homocysteine stimulates superoxide anion elevation and NADPH oxidase activation. These effects are significantly decreased by GF109203X and U73122, suggesting the involvement of PKC in NADPH oxidase activation. Homocysteine induces formation of the peroxynitrite biomarker nitrotyrosine. Taken together these results suggest that the homocysteine-mediated responses leading to nitric oxide impairment are mainly coupled to PKC activation. Thus homocysteine stimulates platelet aggregation and decreases nitric oxide bioavailability.

Topics: Analysis of Variance; Blood Platelets; Citrulline; Cyclic GMP; Enzyme Activation; Enzyme Inhibitors; Homocysteine; Humans; Microscopy, Fluorescence; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphorylation; Protein Kinase C; Superoxides; Tyrosine

Endothelial dysfunction occurs in heart disease and may reduce functional capacity via attenuations in peripheral blood flow. Dietary decosahexaenoic acid (DHA) may improve this dysfunction, but the mechanism is unknown. This study determined if DHA enhances expression and activity of eNOS in cultured human coronary artery endothelial cells (HCAEC). HCAEC from 4 donors were treated with 5 nM, 50 nM, or 1 microM DHA for 7 days to model chronic DHA exposure. A trend for increased expression of endothelial nitric oxide synthase (eNOS) and phospho-eNOS was observed with 5 and 50 nM DHA. DHA also enhanced expression of 2 proteins instrumental in activation of eNOS: phospho-Akt (5 and 50 nM) and HSP90 (50 nM and 1 microM). Vascular endothelial growth factor-induced activation of Akt increased NOx in treated (50 nM DHA) versus untreated HCAEC (9.2 +/- 1.0 vs 3.3 +/- 1.1 micromol/microg protein/microL). Findings suggest that DHA enhances eNOS and Akt activity, augments HSP90 expression, and increases NO bioavailability in response to Akt kinase activation.

Topics: Analysis of Variance; Blotting, Western; Cells, Cultured; Coronary Vessels; Cyclic GMP; Dietary Supplements; Docosahexaenoic Acids; Dose-Response Relationship, Drug; Endothelial Cells; Female; HSP90 Heat-Shock Proteins; Humans; Male; Nitric Oxide Synthase Type III; Phosphorylation; Proto-Oncogene Proteins c-akt; Time Factors; Tyrosine; Vascular Endothelial Growth Factors

The vascular mediator, nitric oxide regulates vascular smooth muscle cell proliferation and can react with superoxide to form peroxynitrite, a highly reactive free radical. The intracellular mechanisms by which nitric oxide and peroxynitrite inhibit smooth muscle cell growth remain undefined, as is the potential role of peroxynitrite formation in the antiproliferative effects of nitric oxide. We sought to define the intracellular effects and signaling mechanisms of nitric oxide and peroxynitrite in smooth muscle cells.. Cultured rat aortic smooth muscle cells were treated with exogenous nitric oxide or peroxynitrite and inhibitors of nitric oxide and redox signaling pathways. Cell growth, DNA synthesis, apoptosis, cyclic guanosine 3'-5' monophosphate (cGMP) levels, poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity, and cytotoxicity were assayed. Peroxynitrite formation was determined by nitrotyrosine immunoblotting. Vasoreactivity was assessed in isolated rat aortic rings after treatment with nitric oxide/peroxynitrite and redox agents.. Both exogenous nitric oxide and peroxynitrite decreased cell growth and DNA synthesis of cultured rat aortic smooth muscle cells, but peroxynitrite-induced growth arrest was irreversible and associated with apoptosis and cytotoxicity. Inhibition of guanylate cyclase, PARP activity, mitogen-activated protein kinase, or bypass of ornithine decarboxylase did not reverse growth arrest by nitric oxide. The antioxidants N-acetylcysteine, ascorbate, and glutathione selectively reversed growth inhibition by nitric oxide but not by peroxynitrite. Antioxidants did not impair nitric oxide-induced cGMP generation in smooth muscle cells or nitric oxide-induced vasodilatation of isolated aortic rings. Nitric oxide treatment did not result in peroxynitrite formation and augmentation of superoxide levels did not induce peroxynitrite-like effects. Peroxynitrite-induced cytotoxicity and apoptosis were not reversed by antioxidants or PARP inhibition, because peroxynitrite activated PARP in J774 macrophages but failed to activate PARP in smooth muscle cells.. Exogenous nitric oxide induces reversible cytostasis in smooth muscle cells by a redox-sensitive mechanism independent of peroxynitrite formation and distinct from the nitric oxide vasodilating mechanism. Peroxynitrite does not activate PARP selectively in smooth muscle cells and induces redox-independent smooth muscle cell cytotoxicity and apoptosis. Thus, the antiproliferative effects of nitric oxide and peroxynitrite on smooth muscle cells use divergent intracellular pathways with distinct redox sensitivities. These findings are relevant to the pathogenesis of vascular disease and the potential application of nitric oxide-based therapy for vascular disease.. Vascular smooth muscle cell proliferation is an important component of atherosclerosis, vein graft failure, and arterial restenosis, and is known to be regulated by the vascular signaling molecule nitric oxide. Nitric oxide can combine with the free radical superoxide to form the unstable metabolite peroxynitrite, which has been detected in human vascular lesions. This study examines the role of peroxynitrite in mediating the antiproliferative effects of nitric oxide. We identify important differences in the effects and intracellular mechanisms of nitric oxide and peroxynitrite in regulating vascular smooth muscle cell proliferation and programmed cell death. Defining the differential effects of these free radicals in vascular cells is important to our understanding of the pathogenesis of vascular disease and the development of novel therapy aimed at treating proliferative vascular lesions.

Topics: Animals; Antioxidants; Apoptosis; Cell Proliferation; Cell Survival; Cells, Cultured; Cyclic GMP; DNA Replication; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide; Nitric Oxide Donors; Oxidation-Reduction; Peroxynitrous Acid; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rats; Signal Transduction; Superoxides; Time Factors; Tyrosine; Vasodilation

The Notch signaling pathway appears to perform an important function in inflammation. Here, we present evidence to suggest that lipopolysaccharide (LPS) suppresses Notch signaling via the direct modification of Notch by the nitration of tyrosine residues in macrophages. In the RAW264.7 macrophage cell line and in rat primary alveolar macrophages, LPS was found to inhibit Notch1 intracellular domain (Notch1-IC) transcription activity, which could then be rescued by treatment with N(G)-nitro-l-arginine, a nitric oxide synthase (NOS) inhibitor. Nitric oxide (NO), which was produced in cells that stably express endothelial NOS (eNOS) and brain NOS (bNOS), also induced the inhibition of Notch1 signaling. The NO-induced inhibition of Notch1 signaling remained unchanged after treatment with 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylyl-cyclase inhibitor, and was not found to be mimicked by 8-bromo-cyclic GMP in the primary alveolar macrophages. With regards to the control of Notch signaling, NO appears to have a significant negative influence, via the nitration of Notch1-IC, on the binding that occurs between Notch1-IC and RBP-Jk, both in vitro and in vivo. By intrinsic fluorescence, we also determined that nitration could mediate conformational changes of Notch1-IC. The substitution of phenylalanine for tyrosine at residue 1905 in Notch1-IC abolished the nitration of Notch1-IC by LPS. Overall, our data suggest that an important relationship exists between LPS-mediated inflammation and the Notch1 signaling pathway, and that this relationship intimately involves the nitration of Notch1-IC tyrosine residues.

Topics: Animals; Cyclic GMP; Down-Regulation; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Lipopolysaccharides; Macrophages; Mice; NIH 3T3 Cells; Nitric Oxide; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Protein Transport; Rats; Rats, Sprague-Dawley; Receptor, Notch1; Signal Transduction; Subcellular Fractions; Transcriptional Activation; Tyrosine

Although the role of nitric oxide (NO) in peripheral glucose uptake has been thoroughly described, little is known regarding the alterations in NO metabolism during the early onset of insulin resistance. During this study we investigated the alterations in NO synthesis and bioavailability in a model for dietary modulations of insulin sensitivity. For 6 weeks, rats were fed a standard diet (C), a high-sucrose diet inducing insulin resistance (HS), or high-sucrose diets supplemented with cysteine, which endowed protection against the high-sucrose-induced insulin resistance (Ti). Several markers of NO synthesis and bioavailability were assessed and confronted with markers of insulin sensitivity. After 5 weeks, although urinary cGMP excretion did not differ between the groups, insulin resistance in HS rats was associated with both a significant increase in NO oxidation, as determined by plasma nitrotyrosine concentrations, and in the inducible NO synthase (iNOS)/endothelial NO synthase (iNOS/eNOS) mRNA ratio in skeletal muscle compared with C rats. These alterations were prevented in rats fed the cysteine-rich diets. NO production, as assessed by urinary 15NO3* excretion following a [15N2-(guanido)]-arginine intra-venous bolus, independently and significantly correlated with insulin sensitivity but did not significantly differ between C, HS, and Ti rats; neither did the aortic eNOS protein expression or skeletal muscle insulin-induced eNOS activation. Our results indicate that in this model of dietary modulations of insulin sensitivity (i) NO production accounts for part of total inter-individual variation in insulin sensitivity, but (ii) early diet-related changes in insulin sensitivity are accompanied by changes in NO bioavailability.

Topics: Animals; Aorta; Biomarkers; Cyclic GMP; Cysteine; Diet; Diet Therapy; Disease Models, Animal; Enzyme Activation; Gene Expression Regulation, Enzymologic; Insulin Resistance; Male; Muscle, Skeletal; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Sucrose; Sweetening Agents; Tyrosine

The beneficial effects of reperfusion therapies have been limited by the amount of ischemic damage that occurs before reperfusion. To enable development of interventions to reduce cell injury, our research has focused on understanding mechanisms involved in cardiac cell death after ischemia/reperfusion (I/R) injury. In this context, our laboratory has been investigating the role of the receptor for advanced-glycation end products (RAGE) in myocardial I/R injury.. In this study we tested the hypothesis that RAGE is a key modulator of I/R injury in the myocardium. In ischemic rat hearts, expression of RAGE and its ligands was significantly enhanced. Pretreatment of rats with sRAGE, a decoy soluble part of RAGE receptor, reduced ischemic injury and improved functional recovery of myocardium. To specifically dissect the impact of RAGE, hearts from homozygous RAGE-null mice were isolated, perfused, and subjected to I/R. RAGE-null mice were strikingly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH, improved functional recovery, and increased adenosine triphosphate (ATP). In rats and mice, activation of the RAGE axis was associated with increases in inducible nitric oxide synthase expression and levels of nitric oxide, cyclic guanosine monophosphate (cGMP), and nitrotyrosine.. These findings demonstrate novel and key roles for RAGE in I/R injury in the heart. The findings also demonstrate that the interaction of RAGE with advanced-glycation end products affects myocardial energy metabolism and function during I/R.

Topics: Animals; Cyclic GMP; Energy Metabolism; Male; Mice; Mice, Knockout; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Synthase Type II; Rats; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Tyrosine; Up-Regulation

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.

Topics: Algal Proteins; Aniline Compounds; Antibodies, Anti-Idiotypic; Cells, Cultured; Cyclic GMP; Fluoresceins; Fungal Proteins; Molsidomine; Nicotiana; Nitric Oxide; Peroxynitrous Acid; Phytophthora; Proteins; Reactive Oxygen Species; Superoxides; Tyrosine; Uric Acid

The organophosphate insecticide mevinphos (Mev) acts on the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates, to elicit phasic cardiovascular responses via nitric oxide (NO) generated by NO synthase (NOS) I and II. We evaluated the contribution of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade and peroxynitrite in this process. PKG expression in ventrolateral medulla of Sprague-Dawley rats manifested an increase during the sympathoexcitatory phase (Phase I) of cardiovascular responses induced by microinjection of Mev bilaterally into the RVLM that was antagonized by co-administration of 7-nitroindazole or Nomega-propyl-L-arginine, two selective NOS I inhibitors or 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (ODQ), a selective sGC antagonist. Co-microinjection of ODQ or two PKG inhibitors, KT5823 or Rp-8-Br-cGMPS, also blunted the Mev-elicited sympathoexcitatory effects. However, the increase in nitrotyrosine, a marker for peroxynitrite, and the sympathoinhibitory circulatory actions during Phase II Mev intoxication were antagonized by co-administration of S-methylisothiourea, a selective NOS II inhibitor, Mn(III)-tetrakis-(4-benzoic acid) porphyrin, a superoxide dismutase mimetic, 5,10,15,20-tetrakis-N-methyl-4'-pyridyl)-porphyrinato iron (III), a peroxynitrite decomposition catalyst, or L-cysteine, a peroxynitrite scavenger. We conclude that sGC/cGMP/PKG cascade and peroxynitrite formation may participate in Mev-induced phasic cardiovascular responses as signals downstream to NO generated respectively by NOS I and II in the RVLM.

Topics: Animals; Blood Pressure; Blotting, Western; Cardiovascular Physiological Phenomena; Cholinesterase Inhibitors; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Heart Rate; Male; Medulla Oblongata; Mevinphos; Microinjections; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Rats; Spectrum Analysis; Superoxides; Time Factors; Tyrosine

The goal of this investigation was to explore the mechanism by which NOS and NO serve to regulate events linked to chondrocyte terminal differentiation. NOS isoform expression and NO adducts in chick growth cartilage were detected by immunohistochemistry and Western blot analysis. All NOS isoforms were expressed in chick growth plate chondrocytes with the highest levels present in the hypertrophic region. The enzymes were active since nitrosocysteine and nitrotyrosine residues were detected in regions of the epiphysis with the highest levels of NOS expression. Maturing chick sternal chondrocytes evidenced an increase in NO release and a rise in NOS protein levels. When treated with NOS inhibitors, there was a decrease in the alkaline phosphatase activity of the hypertrophic cells. On the other hand, NO donors caused a small but significant elevation in alkaline phosphatase activity. Transient transfections of chondrocytes with an endothelial NOS isoform caused an increase in collagen type X promoter activity. Induction of both collagen type X expression and alkaline phosphatase activity was blocked by inhibitors of the cGMP pathway. These findings indicate that NO is generated by three NOS isoforms in terminally differentiated chondrocytes. The expression of NOS and the generation of NO enhanced maturation by upregulating alkaline phosphatase and collagen type X expression. Since expression of these two determinants was blocked by inhibitors of the cGMP pathway, it is concluded that NO metabolism is required for development of the mature chondrocyte phenotype.

Topics: Alkaline Phosphatase; Animals; Blotting, Western; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chick Embryo; Chondrocytes; Collagen Type X; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cysteine; Gene Expression; Growth Plate; Guanylate Cyclase; Immunohistochemistry; Isoenzymes; Luciferases; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Promoter Regions, Genetic; Recombinant Fusion Proteins; S-Nitrosoglutathione; S-Nitrosothiols; Sternum; Transfection; Tretinoin; Tyrosine

Recent studies suggest that adipose tissue hormone, leptin, is involved in the pathogenesis of arterial hypertension. However, the mechanism of hypertensive effect of leptin is incompletely understood. We investigated whether antioxidant treatment could prevent leptin-induced hypertension. Hyperleptinemia was induced in male Wistar rats by administration of exogenous leptin (0.25 mg/kg twice daily s.c. for 7 days) and separate groups were simultaneously treated with superoxide scavenger, tempol, or NAD(P)H oxidase inhibitor, apocynin (2 mM in the drinking water). After 7 days, systolic blood pressure was 20.6% higher in leptin-treated than in control animals. Both tempol and apocynin prevented leptin-induced increase in blood pressure. Plasma concentration and urinary excretion of 8-isoprostanes increased in leptin-treated rats by 66.9% and 67.7%, respectively. The level of lipid peroxidation products, malonyldialdehyde + 4-hydroxyalkenals (MDA+4-HNE), was 60.3% higher in the renal cortex and 48.1% higher in the renal medulla of leptin-treated animals. Aconitase activity decreased in these regions of the kidney following leptin administration by 44.8% and 45.1%, respectively. Leptin increased nitrotyrosine concentration in plasma and renal tissue. Urinary excretion of nitric oxide metabolites (NO(x)) was 57.4% lower and cyclic GMP excretion was 32.0% lower in leptin-treated than in control group. Leptin decreased absolute and fractional sodium excretion by 44.5% and 44.7%, respectively. Co-treatment with either tempol or apocynin normalized 8-isoprostanes, MDA+4-HNE, aconitase activity, nitrotyrosine, as well as urinary excretion of NO(x), cGMP and sodium in rats receiving leptin. These results indicate that oxidative stress-induced NO deficiency is involved in the pathogenesis of leptin-induced hypertension.

Topics: Acetophenones; Aconitate Hydratase; Aldehydes; Animals; Antioxidants; Blood Pressure; Body Weight; Creatine; Cyclic GMP; Cyclic N-Oxides; Drinking; Eating; Hypertension; Isoprostanes; Kidney; Leptin; Male; Malondialdehyde; Natriuresis; Nitric Oxide; Rats; Rats, Wistar; Reactive Nitrogen Species; Sodium; Spin Labels; Tyrosine

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.

Topics: Adolescent; Adult; Blotting, Western; Cell Survival; Chemotaxis, Leukocyte; Cyclic GMP; Eosinophils; Female; Humans; In Vitro Techniques; Male; Middle Aged; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Nitric Oxide Donors; Nitroprusside; Penicillamine; Tetrazolium Salts; Thiazoles; Tyrosine

We measured changes in nitric oxide (NO) concentration in the cerebral cortex during experimental carbon monoxide (CO) poisoning and assessed the role for N-methyl-d-aspartate receptors (NMDARs), a glutamate receptor subtype, with progression of CO-mediated oxidative stress. Using microelectrodes, NO concentration was found to nearly double to 280 nM due to CO exposure, and elevations in cerebral blood flow, monitored as laser Doppler flow (LDF), were found to loosely correlate with NO concentration. Neuronal nitric oxide synthase (nNOS) activity was the cause of the NO elevation based on the effects of specific NOS inhibitors and observations in nNOS knockout mice. Activation of nNOS was inhibited by the NMDARs inhibitor, MK 801, and by the calcium channel blocker, nimodipine, thus demonstrating a link to excitatory amino acids. Cortical cyclic GMP concentration was increased due to CO poisoning and shown to be related to NO, versus CO, mediated guanylate cyclase activation. Elevations of NO were inhibited when rats were infused with superoxide dismutase and in rats depleted of platelets or neutrophils. When injected with MK 801 or 7-nitroindazole, a selective nNOS inhibitor, rats did not exhibit CO-mediated nitrotyrosine formation, myeloperoxidase (MPO) elevation (indicative of neutrophil sequestration), or impaired learning. Similarly, whereas CO-poisoned wild-type mice exhibited elevations in nitrotyrosine and myeloperoxidase, these changes did not occur in nNOS knockout mice. We conclude that CO exposure initiates perivascular processes including oxidative stress that triggers activation of NMDA neuronal nNOS, and these events are necessary for the progression of CO-mediated neuropathology.

Topics: Animals; Brain Chemistry; Calcium Channel Blockers; Carbon Monoxide Poisoning; Cyclic GMP; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Indazoles; Laser-Doppler Flowmetry; Male; Maze Learning; Mice; Mice, Knockout; Microelectrodes; Neurons; Neurotoxicity Syndromes; Neutropenia; Neutrophils; NG-Nitroarginine Methyl Ester; Nimodipine; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Platelet Count; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Tyrosine

We investigated the role of nitric oxide (NO) on mitochondrial complexes activity, following short-term non-desensitizing activation of AMPA receptors with kainate (KA) plus cyclothiazide (CTZ), in cultured rat hippocampal neurons. In these conditions, we observed a decrease in the activity of mitochondrial complexes I, II/III, and IV. A selective neuronal nitric oxide synthase inhibitor, 7-Nitroindazole, prevented the decrease in the activity of mitochondrial complex I, but not for the other complexes. Exposure to KA plus CTZ also increased cyclic GMP levels significantly, and led to increased levels of 3-nitrotyrosine, a biomarker for peroxynitrite production. Taken together, our results suggest that non-desensitizing activation of AMPA receptors causes inhibition of mitochondrial complex I via peroxynitrite.

Topics: Analysis of Variance; Animals; Antihypertensive Agents; Benzothiadiazines; Cells, Cultured; Cyclic GMP; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Immunohistochemistry; Kainic Acid; Microtubule-Associated Proteins; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peroxynitrous Acid; Proton Pumps; Quinoxalines; Rats; Receptors, AMPA; Tyrosine

Increased superoxide anion (O(2)(-).) may contribute to vascular dysfunction in aging. In aged cavernosal tissue, lucigenin-enhanced chemiluminescence demonstrated a threefold increase in superoxide formation, and the oxidative fluorescent probe hydroethidine indicated higher superoxide levels throughout the aged penis. This increase in superoxide was associated with impaired cavernosal nerve-mediated and agonist-induced erectile responses, increased nitrotyrosine staining, and lower cGMP levels, but no compensatory change in cavernosal extracellular (EC)-superoxide dismutase (EC-SOD) mRNA or protein. In vivo adenoviral (Ad) gene transfer of EC-SOD to the penis resulted in higher expression of EC-SOD mRNA, protein, SOD activity, cGMP levels, and lower nitrotyrosine staining. Transfection with AdCMVEC-SOD resulted in a significant increase in erectile response to cavernosal nerve stimulation, ACh, and zaprinast to a magnitude similar to young rats. These data provide evidence in support of the hypothesis that erectile dysfunction associated with aging is related in part to an increase in cavernosal O(2)(-). formation. Gene-transfer of EC-SOD reduces superoxide formation and restores age-associated erectile function and may represent a novel therapeutic target for the treatment of erectile dysfunction.

Topics: Adenoviridae; Aging; Animals; beta-Galactosidase; Cell Line; Cyclic GMP; Genetic Vectors; Humans; Male; Microscopy, Confocal; Penile Erection; Penis; Rats; Rats, Inbred BN; Recombinant Fusion Proteins; RNA, Messenger; Superoxide Dismutase; Superoxides; Transfection; Tyrosine

In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Topics: Animals; Aorta; Apolipoproteins E; Arteriosclerosis; Ascorbic Acid; Biopterins; Cyclic AMP; Cyclic GMP; Dietary Supplements; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; In Vitro Techniques; Lipids; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Superoxides; Time; Tyrosine; Vasomotor System; Vitamin E

Myocardial ischemia and reperfusion induced by cardioplegic arrest subjects the heart to free radical-mediated stress. The purpose of our study was to investigate the effect of cardioplegia-induced ischemia and reperfusion on myocardial formation and distribution of (1) nitrotyrosine as an indicator for peroxynitrite-mediated tissue injury resulting from increased nitric oxide release and (2) 8-isoprostane as an indicator for oxygen-derived free radical-mediated lipid peroxidation.. In 10 patients undergoing coronary artery operations (64 +/- 6 [mean +/- SD] years, 3 women and 7 men) subjected to cardiopulmonary bypass and intermittent cold blood cardioplegia, we collected transmural left ventricular biopsy specimens before and at the end of cardiopulmonary bypass. Specimens were cut at 10 micro m and subjected to immunocytochemical staining against the nitric oxide-producing enzyme constitutive nitric oxide synthase, cyclic guanosine monophosphate (intracellular second messenger of nitric oxide), nitrotyrosine, and 8-isoprostane by using polyclonal antibodies. For global left ventricular function determination, we measured the fractional area of contraction using transesophageal echocardiography.. Nitric oxide synthase activity in cardiac myocytes increased from 34 +/- 10 gray units before cardiopulmonary bypass to 47 +/- 12 gray units at the end of bypass (P =.015), and all hearts showed increased cyclic guanosine monophosphate content in both myocytes and endothelial cells at the end of bypass. The number of nitrotyrosine-positive capillaries increased from 36 +/- 29/mm(2) before bypass to 82 +/- 47/mm(2) at the end of bypass (P =.012), and 8-isoprostane-positive capillaries increased from 92 +/- 72/mm(2) before bypass to 209 +/- 108/mm(2) at the end of bypass (P =.005). The fractional area of contraction was 53% +/- 12% before bypass and 56% +/- 12% after bypass (P =.47) but was slightly decreased to 45% +/- 14% at 4 hours after bypass (P =.121).. Our data show that cardioplegia-induced myocardial ischemia and reperfusion is associated with nitrotyrosine and 8-isoprostane formation mainly in the coronary endothelium, indicating injury mediated by both peroxynitrite and oxygen-derived free radicals. Because nitric oxide synthase activation was accompanied with increased cyclic guanosine monophosphate, these data suggest that direct effects of nitric oxide on cardiac myocytes, as well as nitric oxide-mediated coronary endothelial injury, might contribute to injury associated with cardioplegia and cardiopulmonary bypass.

Topics: Coronary Vessels; Cyclic GMP; Dinoprost; Endothelium, Vascular; F2-Isoprostanes; Female; Heart Arrest, Induced; Humans; Male; Middle Aged; Myocardial Reperfusion Injury; Nitric Oxide; Reactive Oxygen Species; Tyrosine

Hyperhomocysteinemia is associated with endothelial dysfunction, although the underlying mechanism is unknown. Previous studies have shown that nitric oxide (NO) plays an important role in the regulation of systemic and renal hemodynamics. This study investigated whether hyperhomocysteinemia induces renal oxidative stress and promotes renal dysfunction involving disturbances of the NO-pathway in Wistar rats. During 8 wk, control (C) and hyperhomocysteinemic (HYC) groups had free access to tap water and homocysteine-thiolactone (HTL, 50 mg/kg per d), respectively. At 8 wk, plasma homocysteine concentration, renal superoxide anion (O(2)), nitrotyrosine, and nitrite+nitrate levels, and renal function were measured. To assess NO involvement, the responses to L-Arginine (L-Arg, 300 mg/kg) and N(G)-nitro-L-arginine-methyl-ester (L-NAME, 20 microg/kg per min for 60 min) were analyzed. The HYC group showed higher homocysteine concentration (7.6 +/- 1.7 versus 4.9 +/- 1.0 micromol/L; P < 0.001), (O(2) production (157.92 +/- 74.46 versus 91.17 +/- 29.03 cpm. 10(3)/mg protein), and nitrite+nitrate levels (33.4 +/- 5.1 versus 11.7 +/- 4.3 micro mol/mg protein; P < 0.001) than the control group. Western blot analyses showed a nitrotyrosine mass 46% higher in the HYC group than in the controls. Furthermore, the HYC group showed lower GFR, renal plasma flow (RPF), and higher renal vascular resistance (RVR) than the controls. After L-Arg administration, the responses of GFR, RPF, and RVR were attenuated by 36%, 40%, and 50%, respectively; after L-NAME, the responses of RPF and RVR were exaggerated by 79% and 112%, respectively. This suggests a reduced NO bioavailability to produce vasodilation and an enhanced sensitivity to NO inhibition. In conclusion, hyperhomocysteinemia induces oxidative stress, NO inactivation, and renal dysfunction involving disturbances on the NO-pathway.

Topics: Animals; Cyclic GMP; Homocysteine; Hyperhomocysteinemia; Kidney Diseases; Male; Nitric Oxide; Oxidative Stress; Rats; Rats, Wistar; Renal Circulation; Thiobarbituric Acid Reactive Substances; Tyrosine

Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 micromol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O2- was not significantly reduced by 10 micromol/L oxypurinol or 30 micromol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 micromol/L) as well as the O2- scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 micromol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O2- scavenger Tiron (10 mmol/L), concomitant with 1 micromol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O2-. Incubation with 100 micromol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. These findings suggest that CsA activates a NADPH oxidase that releases O2- and disrupts the bradykinin/soluble guanylate cyclase pathway, probably by binding with NO to form peroxynitrite (ONOO-).

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Acetylcysteine; Animals; Antioxidants; Blotting, Western; Bradykinin; Cyclic GMP; Cyclosporine; Enzyme Activation; Free Radical Scavengers; GTP-Binding Proteins; Guanylate Cyclase; LLC-PK1 Cells; NADPH Oxidases; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Onium Compounds; Phospholipases; Quercetin; Receptor, Bradykinin B2; Receptors, Bradykinin; Signal Transduction; Solubility; Superoxides; Swine; Tyrosine; Vitamin K 3

Peroxisome proliferator-activated receptor (PPAR) signaling pathways have been reported to exert anti-inflammatory effects and attenuate atherosclerosis formation. However, the mechanisms responsible for their anti-inflammatory and antiatherosclerotic effects remain largely unknown. The present study tested the hypothesis that a PPARgamma agonist may exert significant endothelial protection by antioxidative and antinitrative effects.. Male New Zealand White rabbits were randomized to receive a normal (control) or a high-cholesterol diet and treated with vehicle or rosiglitazone (a PPARgamma agonist) 3 mg x kg(-1) x d(-1) for 5 weeks beginning 3 weeks after the high-cholesterol diet. At the end of 8 weeks of a high-cholesterol diet, the rabbits were killed, and the carotid arteries were isolated. Bioactive nitric oxide was determined functionally (endothelium-dependent vasodilatation) and biochemically (the phosphorylation of vasodilator-stimulated phosphoprotein, or P-VASP). Vascular superoxide production, PPARgamma, gp91phox, and inducible nitric oxide synthase (iNOS) expression, and vascular ONOO- formation were determined. Hypercholesterolemia caused severe endothelial dysfunction and reduced P-VASP, despite a marked increase in iNOS expression and total NOx production. Treatment with rosiglitazone enhanced PPARgamma expression, improved endothelium-dependent vasodilatation, preserved P-VASP, suppressed gp91phox and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation.. The PPARgamma agonist rosiglitazone exerted a significant vascular protective effect in hypercholesterolemic rabbits, most likely by attenuation of oxidative and nitrative stresses. The endothelial protective effects of PPARgamma agonists may reduce leukocyte accumulation in vascular walls and contribute to their antiatherosclerotic effect.

Topics: Animals; Antioxidants; Blood Vessels; Carotid Arteries; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Hypercholesterolemia; In Vitro Techniques; Lipids; Male; Nitrates; Nitric Oxide; Nitrites; Rabbits; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Signal Transduction; Thiazolidinediones; Transcription Factors; Tyrosine; Vasodilation

Platelets play an important role in the development of vascular disease, while vegetarian diets, which are rich in inorganic nitrate, protect against it. This study was performed to assess the effect of potassium nitrate (KNO(3)) ingestion on platelet function in humans. Oral KNO(3) (2 mmol) was given to healthy volunteers and its effect on platelet function assessed by measuring the aggregant effect of collagen. Blood samples were taken for measurement of plasma S-nitrosothiols (RSNO) and platelet cyclic GMP and nitrotyrosine levels. Gastric juice samples were taken for measurement of RSNO. In a separate study, the effect of oral KNO(3) on portal RSNO levels in patients with intrahepatic porto-systemic shunts was assessed. KNO(3) caused a significant increase in gastric RSNO levels, from 0.46 +/- 0.06 to 3.62 +/- 2.82 microM (t(max) 45 min; P < 0.001), and significantly inhibited platelet function (t(max) 60 min; P < 0.001). There was no effect on systemic or portal RSNO, platelet cGMP or platelet nitrotyrosine levels. Oral KNO(3) inhibits platelet aggregation. The time course suggests that gastric RSNO production may be involved in this effect. The protection against vascular events associated with a high intake of vegetables may be due to their high nitrate content.

Topics: Administration, Oral; Adolescent; Adult; Collagen; Cyclic GMP; Diet, Vegetarian; Female; Gastric Juice; Humans; Kinetics; Male; Nitrates; Platelet Aggregation; Platelet Function Tests; S-Nitrosothiols; Tyrosine

We investigated putative mechanisms by which nitric oxide modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in epithelial cells. Immunoprecipitation followed by Western blotting, as well as immunocytochemical and cell surface biotinylation measurements, showed that incubation of both stably transduced (HeLa) and endogenous CFTR expressing (16HBE14o-, Calu-3, and mouse tracheal epithelial) cells with 100 microm diethylenetriamine NONOate (DETA NONOate) for 24-96 h decreased both intracellular and apical CFTR levels. Calu-3 and mouse tracheal epithelial cells, incubated with DETA NONOate but not with 100 microm 8-bromo-cGMP for 96 h, exhibited reduced cAMP-activated short circuit currents when mounted in Ussing chambers. Exposure of Calu-3 cells to nitric oxide donors resulted in the nitration of a number of proteins including CFTR. Nitration was augmented by proteasome inhibition, suggesting a role for the proteasome in the degradation of nitrated proteins. Our studies demonstrate that levels of nitric oxide that are likely to be encountered in the vicinity of airway cells during inflammation may nitrate CFTR resulting in enhanced degradation and decreased function. Decreased levels and function of normal CFTR may account for some of the cystic fibrosis-like symptoms that occur in chronic inflammatory lung diseases associated with increased NO production.

Topics: Animals; Cell Line; Cell Membrane; Chlorides; Cyclic AMP; Cyclic GMP; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; HeLa Cells; Humans; Mice; Multienzyme Complexes; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Proteasome Endopeptidase Complex; Reactive Nitrogen Species; Reactive Oxygen Species; Respiratory Mucosa; Trachea; Tyrosine

Nitric oxide (NO) is produced by a group of synthase enzymes (NOS). By means of different pathways, NO exerts several functions in benign and malignant human bladder tissues. The current paper describes the NO/guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) and the NO/oxidative pathways in human bladder tissues.. Bladder carcinoma tissues were collected from 18 patients by transurethral resection procedures. Normal benign vesical tissue specimens from a further eight patients with benign diseases served as controls. Immunohistochemistry was conducted for localization of sGC, cGMP, and nitrotyrosine in benign and malignant vesical tissues, evaluating two-three tissue sections per patient.. Positive immunolabeling for sGC and cGMP was detected in vascular endothelial cells of normal and malignant vesical tissues. Those signals were most intense in bladder carcinoma tissues. Immunolabeling for sGC and cGMP was also detected in normal urothelial cells. In bladder carcinoma cells, a heterogeneous immunolabeling for sGC and cGMP was seen, with a wide spectrum of signal intensity. Positive immunostaining for sGC and cGMP was also observed in stromal round cells in benign and malignant bladder tissues. Immunolabeling for nitrotyrosine was mainly observed in endothelial cells, with a much stronger immunolabeling in bladder carcinoma tissues compared to normal benign controls. A weak immunolabeling for nitrotyrosine was also observed in bladder carcinoma cells. Normal urothelial cells did not show such nitrotyrosine expression.. The current report provides evidences that NO play several roles through different pathways in benign and malignant vesical tissues. The influences generated by NO molecules can be divided into cGMP-mediated effects (those resulting from the NO/sGC/cGMP pathway) and non-cGMP-mediated effects (those resulting from the NO/oxidative pathway). Increased angiogenesis is a cGMP-mediated effect, while nitrotyrosine production is a non cGMP-mediated oxidative effect. Such an NO/oxidative pathway is observed more often in bladder carcinoma.

Topics: Aged; Blotting, Western; Carcinoma; Cell Transformation, Neoplastic; Cyclic GMP; Free Radical Scavengers; Guanylate Cyclase; Humans; Immunohistochemistry; Neovascularization, Pathologic; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Tyrosine; Urinary Bladder Neoplasms

Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO(2)). The effect of LNO(2) on human platelets was examined to define how nitrated lipids might behave in vivo. LNO(2), but not LA or 3-nitrotyrosine, dose dependently (0.5-10 microm) inhibited thrombin-mediated aggregation of washed human platelets, with concomitant attenuation of P-selectin expression and selective phosphorylation of VASP at the cAMP-dependent protein kinase selective site, serine 157. LNO(2) caused slight mobilization of calcium (Ca(2+)) from intracellular stores but significantly inhibited subsequent thrombin-stimulated Ca(2+) elevations. LNO(2) did not elevate platelet cGMP, and its effects were not blocked with inhibitors of NO signaling (oxyhemoglobin, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. 2-fold elevations in cAMP were found following LNO(2) treatment of platelets, and the adenylyl cyclase inhibitors 2',5'-dideoxyadenosine and SQ22536 partially restored thrombin-stimulated aggregation. Finally, LNO(2) significantly inhibited cAMP hydrolysis to AMP by platelet lysates. These data implicate cAMP in the anti-aggregatory action of LNO(2). The platelet inhibitory actions of LNO(2) indicate that nitration reactions that occur following NO generation in an oxidizing environment can alter the activity of lipids and lend insight into mechanisms by which NO-derived species may modulate the progression of vascular injury.

Topics: 1-Methyl-3-isobutylxanthine; Blood Platelets; Calcium; Calcium Signaling; Cyclic AMP; Cyclic GMP; Humans; In Vitro Techniques; Kinetics; Linoleic Acid; Linoleic Acids; Nitro Compounds; Phosphoproteins; Phosphorylation; Platelet Activation; Platelet Aggregation; Thrombin; Tyrosine; Vasodilator Agents

Loss of the modulatory role of the endothelium may be a critical initial factor in the development of diabetic vascular diseases. Exposure of human aortic endothelial cells (HAECs) to high glucose (30 or 44 mmol/l) for 7-10 days significantly increased the release of superoxide anion in response to the calcium ionophore A23187. Nitrate, a breakdown product of peroxynitrite (ONOO(-)), was substantially increased in parallel with a decline in cyclic guanosine monophosphate (GMP). Using immunochemical techniques and high-performance liquid chromatography, an increase in tyrosine nitration of prostacyclin (PGI(2)) synthase (PGIS) associated with a decrease in its activity was found in cells exposed to high glucose. Both the increase in tyrosine nitration and the decrease in PGIS activity were lessened by decreasing either nitric oxide or superoxide anion, suggesting that ONOO(-) was responsible. Furthermore, SQ29548, a thromboxane/prostaglandin (PG) H(2) (TP) receptor antagonist, significantly reduced the increased endothelial cell apoptosis and the expression of soluble intercellular adhesion molecule-1 that occurred in cells exposed to high glucose, without affecting the decrease in PGIS activity. Thus, exposure of HAECs to high glucose increases formation of ONOO(-), which causes tyrosine nitration and inhibition of PGIS. The shunting of arachidonic acid to the PGI(2) precursor PGH(2) or other eicosanoids likely results in TP receptor stimulation. These observations can explain several abnormalities in diabetes, including 1) increased free radicals, 2) decreased bioactivity of NO, 3) PGI(2) deficiency, and 4) increased vasoconstriction, endothelial apoptosis, and inflammation via TP receptor stimulation.

Topics: Aorta; Apoptosis; Cell Adhesion Molecules; Cells, Cultured; Cyclic GMP; DNA Fragmentation; Endothelium, Vascular; Glucose; Humans; Nitrates; Peroxynitrous Acid; Prostaglandin-Endoperoxide Synthases; Receptors, Prostaglandin; Receptors, Thromboxane A2, Prostaglandin H2; Superoxides; Tyrosine

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.

Topics: Animals; Cattle; Cells, Cultured; Cyclic GMP; Diabetes Complications; Diabetes Mellitus; Diabetic Retinopathy; Endothelium, Vascular; Glucose; Guanidines; Hyperglycemia; Immunohistochemistry; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Retina; Streptozocin; Tyrosine

Deprivation of trophic factors induces expression of neuronal nitric oxide synthase (NOS) and nitric oxide production in cultured motor neurons, leading to apoptosis. Motor neuron apoptosis requires the simultaneous production of nitric oxide and superoxide and is associated with increased nitrotyrosine immunoreactivity. Nitric oxide also stimulates cyclic guanosine 5' monophosphate (cGMP) synthesis, which enhances the survival of motor neurons treated with brain derived trophic factor (BDNF). Here we report that cGMP analogs blocked neuronal NOS induction, nitrotyrosine accumulation, and prevented apoptosis for up to 3 day of motor neurons deprived of trophic factors. Low concentrations of exogenous nitric oxide (<100 nM), which are not toxic for BDNF-treated cultures, reversed the protective effect of cGMP. These results suggest that elevation of cGMP could decrease nitric oxide production, and thereby preventing motor neuron apoptosis.

Topics: Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Cells, Cultured; Cyclic GMP; Embryo, Mammalian; Free Radical Scavengers; Growth Substances; Immunohistochemistry; Motor Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Rats; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Spinal Cord; Tyrosine

The aim of this study is to investigate the status of oxidative stress and nitric oxide related parameters in type II diabetes mellitus (DM) patients in which heart disease, atherosclerosis, retinopathy, and nephropathy commonly occur, and also to determine the effect of glycemic control on these parameters.. Erythrocyte copper zinc-superoxide dismutase (CuZn-SOD), erythrocyte and plasma selenium dependent glutathione peroxidase (Se-GPx), erythrocyte catalase (CAT) activities, erythrocyte and plasma thiobarbituric acid reactive substances (TBARS) levels; nitrite/nitrate (NO(2)(-)/NO(3)(-)), cyclic guanosine monophosphate (cGMP) and nitrotyrosine levels in plasma of type II DM patients were measured.. Erythrocyte CuZn-SOD activities in type II DM were significantly higher than those of the control subjects (p < 0.05). TBARS levels in type II DM were significantly higher than the control subjects (p < 0.001). Plasma NO(2)(-)/NO(3)(-) levels in type II DM patients both during poor glycemic control and after three months of oral antidiabetic treatment were significantly higher than those of the control subjects (p < 0.001). Plasma cGMP levels in type II DM patients during poor glycemic control were significantly lower than those of control subjects (p < 0.001).. These results indicate that oxidative status and nitric oxide metabolism are affected in type II DM patients. We found high CuZn-SOD activity in type II DM patients. This increased activity could not protect the patients against the reactive oxygen species (ROS), since lipid peroxidation (defined by erythrocyte and plasma TBARS levels) still occurs in DM patients. After the therapy with oral antidiabetic agents for three months, erythrocyte SE-GPx and CAT activities were found to be decreased below the control values. Our results suggested that the low cGMP levels in the study may be a good marker of endothelium dysfunction in DM.

Topics: Adult; Aged; Case-Control Studies; Catalase; Cyclic GMP; Diabetes Mellitus, Type 2; Erythrocytes; Female; Glutathione Peroxidase; Humans; Hyperglycemia; Hypoglycemic Agents; Male; Middle Aged; Nitrates; Nitric Oxide; Nitrites; Oxidative Stress; Reactive Oxygen Species; Selenium; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Time Factors; Tyrosine

We investigated whether impaired platelet-derived nitric oxide (PDNO) bioactivity and augmented platelet aggregability in chronic smokers are related to the imbalance of the intraplatelet redox state through increased oxidative stress.. Chronic smoking impairs PDNO release and augments platelet aggregability. However, their mechanisms are unknown.. Collagen-induced PDNO release, platelet aggregation, plasma and intraplatelet vitamin C and reduced glutathione (GSH), intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) and intraplatelet nitrotyrosine production, which is a marker of the peroxynitrite formation, were measured in 11 chronic smokers and 10 age-matched nonsmokers.. Release of PDNO and levels of intraplatelet cGMP were lower, and platelet aggregation was greater, in smokers than in nonsmokers. Intraplatelet vitamin C and GSH levels were lower in smokers than in nonsmokers. Intraplatelet nitrotyrosine production was greater in smokers than in nonsmokers. Next, we investigated the effects of oral vitamin C administration (2 g). After vitamin C administration, intraplatelet vitamin C levels were increased and not different at 2 h between the two groups. Then, PDNO release, intraplatelet cGMP levels and platelet aggregation in smokers were restored to the levels of nonsmokers. In smokers, PDNO release and consumption of GSH during platelet aggregation were inversely correlated, and consumption was much less after vitamin C administration. Vitamin C administration decreased intraplatelet nitrotyrosine production in smokers.. Impaired PDNO bioactivity and augmented platelet aggregability may be caused by an imbalance of the intraplatelet redox state through increased oxidative stress in smokers.

Topics: Administration, Oral; Adult; Antioxidants; Ascorbic Acid; Biological Availability; Blood Platelets; Case-Control Studies; Chronic Disease; Cyclic GMP; Free Radical Scavengers; Glutathione; Humans; Male; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Peroxynitrous Acid; Platelet Aggregation; Smoking; Time Factors; Tyrosine

Platelet-derived nitric oxide inhibits platelet aggregation via constitutive NO synthase (NOS). Tetrahydrobiopterin (BH(4)), a cofactor of NOS, augments NO formation, whereas its deficiency decreases NO bioactivity and increases superoxide generation by NOS. The roles of intraplatelet BH(4) in platelet aggregation and thrombus formation, however, are unknown. Accordingly, we investigated whether intraplatelet BH(4) is involved in regulating cyclic flow variations (CFVs) and platelet aggregation in a canine model with stenosed and endothelium-injured coronary arteries that mimics acute coronary syndromes in humans.. After developing CFVs, dogs received saline or BH(4) (10 or 30 mg/kg) intravenously. Intraplatelet BH(4) and cGMP levels were decreased and intraplatelet nitrotyrosine production was increased during CFVs. ADP- and U46619-induced ex vivo platelet aggregation and platelet P-selectin expression were augmented during CFVs. BH(4) administration restored intraplatelet BH(4) and cGMP levels and decreased intraplatelet nitrotyrosine production, resulting in reduced CFVs and inhibited ex vivo platelet aggregation and platelet P-selectin expression. CFVs again developed after N(G)-monomethyl-L-arginine, an inhibitor of NOS, in BH(4)-treated dogs. Ex vivo platelet NOS activity at baseline, during CFVs, and after BH(4) administration did not differ.. Intraplatelet BH(4) may play an important role in regulating thrombus formation by modulating platelet-derived nitric oxide and superoxide generation by platelet NOS.

Topics: Animals; Biopterins; Blood Platelets; Coronary Thrombosis; Cyclic GMP; Dogs; Enzyme Inhibitors; Hemodynamics; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; P-Selectin; Platelet Aggregation; Superoxides; Tyrosine

We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca(2+) signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca(2+) ([Ca(2+)](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 microM SNAP caused concentration-dependent elevation of [Ca(2+)](i) with an optimal effect at 50 microM. This was probably induced by NO itself, because no change in [Ca(2+)](i) was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (>/=200 microM) attenuated the Ca(2+) response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induced Ca(2+) transients were abolished at 800 microM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca(2+)](i), and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca(2+) homeostasis, and also affect cell migration, e.g., through effects on F-actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration.

Topics: Actins; Calcium; Calcium Signaling; Cell Adhesion; Cell Movement; Cell Size; Cyclic GMP; Dose-Response Relationship, Drug; Homeostasis; Humans; Indicators and Reagents; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Penicillamine; Tyrosine

In the present study, nitric oxide (NO)-related substances, namely NO synthase (NOS), L-citrulline, cGMP and nitrotyrosine, have been localized in the quail ovary, using NADPH-diaphorase staining and immunohistochemical methods. The results indicate the presence of the NOS isoforms, showing distinct cell-specific distribution patterns in the quail ovary. Inducible NOS is primarily present in leukocytes, endothelial NOS in granulosa cells, and neuronal NOS in nerve cells, oocytes, interstitial cells and granulosa cells of pre-hierarchal follicles and of the germinal disc region of pre-ovulatory follicles. NOS activity, indicated by the presence of L-citrulline, is observed in oocytes, nerve cells, interstitial cells and a few granulosa cells of pre-hierarchal follicles. Detection of accumulated cGMP indicates that granulosa cells of pre-hierarchal and of pre- and post-ovulatory follicles, the theca interna of pre-ovulatory follicles, and oocytes are main targets of NO. Nitrotyrosine, a marker of peroxynitrite activity, is mainly localized in atretic follicles and in post-ovulatory follicles. It is concluded that the quail ovary possesses a NO/NOS system, and that NO may be considered as a mediator involved in various ovarian processes, including atresia.

Topics: Animals; Citrulline; Coturnix; Cyclic GMP; Female; Isoenzymes; NADPH Dehydrogenase; Nitric Oxide; Nitric Oxide Synthase; Ovarian Follicle; Ovary; Tyrosine

The nose contributes the large amount of nitric oxide (NO) to exhaled air. NO is a mediator of vasodilation and yields peroxynitrite (ONOO-) by reacting with superoxide (O2-). ONOO attacks tyrosine residues to form nitrotyrosine.. The aim of this study was to examine the pathophysiological role of NO in nasal mucosa in patients with perennial nasal allergy.. We measured nitrite and nitrate (NO2-/NO3-) and 3',5'-guanosine monophosphate (cyclic GMP) in nasal lavage fluid, and also measured haemoglobin concentration in nasal mucosa as an indicator of blood volume in the patients and healthy volunteers. The deleterious role of NO was also investigated by measuring nitrotyrosine in nasal mucosa.. The NO2-/NO3- concentration in the nasal lavage fluid was 39.5+/-2.8 microM in healthy volunteers (n=40), 42.4+/-3.0 microM in patients with mild allergy (mild group, n=32), and 88.7+/-6.6 microM in patients with severe allergy (severe group, n=61). In the patients whose symptoms were improved with treatment, NO2-/NO3- levels decreased to 45.7+/-10.4 microM. The concentration of cyclic GMP in nasal lavage fluid was higher in the severe group than in the healthy volunteers. The mucosal haemoglobin index was 88+/-4 in the healthy volunteers, 67+/-4 in the mild group, and 53+/-2 in the severe group. The formation of nitrotyrosine was expressed 0.58+/-10% to total tyrosine in the severe group (n=11), but was not found in non-allergy patients (n=9).. The production of NO was increased in patients with perennial nasal allergy, but the blood flow in the nasal mucosa of patients was reduced. Nitrotyrosine formation suggests that there is a process of ONOO(-)-induced damage in mucosa of patients with the perennial nasal allergy and this damage may limit the dilatation of blood vessels, despite the presence of excessive NO.

Topics: Adult; Cyclic GMP; Hemoglobins; Humans; Nasal Lavage Fluid; Nasal Mucosa; Nitrates; Nitric Oxide; Nitrites; Rhinitis, Allergic, Perennial; Tyrosine

Our understanding of how human glial cells are induced to produce nitric oxide and how the production is regulated may allow us to better design therapeutic strategies for treating inflammatory diseases of the central nervous system in man. Cultures of human fetal astrocytes and microglia produce inducible nitric oxide synthase and nitric oxide in response to Interferon gamma and Interleukin 1 beta. The mRNA for the enzyme was induced by 2 h and returned to baseline by day 2; the protein was expressed by 24 h and was present in cells for the entire 7 days of culture. Nitric oxide was not seen in cell supernatants until day 3 reaching a peak by day 7. Footprints of nitric oxide production such as NADPH diaphorase and nitrotyrosine staining as well as cGMP production were not significantly above background until day 3 to day 4, rising steadily until day 7. These data suggest that while the type II nitric oxide synthase is induced in human glial cells within 24 h of stimulation, it is not a functionally active enzyme until 48-72 h later, implying that there is a posttranslational regulation of the enzyme limiting nitric oxide production in these cells.

Topics: Brain; Cells, Cultured; Cyclic GMP; Enzyme Induction; Gene Expression Regulation, Developmental; Humans; Interferon-gamma; Interleukin-1; Isoenzymes; Kinetics; NADPH Dehydrogenase; Neuroglia; Nitric Oxide; Nitric Oxide Synthase; Tyrosine

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Topics: Animals; Benzoates; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitrites; Serum Albumin, Bovine; Swine; Tyrosine

The aim of this investigation was to identify the mechanism by which nitric oxide inhibits neutrophil beta 2 integrin dependent adherence. Isolated rat neutrophils from blood and peritoneal exudates were exposed for 2 min to nitric oxide generated by diethylamine-NO at rates between 1.6 and 138 nmol/min. Exposure to nitric oxide at rates less than 14 nmol/min had no effect on adherence. Exposure to 14 to 56 nmol nitric oxide/min inhibited beta 2 integrin dependent adherence to endothelial cells, nylon columns, and fibrinogen-coated plates, but higher concentrations had no significant effect on adherence. Adherence by beta 2 integrins could be restored by incubating cells with dithioerythritol, phorbol 12-myristate 13-acetate, or 8-bromo cyclic GMP. Elevations in cellular cyclic GMP concentration were associated with adherence, but this did not occur after cells were exposed to concentrations of nitric oxide that inhibited beta 2 integrin-dependent adherence. Elevations in cyclic GMP did occur after cells were incubated with dithioerythritol or phorbol 12-myristate 13-acetate. Concentrations of nitric oxide that inhibited beta 2 integrin-dependent adherence also inhibited catalytic activity of membrane associated guanylate cyclase and binding of atrial natriuretic peptide, but were insufficient to activate cytosolic guanylate cyclase. Nitric oxide did not inhibit neutrophil oxidative burst or degranulation, nor effect beta 2 integrin expression or adherence that did not depend on beta 2 integrins, nor cause oxidative stress identified in terms of cellular glutathione concentration or protein nitrotyrosine. The results indicate that nitric oxide inhibited beta 2 integrins in a concentration-dependent fashion by inhibiting cell-surface transduction of signals linked to the activity of membrane-bound guanylate cyclase. The inhibitory effect could be overcome by providing cells with cyclic GMP exogenously or by stimulating cytosolic guanylate cyclase.

Topics: Animals; Atrial Natriuretic Factor; CD18 Antigens; Cell Adhesion; Cell Degranulation; Cyclic GMP; Glutathione; Guanylate Cyclase; Hydrazines; Male; Neutrophils; Nitric Oxide; Nitrogen Oxides; Protein Kinase C; Rats; Rats, Wistar; Respiratory Burst; Tetradecanoylphorbol Acetate; Tyrosine