sepiapterin and 3-nitrotyrosine

Peroxynitrite (ONOO(-)) contributes to coronary microvascular dysfunction in diabetes mellitus (DM). We hypothesized that in DM, ONOO(-) interferes with the function of coronary endothelial caveolae, which plays an important role in nitric oxide (NO)-dependent vasomotor regulation. Flow-mediated dilation (FMD) of coronary arterioles was investigated in DM (n = 41) and non-DM (n = 37) patients undergoing heart surgery. NO-mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO(-) scavenger, iron-(III)-tetrakis(N-methyl-4'pyridyl)porphyrin-pentachloride, or uric acid, whereas exogenous ONOO(-) reduced FMD in non-DM subjects. Immunoelectron microscopy demonstrated an increased 3-nitrotyrosine formation (ONOO(-)-specific protein nitration) in endothelial plasma membrane in DM, which colocalized with caveolin-1 (Cav-1), the key structural protein of caveolae. The membrane-localized Cav-1 was significantly reduced in DM and also in high glucose-exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO(-) reduced caveolae number. Correspondingly, pharmacological (methyl-β-cyclodextrin) or genetic disruption of caveolae (Cav-1 knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase (NOS) cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM patients. Thus, we propose that ONOO(-) selectively targets and disrupts endothelial caveolae, which contributes to NOS uncoupling, and, hence, reduced NO-mediated coronary vasodilation in DM patients.

Topics: Aged; Animals; Arterioles; beta-Cyclodextrins; Caveolae; Caveolin 1; Cells, Cultured; Diabetes Mellitus; Endothelial Cells; Endothelium, Vascular; Female; Humans; Male; Mice; Mice, Knockout; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type III; Peroxynitrous Acid; Pterins; Regional Blood Flow; Tyrosine; Vasodilation

1. Uncoupling of nitric oxide synthase (NOS) has been implicated in the pathogenesis of left ventricular (LV) dysfunction in diabetes mellitus. In the present study, we investigated the role of NOS uncoupling in oxidative/nitrosative stress and LV dysfunction in the diabetic mouse heart. 2. Diabetes was induced in wild-type (WT), endothelial (e) NOS knockout (eNOS(-/-)), inducible (i) NOS knockout (iNOS(-/-)) and neuronal (n) NOS knockout (nNOS(-/-)) mice by streptozotocin (STZ) treatment. 3. In the diabetic heart, iNOS, but not eNOS or nNOS, expression was increased. Levels of malondialdehyde (MDA), 4-hydroxy-noneal (HNE) and nitrotyrosine (NT), as markers of oxidative/nitrosative stress, were increased in the diabetic mouse heart, but the increase in oxidative/nitrosative stress was significantly repressed in the iNOS(-/-) diabetic mouse heart. Levels of nitrite and nitrate (NO(x)), as an index of nitric oxide, bioavailability were significantly decreased in the iNOS(-/-) diabetic mouse heart. 4. Oral administration of sepiapterin (10 mg/kg per day), a precursor of tetrahydrobiopterin (BH(4)), significantly increased BH(4) and the BH(4)/BH(2) ratio in diabetic mouse heart. Similarly, sepiapterin inhibited the formation of HNE, MDA and NT in diabetic hearts from all three genotypes, but the increase in NO(x) following sepiapterin treatment was significantly attenuated in the iNOS(-/-) diabetic mouse heart. Percentage fractional shortening (FS), evaluated by echocardiography, decreased significantly in all genotypes of diabetic mice. Sepiapterin significantly increased percentage FS in diabetic mice, except in iNOS(-/-) mice. 5. These results suggest that sepiapterin inhibits uncoupling of NOS and improves LV function presumably by increasing iNOS-derived nitric oxide in the diabetic heart.

Topics: Animals; Biopterins; Cardiotonic Agents; Coenzymes; Diabetes Mellitus, Experimental; Diabetic Cardiomyopathies; Enzyme Inhibitors; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Pterins; Tyrosine; Ventricular Dysfunction, Left; Ventricular Function, Left

Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI, depending on its coupling status. MI was created in wild-type, iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared with other genotypes of mice 4 wk after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH(4)) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH(2)), BH(4), and BH(4)-to-BH(2) ratio in the infarcted but not sham-operated heart. The increase in BH(4)-to-BH(2) ratio was associated with inhibition of nitrotyrosine formation and an increase in nitrite plus nitrate. However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in wild-type, eNOS(-/-), and nNOS(-/-) but not iNOS(-/-) mice. N(ω)-nitro-L-arginine methyl ester abrogated sepiapterin-induced increase in nitrite plus nitrate and angiogenesis and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH(4) synthesis and increasing bioavailable nitric oxide predominantly derived from iNOS.

Topics: Administration, Oral; Angiogenesis Inducing Agents; Animals; Biopterins; Blood Pressure; Cardiotonic Agents; Disease Models, Animal; Enzyme Inhibitors; Fibrosis; GTP Cyclohydrolase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pterins; Recovery of Function; Stroke Volume; Time Factors; Tyrosine; Ultrasonography; Ventricular Function, Left

Tetrahydrobiopterin (BH(4)), a cofactor of inducible nitric-oxide synthase (iNOS), is an important post-translational regulator of NO bioactivity. We examined whether treatment of cardiac allograft recipients with sepiapterin [S-(-)-2-amino-7,8-dihydro-6-(2-hydroxy-1-oxopropyl)-4-(1H)-pteridinone], a precursor of BH(4), inhibited acute rejection and apoptosis in cardiac transplants. Heterotopic cardiac transplantation was performed in Wistar-Furth donor to Lewis recipient strain rats. Recipients were treated daily after transplantation with 10 mg/kg sepiapterin. Grafts were harvested on post-transplant day 6 for analysis of BH(4) (high-performance liquid chromatography), expression of inflammatory cytokines (reverse transcription- and real-time polymerase chain reaction), iNOS (Western blots), and NO (Griess reaction and NO analyzer). Histological rejection grade was scored, and graft function was determined by echocardiography. Apoptosis, protein nitration, and oxidative stress were determined by immunohistochemistry. Treatment of allografts with sepiapterin increased cardiac BH(4) levels by 3-fold without changing protein levels of GTP cyclohydrolase, the enzyme that regulates de novo BH(4) synthesis. Sepiapterin decreased inflammatory cell infiltrate and significantly inhibited histological rejection scores and apoptosis similar in magnitude to cyclosporine. Sepiapterin also decreased nitrative and oxidative stress. Sepiapterin caused a smaller increase in left ventricular mass versus untreated allografts but without improving fractional shortening. Sepiapterin did not alter tumor necrosis factor-alpha and interferon-gamma expression, whereas it decreased interleukin (IL)-2 expression. Sepiapterin did not change total iNOS protein or monomer levels, or plasma and tissue NO metabolites levels. It is concluded that the mechanism(s) of antirejection are due in part to decreased apoptosis, protein nitration, and oxidation of cardiomyocytes, which seems to be mediated at the immune level by limiting inflammatory cell infiltration via decreased IL-2-mediated T-lymphocyte expansion.

Topics: Aldehydes; Animals; Apoptosis; Arginase; Biopterins; Cyclosporine; Cytokines; Echocardiography; Gene Expression; Graft Rejection; GTP Cyclohydrolase; Heart Transplantation; Heart Ventricles; Immunosuppressive Agents; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type II; Protein Processing, Post-Translational; Pterins; Rats; Rats, Inbred Lew; Rats, Inbred WF; Transplantation, Homologous; Transplantation, Isogeneic; Tyrosine

Endothelial nitric oxide synthase (eNOS) uncoupling is a mechanism that leads to endothelial dysfunction. Previously, we reported that shear stress-induced release of nitric oxide in vessels of aged rats was significantly reduced and was accompanied by increased production of superoxide (18, 27). In the present study, we investigated the influence of aging on eNOS uncoupling. Mesenteric arteries were isolated from young (3 mo) and aged (24 mo) C57 BL/6J mice. The expression of eNOS protein in young vs. aged mice was not significantly different. However, the aged mice had remarkable increases in the ratio of eNOS monomers to dimers and N(omega)-nitro-l-arginine methyl ester-inhibitable superoxide formation. The level of nitrotyrosine in the total protein and precipitated eNOS of aged vessels was increased compared with that in young vessels. HPLC analysis indicated a reduced level of tetrahydrobiopterin (BH4), an essential cofactor for eNOS, in the mesenteric arteries of aged mice. Quantitative PCR results implied that the diminished BH4 may result from the decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, enzymes involved in BH4 biosynthesis. When isolated and cannulated second-order mesenteric arteries (approximately 150 microm) from aged mice were treated with sepiapterin, acetylcholine-induced, endothelium-dependent vasodilation improved significantly, which was accompanied by stabilization of the eNOS dimer. These data suggest that eNOS uncoupling and increased nitrosylation of eNOS, decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, and subsequent reduced BH4 bioavailability may be important contributors of endothelial dysfunction in aged vessels.

Topics: Acetylcholine; Age Factors; Aging; Alcohol Oxidoreductases; Animals; Biopterins; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Enzyme Stability; GTP Cyclohydrolase; Male; Mesenteric Arteries; Mice; Mice, Inbred C57BL; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Oxidation-Reduction; Protein Multimerization; Pterins; Superoxides; Time Factors; Tyrosine; Vasodilation; Vasodilator Agents

Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Cell Survival; Dopamine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Mice; Mice, Inbred C57BL; Neurochemistry; Neurotoxins; Nitric Oxide Donors; Nitric Oxide Synthase; Nitro Compounds; Organ Culture Techniques; Oxidative Stress; Parkinson Disease; Proteins; Pterins; Reactive Nitrogen Species; Substantia Nigra; Tyrosine; Tyrosine 3-Monooxygenase; Uncoupling Agents

Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO. bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH4) or the BH4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO. therapy in I-R. Our data indicate that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of the eNOS enzyme with cofactor therapy is beneficial.

Topics: Animals; Biological Availability; Biopterins; Diabetes Mellitus, Type 2; Drug Synergism; Genetic Therapy; Liver; Metalloporphyrins; Mice; Mice, Inbred Strains; Mice, Transgenic; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Nitrites; Phenotype; Phosphorylation; Pterins; Reperfusion Injury; Severity of Illness Index; Tyrosine

Increased homocysteine is associated with the pregnancy complication preeclampsia and with later-life cardiovascular disease. Although elevated homocysteine persists after pregnancy, the vascular changes of preeclampsia abate with delivery, and cardiovascular disease occurs decades later. This suggests the vasculature during pregnancy may manifest increased sensitivity to homocysteine. We used the cystathionine-beta synthase (CBS)-deficient transgenic mouse to investigate whether hyperhomocysteinemia would differentially affect vascular function in nonpregnant and pregnant animals. Mesenteric arteries from nonpregnant and midpregnant (14 to 16 days) wild-type, heterozygous, and homozygous CBS-deficient transgenic mice were investigated for their response to vasoconstriction, endothelial-dependent, and endothelial-independent relaxation using an isometric wire myograph system. Endothelial-dependent vasodilation was similar in arteries from nonpregnant heterozygous and wild-type mice. In contrast, endothelial-dependent relaxation was reduced significantly in arteries from pregnant heterozygous animals compared with wild-type mice. Inhibition of NO synthesis blunted relaxation in arteries from pregnant wild-type but not pregnant heterozygous mice. Endothelial-dependent relaxation was restored by in vitro pretreatment with the tetrahydrobiopterin precursor sepiapterin. These data indicate that in pregnant mice, endothelial-dependent vasodilation is more sensitive to the effect of increased homocysteine than arteries from nonpregnant mice. This effect appears to result from a loss in NO-mediated relaxation that may be mediated by the oxidative inactivation of the NO synthase cofactor tetrahydrobiopterin.

Topics: Animals; Cystathionine beta-Synthase; Endothelium, Vascular; Female; Hyperhomocysteinemia; Mesenteric Arteries; Mice; Mice, Knockout; Mice, Transgenic; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Phenylephrine; Pregnancy; Pregnancy Complications; Pterins; Tyrosine; Vasodilation