7-8-dihydrobiopterin and Disease-Models--Animal

Obesity is a serious health problem associated with the pathogenesis of various metabolic diseases. Nitric Oxide (NO) plays an important role in kidney function and altered NO levels have been associated with the pathogenesis of obesity. Therefore, we aimed to study whether an early stage of obesity contributes with progression of renal failure through further NO impairment.. Male C57BL/6 mice were fed with a high-fat diet (HFD) or a normal diet (ND) during 2 weeks. All mice underwent either sham surgery (sham) or 5/6 nephrectomy (Np). One group of HFD Np mice was treated with antioxidants plus L-arginine. Kidney damage parameters were assessed and eNOS metabolism was evaluated.. Mice on a HFD increased body weight, eNOS protein and mRNA expression, and radical oxygen species (ROS). Urine nitrites excretion, urine volume, and plasma BH4 were decreased. In HFD mice, 5/6 Np further increased BH2 and urine protein concentration, ROS levels, and eNOS mRNA expression. The decrease in BH4 plasma levels and urine nitrites excretion was accentuated. NO synthesis stimulation with the antioxidants + L-arginine treatment prevented all these changes.. The early changes in NO metabolism are associated with an early stage of obesity. This effect on NO potentiates kidney damage development.

Topics: Animals; Antioxidants; Biomarkers; Biopterins; Diet, High-Fat; Disease Models, Animal; Disease Progression; Down-Regulation; Kidney; Male; Mice, Inbred C57BL; Nephrectomy; Nitric Oxide; Nitric Oxide Synthase Type III; Obesity; Oxidative Stress; Proteinuria; Reactive Oxygen Species; Renal Insufficiency; Risk Factors; RNA, Messenger; Weight Gain

Adequate production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) requires eNOS coupling promoted by tetrahydrobiopterin (BH(4)). Under pathological conditions such as hypertension, BH(4) is diminished, avoiding eNOS coupling. When eNOS is "uncoupled", it yields a superoxide anion instead of NO. Peroxisome proliferator activated receptors (NR1C) are a family of nuclear receptors activated by ligand. Clofibrate, a member of a hypolipidemic class of drugs, acts by activating the alpha isoform of NR1C. To determine the participation of NR1C1 activation in BH(4) and dihydrobiopterin (BH(2)) metabolism and its implications on eNOS coupling in hypertension, we performed aortic coarctation (AoCo) at inter-renal level on male Wistar rats in order to have a hypertensive model. Rats were divided into the following groups: Sham+vehicle (Sham-V); AoCo+vehicle (AoCo-V); Sham+clofibrate (Sham-C), and AoCo+clofibrate (AoCo-C). Clofibrate (7 days) increased eNOS coupling in the AoCo-C group compared with AoCo-V. Clofibrate also recovered the BH(4):BH(2) ratio in control values and prevented the rise in superoxide anion production, lipoperoxidation, and reactive oxygen species production. In addition, clofibrate increased GTP cyclohydrolase-1 (GTPCH-1) protein expression, which is related with BH(4) recovered production. NR1C1 stimulation re-establishes eNOS coupling, apparently through recovering the BH(4):BH(2) equilibrium and diminishing oxidative stress. Both can contribute to high blood pressure attenuation in hypertension secondary to AoCo.

Topics: Animals; Biopterins; Clofibrate; Disease Models, Animal; GTP Cyclohydrolase; Hypertension; Hypolipidemic Agents; Lipid Peroxidation; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; PPAR alpha; Rats; Rats, Wistar; Reactive Oxygen Species; Superoxides

Previously we showed that Brown Norway (BN/Mcw) rats are more resistant to myocardial ischemia-reperfusion (I/R) injury than Dahl S (SS/Mcw) rats due to increased nitric oxide (x NO) generation secondary to increased heat shock protein 90 (HSP90) association with endothelial nitric oxide synthase (NOS3). Here we determined whether increased resistance to I/R injury in BN/Mcw hearts is also related to tetrahydrobiopterin (BH(4)) and GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for BH(4) synthesis. We observed that BH(4) supplementation via sepiapterin (SP) and inhibition of GCH-1 via 2,4-diamino-6-hydroxypyrimidine (DAHP) differentially modulate cardioprotection and that SP alters the association of HSP90 with NOS3. BH(4) levels were significantly higher and 7,8-dihydrobiopterin (BH(2)) levels were significantly lower in BN/Mcw than in SS/Mcw hearts. The BH(4)-to-BH(2) ratio in BN/Mcw was more than two times that in SS/Mcw hearts. After I/R, BH(4) decreased and BH(2) increased in hearts from both strains compared with their preischemia levels. However, the increase in BH(2) in SS/Mcw hearts was significantly higher than in BN/Mcw hearts. Real-time PCR revealed that BN/Mcw hearts contained more GCH-1 transcripts than SS/Mcw hearts. SP increased recovery of left ventricular developed pressure (rLVDP) following I/R as well as decreased superoxide (O(2)(x-)) and increased x NO in SS/Mcw hearts but not in BN/Mcw hearts. DAHP decreased rLVDP as well as increased O(2)(x-) and decreased x NO in BN/Mcw hearts compared with controls but not in SS/Mcw hearts. SP increased the association of HSP90 with NOS3. These data indicate that BH(4) mediates resistance to I/R by acting as a cofactor and enhancing HSP90-NOS3 association.

Topics: Animals; Biopterins; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; GTP Cyclohydrolase; HSP90 Heat-Shock Proteins; Hypoxanthines; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase Type III; Pterins; Rats; Rats, Inbred BN; Rats, Inbred Dahl; RNA, Messenger; Species Specificity; Superoxides; Ventricular Function, Left; Ventricular Pressure

5,6,7,8-Tetrahydrobiopterin (BH(4)) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH(4), in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH(4) insufficiency, but rather results from a diminished ratio of BH(4) vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH(2)). In support of this hypothesis, [(3)H]BH(4) binding studies revealed that BH(4) and BH(2) bind eNOS with equal affinity (K(d) approximately 80 nM) and BH(2) can rapidly and efficiently replace BH(4) in preformed eNOS-BH(4) complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH(2) levels increased from undetectable to 40% of total biopterin. This BH(2) accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH(4) supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH(4):BH(2) and not absolute intracellular levels of BH(4). Reciprocally, superoxide production was found to negatively correlate with intracellular BH(4):BH(2). Hyperglycemia-associated BH(4) oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH(4):BH(2), rather than BH(4) depletion per se, as the molecular trigger for NO insufficiency in diabetes.

Topics: Animals; Biopterins; Blood Glucose; Cell Line; Diabetes Mellitus, Type 2; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Glucose; Glutathione; Mice; Mitochondria; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidation-Reduction; Protein Binding; Rats; Rats, Zucker; Superoxides; Time Factors; Tritium

NO synthase (NOS) can paradoxically contribute to the production of reactive oxygen species when l-arginine or the cofactor R-tetrahydrobiopterin (BH(4)) becomes limited. The present study examined whether NOS contributes to superoxide production in kidneys of hypertensive Dahl salt-sensitive (SS) rats compared with an inbred consomic control strain (SS-13(BN)) and tested the hypothesis that elevated dihydrobiopterin (BH(2)) levels are importantly involved in this process. This was assessed by determining the effects of l-nitroarginine methyl ester (l-NAME) inhibition of NOS on superoxide production and by comparing tissue concentrations of BH(4) and BH(2). A reverse-phase high-performance liquid chromatography method was applied for direct measurements of BH(4) and BH(2) using (S)-tetrahydrobiopterin as an internal standard. Superoxide concentrations were measured in vivo from medullary microdialysis fluid using dihydroethidine and in vitro using lucigenin. The results indicate the following: (1) that superoxide levels were elevated in the outer medulla of SS rats fed a 4% salt diet and could be inhibited by l-NAME. In contrast, l-NAME resulted in elevated superoxide production in consomic SS-13(BN) rats because of higher NOS activity; (2) SS rats showed a reduced ratio of BH(4)/BH(2) in the outer medulla that was driven by increased concentrations of BH(2); and (3) lower superoxide dismutase and catalase activities contributed to elevated reactive oxygen species in SS samples. Based on the shift of BH(4) to BH(2) and the observation of l-NAME inhibitable superoxide production, we conclude that NOS uncoupling occurs in the renal medulla of hypertensive SS rats fed a high-salt diet.

Topics: Animals; Biopterins; Disease Models, Animal; Enzyme Inhibitors; Hypertension; Kidney; Kidney Medulla; Male; NG-Nitroarginine Methyl Ester; Oxidative Stress; Rats; Rats, Inbred Dahl; Superoxides

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

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg x kg-1 x day-1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

Topics: Acetylcholine; Administration, Oral; Animals; Biopterins; Diabetes Mellitus; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Endothelium, Vascular; GTP Cyclohydrolase; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Artery, Inferior; Mice; Mice, Inbred C57BL; Neopterin; Oxidative Stress; Phenylephrine; Pterins; Vasoconstriction; Vasodilation