Recoupling the cardiac nitric oxide synthases: tetrahydrobiopterin synthesis and recycling.
ABSTRACT: Nitric oxide (NO), a key regulator of cardiovascular function, is synthesized from L-arginine and oxygen by the enzyme nitric oxide synthase (NOS). This reaction requires tetrahydrobiopterin (BH4) as a cofactor. BH4 is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GTPCH) and recycled from 7,8-dihydrobiopterin (BH2) by dihydrofolate reductase. Under conditions of low BH4 bioavailability relative to NOS or BH2, oxygen activation is "uncoupled" from L-arginine oxidation, and NOS produces superoxide (O (2) (-) ) instead of NO. NOS-derived superoxide reacts with NO to produce peroxynitrite (ONOO(-)), a highly reactive anion that rapidly oxidizes BH4 and propagates NOS uncoupling. BH4 depletion and NOS uncoupling contribute to overload-induced heart failure, hypertension, ischemia/reperfusion injury, and atrial fibrillation. L-arginine depletion, methylarginine accumulation, and S-glutathionylation of NOS also promote uncoupling. Recoupling NOS is a promising approach to treating myocardial and vascular dysfunction associated with heart failure.
Project description:Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.
Project description:Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions.In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH.FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH.FA might serve as a novel therapeutic option combating PH.
Project description:Here, evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast with normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on high-performance liquid chromatography analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid, and head and neck tumors compared with normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling, including increased cGMP-dependent protein kinase (PKG) activity, decreased ?-catenin expression, and TCF4 promoter activity, and reduced NF-?B promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by a clonogenic assay, Ki67 staining, and 2[18F]fluoro-2-deoxy-D-glucose-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and, as a consequence, NOS activity generates more peroxynitrite and superoxide anion than nitric oxide, resulting in important tumor growth-promoting and antiapoptotic signaling properties.The synthetic BH4, Kuvan, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction, suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth.
Project description:Tetrahydrobiopterin (BH4) is well-known as a cofactor of phenylalanine hydroxylase (PAH) and nitric oxide synthase (NOS), but its exact role in lipogenesis is unclear. In this study, the GTP cyclohydrolase I (GTPCH) gene was overexpressed to investigate the role of BH4 in lipogenesis in oleaginous fungus Mortierella alpina. Transcriptome data analysis reveal that GTPCH expression was upregulated when nitrogen was exhausted, resulting in lipid accumulation. Significant changes were also found in the fatty acid profile of M. alpina grown on medium that contained a GTPCH inhibitor relative to that of M. alpina grown on medium that lacked the inhibitor. GTPCH overexpression in M. alpina (the MA-GTPCH strain) led to a sevenfold increase in BH4 levels and enhanced cell fatty acid synthesis and poly-unsaturation. Increased levels of nicotinamide adenine dinucleotide phosphate (NADPH) and upregulated expression of NADPH-producing genes in response to enhanced BH4 levels were also observed, which indicate a novel aspect of the NADPH regulatory mechanism. Increased BH4 levels also enhanced phenylalanine hydroxylation and nitric oxide synthesis, and the addition of an NOS or a PAH inhibitor in the MA-GTPCH and control strain cultures decreased fatty acid accumulation, NADPH production, and the transcript levels of NADPH-producing genes. Our research suggests an important role of BH4 in lipogenesis and that the phenylalanine catabolism and arginine-nitric oxide pathways play an integrating role in translating the effects of BH4 on lipogenesis by regulating the cellular NADPH pool. Thus, our findings provide novel insights into the mechanisms of efficient lipid biosynthesis regulation in oleaginous microorganisms and lay a foundation for the genetic engineering of these organisms to optimize their dietary fat yield.
Project description:The cofactor tetrahydrobiopterin (BH4) is a critical regulator of endothelial NOS (eNOS) function, eNOS-derived NO and ROS signalling in vascular physiology. To determine the physiological requirement for de novo endothelial cell BH4 synthesis for the vasomotor function of resistance arteries, we have generated a mouse model with endothelial cell-specific deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme for BH4 biosynthesis, and evaluated BH4-dependent eNOS regulation, eNOS-derived NO and ROS generation.The reactivity of mouse second-order mesenteric arteries was assessed by wire myography. High performance liquid chromatography was used to determine BH4, BH2 and biopterin. Western blotting was used for expression analysis.Gch1fl/fl Tie2cre mice demonstrated reduced GTPCH protein and BH4 levels in mesenteric arteries. Deficiency in endothelial cell BH4 leads to eNOS uncoupling, increased ROS production and loss of NO generation in mesenteric arteries of Gch1fl/fl Tie2cre mice. Gch1fl/fl Tie2cre mesenteric arteries had enhanced vasoconstriction to U46619 and phenylephrine, which was abolished by L-NAME. Endothelium-dependent vasodilatations to ACh and SLIGRL were impaired in mesenteric arteries from Gch1fl/fl Tie2cre mice, compared with those from wild-type littermates. Loss of eNOS-derived NO-mediated vasodilatation was associated with increased eNOS-derived H2 O2 and cyclooxygenase-derived vasodilator in Gch1fl/fl Tie2cre mesenteric arteries.Endothelial cell Gch1 and BH4-dependent eNOS regulation play pivotal roles in maintaining vascular homeostasis in resistance arteries. Therefore, targeting vascular Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of microvascular dysfunction in patients with cardiovascular disease.
Project description:In diabetes, endothelial nitric oxide synthase (eNOS) produces superoxide anion rather than nitric oxide, referred to as "eNOS uncoupling," which may contribute to endothelial dysfunction, albuminuria, and diabetic nephropathy. Reduced levels of endothelium-derived tetrahydrobiopterin (BH4), an essential cofactor for eNOS, promote eNOS uncoupling. Accelerated degradation of guanosine triphosphate cyclohydrolase I (GTPCH I), the rate-limiting enzyme in BH4 biosynthesis, also occurs in diabetes, suggesting that GTPCH I may have a role in diabetic microvascular disease. Here, we crossed endothelium-dominant GTPCH I transgenic mice with Ins2(+/Akita) diabetic mice and found that endothelial overexpression of GTPCH I led to higher levels of intrarenal BH4 and lower levels of urinary albumin and reactive oxygen species compared with diabetic control mice. Furthermore, GTPCH I overexpression attenuated the hyperpermeability of macromolecules observed in diabetic control mice. In addition, we treated Ins2(+/Akita) mice with metformin, which activates AMP-activated protein kinase (AMPK) and thereby slows the degradation of GTPCH I; despite blood glucose levels that were similar to untreated mice, those treated with metformin had significantly less albuminuria. Similarly, in vitro, treating human glomerular endothelial cells with AMPK activators attenuated glucose-induced reductions in phospho-AMPK, GTPCH I, and coupled eNOS. Taken together, these data suggest that maintenance of endothelial GTPCH I expression and the resulting improvement in BH4 biosynthesis ameliorate diabetic nephropathy.
Project description:Endothelial progenitor cells (EPCs) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. Endothelial nitric oxide synthase (eNOS) critically regulates EPC mobilization and function but is uncoupled in salt-sensitive hypertension because of the reduced cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 de novo synthesis, protects EPCs and its function in deoxycorticosterone acetate (DOCA)-salt mice. EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), GTPCH transgenic DOCA-salt, and BH4-deficient hph-1 mice. In WT DOCA-salt and hph-1 mice, EPCs were significantly decreased with impaired angiogenesis and adhesion, which were restored in Tg-GCH DOCA-salt mice. Superoxide (O??) and nitric oxide (NO) levels in EPCs were elevated and reduced, respectively, in WT DOCA-salt and hph-1 mice; both were rescued in Tg-GCH DOCA-salt mice. eNOS(-/-)/GCH(+/-) hybrid mice demonstrated that GTPCH preserved the circulating EPC number, reduced intracellular O?? in EPCs, and ameliorated EPC dysfunction independent of eNOS in DOCA-salt hypertension. Secreted thrombospondin-1 (TSP-1; a potent angiogenesis inhibitor) from EPCs was elevated in WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mice. In vitro treatment with BH4, polyethylene glycol-superoxide dismutase (PEG-SOD), or Nomega-nitro-L-arginine (L-NNA) significantly augmented NO and reduced TSP-1 and O?? levels from EPCs of WT DOCA-salt mice. These results demonstrated, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertensive mice, at least in part, via suppressing TSP-1 expression and oxidative stress.
Project description:Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (<6 months) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous iron-nitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function.
Project description: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.
Project description:HMG-CoA reductase inhibitors have been shown to upregulate GTP cyclohydrolase I (GTPCH-I), the key enzyme for tetrahydrobiopterin de novo synthesis and to normalize tetrahydrobiopterin levels in hyperglycemic endothelial cells. We sought to determine whether in vivo treatment with the HMG-CoA reductase inhibitor atorvastatin is able to upregulate the GTPCH-I, to recouple eNOS and to normalize endothelial dysfunction in an experimental model of diabetes mellitus.In male Wistar rats, diabetes was induced by streptozotocin (STZ, 60 mg/kg). In STZ rats, atorvastatin feeding (20 mg/kg/d, 7 weeks), normalized vascular dysfunction as analyzed by isometric tension studies, levels of circulating endothelial progenitor cells (FACS-analysis), superoxide formation (assessed by lucigenin-enhanced chemiluminescence and dihydroethidium staining), vascular levels of the phosphorylated vasodilator-stimulated phosphoprotein (P-VASP), tyrosine nitration of the prostacyclin synthase, expression of GTPCH-I, dihydrofolate reductase and eNOS, translocation of regulatory NADPH oxidase subunits rac1, p47phox and p67phox (assessed by Western blot) and vascular tetrahydrobiopterin levels as measured by HPLC. Dihydroethidine staining revealed that the reduction of vascular superoxide was at least in part due to eNOS recoupling.HMG-CoA reductase inhibition normalizes endothelial function and reduces oxidative stress in diabetes by inhibiting activation of the vascular NADPH oxidase and by preventing eNOS uncoupling due to an upregulation of the key enzyme of tetrahydrobiopterin synthesis, GTPCH-I.