RESUMO
Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic refractory wounds. Endothelial nitric oxide synthase (eNOS), which critically regulates the mobilization and function of EPCs, is uncoupled in diabetes due to decreased cofactor tetrahydrobiopterin (BH4). We tested whether GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 synthesis, preserves EPC function in type 1 diabetic mice. Type 1 diabetes was induced in wild-type (WT) and GTPCH I transgenic (Tg-GCH) mice by intraperitoneal injection of streptozotocin (STZ). EPCs were isolated from the peripheral blood and bone marrow of WT, Tg-GCH, and GTPCH I-deficient hph-1 mice. The number of EPCs was significantly lower in STZ-WT mice and hph-1 mice and was rescued in STZ Tg-GCH mice. Furthermore, GTPCH I overexpression improved impaired diabetic EPC migration and tube formation. EPCs from WT, Tg-GCH, and STZ-Tg-GCH mice were administered to diabetic excisional wounds and accelerated wound healing significantly, with a concomitant augmentation of angiogenesis. Flow cytometry measurements showed that intracellular nitric oxide (NO) levels were reduced significantly in STZ-WT and hph-1 mice, paralleled by increased superoxide anion levels; both were rescued in STZ-Tg-GCH mice. Western blot analysis revealed that thrombospondin-1 (TSP-1) was significantly upregulated in the EPCs of STZ-WT mice and hph-1 mice and suppressed in STZ-treated Tg-GCH mice. Our results demonstrate that the GTPCH I/BH4 pathway is critical to preserve EPC quantity, function, and regenerative capacity during wound healing in type 1 diabetic mice at least partly through the attenuation of superoxide and TSP-1 levels and augmentation of NO level.
Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Células Endoteliais/efeitos dos fármacos , GTP Cicloidrolase/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Estresse Oxidativo/genética , Trombospondina 1/metabolismo , Cicatrização/genética , Animais , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Proliferação de Células , Regulação para Baixo/genética , Células Endoteliais/patologia , Células Endoteliais/fisiologia , GTP Cicloidrolase/genética , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cicatrização/efeitos dos fármacosRESUMO
The enzymes 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS) and GTP cyclohydrolase II (GCHII) catalyze the initial steps of both branches of the bacterial riboflavin-biosynthesis pathway. The structures and molecular mechanisms of DHBPS and GCHII as separate polypeptides are known; however, their organization and molecular mechanism as a bifunctional enzyme are unknown to date. Here, the crystal structure of an essential bifunctional DHBPS/GCHII enzyme from Mycobacterium tuberculosis (Mtb-ribA2) is reported at 3.0â Å resolution. The crystal structure revealed two conformationally different molecules of Mtb-ribA2 in the asymmetric unit that form a dimer via their GCHII domains. Interestingly, analysis of the crystal packing revealed a long `helical-like oligomer' formed by DHBPS and GCHII functional homodimers, thus generating an `open-ended' unit-cell lattice. However, size-exclusion chromatography studies suggest that Mtb-ribA2 exists as a dimer in solution. To understand the discrepancy between the oligomerization observed in solution and in the crystal structure, the DHBPS (Mtb-DHBPS) and GCHII (Mtb-GCHII) domains of Mtb-ribA2 have been cloned, expressed and purified as His-tagged proteins. Size-exclusion chromatography studies indicated that Mtb-GCHII is a dimer while Mtb-DHBPS exists as a monomer in solution. Moreover, kinetic studies revealed that the GCHII activities of Mtb-ribA2 and Mtb-GCHII are similar, while the DHBPS activity of Mtb-ribA2 is much higher than that of Mtb-DHBPS alone. Taken together, the results strongly suggest that Mtb-ribA2 exists as a dimer formed through its GCHII domains and requires full-length Mtb-ribA2 for optimal DHBPS activity.
Assuntos
Proteínas de Bactérias/química , GTP Cicloidrolase/química , Transferases Intramoleculares/química , Enzimas Multifuncionais/química , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X , GTP Cicloidrolase/fisiologia , Transferases Intramoleculares/fisiologia , Enzimas Multifuncionais/fisiologia , Multimerização Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury-evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.
Assuntos
Biopterinas/análogos & derivados , GTP Cicloidrolase/fisiologia , Limiar da Dor/fisiologia , Dor/fisiopatologia , Adulto , Oxirredutases do Álcool/antagonistas & inibidores , Animais , Biopterinas/fisiologia , Inibidores Enzimáticos/farmacologia , GTP Cicloidrolase/antagonistas & inibidores , Haplótipos , Humanos , Manejo da Dor , Estudos Prospectivos , RatosRESUMO
GTP cyclohydrolase I (GCH), an oligomeric protein composed of 10 identical subunits, is required for the synthesis of neurotransmitters; mutations in GCH are associated with dopa-responsive dystonia (DRD) and hyperphenylalaninemia. Mutated GCH proteins are unstable and prone to dominant-negative effect. We show herein that expression of the GCH mutant GCH-201E or the splicing variant GCH-II caused intracellular inclusion bodies. When Hsp27 was expressed together with the GCH mutants, Hsp27 expression decreased the formation of inclusion bodies by GCH (as assessed by immunofluorescence) and decreased the amount of insoluble GCH mutant proteins (as assessed by Western blot). Transfection of pcDNA-Hsp27-S3D, a phosphorylation-mimicry Hsp27 mutant, was more effective at the mutated GCH proteins than transfection with pcDNA-Hsp27, but okadaic acid, a phosphatase inhibitor, enhanced the effect of pcDNA-Hsp27. Hsp27-S3D also abolished the dominant-negative action of GCH-II. The mutated GCH proteins interacted with the wild-type GCH protein; the inclusion bodies were positive for lysosomal marker LAMP1, soluble in 2% SDS, and were not ubiquitinated. Phophorlyated Hsp27 also decreased the inclusion body formation by the huntingtin polyglutamines. Therefore, diseases involving mutated oligomeric proteins would be manageable by chaperone therapies.
Assuntos
GTP Cicloidrolase/fisiologia , Proteínas de Choque Térmico/farmacologia , Corpos de Inclusão/efeitos dos fármacos , Proteínas de Neoplasias/farmacologia , Animais , Células Cultivadas , Cricetinae , Imunofluorescência , GTP Cicloidrolase/genética , Proteínas de Choque Térmico HSP27 , Chaperonas Moleculares , Mutação , Ácido Okadáico/farmacologia , Fosforilação , TransfecçãoRESUMO
Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.
Assuntos
Biopterinas/análogos & derivados , GTP Cicloidrolase/fisiologia , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Vitiligo/metabolismo , Biópsia , Biopterinas/biossíntese , Estudos de Casos e Controles , Catalase/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/fisiologia , Ativação Enzimática/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/patologia , Retroalimentação Fisiológica/efeitos dos fármacos , GTP Cicloidrolase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxirredução/efeitos dos fármacos , Vitiligo/patologiaRESUMO
Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.
Assuntos
Biopterinas/análogos & derivados , GTP Cicloidrolase/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Óxido Nítrico/biossíntese , Tuberculose/imunologia , Animais , Biopterinas/genética , Biopterinas/metabolismo , Biopterinas/fisiologia , Sobrevivência Celular , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Increased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate-cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states.
Assuntos
Biopterinas/análogos & derivados , Biopterinas/fisiologia , Diabetes Mellitus Experimental/metabolismo , Endotélio Vascular/fisiologia , GTP Cicloidrolase/fisiologia , Óxido Nítrico/fisiologia , Animais , Diabetes Mellitus Experimental/terapia , GTP Cicloidrolase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Estresse Oxidativo , RNA Mensageiro/análise , Estreptozocina , Superóxidos/metabolismoRESUMO
Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O2*-) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2*- rather than NO, possibly because of a mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS coupling in vivo remains undefined because no studies have investigated the regulation of eNOS by BH4 in the absence of vascular disease states that cause pathological oxidative stress through multiple mechanisms. We investigated the stoichiometry of BH4-eNOS interactions in vivo by crossing endothelial-targeted eNOS transgenic (eNOS-Tg) mice with mice overexpressing endothelial GTP cyclohydrolase 1 (GCH-Tg), the rate-limiting enzyme in BH4 synthesis. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with wild type. The ratio of eNOS dimer:monomer was significantly reduced in aortas from eNOS-Tg mice compared with wild-type mice but restored to normal in eNOS/GCH-Tg mice. NO synthesis was elevated by 2-fold in GCH-Tg and eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with wild type. Aortic BH4 levels were elevated in GCH-Tg and maintained in eNOS/GCH-Tg mice but depleted in eNOS-Tg mice compared with wild type. Aortic and cardiac O2*- production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME), suggesting O2*- production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O2*- production was similar to wild type, and L-NAME had no effect, indicating preserved eNOS coupling. These data indicate that eNOS coupling is directly related to eNOS-BH4 stoichiometry even in the absence of a vascular disease state. Endothelial BH4 availability is a pivotal regulator of eNOS activity and enzymatic coupling in vivo.
Assuntos
Biopterinas/análogos & derivados , Endotélio Vascular/fisiologia , GTP Cicloidrolase/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Animais , Biopterinas/análise , Biopterinas/fisiologia , Células Cultivadas , Dimerização , Endotélio Vascular/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo III , Superóxidos/metabolismoRESUMO
Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH4), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH4 levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH4 biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear. We sought to investigate the signaling pathways whereby cytokines induce GTPCH I expression in human umbilical vein endothelial cells (HUVECs). Interferon-gamma (IFN-gamma) induced endothelial cell GTPCH I protein and BH4 modestly, whereas high-level induction required combinations of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha). In the presence of IFN-gamma, TNF-alpha increased GTPCH I mRNA in a manner dependent on nuclear factor-kappaB (NF-kappaB), as this effect was abrogated by overexpression of a dominant-negative IkappaB construct. HUVEC IFN-gamma treatment resulted in signal transducer and activator of transcription 1 (Stat1) activation and DNA binding in a Jak2-dependent manner, as this was inhibited by AG490. Conversely, overexpression of Jak2 effectively substituted for IFN-gamma in supporting TNF-alpha-mediated GTPCH I induction. The role of IFN-gamma was also Stat1-dependent as Stat1-null cells exhibited no GTPCH I induction in response to cytokines. However, Stat1 activation with oncostatin M failed to support TNF-alpha-mediated GTPCH I induction because of concomitant Stat3 activation. Consistent with this notion, siRNA-mediated Stat3 gene silencing allowed oncostatin M to substitute for IFN-gamma in this system. These data implicate both NF-kappaB and Stat1 in endothelial cell cytokine-stimulated GTPCH I induction and highlight the role of Stat3 in modulating Stat1-supported gene transcription. Thus, IFN-gamma and TNF-alpha exert distinct but cooperative roles for BH4 biosynthesis in endothelium that may have important implications for vascular function during vascular inflammation.
Assuntos
Biopterinas/análogos & derivados , Biopterinas/biossíntese , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , GTP Cicloidrolase/fisiologia , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/fisiologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/metabolismo , Indução Enzimática/efeitos dos fármacos , GTP Cicloidrolase/antagonistas & inibidores , GTP Cicloidrolase/biossíntese , GTP Cicloidrolase/genética , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Proteínas I-kappa B/farmacologia , Interferon gama/farmacologia , Interferon gama/fisiologia , Interleucina-1/farmacologia , Janus Quinase 2 , Camundongos , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Oncostatina M , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transativadores/antagonistas & inibidores , Transativadores/fisiologia , Transfecção , Fator de Necrose Tumoral alfa/fisiologia , Tirfostinas/farmacologia , Veias UmbilicaisRESUMO
Novel single nucleotide polymorphisms within Parkinson's disease (PD) can predict disease risk, but their influence on clinical, cognitive, and neurobiological indices remains unexplored. We investigated differences between functional polymorphisms at RS11158026 coding for guanosine triphosphate cyclohydrolase-1 (GCH1), an essential enzyme for dopamine production in nigrostriatal cells. Among newly diagnosed, untreated PD subjects and age-matched controls from the Parkinson's Progression Markers Initiative, T allele carriers showed higher PD risk (odds ratio = 1.23, p = 0.048), earlier age of onset by 5 years (p = 0.003), and lower striatal dopamine reuptake transporter uptake (p = 0.003). Carriers also had increased cerebrospinal fluid α-synuclein (p = 0.016), worse motor function (p = 0.041), anxiety (p = 0.038), and executive function (p < 0.001). Strikingly, these effects were only in younger T carriers (<50 years), where aging quells the effects of these genetic factors. This suggests GCH1 variants affect early PD risk through altered dopamine uptake, and aging alters how genetic factors contribute to disease development. Future studies should investigate how aging modifies genotypes' contributions on PD risk and sequelae.
Assuntos
Envelhecimento/fisiologia , Dopamina/metabolismo , GTP Cicloidrolase/genética , Doença de Parkinson/genética , Polimorfismo Genético , Idoso , Alelos , Progressão da Doença , Função Executiva , Feminino , GTP Cicloidrolase/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/líquido cefalorraquidiano , Doença de Parkinson/metabolismo , Doença de Parkinson/psicologia , Índice de Gravidade de Doença , alfa-Sinucleína/líquido cefalorraquidianoRESUMO
BACKGROUND: Pulmonary hypertension is a fatal disease characterized by vasoconstriction and vascular remodeling. Loss of endothelial nitric oxide bioavailability is implicated in pulmonary hypertension pathogenesis. Recent evidence suggests that the cofactor tetrahydrobiopterin (BH4) is an important regulator of nitric oxide synthase enzymatic function. METHODS AND RESULTS: In the hph-1 mouse with deficient BH4 biosynthesis, BH4 deficiency caused pulmonary hypertension, even in normoxic conditions, and greatly increased susceptibility to hypoxia-induced pulmonary hypertension. In contrast, augmented BH4 synthesis in the endothelium, by targeted transgenic overexpression of GTP-cyclohydrolase I (GCH), prevented hypoxia-induced pulmonary hypertension. Furthermore, specific augmentation of endothelial BH4 in hph-1 mice by crossing with GCH transgenic mice rescued pulmonary hypertension induced by systemic BH4 deficiency. Lung BH4 availability controlled pulmonary vascular tone, right ventricular hypertrophy, and vascular structural remodeling in a dose-dependent manner in both normoxia and hypoxia. Furthermore, BH4 availability had striking effects on the immediate vasoconstriction response to acute hypoxia. These effects of BH4 were mediated through the regulation of nitric oxide compared with superoxide synthesis by endothelial nitric oxide synthase. CONCLUSIONS: Endothelial BH4 availability is essential for maintaining pulmonary vascular homeostasis, is a critical mediator in the pathogenesis of pulmonary hypertension, and is a novel therapeutic target.
Assuntos
Biopterinas/análogos & derivados , Endotélio Vascular/química , Hipertensão Pulmonar/etiologia , Animais , Biopterinas/deficiência , Biopterinas/fisiologia , GTP Cicloidrolase/genética , GTP Cicloidrolase/fisiologia , Homeostase , Hipertrofia Ventricular Direita , Hipóxia/complicações , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/metabolismo , Circulação Pulmonar/fisiologia , VasoconstriçãoRESUMO
Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a key signaling molecule in vascular homeostasis. Loss of NO bioavailability due to reduced synthesis and increased scavenging by reactive oxygen species is a cardinal feature of endothelial dysfunction in vascular disease states. The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of eNOS activity: when BH4 availability is limiting, eNOS no longer produces NO but instead generates superoxide. In vascular disease states, there is oxidative degradation of BH4 by reactive oxygen species. However, augmentation of BH4 concentrations in vascular disease by pharmacological supplementation, by enhancement of its rate of de novo biosynthesis or by measures to reduce its oxidation, has been shown in experimental studies to enhance NO bioavailability. Thus, BH4 represents a potential therapeutic target in the regulation of eNOS function in vascular disease.
Assuntos
Biopterinas/análogos & derivados , Biopterinas/fisiologia , Coenzimas/fisiologia , Óxido Nítrico Sintase/metabolismo , Animais , Disponibilidade Biológica , Biopterinas/uso terapêutico , Diabetes Mellitus/enzimologia , Indução Enzimática , GTP Cicloidrolase/fisiologia , Humanos , Hipercolesterolemia/enzimologia , Hipertensão/enzimologia , Camundongos , Camundongos Mutantes , Modelos Animais , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Oxirredução , Estresse Oxidativo , Pterinas/uso terapêutico , Coelhos , Ratos , Ratos Endogâmicos , Superóxidos/metabolismo , Doenças Vasculares/metabolismoRESUMO
OBJECTIVE: Increased production of reactive oxygen species and loss of endothelial nitric oxide (NO) bioactivity are key features of vascular disease states such as atherosclerosis. Tetrahydrobiopterin (BH4) is a required cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS); pharmacologic studies suggest that reduced BH4 availability may be an important mediator of endothelial dysfunction in atherosclerosis. We aimed to investigate the importance of endothelial BH4 availability in atherosclerosis using a transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GTPCH). METHODS AND RESULTS: Transgenic mice were crossed into an ApoE knockout (ApoE-KO) background and fed a high-fat diet for 16 weeks. Compared with ApoE-KO controls, transgenic mice (ApoE-KO/GCH-Tg) had higher aortic BH4 levels, reduced endothelial superoxide production and eNOS uncoupling, increased cGMP levels, and preserved NO-mediated endothelium dependent vasorelaxations. Furthermore, aortic root atherosclerotic plaque was significantly reduced in ApoE-KO/GCH-Tg mice compared with ApoE-KO controls. CONCLUSIONS: These findings indicate that BH4 availability is a critical determinant of eNOS regulation in atherosclerosis and is a rational therapeutic target to restore NO-mediated endothelial function and reduce disease progression.
Assuntos
Doenças da Aorta/fisiopatologia , Apolipoproteínas E/deficiência , Arteriosclerose/fisiopatologia , Biopterinas/análogos & derivados , Biopterinas/biossíntese , Coenzimas/biossíntese , Endotélio Vascular/fisiopatologia , GTP Cicloidrolase/fisiologia , Animais , Aorta/metabolismo , Doenças da Aorta/metabolismo , Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Biopterinas/fisiologia , Coenzimas/fisiologia , Cruzamentos Genéticos , GMP Cíclico/metabolismo , Dieta Aterogênica , Endotélio Vascular/metabolismo , GTP Cicloidrolase/biossíntese , GTP Cicloidrolase/genética , Humanos , Hiperlipoproteinemia Tipo II/complicações , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo IV/complicações , Hiperlipoproteinemia Tipo IV/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico/biossíntese , Especificidade de Órgãos , Receptor TIE-2/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Superóxidos/metabolismo , Vasodilatação/fisiologiaRESUMO
In insects, pigment patterns are formed by melanin, ommochromes, and pteridines. Here, the effects of pteridine synthesis on melanin formation were studied using 4th instar larvae of a wild-type silkworm strain, dazao (Bombyx mori), with normal color and markings. Results from injected larvae and in vitro integument culture indicated that decreased activity of guanosine triphosphate cyclohydrolase I (GTP-CH I, a rate-limiting enzyme for pteridine synthesis), lowers BH4 (6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a production correlated with GTP-CH I activity) levels and eliminates markings and coloration. The conversion of phenylalanine and tyrosine to melanin was prevented when GTP-CH I was inhibited. When BH4 was added, phenylalanine was converted to tyrosine, and the tyrosine concentration increased. Tyrosine was then converted to melanin to create normal markings and coloration. Decreasing GTP-CH I activity did not affect L-DOPA (3,4-l-dihydroxyphenylalanine). GTP-CH I affected melanin synthesis by generating the BH4 used in two key reaction steps: (1) conversion of phenylalanine to tyrosine by PAH (phenylalanine hydroxylase) and (2) conversion of tyrosine to L-DOPA by TH (tyrosine hydroxylase). Expression profiles of BmGTPCH Ia, BmGTPCH Ib, BmTH, and BmPAH in the integument were consistent with the current findings.
Assuntos
Bombyx/enzimologia , GTP Cicloidrolase/fisiologia , Proteínas de Insetos/fisiologia , Melaninas/biossíntese , Fenilalanina Hidroxilase/fisiologia , Tirosina 3-Mono-Oxigenase/fisiologia , Animais , Vias Biossintéticas , Bombyx/crescimento & desenvolvimento , Guanosina Trifosfato , Muda , Pigmentação da Pele , Técnicas de Cultura de TecidosRESUMO
Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes. The ribBA and ribC loci complemented corresponding E. coli rib mutants. S. meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin. Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain. No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N. These results support the importance of extracellular riboflavin for alfalfa root colonization by S. meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.
Assuntos
GTP Cicloidrolase/genética , Transferases Intramoleculares/genética , Medicago sativa/fisiologia , Riboflavina Sintase/genética , Riboflavina/biossíntese , Sinorhizobium/fisiologia , Simbiose/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , GTP Cicloidrolase/fisiologia , Transferases Intramoleculares/fisiologia , Medicago sativa/microbiologia , Dados de Sequência Molecular , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Riboflavina/fisiologia , Riboflavina Sintase/fisiologia , Homologia de Sequência de Aminoácidos , Sinorhizobium/genética , Simbiose/genéticaRESUMO
Computer simulation analysis of the experimental data showed that Cl'-keto PH4 and C2'-keto PH4 were sequential intermediates of the reduction of PPH4 to BH4 by SPR and that the reaction in the last step of the BH4 biosynthesis proceeds as follows: PPH4-->Cl'-keto PH4-->C2'-keto PH4-->BH4 Cl'-keto PH4 is not detectable in the reduction of PPH4 by the purified SPR at neutral pH since isomerization (-->) [8] may occur far more rapidly than the first reduction (-->).
Assuntos
Oxirredutases do Álcool/fisiologia , Biopterinas/análogos & derivados , Simulação por Computador , GTP Cicloidrolase/fisiologia , Modelos Biológicos , Fósforo-Oxigênio Liases , Animais , Biopterinas/biossínteseRESUMO
Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and NO generation. Augmentation of BH4 levels can prevent eNOS uncoupling and can improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele (GCH1(fl/fl)) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1(fl/fl)Tie2cre mice demonstrated virtually absent endothelial NO bioactivity and significantly greater O2 (â¢-) production. GCH1(fl/fl)Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1(fl/fl)Tie2cre aortas were, in part, mediated by eNOS-derived hydrogen peroxide (H2O2), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1(fl/fl)Tie2cre aortas. GCH1(fl/fl)Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalized by NOS inhibitor, NG-nitro-L-arginine methyl ester. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure and identify endothelial cell BH4 as a pivotal regulator of NO versus H2O2 as alternative eNOS-derived endothelial-derived relaxing factors.
Assuntos
Biopterinas/análogos & derivados , Pressão Sanguínea/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , GTP Cicloidrolase/fisiologia , Acetilcolina/farmacologia , Animais , Biopterinas/genética , Biopterinas/fisiologia , Pressão Sanguínea/genética , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Feminino , GTP Cicloidrolase/deficiência , GTP Cicloidrolase/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oligopeptídeos/farmacologia , Oxigênio/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologiaAssuntos
Dano ao DNA/genética , Enzimas Reparadoras do DNA , Mutação/genética , Precursores de Ácido Nucleico , 8-Hidroxi-2'-Desoxiguanosina , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Enzimas Reparadoras do DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Nucleotídeos de Desoxiguanina/genética , Nucleotídeos de Desoxiguanina/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/fisiologia , Proteínas de Escherichia coli/fisiologia , GTP Cicloidrolase/fisiologia , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Pirofosfatases/fisiologiaAssuntos
Biopterinas/análogos & derivados , Biopterinas/biossíntese , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , GTP Cicloidrolase/fisiologia , Adaptação Fisiológica , Animais , Proteínas de Ligação a DNA/fisiologia , Indução Enzimática , GTP Cicloidrolase/antagonistas & inibidores , Humanos , Interferon gama/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Proteínas/fisiologia , Fator de Transcrição STAT1 , Transdução de Sinais/fisiologia , Transativadores/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
AIMS: Clinical markers of cardiac autonomic function, such as heart rate and response to exercise, are important predictors of cardiovascular risk. Tetrahydrobiopterin (BH4) is a required cofactor for enzymes with roles in cardiac autonomic function, including tyrosine hydroxylase and nitric oxide synthase. Synthesis of BH4 is regulated by GTP cyclohydrolase I (GTPCH), encoded by GCH1. Recent clinical studies report associations between GCH1 variants and increased heart rate, but the mechanistic importance of GCH1 and BH4 in autonomic function remains unclear. We investigate the effect of BH4 deficiency on the autonomic regulation of heart rate in the hph-1 mouse model of BH4 deficiency. METHODS AND RESULTS: In the hph-1 mouse, reduced cardiac GCH1 expression, GTPCH enzymatic activity, and BH4 were associated with increased resting heart rate; blood pressure was not different. Exercise training decreased resting heart rate, but hph-1 mice retained a relative tachycardia. Vagal nerve stimulation in vitro induced bradycardia equally in hph-1 and wild-type mice both before and after exercise training. Direct atrial responses to carbamylcholine were equal. In contrast, propranolol treatment normalized the resting tachycardia in vivo. Stellate ganglion stimulation and isoproterenol but not forskolin application in vitro induced a greater tachycardic response in hph-1 mice. ß1-adrenoceptor protein was increased as was the cAMP response to isoproterenol stimulation. CONCLUSION: Reduced GCH1 expression and BH4 deficiency cause tachycardia through enhanced ß-adrenergic sensitivity, with no effect on vagal function. GCH1 expression and BH4 are novel determinants of cardiac autonomic regulation that may have important roles in cardiovascular pathophysiology.