RESUMEN
BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.
Asunto(s)
Ciclinas , Hipertensión Arterial Pulmonar , Humanos , Ratones , Animales , Ciclinas/metabolismo , Hipertensión Arterial Pulmonar/metabolismo , Cisteína/metabolismo , Células Endoteliales/metabolismo , Proliferación Celular , Arteria Pulmonar/metabolismo , Fosforilación , Puntos de Control del Ciclo Celular , Ciclina D/metabolismo , Células Cultivadas , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismoRESUMEN
Group 3 pulmonary hypertension (PH), which occurs secondary to hypoxia lung diseases, is one of the most common causes of PH worldwide and has a high unmet clinical need. A deeper understanding of the integrative pathological and adaptive molecular mechanisms within this group is required to inform the development of novel drug targets and effective treatments. The production of oxidants is increased in PH Group 3, and their pleiotropic roles include contributing to disease progression by promoting prolonged hypoxic pulmonary vasoconstriction and pathological pulmonary vascular remodeling, but also stimulating adaptation to pathological stress that limits the severity of this disease. Inflammation, which is increasingly being viewed as a key pathological feature of Group 3 PH, is subject to complex regulation by redox mechanisms and is exacerbated by, but also augments oxidative stress. In this review, we investigate aspects of this complex crosstalk between inflammation and oxidative stress in Group 3 PH, focusing on the redox-regulated transcription factor NF-κB and its upstream regulators toll-like receptor 4 and high mobility group box protein 1. Ultimately, we propose that the development of specific therapeutic interventions targeting redox-regulated signaling pathways related to inflammation could be explored as novel treatments for Group 3 PH.
Asunto(s)
Hipertensión Pulmonar , Humanos , Hipoxia , Inflamación , Oxidación-Reducción , Estrés OxidativoRESUMEN
BACKGROUND: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans. METHODS: In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting. RESULTS: Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men. CONCLUSIONS: These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.
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Presión Sanguínea/efectos de los fármacos , Genómica/métodos , Hipertensión/tratamiento farmacológico , Proteínas de la Membrana/uso terapéutico , Fosfoproteínas/uso terapéutico , Fosforilación/fisiología , Animales , Humanos , Hipertensión/fisiopatología , Masculino , Proteínas de la Membrana/farmacología , Ratones , Fosfoproteínas/farmacologíaRESUMEN
Nitro-oleate (10-nitro-octadec-9-enoic acid), which inhibits soluble epoxide hydrolase (sEH) by covalently adducting to C521, increases the abundance of epoxyeicosatrienoic acids (EETs) that can be health promoting, for example by lowering blood pressure or their anti-inflammatory actions. However, perhaps consistent with their impact on angiogenesis, increases in EETs may exacerbate progression of some cancers. To assess this, Lewis lung carcinoma (LLc1) cells were exposed to oleate or nitro-oleate, with the latter inhibiting the hydrolase and increasing their proliferation and migration in vitro. The enhanced proliferation induced by nitro-oleate was EET-dependent, being attenuated by the ETT-receptor antagonist 14,15-EE-5(Z)-E. LLc1 cells were engineered to stably overexpress wild-type or C521S sEH, with the latter exhibiting resistance to nitro-oleate-dependent hydrolase inhibition and the associated stimulation of tumor growth in vitro or in vivo. Nitro-oleate also increased migration in endothelial cells isolated from wild-type (WT) mice, but not those from C521S sEH knock-in (KI) transgenic mice genetically modified to render the hydrolase electrophile-resistant. These observations were consistent with nitro-oleate promoting cancer progression, and so the impact of this electrophile was examined in vivo again, but this time comparing growth of LLc1 cells expressing constitutive levels of wild-type hydrolase when implanted into WT or KI mice. Nitro-oleate inhibited tumor sEH (P < 0.05), with a trend for elevated plasma 11(12)-EET/DHET and 8(9)EET/DHET (dihydroxyeicosatrienoic acid) ratios when administered to WT, but not KI, mice. Although in vitro studies with LLc1 cells supported a role for nitro-oleate in cancer cell proliferation, it failed to significantly stimulate tumor growth in WT mice implanted with the same LLc1 cells in vivo, perhaps due to its well-established anti-inflammatory actions. Indeed, pro-inflammatory cytokines were significantly down-regulated in nitro-oleate treated WT mice, potentially countering any impact of the concomitant inhibition of sEH.
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Epóxido Hidrolasas , Neoplasias , Alquenos , Animales , Células Endoteliales , Epóxido Hidrolasas/genética , Inflamación , RatonesRESUMEN
Chronic hypoxia causes pulmonary hypertension (PH), vascular remodeling, right ventricular (RV) hypertrophy, and cardiac failure. Protein kinase G Iα (PKGIα) is susceptible to oxidation, forming an interprotein disulfide homodimer associated with kinase targeting involved in vasodilation. Here we report increased disulfide PKGIα in pulmonary arteries from mice with hypoxic PH or lungs from patients with pulmonary arterial hypertension. This oxidation is likely caused by oxidants derived from NADPH oxidase-4, superoxide dismutase 3, and cystathionine γ-lyase, enzymes that were concomitantly increased in these samples. Indeed, products that may arise from these enzymes, including hydrogen peroxide, glutathione disulfide, and protein-bound persulfides, were increased in the plasma of hypoxic mice. Furthermore, low-molecular-weight hydropersulfides, which can serve as "superreductants" were attenuated in hypoxic tissues, consistent with systemic oxidative stress and the oxidation of PKGIα observed. Inhibiting cystathionine γ-lyase resulted in decreased hypoxia-induced disulfide PKGIα and more severe PH phenotype in wild-type mice, but not in Cys42Ser PKGIα knock-in (KI) mice that are resistant to oxidation. In addition, KI mice also developed potentiated PH during hypoxia alone. Thus, oxidation of PKGIα is an adaptive mechanism that limits PH, a concept further supported by polysulfide treatment abrogating hypoxia-induced RV hypertrophy in wild-type, but not in the KI, mice. Unbiased transcriptomic analysis of hypoxic lungs before structural remodeling identified up-regulation of endothelial-to-mesenchymal transition pathways in the KI compared with wild-type mice. Thus, disulfide PKGIα is an intrinsic adaptive mechanism that attenuates PH progression not only by promoting vasodilation but also by limiting maladaptive growth and fibrosis signaling.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Hipertensión Pulmonar/patología , Hipoxia/complicaciones , Arteria Pulmonar/patología , Adulto , Animales , Línea Celular , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/química , Cistationina gamma-Liasa/antagonistas & inhibidores , Cistationina gamma-Liasa/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Disulfuros/química , Femenino , Fibrosis , Técnicas de Sustitución del Gen , Humanos , Hipertensión Pulmonar/sangre , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/prevención & control , Hipoxia/sangre , Hipoxia/tratamiento farmacológico , Pulmón/irrigación sanguínea , Pulmón/patología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Oxidantes/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sulfuros/administración & dosificación , Sulfuros/sangre , Sulfuros/metabolismo , Regulación hacia Arriba , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
The kinase p38α MAPK (p38α) plays a pivotal role in many biological processes. p38α is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38α. p38α was activated and formed a disulfide-bound heterodimer with MAP2K3 (MKK3) in rat cardiomyocytes and isolated hearts exposed to H2O2 This disulfide heterodimer was sensitive to reduction by mercaptoethanol and was enhanced by the thioredoxin-reductase inhibitor auranofin. We predicted that Cys-119 or Cys-162 of p38α, close to the known MKK3 docking domain, were relevant for these redox characteristics. The C119S mutation decreased whereas the C162S mutation increased the dimer formation, suggesting that these two Cys residues act as vicinal thiols, consistent with C119S/C162S being incapable of sensing H2O2 Similarly, disulfide heterodimer formation was abolished in H9C2 cells expressing both MKK3 and p38α C119S/C162S and subjected to simulated ischemia and reperfusion. However, the p38α C119S/C162S mutants did not exhibit appreciable alteration in activating dual phosphorylation. In contrast, the anti-inflammatory agent 10-nitro-oleic acid (NO2-OA), a component of the Mediterranean diet, reduced p38α activation and covalently modified Cys-119/Cys-162, probably obstructing MKK3 access. Moreover, NO2-OA reduced the dephosphorylation of p38α by hematopoietic tyrosine phosphatase (HePTP). Furthermore, steric obstruction of Cys-119/Cys-162 by NO2-OA pretreatment in Langendorff-perfused murine hearts prevented the p38-MKK3 disulfide dimer formation and attenuated H2O2-induced contractile dysfunction. Our findings suggest that cysteine residues within p38α act as redox sensors that can dynamically regulate the association between p38 and MKK3.
Asunto(s)
Cistina/metabolismo , Ventrículos Cardíacos/enzimología , MAP Quinasa Quinasa 3/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Modelos Moleculares , Miocitos Cardíacos/enzimología , Estrés Oxidativo , Sustitución de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Cisteína/química , Cisteína/metabolismo , Cistina/química , Activación Enzimática , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , Técnicas In Vitro , MAP Quinasa Quinasa 3/química , MAP Quinasa Quinasa 3/genética , Masculino , Ratones Endogámicos C57BL , Proteína Quinasa 14 Activada por Mitógenos/química , Proteína Quinasa 14 Activada por Mitógenos/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Conformación Proteica , Multimerización de Proteína , Ratas Wistar , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Protein kinase G (PKG) Iα is the end-effector kinase that mediates nitric oxide (NO)-dependent and oxidant-dependent vasorelaxation to maintain blood pressure during health. A hallmark of cardiovascular disease is attenuated NO production, which in part is caused by NO Synthase (NOS) uncoupling, which in turn increases oxidative stress because of superoxide generation. NOS uncoupling promotes PKG Iα oxidation to the interprotein disulfide state, likely mediated by superoxide-derived hydrogen peroxide, and because the NO-cyclic guanosine monophosphate (cGMP) pathway otherwise negatively regulates oxidation of the kinase to its active disulfide dimeric state. Diet-induced obesity is associated with NOS uncoupling, which may in part contribute to the associated cardiovascular dysfunction due to exacerbated PKG Iα disulfide oxidation to the disulfide state. This is a rational hypothesis because PKG Iα oxidation is known to significantly contribute to heart failure that arises from chronic myocardial oxidative stress. METHODS AND RESULTS: Bovine arterial endothelial cells (BAECs) or smooth muscle cells (SMCs) were exposed to drugs that uncouple NOS. These included 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) which promotes its S-glutathiolation, 4-diamino-6-hydroxy-pyrimidine (DAHP) which inhibits guanosine-5'-triphosphate-cyclohydrolase 2 to prevent BH4 synthesis or methotrexate (MTX) which inhibits the regeneration of BH4 from BH2 by dihydrofolate reductase. While all the drugs mentioned above induced robust PKG Iα disulfide dimerization in cells, exposure of BAECs to NOS inhibitor L-NMMA did not. Increased PKG Iα disulfide formation occurred in hearts and aortae from mice treated in vivo with DAHP (10mM in a drinking water for 3 weeks). Redox-dead C42S PKG Iα knock-in (KI) mice developed less pronounced cardiac posterior wall hypertrophy and did not develop cardiac dysfunction, assessed by echocardiography, compared to the wild-type (WT) mice after chronic DAHP treatment. WT or KI mice were then subjected to a diet-induced obesity protocol by feeding them with a high fat Western-type diet (RM 60% AFE) for 27 weeks, which increased body mass, adiposity, plasma leptin, resistin and glucagon levels comparably in each genotype. Obesity-induced hypertension, assessed by radiotelemetry, was mild and transient in the WT, while the basally hypertensive KI mice were resistant to further increases in blood pressure following high fat feeding. Although the obesogenic diet caused mild cardiac dysfunction in the WT but not the KI mice, gross changes in myocardial structure monitored by echocardiography were not apparent in either genotype. The level of cyclic guanosine monophosphate (cGMP) was decreased in the aortae of WT and KI mice following high fat feeding. PKG Iα oxidation was not evident in the hearts of WT mice fed a high fat diet. CONCLUSIONS: Despite robust evidence for PKG Iα oxidation during NOS uncoupling in cell models, it is unlikely that PKG Iα oxidation occurs to a significant extent in vivo during diet-induced obesity and so is unlikely to mediate the associated cardiovascular dysfunction.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Dieta Alta en Grasa/efectos adversos , Hipertensión/genética , Óxido Nítrico Sintasa de Tipo III/genética , Obesidad/genética , Especies Reactivas de Oxígeno/metabolismo , Desacopladores/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Carmustina/farmacología , Bovinos , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación de la Expresión Génica , Glucagón/genética , Glucagón/metabolismo , Hipertensión/etiología , Hipertensión/metabolismo , Hipertensión/patología , Leptina/genética , Leptina/metabolismo , Metotrexato/farmacología , Ratones , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Oxidación-Reducción , Estrés Oxidativo , Resistina/genética , Resistina/metabolismo , Transducción de Señal , Azúcares Ácidos/farmacologíaRESUMEN
The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank-Starling response.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Diástole/fisiología , Ventrículos Cardíacos/enzimología , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Animales , Fenómenos Biomecánicos , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Gasto Cardíaco/fisiología , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Disulfuros/química , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Ventrículos Cardíacos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica/fisiología , Miocardio/citología , Miocitos Cardíacos/citología , Técnicas de Cultivo de Órganos , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Cultivo Primario de Células , Serina/metabolismo , Especificidad por SustratoRESUMEN
Angiogenesis is essential for tissue development, wound healing and tissue perfusion, with its dysregulation linked to tumorigenesis, rheumatoid arthritis and heart disease. Here we show that pro-angiogenic stimuli couple to NADPH oxidase-dependent generation of oxidants that catalyse an activating intermolecular-disulphide between regulatory-RIα subunits of protein kinase A (PKA), which stimulates PKA-dependent ERK signalling. This is crucial to blood vessel growth as 'redox-dead' Cys17Ser RIα knock-in mice fully resistant to PKA disulphide-activation have deficient angiogenesis in models of hind limb ischaemia and tumour-implant growth. Disulphide-activation of PKA represents a new therapeutic target in diseases with aberrant angiogenesis.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Neovascularización Fisiológica/genética , Animales , Aorta/fisiología , Bovinos , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Células Endoteliales , Técnicas de Sustitución del Gen , Miembro Posterior , Inmunoprecipitación , Isquemia , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/irrigación sanguínea , Oxidación-Reducción , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Sepsis and sepsis-associated multiorgan failure represent the major cause of mortality in intensive care units worldwide. Cardiovascular dysfunction, a key component of sepsis pathogenesis, has received much research interest, although research translatability remains severely limited. There is a critical need for more comprehensive preclinical sepsis models, with more clinically relevant end points, such as microvascular perfusion. The purpose of this study was to compare microcirculatory blood flow measurements, using a novel application of laser speckle contrast imaging technology, with more traditional hemodynamic end points, as part of a multiparameter monitoring system in preclinical models of sepsis. Our aim, in measuring mesenteric blood flow, was to increase the prognostic sensitivity of preclinical studies. In two commonly used sepsis models (cecal ligation and puncture, and lipopolysaccharide), we demonstrate that blood pressure and cardiac output are compromised postsepsis, but subsequently stabilize over the 24-h recording period. In contrast, mesenteric blood flow continuously declines in a time-dependent manner and in parallel with the development of metabolic acidosis and organ dysfunction. Importantly, these microcirculatory perturbations are reversed by fluid resuscitation, a mainstay intervention associated with improved outcome in patients. These data suggest that global hemodynamics are maintained at the expense of the microcirculation and are, therefore, not sufficiently predictive of outcome. We demonstrate that microcirculatory blood flow is a more sensitive biomarker of sepsis syndrome progression and believe that incorporation of this biomarker into preclinical models will facilitate sophisticated proof-of-concept studies for novel sepsis interventions, providing more robust data on which to base future clinical trials.
Asunto(s)
Biomarcadores/sangre , Microcirculación/fisiología , Flujo Sanguíneo Regional/fisiología , Sepsis/patología , Animales , Presión Sanguínea/fisiología , Gasto Cardíaco/fisiología , Ciego/patología , Modelos Animales de Enfermedad , Hemodinámica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , PronósticoRESUMEN
Dysregulated blood pressure control leading to hypertension is prevalent and is a risk factor for several common diseases. Fully understanding blood pressure regulation offers the possibility of developing rationale therapies to alleviate hypertension and associated disease risks. Although hydrogen sulfide (H2S) is a well-established endogenous vasodilator, the molecular basis of its blood-pressure lowering action is incompletely understood. H2S-dependent vasodilation and blood pressure lowering in vivo was mediated by it catalyzing formation of an activating interprotein disulfide within protein kinase G (PKG) Iα. However, this oxidative activation of PKG Iα is counterintuitive because H2S is a thiol-reducing molecule that breaks disulfides, and so it is not generally anticipated to induce their formation. This apparent paradox was explained by H2S in the presence of molecular oxygen or hydrogen peroxide rapidly converting to polysulfides, which have oxidant properties that in turn activate PKG by inducing the disulfide. These observations are relevant in vivo because transgenic knockin mice in which the cysteine 42 redox sensor within PKG has been systemically replaced with a redox-dead serine residue are resistant to H2S-induced blood pressure lowering. Thus, a primary mechanism by which the reductant molecule H2S lowers blood pressure is mediated somewhat paradoxically by the oxidative activation of PKG.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Sulfuro de Hidrógeno/farmacología , Hipertensión/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sustancias ReductorasRESUMEN
Oxidative post-translational modifications of proteins resulting from events that increase cellular oxidant levels play important roles in physiological and pathophysiological processes. Evaluation of alterations to protein redox states is increasingly common place because of methodological advances that have enabled detection, quantification and identification of such changes in cells and tissues. This mini-review provides a synopsis of biochemical methods that can be utilized to monitor the array of different oxidative and electrophilic modifications that can occur to protein thiols and can be important in the regulatory or maladaptive impact oxidants can have on biological systems. Several of the methods discussed are valuable for monitoring the redox state of established redox sensing proteins such as Keap1.
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Proteínas/metabolismo , Compuestos de Sulfhidrilo/química , Cisteína/química , Cisteína/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Humanos , Nitrosación , Oxidación-Reducción , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Soluble epoxide hydrolase (sEH) is inhibited by electrophilic lipids by their adduction to Cys521 proximal to its catalytic center. This inhibition prevents hydrolysis of the enzymes' epoxyeicosatrienoic acid (EET) substrates, so they accumulate inducing vasodilation to lower blood pressure (BP). We generated a Cys521Ser sEH redox-dead knockin (KI) mouse model that was resistant to this mode of inhibition. The electrophilic lipid 10-nitro-oleic acid (NO2-OA) inhibited hydrolase activity and also lowered BP in an angiotensin II-induced hypertension model in wild-type (WT) but not KI mice. Furthermore, EET/dihydroxy-epoxyeicosatrienoic acid isomer ratios were elevated in plasma from WT but not KI mice following NO2-OA treatment, consistent with the redox-dead mutant being resistant to inhibition by lipid electrophiles. sEH was inhibited in WT mice fed linoleic acid and nitrite, key constituents of the Mediterranean diet that elevates electrophilic nitro fatty acid levels, whereas KIs were unaffected. These observations reveal that lipid electrophiles such as NO2-OA mediate antihypertensive signaling actions by inhibiting sEH and suggest a mechanism accounting for protection from hypertension afforded by the Mediterranean diet.
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Dieta Mediterránea , Epóxido Hidrolasas/metabolismo , Ácidos Grasos/metabolismo , Hipertensión/dietoterapia , Hipertensión/prevención & control , Angiotensina II/farmacología , Animales , Presión Sanguínea , Cardiomegalia/dietoterapia , Cardiomegalia/prevención & control , Celulasa , Modelos Animales de Enfermedad , Epóxido Hidrolasas/genética , Técnicas de Sustitución del Gen , Hipertensión/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Nitratos/metabolismo , Nitritos/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
The prevalence of obesity among pregnant women is increasing. Evidence from human cohort studies and experimental animals suggests that offspring cardiovascular and metabolic function is compromised through early life exposure to maternal obesity. Previously, we reported that juvenile offspring of obese rats develop sympathetically mediated hypertension associated with neonatal hyperleptinemia. We have now addressed the hypothesis that neonatal exposure to raised leptin in the immediate postnatal period plays a causal role. Pups from lean Sprague-Dawley rats were treated either with leptin (3 mg/kg IP) or with saline twice daily from postnatal day 9 to 15 to mimic the exaggerated postnatal leptin surge observed in offspring of obese dams. Cardiovascular function was assessed by radiotelemetry at 30 days, and 2 and 12 months. In juvenile (30 days) leptin-treated rats, hearts were heavier and night-time (active period) systolic blood pressure was raised (mm Hg; mean ± SEM: male leptin-treated, 132 ± 1 versus saline-treated, 119 ± 1, n=6, P<0.05; female leptin-treated, 132 ± 2 versus saline-treated, 119 ± 1, n=6, P<0.01), and the pressor response to restraint stress and leptin challenge increased compared with saline-treated rats. Heart rate variability demonstrated an increased low:high frequency ratio in 30-day leptin-treated animals, indicative of heightened sympathetic efferent tone. Echocardiography showed altered left ventricular structure and systolic function in 30-day female leptin versus saline-treated rats. These disorders persisted to adulthood. In isolated hearts, contractile function was impaired at 5 months in male leptin-treated rats. Exogenously imposed hyperleptinemia in neonatal rats permanently influences blood pressure and cardiac structure and function.
Asunto(s)
Tejido Adiposo/metabolismo , Corazón/fisiopatología , Hipertensión/metabolismo , Leptina/sangre , Miocardio/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/fisiopatología , Animales , Animales Recién Nacidos , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Sistema Cardiovascular/efectos de los fármacos , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/fisiopatología , Femenino , Corazón/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Hipertensión/fisiopatología , Leptina/farmacología , Masculino , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Obesidad/metabolismo , Obesidad/fisiopatología , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
Aberrant proliferation of mesangial cells (MCs) is a key finding in progressive glomerular disease. TH1177 is a small molecule that has been shown to inhibit low-voltage activated T-type Ca(2+) channels (TCCs). The current study investigates the effect of TH1177 on MC proliferation in vitro and in vivo. The effect of Ca(2+) channel inhibition on primary rat MC proliferation in vitro was studied using the microculture tetrazolium assay and by measuring bromodeoxyuridine incorporation. In vivo, rats with Thy1 nephritis were treated with TH1177 or vehicle. Glomerular injury and average glomerular cell number were determined in a blinded fashion. Immunostaining for Ki-67 and phosphorylated ERK were also performed. The expression of TCC isoforms in healthy and diseased tissue was investigated using quantitative real-time PCR. TCC blockade caused a significant reduction in rat MC proliferation in vitro, whereas L-type inhibition had no effect. Treatment of Thy1 nephritis with TH1177 significantly reduced glomerular injury (P < 0.005) and caused a 49% reduction in glomerular cell number (P < 0.005) compared to the placebo. TH1177 also reduced Ki-67-positive and pERK-positive cells per glomerulus by 52% (P < 0.01 and P < 0.005, respectively). These results demonstrate that TH1177 inhibits MC proliferation in vitro and in vivo, supporting the hypothesis that TCC inhibition may be a useful strategy for studying and modifying MC proliferative responses to injury.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/efectos de los fármacos , Glomerulonefritis/tratamiento farmacológico , Células Mesangiales/patología , Pirrolidinas/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Creatinina/metabolismo , Relación Dosis-Respuesta a Droga , Glomerulonefritis/patología , Corteza Renal/metabolismo , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Masculino , Níquel/farmacología , Proteinuria/inducido químicamente , Ratas , Ratas Wistar , Antígenos Thy-1/metabolismo , Verapamilo/farmacologíaRESUMEN
Sepsis is a common life-threatening clinical syndrome involving complications as a result of severe infection. A cardinal feature of sepsis is inflammation that results in oxidative stress. Sepsis in wild-type mice induced oxidative activation of cGMP-dependent protein kinase 1 alpha (PKG Iα), which increased blood vessel dilation and permeability, and also lowered cardiac output. These responses are typical features of sepsis and their combined effect is a lowering of blood pressure. This hypotension, a hallmark of sepsis, resulted in underperfusion of end organs, resulting in their damage. A central role for PKG Iα oxidative activation in injury is supported by oxidation-resistant Cys42Ser PKG Iα knock-in mice being markedly protected from these clinical indices of injury during sepsis. We conclude that oxidative activation of PKG Iα is a key mediator of hypotension and consequential organ injury during sepsis.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Hipotensión/fisiopatología , Insuficiencia Multiorgánica/fisiopatología , Sepsis/fisiopatología , Sustitución de Aminoácidos , Animales , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Activación Enzimática/genética , Hipotensión/enzimología , Hipotensión/genética , Immunoblotting , L-Lactato Deshidrogenasa/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Insuficiencia Multiorgánica/enzimología , Insuficiencia Multiorgánica/genética , Oxidación-Reducción , Sepsis/enzimología , Sepsis/genéticaRESUMEN
Protein kinase G (PKG) is activated by nitric oxide (NO)-induced cGMP binding or alternatively by oxidant-induced interprotein disulfide formation. We found preactivation with cGMP attenuated PKG oxidation. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) blockade of cGMP production increased disulfide PKG to 13 ± 2% and 29±4% of total in aorta and mesenteries, respectively. This was potentially anomalous, because we observed 2.7-fold higher NO levels in aorta than mesenteries; consequently, we had anticipated that ODQ would induce more disulfide in the conduit vessel. ODQ also constricted aorta, whereas it had no effect on mesenteries. Thus, mesenteries, but not aorta, can compensate for loss of NO-cGMP by recruiting disulfide activation of PKG. Mechanistically, this is explained by loss of cGMP allowing disulfide formation in response to basal oxidant production. Why aorta treated with ODQ generated less PKG disulfide that is insufficient to induce vasoconstriction was unclear. One potential explanation, especially because aorta were much less sensitive than mesenteries to exogenous H(2)O(2)-induced relaxation (EC(50)=205 ± 24 and 33 ± 2 µmol/L, respectively) was that conduit vessels may have higher peroxidase capacity. Indeed, we found that aorta express 49 ± 22% and 80 ± 25% more peroxiredoxin and thioredoxin, respectively, than mesenteries, and their 2-Cys peroxiredoxin peroxidatic cysteines were also less sensitive to hyperoxidation. The higher peroxidase capacity of aortas would explain their constriction during cGMP removal and their insensitivity to H(2)O(2)-induced relaxation compared with mesenteries. In summary, cGMP binding to PKG induces a state that is resistant to disulfide formation. Consequently, cGMP depletion sensitizes PKG to oxidation; this happens to a lesser extent in aortas than in mesenteries, because the conduit vessels generate more NO and express more peroxiredoxin.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Disulfuros/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiología , Sitios de Unión/genética , Presión Sanguínea/efectos de los fármacos , Células Cultivadas , GMP Cíclico/farmacología , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Peróxido de Hidrógeno/farmacología , Immunoblotting , Técnicas In Vitro , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Mutación , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Oxadiazoles/farmacología , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Fosforilación/efectos de los fármacos , Quinoxalinas/farmacología , Ratas , Espermina/análogos & derivados , Espermina/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Although nitroglycerin has remained in clinical use since 1879, the mechanism by which it relaxes blood vessels to lower blood pressure remains incompletely understood. Nitroglycerin undergoes metabolism that generates several reaction products, including oxidants, and this bioactivation process is essential for vasodilation. Protein kinase G (PKG) mediates classic nitric oxide-dependent vasorelaxation, but the 1α isoform is also independently activated by oxidation that involves interprotein disulfide formation within this homodimeric protein complex. We hypothesized that nitroglycerin-induced vasodilation is mediated by disulfide activation of PKG1α. METHODS AND RESULTS: Treating smooth muscle cells or isolated blood vessels with nitroglycerin caused PKG1α disulfide dimerization. PKG1α disulfide formation was increased in wild-type mouse aortas by in vivo nitroglycerin treatment, but this oxidation was lost as tolerance developed. To establish whether kinase oxidation underlies nitroglycerin-induced vasodilation in vivo, we used a Cys42Ser PKG1α knock-in mouse that cannot transduce oxidant signals because it does not contain the vital redox-sensing thiol. This redox-dead knock-in mouse was substantively deficient in hypotensive response to nitroglycerin compared with wild-type littermates as measured in vivo by radiotelemetry. Resistance blood vessels from knock-ins were markedly less sensitive to nitroglycerin-induced vasodilation (EC(50)=39.2 ± 10.7 µmol/L) than wild-types (EC(50)=12.1 ± 2.9 µmol/L). Furthermore, after ≈24 hours of treatment, wild-type controls stopped vasodilating to nitroglycerin, and the vascular sensitivity to nitroglycerin was decreased, whereas this tolerance phenomenon, which routinely hampers the management of hypertensive patients, was absent in knock-ins. CONCLUSIONS: PKG1α disulfide formation is a significant mediator of nitroglycerin-induced vasodilation, and tolerance to nitroglycerin is associated with loss of kinase oxidation.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Hipertensión , Nitroglicerina/farmacología , Sustitución de Aminoácidos/genética , Animales , Aorta/citología , Células Cultivadas , Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Proteínas Quinasas Dependientes de GMP Cíclico/química , Dimerización , Disulfuros/química , Disulfuros/metabolismo , Técnicas de Sustitución del Gen , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Hipertensión/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Músculo Liso Vascular/citología , Oxidación-Reducción/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Compuestos de Sulfhidrilo/metabolismo , Telemetría , Vasodilatación/efectos de los fármacos , Vasodilatación/genética , Vasodilatadores/farmacologíaRESUMEN
Blood pressure regulation is crucial for the maintenance of health, and hypertension is a risk factor for myocardial infarction, heart failure, stroke and renal disease. Nitric oxide (NO) and prostacyclin trigger well-defined vasodilator pathways; however, substantial vasorelaxation in response to agents such as acetylcholine persists when the synthesis of these molecules is prevented. This remaining vasorelaxation activity, termed endothelium-derived hyperpolarizing factor (EDHF), is more prevalent in resistance than in conduit blood vessels and is considered a major mechanism for blood pressure control. Hydrogen peroxide (H2O2) has been shown to be a major component of EDHF in several vascular beds in multiple species, including in humans. H2O2 causes the formation of a disulfide bond between the two α subunits of protein kinase G I-α (PKGI-α), which activates the kinase independently of the NO-cyclic guanosine monophosphate (cGMP) pathway and is coupled to vasodilation. To test the importance of PKGI-α oxidation in the EDHF mechanism and blood pressure control in vivo, we generated a knock-in mouse expressing only a C42S 'redox-dead' version of PKGI-α. This amino acid substitution, a single-atom change (an oxygen atom replacing a sulfur atom), blocked the vasodilatory action of H2O2 on resistance vessels and resulted in hypertension in vivo.