RESUMEN
Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of ß-adrenergic receptors. Because BAT therapies based on cold exposure or ß-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.
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Adenosina/metabolismo , Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Receptor de Adenosina A2A/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Agonistas del Receptor de Adenosina A2/farmacología , Tejido Adiposo Pardo/efectos de los fármacos , Animales , Células Cultivadas , Cricetinae , Dieta , Humanos , Masculino , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Fenetilaminas/farmacologíaRESUMEN
Brown adipose tissue (BAT) was previously regarded as a special type of fat relevant only for defending hibernating animals and newborns against a cold environment. Recently, BAT has received considerable attention following its (re)discovery in humans. Using glucose tracers, multiple laboratories independently found metabolically active BAT in adults. The enormous metabolic powers of BAT in animal models could make it an attractive target for antiobesity therapies in humans. Here, we review the present knowledge on the role of BAT in energy homeostasis and metabolism, focusing on signaling pathways and potential targets for novel therapeutics. We also shine light on ongoing debates, including those about the true color of brown fat in adults, as well as on the requirements for translation of basic research on BAT into clinical medicine.
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Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Obesidad/tratamiento farmacológico , Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/metabolismo , Adipocitos Blancos/efectos de los fármacos , Adipocitos Blancos/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/fisiopatología , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/fisiopatología , Animales , Descubrimiento de Drogas , Metabolismo Energético/efectos de los fármacos , Humanos , MicroARNs/metabolismo , Terapia Molecular Dirigida , Obesidad/genética , Obesidad/metabolismo , Obesidad/fisiopatología , Fenotipo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
The second messenger cyclic guanosine monophosphate (cGMP) is a key mediator in physiological processes such as vascular tone, and its essential involvement in pathways regulating metabolism has been recognized in recent years. Here, we focus on the fundamental role of cGMP in brown adipose tissue (BAT) differentiation and function. In contrast to white adipose tissue (WAT), which stores energy in the form of lipids, BAT consumes energy stored in lipids to generate heat. This so-called non-shivering thermogenesis takes place in BAT mitochondria, which express the specific uncoupling protein 1 (UCP1). The energy combusting properties of BAT render it a promising target in antiobesity strategies in which BAT could burn the surplus energy that has accumulated in obese and overweight individuals. cGMP is generated by guanylyl cyclases upon activation by nitric oxide or natriuretic peptides. It affects several downstream molecules including cGMP-receptor proteins such as cGMP-dependent protein kinase and is degraded by phosphodiesterases. The cGMP pathway contains several signaling molecules that can increase cGMP signaling, resulting in activation and recruitment of brown adipocytes, and hence can enhance the energy combusting features of BAT. In this review we highlight recent results showing the physiological significance of cGMP signaling in BAT, as well as pharmacological options targeting cGMP signaling that bear a high potential to become BAT-centered therapies for the treatment of obesity.
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Tejido Adiposo Pardo/fisiología , GMP Cíclico/fisiología , Adipocitos/fisiología , Tejido Adiposo Pardo/efectos de los fármacos , Animales , Diabetes Mellitus/tratamiento farmacológico , Humanos , Mitocondrias/fisiología , Obesidad/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
With more than half a billion individuals affected worldwide, obesity has reached pandemic proportions. Development of "brown-like" or "brite" adipocytes within white adipose tissue (WAT) has potential antiobesity and insulin-sensitizing effects. We investigated the role of cyclic GMP (cGMP) signaling, focusing on cGMP-dependent protein kinase I (PKGI) in WAT. PKGI is expressed in murine WAT, primary adipocytes, and 3T3-L1. Treatment of adipocytes with cGMP resulted in increased adipogenesis, with a 54% increase in expression of peroxisome proliferator-activated receptor-γ. Lentiviral overexpression of PKGI further increased adipogenesis, whereas loss of PKGI significantly reduced adipogenic differentiation. In addition to adipogenic effects, PKGI had an antihypertrophic and anti-inflammatory effect via RhoA phosphorylation and reduction of proinflammatory adipokine expression. Moreover, PKGI induced a 4.3-fold increase in abundance of UCP-1 and the development of a brown-like thermogenic program in primary adipocytes. Notably, treatment of C57BL/6 mice with phosphodiesterase inhibitor sildenafil (12 mg/kg/d) for 7 d caused 4.6-fold increase in uncoupling protein-1 expression and promoted establishment of a brown fat cell-like phenotype ("browning") of WAT in vivo. Taken together, PKGI is a key regulator of cell size, adipokine secretion and browning of white fat depots and thus could be a valuable target in developing novel treatments for obesity.
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Adipogénesis , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , GMP Cíclico/metabolismo , Células 3T3-L1/citología , Adipocitos/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Blanco/citología , Animales , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Canales Iónicos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Obesidad/metabolismo , Transducción de Señal , Proteína Desacopladora 1RESUMEN
A microfluidic polymer chip for the self-assembly of DNA conjugates through DNA-directed immobilization is developed. The chip is fabricated from two parts, one of which contains a microfluidic channel produced from poly(dimethylsiloxane) (PDMS) by replica-casting technique using a mold prepared by photolithographic techniques. The microfluidic part is sealed by covalent bonding with a chemically activated glass slide containing a DNA oligonucleotide microarray. The dimension of the PDMS-glass microfluidic chip is equivalent to standard microscope slides (76 x 26 mm(2)). The DNA microarray surface inside the microfluidic channels is configured through conventional spotting, and the resulting DNA patches can be conveniently addressed with compounds containing complementary DNA tags. To demonstrate the utility of the addressable surface within the microfluidic channel, DNA-directed immobilization (DDI) of DNA-modified gold nanoparticles (AuNPs) and DNA-conjugates of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) are carried out. DDI of AuNPs is used to demonstrate site selectivity and reversibility of the surface-modification process. In the case of the DNA-enzyme conjugates, the patterned assembly of the two enzymes allows the establishment and investigation of the coupled reaction of GOx and HRP, with particular emphasis on surface coverage and lateral flow rates. The results demonstrate that this addressable chip is well suited for the generation of fluidically coupled multi-enzyme microreactors.
Asunto(s)
Técnicas Biosensibles/instrumentación , ADN/química , Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Adsorción , ADN/ultraestructura , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of the IC50 value for LS104. In line with these data, combination treatment using LS104 plus an ATP-competitive JAK2 inhibitor (JAK inhibitor I) led to synergistically increased apoptosis in JAK2V617F-positive cells. Furthermore, LS104 strongly inhibited cytokine-independent growth of endogenous erythroid colonies isolated from patients with JAK2V617F-positive MPD in vitro, whereas there was no significant effect on growth of myeloid colonies obtained from normal controls. Based on these data, we have recently started a phase I clinical trial of LS104 for patients with JAK2V617F-positive MPDs. To the best of our knowledge, this is the first report on a non-ATP-competitive kinase inhibitor being tested in a clinical trial.
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Acrilonitrilo/análogos & derivados , Adenosina Trifosfato/metabolismo , Apoptosis , Janus Quinasa 2/antagonistas & inhibidores , Trastornos Mieloproliferativos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estirenos/farmacología , Acrilonitrilo/farmacología , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Janus Quinasa 2/metabolismo , Células K562 , Ratones , Trastornos Mieloproliferativos/tratamiento farmacológico , Fosforilación , Transducción de SeñalRESUMEN
Cancer research of immune-modulating mechanisms mainly addresses the role of tumor-infiltrating immune cells. Mechanisms modulating the adaptive immune system at the primary activation site - the draining lymph node (LN) - are less investigated. Here we present tumor-caused histomorphological changes in tumor draining LNs of breast cancer patients, dependent on the localization (sentinel LN vs. non-sentinel LN), the tumor size, the intrinsic subtype and nodal metastatic status. The quantitative morphological study was conducted in breast cancer patients with at least one sentinel LN and no neoadjuvant therapy. All LNs were annotated considering to their topographical location, stained for IgD/H&E, digitized and quantitatively analyzed. In 206 patients, 394 sentinels and 940 non-sentinel LNs were categorized, comprising 40758 follicles and 7074 germinal centers. Subtype specific immunomorphological patterns were detectable: Follicular density was higher in LNs of Her2 enriched hormone receptor positive and triple-negative breast cancers whereas hormone receptor positive breast cancers showed more macrophage infiltrations in the LN cortex. Follicles are rounder in metastatic LNs and non-sentinel LNs. The identified immunomorphological changes reflect different underlying immunomodulations taking place in the tumor-draining LNs and should therefore be considered as possible prognostic and predictive markers for LN metastasis and therapy associated immunomodulation.
Asunto(s)
Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Inmunomodulación , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Femenino , Centro Germinal , Humanos , Inmunohistoquímica , Inmunofenotipificación , Ganglios Linfáticos/metabolismo , Metástasis Linfática , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Biopsia del Ganglio Linfático CentinelaRESUMEN
Current worldwide figures suggest that obesity is pandemic. Understanding the underlying molecular mechanisms would help develop viable anti-obesity therapies. Here, we assess the influence of obesity-induced inflammation on white adipocyte cyclic guanosine monophosphate (cGMP) signaling, which is beneficial for adipocyte differentiation and thermogenesis. We find that murine gonadal and not inguinal fat is prone to obesity-induced inflammation. Correspondingly, the cGMP cascade is dysregulated in gonadal but not in inguinal fat of obese mice. Analysis of two independent human cohorts reveals a defective cGMP pathway only in visceral fat of obese subjects. Congruently, cGMP signaling is dysregulated in tumor necrosis factor α (TNF-α)-treated primary white adipocytes. TNF-α-mediated suppression of sGCß1 is mediated via NF-κB, whereas PKG is repressed by JNK signaling. Additionally, TNF-α-activated JNK signaling suppresses PPARγ and aP2. Taken together, the intensity of obesity-induced inflammation dictates the amplitude of cGMP signaling dysregulation in white adipocytes through distinct pathways.
Asunto(s)
GMP Cíclico/metabolismo , Inflamación/metabolismo , Inflamación/patología , Obesidad/metabolismo , Obesidad/patología , Transducción de Señal , Adipogénesis/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Femenino , Gónadas/metabolismo , Guanilato Ciclasa/metabolismo , Humanos , Inflamación/complicaciones , Conducto Inguinal/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Biológicos , FN-kappa B/metabolismo , Obesidad/complicaciones , Fenotipo , Transducción de Señal/efectos de los fármacos , Solubilidad , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity.
RESUMEN
Helminths induce type 2 immune responses and establish an anti-inflammatory milieu in their hosts. This immunomodulation was previously shown to improve diet-induced insulin resistance which is linked to chronic inflammation. In the current study, we demonstrate that infection with the filarial nematode Litomosoides sigmodontis increased the eosinophil number and alternatively activated macrophage abundance within epididymal adipose tissue (EAT) and improved glucose tolerance in diet-induced obese mice in an eosinophil-dependent manner. L. sigmodontis antigen (LsAg) administration neither altered the body weight of animals nor adipose tissue mass or adipocyte size, but it triggered type 2 immune responses, eosinophils, alternatively activated macrophages, and type 2 innate lymphoid cells in EAT. Improvement in glucose tolerance by LsAg treatment remained even in the absence of Foxp3+ regulatory T cells. Furthermore, PCR array results revealed that LsAg treatment reduced inflammatory immune responses and increased the expression of genes related to insulin signaling (Glut4, Pde3b, Pik3r1, and Hk2) and fatty acid uptake (Fabp4 and Lpl). Our investigation demonstrates that L. sigmodontis infection and LsAg administration reduce diet-induced EAT inflammation and improve glucose tolerance. Helminth-derived products may, therefore, offer new options to improve insulin sensitivity, while loss of helminth infections in developing and developed countries may contribute to the recent increase in the prevalence of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/inmunología , Eosinófilos/inmunología , Filariasis/inmunología , Filarioidea/inmunología , Macrófagos/inmunología , Obesidad/inmunología , Células Th2/inmunología , Animales , Antígenos Helmínticos/inmunología , Dieta , Modelos Animales de Enfermedad , Humanos , Resistencia a la Insulina/genética , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genéticaRESUMEN
The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and anti-inflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC.
Asunto(s)
Guanilato Ciclasa/metabolismo , Hemo/metabolismo , Riñón/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Presión Sanguínea/fisiología , GMP Cíclico/metabolismo , Tasa de Filtración Glomerular/fisiología , Masculino , Óxido Nítrico/metabolismo , Ratas , Ratas Endogámicas Dahl , Guanilil Ciclasa SolubleRESUMEN
Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimulator BAY 41-8543 enhances lipid uptake into BAT and increases whole-body energy expenditure, whereas ablation of the haeme-containing ß1-subunit of sGC severely impairs BAT function. Notably, the sGC stimulator enhances differentiation of human brown adipocytes as well as induces 'browning' of primary white adipocytes. Taken together, our data suggest that sGC is a potential pharmacological target for the treatment of obesity and its comorbidities.
Asunto(s)
Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Morfolinas/uso terapéutico , Obesidad/tratamiento farmacológico , Pirimidinas/uso terapéutico , Adipocitos/efectos de los fármacos , Animales , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Morfolinas/farmacología , Obesidad/prevención & control , Pirimidinas/farmacología , Termogénesis , Pérdida de PesoRESUMEN
Essential physiological homeostatic processes such as vascular tone, fluid balance, cardiorenal function, and sensory processes are regulated by the second messenger cyclic guanosine 3', 5'-monophosphate (cGMP). Dysregulation of cGMP-dependent pathways plays an important role in cardiovascular diseases such as hypertension, pulmonary hypertension, heart failure, or erectile dysfunction. Thus, the cGMP pathway consisting of the cGMP-generating guanylyl cyclases, protein kinases, and phosphodiesterases (PDE) has evolved to an important drug target and is the focus of a wide variety of research fields ranging from unraveling mechanisms on the molecular level to understanding the regulation of physiological and pathophysiological processes by cGMP. Based on the results from basic and preclinical research, therapeutic drugs have been developed which modulate the cGMP pathway and are investigated in clinical trials. Riociguat, a nitric oxide (NO)-independent soluble guanylyl cyclase stimulator; recombinant B-type natriuretic peptide (BNP); or recombinant atrial natriuretic peptide (ANP) and PDE5 inhibitors are cGMP-modulating drugs that are already available for the treatment of pulmonary hypertension, acute heart failure, and erectile dysfunction, respectively. The latest results from basic to clinical research on cGMP were presented on the 6th International Conference on cGMP in Erfurt, Germany, and are summarized in this article.
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GMP Cíclico/metabolismo , Animales , Bacterias/metabolismo , Guanilato Ciclasa/química , Guanilato Ciclasa/metabolismo , Humanos , Péptidos Natriuréticos/metabolismo , Óxido Nítrico/metabolismo , Conformación Proteica , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa SolubleRESUMEN
The second messenger cyclic guanosine monophosphate (cGMP) mediates the physiological effects of nitric oxide and natriuretic peptides in a broad spectrum of tissues and cells. So far, the major focus of research on cGMP lay on the cardiovascular system. Recent evidence suggests that cGMP also plays a major role in the regulation of cellular and whole-body metabolism. Here, we focus on the role of cGMP in adipose tissue. In addition, other organs important for the regulation of metabolism and their regulation by cGMP are discussed. Targeting the cGMP signaling pathway could be an exciting approach for the regulation of energy expenditure and the treatment of obesity.
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GMP Cíclico/metabolismo , Tejido Adiposo/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Metabolismo Energético , Humanos , Hígado/metabolismo , Páncreas/metabolismo , Transducción de SeñalRESUMEN
In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.
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Fluorescencia , Guanilato Ciclasa/metabolismo , Hemo/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Benzoatos/farmacología , Células CHO , Cricetinae , Cricetulus , GMP Cíclico/metabolismo , Cisteína/genética , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Colorantes Fluorescentes/química , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Humanos , Datos de Secuencia Molecular , Mutación , Óxido Nítrico/metabolismo , Oxadiazoles/farmacología , Oxazinas/farmacología , Oxidación-Reducción/efectos de los fármacos , Ingeniería de Proteínas/métodos , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Rotenona/farmacología , Transducción de Señal/efectos de los fármacos , Guanilil Ciclasa Soluble , Espectrometría de FluorescenciaRESUMEN
OBJECTIVES: The nitric oxide-soluble guanylate cyclase (sGC)-cGMP signal transduction pathway is impaired in different cardiovascular diseases, including pulmonary hypertension, heart failure and arterial hypertension. Riociguat is a novel stimulator of soluble guanylate cyclase (sGC). However, little is known about the effects of sGC stimulators in experimental models of hypertension. We thus investigated the cardio-renal protective effects of riociguat in low-renin and high-renin rat models of hypertension. METHODS: The vasorelaxant effect of riociguat was tested in vitro on isolated saphenous artery rings of normal and nitrate tolerant rabbits. The cardiovascular in-vivo effects of sGC stimulation were evaluated in hypertensive renin-transgenic rats treated with the nitric oxide-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (high-renin model) and in rats with 5/6 nephrectomy (low-renin model). RESULTS: In both animal models, riociguat treatment improved survival and normalized blood pressure. Moreover, in the L-NAME study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight and lower cardiac interstitial fibrosis, and reduced renal target organ damage as indicated by lower plasma creatinine and urea, less glomerulosclerosis and less renal interstitial fibrosis. In the 5/6 nephrectomy study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight, lower myocyte diameter and lower arterial media/lumen ratio, and reduced renal target organ damage as indicated by improved creatinine clearance and less renal interstitial fibrosis. CONCLUSION: We demonstrate for the first time that the novel sGC stimulator riociguat shows in two independent models of hypertension a potent protection against cardiac and renal target organ damage.
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Guanilato Ciclasa/metabolismo , Corazón/efectos de los fármacos , Hipertensión/prevención & control , Riñón/efectos de los fármacos , Óxido Nítrico/metabolismo , Renina/fisiología , Animales , Animales Modificados Genéticamente , Antihipertensivos/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Guanilato Ciclasa/antagonistas & inhibidores , Hipertensión/inducido químicamente , Hipertensión/enzimología , Riñón/patología , Longevidad/efectos de los fármacos , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocardio/patología , NG-Nitroarginina Metil Éster/toxicidad , Nefrectomía , Nefritis Intersticial/inducido químicamente , Nefritis Intersticial/patología , Nefritis Intersticial/prevención & control , Pirazoles/farmacología , Pirimidinas/farmacología , Conejos , Ratas , Ratas Wistar , Renina/efectos de los fármacos , Transducción de SeñalRESUMEN
The ubiquitously expressed nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in signal transduction. Binding of NO to the N-terminal prosthetic heme moiety of sGC results in approximately 200-fold activation of the enzyme and an increased conversion of GTP into the second messenger cGMP. sGC exists as a heterodimer the dimerization of which is mediated mainly by the central region of the enzyme. In the present work, we constructed deletion mutants within the predicted dimerization region of the sGC alpha(1)- and beta(1)-subunit to precisely map the sequence segments crucial for subunit dimerization. To track mutation-induced alterations of sGC dimerization, we used a bimolecular fluorescence complementation approach that allows visualizing sGC heterodimerization in a noninvasive manner in living cells. Our study suggests that segments spanning amino acids alpha(1)363-372, alpha(1)403-422, alpha(1)440-459, beta(1)212-222, beta(1)304-333, beta(1)344-363, and beta(1)381-400 within the predicted dimerization region are involved in the process of heterodimerization and therefore in the expression of functional sGC.
Asunto(s)
Guanilato Ciclasa/química , Receptores Citoplasmáticos y Nucleares/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Dimerización , Fluorescencia , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Eliminación de Secuencia , Guanilil Ciclasa Soluble , Espectrometría de Fluorescencia/métodosRESUMEN
Pex19p is required for the topogenesis of peroxisomal membrane proteins (PMPs). Here we have demonstrated that Pex19p is also required for the peroxisomal targeting and stability of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have demonstrated that Pex17p is associated with the peroxisomal Pex13p-Pex14p complex as well as with Pex19p. We have identified the corresponding binding sites for Pex14p and Pex19p and demonstrated that a specific loss of the Pex19p interaction resulted in mistargeting of Pex17p. We have shown that a construct consisting only of the Pex19p- and Pex14p-binding sites of Pex17p is sufficient to direct an otherwise cytosolic reporter protein to the peroxisomal membrane in a Pex19p-dependent manner. Our data show that the function of Pex19p as chaperone or import receptor is not restricted to integral membrane proteins but may also include peripheral PMPs. As a consequence of our data, the previous definition of a targeting signal for PMPs (mPTS) as a Pex19p-binding motif in conjunction with a transmembrane segment should be extended to regions comprising a Pex19p-binding motif and a peroxisomal anchor sequence.