RESUMEN
The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure-function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF-RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand-receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor-synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Proteínas Bacterianas/química , Burkholderia/metabolismo , Cristalización , Cristalografía por Rayos X , GMP Cíclico/biosíntesis , Ácidos Láuricos/química , Ácidos Láuricos/metabolismo , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Percepción de QuorumRESUMEN
The ability of many pathogenic bacteria to grow as a biofilm results in lower susceptibility to antibiotic treatments and to the host immune response, thus leading to the development of chronic infections. The understanding that biofilms can play an important role in bacterial virulence has prompted the search for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we present two examples of target-based microbiological screenings for antimicrobials endowed with anti-biofilm activity, aimed respectively at the inhibition of the signal molecule cyclic di-GMP and of quorum sensing.
Asunto(s)
Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Vías Biosintéticas/efectos de los fármacos , GMP Cíclico/análogos & derivados , GMP Cíclico/biosíntesis , Evaluación Preclínica de Medicamentos , Percepción de Quorum/efectos de los fármacosRESUMEN
Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx(5cv) mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Distrofina/genética , Hemo-Oxigenasa 1/biosíntesis , Distrofia Muscular de Duchenne/tratamiento farmacológico , Animales , GMP Cíclico/biosíntesis , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Distrofina/deficiencia , Hemo-Oxigenasa 1/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Purinas/farmacología , ARN Mensajero/genética , Transducción de Señal/genética , Citrato de Sildenafil , Sulfonas/farmacología , Regulación hacia Arriba , Pez Cebra/genéticaRESUMEN
Human skin contains photolabile nitric oxide (NO) derivates such as nitrite and S-nitrosothiols, which upon UVA radiation decompose under high-output NO formation and exert NO-specific biological responses such as increased local blood flow or reduced blood pressure. To avoid the injurious effects of UVA radiation, we here investigated the mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic NO generation from photolabile nitric oxide derivates in human skin in vitro and in vivo. As quantified by chemiluminescence detection (CLD), at physiological pH blue light at 420 or 453 nm induced a significant NO formation from S-nitrosoalbumin and also from aqueous nitrite solutions by a to-date not entirely identified Cu(1+)-dependent mechanism. As detected by electron paramagnetic resonance spectrometry in vitro with human skin specimens, blue light irradiation significantly increased the intradermal levels of free NO. As detected by CLD in vivo in healthy volunteers, irradiation of human skin with blue light induced a significant emanation of NO from the irradiated skin area as well as a significant translocation of NO from the skin surface into the underlying tissue. In parallel, blue light irradiation caused a rapid and significant rise in local cutaneous blood flow as detected noninvasively by using micro-light-guide spectrophotometry. Irradiation of human skin with moderate doses of blue light caused a significant increase in enzyme-independent cutaneous NO formation as well as NO-dependent local biological responses, i.e., increased blood flow. The effects were attributed to blue-light-induced release of NO from cutaneous photolabile NO derivates. Thus, in contrast to UVA, blue-light-induced NO generation might be therapeutically used in the treatment of systemic and local hemodynamic disorders that are based on impaired physiological NO production or bioavailability.
Asunto(s)
Óxido Nítrico/biosíntesis , Nitritos/química , S-Nitrosotioles/química , Piel/metabolismo , Piel/efectos de la radiación , Adulto , Animales , Línea Celular Tumoral , Cobre/química , GMP Cíclico/biosíntesis , GMP Cíclico/química , Femenino , Humanos , Luz , Luminiscencia , Masculino , Óxido Nítrico/sangre , Óxido Nítrico/química , Compuestos Nitrosos/química , Fototerapia/métodos , Ratas , Albúmina Sérica Bovina/químicaRESUMEN
Ingestion of vegetables rich in inorganic nitrate has emerged as an effective method, via the formation of a nitrite intermediate, for acutely elevating vascular NO levels. As such a number of beneficial effects of dietary nitrate ingestion have been demonstrated including the suggestion that platelet reactivity is reduced. In this study we investigated whether inorganic nitrate supplementation might also reduce platelet reactivity in healthy volunteers and have determined the mechanisms involved in the effects seen. We conducted two randomised crossover studies each in 24 (12 of each sex) healthy subjects assessing the acute effects of dietary nitrate (250 ml beetroot juice) or potassium nitrate capsules (KNO3, 8 mmol) vs placebo control on platelet reactivity. Inorganic nitrate ingested either from a dietary source or via supplementation raised circulating nitrate and nitrite levels in both sexes and attenuated ex vivo platelet aggregation responses to ADP and, albeit to a lesser extent, collagen but not epinephrine in male but not female volunteers. These inhibitory effects were associated with a reduced platelet P-selectin expression and elevated platelet cGMP levels. In addition, we show that nitrite reduction to NO occurs at the level of the erythrocyte and not the platelet. In summary, our results demonstrate that inorganic nitrate ingestion, whether via the diet or through supplementation, causes a modest decrease in platelet reactivity in healthy males but not females. Our studies provide strong support for further clinical trials investigating the potential of dietary nitrate as an adjunct to current antiplatelet therapies to prevent atherothrombotic complications. Moreover, our observations highlight a previously unknown sexual dimorphism in platelet reactivity to NO and intimate a greater dependence of males on the NO-soluble guanylate cyclase pathway in limiting thrombotic potential.
Asunto(s)
Plaquetas/metabolismo , Nitratos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Compuestos de Potasio/farmacología , Adolescente , Adulto , Beta vulgaris , Enfermedades Cardiovasculares/tratamiento farmacológico , Colágeno/farmacología , Estudios Cruzados , GMP Cíclico/biosíntesis , Dieta , Suplementos Dietéticos , Epinefrina/farmacología , Eritrocitos/metabolismo , Femenino , Guanilato Ciclasa/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Nitratos/administración & dosificación , Óxido Nítrico/metabolismo , Selectina-P/biosíntesis , Inhibidores de Agregación Plaquetaria/administración & dosificación , Compuestos de Potasio/administración & dosificación , Factores Sexuales , Verduras , Adulto JovenRESUMEN
In addition to its role as a carboxylase cofactor, biotin modifies gene expression and has manifold effects on systemic processes. Several studies have shown that biotin supplementation reduces hypertriglyceridemia. We have previously reported that this effect is related to decreased expression of lipogenic genes. In the present work, we analyzed signaling pathways and posttranscriptional mechanisms involved in the hypotriglyceridemic effects of biotin. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg of free biotin/kg diet, respectively for 8 weeks after weaning. The abundance of mature sterol regulatory element-binding protein (SREBP-1c), fatty-acid synthase (FAS), total acetyl-CoA carboxylase-1 (ACC-1) and its phosphorylated form, and AMP-activated protein kinase (AMPK) were evaluated in the liver. We also determined the serum triglyceride concentrations and the hepatic levels of triglycerides and cyclic GMP (cGMP). Compared to the control group, biotin-supplemented mice had lower serum and hepatic triglyceride concentrations. Biotin supplementation increased the levels of cGMP and the phosphorylated forms of AMPK and ACC-1 and decreased the abundance of the mature form of SREBP-1c and FAS. These data provide evidence that the mechanisms by which biotin supplementation reduces lipogenesis involve increased cGMP content and AMPK activation. In turn, these changes lead to augmented ACC-1 phosphorylation and decreased expression of both the mature form of SREBP-1c and FAS. Our results demonstrate for the first time that AMPK is involved in the effects of biotin supplementation and offer new insights into the mechanisms of biotin-mediated hypotriglyceridemic effects.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Biotina/farmacología , GMP Cíclico/biosíntesis , Suplementos Dietéticos , Hipertrigliceridemia/dietoterapia , Hipertrigliceridemia/enzimología , Hipolipemiantes/farmacología , Proteínas Quinasas Activadas por AMP/genética , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipertrigliceridemia/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Lipogénesis/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/sangreRESUMEN
Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.
Asunto(s)
Encéfalo/metabolismo , D-Aminoácido Oxidasa/antagonistas & inhibidores , Memoria a Corto Plazo/fisiología , Agitación Psicomotora/tratamiento farmacológico , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , GMP Cíclico/análisis , GMP Cíclico/biosíntesis , D-Aminoácido Oxidasa/metabolismo , D-Aminoácido Oxidasa/fisiología , Evaluación Preclínica de Medicamentos , Electroencefalografía , Habituación Psicofisiológica/efectos de los fármacos , Habituación Psicofisiológica/fisiología , Harmalina/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Memoria a Corto Plazo/efectos de los fármacos , Mescalina/farmacología , Ratones , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Modelos Biológicos , Modelos Químicos , Terapia Molecular Dirigida , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Prurito/inducido químicamente , Prurito/prevención & control , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Filtrado Sensorial/efectos de los fármacos , Filtrado Sensorial/fisiología , Serina/sangre , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1ß, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.
Asunto(s)
Adenosina Trifosfato/metabolismo , Biotecnología/métodos , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Guanosina Monofosfato/metabolismo , Guanilato-Quinasas/metabolismo , Factores Inmunológicos/biosíntesis , Nucleósido-Difosfato Quinasa/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Animales , Línea Celular , Cromatografía por Intercambio Iónico , Clonación Molecular , GMP Cíclico/biosíntesis , GMP Cíclico/farmacología , Escherichia coli , Proteínas de Escherichia coli/genética , Guanilato-Quinasas/genética , Factores Inmunológicos/farmacología , Interleucina-1beta/biosíntesis , Interleucina-1beta/inmunología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Espectroscopía de Resonancia Magnética , Ratones , Mutación , Nucleósido-Difosfato Quinasa/genética , Liasas de Fósforo-Oxígeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformación Bacteriana , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Guanylate cyclases constitute a gene family of enzymes that synthesize the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) and play important roles in diverse physiological functions. Here we report a novel, simple and highly sensitive method for measurement intracellular cGMP concentrations using a cAMP-responsive element (CRE) and cGMP-dependent protein kinase (cGK). Transient transfection of the CRE reporter plasmid, encoding a luciferase reporter gene under the control of a modified promoter containing a CRE, and a cGK expression vector into HEK293 cells followed by treatment with 8-bromo-cGMP showed a dose dependent increase in luciferase activity. Moreover, HEK293 cells expressing GC-A or GC-B natriuretic peptide receptors and harboring this reporter system responded to specific ligands in a dose dependent manner. Our results indicate that this reporter gene method enables high throughput screening of receptor-type GC selective agonists in the treatment of cardiovascular diseases and homeostatic dysfunctions.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/biosíntesis , GMP Cíclico/análisis , Receptores Acoplados a la Guanilato-Ciclasa/agonistas , GMP Cíclico/análogos & derivados , GMP Cíclico/biosíntesis , GMP Cíclico/genética , GMP Cíclico/farmacología , Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Evaluación Preclínica de Medicamentos , Genes Reporteros/efectos de los fármacos , Células HEK293 , Humanos , Elementos de Respuesta , Transducción de SeñalRESUMEN
OBJECTIVES: To investigate further the role of phosphodiesterase (PDE) isoenzymes in the control of human seminal vesicle (SV) smooth muscle contractility, we examined the functional responses of isolated SV tissue to various PDE inhibitors. It has been suggested that the application of inhibitors of the PDE type 5 may facilitate SV smooth muscle relaxation and, subsequently, retard ejaculatory response. METHODS: Using the organ bath technique, strip preparations of human SV were exposed for 5 minutes to 1 µM of the PDE inhibitors milrinone (PDE3 inhibitor), rolipram, Ro 20-1724 (PDE4 inhibitors), and sildenafil (PDE5 inhibitor). Norepinephrine (NE, alpha agonist) was then added (0,1 µM, 1 µM, and 10 µM) and isometric responses were recorded. A contraction-response curve to NE in the absence of PDE inhibitors was also generated. Drug effects on the production of cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP) were measured by means of radioimmunometric assays. RESULTS: The contraction induced by NE was effectively antagonized by 1 µM of rolipram (83.3% inhibition), Ro 20-1724 (72.3% inhibition), sildenafil (41.6% inhibition), and milrinone (37.5% inhibition). The inhibition of force generation was paralleled by a 1.6-fold to 2.8-fold increase in tissue cyclic AMP (induced by milrinone, rolipram, Ro 20-1724), and a 12-fold rise in cyclic GMP (induced by sildenafil). CONCLUSION: The findings demonstrate that PDE inhibitors can counteract the contraction of human SV mediated by alpha-adrenergic receptors and enhance levels of cyclic nucleotides. This might be of importance with regard to the identification of new options for the pharmacological treatment of premature ejaculation.
Asunto(s)
AMP Cíclico/biosíntesis , GMP Cíclico/biosíntesis , Relajación Muscular/efectos de los fármacos , Norepinefrina/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Vesículas Seminales/efectos de los fármacos , 4-(3-Butoxi-4-metoxibencil)-2-imidazolidinona/farmacología , Anciano , Colforsina/farmacología , Evaluación Preclínica de Medicamentos , Eyaculación/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Milrinona/farmacología , Nitroprusiato/farmacología , Piperazinas/farmacología , Purinas/farmacología , Receptores Adrenérgicos alfa/fisiología , Rolipram/farmacología , Vesículas Seminales/metabolismo , Citrato de Sildenafil , Sulfonas/farmacologíaRESUMEN
A computational model of human cones for intensities ranging from 1 td up to full bleaching levels is presented. The model conforms well with measurements made in primate horizontal cells, follows Weber's law at high intensities, and performs range compression consistent with what is known of cones in other vertebrates. The model consists entirely of processes with a clear physiological interpretation: pigment bleaching, saturation of cGMP hydrolysis, calcium feedback on cGMP synthesis, and a nonlinear membrane. The model is implemented according to a very fast computational scheme useful for simulations, and sample programs in Matlab and Fortran are provided as supplementary material.
Asunto(s)
Simulación por Computador , Luz , Modelos Neurológicos , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Adaptación Fisiológica , Animales , Calcio/metabolismo , Sensibilidad de Contraste/fisiología , GMP Cíclico/biosíntesis , GMP Cíclico/metabolismo , Retroalimentación Fisiológica , Humanos , Hidrólisis , Macaca , Membranas/fisiología , Dinámicas no Lineales , Estimulación Luminosa/métodos , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Horizontales de la Retina/fisiología , Pigmentos Retinianos/fisiologíaRESUMEN
The butanol extract of Phellinus igniarius (BPI) induced relaxation of the phenylephrin e-precontracted rat aorta in a dose-dependent manner, and its effect was abolished by the removal of functional endothelium. Pretreatment of the aortic tissues with N(G)-nitro-L-arginine methyl ester (L-NAME), methylene blue, or 1H-[1,2,4]-oxadiazole-[4,3-alpha]-quinoxalin1-one (ODQ) inhibited the vascular relaxation induced by BPI. BPI-induced vascular relaxations were also markedly attenuated by the addition of verapamil or diltiazem, while the relaxant effect of BPI was not blocked by pretreatment with indomethacine, glibenclamide, tetraethylammonium (TEA), atropine, or propranolol. Incubation of endothelium-intact rat aorta with BPI increased the production of cGMP in a dose-dependent manner. These results suggest that BPI dilates vascular smooth muscle via endothelium-dependent nitric oxide-cGMP signaling pathway, with the possible involvement of L-type Ca(2+) channels.
Asunto(s)
Aorta/efectos de los fármacos , Endotelio Vascular/fisiología , Polyporaceae/química , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Aorta/metabolismo , Aorta/fisiología , Atropina/farmacología , Butanoles/química , GMP Cíclico/biosíntesis , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Gliburida/farmacología , Técnicas In Vitro , Indometacina/farmacología , Masculino , Azul de Metileno/farmacología , NG-Nitroarginina Metil Éster/farmacología , Oxadiazoles/farmacología , Propranolol/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Tetraetilamonio/farmacología , Vasodilatación/fisiología , Vasodilatadores/aislamiento & purificación , Verapamilo/farmacologíaRESUMEN
Polyphenolic grapevine components involved in plant resistance against pathogens possess various pharmacological properties that include nitric oxide (NO)-dependent vasodilation and anti-inflammatory and free radical scavenging activities, which may explain the protective effect of moderate red wine consumption against cardiovascular disease. The aim of this work was (a) to verify the possibility that preharvest treatments of grapevine with a plant activator, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), could lead to an enriched nutraceutical potential of wine and (b) to characterize the profile of metabolites responsible for pharmacological activity. Plant spraying at the end of veraison, with a water suspension of BTH (0.3 mM), led to increased whole anthocyanin content as confirmed by HPLC comparative analysis. Extracts from berry skins of BTH-treated grapevines caused NO-dependent vasorelaxation, with a concentration-response curve that was significantly shifted to the left of the control non-BTH-treated curve. Moreover, 1:1000 dilutions of berry extracts from BTH-treated plants significantly increased basal production of guanosine 3',5'-cyclic monophosphate (cGMP) in human vascular endothelial cells when compared to the corresponding extracts of untreated plants. These results show that BTH treatment increases anthocyanin content of grape extracts, as well as their ability to induce NO-mediated vasoprotection. No increase of anthocyanin content was observed in the wine extracts from BTH-treated vines. It is concluded that BTH treatment could be exploited to increase the nutraceutical potential of grapes.
Asunto(s)
Flavonoides/farmacología , Frutas/química , Óxido Nítrico/farmacología , Fenoles/farmacología , Tiadiazoles/administración & dosificación , Vasodilatación/efectos de los fármacos , Vitis/química , Antiinflamatorios/farmacología , Células Cultivadas , GMP Cíclico/biosíntesis , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Depuradores de Radicales Libres/farmacología , Humanos , Extractos Vegetales/farmacología , Polifenoles , Venas Umbilicales , Vitis/efectos de los fármacos , Vitis/crecimiento & desarrollo , Vino/análisisRESUMEN
Quercetin is one of the dietary-derived flavonoids that are held responsible for the beneficial effects of red wine drinking in coronary artery disease known as the "French paradox". We examined whether quercetin modulates endothelial function by influencing Ca2+-activated K+ channels with large conductance (BK(Ca)) in cultured human endothelial cells. Membrane potential and intracellular Ca2+ concentrations of cultured human endothelial cells derived from umbilical cord veins (HUVEC) were measured using the fluorescence dyes DiBAC, and FURA-2, respectively. NO production was examined using a cGMP radioimmunoassay. HUVEC proliferation was analyzed by cell counts and thymidine incorporation. A dose-dependent hyperpolarization of HUVEC was recorded when quercetin was added (5-100 micromol/L). The maximum effect (50 micromol/L) was significantly reduced by the addition of the highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/L), but not by blockers of other Ca2+-activated K+ channels (n = 30; p < 0.05). This BK(Ca)-induced hyperpolarization caused a transmembrane Ca2+ influx, because the quercetin-induced increase of intracellular Ca2+ was blocked by iberiotoxin, or by applying 2-aminoethoxydiphenylborate (100 micromol/L)--an inhibitor of capacitative Ca2+ entry (n = 30; p < 0.05). Quercetin-induced cGMP levels were significantly reduced by the eNOS-inhibitor l-NMMA (300 micromol/L), and by iberiotoxin (n = 10; p < 0.05). Endothelial proliferation was significantly reduced by 56 % when cells were incubated with quercetin (n = 12; p < 0.05). This effect was due to the increased NO production, because it was reversed when the cells were treated with a combination of quercetin and l-NMMA. In conclusion quercetin improves endothelial dysfunction by increasing NO synthesis involving BK(Ca)-dependent membrane hyperpolarization-induced capacitative Ca 2+ entry. Increased NO production is responsible for the quercetin-dependent inhibition of endothelial proliferation.
Asunto(s)
Calcio/metabolismo , Fitoterapia , Plantas Medicinales , Canales de Potasio Calcio-Activados/efectos de los fármacos , Quercetina/farmacología , División Celular/efectos de los fármacos , GMP Cíclico/biosíntesis , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Humanos , Potenciales de la Membrana , Óxido Nítrico/biosíntesis , Canales de Potasio Calcio-Activados/fisiología , Quercetina/administración & dosificación , Quercetina/uso terapéuticoRESUMEN
BACKGROUND: Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. METHODS AND RESULTS: In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272. CONCLUSIONS: In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.
Asunto(s)
Proteínas de Unión al Calcio/agonistas , Hipertensión Pulmonar/tratamiento farmacológico , Óxido Nítrico/uso terapéutico , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Resistencia Vascular/efectos de los fármacos , Vasodilatadores/uso terapéutico , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/toxicidad , Administración por Inhalación , Animales , Presión Sanguínea/efectos de los fármacos , Proteínas de Unión al Calcio/fisiología , Dióxido de Carbono/sangre , Monóxido de Carbono/farmacología , GMP Cíclico/biosíntesis , GMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Proteínas Activadoras de la Guanilato-Ciclasa , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/fisiopatología , Infusiones Intravenosas , NG-Nitroarginina Metil Éster/administración & dosificación , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/administración & dosificación , Óxido Nítrico/farmacología , Oxígeno/sangre , Arteria Pulmonar , Pirazoles/administración & dosificación , Pirazoles/farmacología , Piridinas/administración & dosificación , Piridinas/farmacología , Ovinos , Vasodilatadores/administración & dosificación , Vasodilatadores/farmacología , VigiliaRESUMEN
Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca2+ mobilization is regulated by phospholipase C-2 (PLC-gamma2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca2+ mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca2+ mobilization.
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Plaquetas/efectos de los fármacos , Calcio/metabolismo , Medicina de Hierbas , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Plaquetas/metabolismo , Proteínas Portadoras/farmacología , AMP Cíclico/biosíntesis , GMP Cíclico/biosíntesis , Humanos , Inositol 1,4,5-Trifosfato/biosíntesis , Corea (Geográfico) , Péptidos/farmacología , Fosfolipasa C gamma , Fosforilación , Activación Plaquetaria , Trombina/farmacología , Tromboxano B2/biosíntesis , Fosfolipasas de Tipo C/metabolismoRESUMEN
Considerable evidence suggests that the nitric oxide (NO)/cGMP signaling pathway plays an important role in the expression of reproductive behavior and in gonadotropin-releasing hormone (GnRH) release from the hypothalamus The effects of the NO/cGMP pathway on GnRH release and gene expression have also been examined in GT1 cells. However, it is still controversial whether NO/cGMP signaling facilitates or inhibits GnRH release in these cells. The current study examined the effects of estradiol and progesterone on neuronal NO synthase (nNOS), soluble guanylyl cyclase (sGC), and NO-dependent cGMP production in the preoptic area (POA) and hypothalamus (HYP) as well as in GT1-1 cells. Ovariectomized female rats received vehicle, estradiol benzoate (48 h) and/or progesterone (3-4 h) before preparation of brain slices. GT1-1 cells were incubated with vehicle, estradiol (48 h), progesterone (3-4 h), or with both hormones. The combination of estradiol and progesterone increased the expression of nNOS protein in the POA and HYP. Hormones had little effect on the abundance of sGC. Estradiol and progesterone together greatly enhanced NO-stimulated sGC activity in HYP-POA slices. In GT1-1 cells, NO-stimulated sGC activity was significantly increased by estradiol and progesterone, alone or in combination, but sGC expression was not altered by hormones.
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GMP Cíclico/metabolismo , Estradiol/farmacología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Óxido Nítrico/metabolismo , Progesterona/farmacología , Animales , Línea Celular , GMP Cíclico/biosíntesis , Femenino , Guanilato Ciclasa , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Nitroprusiato/farmacología , Ovariectomía , Área Preóptica/efectos de los fármacos , Área Preóptica/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa SolubleAsunto(s)
Endotelio Vascular/fisiología , Oxigenoterapia Hiperbárica , Neutrófilos/fisiología , Animales , Antígenos CD18/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , GMP Cíclico/biosíntesis , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
1. Nitric oxide (NO) participates, at least in part, to the establishment and maintenance of pain after nerve injury. Therefore, drugs that target the NO/cGMP signaling pathway are of interest for the treatment of human neuropathic pain. Various compounds endowed with NO-releasing properties modulate the expression and function of inducible nitric oxide synthase (iNOS), the key enzyme responsible for sustained NO production under pathological conditions including neuropathic pain. 2. With this background, we synthesized a new chemical entity, [1-(aminomethyl)cyclohexane acetic acid 3-(nitroxymethyl)phenyl ester] NCX8001, which has a NO-releasing moiety bound to gabapentin, a drug currently used for the clinical management of neuropathic pain. We examined the pharmacological profile of this drug with respect to its NO-releasing properties in vitro as well as to its efficacy in treating neuropathic pain conditions (allodynia) consequent to experimental sciatic nerve or spinal cord injuries. 3. NCX8001 (1-30 microm) released physiologically relevant concentrations of NO as it induced a concentration-dependent activation of soluble guanylyl cyclase (EC(50)=5.6 microm) and produced consistent vasorelaxant effects in noradrenaline-precontracted rabbit aortic rings (IC(50)=1.4 microm). 4. NCX8001, but not gabapentin, counteracted in a concentration-dependent fashion lipopolysaccharide-induced overexpression and function of iNOS in RAW264.7 macrophages cell line. Furthermore, NCX8001 also inhibited the release of tumor necrosis factor alpha (TNFalpha) from stimulated RAW264.7 cells. 5. NCX8001 (28-280 micromol x kg(-1), i.p.) reduced the allodynic responses of spinal cord injured rats in a dose-dependent fashion while lacking sedative or motor effects. In contrast, gabapentin (170-580 micromol x kg(-1), i.p.) resulted less effective and elicited marked side effects. 6. NCX8001 alleviated the allodynia-like responses of rats to innocuous mechanical or cold stimulation following lesion of the sciatic nerve. This effect was not shared by equimolar doses of gabapentin. 7. Potentially due to the slow releasing kinetics of NO, NCX8001 alleviated pain-like behaviors in two rat models of neuropathic pain in a fashion that is superior to its parent counterpart gabapentin. This new gabapentin derivative, whose mechanism deserves to be explored further, offers new hopes to the treatment of human neuropathic pain.
Asunto(s)
Acetatos/uso terapéutico , Aminas/uso terapéutico , Conducta Animal/efectos de los fármacos , Ácidos Ciclohexanocarboxílicos/uso terapéutico , Nitratos/uso terapéutico , Óxido Nítrico/metabolismo , Dolor/tratamiento farmacológico , Traumatismos de los Nervios Periféricos , Nervios Periféricos/efectos de los fármacos , Ácido gamma-Aminobutírico/uso terapéutico , Acetatos/química , Acetatos/metabolismo , Acetatos/farmacología , Aminas/metabolismo , Aminas/farmacología , Animales , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Conducta Animal/fisiología , GMP Cíclico/biosíntesis , GMP Cíclico/química , Ácidos Ciclohexanocarboxílicos/metabolismo , Ácidos Ciclohexanocarboxílicos/farmacología , Ciclohexanos/química , Ciclohexanos/farmacología , Ciclohexanos/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Gabapentina , Humanos , Inyecciones Intraperitoneales , Lipopolisacáridos/efectos adversos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Nitratos/metabolismo , Nitratos/farmacología , Células PC12 , Dolor/complicaciones , Dolor/etiología , Dimensión del Dolor/métodos , Nervios Periféricos/fisiopatología , Conejos , Ensayos Clínicos Controlados Aleatorios como Asunto , Ratas , Ratas Sprague-Dawley , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/fisiopatología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/etiología , Traumatismos de la Médula Espinal/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismo , Vasodilatación/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Lentinan (LEN) is an intensively studied beta-glucan that has been shown to have immunostimulating and antitumor functions. However, it currently is not used in veterinary practice and animal production. This study was conducted to investigate the impacts of LEN on broiler splenocyte proliferation, interleukin-2 (IL-2) production, and to explore its effects on receptor signal transduction by determining signaling molecules, including nitric oxide (NO), cytosolic-free Ca2+, cytosolic cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP). The results showed that compared to the control all three measured doses, including 40, 80, and 160 microg/mL LEN, increased splenocyte proliferation and IL-2 production (P < 0.05). Lentinan increased splenocyte NO production and cytosolic-free calcium concentration in a dose-dependent manner (P < 0.001). After incubating splenocytes for 20 min, LEN increased intracellular cAMP and cGMP concentrations at a dose of 80 and 160 microg/mL, respectively. However, after incubating the splenocytes for 60 min, LEN had no effect on cytosolic cAMP and cGMP concentration irrespective of the LEN dosage. Those results indicated that LEN had immunostimulatory effects on splenocytes by increasing splenocyte proliferation and IL-2 production and by activating splenocyte receptor signal transduction.