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1.
Cell ; 148(3): 421-33, 2012 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-22304913

RESUMEN

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Envejecimiento/metabolismo , Restricción Calórica , Transducción de Señal , Estilbenos/administración & dosificación , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Tejido Adiposo Blanco/efectos de los fármacos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Dieta , Intolerancia a la Glucosa/prevención & control , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Modelos Moleculares , Músculo Esquelético/efectos de los fármacos , NAD/metabolismo , Obesidad/prevención & control , Proteínas Quinasas/metabolismo , Resveratrol , Rolipram/administración & dosificación , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sirtuina 1/metabolismo
2.
J Mol Cell Cardiol ; 132: 60-70, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31051182

RESUMEN

Phosphodiesterase type 3 (PDE3) inhibitors block the cAMP hydrolyzing activity of both PDE3 isoforms, PDE3A and PDE3B, which have distinct roles in the heart. Although PDE3 inhibitors improve cardiac function in heart disease patients, they also increase mortality. Nevertheless, PDE3 inhibitors can provide benefit to non-ischemic heart disease patients and are used extensively to treat heart failure in dogs. Since the isoform-dependence of the complex cardiac actions of PDE3 inhibition in diseased hearts remains unknown, we assessed the effects of PDE3 inhibitors as well as gene ablation of PDE3A or PDEB in mice following the induction of non-ischemic heart disease by pressure-overload with transverse-aortic constriction (TAC). As expected, after 6 weeks of TAC, mice exhibited left ventricular contractile dysfunction, dilation, hypertrophy and interstitial fibrosis, in association with increased macrophage numbers, activation of p38 MAPK and elevated PDE3 activity. Chronic PDE3 inhibition with milrinone (MIL), at doses that did not affect either cardiac contractility or arterial blood pressure, profoundly attenuated the adverse ventricular remodeling, reduced macrophage number and diminished p38-MAPK activation induced by TAC. Surprisingly, whole-body ablation of PDE3A, but not PDE3B, provided similar protection against TAC-induced adverse ventricular remodeling, and the addition of MIL to mice lacking PDE3A provided no further protection. Our results support the conclusion that PDE3A plays an important role in adverse cardiac remodeling induced by chronic pressure overload in mice, although the underlying biochemical mechanisms remain to be fully elucidated. The implications of this conclusion on the clinical use of PDE3 inhibitors are discussed.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/fisiología , Cardiopatías/patología , Estrés Mecánico , Remodelación Ventricular , Animales , Cardiopatías/etiología , Cardiopatías/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
3.
Proc Natl Acad Sci U S A ; 112(17): E2253-62, 2015 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-25877153

RESUMEN

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B(-/-) mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B(-/-) mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca(2+)-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B(-/-) heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3-enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/deficiencia , Daño por Reperfusión Miocárdica , Miocardio/enzimología , Animales , Caveolina 3/genética , Caveolina 3/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/farmacología , Poro de Transición de la Permeabilidad Mitocondrial , Infarto del Miocardio/enzimología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/patología , Inhibidores de Fosfodiesterasa/farmacología , Quinolonas/farmacología
4.
J Immunol ; 195(6): 2763-73, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26268658

RESUMEN

Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.


Asunto(s)
Hemo-Oxigenasa 1/sangre , Metaloproteinasa 1 de la Matriz/sangre , Estrés Oxidativo/fisiología , Tuberculosis Pulmonar/patología , Adulto , Anciano , Biomarcadores/sangre , Brasil , Femenino , Hemo-Oxigenasa 1/metabolismo , Humanos , India , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de Unión a TGF-beta Latente/sangre , Pulmón/microbiología , Pulmón/patología , Macrófagos/microbiología , Macrófagos/patología , Masculino , Metaloproteinasa 1 de la Matriz/biosíntesis , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Factor de Transcripción AP-1/metabolismo , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Estados Unidos , Adulto Joven
5.
Biochem J ; 473(22): 4205-4225, 2016 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-27647936

RESUMEN

Oxidative stress plays a pivotal role in pathogenesis of cardiovascular diseases and diabetes; however, the roles of protein kinase A (PKA) and human phosphodiesterase 3A (hPDE3A) remain unknown. Here, we show that yeast expressing wild-type (WT) hPDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous hydrogen peroxide (H2O2), respectively. H2O2-stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A (Oxidative stress Sensitive 1, OxiS1), compared with yeast expressing WT hPDE3A (Oxidative stress Resistant 1, OxiR1). In OxiR1, YAP1 and YAP1-dependent antioxidant genes were up-regulated, accompanied by a reduction in thioredoxin peroxidase. In OxiS1, expression of YAP1 and YAP1-dependent genes was impaired, and the thioredoxin system malfunctioned. H2O2 increased cyclic adenosine monophosphate (cAMP)-hydrolyzing activity of WT hPDE3A, but not K13R hPDE3A, through PKA-dependent phosphorylation of hPDE3A, which was correlated with its ubiquitinylation. The changes in antioxidant gene expression did not directly correlate with differences in cAMP-PKA signaling. Despite differences in their capacities to hydrolyze cAMP, total cAMP levels among OxiR1, OxiS1, and mock were similar; PKA activity, however, was lower in OxiS1 than in OxiR1 or mock. During exposure to H2O2, however, Sch9p activity, a target of Rapamycin complex 1-regulated Rps6 kinase and negative-regulator of PKA, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol, with PKA activation. In OxiS1, Sch9p activity was unchanged during exposure to H2O2, consistent with reduced activation of PKA. These results suggest that, during oxidative stress, TOR-Sch9 signaling might regulate PKA activity, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Saccharomyces cerevisiae/enzimología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/farmacología , Inmunoprecipitación , Microscopía Fluorescente , Modelos Biológicos , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
6.
J Am Soc Nephrol ; 27(5): 1312-20, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26374610

RESUMEN

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1- or Pde3-knockout mice on the Pkd2(-/WS25) background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling (PDE1), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) -driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2(-/WS25) mice, knockout of Pde1a, Pde1c, or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase A-phosphorylated (Ser133) cAMP-responsive binding protein (P-CREB), activating transcription factor-1, and CREB-induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2(-/) (WS25);Pde1a(-/-), Pkd2(-) (/WS25);Pde1c(-/-), and Pkd2(-/) (WS25);Pde3a(-/-) kidneys. The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2(-/WS25);Pde3a(-/-) mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium- and calmodulin-dependent PDEs (PDE1A and PDE1C) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR-driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/fisiología , Enfermedades Renales Poliquísticas/enzimología , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Enfermedades Renales Poliquísticas/etiología , Índice de Severidad de la Enfermedad
7.
J Biol Chem ; 290(11): 6763-76, 2015 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-25593322

RESUMEN

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Miocardio/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas de Anclaje a la Quinasa A/análisis , Proteínas de Anclaje a la Quinasa A/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/análisis , Humanos , Miocardio/citología , Miocardio/enzimología , Miocardio/ultraestructura , Fosforilación , Mapas de Interacción de Proteínas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/análisis
8.
Am J Respir Crit Care Med ; 191(9): 990-1000, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25692941

RESUMEN

RATIONALE: Although lipids, apolipoproteins, and lipoprotein particles are important modulators of inflammation, varying relationships exist between these parameters and asthma. OBJECTIVES: To determine whether serum lipids and apolipoproteins correlate with the severity of airflow obstruction in subjects with atopy and asthma. METHODS: Serum samples were obtained from 154 atopic and nonatopic subjects without asthma, and 159 subjects with atopy and asthma. Serum lipid and lipoprotein levels were quantified using standard diagnostic assays and nuclear magnetic resonance (NMR) spectroscopy. Airflow obstruction was assessed by FEV1% predicted. MEASUREMENTS AND MAIN RESULTS: Serum lipid levels correlated with FEV1 only in the subjects with atopy and asthma. Serum levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) were positively correlated with FEV1 in subjects with atopy and asthma, whereas a negative correlation existed between FEV1 and serum levels of triglycerides, low-density lipoprotein (LDL) cholesterol, apolipoprotein B (apoB), and the apoB/apoA-I ratio. NMR spectroscopy identified a positive correlation between FEV1 and HDLNMR particle size, as well as the concentrations of large HDLNMR particles and total IDLNMR (intermediate-density lipoprotein) particles in subjects with atopy and asthma. In contrast, LDLNMR particle size and concentrations of LDLNMR and VLDLNMR (very-low-density lipoprotein) particles were negatively correlated with FEV1 in subjects with atopy and asthma. CONCLUSIONS: In subjects with atopy and asthma, serum levels of apoA-I and large HDLNMR particles are positively correlated with FEV1, whereas serum triglycerides, LDL cholesterol, and apoB are associated with more severe airflow obstruction. These results may facilitate future studies to assess whether apoA-I and large HDLNMR particles can reduce airflow obstruction and disease severity in asthma.


Asunto(s)
Apolipoproteína A-I/sangre , Asma/sangre , Asma/fisiopatología , HDL-Colesterol/sangre , Volumen Espiratorio Forzado , Hipersensibilidad Inmediata/sangre , Hipersensibilidad Inmediata/fisiopatología , Adulto , Obstrucción de las Vías Aéreas/sangre , Obstrucción de las Vías Aéreas/fisiopatología , Asma/complicaciones , Femenino , Humanos , Hipersensibilidad Inmediata/complicaciones , Masculino , Persona de Mediana Edad
9.
Proc Natl Acad Sci U S A ; 110(49): 19778-83, 2013 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-24248367

RESUMEN

Inhibitors of cyclic nucleotide phosphodiesterase (PDE) PDE3A have inotropic actions in human myocardium, but their long-term use increases mortality in patients with heart failure. Two isoforms in cardiac myocytes, PDE3A1 and PDE3A2, have identical amino acid sequences except for a unique N-terminal extension in PDE3A1. We expressed FLAG-tagged PDE3A1 and PDE3A2 in HEK293 cells and examined their regulation by PKA- and PKC-mediated phosphorylation. PDE3A1, which is localized to intracellular membranes, and PDE3A2, which is cytosolic, were phosphorylated at different sites within their common sequence. Exposure to isoproterenol led to phosphorylation of PDE3A1 at the 14-3-3-binding site S312, whereas exposure to PMA led to phosphorylation of PDE3A2 at an alternative 14-3-3-binding site, S428. PDE3A2 activity was stimulated by phosphorylation at S428, whereas PDE3A1 activity was not affected by phosphorylation at either site. Phosphorylation of PDE3A1 by PKA and of PDE3A2 by PKC led to shifts in elution on gel-filtration chromatography consistent with increased interactions with other proteins, and 2D electrophoresis of coimmunoprecipitated proteins revealed that the two isoforms have distinct protein interactomes. A similar pattern of differential phosphorylation of endogenous PDE3A1 and PDE3A2 at S312 and S428 is observed in human myocardium. The selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites through different signaling pathways, along with the different functional consequences of phosphorylation for each isoform, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium, and raise the possibility that isoform-selective inhibition may allow inotropic responses without an increase in mortality.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de Fosfodiesterasa 3/farmacología , Proteínas 14-3-3/genética , Sitios de Unión/genética , Cromatografía en Gel , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis en Gel Bidimensional , Activación Enzimática/fisiología , Células HEK293 , Humanos , Inmunoprecipitación , Isoenzimas/metabolismo , Isoproterenol/farmacología , Inhibidores de Fosfodiesterasa 3/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo
10.
Circ Res ; 112(2): 289-97, 2013 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-23168336

RESUMEN

RATIONALE: cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. OBJECTIVE: To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. METHODS AND RESULTS: Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A(-/-), but not PDE3B(-/-), mice. Furthermore, PDE3 inhibition had no effect on PDE3A(-/-) hearts but increased contractility in wild-type (as expected) and PDE3B(-/-) hearts to levels indistinguishable from PDE3A(-/-). The enhanced contractility in PDE3A(-/-) hearts was associated with cAMP-dependent elevations in Ca(2+) transient amplitudes and increased sarcoplasmic reticulum (SR) Ca(2+) content, without changes in L-type Ca(2+) currents of cardiomyocytes, as well as with increased SR Ca(2+)-ATPase type 2a activity, SR Ca(2+) uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A(-/-) hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. CONCLUSIONS: Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca(2+) content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a-phospholamban-PDE3A.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/fisiología , Corazón/fisiología , Contracción Miocárdica/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/fisiología
11.
Circ Res ; 109(9): 1024-1030, 2011 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-21903937

RESUMEN

RATIONALE: Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-γ-dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a). OBJECTIVE: The goal of this study was to determine whether PDE4D regulates basal cardiac contractility. METHODS AND RESULTS: At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D(-/-)) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D(-/-) cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor R(p)-adenosine-3',5' cyclic monophosphorothioate (R(p)-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D(-/-) cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D(-/-) preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D(-/-). The functional changes in PDE4D(-/-) myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D(-/-) cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor. CONCLUSIONS: PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.


Asunto(s)
Canales de Calcio Tipo L/fisiología , Calcio/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Femenino , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Miocitos Cardíacos/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo
12.
Nature ; 445(7129): 771-5, 2007 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-17220874

RESUMEN

Regulatory CD4+ T cells (Tr cells), the development of which is critically dependent on X-linked transcription factor Foxp3 (forkhead box P3), prevent self-destructive immune responses. Despite its important role, molecular and functional features conferred by Foxp3 to Tr precursor cells remain unknown. It has been suggested that Foxp3 expression is required for both survival of Tr precursors as well as their inability to produce interleukin (IL)-2 and independently proliferate after T-cell-receptor engagement, raising the possibility that such 'anergy' and Tr suppressive capacity are intimately linked. Here we show, by dissociating Foxp3-dependent features from those induced by the signals preceding and promoting its expression in mice, that the latter signals include several functional and transcriptional hallmarks of Tr cells. Although its function is required for Tr cell suppressor activity, Foxp3 to a large extent amplifies and fixes pre-established molecular features of Tr cells, including anergy and dependence on paracrine IL-2. Furthermore, Foxp3 solidifies Tr cell lineage stability through modification of cell surface and signalling molecules, resulting in adaptation to the signals required to induce and maintain Tr cells. This adaptation includes Foxp3-dependent repression of cyclic nucleotide phosphodiesterase 3B, affecting genes responsible for Tr cell homeostasis.


Asunto(s)
Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Linaje de la Célula , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Femenino , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Homeostasis , Interleucina-12/inmunología , Interleucina-12/metabolismo , Masculino , Ratones , Transducción de Señal , Linfocitos T Reguladores/inmunología
13.
J Biol Chem ; 286(29): 26238-49, 2011 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-21632535

RESUMEN

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²59) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G0/G1 cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G1 to S cell cycle progression.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/deficiencia , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Eliminación de Gen , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Animales , Biocatálisis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN/biosíntesis , Fosfatasa 1 de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , ARN Interferente Pequeño/genética
14.
Biochem Biophys Res Commun ; 425(4): 812-7, 2012 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-22892131

RESUMEN

The incretin - glucose-dependent insulinotropic polypeptide (GIP) - and the pro-inflammatory cytokine osteopontin are known to have important roles in the regulation of adipose tissue functions. In this work we show that GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. The GIP-induced increase in osteopontin expression was inhibited by the NFAT (the transcription factor nuclear factor of activated T-cells) inhibitor A-285222. Also, the NFAT kinase glycogen synthase kinase (GSK) 3 was upregulated by GIP. To test whether cAMP might be involved in GIP-mediated effects on osteopontin a number of strategies were used. Thus, the ß3-adrenergic receptor agonist CL316,243 stimulated osteopontin expression, an effects which was mimicked by OPC3911, a specific inhibitor of phosphodiesterase 3. Furthermore, treatment of phosphodiesterase 3B knock-out mice with CL316,243 resulted in a dramatic upregulation of osteopontin in adipose tissue which was not the case in wild-type mice. In summary, we delineate mechanisms by which GIP stimulates osteopontin in adipocytes. Given the established link between osteopontin and insulin resistance, our data suggest that GIP by stimulating osteopontin expression, also could promote insulin resistance in adipocytes.


Asunto(s)
Adipocitos/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Polipéptido Inhibidor Gástrico/fisiología , Lipogénesis/fisiología , Factores de Transcripción NFATC/fisiología , Osteopontina/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Animales , Polipéptido Inhibidor Gástrico/farmacología , Insulina/metabolismo , Lipogénesis/efectos de los fármacos , Masculino , Ratones , Factores de Transcripción NFATC/antagonistas & inhibidores , Osteopontina/genética , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley
15.
Proc Natl Acad Sci U S A ; 106(15): 6158-63, 2009 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-19332778

RESUMEN

ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.


Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/aislamiento & purificación , Citosol/efectos de los fármacos , Citosol/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/aislamiento & purificación , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Inhibidores de Fosfodiesterasa 3 , Unión Proteica , ARN Interferente Pequeño/genética
17.
Biochem J ; 424(3): 399-410, 2009 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-19747167

RESUMEN

In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta3-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KD of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained 32P-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta3-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KD. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.


Asunto(s)
Adipocitos/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Dioxoles/farmacología , Insulina/farmacología , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Western Blotting , Caveolas/efectos de los fármacos , Caveolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Retículo Endoplásmico/enzimología , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Aparato de Golgi/enzimología , Lipólisis/efectos de los fármacos , Sustancias Macromoleculares/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato
18.
J Clin Invest ; 116(12): 3240-51, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17143332

RESUMEN

Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , Metabolismo Energético/genética , Homeostasis/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Adiponectina/metabolismo , Animales , Western Blotting , Catecolaminas/metabolismo , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Metabolismo Energético/fisiología , Femenino , Homeostasis/fisiología , Inmunohistoquímica , Insulina/metabolismo , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Lipólisis/genética , Lipólisis/fisiología , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Tiempo , Triglicéridos/metabolismo
19.
Biochim Biophys Acta ; 1773(4): 584-92, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17320989

RESUMEN

Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adipocitos/enzimología , Hepatocitos/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Adipocitos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Activación Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Insulina/farmacología , Lipólisis/efectos de los fármacos , Neoplasias Hepáticas Experimentales/enzimología , Masculino , Toxinas Marinas , Ratones , Datos de Secuencia Molecular , Oxazoles/farmacología , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología
20.
Pediatr Res ; 64(5): 477-81, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18552705

RESUMEN

A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. After PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE)-mediated degradation. We used ductus from immature (n = 25) and mature (n = 21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activity that contributes to the decreased sensitivity of the late-gestation ductus arteriosus to vasodilators like PGE2.


Asunto(s)
Conducto Arterial/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Vasodilatación , Animales , Aorta/embriología , Aorta/enzimología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Conducto Arterial/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Edad Gestacional , Hidrólisis , Isoenzimas , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/genética , ARN Mensajero/metabolismo , Ovinos , Regulación hacia Arriba , Vasodilatación/efectos de los fármacos
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