RESUMEN
By catalyzing hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), cyclic nucleotide phosphodiesterases are critical regulators of their intracellular concentrations and their biological effects. As these intracellular second messengers control many cellular homeostatic processes, dysregulation of their signals and signaling pathways initiate or modulate pathophysiological pathways related to various disease states, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication, chronic obstructive pulmonary disease, and psoriasis. Alterations in expression of PDEs and PDE-gene mutations (especially mutations in PDE6, PDE8B, PDE11A, and PDE4) have been implicated in various diseases and cancer pathologies. PDEs also play important role in formation and function of multimolecular signaling/regulatory complexes, called signalosomes. At specific intracellular locations, individual PDEs, together with pathway-specific signaling molecules, regulators, and effectors, are incorporated into specific signalosomes, where they facilitate and regulate compartmentalization of cyclic nucleotide signaling pathways and specific cellular functions. Currently, only a limited number of PDE inhibitors (PDE3, PDE4, PDE5 inhibitors) are used in clinical practice. Future paths to novel drug discovery include the crystal structure-based design approach, which has resulted in generation of more effective family-selective inhibitors, as well as burgeoning development of strategies to alter compartmentalized cyclic nucleotide signaling pathways by selectively targeting individual PDEs and their signalosome partners.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/fisiología , 3',5'-GMP Cíclico Fosfodiesterasas/fisiología , Transducción de Señal/efectos de los fármacos , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/efectos de los fármacos , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-GMP Cíclico Fosfodiesterasas/efectos de los fármacos , Animales , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Transducción de Señal/fisiologíaRESUMEN
The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic ß-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Transducción de Señal , Adipocitos/metabolismo , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/química , Humanos , Insulina/metabolismo , Ligandos , Unión ProteicaRESUMEN
BACKGROUND: Agents that target pro-inflammatory cytokines may be useful in pulmonary sarcoidosis. OBJECTIVE: To determine effectiveness of a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, pentoxifylline (POF). DESIGN: Randomized, double-blind, placebo-controlled trial, SETTING: Clinical Research Center, National Institutes of Health. PATIENTS: 27 patients with biopsy-confirmed pulmonary sarcoidosis receiving prednisone. INTERVENTION: Placebo or POF (1200-2000 mg/day) for 10 months, as prednisone was tapered. MEASUREMENTS: Primary endpoints: sustained improvement in two or more pulmonary function parameters, or a combination of one pulmonary function parameter and dyspnea. RESULTS: Except for one patient, primary endpoints were not reached in POF-treated patients. Therefore, a post hoc analysis was performed. The observed relative risk reduction for flares associated with POF treatment was 54.9% (95% CI 0.21, 0.89) and the absolute risk reduction was 50.6% (95% CI 0.22, 0.80). Compared to placebo treatment, in the POF group, the mean prednisone dose was lower at 8 and 10 months (p = 0.007 and 0.01 respectively), and there was a trend towards less prednisone usage over the entire study period (p = 0.053), as determined by cumulative change analysis. CONCLUSIONS: Although our exploratory post hoc analysis suggested that POF reduced flares and had steroid-sparing effects, given the study limitations, definitive conclusions cannot be drawn regarding the efficacy of POF in pulmonary sarcoidosis. In addition, gastrointestinal side-effects, at the doses used, would seem to limit the use of POF in treating pulmonary sarcoidosis. Overall, however, this trial may provide a basis for using more specific, better-tolerated, PDE inhibitors in future clinical trials.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Pentoxifilina/uso terapéutico , Inhibidores de Fosfodiesterasa/uso terapéutico , Adolescente , Adulto , Anciano , Biopsia , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Flujo Espiratorio Forzado/efectos de los fármacos , Humanos , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Masculino , Persona de Mediana Edad , Pentoxifilina/administración & dosificación , Inhibidores de Fosfodiesterasa/administración & dosificación , Pruebas de Función Respiratoria , Resultado del Tratamiento , Adulto JovenAsunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Animales , Dominio Catalítico , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Regulación de la Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismoRESUMEN
A recent preliminary (unpublished) study showed that phosphodiesterase (PDE) 3A and 3B are expressed in rat submandibular glands. Here, PDE3 activity was detected in homogenates of rat submandibular gland acinar epithelial (SMIE) cells, but not rat A5 (epithelial duct) cells. Most of the PDE3 activity in SMIE cells was recovered in the particulate fraction. Only PDE3B mRNA was detected by reverse transcription-polymerase chain reaction in RNA from SMIE cells. The nucleotide sequence of the fragment was identical to the sequence of rat PDE3B. The PDE3 specific inhibitor, OPC3689 (10 and 50 microM), inhibited the growth of SMIE cells (19 and 63%), but not A5 cells. As the submandibular gland contains many types of cells, these results indicate that PDE3B may regulate a cAMP pool that is important in submandibular gland acinar epithelial cell function.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/biosíntesis , Glándula Submandibular/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Animales , Línea Celular , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Células Epiteliales/enzimología , Isoformas de Proteínas , ARN Mensajero/análisis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Submandibular/citologíaRESUMEN
Phosphodiesterase (PDE) 3s have been characterized in human neoplastic submandibular gland intercalated duct HSG cells. There have been no reports on PDE3 in malignant salivary gland cells. PDE3 activity was detected in homogenates of HSG cells. About 75% of PDE3 activity in HSG cells was recovered in supernatant fractions and 25% in particulate fractions. PDE3A and 3B mRNAs were detected by reverse transcription-polymerase chain reaction in RNA from HSG cells. The nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. The PDE3-specific inhibitor, cilostamide, inhibited the growth of HSG cells. Our results indicate that PDE3s may be important in the growth of HSG cells. PDE3 thus appears to be a potential new target for antiproliferative therapies.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Neoplasias de la Glándula Submandibular/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/genética , División Celular/efectos de los fármacos , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Quinolonas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rolipram/farmacología , Neoplasias de la Glándula Submandibular/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
We have identified a new cyclic-nucleotide phosphodiesterase isoform, PDE3A, and cloned its cDNA from cultured aortic myocytes. The nucleotide sequence of its coding region is similar to that of the previously cloned myocardial isoform except for the absence of the initial 300-400 nt that are present in the latter, as confirmed by reverse-transcriptase-mediated PCR, 5' rapid amplification of cDNA ends and a ribonuclease protection assay. Expression in Spodoptera frugiperda (Sf9) cells yields a protein with catalytic activity and inhibitor sensitivity typical of the PDE3 family. The recombinant protein's molecular mass of approx. 131 kDa is compatible with translation from an ATG sequence corresponding to nt 436-438 of the myocardial PDE3A coding region. Antibodies against residues 424-460 (nt 1270-1380) and 1125-1141 (nt 3373-3423) of the myocardial isoform react with an approx. 118 kDa band in Western blots of homogenates of human aortic myocytes, whereas antibodies against residues 29-42 (nt 85-126) do not react with any bands in these homogenates. Our results suggest that a vascular smooth-muscle isoform ('PDE3A2') is a product of the same gene as the longer myocardial ('PDE3A1') and the shorter placental ('PDE3A3') isoforms and is generated pre-translationally in a manner that results in the absence of the 145 N-terminal amino acids of PDE3A1.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Músculo Liso Vascular/enzimología , Porcinos/genética , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/química , Secuencia de Aminoácidos , Animales , Anticuerpos , Aorta/citología , Aorta/enzimología , Western Blotting , Catálisis , Células Cultivadas , Clonación Molecular , Exones/genética , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Peso Molecular , Músculo Liso Vascular/citología , Miocardio/enzimología , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de SecuenciaAsunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/química , Adipocitos/metabolismo , Animales , Plaquetas/metabolismo , Dominio Catalítico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Citocinas/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Humanos , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Isoproterenol/metabolismo , Hígado/metabolismo , Modelos Biológicos , Familia de Multigenes , Fosforilación , Isoformas de Proteínas , Distribución TisularRESUMEN
Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adipocitos/enzimología , Retículo Endoplásmico/enzimología , Membranas Intracelulares/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Células 3T3 , Adipocitos/ultraestructura , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Línea Celular , Chlorocebus aethiops , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Cartilla de ADN , Aparato de Golgi/enzimología , Humanos , Isoenzimas/análisis , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , TransfecciónRESUMEN
Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba-, F/Bb-, F/Bc-) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of insulin-like growth factor-1 (IGF-1), activities of PKB, cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) cells, and F/B and F/B- cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities approximately 2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity approximately 10-fold and PDE3B phosphorylation and activity ( approximately 4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased ( approximately 10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B- cells, IGF-1 activated PDE4, not PDE3B, suggesting that kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Células Madre Hematopoyéticas/enzimología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Timidina/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Línea Celular , AMP Cíclico/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Vectores Genéticos , Células Madre Hematopoyéticas/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Ratones , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transfección , Proteína Letal Asociada a bclRESUMEN
Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Tejido Adiposo/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor de Insulina/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/aislamiento & purificación , Tejido Adiposo/enzimología , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Activación Enzimática , Humanos , Insulina/farmacología , Insulina/fisiología , Proteínas Sustrato del Receptor de Insulina , Peso Molecular , Fosfatidilinositol 3-Quinasas/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor de Insulina/efectos de los fármacos , Transducción de SeñalRESUMEN
The N-terminal portion of phosphodiesterase (PDE) 3 was arbitrarily divided into region 1 (amino acids 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, and region 2 (amino acids 301-500), with a smaller hydrophobic domain ( approximately 50 residues). To analyze these regions, full-length human (H)PDE3A and mouse (M)PDE3B and a series of N-terminal truncated mutants were synthesized in Sf9 cells. Activities of HPDE3A, H3A-Delta189, MPDE3B, and M3B-Delta196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-Delta321 and M3B-Delta302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-Delta397 and H3A-Delta457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-Delta510 and M3B-Delta604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-Delta196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-Delta607, H3A-Delta721, and M3B-Delta604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixazinone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with intracellular membranes, as well for self-association or aggregation during gel filtration and sensitivity to a specific inhibitor.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Isoenzimas/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/química , Animales , Western Blotting , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Humanos , Isoenzimas/química , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Conformación Proteica , Quinazolinas/farmacología , Eliminación de Secuencia , Solubilidad , Relación Estructura-ActividadRESUMEN
We have used murine 3T3-L1 cells, which differentiate in culture and acquire morphological and biochemical features of mature adipocytes, as a model for studying the expression of cyclic-nucleotide phosphodiesterase (PDE) 3B activity, protein and mRNA during differentiation and during long-term treatment of the cells with tumour necrosis factor alpha (TNF-alpha), a cytokine associated with insulin resistance, and a cAMP analogue, N(6),2'-O-dibutyryl cAMP (dbcAMP). PDE3B activity, protein and mRNA could be detected 4 days after the initiation of differentiation of 3T3-L1 preadipocytes. Treatment of 3T3-L1 adipocytes with 10 ng/ml TNF-alpha for 24 h produced a maximal (50%) decrease in PDE3B activity, protein and mRNA, which was well correlated with both activation of protein kinase A (PKA) and stimulation of lipolysis, presumably reflecting an increase in intracellular cAMP concentration. To investigate the effect of cAMP on PDE3B we treated 3T3-L1 adipocytes with dbcAMP. After 4 h with 0.5 mM dbcAMP, PDE3B activity was decreased by 80%, which was also correlated with a decrease in PDE3B protein and mRNA. This effect was abolished in the presence of N-[2-(bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide] (H-89), a specific PKA inhibitor. We conclude that the lipolytic effect of TNF-alpha involves the down-regulation of PDE3B, which is associated with increased activation of PKA, presumably owing to increased levels of cAMP. In addition, the PKA activation induced by dbcAMP resulted in the down-regulation of PDE3B. These results, which suggest that PDE3B is a novel target for long-term regulation by TNF-alpha and cAMP, could contribute to the understanding of the mechanisms of insulin resistance.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adipocitos/enzimología , AMP Cíclico/farmacología , Regulación hacia Abajo/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3 , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , RatonesRESUMEN
Phosphodiesterase type 3 isoforms, PDE3A and 3B, are expressed primarily in cardiovascular and adipose tissues, respectively. We previously reported a shorter transcript of 4.4-kb PDE3A which is predominantly transcribed in human placenta, whereas a full-length 7. 6-kb transcript corresponding to the cardiac PDE3A cDNA has not been characterized. Due to unfortunate circumstances created by changes in PDE3 nomenclature, PDE3B gene structure previously reported used PDE3A in its title. Here, we describe PDE3A gene structure, which comprises 16 exons spanning over 130 kb on chromosome 12p12. Two PDE3 isoforms share similar gene organization, but localize to different chromosomes. The most distal transcription initiation site of the PDE3A gene is approximately 1071 bases upstream of the ATG site, suggesting that exon 1 consists of 1071 and 960 bp of untranslated and translated sequences, respectively. The proximal 5'-flanking region, which does not contain TATA-like sequences, exhibited weak but significant promoter activity. Results suggest potential involvement of distal promoter/enhancer and translational regulation for expression of the 7.6-kb transcript.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , Adipocitos/enzimología , Isoenzimas/genética , Miocardio/enzimología , Secuencia de Bases , Dominio Catalítico/genética , Mapeo Cromosómico , Cromosomas Humanos Par 12/genética , Clonación Molecular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Cartilla de ADN/genética , Exones , Femenino , Humanos , Hibridación Fluorescente in Situ , Intrones , Datos de Secuencia Molecular , Embarazo , Distribución Tisular , Transcripción GenéticaRESUMEN
Intracellular cyclic AMP, determined in part by cyclic nucleotide phosphodiesterases (PDEs), regulates proliferation and immune functions in lymphoid cells. Total PDE, PDE3, and PDE4 activities were measured in phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC-PHA), normal natural killer (NK) cells, Jurkat and Kit225-K6 leukemic T-cells, T-cell lines transformed with human T-lymphotropic virus (HTLV)-I (a retrovirus that causes adult T-cell leukemia/lymphoma) and HTLV-II (a nonpathogenic retrovirus), normal B-cells, and B-cells transformed with Epstein-Barr virus (EBV). All cells exhibited PDE3 and PDE4 activities but in different proportions. In EBV-transformed B cells, PDE4 was much higher than PDE3. HTLV-I+ T-cells differed significantly from other T-lymphocyte-derived cells in also having a higher proportion of PDE4 activities, which apparently were not related to selective induction of any one PDE4 mRNA (judged by reverse transcription-polymerase chain reaction) or expression of the HTLV-I regulatory protein Tax. In MJ cells (an HTLV-I+ T-cell line), Jurkat cells, and PBMC-PHA cells, the tyrosine kinase inhibitor herbimycin A strongly inhibited PDE activity. Growth of MJ cells was inhibited by herbimycin A and a protein kinase C (PKC) inhibitor, and was arrested in G1 by rolipram, a specific PDE4 inhibitor. Proliferation of several HTLV-I+ T-cell lines, PBMC-PHA, and Jurkat cells was inhibited differentially by forskolin (which activates adenylyl cyclase), the selective PDE inhibitors cilostamide and rolipram, and the nonselective PDE inhibitors pentoxifylline and isobutyl methylxanthine. These results suggest that PDE4 isoforms may be functionally up-regulated in HTLV-I+ T-cells and may contribute to the virus-induced proliferation, and that PDEs could be therapeutic targets in immune/inflammatory and neoplastic diseases.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Transformación Celular Viral , Virus Linfotrópico T Tipo 1 Humano/fisiología , Linfocitos/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Adulto , Linfocitos B/enzimología , Benzoquinonas , División Celular/efectos de los fármacos , Línea Celular Transformada/enzimología , Colforsina/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Inhibidores Enzimáticos/farmacología , Productos del Gen tax/biosíntesis , Productos del Gen tax/metabolismo , Humanos , Interleucina-2/metabolismo , Células Jurkat/enzimología , Células Asesinas Naturales/enzimología , Lactamas Macrocíclicas , Leucocitos Mononucleares/enzimología , Linfocitos/virología , Inhibidores de Proteínas Quinasas , Quinonas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rifabutina/análogos & derivados , Linfocitos T/enzimologíaRESUMEN
The present study was undertaken to characterise the phosphodiesterases (PDEs) present in brown adipose tissue (BAT) of Zucker rat pups and to determine whether the capacity for degradation of cyclic nucleotides was affected by the fatty genotype. Regardless of the genotype, PDE2-4 contributed to total PDE activity, the PDE3 activity equalling the sum of PDE2 and 4 activities. In fa/fa compared to Fa/fa rats, (a) PDE2 activity was significantly increased, (b) Western blot analysis of PDE2 revealed two signals at 71 and 105 kDa, with changes in protein being in good parallelism with changes in activity, (c) the PDE2 mRNA concentration was also significantly increased. In good agreement, the cGMP concentration was decreased in BAT from fa/fa pups.
Asunto(s)
Tejido Adiposo Pardo/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Proteínas/genética , Animales , Animales Recién Nacidos , Northern Blotting , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Citosol/enzimología , Femenino , Regulación de la Expresión Génica , Immunoblotting , Isoenzimas/genética , Isoenzimas/metabolismo , Leptina , Masculino , Obesidad/genética , Hidrolasas Diéster Fosfóricas/genética , Ratas , Ratas ZuckerRESUMEN
Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosphorylated in response to insulin and hormones that increase cAMP. In order to study serine/threonine protein phosphatases involved in the regulation of rat adipocyte PDE3B, we investigated the phosphorylation and activation of PDE3B in vivo in response to phosphatase inhibitors and the dephosphorylation and deactivation of PDE3B in vitro by phosphatases purified from rat adipocyte homogenates. Okadaic acid and calyculin A induced dose- and time-dependent activation of PDE3B. Maximal effects were obtained after 30 min using 1 microM okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce activation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibited protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 microM) reduced PP2A activity by approx. 50% but did not affect PP1 activity, and 1 microM tautomycin reduced PP1 activity by approx. 60% but PP2A activity by only 11%. This indicates an important role for PP2A in the regulation of PDE3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PP1 during MonoQ chromatography. As compared with insulin, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- and 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, the site known to be phosphorylated on PDE3B in response to insulin and isoproterenol (isoprenaline), as well as on sites not identified previously. In summary, PP2A seems to be involved in the regulation of PDE3B in vivo and can act as a PDE3B phosphatase in vitro. In comparison with insulin, calyculin A induced a dramatic activation of PDE3B and both calyculin A and okadaic acid induced phosphorylation on additional sites, which could have a role in signalling pathways not yet identified.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adipocitos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fosfoproteínas Fosfatasas/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Adipocitos/enzimología , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Activación Enzimática , Insulina/farmacología , Toxinas Marinas , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Mapeo Peptídico , Fosforilación , RatasRESUMEN
Vanadate and peroxovanadate (pV), potent inhibitors of tyrosine phosphatases, mimic several of the metabolic actions of insulin. Here we compare the mechanisms for the anti-lipolytic action of insulin, vanadate and pV in rat adipocytes. Vanadate (5 mM) and pV (0.01 mM) inhibited lipolysis induced by 0.01-1 microM isoprenaline, vanadate being more and pV less efficient than insulin (1 nM). A loss of anti-lipolytic effect of pV was observed by increasing the concentration of isoprenaline and/or pV. pV induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 to a greater extent than insulin, whereas vanadate affected these components little if at all. In addition, only a higher concentration (0.1 mM) of pV induced the tyrosine phosphorylation of p85, the 85 kDa regulatory subunit of phosphoinositide 3-kinase (PI-3K). Vanadate activated PI-3K-independent (in the presence of 10 nM isoprenaline) and PI-3K-dependent (in the presence of 100 nM isoprenaline) anti-lipolytic pathways, both of which were found to be independent of phosphodiesterase type 3B (PDE3B). pV (0.01 mM), like insulin, activated PI-3K- and PDE3B-dependent pathways. However, the anti-lipolytic pathway of 0.1 mM pV did not seem to require insulin receptor substrate-1-associated PI-3K and was found to be partly independent of PDE3B. Vanadate and pV (only at 0.01 mM), like insulin, decreased the isoprenaline-induced activation of cAMP-dependent protein kinase. Overall, these results underline the complexity and the diversity in the mechanisms that regulate lipolysis.