RESUMEN
Angiogenesis is physiologically essential for embryogenesis and development and reinitiated in adult animals during tissue growth and repair. Forming new vessels from the walls of existing vessels occurs as a multistep process coordinated by sprouting, branching, and a new lumenized network formation. However, little is known regarding the molecular mechanisms that form new tubular structures, especially molecules regulating the proper network density of newly formed capillaries. This study conducted microarray analyses in human primary microvascular endothelial cells (HMVECs) plated on Matrigel. The RAPGEF4 gene that encodes exchange proteins directly activated by cAMP 2 (EPAC2) proteins was increased in Matrigel-driven tubulogenesis. Tube formation was suppressed by the overexpression of EPAC2 and enhanced by EPAC2 knockdown in endothelial cells. Endothelial cell morphology was changed to round cell morphology by EPAC2 overexpression, while EPAC2 knockdown showed an elongated cell shape with filopodia-like protrusions. Furthermore, increased EPAC2 inhibited endothelial cell migration, and ablation of EPAC2 inversely enhanced cell mobility. These results suggest that EPAC2 affects the morphology and migration of microvascular endothelial cells and is involved in the termination and proper network formation of vascular tubes.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Endotelio Vascular/crecimiento & desarrollo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Morfogénesis , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Movimiento Celular , Forma de la Célula , Células Cultivadas , Colágeno , Combinación de Medicamentos , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Humanos , Laminina , Proteoglicanos , SeudópodosRESUMEN
The cAMP analogue 8-Br-cAMP-AM (8-Br) confers marked protection against global ischaemia/reperfusion of isolated perfused heart. We tested the hypothesis that 8-Br is also protective under clinically relevant conditions (regional ischaemia) when applied either before ischemia or at the beginning of reperfusion, and this effect is associated with the mitochondrial permeability transition pore (MPTP). 8-Br (10 µM) was administered to Langendorff-perfused rat hearts for 5 min either before or at the end of 30 min regional ischaemia. Ca2+-induced mitochondria swelling (a measure of MPTP opening) and binding of hexokinase II (HKII) to mitochondria were assessed following the drug treatment at preischaemia. Haemodynamic function and ventricular arrhythmias were monitored during ischaemia and 2 h reperfusion. Infarct size was evaluated at the end of reperfusion. 8-Br administered before ischaemia attenuated ventricular arrhythmias, improved haemodynamic function, and reduced infarct size during ischaemia/reperfusion. Application of 8-Br at the end of ischaemia protected the heart during reperfusion. 8-Br promoted binding of HKII to the mitochondria and reduced Ca2+-induced mitochondria swelling. Thus, 8-Br protects the heart when administered before regional ischaemia or at the beginning of reperfusion. This effect is associated with inhibition of MPTP via binding of HKII to mitochondria, which may underlie the protective mechanism.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/administración & dosificación , Fármacos Cardiovasculares/administración & dosificación , Mitocondrias Cardíacas/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Esquema de Medicación , Hemodinámica/efectos de los fármacos , Hexoquinasa/metabolismo , Preparación de Corazón Aislado , Masculino , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Dilatación Mitocondrial/efectos de los fármacos , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas Wistar , Transducción de Señal , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
The steroidogenic acute regulatory (StAR) protein performs the delivery of cholesterol from the outer to inner mitochondrial membrane. This is considered the rate-limiting step of acute steroid production, widely studied in mammals. However, there are only few reports regarding the characterization and expression of StAR protein in non-mammalian vertebrates. In this study, StAR protein sequence of Rhinella arenarum has been characterized and deduced from interrenal and testis cDNA sequences. StAR encodes a 285 amino acid protein with a conserved domain containing putative lipid binding sites. In vitro incubations showed that expression of StAR mRNA in testis, determined by qPCR, and testosterone synthesis determined by radioimmunoassay were stimulated after treatment with hCG and 8Br-cAMP. However, StAR mRNA expression results obtained with hCG show a higher stimulation than those obtained with 8Br-cAMP, even though steroidogenic production is the same with both treatments.
Asunto(s)
Anuros/metabolismo , Fosfoproteínas/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Secuencia de Aminoácidos , Andrógenos/biosíntesis , Animales , Gonadotropina Coriónica/farmacología , Masculino , Fosfoproteínas/química , Fosfoproteínas/genética , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
Acephate is an organophosphate pesticide. It is widely used. However, whether it inhibits androgen synthesis and metabolism remains unclear. In the current study, we investigated the effect of acephate on the inhibition of androgen synthetic and metabolic pathways in rat immature Leydig cells after 3-h culture. Acephate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.5 µM. It significantly inhibited luteinizing hormone and 8-Br-cAMP stimulated androgen output at 50 µM. It significantly inhibited progesterone-mediated androgen output at 50 µM. Further study demonstrated that acephate down-regulated the expression of Hsd3b1 and its protein at ≥ 0.5 µM, Lhcgr at 5 µM and Star at 50 µM. Acephate directly blocked rat testicular HSD3B1 activity at 50 µM. Acephate did not affect other androgen synthetic and metabolic enzyme activities as well as ROS production, proliferation, and apoptosis of immature Leydig cells. In conclusion, acephate targets LHCGR, STAR, and HSD3B1, thus blocking androgen synthesis in rat immature Leydig cells and HSD3B1 is being the most sensitive target of acephate.
Asunto(s)
Andrógenos/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Compuestos Organotiofosforados/farmacología , Fosforamidas/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/antagonistas & inhibidores , Hormona Luteinizante/metabolismo , Masculino , Complejos Multienzimáticos/antagonistas & inhibidores , Progesterona/farmacología , Progesterona Reductasa/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptores de HL/antagonistas & inhibidores , Esteroide Isomerasas/antagonistas & inhibidores , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Liasas de Fósforo-Oxígeno/genética , Pseudomonas putida/enzimología , Pseudomonas putida/genética , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , GMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Regiones Promotoras Genéticas , Sistemas de Mensajero SecundarioRESUMEN
A better understanding of the molecular mechanisms that participate in the development and clinical manifestations of schizophrenia can lead to improve our ability to diagnose and treat this disease. Previous data strongly associated the levels of deregulated ADAMTS2 expression in peripheral blood mononuclear cells (PBMCs) from patients at first episode of psychosis (up) as well as in clinical responders to treatment with antipsychotic drugs (down). In this current work, we performed an independent validation of such data and studied the mechanisms implicated in the control of ADAMTS2 gene expression. Using a new cohort of drug-naïve schizophrenia patients with clinical follow-up, we confirmed that the expression of ADAMTS2 was highly upregulated in PBMCs at the onset (drug-naïve patients) and downregulated, in clinical responders, after treatment with antipsychotics. Mechanistically, ADAMTS2 expression was activated by dopaminergic signalling (D1-class receptors) and downstream by cAMP/CREB and mitogen-activated protein kinase (MAPK)/ERK signalling. Incubation with antipsychotic drugs and selective PKA and MEK inhibitors abrogated D1-mediated activation of ADAMTS2 in neuronal-like cells. Thus, D1 receptors signalling towards CREB activation might participate in the onset and clinical responses to therapy in schizophrenia patients, by controlling ADAMTS2 expression and activity. The unbiased investigation of molecular mechanisms triggered by antipsychotic drugs may provide a new landscape of novel targets potentially associated with clinical efficacy.
Asunto(s)
Proteínas ADAMTS/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dopamina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Esquizofrenia/fisiopatología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Proteínas ADAMTS/genética , Animales , Antipsicóticos/farmacología , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Humanos , Leucocitos Mononucleares/metabolismo , Ratones , Fosforilación , Esquizofrenia/genética , Esquizofrenia/metabolismo , Transducción de SeñalRESUMEN
Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the "pool" of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Células Endoteliales/metabolismo , Neovascularización Patológica/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animales , AMP Cíclico/genética , Humanos , Ratones , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Inhibidores de Fosfodiesterasa 3/farmacologíaRESUMEN
Pharmacological agents that raise cAMP and activate protein kinase A (PKA) stimulate 26S proteasome activity, phosphorylation of subunit Rpn6, and intracellular degradation of misfolded proteins. We investigated whether a similar proteasome activation occurs in response to hormones and under various physiological conditions that raise cAMP. Treatment of mouse hepatocytes with glucagon, epinephrine, or forskolin stimulated Rpn6 phosphorylation and the 26S proteasomes' capacity to degrade ubiquitinated proteins and peptides. These agents promoted the selective degradation of short-lived proteins, which are misfolded and regulatory proteins, but not the bulk of cell proteins or lysosomal proteolysis. Proteasome activities and Rpn6 phosphorylation increased similarly in working hearts upon epinephrine treatment, in skeletal muscles of exercising humans, and in electrically stimulated rat muscles. In WT mouse kidney cells, but not in cells lacking PKA, treatment with antidiuretic hormone (vasopressin) stimulated within 5-minutes proteasomal activity, Rpn6 phosphorylation, and the selective degradation of short-lived cell proteins. In livers and muscles of mice fasted for 12-48 hours cAMP levels, Rpn6 phosphorylation, and proteasomal activities increased without any change in proteasomal content. Thus, in vivo cAMP-PKA-mediated proteasome activation is a common cellular response to diverse endocrine stimuli and rapidly enhances the capacity of target tissues to degrade regulatory and misfolded proteins (e.g., proteins damaged upon exercise). The increased destruction of preexistent regulatory proteins may help cells adapt their protein composition to new physiological conditions.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animales , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Epinefrina/farmacología , Glucagón/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Riñón , Masculino , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Proteolisis/efectos de los fármacos , Deficiencias en la Proteostasis/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteínas Ubiquitinadas/metabolismoRESUMEN
Glucose metabolism stimulates cell division control protein 42 homolog (Cdc42)-p21-activated kinase (Pak1) activity and initiates filamentous actin (F-actin) cytoskeleton remodeling in pancreatic ß-cells so that cytoplasmic secretory granules can translocate to the plasma membrane where insulin exocytosis occurs. Since glucose metabolism also generates cAMP in ß-cells, the cross talk of cAMP signaling with Cdc42-Pak1 activation might be of fundamental importance to glucose-stimulated insulin secretion (GSIS). Previously, the type-2 isoform of cAMP-regulated guanine nucleotide exchange factor 2 (Epac2) was established to mediate a potentiation of GSIS by cAMP-elevating agents. Here we report that nondiabetic human islets and INS-1 832/13 ß-cells treated with the selective Epac activator 8-pCPT-2'-O-Me-cAMP-AM exhibited Cdc42-Pak1 activation at 1 mmol/L glucose and that the magnitude of this effect was equivalent to that which was measured during stimulation with 20 mmol/L glucose in the absence of 8-pCPT-2'-O-Me-cAMP-AM. Conversely, the cAMP antagonist Rp-8-Br-cAMPS-pAB prevented glucose-stimulated Cdc42-Pak1 activation, thereby blocking GSIS while also increasing cellular F-actin content. Although islets from donors with type 2 diabetes had profound defects in glucose-stimulated Cdc42-Pak1 activation and insulin secretion, these defects were rescued by the Epac activator so that GSIS was restored. Collectively, these findings indicate an unexpected role for cAMP as a permissive or direct metabolic coupling factor in support of GSIS that is Epac2 and Cdc42-Pak1 regulated.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/química , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Línea Celular , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ratas , Tionucleótidos/química , Tionucleótidos/farmacologíaRESUMEN
The migration of cells according to a diffusible chemical signal in their environment is called chemotaxis, and the slime mold Dictyostelium discoideum is widely used for the study of eukaryotic chemotaxis. Dictyostelium must sense chemicals, such as cAMP, secreted during starvation to move towards the sources of the signal. Previous work demonstrated that the gskA gene encodes the Dictyostelium homologue of glycogen synthase kinase 3 (GSK3), a highly conserved serine/threonine kinase, which plays a major role in the regulation of Dictyostelium chemotaxis. Cells lacking the GskA substrates Daydreamer and GflB exhibited chemotaxis defects less severe than those exhibited by gskA- (GskA null) cells, suggesting that additional GskA substrates might be involved in chemotaxis. Using phosphoproteomics we identify the GskA substrates PdeD, dynacortin and SogA and characterize the phenotypes of their respective null cells in response to the chemoattractant cAMP. All three chemotaxis phenotypes are defective, and in addition, we determine that carboxylesterase D2 is a common downstream effector of GskA, its direct substrates PdeD, GflB and the kinases GlkA and YakA, and that it also contributes to cell migration. Our findings identify new GskA substrates in cAMP signalling and break down the essential role of GskA in myosin II regulation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quimiotaxis/fisiología , Dictyostelium/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Protozoarias/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Proteínas de Ciclo Celular/genética , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Secuencia Kelch , Hidrolasas Diéster Fosfóricas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Transducción de Señal/fisiologíaRESUMEN
NADPH oxidase Nox4-derived reactive oxygen species (ROS) play important roles in renal fibrosis. Our previous study demonstrated that intermedin (IMD) alleviated unilateral ureteral obstruction (UUO)-induced renal fibrosis by inhibition of ROS. However, the precise mechanisms remain unclear. Herein, we investigated the effect of IMD on Nox4 expression and NADPH oxidase activity in rat UUO model, and explored if these effect were achieved through cAMP-PKA pathway, the important post-receptor signal transduction pathway of IMD, in TGF-ß1-stimulated rat proximal tubular cell (NRK-52E). Renal fibrosis was induced by UUO. NRK-52E was exposed to rhTGF-ß1 to establish an in vitro model of fibrosis. IMD was overexpressed in the kidney and in NRK-52E by IMD gene transfer. We studied UUO-induced ROS by measuring dihydroethidium levels and lipid peroxidation end-product 4-hydroxynonenal expression. Nox4 expression in the obstructed kidney of UUO rat or in TGF-ß1-stimulated NRK-52E was measured by quantitative RT-PCR and Western blotting. We analyzed NADPH oxidase activity using a lucigenin-enhanced chemiluminescence system. We showed that UUO-stimulated ROS production was remarkably attenuated by IMD gene transfer. IMD overexpression inhibited UUO-induced up-regulation of Nox4 and activation of NADPH oxidase. Consistent with in vivo results, TGF-ß1-stimulated increase in Nox4 expression and NADPH oxidase activity was blocked by IMD. In NRK-52E, these beneficial effects of IMD were abolished by pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89), a PKA inhibitor, and mimicked by a cell-permeable cAMP analog dibutyl-cAMP. Our results indicate that IMD exerts anti-oxidant effects by inhibition of Nox4, and the effect can be mediated by cAMP-PKA pathway.
Asunto(s)
Adrenomedulina/metabolismo , AMP Cíclico/metabolismo , Enfermedades Renales/patología , Riñón/patología , NADPH Oxidasa 4/metabolismo , Neuropéptidos/metabolismo , Estrés Oxidativo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Adrenomedulina/genética , Aldehídos/metabolismo , Animales , Línea Celular , AMP Cíclico/análogos & derivados , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Etidio/análogos & derivados , Etidio/metabolismo , Fibrosis , Técnicas de Transferencia de Gen , Isoquinolinas/farmacología , Enfermedades Renales/etiología , Peroxidación de Lípido , Masculino , Neuropéptidos/genética , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Sulfonamidas/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia ArribaRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenetic disease characterized by the progressive development of renal cysts with further need for effective therapy. Here our aim was to investigate the effect of Ganoderma triterpenes (GT) on the development of kidney cysts. Importantly, GT attenuated cyst development in two mouse models of ADPKD with phenotypes of severe cystic kidney disease. Assays for tubulogenesis showed that GT promoted epithelial tubule formation in MDCK cells, suggesting a possible effect on epithelial cell differentiation. The role of GT in regulating key signaling pathways involved in the pathogenesis of PKD was further investigated by immune blotting. This showed that GT specifically downregulated the activation of the Ras/MAPK signaling pathway both in vitro and in vivo without detectable effect on the mTOR pathway. This mechanism may be involved in GT downregulating intracellular cAMP levels. Screening of 15 monomers purified from GT for their effects on cyst development indicated that CBLZ-7 (ethyl ganoderate C2) had a potent inhibitory effect on cyst development in vitro. Additionally, like GT, CBLZ-7 was able to downregulate forskolin-induced activation of the Ras/MAPK pathway. Thus, GT and its purified monomer CBLZ-7 may be potential therapeutic regents for treating ADPKD.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Ganoderma/química , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Triterpenos/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Colforsina/farmacología , Quistes/tratamiento farmacológico , Modelos Animales de Enfermedad , Regulación hacia Abajo , Células Epiteliales/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Riñón Poliquístico Autosómico Dominante/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Canales Catiónicos TRPP/genética , Triterpenos/aislamiento & purificación , Triterpenos/uso terapéutico , Proteínas ras/metabolismoRESUMEN
The present study was performed to determine the effect of Zn exposure influencing endoplasmic reticulum (ER) stress, explore the underlying molecular mechanism of Zn-induced hepatic lipolysis in a fish species of significance for aquaculture, yellow catfish Pelteobagrus fulvidraco. We found that waterborne Zn exposure evoked ER stress and unfolded protein response (UPR), and activated cAMP/PKA pathway, and up-regulated hepatic lipolysis. The increase in ER stress and lipolysis were associated with activation of cAMP/PKA signaling pathway. Zn also induced an increase in intracellular Ca2+ level, which could be partially prevented by dantrolene (RyR receptor inhibitor) and 2-APB (IP3 receptor inhibitor), demonstrating that the disturbed Ca2+ homeostasis in ER contributed to ER stress and dysregulation of lipolysis. Inhibition of ER stress by PBA attenuated UPR, inhibited the activation of cAMP/PKA pathway and resulted in down-regulation of lipolysis. Inhibition of protein kinase RNA-activated-like ER kinase (PERK) by GSK2656157 and inositol-requiring enzyme (IRE) by STF-083010 differentially influenced Zn-induced changes of lipid metabolism, indicating that PERK and IRE pathways played different regulatory roles in Zn-induced lipolysis. Inhibition of PKA by H89 blocked the Zn-induced activation of cAMP/PKA pathway with a concomitant inhibition of ER stress-mediated lipolysis. Taken together, our findings highlight the importance of the ER stress-cAMP/PKA axis in Zn-induced lipolysis, which provides new insights into Zn toxicology in fish and probably in other vertebrates.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Sustancias Peligrosas/toxicidad , Lipólisis/fisiología , Zinc/toxicidad , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Animales , Bagres , Regulación hacia Abajo , Retículo Endoplásmico , Homeostasis , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Hígado/fisiología , Transducción de Señal/efectos de los fármacos , Sulfonamidas , TiofenosRESUMEN
ß2-receptor agonists are used in the treatment of inflammatory obstructive lung diseases asthma and COPD as a symptomatic remedy, but they have been suggested to possess anti-inflammatory properties, also. ß2-receptor activation is considered to lead to the activation of ERK pathway through G-protein- and cAMP-independent mechanisms. In this study, we investigated the effects of ß2-receptor agonists salbutamol and terbutaline on the production of inflammatory factors in macrophages. We found that ß2-receptor agonists inhibited LPS-induced ERK phosphorylation and the production of MCP-1. A chemical cAMP analog 8-Br-cAMP also inhibited ERK phosphorylation and TNF and MCP-1 release. As expected, MAPK/ERK kinase (MEK)1/2 inhibitor PD0325901 inhibited ERK phosphorylation and suppressed both TNF and MCP-1 production. In conclusion, we suggest that ß2-receptor agonists salbutamol and terbutaline inhibit inflammatory gene expression partly by a mechanism dependent on cAMP leading to the inhibition of ERK signaling in macrophages. Observed anti-inflammatory effects of ß2-receptor agonists may contribute to the clinical effects of these drugs.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Albuterol/farmacología , Citocinas/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Terbutalina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Benzamidas/farmacología , Línea Celular , Quimiocina CCL2/metabolismo , AMP Cíclico/metabolismo , Citocinas/genética , Difenilamina/análogos & derivados , Difenilamina/farmacología , Factores Inmunológicos/farmacología , Mediciones Luminiscentes , Ratones , Fosforilación/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.
Asunto(s)
Membrana Celular/química , Plexo Coroideo/metabolismo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Líquido Intracelular/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/administración & dosificación , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animales , Encéfalo/fisiología , Membrana Celular/metabolismo , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/enzimología , Líquido Cefalorraquídeo/fisiología , Plexo Coroideo/química , Plexo Coroideo/citología , Plexo Coroideo/ultraestructura , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/efectos adversos , Citometría de Flujo , Líquido Intracelular/enzimología , Líquido Intracelular/metabolismo , Macrólidos/administración & dosificación , Macrólidos/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Sodio/metabolismo , Tionucleótidos/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP) reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells.
Asunto(s)
Astrocitos/patología , Neoplasias Encefálicas/patología , Diferenciación Celular , AMP Cíclico/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Glucólisis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Astrocitos/metabolismo , Astrocitos/ultraestructura , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/ultraestructura , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Perfilación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioblastoma/genética , Glioblastoma/ultraestructura , Glucólisis/efectos de los fármacos , Humanos , Biogénesis de Organelos , Fosforilación Oxidativa/efectos de los fármacos , Proteómica , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The main purpose of our work was to evaluate the effects of different cyclic adenosine monophosphate analogs on thyroid cancer-derived cell lines. In particular we studied 8-chloroadenosine-3',5'-cyclic monophosphate, the most powerful cyclic adenosine monophosphate analog, and the protein kinase A I-selective combination of 8-hexylaminoadenosine-3',5'cyclic monophosphate and 8-piperidinoadenosine-3',5'-cyclic monophosphate. The cyclic adenosine monophosphate/protein kinase A pathway plays a fundamental role in the regulation of thyroid cells growth. Site-selective cyclic adenosine monophosphate analogs are a class of cyclic adenosine monophosphate-derivate molecules that has been synthesized to modulate protein kinase A activity. Although the cyclic adenosine monophosphate/protein kinase A pathway plays a fundamental role in the regulation of thyroid cells proliferation, there are currently no studies exploring the role of cyclic adenosine monophosphate analogs in thyroid cancer. We evaluated the effects on cell proliferation, apoptosis activation and alterations of different intracellular pathways using 3-(4,5-dimetylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytofluorimetry, western blotting, and kinase inhibitors. Our results show that both compounds have antiproliferative potential. Both treatments were able to modify protein kinase A RI/RII ratio, thus negatively influencing cancer cells growth. Moreover, the two treatments differentially modulated various signaling pathways that regulate cell proliferation and apoptosis. Both treatments demonstrated interesting characteristics that prompt further studies aiming to understand the intimate interaction between different intracellular pathways and possibly develop novel anticancer therapies for undifferentiated thyroid cancer.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , AMP Cíclico/análogos & derivados , Transducción de Señal/efectos de los fármacos , Glándula Tiroides/efectos de los fármacos , Neoplasias de la Tiroides/patología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , AMP Cíclico/farmacología , Humanos , Fosforilación/efectos de los fármacos , Glándula Tiroides/patologíaRESUMEN
OBJECTIVE: To explore the effects of MCA-MAO on the cAMP pathway in rats with cerebral hemorrhage. MATERIALS AND METHODS: Forty SD male rats were randomly divided into four groups: the sham operation group (n=10), the model group (n=10), the negative control group (n=10) and the experimental group (n=10). To prepare rat models for cerebral hemorrhage, autogenous femoral arterial blood was injected into the caudate nucleus. In the case of rats in sham operation group, normal saline was injected into the caudate nucleus. Rats in the negative control group received a proper amount of saline via an injection into the abdominal cavity. Rats in the experimental group were injected with 500 µL/kg MCA-MAO into the abdominal cavity. Five rats from each group were executed after 1 to 3 days, the water contents of gray and white matters were detected using far infrared moisture analyzer, the MAO activity was measured by the histochemical method. The cAMP level was measured by radio-immunity method and the protein kinase A (PKA) level was measured by Western blot. cAMP response element binding (CREB) mRNA expression level was detected by RT-PCR. RESULTS: Water content, MAO activity, cAMP, PKA, and CREB mRNA expression levels in the model, and the negative control groups were significantly higher than those of the sham operation and the experimental groups, the differences were statistically significant (p<0.05). CONCLUSIONS: MAO may mediate the pathophysiological process of hemorrhage via cAMP signaling pathway.
Asunto(s)
Hemorragia Cerebral , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Monoaminooxidasa , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Animales , Masculino , RatasRESUMEN
UNLABELLED: Exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) are intracellular receptors for cAMP. Although PKA and its downstream effectors have been studied extensively in the context of drug addiction, whether and how Epac regulates cellular and behavioral effects of drugs of abuse remain essentially unknown. Epac is known to regulate AMPA receptor (AMPAR) trafficking. Previous studies have shown that a single cocaine exposure in vivo leads to an increase in GluA2-lacking AMPARs in dopamine neurons of the ventral tegmental area (VTA). We tested the hypothesis that Epac mediates cocaine-induced changes in AMPAR subunit composition in the VTA. We report that a single cocaine injection in vivo in wild-type mice leads to inward rectification of EPSCs and renders EPSCs sensitive to a GluA2-lacking AMPAR blocker in VTA dopamine neurons. The cocaine-induced increase in GluA2-lacking AMPARs was absent in Epac2-deficient mice but not in Epac1-deficient mice. In addition, activation of Epac with the selective Epac agonist 8-CPT-2Me-cAMP (8-CPT) recapitulated the cocaine-induced increase in GluA2-lacking AMPARs, and the effects of 8-CPT were mediated by Epac2. We also show that conditioned place preference to cocaine was impaired in Epac2-deficient mice and in mice in which Epac2 was knocked down in the VTA but was not significantly altered in Epac1-deficient mice. Together, these results suggest that Epac2 is critically involved in the cocaine-induced change in AMPAR subunit composition and drug-cue associative learning. SIGNIFICANCE STATEMENT: Addictive drugs, such as cocaine, induce long-lasting adaptions in the reward circuits of the brain. A single intraperitoneal injection of cocaine leads to changes in the composition and property of the AMPAR that carries excitatory inputs to dopamine neurons. Here, we provide evidence that exchange protein directly activated by cAMP (Epac), a cAMP sensor protein, is required for the cocaine-induced changes of the AMPAR. We found that the effects of cocaine were mimicked by activation of Epac but were blocked by genetic deletion of Epac. Furthermore, cocaine-cue associative learning was impaired in mice lacking Epac. These findings uncovered a critical role of Epac in regulating the cellular and behavioral actions of cocaine.
Asunto(s)
Cocaína/farmacología , Receptores AMPA/efectos de los fármacos , Área Tegmental Ventral/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Animales , AMP Cíclico/análogos & derivados , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Recompensa , Sinapsis , Tionucleótidos , Área Tegmental Ventral/citologíaRESUMEN
In most conditions, glucose is the best carbon source for E. coli: it provides faster growth than other sugars, and is consumed first in sugar mixtures. Here we identify conditions in which E. coli strains grow slower on glucose than on other sugars, namely when a single amino acid (arginine, glutamate, or proline) is the sole nitrogen source. In sugar mixtures with these nitrogen sources, E. coli still consumes glucose first, but grows faster rather than slower after exhausting glucose, generating a reversed diauxic shift. We trace this counterintuitive behavior to a metabolic imbalance: levels of TCA-cycle metabolites including α-ketoglutarate are high, and levels of the key regulatory molecule cAMP are low. Growth rates were increased by experimentally increasing cAMP levels, either by adding external cAMP, by genetically perturbing the cAMP circuit or by inhibition of glucose uptake. Thus, the cAMP control circuitry seems to have a 'bug' that leads to slow growth under what may be an environmentally rare condition.