RESUMEN
Some patients with psychiatric disorders show hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. This may be due to an impaired feedback inhibition and can be seen through increased levels of circulating cortisol. Here a protocol is described to mimic this situation by subcutaneous implantation of corticosterone pellets in mice. We also present characterization of the model by looking at effects on neuronal proliferation in hippocampus, one of the main tissues known to be affected by HPA axis hyper-activation. Such tissues could be used in analyses by proteomic platforms.
Asunto(s)
Corticosterona/toxicidad , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional/métodos , Hipocampo/efectos de los fármacos , Trastornos Mentales/inducido químicamente , Proteómica/métodos , Animales , Corticosterona/administración & dosificación , Corticosterona/sangre , Implantes de Medicamentos , Hipocampo/química , Trastornos Mentales/metabolismo , Trastornos Mentales/fisiopatología , Ratones , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/fisiología , Receptores de Mineralocorticoides/agonistas , Receptores de Mineralocorticoides/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin-proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program.
Asunto(s)
Autofagia/efectos de los fármacos , Dexametasona/toxicidad , Glucocorticoides/toxicidad , Mitocondrias Musculares/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Atrofia Muscular/inducido químicamente , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Línea Celular , Relación Dosis-Respuesta a Droga , Dinaminas/genética , Dinaminas/metabolismo , Proteínas de Choque Térmico/deficiencia , Proteínas de Choque Térmico/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Mitofagia/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Proteínas/genética , Proteínas/metabolismo , Quinazolinonas/farmacología , Interferencia de ARN , Ratas , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Proteína Sequestosoma-1 , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively. RESULTS: Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively. CONCLUSIONS: Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.
Asunto(s)
Aziridinas/farmacología , Compuestos de Amonio Cuaternario/farmacología , Bazo/efectos de los fármacos , Proteínas de Dominio T Box/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Acetatos , Animales , Factor de Transcripción GATA3/biosíntesis , Factor de Transcripción GATA3/inmunología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Mifepristona/farmacología , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inhibidores , Bazo/inmunología , Proteínas de Dominio T Box/biosíntesis , Linfocitos T/inmunología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Balance Th1 - Th2/efectos de los fármacos , Células Th2/efectos de los fármacos , Células Th2/inmunología , Tiramina/análogos & derivadosRESUMEN
The introduction of a hemisuccinate group at the 21-position of the passive antiglucocorticoid 21OH-6,19OP leads to a compound (21HS-6,19OP) with a notable activity profile toward the glucocorticoid receptor (GR). In contrast to the parent steroid, 21HS-6,19OP behaves as a pure agonist of GR activity in direct transactivation assays. However, the apoptotic effects of 21HS-6,19OP show that the effect depends on cell type: while 21HS-6,19OP is a pure agonist in L929 mouse fibroblasts, in thymocytes 21HS-6,19OP had significant antiglucocorticoid activity. This tissue-specific activity makes 21HS-6,19OP a novel selective GR modulator. To investigate the molecular basis of action of 21HS-6,19OP, we carried out molecular dynamics simulations (6 ns) of the GR ligand binding domain (LBD) complexed with 21HS-6,19OP. Our results indicate that the hemisuccinate moiety may play a key role in stabilizing the active conformation of the receptor dimerization interface, reverting the changes observed with the antagonist 21OH-6,19OP. Other changes in regions of the GR related to cofactor recruitment (possibly tissue-specific), could explain this particular activity profile.
Asunto(s)
Progesterona/análogos & derivados , Receptores de Glucocorticoides/agonistas , Animales , Apoptosis , Células COS , Chlorocebus aethiops , Simulación por Computador , Dimerización , Genes Reporteros , Humanos , Ratones , Progesterona/química , Progesterona/farmacología , Progestinas/química , Progestinas/farmacología , Receptores de Glucocorticoides/química , Relación Estructura-ActividadRESUMEN
AIM: To investigate the severity of acute pancreatitis (AP) is associated to the intensity of leukocyte activation, inflammatory up-regulation and microcirculatory disruption associated to ischemia-reperfusion injury. Microvascular integrity and inhibition of pro-inflammatory mediators are key-factors in the evolution of AP. Relaxin is an insulin-like hormone that has been attributed vasorelaxant properties via the nitric oxide pathway while behaving as a glucocorticoid receptor agonist. METHODS: AP was induced by the bilio-pancreatic duct-outlet-exclusion closed-duodenal-loops model. Treatment with relaxin was done at different time-points. Nitric oxide synthase inhibition by L-NAME and glucocorticoid receptor (GR) blockage by mifepristone was considered. AP severity was assessed by biochemical and histopathological analyses. RESULTS: Treatment with relaxin reduced serum amylase, lipase, C-reactive protein, IL-6, IL-10, hsp72, LDH and 8-isoprostane as well as pancreatic and lung myeloperoxidase. Acinar and fat necrosis, hemorrhage and neutrophil infiltrate were also decreased. ATP depletion and ADP/ATP ratio were reduced while caspases 2-3-8 and 9 activities were increased. L-NAME and mifepristone decreased the efficiency of relaxin. CONCLUSION: Relaxin resulted beneficial in the treatment of AP combining the properties of a GR agonist while preserving the microcirculation and favoring apoptosis over necrosis.
Asunto(s)
Pancreatitis/tratamiento farmacológico , Pancreatitis/fisiopatología , Relaxina/farmacología , Enfermedad Aguda , Animales , Apoptosis , Proteínas Sanguíneas/análisis , Proteína C-Reactiva/análisis , Caspasas/fisiología , Modelos Animales de Enfermedad , Inflamación , Pulmón/fisiopatología , Masculino , Óxido Nítrico/fisiología , Páncreas/irrigación sanguínea , Páncreas/fisiopatología , Ratas , Ratas Wistar , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/fisiología , Relaxina/fisiología , Transducción de Señal , VasodilataciónRESUMEN
We studied the glucocorticoid response to the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (DeltaHOP) in several cell types and correlated its biological effect with the ability of the glucocorticoid receptor (GR) to be retained in the nuclear compartment. We observed that the DeltaHOP-transformed GR was diffusely distributed in the nucleus compared to the discrete structures observed for the dexamethasone (DEX)-transformed GR. Despite the fact that the receptor was entirely nuclear upon binding of each steroid and exhibited identical nuclear export rates, a greater amount of DeltaHOP-transformed GR was recovered in the cytoplasmic fraction after hypotonic cell lysis. Furthermore, accelerated nuclear export of GR was evidenced in digitonin-permeabilized cells treated with ATP and molybdate. Inasmuch as limited trypsinization of DEX-GR and DeltaHOP-GR complexes yielded different proteolytic products, we conclude that GR undergoes a differential conformational change upon binding of each ligand. We propose that these conformational differences may consequently lead to changes of stability in the interaction of the GR with chromatin. Therefore, the dynamic exchange of liganded GR with chromatin is likely to have significant consequences for the observed pleiotropic physiological responses triggered by glucocorticoid ligands, not only in different tissues but also in the same cell type.
Asunto(s)
Compartimento Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Células Eucariotas/efectos de los fármacos , Glucocorticoides/farmacología , Receptores de Glucocorticoides/agonistas , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Compartimento Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Dexametasona/metabolismo , Dexametasona/farmacología , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Células Eucariotas/citología , Células Eucariotas/metabolismo , Femenino , Glucocorticoides/metabolismo , Hidroxiprogesteronas/metabolismo , Hidroxiprogesteronas/farmacología , Masculino , Ratones , Conformación Molecular , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Proteína bcl-XRESUMEN
In previous studies, we have shown that male Wistar rats exposed to a single inescapable stressor session (15 min restraint) exhibited 24 h later an anxiogenic-like behavior in the elevated plus-maze (EPM), which was reversed by inhibition of corticosterone (CS) synthesis with metyrapone (75 mg/kg i.p.) 3 h before stress. Since CS binds to two central corticosteroid receptors, the mineralocorticoid (MR) and the glucocorticoid (GR) receptors, involvement of MR and GR in the modulation of anxiogenic responses was assessed in the EPM. Administration of the GR agonist dexamethasone (Dex, 1.25 microg/kg s.c.) to metyrapone-pretreated rats 1 h before restraint restored the anxiogenic-like response induced by the stressor. Removal of the adrenals also inhibited the anxiogenic-like effect, which was restored by either Dex (1.25 microg/kg s.c.), the MR agonist deoxycorticosterone (0.8 mg/kg s.c.) or CS, the common endogenous agonist of MR and GR (5 mg/kg s.c.) administered 1 h before stress. Intracerebroventricular infusion to intact animals 15 min before restraint of either a selective GR antagonist (A-GR, RU 38486, 100 ng/2 microl), a selective MR antagonist (A-MR, RU 28318, 100 ng/2 microl) or a combination of A-GR and A-MR (100 ng of each one/2 microl), abolished the stress-induced anxiogenic-like effect. The present findings indicate that both MR and GR are involved in the long-term CS modulation of the anxiety response induced by restraint. Both receptors mediate CS effects in an independent manner.
Asunto(s)
Ansiedad/fisiopatología , Ansiedad/psicología , Receptores de Glucocorticoides/fisiología , Receptores de Mineralocorticoides/fisiología , Estrés Psicológico/fisiopatología , Estrés Psicológico/psicología , Adrenalectomía , Animales , Antiinflamatorios/farmacología , Corticosterona/sangre , Corticosterona/farmacología , Dexametasona/farmacología , Inyecciones Intraventriculares , Masculino , Metirapona/farmacología , Mifepristona/farmacología , Antagonistas de Receptores de Mineralocorticoides/farmacología , Ratas , Ratas Wistar , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Mineralocorticoides/agonistas , Restricción Física , Espironolactona/análogos & derivados , Espironolactona/farmacologíaRESUMEN
Evidence exists that the spinal cord is a glucocorticoid-responsive tissue, and glucocorticoids have beneficial effects in cases of spinal cord injury. Using sham-operated rats, spinal cord transected (TRX) rats, and TRX animals receiving dexamethasone (DEX) 5 min or 24 h post-lesion, we have examined the following GC-sensitive parameters 6 h after DEX treatment: (1) binding of glutamate to NMDA-sensitive receptors; (2) the activity of ornithine decarboxylase (ODC); and (3) levels of polyamines. We found that glutamate binding in the dorsal horn (Laminae 1-2) and central canal were upregulated in TRX rats, whereas DEX had an additional stimulatory effect. 24 h post-lesion, glutamate binding was unmodified in TRX or TRX+DEX rats. ODC activity was increased 10-fold in rats killed on the day of transection but only 2-fold 24 h post-lesion. DEX reduced ODC activity on transection day but highly increased it when given 24 h after surgery. The content of the polyamines spermidine and spermine were unchanged after TRX or DEX treatment, in contrast to putrescine which increased in TRX rats and further increased in TRX+DEX rats when measured the day post-lesion. Thus, parallel increases in ODC and putrescine 1 day after the lesion, suggest that glucocorticoid effects on growth responses due to polyamines may develop at a late period. The changes of glutamate binding in the dorsal horn and central canal due to early glucocorticoid treatment, further suggest hormonal modulation of neurotransmission in sensitive areas of the deafferented spinal cord.