RESUMO
Chondrocytes, which are embedded within the growth-plate or the intervertebral disc, are sensitive to environmental stresses, such as inflammation and hypoxia. However, little is known about the molecular signaling pathways underlying hypoxia-induced mitochondrial dysfunction and apoptosis in chondrocytes. We first examined the apoptosis, caspase-3 activity, and apoptosis-associated markers in human chondrocyte cell line C28/I2 under normoxia or hypoxia. We then investigated mitochondrial dysfunction and the activation of cyclic adenosine monophosphate response element-binding protein (CREB) signaling in the same human chondrocyte cell line. Our results indicated that hypoxia induced apoptosis and reduced CREB phosphorylation in chondrocytes. Upregulated mitochondrial superoxide and reactive oxygen species levels, and reduced mitochondrial membrane potential and complex IV activity were observed in hypoxia-treated C28/I2 cells. In conclusion, the present study confirmed reduced CREB phosphorylation, apoptosis induction, and mitochondrial dysfunction in the hypoxia-treated chondrocyte cells. This implies the key role played by CREB signaling in hypoxia-induced mitochondrial dysfunction and apoptosis in chondrocytes.
Assuntos
Condrócitos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Mitocôndrias/metabolismo , Apoptose/genética , Hipóxia Celular/genética , Linhagem Celular , Condrócitos/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/patologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxidos/metabolismoRESUMO
AIMS: The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with ß1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. MAIN METHODS: Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. KEY FINDINGS: Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. SIGNIFICANCE: Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis.
Assuntos
NF-kappa B/biossíntese , Norepinefrina/farmacologia , Glândula Pineal/efeitos dos fármacos , Animais , Arilalquilamina N-Acetiltransferase/biossíntese , Arilalquilamina N-Acetiltransferase/metabolismo , Arilalquilamina N-Acetiltransferase/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Masculino , Melatonina/biossíntese , Melatonina/fisiologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Técnicas de Cultura de Órgãos , Glândula Pineal/metabolismo , Glândula Pineal/fisiologia , Pirrolidinas/farmacologia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Salicilato de Sódio/farmacologia , Tiocarbamatos/farmacologiaRESUMO
Our group previously demonstrated that short-term treatment with a standardized extract of Ginkgo biloba (EGb) changed fear-conditioned memory by modulating gene expression in the hippocampus, amygdaloid complex and prefrontal cortex. Although there are few controlled studies that support the long-term use of EGb for the prevention and/or treatment of memory impairment, the chronic use of Ginkgo is common. This study evaluated the effects of chronic treatment with EGb on the conditioned emotional response, assessed by the suppression of ongoing behavior and in the modulation of gene and protein expression. Male adult Wistar rats were treated over 28days and assigned to five groups (n=10) as follows: positive control (4mgkg(-1) Diazepam), negative control (12% Tween 80), EGb groups (0.5 and 1.0gkg(-1)) and the naïve group. The suppression of the licking response was calculated for each rat in six trials. Our results provide further evidence for the efficacy of EGb on memory. For the first time, we show that long-term treatment with the highest dose of EGb improves the fear memory and suggests that increased cAMP-responsive element-binding protein (CREB)-1 and glial fibrillary acidic protein (GFAP) mRNA and protein (P<0.001) in the dorsal hippocampus and amygdaloid complex and reduced growth and plasticity-associated protein 43 (GAP-43) (P<0.01) in the hippocampus are involved in this process. The fear memory/treatment-dependent changes observed in our study suggest that EGb might be effective for memory enhancement through its effect on the dorsal hippocampus and amygdaloid complex.
Assuntos
Tonsila do Cerebelo/fisiologia , Condicionamento Operante/efeitos dos fármacos , Ginkgo biloba/química , Hipocampo/fisiologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Interpretação Estatística de Dados , Diazepam/farmacologia , Medo/efeitos dos fármacos , Proteína GAP-43/biossíntese , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/genética , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Imuno-Histoquímica , Masculino , Memória/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos WistarRESUMO
We studied the effects of tutin, a sesquiterpenoid obtained from Coriaria ruscifolia subspecie ruscifolia, a native poisonous Chilean plant, on spinal glycine receptors using patch clamp recordings. In addition, cytosolic Ca(2+) transients and activation of cAMP response element-binding protein (CREB) were measured in the presence of tutin. Application of tutin (1-1000 microM) inhibited the glycinergic evoked current in a concentration-dependent manner. Moreover, the frequency of spontaneous Ca(2+) spikes and spontaneous synaptic activity (AMPAergic events) was augmented and correlated with an increase in phosphorylated CREB levels, suggesting an enhancement in neuronal excitability. These results may explain the toxic effects of the plant characterized by seizures and convulsions with subsequent coma and death seen in humans and mice.