RESUMO
INTRODUCTION AND OBJECTIVES: Pulmonary regurgitation (PR) is a frequent complication after repair of congenital heart disease. Lymphocyte expression of adrenoceptors (ß1 and ß2) and kinases (GRK2, GRK3, and GRK5) reflects the neurohumoral changes that occur in heart failure (HF). The main objective of this study was to describe the gene expression of these molecules in circulating lymphocytes in patients with severe PR. METHODS: A prospective study was conducted to analyze lymphocyte expression of these molecules in patients with severe PR and compare it with expression in healthy controls and patients with advanced HF. RESULTS: We studied 35 patients with severe PR, 22 healthy controls, and 13 patients with HF. Multiple comparisons analysis showed that ß2-adrenoceptor gene expression levels were higher in the control group than in patients in the PR and HF groups and that expression in the latter 2 groups was similar (748.49 [rank 1703.87] vs 402.80 [rank 1210.81] vs 287.46 [rank 685.69] P = .001). Similar findings were obtained in gene expression of GRK2 (760.89 [rank 1169.46] vs 445.17 [rank 1190.69] vs 284.09 [rank 585.27] P < .001). There were no differences in expression levels of these molecules according to clinical variables in patients with PR. CONCLUSIONS: The gene expression pattern of GRK2 and ß2-adrenoceptor as molecular markers of cardiac dysfunction was altered in patients with severe PR compared with controls and was similar to expression in patients with advanced HF.
Assuntos
Quinases de Receptores Acoplados a Proteína G/genética , Regulação da Expressão Gênica , Insuficiência da Valva Pulmonar/genética , RNA/genética , Receptores Adrenérgicos/genética , Adulto , Doença Crônica , Feminino , Seguimentos , Quinases de Receptores Acoplados a Proteína G/biossíntese , Humanos , Imagem Cinética por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Insuficiência da Valva Pulmonar/diagnóstico , Insuficiência da Valva Pulmonar/metabolismo , Receptores Adrenérgicos/biossínteseRESUMO
G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans, and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Quinases de Receptores Acoplados a Proteína G/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Monoaminoxidase/metabolismo , Serotonina/metabolismo , Transdução de Sinais/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Monoaminoxidase/genética , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/genéticaRESUMO
Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck cancers with high invasiveness and poor prognosis. To identify targeted therapeutics against metastasis, a better understanding of the regulation of HSCC cell invasion is needed. In recent years, G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through phosphorylation of the activated form of G protein coupled receptors (GPCRs). However, there is little information regarding GRKs expression in HSCC. In the present study, we examined GRK6 expression in HSCC and also assessed the possible cause of its aberrant expression, as well as its clinical significance. Real-time quantitative PCR (qPCR) and western blotting were performed to analyze the expression of GRK6 in HSCC tissues and corresponding non-malignant tissues. Subsequently, paired HSCC and corresponding non-malignant tissues were evaluated for the methylation status of GRK6 gene promoter using methylation-specific PCR (MSP). Furthermore, we investigated the methylation status and the clinicopathological significance of GRK6 in 45 paired HSCC and corresponding non-malignant tissues. The suppression of GRK6 in hypopharyngeal cell line FaDu by GRK6-shRNA lentivirus transfection was utilized to detect the role of GRK6 in hypopharyngeal cancer progression. Our results showed that the expression of GRK6 mRNA and protein was significantly lower in HSCC than in corresponding adjacent non-tumor tissues, and this downregulation was found to be in accordance with aberrant methylation of the gene. Hypermethylation of the gene was observed in 77.8% (35/45) of the HSCC tissues, while it was found in only 42.2% (19/45) of the corresponding non-malignant tissues. GRK6 methylation was related to depth of tumor invasion and TNM stage. Upon treatment with 5-aza-2'-deoxycytidine, GRK6 expression was upregulated in FaDu cells, and cell invasion was signinficantly inhibited. Furthermore, the suppression of GRK6 by shRNA transfection enhanced FaDu cells invasion. Our results indicate that the aberrant methylation of GRK6 gene promoter may underlie its downregulation in HSCC, and may play an important role in the metastasis of HSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Quinases de Receptores Acoplados a Proteína G/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Hipofaríngeas/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Metilação de DNA/efeitos dos fármacos , Decitabina , Regulação para Baixo , Quinases de Receptores Acoplados a Proteína G/antagonistas & inibidores , Quinases de Receptores Acoplados a Proteína G/biossíntese , Quinases de Receptores Acoplados a Proteína G/fisiologia , Humanos , Neoplasias Hipofaríngeas/mortalidade , Neoplasias Hipofaríngeas/patologia , Invasividade Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Prognóstico , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genéticaRESUMO
l-DOPA therapy in Parkinson's disease often results in side effects such as l-DOPA-induced dyskinesia (LID). Our previous studies demonstrated that defective desensitization of dopamine receptors caused by decreased expression of G protein-coupled receptor kinases (GRKs) plays a role. Overexpression of GRK6, the isoform regulating dopamine receptors, in parkinsonian rats and monkeys alleviated LID and reduced LID-associated changes in gene expression. Here we show that 2-fold lentivirus-mediated overexpression of GRK6 in the dopamine-depleted striatum in rats unilaterally lesioned with 6-hydroxydopamine ameliorated supersensitive ERK response to l-DOPA challenge caused by loss of dopamine. A somewhat stronger effect of GRK6 was observed in drug-naïve than in chronically l-DOPA-treated animals. GRK6 reduced the responsiveness of p38 MAP kinase to l-DOPA challenge rendered supersensitive by dopamine depletion. The JNK MAP kinase was unaffected by loss of dopamine, chronic or acute l-DOPA, or GRK6. Overexpressed GRK6 suppressed enhanced activity of Akt in the lesioned striatum by reducing elevated phosphorylation at its major activating residue Thr(308). Finally, GRK6 reduced accumulation of ΔFosB in the lesioned striatum, the effect that paralleled a decrease in locomotor sensitization to l-DOPA in GRK6-expressing rats. The results suggest that elevated GRK6 facilitate desensitization of DA receptors, thereby normalizing of the activity of multiple signaling pathways implicated in LID. Thus, improving the regulation of dopamine receptor function via the desensitization mechanism could be an effective way of managing LID.
Assuntos
Dopaminérgicos/farmacologia , Quinases de Receptores Acoplados a Proteína G/biossíntese , Levodopa/farmacologia , Neostriado/metabolismo , Neostriado/fisiopatologia , Doença de Parkinson Secundária/metabolismo , Doença de Parkinson Secundária/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dopamina/deficiência , Discinesia Induzida por Medicamentos/psicologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neostriado/efeitos dos fármacos , Doença de Parkinson Secundária/induzido quimicamente , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , Ratos , Ratos Sprague-DawleyRESUMO
Nerve injury and inflammation can both induce neuropathic pain via the production of pro-inflammatory cytokines. In the process, G protein-coupled receptors (GPCRs) were involved in pain signal transduction. GPCR kinase (GRK) 6 is a member of the GRK family that regulates agonist-induced desensitization and signaling of GPCRs. However, its expression and function in neuropathic pain have not been reported. In this study, we performed a chronic constriction injury (CCI) model in adult male rats and investigated the dynamic change of GRK6 expression in spinal cord. GRK6 was predominantly expressed in the superficial layers of the lumbar spinal cord dorsal horn neurons and its expression was decreased bilaterally following induction of CCI. The changes of GRK6 were mainly in IB4 and P substrate positive areas in spinal cord dorsal horn. And over-expression of GRK6 in spinal cord by lentivirus intrathecal injection attenuated the pain response induced by CCI. In addition, the level of TNF-α underwent the negative pattern of GRK6 in spinal cord. And neutralized TNF-α by antibody intrathecal injection up-regulated GRK6 expression and attenuated the mechanical allodynia and heat hyperalgesia in CCI model. All the data indicated that down-regulation of neuronal GRK6 expression induced by cytokine may be a potential mechanism that contributes to increasing neuronal signaling in neuropathic pain.
Assuntos
Quinases de Receptores Acoplados a Proteína G/fisiologia , Neuralgia/enzimologia , Traumatismos da Medula Espinal/enzimologia , Animais , Constrição , Quinases de Receptores Acoplados a Proteína G/biossíntese , Hiperalgesia/fisiopatologia , Masculino , Neuralgia/fisiopatologia , Células do Corno Posterior/enzimologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The melanocortin 1 receptor (MC1R), a G(s) protein-coupled receptor, has an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human ß-defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-melanocyte-stimulating hormone (α-MSH))-induced increase in the activities of adenylate cyclase and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C, increased, whereas exposure to UV radiation reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, whereas continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6, as well as ß-arrestin 1. Therefore, in addition to the MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV.
Assuntos
Proteína Agouti Sinalizadora/farmacologia , Melanocortinas/farmacologia , Melanócitos/efeitos dos fármacos , Receptor Tipo 1 de Melanocortina/agonistas , Receptor Tipo 1 de Melanocortina/antagonistas & inibidores , alfa-MSH/farmacologia , beta-Defensinas/farmacologia , Adenilil Ciclases/metabolismo , Arrestinas/biossíntese , Células Cultivadas , Colforsina/farmacologia , Quinases de Receptores Acoplados a Proteína G/biossíntese , Humanos , Melanócitos/metabolismo , Melanócitos/efeitos da radiação , Monofenol Mono-Oxigenase/metabolismo , Proteína Quinase C/metabolismo , Receptor Tipo 1 de Melanocortina/biossíntese , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/fisiologia , Pigmentação da Pele/efeitos da radiação , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Raios Ultravioleta , beta-Arrestina 1 , beta-ArrestinasRESUMO
Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting a powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed a reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. A reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/deficiência , Quinase 3 de Receptor Acoplado a Proteína G/deficiência , Quinase 5 de Receptor Acoplado a Proteína G/deficiência , Quinases de Receptores Acoplados a Proteína G/deficiência , Córtex Pré-Frontal/metabolismo , Transtornos Psicóticos/genética , Esquizofrenia/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Arrestinas/biossíntese , Arrestinas/deficiência , Arrestinas/genética , Estudos de Coortes , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/biossíntese , Quinase 3 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/biossíntese , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/biossíntese , Quinases de Receptores Acoplados a Proteína G/genética , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/fisiopatologia , Transtornos Psicóticos/metabolismo , Transtornos Psicóticos/fisiopatologia , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia , Adulto JovemRESUMO
Animals with the neonatal ventral hippocampal lesion (NVHL) demonstrate altered responsiveness to stress and various drugs reminiscent of that in schizophrenia. Post-pubertal onset of abnormalities suggests the possibility of sex differences in NVHL effects that may model sex differences in schizophrenia. Here we demonstrate that novelty- and MK-801-induced hyperactivity is evident in both male and female NVHL rats, whereas only NVHL males were hyperactive in response to apomorphine. Next, we examined the sex- and NVHL-dependent differences in the activity of the ERK and Akt pathways. The basal activity of both pathways was higher in females than in males. NVHL reduces the level of phosphorylation of ERK1/2, Akt, and GSK-3 in both sexes, although males show more consistent down-regulation. Females had higher levels of G-protein-coupled kinases [G-protein-coupled receptor kinase (GRK)] 3 and 5, whereas the concentrations of other GRKs and arrestins were the same. In the nucleus accumbens, the concentration of GRK5 in females was elevated by NVHL to the male level. The data demonstrate profound sex differences in the expression and activity of signalling molecules that may underlie differential susceptibility to schizophrenia.