RESUMO
OBJECTIVE: Obstructive Sleep Apnoea Syndrome (OSAS) is a respiratory disorder characterized by recurrent airflow obstruction caused by total or partial collapse of the upper airway. OSAS is an established independent factor of cardiovascular risk together with other risk factors such as smoking and increased lipids. The aim of our study was to measure serum levels of aldosterone and renin in OSAS patients that did not suffer from arterial hypertension and compare them to matched healthy subjects in order to reveal the impact of chronic intermittent hypoxia on the renin-angiotensin-aldosterone system. PATIENTS AND METHODS: The patients that enrolled in this study were 19 OSAS patients who had undergone overnight polysomnography and had an Apnoea Hypopnoea Index (AHI) greater than 10 events/hour. They were compared to 20 healthy non-OSAS closely matched controls. Serum aldosterone and direct renin concentration were measured by radioimmunoassay. RESULTS: Aldosterone concentration follows a diurnal variation; therefore, all blood samples were obtained at the same time (6 AM). There were no significant differences in serum aldosterone levels between the two studied groups of OSAS patients and the healthy subjects group (140.6 pg/ml ± 25.2 vs. 133.2 pg/ml ± 18.5 with p = 0.223). Similar were the results for the renin levels (25.0 ± 6.9 vs. 24.9 ± 4.4 with p = 0.360). CONCLUSIONS: Our study suggests that patients with OSAS, but without existing hypertension have aldosterone and renin levels similar to healthy subjects. According to our findings a direct connection between OSAS and the development of arterial hypertension may not be established via sympathetic system activation.
Assuntos
Aldosterona/sangue , Renina/sangue , Apneia Obstrutiva do Sono/sangue , Apneia Obstrutiva do Sono/diagnóstico , Adulto , Biomarcadores/sangue , Ritmo Circadiano/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polissonografia/métodos , Sistema Renina-Angiotensina/fisiologia , Fatores de Risco , Apneia Obstrutiva do Sono/fisiopatologiaRESUMO
BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor and bronchoconstrictor but it has been shown to have also proinflammatory properties. Its ability to attract inflammatory cells in its site of production, upregulates the synthesis of adhesion molecules and stimulates the release of cytokines. The fact that cytokines have the ability to induce its synthesis and release, creates a dynamic loop for self-preservation and augmentation of the airway inflammation in Chronic Obstructive Pulmonary Disease (COPD), even after the ceasing of the noxious stimulus, i.e., cigarette smoke. Therefore, functional polymorphisms that may lead to increased levels of ET-1 may also cause an increased susceptibility to COPD development. MATERIALS AND METHODS: We analyzed the longitudinal effect on lung function of two ET-1 gene polymorphisms in a population of 190 smokers (95 non-COPD and 95 COPD smokers). The two polymorphisms involved an insertion polymorphism (+138 adenine insertion 3A/4A, 138bp downstream from the transcription start site, exon 1) and a single nucleotide transversion polymorphism on exon 5 (G/T, Lys198Asn). A total of 190 subjects were enrolled in the study for each polymorphism and were followed for 3 years by annual spirometry sessions. RESULTS: The adjusted annual decline of forced expiratory volume in 1 second (dFEV1) was greater for those having at least one copy of the mutated gene ins/delA compared to those with the wild type allele both in the non-COPD smokers group (mean difference in dFEV, of 19.4 ml/year, p = 0.004) and COPD smokers (mean difference in dFEV1 of 11.15 ml/year, p = 0.003). On the contrary, those heterozygous for the Lys198Asn polymorphism were found to have a slower decline in FEV1 compared to those homozygous for the wild type allele. The non-COPD smokers group had a gain-in-loss of 11,24 ml/year (p < 0.001) while the COPD-smokers group had a slower decline of 11.42 ml/year (p = 0.002). Those homozygous for the polymorphisms examined show an even greater deviation from those with the wild type allele but due to the small number comprising their group, the results don't have enough statistical power. Though, they still show the trend of the effect the polymorphisms have on annual FEV1 decline. CONCLUSIONS: The present data shows that ET-1 and its functional polymorphisms may be implicated in COPD phenotype and severity.
Assuntos
Endotelina-1/genética , Polimorfismo Genético , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Adulto , Idoso , Alelos , Éxons , Feminino , Seguimentos , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Projetos Piloto , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Índice de Gravidade de Doença , EspirometriaRESUMO
Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclo Celular/fisiologia , Sistema Nervoso Central/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Células-Tronco/fisiologia , Animais , Antimetabólitos , Bromodesoxiuridina , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Regulação para Baixo/fisiologia , Feminino , Imunofluorescência , Geminina , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Plasmídeos/genética , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Advances in molecular biology and functional genomics have demonstrated that the "one gene-one phenotype-one drug" paradigm, that has dominated pharmaceutical industry and clinical pharmacology thinking, is too simplistic for management of complex polygenic traits. The traditional highly specific drugs with unique target have proven their clinical usefulness. However, they do not always display the required efficacy versus side-effect profile, in major part because polygenic traits are determined by redundant mechanisms. Simultaneously modulating multiple targets may enhance therapeutic efficacy in the treatment of a range of disorders. Multi-targeting can be achieved by the combination of different drugs having specific single target activity. This approach introduces potential problems with pharmacokinetic interactions, toxicity and patient compliance. High efficacy can be achieved, alternatively, by administering selectively non-selective drugs with complex pharmacological profiles directed towards various molecular targets and affording pleiotropic actions. Dual- or multiple-ligands can be discovered accidentally, but can also be rationally designed according to validated medicinal chemical approaches. The merits of multiple-target versus single-target approaches for cardiovascular disease traits are assessed in the present review. The main aim is to make evident the molecular biological basis of the possibility for targeting multiple sites and the subsequently emerging strategies for interventions with superior clinical value by harnessing receptor tyrosine kinases (RTKs) such as VEGFR, PDGFR, bFGFR, as well as G protein-coupled receptors (GPCRs). The premises for lead discovery in this new area and the challenges of medicinal chemistry behind the rational design of multitasked ligands are also discussed.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Neovascularização Patológica/tratamento farmacológicoRESUMO
Alpha 2-adrenergic receptors (alpha(2)-ARs) have a widespread distribution in the central nervous system (CNS) and affect a number of biochemical and behavioral functions, including stimulation of prefrontal cortex (PFC) and cognitive function. In addition to its role as a classical neurotransmitter, norepinephrine (NE) has been recently shown to exert an important influence on the plasticity in areas of the brain where neurogenesis persists in the adult, notably the subgranular zone (SGZ) within the dentate gyrus of the hippocampus and the olfactory bulb (OB). In regulating adult neurogenesis, the noradrenergic system is functionally integrated with chronic stress and depression. Chronic stress, depression, or depletion of NE in vivo suppress, and antidepressant treatments induce hippocampal neurogenesis by down- or upregulating, respectively, cell proliferation. In the present study we show that alpha(2)-AR subtypes promote the differentiation rather than cell proliferation of PC12 cells. It is conceivable that alpha(2)-ARs might contribute neurotrophic actions in vivo synergistically or in permutation with other neurotrophic factors.
Assuntos
Replicação do DNA/fisiologia , Neuritos/fisiologia , Neurônios/fisiologia , Receptores Adrenérgicos alfa 2/genética , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Epinefrina/farmacologia , Humanos , Neurônios/ultraestrutura , Células PC12 , Ratos , Receptores Adrenérgicos alfa 2/metabolismo , TransfecçãoRESUMO
Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. The purpose of the present study was to evaluate the effect of methylene blue in chick chorioallantoic membrane angiogenesis model in vivo. In this well characterized model, methylene blue inhibited angiogenesis in a concentration-dependent manner. In addition, when methylene blue was combined with sodium nitroprusside, a spontaneous generator of nitric oxide, an inhibition of angiogenesis was evident which was comparable with that observed by the application of methylene blue alone. Sodium nitroprusside, alone, caused a significant inhibition in basal angiogenesis. These results provide evidence that methylene blue inhibits angiogenesis independently of nitric oxide pathway and suggest that methylene blue may be useful for treating angiogenesis-dependent human diseases.
Assuntos
Membrana Corioalantoide/irrigação sanguínea , Inibidores Enzimáticos/farmacologia , Azul de Metileno/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/fisiologia , Animais , Embrião de Galinha , Relação Dose-Resposta a DrogaRESUMO
Pharmacogenomics would be instrumental for the realization of personalized medicine in coming decades. Efforts are evident to clarify the potential bioethical, societal, and legal implications of key pharmacogenomics-based technologies projected to be soon introduced into the core practice of medicine. In sharp contrast, a lack of sufficient attention to educational aspects of pharmacogenomics, both for professionals and for society at large, is evident. In order to contribute to this discussion, a 'Pharmacogenomics Education Forum' was held on October 2, 2004 during the 3rd Annual Meeting of the International Society of Pharmacogenomics (ISP) at Santorini, Greece. The participants, members of the ISP Pharmacogenomics Education Forum, after deliberate discussions, proposed a document of 'Background Statement' and 'Recommendations and Call for Action' addressed to Deans of Education at Medical, Pharmaceutical, and Health Schools globally. This document has been considered by the education committee of the International Society of Pharmacogenomics and the result is presented here. We hope that this call would be listened to, and soon followed by beneficial action, ultimately leading to enhanced implementation of personalized medicine into core medical education and practice.
Assuntos
Currículo/normas , Guias como Assunto , Cooperação Internacional , Farmacogenética/educação , Escolas para Profissionais de Saúde/normas , Sociedades MédicasRESUMO
Adrenoceptors (ARs) consist of nine subtypes (alpha(1A)-, alpha(1B)-, alpha(1D)-, beta(1)-, beta(2)-, beta(3)-, alpha(2A)-, alpha(2B)- and alpha(2C)-AR), which are involved in a wide spectrum of physiological functions and are the site of action for a considerable percentage of currently prescribed therapeutics. With the exception of alpha(1D), all AR subtypes are polymorphic with genetic variations in the coding and non-coding regions. This review discusses the biochemical consequences of these genetic variations and their impact in receptor function, disease pathophysiology, and drug response. Pharmacogenomic principles that have been discovered are also discussed.
Assuntos
Farmacogenética/métodos , Receptores Adrenérgicos/genética , Animais , Variação Genética/genética , Humanos , Farmacogenética/tendências , Polimorfismo Genético , Receptores Adrenérgicos/fisiologiaRESUMO
1. Rat foetal liver contains large amounts of alpha2-adrenoceptors. The present work aimed to identify the receptor subtype and the cell type accounting for high expression and to clarify the mechanisms responsible for the sharp decrease in hepatic receptivity occurring during the late stage of foetal development. 2. Binding experiments indicated that the density of alpha2-adrenoceptors in the foetal liver (embryonic day 18; 615+/-155 fmol mg(-1) of protein) is 18 fold higher than in newborn or adult (35.2+/-4.3 fmol mg(-1)). A high amount of receptor is also found in the placenta (443+/-53 fmol mg(-1)). In both tissues, the rank order of antagonists to inhibit radioligand binding matched the pharmacological profile of the alpha2B-adrenoceptor and exclusively RNG transcripts were detected by RNase protection assays. 3. Isolation of cell fractions from foetal liver showed that alpha2B-adrenoceptor is primarily expressed by haematopoietic cells. Consistent with this view, the receptor is found to be abundant in foetal blood, carried by reticulocytes. The expression in blood gradually declines to zero at 3 weeks of age and it is not recovered following induction of reticulocytosis in adults. 4. In foetal reticulocytes, a low proportion of the receptor population is coupled to G-protein. The alpha2-agonist UK14304 has a marginal effect on cyclic AMP level but significantly increases arachidonic acid release. The function of the receptor remains to be elucidated. However, together with observations on alpha2B-knockout mice, the current finding strongly suggests a role for alpha2B-adrenoceptor during foetal haematopoiesis in rodents.
Assuntos
Linhagem da Célula , Eritrócitos/metabolismo , Fígado/embriologia , Fígado/metabolismo , Placenta/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas alfa-Adrenérgicos/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Animais Recém-Nascidos , Ácido Araquidônico/metabolismo , Ligação Competitiva , AMP Cíclico/metabolismo , Eritrócitos/citologia , Eritropoetina/farmacologia , Hematopoese/efeitos dos fármacos , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Pulmão/metabolismo , Camundongos , RNA/genética , RNA/metabolismo , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 2/genética , Contagem de Reticulócitos , Baço/metabolismo , Especificidade por SubstratoRESUMO
The heterologous regulation of the alpha2C-adrenergic receptor (alpha2C-AR) was investigated in the HepG2 cell line. Binding of [(3)H]MK912 (alpha2-antagonist) to membranes from cells submitted to various treatments showed that exposure to insulin, phorbol 12-myristate 13-acetate, or dexamethasone did not affect receptor density. On the other hand, treatment with forskolin resulted in a large reduction of alpha2C-AR number. The effect of forskolin was mimicked by 8-br-cAMP and was abolished by the protein kinase A inhibitor, H89. The action of cAMP was slow (t(1/2) = 23 h), dose-dependent, and additive to the receptor down-regulation elicited by the alpha2-agonist, UK14304. Furthermore, the diminution of receptor was not caused by an increased rate of its degradation but resulted from a decrease in the steady state amounts of alpha2C4-mRNA. As assessed by experiments in the presence of actinomycin D, the stability of alpha2C4-mRNA was not affected by 8-br-cAMP or forskolin. By contrast, the activity of a luciferase construct containing the entire promoter region of the alpha2C4 gene (1.9 kilobase pairs) was inhibited, indicating that the primary mechanism of action of the two compounds is at the transcriptional level. Deletions in the 5'-end of this construct showed that the elements responsible for cAMP responsiveness lie within a 242-base-pair fragment of the gene promoter (nucleotides -236/+6 relative to transcription start). Band-shift experiments indicated that nuclear factors bind to this region in a cAMP-dependent manner. The determination of the actual cis- and trans-acting elements involved will be the object of future investigation, but the present study provides evidence for transcriptional regulation of human alpha2C-AR by cAMP.
Assuntos
AMP Cíclico/farmacologia , Regulação para Baixo/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Colforsina/farmacologia , Humanos , Cinética , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/biossíntese , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9 - 12 of chick embryo development. After that period, vascular density remains constant. Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT - PCR), increased from the 8th day reaching a maximum (70% increase) at days 10 - 11. NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process. British Journal of Pharmacology (2000) 129, 207 - 213
Assuntos
Alantoide/enzimologia , Córion/enzimologia , Neovascularização Fisiológica/fisiologia , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Alantoide/metabolismo , Animais , Embrião de Galinha , Córion/metabolismo , Inibidores Enzimáticos/farmacologia , Lipopolissacarídeos/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Radiation-induced esophagitis often results in treatment interruption, which may severely affect the probability of control of the local disease in patients undergoing chest radiotherapy (RT). No effective regimen that would reduce the incidence and severity of this complication has been identified up to now. Although acceleration of oral mucosal healing using topical recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) has been reported, the mechanism of such an interaction remains obscure. Effective topical application of rhGM-CSF for the treatment of radiation-induced esophagitis has never been reported in the past. In pharmacological studies, we observed that glycerol exerts a remarkable stabilizing effect on rhGM-CSF immunoreactivity. After studying the kinetics of esophageal emptying with nuclear imaging, we proposed a rhGM-CSF regimen that could be applied for topical treatment of esophagitis during RT. The regimen was applied for 5 consecutive days in a cohort of 36 patients undergoing chest RT, immediately after the documentation of grade 3 esophagitis. RT was not interrupted. Mucosal biopsies were performed endoscopically and examined immunohistochemically. Regression of dysphagia to grade 0/1 was observed in 19 of 36 (52%) patients, whereas grade 2 dysphagia persisted in 12 of 36 (33%) patients. Progression of dysphagia was seen in 5 of 36 (14%) patients. Recurrence of severe esophagitis within 5-8 days after rhGM-CSF therapy was observed in 7 of 31 (22%) patients with initial response to rhGM-CSF. Four of these patients presented significant improvement of symptomatology after additional rhGM-CSF medication. In immunohistochemical studies, active intraepithelial neovascularization and thymidine phosphorylase and vascular endothelial growth factor overexpression were observed in the damaged epithelium, which was not accompanied by macrophage or neutrophil infiltration. We conclude that rhGM-CSF topical therapy (p.o. administration) exerts a significant therapeutic effect against RT-induced esophagitis. The rhGM-CSF mucosa healing effect is probably due to its direct angiogenic activity and/or to the potentiation of the activity of other angiogenic factors released by the damaged epithelium.
Assuntos
Esofagite/tratamento farmacológico , Esofagite/etiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Lesões por Radiação/tratamento farmacológico , Lesões por Radiação/etiologia , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Estabilidade de Medicamentos , Esofagite/patologia , Excipientes , Feminino , Glicerol/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacocinética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Radioterapia/efeitos adversos , Proteínas RecombinantesRESUMO
Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/metabolismo , Genes Reguladores , Regiões Promotoras Genéticas/genética , Receptores Adrenérgicos alfa 2/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Neoplasias do Colo/metabolismo , Sequência Consenso , Sequência Conservada , Humanos , Insulinoma/metabolismo , Dados de Sequência Molecular , Fatores de Transcrição NFI , Proteínas Nucleares , Neoplasias Pancreáticas/metabolismo , Ratos , Receptores Adrenérgicos alfa 2/metabolismo , TATA Box , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteína 1 de Ligação a Y-BoxRESUMO
Epinephrine stimulation of rat alpha 2D, alpha 2B, and alpha 2C adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B approximately CHO-2D much greater than CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive Gi proteins. Contrary to what has been reported for the alpha 2A subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21ras in the CHO transfectants. Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha 2 receptor subtypes. These results show that in CHO cells, the different alpha 2 adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21ras-independent way.
Assuntos
Proteína Oncogênica p21(ras)/metabolismo , Proteínas Quinases/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática , Fosforilação , Ratos , Receptores Adrenérgicos alfa 2/genéticaRESUMO
Previously, we have reported two major alpha 2-adrenergic receptor transcripts in rat brain of 3.8 and 3.0 kb and the cloning and characterization of the rat brain complementary DNA (cDNA) (RB alpha 2C) specific for the 3.0-kb messenger RNA. In this report, we used rat brain cDNAs specific for the 3.0 and 3.8 kb transcripts, which encode the alpha 2C- and alpha 2A-adrenergic receptors, respectively, and the RNG alpha 2 cDNA, which encodes for the nonglycosylated alpha 2B-adrenergic receptor in rat, to study tissue-specific expression of the three alpha 2-adrenergic receptor genes in rat. To eliminate cross-hybridization of probes with transcripts from other alpha 2 genes, we subcloned fragments that encode for the highly divergent third cytoplasmic loop of each rat alpha 2-adrenergic receptor cDNA and used RNase protection analysis to detect specific transcripts. We show that the three rat alpha 2-adrenergic receptor genes have diverse patterns of tissue expression, and although transcripts specific for each alpha 2-adrenergic receptor gene are found in brain and kidney, the levels of expression of each subtype differ in these tissues. We speculate on the significance of tissue-specific expression of the alpha 2-adrenergic receptor genes.
Assuntos
Expressão Gênica , Receptores Adrenérgicos alfa/genética , Animais , Sondas Moleculares , Hibridização de Ácido Nucleico , RNA/metabolismo , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa/metabolismo , Ribonucleases , Distribuição Tecidual , Transcrição GênicaRESUMO
We have isolated a cDNA clone (RB alpha 2B) and its homologous gene (GR alpha 2B) encoding an alpha 2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha 2-C4) and divergent from the rat kidney nonglycosylated alpha 2B subtype (RNG alpha 2). Transient expression of RB alpha 2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine greater than yohimbine greater than prazosin greater than oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha 2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR alpha 2B may be transcriptionally active. These findings show that rat brain expresses an alpha 2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha 2B subtype. Thus the rat expresses at least two divergent alpha 2B-adrenergic receptors.
Assuntos
Encéfalo/metabolismo , Receptores Adrenérgicos alfa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Expressão Gênica , Biblioteca Genômica , Humanos , Cinética , Dados de Sequência Molecular , Poli A/genética , RNA/genética , Sondas RNA , RNA Mensageiro , Ratos , Receptores Adrenérgicos alfa/metabolismo , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transfecção , Ioimbina/metabolismoRESUMO
We used a human platelet alpha 2-adrenergic receptor probe to study the tissue distribution and messenger RNA (mRNA) forms of the rat alpha 2-adrenergic receptor. Under stringent conditions of hybridization and washing, we detected an mRNA species of 3.8 kb. The abundance of this form follows the order spleen, kidney, brain stem and cortex, and skeletal muscle and lung and is consistent with the reported abundance and tissue distribution of the alpha 2 receptor activity. A 3.0 kb mRNA form was also detected in cerebral cortex and brain stem and a 4.1 kb mRNA form was observed in kidney under less stringent hybridization conditions. The tissue distribution of the 3.0 kb form is different from that of alpha 1- and beta-adrenergic receptors and the D2 dopaminergic receptor. The mRNA analysis combined with Southern blot analysis of rat and human genomic DNA indicate that: 1) in addition to a 3.8 kb rat alpha 2-adrenergic receptor transcript, there are other mRNA forms in the rat that do not correspond to previously described adrenergic receptor mRNA species and 2) more than one alpha 2-adrenergic receptor gene in the rat is expressed in a tissue-specific manner.
Assuntos
RNA Mensageiro/metabolismo , Receptores Adrenérgicos alfa/genética , Animais , Masculino , Ratos , Ratos Endogâmicos , Transcrição GênicaRESUMO
We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure: function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.
Assuntos
Plaquetas/metabolismo , Expressão Gênica , Receptores Adrenérgicos alfa/genética , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Ligação Competitiva , Plaquetas/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , RNA Mensageiro/análise , Receptores Adrenérgicos alfa/metabolismo , TransfecçãoAssuntos
Genes Virais , Oncogenes , Proto-Oncogenes , Sequência de Aminoácidos , Animais , Vírus da Leucose Aviária/genética , Sequência de Bases , Galinhas/genética , Clonagem Molecular , DNA Viral , Humanos , Camundongos , Dados de Sequência Molecular , Proteína Oncogênica p55(v-myc) , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc , RNA Mensageiro/análise , Mapeamento por Restrição , Proteínas dos Retroviridae/genética , Homologia de Sequência do Ácido Nucleico , Truta/genéticaRESUMO
The avian carcinoma virus MH2 contains a hybrid gene delta gag-mht with a contiguous open reading frame of 2682 base pairs as well as v-myc and avian helper virus-related sequences. delta gag is a partial retroviral core protein gene while v-mht and v-myc are cell-drived sequences. The v-mht sequence can be divided into two regions: the v-raf-related region at its 3' end contains 969 nucleotides which are 94% related as amino acid sequence to the onc-specific v-raf sequence of murine sarcoma virus 3611 (MSV 3611), and the v-mht-specific region at its 5' end contains 173 nucleotides which are unrelated to either MSV 3611 or avian helper virus sequences. To study the origin of the v-mht-specific sequences, the 5' region of the proto-mht/raf gene was molecularly cloned from a phage lambda library containing genomic chicken sequences. Nucleic acid hybridization, heteroduplex and DNA sequence analyses indicate that the v-mht-specific sequences are encoded in three exons. The first and second exons are separated by a 3.4-kb intron while the second and third exons are separated by a 90-bp intron. The last 14 bp of the third exon are shared with v-raf and thus represent the start of v-raf-related sequences. The junction between v-mht-unrelated and related cellular sequences occurs within the first exon. There is no homology between the v-mht-unrelated sequences and the retroviral helper sequences indicating that the viral transduction of the proto-mht/raf sequences occurred through illegitimate recombination. The predominant v-mht-related messenger RNA (4.0 kb) hybridizes to several noncontiguous regions on the molecularly cloned cellular proto-mht/raf DNA indicating that the proto-mht/raf gene is distributed over at least 10 kb of DNA in the chicken genome. Thus the v-mht oncogene is a subset of its normal cellular homolog in that it lacks intervening sequences and possibly lacks 5'-coding sequences.