RESUMO
Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.
Assuntos
Hormônios Peptídicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cálcio/metabolismo , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Grelina , Humanos , Ligantes , Dados de Sequência Molecular , Hormônios Peptídicos/genética , Hormônios Peptídicos/farmacologia , Hipófise/citologia , Hipófise/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , TransfecçãoRESUMO
AIM: This study was designed to characterize the endothelin pathway in an immortalized human adenocarcinoma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC). METHODS: The release of ET-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin isoforms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-1a, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody. RESULTS: Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in both cell lines phosphoramidon produced a concentration-dependent inhibition of ET-1 release and an enhanced accumulation of big-ET-1. Both HUVEC and A549 cells express the mRNAs for ppET-1, ET-A, and ET-B receptor subtypes and ECE-1 (isoforms ECE-1b, c and/or d). In addition, in HUVEC the mRNAs for ppET-2 and for the isoform ECE-1a were also detected. In A549 cells, ECE-1 had a preferential subcellular localization in the membrane fraction but was not detected in the cytosol. CONCLUSION: Both A549 and HUVEC produce and release endothelin-1 through a specific enzymatic pathway, whether or not ECE-1 is the only enzyme involved remains to be determined. A549 might be used as a screening assay for drug discovery such as for inhibitors of endothelin-1 release.
Assuntos
Ácido Aspártico Endopeptidases/biossíntese , Endotelina-1/metabolismo , Endotelinas/biossíntese , Endotélio Vascular/metabolismo , Precursores de Proteínas/biossíntese , Veias Umbilicais/metabolismo , Adenocarcinoma/patologia , Ácido Aspártico Endopeptidases/genética , Células Cultivadas , Enzimas Conversoras de Endotelina , Endotelinas/genética , Endotélio Vascular/citologia , Glicopeptídeos/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Metaloendopeptidases , Precursores de Proteínas/genética , RNA Mensageiro/biossíntese , Células Tumorais Cultivadas , Veias Umbilicais/citologiaRESUMO
The effects of cAMP-elevating agents on antigen-induced IL-5 (interleukin-5) messenger RNA expression and protein production were examined in vitro in an antigen-driven system of splenocytes from ovalbumin sensitized BALB/c mice. IL-5 production was inhibited by rolipram, a type 4 phosphodiesterase (PDE4) inhibitor, dose-dependently (maximally at 10(-5) M) and by dibutyryl-cAMP (db-cAMP) (3 x 10(-4) M), but not by the type 3 and type 5 PDE inhibitors milrinone and zaprinast (10(-5) M), respectively. Forskolin (10(-5) M), an adenylate cyclase activator, was noninhibitory alone but potentiated inhibition by rolipram. Inhibition was associated with a decrease in IL-5 mRNA expression. Cycloheximide 10(-6) M and actinomycin 2 micrograms/ml abolished IL-5 production and mRNA expression. We conclude that in splenocytes from sensitized mice, IL-5 production and mRNA expression depend on antigen stimulation. The time course of IL-5 protein production is closely related to IL-5 mRNA expression and depends on de novo protein synthesis. db-cAMP and a selective PDE4 inhibitor, alone or in combination with forskolin, are the only cAMP-elevating agents that dose-dependently inhibited antigen-induced IL-5 mRNA expression and protein production. These results are in agreement with in vivo inhibition by a selective PDE4 inhibitor of antigen-induced pulmonary eosinophil infiltration and IL-5 production in sensitized mice, and they suggest that PDE4 inhibitors have potential for treating respiratory allergy.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases , Antígenos/imunologia , AMP Cíclico/fisiologia , Interleucina-5/genética , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/fisiologia , Animais , Asma/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ovalbumina/imunologia , Inibidores da Síntese de Proteínas/farmacologia , Pirrolidinonas/farmacologia , RNA Mensageiro/genética , Rolipram , Baço/citologiaRESUMO
A cDNA clone for dopa decarboxylase (EC 4.1.1.28) has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5' end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Drosophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5' untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3' untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5' untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.
Assuntos
Dopa Descarboxilase/genética , Neurônios/enzimologia , Splicing de RNA , RNA Mensageiro/genética , Neoplasias das Glândulas Suprarrenais/enzimologia , Neoplasias das Glândulas Suprarrenais/genética , Glândulas Suprarrenais/enzimologia , Animais , Sequência de Bases , Encéfalo/enzimologia , Biblioteca Gênica , Humanos , Rim/enzimologia , Fígado/enzimologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Especificidade de Órgãos , Feocromocitoma/enzimologia , Feocromocitoma/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos EndogâmicosRESUMO
The L-DOPA decarboxylase mRNA levels were determined by a sensitive S1 nuclease method in four organs and one tumor of adult rat. S1 mapping analysis, with probes corresponding to the mRNA coding region, showed that this region is conserved in all L-DOPA decarboxylase mRNA of neuronal and non-neuronal tissues. The mRNA was not very abundant; its representation varies approximately from 0.00035% of total RNA in the mid brain to 0.013% of total mRNA in the pheochromocytoma. A strong correlation between mRNA level and enzyme amount was observed (correlation coefficient = 0.99). The results indicate that the level of mRNA is a primary factor determining the L-DOPA decarboxylase level.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Dopa Descarboxilase/metabolismo , RNA Mensageiro/análise , Neoplasias das Glândulas Suprarrenais/enzimologia , Glândulas Suprarrenais/enzimologia , Animais , Encéfalo/enzimologia , Dopa Descarboxilase/genética , Rim/enzimologia , Fígado/enzimologia , Feocromocitoma/enzimologia , RNA Neoplásico/análise , Ratos , Distribuição Tecidual , Células Tumorais Cultivadas/enzimologiaRESUMO
Total poly (A+) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A+ mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , Dopa Descarboxilase/genética , Feocromocitoma/enzimologia , RNA Mensageiro/análise , Animais , Dopa Descarboxilase/biossíntese , Técnicas In Vitro , Rim/enzimologia , Poli A/análise , Testes de Precipitina , Ratos , SuínosRESUMO
L-DOPA decarboxylase was purified from rat pheochromocytoma. Tryptic digestion of this enzyme permitted obtaining fourteen peptides. The comparison of the sequence of L-DOPA decarboxylase from other species with one of these peptides demonstrates a great preservation of this protein.