RESUMO
Acrodysostosis refers to a group of rare skeletal dysplasias that share in common characteristic clinical and radiological features including brachydactyly, facial dysostosis, and nasal hypoplasia. In the past, the term acrodysostosis has been used to describe patients with heterogeneous phenotypes, including, in some cases, patients that today would be given alternative diagnoses. The recent finding that mutations impairing the cAMP binding to PRKAR1A are associated with "typical" acrodysostosis and hormonal resistance initiates the era where this group of disorders can be categorized on a genetic basis. In this review, we will first discuss the clinical, radiologic, and metabolic features of acrodysostosis, emphasizing evidence that several forms of the disease are likely to exist. Second, we will describe recent results explaining the pathogenesis of acrodysostosis with hormonal resistance (ADOHR). Finally, we will discuss the similarities and differences observed comparing patients with ADOHR and other diseases resulting from defects in the PTHR1 signaling pathway, in particular, pseudohypoparathyroidism type 1a and pseudopseudohypoparathyroidism.
Assuntos
Disostoses/genética , Disostoses/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Animais , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Disostoses/diagnóstico por imagem , Disostoses/etiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/etiologia , Mutação/genética , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/etiologia , Fenótipo , Radiografia , Transdução de Sinais/genéticaRESUMO
The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.
Assuntos
Alanina/genética , Antitrombina III/genética , Mutação , Adulto , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA/análise , DNA Polimerase I/metabolismo , Eletroforese em Gel de Ágar , Feminino , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome.
Assuntos
Síndrome de ACTH Ectópico/metabolismo , Pró-Opiomelanocortina/genética , Receptores de Vasopressinas/genética , Adenoma/metabolismo , Sequência de Bases , Neoplasias Brônquicas/metabolismo , Tumor Carcinoide/metabolismo , Humanos , Dados de Sequência Molecular , Fenótipo , Neoplasias Hipofisárias/metabolismo , RNA Mensageiro/análise , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
The V2 vasopressin and the AT1A angiotensin II receptors are respectively coupled to the adenylyl cyclase and the phosphoinositide pathways. The cross-talk between these two receptors and their transduction pathways were investigated in CHO cells transfected with cDNA of both AT1A and V2 receptors. In these cells, angiotensin II induced an increase in intracellular calcium, and vasopressin a rise in intracellular cAMP accumulation. The simultaneous addition of angiotensin II and vasopressin potentiated the production of cAMP by the V2 receptor. This potentiation was dose-dependent and, at a concentration of 10(-7) M angiotensin II, the accumulation of cAMP was 4-fold greater than that induced by 10(-7) M vasopressin alone. Such cross-talk occurred in the presence and absence of cyclic nucleotide phosphodiesterase inhibitors, indicating that inhibition of phosphodiesterase activity was not the principal cause of potentiation. This was confirmed by the absence of calcium-inhibitable isoforms of phosphodiesterases in CHO cells. The addition of angiotensin II to forskolin, which stimulates the adenylyl cyclase, did not modify the production of cAMP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), partially mimicked, and staurosporine, an inhibitor of PKC, partially inhibited the effect of angiotensin II on vasopressin. Chelation of intracellular calcium with BAPTA-AM markedly reduced the potentiation of V2 receptor by angiotensin II. However, increase in intracellular calcium with thapsigargin did not modify the cAMP accumulation induced by vasopressin. It was concluded that, in CHO cells, activation of the AT1A receptor by angiotensin II potentiates the V2 receptor through activation of protein kinase C in the presence of intracellular calcium at a step located between the receptor and the adenylyl cyclase.
Assuntos
Angiotensina II/metabolismo , AMP Cíclico/metabolismo , Receptores de Angiotensina/metabolismo , Receptores de Vasopressinas/metabolismo , Vasopressinas/metabolismo , Angiotensina II/farmacologia , Animais , Células CHO , Cálcio/metabolismo , Cálcio/farmacologia , Colforsina/metabolismo , Colforsina/farmacologia , Cricetinae , Líquido Intracelular/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteína Quinase C/metabolismo , Receptor Tipo 1 de Angiotensina , Receptores de Angiotensina/genética , Receptores de Vasopressinas/genética , TransfecçãoRESUMO
The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus with the enhanced green fluorescent protein (EGFP), and the corresponding chimeric cDNA was expressed in HEK-293 cells. This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies. Therefore, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological observation of both the fluorescent receptor and its cognate Galphaq/11 protein, identified by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification of the cellular distribution of these proteins before and after the binding of different agonist or antagonist ligands. These observations result in several conclusions: 1) Expression of increasing amounts of the AT(1A) receptor at the cell surface is associated with a progressive recruitment of the cytosolic Galphaq/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a Galphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Galphaq/11 protein cellular translocation is dependent on receptor internalization process, but not G protein coupling and signal transduction mechanisms, as assessed by pharmacological data using agonists and antagonists and the characterization of AT(1A) receptor mutants (D(74)N and Delta329) for which the coupling and internalization functions are modified.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Luminescentes/genética , Receptores de Angiotensina/fisiologia , Angiotensina II/farmacologia , Linhagem Celular , Membrana Celular/química , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas Heterotriméricas de Ligação ao GTP/análise , Humanos , Rim/ultraestrutura , Cinética , Microscopia Confocal , Mutagênese , Mutação Puntual , Reação em Cadeia da Polimerase , Receptor Tipo 1 de Angiotensina , Receptores de Angiotensina/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Insulin stimulates the autophosphorylation of the beta-subunit of the insulin receptor (IR) on tyrosine residues. Mutations which compromise IR autophosphorylation in vivo result in a decrease of the insulin-activated uptake of 2-deoxyglucose. These results are consistent with previous results which implicate IR autophosphorylation in the generation of the insulin response by cells. To further explore the specificity of the IR tyrosine phosphokinase (TPK) domain in IR function, we have altered the human IR (hIR) cDNA to encode truncated insulin-independent TPKs, which are expressed in chinese hamster ovary (CHO) cells as either membrane-anchored or cytosolic proteins. Both mutant hIRs exhibit TPK activity in vitro, although the cytosolic form is approximately 20 times more active. The carbohydrate moiety of the membrane-anchored form is of the high mannose type, consistent with an intracellular localization for this mutant hIR. The two mutant hIRs mediate very different physiological responses in transfected cells: the membrane-anchored, but not the cytosolic, hIR TPK mediates a constitutively elevated (135% the maximum insulin-stimulated response in CHO cells) insulin-independent uptake of 2-deoxyglucose. These results thus suggest that the hIR TPK is in fact specific for this aspect of IR function and, when membrane-associated, can mediate the insulin-independent uptake of 2-deoxyglucose. Neither of these mutant hIRs appears to transform CHO cells.
Assuntos
Citoplasma/ultraestrutura , Desoxiaçúcares/farmacocinética , Desoxiglucose/farmacocinética , Insulina/farmacocinética , Receptor de Insulina/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Membrana Celular/ultraestrutura , Cricetinae , Cricetulus , Feminino , Glicosilação , Humanos , Ovário/citologia , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/análise , Receptor de Insulina/genética , Transfecção , Transformação GenéticaRESUMO
The peptide hormone angiotensin II (AngII) plays a principal role in regulating blood pressure and fluid homeostasis. Most of its known effects are mediated by a guanine nucleotide-regulatory protein (G protein)-coupled receptor pharmacologically defined as the type-1 AngII receptor or AT1. Characterization of cDNA and genomic clones shows that the human AT1 gene contains five exons and encodes two receptor isoforms as a result of alternative splicing. Exon 5 contains the previously characterized open reading frame for AT1, and exons 1 to 3 are alternatively spliced upstream of it to generate several mRNA species, while transcripts containing exon 4 are of minor abundance. In an in vitro translation system, the presence of exon 1 was found to be extremely inhibitory to translation, probably because it can form a stable secondary structure at the RNA level. The alternatively spliced second exon also had a strong inhibitory effect on translation, presumably because it contains a minicistron commencing with an ATG in an optimal context for translation initiation. Exon 2 was similarly inhibitory to protein production in transfected cells, but exon 1 was found to enhance protein synthesis in this system. Transcripts containing exon 3 and 5, which comprise up to one-third of AT1 mRNAs in all tissues examined, encode a receptor with an amino-terminal extension of 32-35 amino acids. These transcripts were translated into a larger receptor isoform in vitro and produced a functional receptor with normal ligand binding and signaling properties in transfected cells.
Assuntos
Processamento Alternativo , Biossíntese de Proteínas , RNA Mensageiro/genética , Receptores de Angiotensina/genética , Sequência de Aminoácidos , Bacteriófago lambda/genética , Sequência de Bases , Sítios de Ligação , DNA Complementar/química , Éxons , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , RNA Mensageiro/análise , RNA Mensageiro/química , Distribuição Tecidual , Transcrição Gênica , TransfecçãoRESUMO
Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.
Assuntos
Insulina/metabolismo , Mutação , Receptor de Insulina/genética , Tirosina/genética , Animais , Linhagem Celular , Cricetinae , Cricetulus , Regulação para Baixo/genética , Feminino , Insulina/farmacologia , Ovário/citologia , Ovário/metabolismo , Ovário/ultraestrutura , Receptor de Insulina/análise , Receptor de Insulina/metabolismo , Transdução Genética , Transfecção , Tirosina/análise , Tirosina/fisiologiaRESUMO
The mas oncogene codes for a GTP binding protein-coupled receptor that determines a physiological response to angiotensin when expressed in Xenopus laevis oocytes or in the neuronal cell line NG115-401L. However, another gene, rat thoracic aorta gene, structurally related to mas, is devoid of any functional similarity with the angiotensin receptor(s). The relationships between the mas-related proteins and the angiotensin receptors were investigated by identifying and characterizing new members of the mas gene family. A new mas-related gene (mrg) was cloned in a human genomic library at low stringency using the mas cDNA as probe. Mrg codes for a seven-hydrophobic-segment receptor that is 35% identical to the mas product and 29% identical to the rat thoracic aorta gene product. Mrg mRNA was not detected in several rat and human adult tissues that normally express the angiotensin II (AII) receptor, and transfections of COS and CHO cells with the mrg gene did not modify the number of AII binding sites. These results indicate that mrg and the human AII receptor genes are not identical. However, injection of mrg mRNA into Xenopus oocytes markedly increased the electrophysiological response to angiotensin peptides, indicating some functional similarities with the mas product. The reduction of the response after defolliculation of the oocyte, together with the full agonist effect of Sar1IIe8AII and the partial agonist effect of Sar1Ala8AII, seem to indicate that mrg interacts with the signaling pathways of the endogenous Xenopus angiotensin receptor to potentiate the response to AII.
Assuntos
Angiotensina II/metabolismo , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores de Angiotensina/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Clonagem Molecular , Cricetinae , Sondas de DNA , Eletrofisiologia , Proteínas de Ligação ao GTP/genética , Biblioteca Genômica , Humanos , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Poli A/genética , Proto-Oncogene Mas , RNA/genética , RNA Mensageiro/genética , Receptores de Angiotensina/fisiologia , Receptores Acoplados a Proteínas G , Transfecção , Xenopus laevisRESUMO
BACKGROUND: MEN1, which is secondary to the mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Most studies demonstrated the absence of direct genotype-phenotype correlations. The existence of a higher risk of death in the Groupe d'étude des Tumeurs Endocrines-cohort associated with a mutation in the JunD interacting domain suggests heterogeneity across families in disease expressivity. This study aims to assess the existence of modifying genetic factors by estimating the intrafamilial correlations and heritability of the six main tumor types in MEN1. METHODS: The study included 797 patients from 265 kindred and studied seven phenotypic criteria: parathyroid and pancreatic neuroendocrine tumors (NETs) and pituitary, adrenal, bronchial, and thymic (thNET) tumors and the presence of metastasis. Intrafamilial correlations and heritability estimates were calculated from family tree data using specific validated statistical analysis software. RESULTS: Intrafamilial correlations were significant and decreased along parental degrees distance for pituitary, adrenal and thNETs. The heritability of these three tumor types was consistently strong and significant with 64% (s.e.m.=0.13; P<0.001) for pituitary tumor, 65% (s.e.m.=0.21; P<0.001) for adrenal tumors, and 97% (s.e.m.=0.41; P=0.006) for thNETs. CONCLUSION: The present study shows the existence of modifying genetic factors for thymus, adrenal, and pituitary MEN1 tumor types. The identification of at-risk subgroups of individuals within cohorts is the first step toward personalization of care. Next generation sequencing on this subset of tumors will help identify the molecular basis of MEN1 variable genetic expressivity.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Neoplasias Brônquicas/genética , Neoplasia Endócrina Múltipla Tipo 1/genética , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Neoplasias das Paratireoides/genética , Neoplasias Hipofisárias/genética , Neoplasias do Timo/genética , Adolescente , Neoplasias das Glândulas Suprarrenais/epidemiologia , Adulto , Distribuição por Idade , Neoplasias Brônquicas/epidemiologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/epidemiologia , Neoplasias Pancreáticas/epidemiologia , Neoplasias das Paratireoides/epidemiologia , Linhagem , Neoplasias Hipofisárias/epidemiologia , Neoplasias do Timo/epidemiologia , Adulto JovemRESUMO
Physiological and ligand binding studies have shown that Angiotensin II (AII) exerts various functions along different segments of the nephron, via the type-1 receptor (AT1R), resulting in the control of glomerular filtration rate (GFR) and water and salt homeostasis. We have used the recently cloned AT1R cDNA to localize, by in situ hybridization, the cells expressing AT1R mRNA in the rat kidney. On serial sections, juxtaglomerular (JG) renin secreting cells, identified by hybridization with a renin cRNA probe, also co-express AT1R mRNA. The co-expression of AT1R and renin mRNAs in the same cells documents visually the direct feedback control of AII on renin secretion. AT1R mRNA was also present in known target cells for AII: proximal convoluted tubule, mesangium and vasa recta.
Assuntos
Sistema Justaglomerular/metabolismo , RNA Mensageiro/metabolismo , Receptores de Angiotensina/genética , Renina/genética , Animais , Hibridização In Situ , Sistema Justaglomerular/citologia , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , RatosRESUMO
The influence of thyroidectomy on the renin-angiotensin system was studied in the rat. From 1-6 weeks after thyroidectomy, PRA and plasma renin substrate (PRS) decreased, but the plasma renin concentration remained unchanged, and the renal renin content increased. T3 injection corrected the changes in the plasma renin-angiotensin system of thyroidectomized rats within 20-40 h. After ethinylestradiol treatment, the PRS in thyroidectomized rats rose in the same proportion as that in normal rats, but remained below the normal level. After binephrectomy, on the other hand, the PRS was high, and PRS levels in normal and thyroidectomized animals were similar. Isoproterenol increased PRA and plasma renin concentration in control animals but had no effect on thyroidectomized rats. From the above results it may be concluded that angiotensinogen production is dependent on thyroid hormones and that renin release depends on beta-adrenergic receptor sensitivity to catecholamines, which is reduced by thyroidectomy. (Endocrinology 108: 647, 1981)
Assuntos
Angiotensinogênio/fisiologia , Angiotensinas/fisiologia , Renina/fisiologia , Tiroxina/farmacologia , Tri-Iodotironina/farmacologia , Animais , Estradiol/farmacologia , Isoproterenol/farmacologia , Masculino , Ratos , Glândula Tireoide/fisiologiaRESUMO
Hepatic storage and secretion of angiotensinogen was studied using rat liver slices and a new direct angiotensinogen RIA. This assay permitted the demonstration of a significant hepatic storage of angiotensinogen, largely underestimated until now by the enzymatic method of angiotensinogen measurement. Angiotensinogen release by rat liver slices was linear with time and was associated with a significant increase in hepatic content of angiotensinogen. The measurement of both release and changes in hepatic content permitted the measurement of de novo synthesis of angiotensinogen by rat liver slices in vitro. Both hepatic content and release of angiotensinogen were decreased by thyroidectomy and increased by ethinyl estradiol, dexamethasone, thyroid hormones, and binephrectomy.
Assuntos
Angiotensinogênio/biossíntese , Angiotensinas/biossíntese , Fígado/metabolismo , Animais , Dexametasona/farmacologia , Etinilestradiol/farmacologia , Fígado/efeitos dos fármacos , Masculino , Nefrectomia , Ratos , Ratos Endogâmicos , Tireoidectomia , Tri-Iodotironina/farmacologiaRESUMO
Angiotensinogen precursors synthesized by rabbit reticulocyte lysate primed with rat liver RNA were compared with angiotensinogen secreted by rat hepatoma cells and rat hepatocytes using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of glycosylation with tunicamycin permitted identification of the nonglycosylated form of secreted angiotensinogen. Whereas angiotensinogen secreted by hepatoma cells and hepatocytes showed electrophoretic heterogeneity (mol wt, 52-62 X 10(3], tunicamycin-treated cells secreted only a single angiotensinogen species [mol wt, 48.3 +/- 0.7 X 10(3) (mean +/- SD)], which could be cleaved by renin. Two putative angiotensinogen precursors were synthesized in the reticulocyte lysate: a major protein of 52.5 +/- 1.0 X 10(3) mol wt and a minor protein of 55.7 +/- 1.3 X 10(3) mol wt. Evidence that these proteins represent separate angiotensinogen precursors includes the following. 1) Both proteins were recognized by five different polyclonal antibodies and two monoclonal antibodies. 2) Both proteins increased in parallel in reticulocyte lysates primed with liver RNA from rats nephrectomized and given hormones that increase liver angiotensinogen production. 3) Both proteins were cleaved by renin to produce a single protein of 47.6 +/- 0.8 X 10(3) mol wt. 4) The des-angiotensin I-angiotensinogen generated by renin treatment of the lysate had an electrophoretic mobility identical to that of des-AI-angiotensinogen produced by renin treatment of nonglycosylated angiotensinogen secreted by tunicamycin-treated hepatoma cells and hepatocytes. These studies suggest that rat liver synthesizes two separate angiotensinogen precursors which may differ only in the size of their prepro sequence. The heterogeneity of secreted angiotensinogen can be fully accounted for by differences in N-glycosylation of asparagine residues of the molecule. Glycosylation of angiotensinogen is not essential for its synthesis, processing, and secretion or its hydrolysis by renin.
Assuntos
Angiotensinogênio/metabolismo , Angiotensinas/metabolismo , Fígado/metabolismo , Precursores de Proteínas/genética , Angiotensinogênio/genética , Animais , Linhagem Celular , Sistema Livre de Células , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Peso Molecular , Biossíntese de Proteínas , Coelhos , Ratos , Ratos Endogâmicos , Reticulócitos/metabolismoRESUMO
The aim of this study was to investigate the mechanisms by which angiotensinogen decreases after adrenalectomy. Plasma angiotensinogen was measured by two different methods: an indirect assay, which measures angiotensin I liberated from the plasma by an excess of renin, and a direct RIA, which measures both angiotensinogen and des-angiotensin I-angiotensinogen. In the normal rat angiotensinogen concentrations were found to be slightly, but not significantly, higher using the direct assay. After adrenalectomy a large discrepancy was observed between the indirect assay, which showed a considerable drop in plasma angiotensinogen levels, and the direct assay, which revealed a small but significant decrease. This discrepancy arose from the presence of a molecule that cross-reacts with angiotensinogen antibodies, and has a more acidic pI in isoelectric focusing than angiotensinogen: des-angiotensin I-angiotensinogen. This molecule accumulates in adrenalectomized rat plasma. The decrease in plasma angiotensinogen levels, measured by the indirect assay, could not be explained by a decrease in angiotensinogen production, as this was unchanged in the in vitro liver slice system, but was caused by an increase in angiotensinogen consumption, due to a rise in the plasma concentration of renin. Renin concentration shows a negative correlation with angiotensinogen (as measured by the indirect assay), and a positive correlation with des-angiotensin I-angiotensinogen level. Moreover, mineralocorticoids were shown to correct both renin and angiotensinogen concentrations, whereas a replacement dose of glucocorticoids (dexamethasone) had no effect on the level of renin or angiotensinogen, as measured by the indirect assay. We conclude that after adrenalectomy, plasma angiotensinogen decreases, due to an increase in renin production. A parallel accumulation of des-angiotensin I-angiotensinogen is observed.
Assuntos
Adrenalectomia , Angiotensinogênio/sangue , Angiotensinas/sangue , Angiotensina I/sangue , Angiotensinogênio/biossíntese , Animais , Desoxicorticosterona/farmacologia , Dexametasona/farmacologia , Focalização Isoelétrica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Fragmentos de Peptídeos/sangue , Radioimunoensaio , Ratos , Ratos Endogâmicos , Renina/sangueRESUMO
The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.
Assuntos
Encéfalo/metabolismo , Receptores da Tireotropina/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/citologia , Células Cultivadas , DNA Complementar/genética , Epêndima/citologia , Epêndima/metabolismo , Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Dados de Sequência Molecular , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores da Tireotropina/genética , Distribuição TecidualRESUMO
Angiotensin II stimulates aldosterone secretion from the adrenal zona glomerulosa and mediates most of its biological effects via G protein-coupled type 1 angiotensin II receptors (AT1). A number of G protein-coupled receptors are constitutively activated as a result of somatic mutations in the gene encoding the protein. It is, therefore, possible that primary hyperaldosteronism caused by an aldosterone-producing adenoma (APA) may be the result of constitutive activation of the AT1 receptor. The 1.1-kilobase coding region (exon 5) of the AT1 receptor gene was analyzed in APA and normal adrenal tissue for the presence of mutations using single stranded conformational polymorphism and sequencing techniques. In 17 APAs, no functional mutations were found that could account for the observed pathophysiology. However, three silent polymorphisms were detected in regions encoding the second extracellular loop, the intracellular arm preceding the COOH terminal, and the 3'-untranslated region. In conclusion, somatic mutations in the coding region of the AT1 receptor gene do not appear to play a role in primary hyperaldosteronism caused by an APA.
Assuntos
Adenoma/genética , Adenoma/metabolismo , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Aldosterona/biossíntese , Mutação , Receptores de Angiotensina/genética , Adenoma/complicações , Neoplasias das Glândulas Suprarrenais/complicações , Glândulas Suprarrenais/metabolismo , Adulto , Idoso , Sequência de Bases , Feminino , Humanos , Hiperaldosteronismo/etiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Valores de ReferênciaRESUMO
The role of angiotensinogen in blood pressure control was assessed in normotensive rats by observing the changes resulting from inhibition by specific rat angiotensinogen antiserum. The antiserum decreased blood pressure in rats on normal sodium as well as sodium-free diets (respectively delta BP = -30 +/- 6 mm Hg and -42 +/- 8 mm Hg). In binephrectomized sodium-replete rats, administration of antiserum did not reduce blood pressure, whereas in sodium-depleted animals it slightly decreased blood pressure by 11 +/- 3 mm Hg. These results suggest that angiotensinogen participates in the regulation of blood pressure in normotensive rats, even in the sodium-replete state.
Assuntos
Angiotensinogênio/fisiologia , Angiotensinas/fisiologia , Pressão Sanguínea , Homeostase , Animais , Rim/fisiologia , Masculino , Ratos , Ratos Endogâmicos , Sódio/sangueRESUMO
Three stable monoclonal antibodies to rat angiotensinogen were obtained by fusing myeloma cells with spleen cells from Balb/c mice injected with pure rat angiotensinogen. They were screened by their binding to pure iodinated angiotensinogen and to insolubilized angiotensinogen in a solid phase assay. The titers of the three antibodies varied from 1/3500 to 1/35000, their dissociation constants from 2.5 X 10(-8) M to 3.8 X 10(-10) M, and the sensitivity of the assay ranged from 200 to 10 pmol of pure angiotensinogen. These monoclonal antibodies did not recognize either angiotensin peptides or angiotensinogen from other species, except for mouse angiotensinogen, which cross-reacted with the different antibodies from 0 to 25%. Rat cerebrospinal fluid angiotensinogen, plasma des-angiotensin I-angiotensinogen, and plasma angiotensinogen were equally recognized by these monoclonal antibodies. Contrary to what was observed for a polyclonal antiserum, the monoclonal antibodies failed to inhibit the renin-angiotensinogen reaction in vitro.