RESUMO
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 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 effects of the estrogen analog mestranol and of the antiestrogen tamoxifen on cell growth and the rate of angiotensinogen production were investigated in HepG2 cells, an hepato-carcinoma cell line of human origin. After 36 h of cell contact with high concentration of mestranol, a (10(-5) M) dose increased by 2-fold the rate of proliferation of HepG2 while reducing angiotensinogen production to below control level. Mestranol at 10(-6) M preferentially stimulated angiotensinogen production 5-fold, whereas cell growth rate was slightly increased. Comparable results were obtained for thymidine uptake in the course of the cell cycle, with a maximum increase for 10(-5) M mestranol, and an increase of angiotensinogen production for 10(-6) M mestranol. At 10(-6) M, tamoxifen acted as a pure antagonist by strongly inhibiting the stimulatory effect of mestranol and reducing angiotensinogen production to below the control level within 60 h. Tamoxifen did not affect the growth rate of HepG2 cells, either when administered alone or together with an equimolar concentration of mestranol.
Assuntos
Angiotensinogênio/biossíntese , Ciclo Celular/efeitos dos fármacos , Fígado/metabolismo , Mestranol/farmacologia , Tamoxifeno/farmacologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Imunofluorescência , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Neoplasias HepáticasRESUMO
Antibodies were raised in rabbit against pure human renin. The antisera obtained are highly specific for human renin versus hog, dog and rat renin. They do not cross-react with acid proteases such as pepsin and human cathepsin D. A direct radioimunoassay is described for human renin in plasma and kidney extracts. 30 to 50 pg of enzyme (2.5 to 4 x 10(-5) Goldblatt units) are detected.
Assuntos
Rim/enzimologia , Renina/análise , Humanos , Radioimunoensaio/métodos , Renina/sangueRESUMO
Arginine-vasopressin (AVP) plays a determinant role in the normal ACTH response to stress in mammals. We cloned a human cDNA coding a 424 amino acid G-protein coupled receptor structurally related to the vasopressin/oxytocin receptor family. When expressed in COS cells, this receptor binds AVP with a high affinity (Kd = 0.55 +/- 0.13 nM) and is functionally coupled to phospholipase C. Competition studies with peptidic or non peptidic AVP analogues reveal that it is pharmacologically distinct from V1a and V2 AVP receptors and therefore it is designated V3. RT-PCR analysis shows that the human V3 receptor is expressed in normal pituitary and also in kidney, but is undetectable in liver, myometrium and adrenal gland. Northern blot analysis reveals a approximately 4.8 kb messenger in human corticotropic pituitary adenomas.
Assuntos
Hipófise/metabolismo , Receptores de Vasopressinas/biossíntese , Adenoma/metabolismo , Sequência de Aminoácidos , Animais , Arginina Vasopressina/farmacologia , Sequência de Bases , Northern Blotting , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Primers do DNA , DNA Complementar , Peixes , Biblioteca Gênica , Humanos , Fosfatos de Inositol/metabolismo , Rim , Dados de Sequência Molecular , Neoplasias Hipofisárias/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores de Ocitocina/química , Receptores de Vasopressinas/química , Receptores de Vasopressinas/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The insulin receptor (IR) is a membrane-bound glycoprotein composed of alpha and beta subunits derived from a common precursor. This processing is observed for both subtypes A and B of the IR and its physiological importance is poorly understood. In order to investigate the functional consequences of the absence of IR precursor cleavage, using site-directed mutagenesis of the hIRB cDNA, we have produced two mutants replacing the sequence Arg-Lys-Arg-Arg by either His-Lys-His-Arg or Arg-Lys-Arg-Ser. These two mutants, stably expressed in CHO, were structurally and functionally characterized in comparison to the wild-type human IR. These mutations result in the production of uncleaved receptors which are expressed normally at the cell surface. These receptors bind insulin with a normal affinity and activate the tyrosine-kinase resulting in normal phosphorylation of the receptors. These uncleaved receptors can mediate both the metabolic and mitogenic effects of insulin. These results provide evidence for a fully functional uncleaved insulin receptor of the B subtype (exon 11 + ) in contrast to the uncleaved A subtype (exon 11 -) described in the literature, which shows a reduced affinity for insulin and cannot therefore correctly transduce the insulin signal.
Assuntos
Receptor de Insulina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetinae , DNA/biossíntese , Éxons , Humanos , Insulina/metabolismo , Insulina/farmacologia , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptor de Insulina/biossíntese , Receptor de Insulina/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0 degree C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.
Assuntos
Córion/enzimologia , Rim/enzimologia , Renina/análise , Células Cultivadas , Eletroforese , Humanos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Peso Molecular , RadioimunoensaioRESUMO
The isolated perfused rat kidney allows a simultaneous kinetic study of both the renal metabolism and the urinary excretion of cortisol and its metabolites in the rat. In this system, cortisol was completely metabolized within 120 minutes. The main renal metabolites of cortisol (cortisone, 20 reduced cortisol and 20 reduced cortisone) were found in the recirculating perfusate and in urine. The formation of these metabolites was quantitatively evaluated and compared to a theoretical model.
Assuntos
Hidrocortisona/metabolismo , Rim/metabolismo , Animais , Cromatografia Gasosa , Técnicas In Vitro , Cinética , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Perfusão , Ratos , Fatores de Tempo , TrítioRESUMO
In the rat model of hypertension induced by a clip on the right renal artery, sparing the left kidney, we compared the efficacity and the endocrine, renal and cardiac effects of classical therapy (CT) of hypertension (Clonidine 0.2 mg/kg and Dihydralazine 15 mg/kg in 2 daily subcutaneous injections and Furosemide 30 mg/kg/day in the drinking water), with inhibition of the angiotensin converting enzyme with a new drug, the S-9490-3 (0.5 mg/kg in one daily administration). The untreated animals (HT: n = 12) had an average systolic blood pressure (SBP) of 215 +/- 32 mmHg. After 1 month' treatment, S-9490-3 (n = 13) lowers SBP to 144 +/- 32 mmHg compared to CT (n = 12) which lowered SBP to only 172 +/- 18 mmHg. The average plasma renin concentrations of the HT animals was four times the normal value (39 +/- 33 ng/ml/h) and both treatment regimes increased it further (S-9490-3: 129 +/- 65 ng/ml/h; CT: 97 +/- 73 ng/ml/h). Angiotensin levels fell in proportion to the increase in renin concentration. Plasma aldosterone was normalised by S-9490-3 (460 +/- 320 pg/ml) but remained as high after CT (850 +/- 650 pg/ml) as in the untreated HT animals (830 +/- 260 pg/ml). Despite the Furosemide, plasma volume increased significantly in the CT group.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Anti-Hipertensivos/uso terapêutico , Clonidina/uso terapêutico , Di-Hidralazina/uso terapêutico , Furosemida/uso terapêutico , Hidralazina/análogos & derivados , Hipertensão Renovascular/tratamento farmacológico , Aldosterona/sangue , Animais , Volume Sanguíneo/efeitos dos fármacos , Quimioterapia Combinada , Ventrículos do Coração/efeitos dos fármacos , Rim/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Perindopril , Ratos , Ratos Endogâmicos , Sistema Renina-Angiotensina/efeitos dos fármacosRESUMO
The cloning of the insulin receptor not only established the primary structure of this membrane-bound glycoprotein but also offered the material necessary for the study of the molecular functions of this tyrosine-kinase receptor using molecular biology tools. This short review will summarize the actual knowledge of the structure, the functions (molecular structure of the binding site, tyrosine-kinase functions and role of autophosphorylation) and of the pathology of this receptor in extreme insulin resistances and type II diabetes.
Assuntos
Receptor de Insulina , Diabetes Mellitus Tipo 1/genética , Humanos , Resistência à Insulina/genética , Receptor de Insulina/química , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
BACKGROUND: Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected. OBJECTIVE: Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit. RESULTS: MLPA allowed identification of exons 3-6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit. CONCLUSIONS: MLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.
Assuntos
Complexo de Carney/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Deleção de Genes , Rearranjo Gênico , Adolescente , Doenças do Córtex Suprarrenal/genética , Doenças do Córtex Suprarrenal/metabolismo , Adulto , Complexo de Carney/metabolismo , Domínio Catalítico , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Éxons , Saúde da Família , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estabilidade Proteica , Adulto JovemAssuntos
Aldosterona/sangue , Renina/sangue , Angiotensina II/sangue , Humanos , Hiperaldosteronismo/sangue , Métodos , Nefrectomia , Postura , Potássio/sangue , RadioimunoensaioRESUMO
Receptor tyrosine kinases play essential roles in cell proliferation and differentiation. We have recently shown that peptides corresponding to the transmembrane domains of the epidermal growth factor (EGF) and ErbB2 receptors inhibit their corresponding receptor activation in cancer cell lines. We extend this observation to cells transfected with chimeric insulin receptors where the transmembrane domain has been replaced by that of the EGF receptor or a mutated Erb2 domain. Peptides corresponding to the transmembrane domains of the EGF receptor and ErbB2 are able to inhibit specifically the autophosphorylation of insulin receptors with the corresponding domain. This inhibitory effect is correlated with the propensity of the different transmembrane domains to self-associate in a genetic reporter assay. Thus, our data strengthen the notion that transmembrane domains are involved in erbB receptor activation, and that these receptors can be modulated by inhibiting protein-protein interactions within the membrane.
Assuntos
Receptores ErbB/antagonistas & inibidores , Peptídeos/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Dimerização , Receptores ErbB/genética , Receptores ErbB/metabolismo , Escherichia coli/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The recent application of recombinant DNA technology to clinical investigation now allows the identification of the molecular alterations responsible for insulin resistance. In this review, the recent knowledge concerning these investigations is reported. Genetic mutations of the insulin gene as the source of insulin resistance have been reported for a long time. More recently a series of mutations of the insulin receptor gene have been identified as the cause of the extreme insulin resistance, observed in rare syndromes, such as type A insulin resistance or leprechaunism. However, it is probable that the majority of the molecular defects causing insulin resistance occur at the postreceptor level. The key proteins involved in the different intracellular signalling pathways of insulin are only partly identified. A better understanding of the mechanisms of insulin action is essential for the identification of corresponding genetic alterations. The investigations concerning the glucose transporter GLUT4 and glucokinase genes are good examples of complex but promising research, which has recently started. Elucidation of the genetic and molecular basis of diseases such as type II diabetes or other states associated with insulin resistance, is the long-term goal.
Assuntos
Resistência à Insulina/genética , Insulina/genética , Proteínas Musculares , Transportador de Glucose Tipo 4 , Humanos , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Receptor de Insulina/genéticaRESUMO
1. Human plasma, amniotic fluid and acidified amniotic fluid were incubated at pH 5.5 with the same concentrations of human plasma renin substrate and rat plasma renin substrate. They produced three to eight times more angiotensin I with human than with rat renin substrate. By contrast, human brain extracts generated 20 times more angiotensin I when incubated with rat plasma renin substrate than with human plasma renin substrate. 2. Serial dilutions of anti-(human renin) antibody inhibited, in a dose-dependent manner, the production of angiotension I when plasma, amniotic fluid and brain extracts were incubated with human plasma renin substrate. They also inhibited the production of angiotensin I when plasma and amniotic fluid were incubated with rat plasma renin substrate. They were ineffective on the angiotensin I generation by human brain extracts acting on rat plasma renin substrate. 3. Affinity chromatography on an haemoglobin-Sepharose gel separated the fraction of brain extract acting on human renin substrate and inhibited by anti-(human renin) antiserum; this was not retained on the gel at pH 3.3. Part of the angiotensin I-forming activity detected by rat renin substrate hydrolysis was not retained on the gel and part was eluted at pH 8.5. These angiotensin I-forming activities did not hydrolyse human renin substrate, and were not neutralized by anti-(human renin) antibody. 4. These results demonstrate that a renin, immunochemically identical with renal, plasma amd amniotic fluid renin, is present in the human brain. Other angiotensin I-forming activity, acting on an heterologous substrate at a more acidic pH, is also present in human brain.
Assuntos
Angiotensina I/biossíntese , Angiotensinas/biossíntese , Encéfalo/enzimologia , Renina/metabolismo , Animais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Cromatografia de Afinidade , Humanos , Concentração de Íons de Hidrogênio , Masculino , Ratos , Renina/imunologiaRESUMO
In order to investigate the cardiac effects of antihypertensive therapies in one-clip two-kidney hypertension in rats, we compared the consequences on myosin isoenzyme profile and on left ventricular hypertrophy of two treatments: one was a new converting enzyme inhibitor (S9490), the second a more standard tripletherapy associating clonidine, dihydralazine and furosemide. The two treatments were initiated 4 weeks after clipping and administered during 5 weeks. During the treatment period average systolic blood pressure was 215 +/- 32 mmHg in the hypertensive untreated group (HC2, n = 12) and 144 +/- 13 mm Hg in the CEI group (HT1, n = 13), which is not significantly different from the value found in the sham-operated group (139 +/- 4 mm Hg, C2, n = 13). Blood pressure was lowered only to 173 +/- 18 mm Hg in the group treated with tripletherapy (HT2, n = 12). The left ventricular weight decreased significantly in the CEI-treated group toward values similar to those of the sham-operated animals (2.2 +/- 0.13 mg/g vs. 1.9 +/- 10 mg/g, respectively NS), whereas it did not change in the tripletherapy group when compared to the untreated hypertensive animals despite the fall in blood pressure. In the hypertensive untreated rats the percentage of V1 isoenzyme of cardiac myosin was lower than in the sham-operated group (42.8 +/- 9.0% vs. 57.5 +/- 7.6% P less than .001). In parallel the V3 form of cardiac myosin increased (24.1 +/- 7.4% vs. 15.7 +/- 4.3%, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anti-Hipertensivos/farmacologia , Hipertensão Renovascular/metabolismo , Isoenzimas/análise , Miocárdio/enzimologia , Miosinas/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal , Cardiomegalia/fisiopatologia , Hipertensão Renovascular/tratamento farmacológico , Masculino , Contração Miocárdica/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Renina/sangue , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
Insulin receptor (IR) is a glycoprotein possessing N-linked oligosaccharide side chains on both alpha and beta subunits. The present study focuses for the first time on the potential contribution of N-linked oligosaccharides of the beta subunit in the processing, structure, and function of the insulin receptor. To investigate this point, a receptor mutant (IR beta N1234) was obtained by stable transfection into Chinese hamster ovary cells of an IR cDNA modified by site-directed mutagenesis on the four potential N-glycosylation sites (Asn-X-Ser/Thr) of the beta subunit. The mutated receptor presents an alpha subunit of 135 kDa, indistinguishable from the wild type alpha subunit, but the beta subunit has a reduced molecular mass (80 kDa instead of 95 kDa) most likely due to the absence of N-glycosylation. Metabolic labeling experiments indicate a normal processing and maturation of this mutated receptor which is normally expressed at the surface of the cells as demonstrated by indirect immunofluorescence. The affinity of the mutant for insulin (Kd = 0.12 nM) is similar to that of the wild type receptor (Kd = 0.12 nM). However, a major defect of the mutated IR tyrosine kinase was assessed both in vitro and in vivo by (i) the absence of insulin-stimulated phosphorylation of the poly(Glu-Tyr) substrate in vitro; (ii) the reduction of the insulin maximal stimulation of the mutated IR autophosphorylation in vitro (2-fold stimulation for the mutant receptor as compared to a 7-fold stimulation for the wild type); and (iii) a more complex alteration of the mutated receptor tyrosine autophosphorylation in vivo (3-fold increase of the basal phosphorylation and a 4-fold simulation of this phosphorylation as compared to the wild type receptor, the phosphorylation of which is stimulated 14-fold by insulin). The physiological consequences of this defect were tested on three classical insulin cellular actions; in Chinese hamster ovary IR beta N1234, glucose transport, glycogen synthesis, and DNA synthesis were all unable to be stimulated by insulin indicating the absence of insulin transduction through this mutated receptor. These data provide the first direct evidence for a critical role of oligosaccharide side chains of the beta subunit in the molecular events responsible for the IR enzymatic activation and signal transduction.
Assuntos
Membrana Celular/fisiologia , Oligossacarídeos/metabolismo , Receptor de Insulina/genética , Transdução de Sinais , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Células CHO , Cricetinae , Cisteína/metabolismo , Imunofluorescência , Glicosilação , Humanos , Insulina/análogos & derivados , Insulina/metabolismo , Radioisótopos do Iodo/metabolismo , Cinética , Substâncias Macromoleculares , Metionina/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Oligossacarídeos/genética , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/biossíntese , Receptor de Insulina/metabolismo , Mapeamento por Restrição , Radioisótopos de Enxofre , TransfecçãoRESUMO
We have prepared and purified a rabbit polyclonal antibody (PcAb) and two mouse monoclonal antibodies (McAbs) against human angiotensinogen. The PcAb (Kd: 4.0 X 10(-12) M) inhibits 50% of the hydrolytic activity of renin on angiotensinogen, at a final dilution of 1:800. The two monoclonal antibodies (Kd: 5.0 X 10(-11) and 9.0 X 10(-13) M) do not inhibit the enzymatic reaction. None of the antibodies showed displacement of 125l-labeled angiotensinogen by angiotensin I, angiotensin II or human tetradecapeptide. The polyclonal antibodies recognize marmoset and baboon angiotensinogen with an affinity 10(3)lower than that of the human angiotensinogen, whereas the McAbs do not recognize primate angiotensinogen. Since the two monoclonal antibodies recognize different epitopes of the human angiotensinogen molecule than the polyclonal antibody, it is therefore possible to use them in various sandwich assays as ELISA. Thus, we have developed a liquid phase radioimmunoassay and an ELISA which allowed to measure human plasma angiotensinogen, under several pathophysiological conditions, and that produced by human hepatocyte cells in culture (HepG2).
Assuntos
Angiotensinogênio/imunologia , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Fígado/análise , Radioimunoensaio , Animais , Carcinoma Hepatocelular , Humanos , Neoplasias Hepáticas , Camundongos , Coelhos , Renina/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
Pituitary corticotropic cells express a specific vasopressin receptor, called V1b or V3, through which vasopressin stimulates corticotropin secretion. We recently cloned a cDNA coding for this receptor and showed that it belongs to the G protein-coupled receptor family. V3 mRNA is readily detected by RT-PCR in normal human pituitaries and corticotropic pituitary adenomas but not in PRL or GH-secreting adenomas, thus demonstrating that, like POMC itself and the CRH receptor, V3 is a marker of the corticotropic phenotype. Nuclease protection experiments suggest that V3 is overexpressed in some corticotropic adenomas, and thus may play a role in tumor development by activating the phospholipase C-signalling pathway. In addition analysis of its expression in nonpituitary neuroendocrine tumors showed a striking association with carcinoids of the lung responsible for the ectopic ACTH syndrome.
Assuntos
Hormônio Adrenocorticotrópico/biossíntese , Fenótipo , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Receptores de Vasopressinas/genética , Expressão Gênica , Humanos , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
Vasopressin (AVP), the antidiuretic hormone, is a cyclic nonapeptide that acts through binding to G protein-coupled specific membrane receptors pharmacologically divided into three subtypes (V1a, V1b, and V2) linked to distinct second messengers. Within the family of human AVP receptors, the V2 AVP receptor has been cloned, but the structure of the human V1a and V1b AVP receptors remains unknown. We report here the structure and functional expression of a human V1a AVP receptor complementary DNA isolated from human liver cDNA libraries. Cloning and sequencing of a full-length clone isolated a 1472-nucleotide sequence encoding a 418-amino acid polypeptide with seven putative transmembrane domains typical of G protein-coupled receptors. Amino acid sequence identity with the rat liver V1a AVP receptor, the human and rat V2 AVP receptors, and the human oxytocin receptor was 72, 36, 37, and 45%, respectively. Functional characterization of the cloned receptor was done by transient expression in COS-7 cells and stable expression in Chinese hamster ovary cells. Localization of the expressed receptor at the cellular surface was illustrated by using the fluorescent linear analog phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 coupled to fluorescein-avidin by dodecabiotin. Competition binding experiments with phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-[125I]Tyr-NH2 and AVP analogs revealed high affinity specific binding sites of the V1a subtype. Saturation binding experiments with [3H]AVP confirmed the presence of a single class of high affinity binding sites. Measurement of AVP-induced inositol phosphate production and calcium mobilization confirmed that the expressed V1a AVP receptor is coupled to phospholipase C via a pertussis toxin-insensitive pathway. Thus, the human V1a AVP receptor belongs to the superfamily of seven-transmembrane segment receptors with a significant sequence identity with the other members of the AVP-oxytocin family of receptors.