RESUMO
We have investigated alterations in beta adrenergic receptor binding sites of rat reticulocytes occurring in animals rendered hypothyroid by thyroidectomy. Beta adrenergic receptor interactions were assessed by measuring the number of (-)[3H]-dihydroalprenolol binding sites and the ability of an agonist to compete for occupancy of the receptors. The number of receptors was significantly reduced in cells from the hypothyroid animals. In addition, there were significant agonist-specific alterations in binding. Using computer assisted curve fitting techniques, it was found that the ability of (-)isoproterenol to stabilize a high affinity guanine nucleotide sensitive "coupled" form of the receptor was impaired. Reticulocytes from hypothyroid animals have, in addition, a reduction in the concentration of the nucleotide regulatory protein as assessed by the number of 42,000 Mr substrates for cholera toxin catalyzed ADP ribosylation. These alterations are associated with reductions in catecholamine and NaF stimulated adenylate cyclase activity. Diminished coupling of beta adrenergic receptors with other regulatory components of the adenylate cyclase system represents a mechanism by which altered thyroid states modulate beta adrenergic receptor function and beta adrenergic responsiveness of tissues.
Assuntos
Adenilil Ciclases/metabolismo , Hipotireoidismo/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos/metabolismo , Reticulócitos/metabolismo , Animais , Ligação Competitiva , Di-Hidroalprenolol/metabolismo , Proteínas de Ligação ao GTP , Isoproterenol/metabolismo , Modelos Biológicos , Ligação Proteica , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/metabolismo , TireoidectomiaRESUMO
Decreased activity of the guanine nucleotide regulatory protein (N) of the adenylate cyclase system is present in cell membranes of some patients with pseudohypoparathyrodism (PHP-Ia) whereas others have normal activity of N (PHP-Ib). Low N activity in PHP-Ia results in a decrease in hormone (H)-stimulatable adenylate cyclase in various tissues, which might be due to decreased ability to form an agonist-specific high affinity complex composed of H, receptor (R), and N. To test this hypothesis, we compared beta-adrenergic agonist-specific binding properties in erythrocyte membranes from five patients with PHP-Ia (N = 45% of control), five patients with PHP-Ib (N = 97%), and five control subjects. Competition curves that were generated by increasing concentrations of the beta-agonist isoproterenol competing with [125I]pindolol were shallow (slope factors less than 1) and were computer fit to a two-state model with corresponding high and low affinity for the agonist. The agonist competition curves from the PHP-Ia patients were shifted significantly (P less than 0.02) to the right as a result of a significant (P less than 0.01) decrease in the percent of beta-adrenergic receptors in the high affinity state from 64 +/- 22% in PHP-Ib and 56 +/- 5% in controls to 10 +/- 8% in PHP-Ia. The agonist competition curves were computer fit to a "ternary complex" model for the two-step reaction: H + R + N in equilibrium HR + N in equilibrium HRN. The modeling was consistent with a 60% decrease in the functional concentration of N, and was in good agreement with the biochemically determined decrease in erythrocyte N protein activity. These in vitro findings in erythrocytes taken together with the recent observations that in vivo isoproterenol-stimulated adenylate cyclase activity is decreased in patients with PHP (Carlson, H. E., and A. S. Brickman, 1983, J. Clin. Endocrinol. Metab. 56:1323-1326) are consistent with the notion that N is a bifunctional protein interacting with both R and the adenylate cyclase. It may be that in patients with PHP-Ia a single molecular and genetic defect accounts for both decreased HRN formation and decreased adenylate cyclase activity, whereas in PHP-Ib the biochemical lesion(s) appear not to affect HRN complex formation.
Assuntos
Adenilil Ciclases/metabolismo , Pseudo-Hipoparatireoidismo/metabolismo , Receptores Adrenérgicos beta/metabolismo , Adolescente , Adulto , Sítios de Ligação , Criança , Membrana Eritrocítica/metabolismo , Feminino , Humanos , Iodo , Masculino , Pessoa de Meia-Idade , Pindolol/metabolismo , Pseudo-Hipoparatireoidismo/sangueRESUMO
Equilibrium binding of 125I-labeled recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) to the blast cells of acute myeloblastic leukemia (AML) revealed the presence of two classes of binding components of high and low affinity, with dissociation constants (Kd) in the range of 5-10 pM and 1-10 nM, respectively. Specificity studies revealed that interleukin-3 (IL-3) could partially inhibit the binding of GM-CSF to AML blasts and to the cells of the leukemic lines M07-E, KG-1, and HL-60. The inhibition of GM-CSF binding by IL-3 was directly dependent on the presence of IL-3 receptors. Analysis of competition curves indicated that the Kd and the number of binding sites per cell of unlabeled and iodinated GM-CSF were identical. In contrast, the inhibition of GM-CSF binding by IL-3 was mediated by IL-3 occupancy of a high affinity receptor only, with the same number of sites as the high affinity GM-CSF receptor but a slightly higher Kd. Despite this competitive binding, IL-3 augmented AML blast proliferation in the presence of GM-CSF, indicating that the two growth factors have converging pathways in supporting blast proliferation. In striking contrast to AML blasts, GM-CSF binding to neutrophils was compatible with the presence of only one class of binding site of intermediate affinity (Kd approximately 100-160 pM). Furthermore, IL-3 does not compete for the binding of GM-CSF to neutrophils.
Assuntos
Fatores Estimuladores de Colônias/metabolismo , Substâncias de Crescimento/metabolismo , Interleucina-3/farmacologia , Leucemia Mieloide Aguda/metabolismo , Sítios de Ligação , Ligação Competitiva , Divisão Celular , Fatores Estimuladores de Colônias/farmacologia , Sinergismo Farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Substâncias de Crescimento/farmacologia , Humanos , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/patologia , Neutrófilos/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Interleucina-3 , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
This report documents the purification and the complete primary structure of bovine aldosterone secretion inhibitory factor precursor (pro-ASIF). ASIF-(1-103) contains at position 69-103 of its carboxy-terminal end the formely identified 35-amino acid biologically active form, hence confirming the endogenous character of ASIF in the adrenal medulla. Compared to atrial natriuretic factor (ANF)-related peptide precursors, bovine ASIF displays 65% homology at the carboxy-terminal while the remaining amino-terminal part shows much more variability. Bovine pro-ASIF exhibits 73% homology with porcine pro-brain natriuretic peptide (BNP), a situation reminiscent of the relationship of pro-ANF in various species. When ANF- and BNP-related COOH-termini of bovine, porcine, human, rat, and chicken are compared, it appears that bovine ASIF and porcine BNP are closely related and belong to the same family which however appears to be much more heterogenous than the ANF-related family. These results strongly suggest that bovine ASIF is encoded by a precursor gene similar to the gene of BNP but different from the one encoding ANF.
Assuntos
Medula Suprarrenal/análise , Neuropeptídeos/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/genética , Sequência de Bases , Bovinos , Galinhas , Genes , Humanos , Dados de Sequência Molecular , Família Multigênica , Neuropeptídeos/isolamento & purificação , Precursores de Proteínas/isolamento & purificação , Ratos , Homologia de Sequência do Ácido Nucleico , SuínosRESUMO
The glucocorticoid receptor (GR) is a hormone-inducible transcription factor which activates transcription of specific genes by binding to a DNA sequence present in the promoters of inducible genes. These glucocorticoid response elements (GREs) have a conserved palindromic sequence. Each half-GRE palindrome binds one subunit of GR. We have assessed the relative affinity of GR monomers and homodimers for GRE and determined whether homodimer formation is rate-limiting for high affinity GRE binding. The in vitro affinity of GRE binding by GR homodimers was approximately 2 x 10(-10) M, whereas it was approximately 1 nM for GR monomers. While homodimer:GRE complexes were very stable, monomer:GRE complexes appeared less stable in vitro. At low receptor concentration, GR preferentially bound GRE as a homodimer. Prior dilution of GR (equilibrium shifted to monomers) before addition to a GRE binding reaction resulted in slower kinetics of binding by comparison to parallel reactions in which concentrated (largely homodimeric) GR was added first. Taken together, these experiments suggest that homodimer formation is rate-limiting for high affinity GRE binding. A GRE mutant which contained only a half-binding site and which was unable to bind GR homodimers was also unable to confer glucocorticoid-inducible transcription. Taken together with previous work, these experiments support the model that GR homodimers are required for hormone-dependent activation of transcription and that receptor homodimer formation is rate-limiting for GRE binding.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores de Glucocorticoides/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/química , Glucocorticoides/fisiologia , Cinética , Metilação , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos/genética , Receptores de Glucocorticoides/química , Sequências Reguladoras de Ácido Nucleico , Transfecção/genéticaRESUMO
Chronic estradiol treatment in vivo has been shown to reduce the density of receptors for angiotensin II (ANG II) in the anterior pituitary lobe (AP). We studied whether estradiol is directly involved in the down-regulation of ANG II receptors, using AP cells in culture. Binding affinity and density of ANG II receptors were measured in disrupted AP cells with the radiolabeled antagonist [125I]Sar1, Ile8-ANG II ([125I]SARILE). Estradiol treatment (10 nM) for either 48 or 96 h caused a marked reduction (approximately 70%) in the density of receptors for ANG II in cultured AP cells, with no change in the dissociation constant of [125I]SARILE (Kd, 0.5 +/- (SE) 0.1 nM). In the AP, specific binding sites for ANG II are present in lactotrophs and ANG II has been shown to release prolactin (PRL). In AP cells treated with estradiol for 48 h, dose-response curves revealed that ANG II still increased PRL release (P less than 0.01). The average net PRL release (ANG II-stimulated minus basal) was greater in estradiol-treated cells than in controls, whereas the half-maximal stimulation dose (ED50) of ANG II was the same (0.07 +/- 0.04 nM). These results suggest that estrogens are directly involved in the modulation of ANG II receptors in the AP, causing marked receptor down-regulation without decreasing target cell responsiveness.
Assuntos
Estradiol/farmacologia , Adeno-Hipófise/metabolismo , Receptores de Angiotensina/efeitos dos fármacos , Receptores de Superfície Celular/efeitos dos fármacos , Animais , Depressão Química , Relação Dose-Resposta a Droga , Feminino , Adeno-Hipófise/efeitos dos fármacos , Prolactina/metabolismo , RatosRESUMO
Recent studies on the regulation of aldosterone biosynthesis have revealed that inhibitors of sodium influx, e.g. amiloride, can inhibit adrenal steroidogenesis with a pharmacological profile suggestive of a Na+/H+ antiport system. We have examined the existence of a Na+/H+ antiport system and its regulation of Na influx and intracellular pH (pHi) in bovine adrenal zona glomerulosa cells. NH4Cl-induced 22Na uptake by zona glomerulosa cells was dose dependently inhibited by ethylisopropylamiloride (EIPA), amiloride, and benzamil with ED50 values of 0.02, 4.30, and 199 microM, respectively. Angiotensin II (AII; 100 nM) caused an initial transient acidification, followed by prolonged alkalinization. The hormone equipotently increased pHi and stimulated aldosterone secretion, with ED50 values of 1.2 and 1.4 nM, respectively. AII-induced alkalinization was suppressed by EIPA, amiloride, and benzamil, with ED50 values of 0.6, 79, and 440 microM, respectively. This increase in pHi induced by AII was dependent upon the extracellular sodium concentration (ED50 values = 2.8 mM) and was blunted in sodium-free medium. AII-stimulated aldosterone synthesis was also inhibited by EIPA, amiloride, and benzamil, with ED50 values of 0.07, 34, and 330 microM, respectively. The time course of activation by angiotensin II on aldosterone secretion was also dependent upon extracellular sodium concentration during a 2-h period. These results document that intracellular pH is regulated through the Na+/H+ exchange system and suggest that the pH change induced by AII might be associated with its regulation of steroidogenesis in bovine adrenal zona glomerulosa cells.
Assuntos
Aldosterona/biossíntese , Angiotensina II/metabolismo , Proteínas de Transporte/farmacologia , Zona Glomerulosa/metabolismo , Aldosterona/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Angiotensina II/farmacologia , Animais , Bovinos , Dimetadiona/análise , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Trocadores de Sódio-Hidrogênio , Fatores de Tempo , Zona Glomerulosa/análise , Zona Glomerulosa/citologiaRESUMO
The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Aldosterona/biossíntese , Neuropeptídeos/farmacologia , Zona Glomerulosa/metabolismo , Aldosterona/metabolismo , Animais , Fator Natriurético Atrial/farmacologia , Bovinos , Células Cultivadas , AMP Cíclico/biossíntese , Humanos , Inositol 1,4,5-Trifosfato/biossíntese , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Peptídeo Intestinal Vasoativo , Zona Glomerulosa/efeitos dos fármacosRESUMO
Somatostatin, at concentrations up to 10(-7) M, does not inhibit the basal release of TSH from primary cultures of rat anterior pituitary cells. The TRH-induced TSH release is however 65% reduced by somatostatin, half-maximal inhibition being measured at 2.5 x 10(-10) M somatostatin. The concentration of TRH giving half-maximal stimulation (ED50) of TSH release is only slightly increased from 1 to 3 x 10(-9) M in the presence of 10(-8) M somatostatin. Somatostatin inhibits by 45-65% both the basal and TRH-induced PRL release of pituitary cells prepared from adult female rats, with half-maximal inhibition at approximately 5 x 10(-10) M somatostatin. The TRH ED50 for PRL release was not significantly affected by somatostatin. Somatostatin (200 mug) has no effect on the basal plasma levels of TSH or PRL in anesthetized male rats treated with estradiol benzoate (EB), hypothyroid rats, or hypothyroid animals treated with EB. The plasma TSH response to TRH is, however, reduced by approximately 75% by somatostatin while the plasma PRL response is not affected by injection of the peptide. The interaction between TRH and somatostatin for both TSH and PRL release is non-competitive and is thus likely to occur at a step subsequent to the binding of the peptides to their specific receptors in both thyrotrophs and mammotrophs.
Assuntos
Adeno-Hipófise/metabolismo , Hipófise/metabolismo , Prolactina/metabolismo , Somatostatina/farmacologia , Hormônio Liberador de Tireotropina/farmacologia , Tireotropina/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estradiol/farmacologia , Feminino , Masculino , Adeno-Hipófise/citologia , Prolactina/sangue , Propiltiouracila/farmacologia , Ratos , Tireotropina/sangue , Tiroxina/farmacologiaRESUMO
Radiolabelled agonist and antagonist binding to porcine anterior pituitary dopaminergic receptor indicates the existence of two affinity forms of the receptor. These two forms of the receptor are modulated by guanine nucleotides. The agonist high affinity form of the receptor is converted to a low affinity form, whereas the antagonist low affinity form is converted to the high affinity form under the influence of guanine nucleotides. Thus, guanine nucleotides appear to reciprocally modulate the same two forms of the receptor discriminated by both agonists and antagonists. These observations may provide insights into the biochemical mechanism by which dopamine exerts its regulation of prolactin release in the anterior pituitary.
Assuntos
Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Adeno-Hipófise/metabolismo , Receptores Dopaminérgicos/metabolismo , Animais , Apomorfina/análogos & derivados , Apomorfina/metabolismo , Ligação Competitiva , Cinética , Receptores Dopaminérgicos/efeitos dos fármacos , Espiperona/metabolismo , SuínosRESUMO
Adrenal steroid hormones potentiate beta-adrenergic actions on the heart. Accordingly, we investigated the effects of adrenalectomy on agonist and antagonist interactions with myocardial beta-adrenergic receptors and adenylate cyclase. The affinity and number of beta-adrenergic receptor sites, both defined by the antagonist (-) [3H]dihydroalprenolol, did not change after adrenalectomy. Computer modelling of agonist (-)-isoproterenol competition curves indicated the presence of two discrete receptor states with high and low affinities. After adrenalectomy, the agonist curves were shifted to the right, and the dissociation constant of the high-affinity state significantly rose from 12 to 48 nM (p < .001), but the dissociation constant of the low affinity state was unchanged. Although basal, maximal isoproterenol-stimulated and fluoride-stimulated adenylate cyclase activities were unaltered, the EC50 for isoproterenol stimulation was increased significantly from 490 to 1500 nM (p <.018). These results suggest that adrenal steroid hormones may regulate the ability of the beta-adrenergic receptors to form a high-affinity "coupled" state, presumably by modulating the interaction of the receptor with nucleotide regulatory proteins.
Assuntos
Glândulas Suprarrenais/fisiologia , Alprenolol/análogos & derivados , Di-Hidroalprenolol/metabolismo , Isoproterenol/metabolismo , Miocárdio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos/metabolismo , Adenilil Ciclases/metabolismo , Adrenalectomia , Animais , Computadores , Técnicas In Vitro , Masculino , Miocárdio/enzimologia , RatosRESUMO
Primary culture of bovine adrenal subcapsular cells was used to investigate direct effects of catecholamines on aldosterone secretion. Cells dispersed with collagenase and DNAse and cultured at high density (1.5-2 million/ml) for 3 days displayed high sensitivity to angiotensin II and ACTH, with an ED50 of 1.4 and 1.5 nM, respectively. Adrenergic agonists elicited a 4- to 6-fold stimulation of aldosterone secretion with potency order (-)isoproterenol greater than (-)epinephrine equals (-)norepinephrine greater than (+)isoproterenol, and corresponding ED50 5, 240, 213, and 3000 nM, respectively. No reproducible inhibition by dopamine of basal or stimulated levels of aldosterone secretion could be detected, but a weak stimulatory effect was sometimes observed at high concentration greater than 10 microM. (-)Isoproterenol stimulation of aldosterone production was potently inhibited by the beta-adrenergic antagonists (-)alprenolol and (+)alprenolol with potencies of 1.8 and 110 nM, respectively. The alpha-adrenergic antagonists prazosin, yohimbine, and phentolamine only weakly inhibited (-)isoproterenol stimulation with potencies of 5, 13, and 140 microM, respectively. The potent beta 2-adrenergic antagonist ICI 118.551 and the weaker beta 1-adrenergic antagonist atenolol were roughly equipotent with potencies of 0.27 and 0.44 microM, respectively. Addition of the phosphodiesterase inhibitor Ro 20-1724 at 10 microM doubled the maximum stimulation effect of (-)isoproterenol without changing the potency of the catecholamine or the basal level of aldosterone secretion, suggesting a potential role of cAMP as a mediator of isoproterenol stimulation. These results indicate the presence of a beta 1-adrenergic receptor stimulating aldosterone secretion in bovine zona glomerulosa cells. The physiological significance of direct beta-adrenergic stimulation of aldosterone production is currently being assessed.
Assuntos
Glândulas Suprarrenais/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Aldosterona/metabolismo , 4-(3-Butoxi-4-metoxibenzil)-2-imidazolidinona/farmacologia , Glândulas Suprarrenais/citologia , Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Animais , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Epinefrina/farmacologia , Hidrocortisona/farmacologia , Isoproterenol/farmacologia , Norepinefrina/farmacologia , Propanolaminas/farmacologia , Prostaglandinas E/farmacologia , Estimulação QuímicaRESUMO
We report the identification and the primary structure of a novel neuroendocrine peptide which inhibits aldosterone secretion. The peptide was isolated from 1.5 x 10(10) cultured bovine chromaffin cells by reversed phase and ion exchange HPLC. It is chromatographically and structurally distinct from atrial natriuretic factor (ANF), which we formely identified in chromaffin tissue. This new Aldosterone Secretion Inhibitory Factor (ASIF) is predominant in cultured chromaffin cells and cross-reacts with ANF receptor sites involved in the inhibition of aldosterone production in zona glomerulosa cells. The sequence of this 35-amino acid peptide includes the shorter fragment isolated from porcine brain confirming its neuropeptide character. ASIF might be involved in concert with ANF in the paracrine regulation of aldosterone secretion and the presence of both peptides in cultured chromaffin cells indicates that this system can be used as a model for studying natriuretic neuropeptide production.
Assuntos
Glândulas Suprarrenais/análise , Aldosterona/metabolismo , Sistema Cromafim/análise , Antagonistas de Receptores de Mineralocorticoides/isolamento & purificação , Neuropeptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial , Bovinos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Dados de Sequência Molecular , Neuropeptídeos/farmacologia , Ensaio Radioligante , Receptores do Fator Natriurético Atrial , Receptores de Superfície Celular , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismoRESUMO
The effect of synthetic atrial natriuretic factor (ANF) on adrenal steroidogenesis has been studied in primary culture of bovine adrenal cells. ANF-(8-33) produced a potent 40-70% inhibition of angiotensin II-, ACTH-, PGE1-, and forskolin-stimulated secretion of aldosterone production from zona glomerulosa cells with an ED50 of 120 pM. An equipotent inhibitory effect of the natriuretic factor on cortisol production was also observed in cultured zona fasciculata cells. Nicotine-stimulated secretion of catecholamines from medullary cells was only slightly inhibited by the factor at doses above 10 nM. [125I]iodo-ANF-(8-33) binding to glomerulosa membranes displayed an apparent affinity of 100-150 pM for specific receptor sites and was not inhibited by angiotensin II or ACTH. Conversely, the natriuretic factor had no affinity for angiotensin II receptor sites. The results demonstrate that part of the natriuretic effect of this new factor might be due to inhibition of adrenal steroidogenesis by action through a distinct receptor.
Assuntos
Corticosteroides/biossíntese , Córtex Suprarrenal/metabolismo , Proteínas/farmacologia , Receptores de Superfície Celular/metabolismo , Aldosterona/biossíntese , Alprostadil , Angiotensina II/farmacologia , Animais , Bovinos , Células Cultivadas , Colforsina , Diterpenos/farmacologia , Hidrocortisona/biossíntese , Natriuréticos , Prostaglandinas E/farmacologia , Receptores do Fator Natriurético AtrialRESUMO
Binding and internalization of [125I]angiotensin II (AII) were studied by morphological and biochemical methods in rats in vivo. Light microscope radioautography demonstrated that [125I]AII binds specifically to adrenal zona glomerulosa (ZG) cells. Ultrastructural radioautographic analysis revealed that [125I]AII binds to the cell surface, clusters in coated pits, is internalized in coated vesicles, and is transported by receptosomes to lysosomes in less than 20 min. Biochemical analysis revealed that as much as 40% of the adrenal radioactive uptake behaves as native [125I]AII as shown by electrophoresis, immunoprecipitation and radioligand binding studies. These results indicate that the effects of AII on the secretion of aldosterone by ZG cells are mediated by cell surface phenomena and not by binding to intracellular organelles involved in steroidogenesis. They also indicate that the half-life of AII bound to receptors and internalized seems to be much longer (min) than in the systemic circulation (sec).
Assuntos
Córtex Suprarrenal/metabolismo , Angiotensina II/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endossomos/metabolismo , Feminino , Cinética , Lisossomos/metabolismo , Microscopia Eletrônica , Ratos , Ratos Endogâmicos , Receptores de Angiotensina/metabolismoRESUMO
The morphological localization of [125I]angiotensin II (AII) in the rat adrenal medulla (AM) was studied by light- and electron-microscopic radioautography in vivo. With light microscopy the presence of binding sites for AII in both norepinephrine-containing (NE) and epinephrine-containing (E) cells was confirmed. With electron microscopy, it was found that AII binds to the cell surface of NE cells, is progressively internalized, and is associated with lysosomes and Golgi complex within 20 min, whereas in E cells AII seems to be internalized earlier and recycled back to the cell surface within 5 min without any appreciable association with intracellular organelles. These results suggest different intracellular pathways for AII in NE and E cells of the rat AM.
Assuntos
Medula Suprarrenal/metabolismo , Angiotensina II/metabolismo , Lisossomos/metabolismo , Norepinefrina/metabolismo , Medula Suprarrenal/ultraestrutura , Animais , Autorradiografia , Epinefrina/metabolismo , Feminino , Complexo de Golgi/metabolismo , Radioisótopos do Iodo , Microscopia Eletrônica , Ratos , Ratos EndogâmicosRESUMO
In our previous work, the existence of the precursor and mature forms of atrial natriuretic factor (ANF) within the bovine chromaffin granules has been reported. To confirm the endogenous character of these peptides, we demonstrate that nicotinic activation and depolarization by KCl increase their co-secretion from cultured chromaffin cells. The increase of intracellular levels of these atrial peptides by phorbol ester is potentiated by addition of forskolin. The release of ANF and their de novo synthesis within the cultured chromaffin cells emphasize the usefulness of this model in the study of the mechanisms of release and storage of these peptides in the neuronal tissues.
Assuntos
Medula Suprarrenal/metabolismo , Fator Natriurético Atrial/biossíntese , Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Medula Suprarrenal/efeitos dos fármacos , Animais , Fator Natriurético Atrial/metabolismo , Bovinos , Células Cultivadas , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Taxa Secretória/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The present report demonstrates the presence in cultured rat aortic smooth muscle cells of a natriuretic factor receptor subtype with a specificity typical of the ANF-R1C (B-clone) receptor subtype. To prove the existence of this receptor subtype in this cell line we show that pCNP-(82-103) is the most potent activator of the intrinsic guanylate cyclase activity, and that [125I]pCNP-(82-103) binds to a specific receptor subtype which is insensitive to the ANF-R2 specific ligand, C-ANF. The investigation of its binding characteristics show the rank potency order of the natriuretic factors in competing for pCNP binding to be pCNP greater than pBNP greater than rANF. Furthermore it was possible to covalently photolabel this receptor subtype with underivatized]125I]pCNP and show that it is composed of a single subunit of 130 kDa with very high specificity for pCNP.
Assuntos
Fator Natriurético Atrial/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Aorta/metabolismo , Células Cultivadas , Guanilato Ciclase/metabolismo , Peptídeo Natriurético Encefálico , Peptídeo Natriurético Tipo C , Ensaio Radioligante , Ratos , Receptores do Fator Natriurético Atrial , Proteínas Recombinantes/metabolismoRESUMO
Specific receptors for atrial natriuretic factor were studied in purified glomeruli, proximal tubules, thick ascending limbs of Henle's loops and collecting ducts from dog kidney. Glomeruli contain the highest concentration of receptor sites (pK = 9.9, Bmax = 200 fmol/mg protein), followed by collecting ducts (pK = 9.4, Bmax = 150 fmol/mg). Low levels of receptor sites were also detectable in thick ascending limbs of Henle's loops (pK = 9.4, Bmax = 36 fmol/mg) while proximal tubules were completely devoid of specific binding sites. These results indicate that the glomeruli appear to be the primary site of interaction of atrial natriuretic factor in kidney cortex but that it might also act in the medulla on lower nephron tubular function.
Assuntos
Rim/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Cães , Glomérulos Renais/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Proximais/metabolismo , Alça do Néfron/metabolismo , Receptores do Fator Natriurético AtrialRESUMO
We report here the regulation of the biosynthesis and the secretion of C-type natriuretic peptide (CNP) in cultured bovine chromaffin cells. The combined treatment with protein kinase A and -C activators induced a 6-fold increase of intracellular levels of CNP-(1-103). The biosynthesized CNP-(1-103) was co-released with its mature forms, typically CNP-(51-103), upon stimulation by nicotine or depolarizing agents. This confirms the neuropeptidic character of this third member of the natriuretic peptide family, which might act as a neuromodulator or neurotransmitter.