RESUMO
NCI-H295, a human adrenocarcinoma cell line, has been proposed as a model system to define the role of the renin-angiotensin system in the regulation of aldosterone production in humans. Because the precise cellular localization of the components of the renin-angiotensin system in human adrenal cortical cells remains unclear, we investigated their localization in this defined cell system. NCI-H295 cells expressed both angiotensinogen and renin as shown by reverse transcriptase polymerase chain reaction and immunohistochemistry. Human angiotensin-converting enzyme (ACE) was not detectable by immunocytochemistry, ACE binding, or reverse transcriptase polymerase chain reaction. However, 3.5 mmol/L K+ stimulated the formation of both angiotensin I and angiotensin II 1. 9- and 2.5-fold, respectively, and increased aldosterone release 3. 0-fold. The K+-induced stimulation of aldosterone release was decreased by captopril and enalaprilat (24% and 26%, respectively) and by the angiotensin type 1 (AT1)-receptor antagonist losartan (28%). Angiotensin II-induced stimulation of aldosterone release was abolished by losartan treatment. Specific [125I]Sar1-angiotensin II binding was detected by receptor autoradiography. The binding of [125I]Sar1-angiotensin II was completely displaced by the AT1 antagonist losartan but not by the AT2 receptor ligand PD 123319, confirming the expression of angiotensin II AT1 receptors in NCI-H295 cells. Our results demonstrate that NCI-H295 cells express most of the components of the renin-angiotensin system. Our failure to detect ACE, however, suggests that the production of angiotensin II in NCI-H295 cells may be ACE independent. NCI-H295 cells are able to produce angiotensin II, and K+ increases aldosterone secretion in part through an angiotensin-mediated pathway. The production of angiotensin II in NCI-H295 cells demonstrates that this human cell line can be useful to characterize the role of locally produced angiotensin II in the regulation of aldosterone release.
Assuntos
Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Potássio/farmacologia , Sistema Renina-Angiotensina/fisiologia , Angiotensinogênio/análise , Captopril/farmacologia , Células Cultivadas , Humanos , Losartan/farmacologia , Peptidil Dipeptidase A/metabolismo , Renina/metabolismoRESUMO
Brain angiotensin II (Ang II) inhibits pituitary prolactin release by an indirect mechanism requiring stimulation of dopamine formation and release. We report that [125I]Sar1-Ang II binding to AT1 receptors and AT1A receptor mRNA expression increase selectively in the dorsomedial arcuate nucleus of 17beta-estradiol-primed ovariectomized rats after treatment with progesterone. In hormone-treated rats, arcuate nucleus AT1A receptor mRNA expression is associated with tyrosine hydroxylase-positive neurons. No AT1A receptor mRNA was detected in tyrosine hydroxylase-positive cells of the arcuate nucleus of intact male rats. Conversely, in the anterior pituitary, where local or circulating Ang II stimulates prolactin release, [125I]Sar1-Ang II binding to AT1 receptors and AT1B receptor mRNA expression are decreased in 17beta-estradiol/progesterone-treated ovariectomized rats. Thus, AT1A receptors in the dorsal arcuate nucleus and AT1B receptors in the anterior pituitary are regulated inversely by estrogen/progesterone treatment, supporting the hypothesis of a dual role for brain and pituitary Ang II on prolactin release. The colocalization of AT1A receptor mRNA and tyrosine hydroxylase in neurons of the arcuate nucleus furthermore indicates that within this area central Ang II acts directly on dopaminergic neurons. These results support the hypothesis that central Ang II inhibits pituitary prolactin release indirectly via modulation of dopaminergic activity in the arcuate nucleus.
Assuntos
Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Dopamina/metabolismo , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Progesterona/farmacologia , Receptores de Angiotensina/biossíntese , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Feminino , Sistema Hipotálamo-Hipofisário/fisiologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Hibridização In Situ , Masculino , Eminência Mediana/efeitos dos fármacos , Eminência Mediana/metabolismo , Proteínas do Tecido Nervoso/genética , Ovariectomia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Prolactina/biossíntese , Prolactina/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina , Receptores de Angiotensina/genética , Caracteres Sexuais , Tirosina 3-Mono-Oxigenase/biossíntese , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
We studied angiotensin II (ANG II) receptor subtype expression in selected brain nuclei and pituitary gland after water deprivation by in vitro receptor autoradiography using 125I-labeled [Sar1]ANG II and by in situ hybridization using 35S-labeled AT1A, AT1B, and AT2 receptor-specific riboprobes. In control rats we found binding to AT1 receptors in the subfornical organ, paraventricular nucleus, median eminence, and anterior pituitary; AT1A mRNA expression in the subfornical organ and paraventricular nucleus; and AT1B mRNA expression in the anterior pituitary. No receptor mRNA was found in the median eminence. AT1 receptors and AT1A receptor mRNA levels were increased in the subfornical organ, and, in the anterior pituitary, AT1 receptors and AT1B receptor mRNA were increased, only after 5 days of water deprivation. No significant changes occurred after 1 or 3 days of water deprivation, and no regulation of ANG II receptor expression was detected in other brain areas. Our results show that prolonged water deprivation selectively regulates AT1 receptor expression and AT1A and AT1B receptor mRNA levels in the subfornical organ and anterior pituitary, respectively, supporting a role for these receptors during sustained dehydration.
Assuntos
Adeno-Hipófise/metabolismo , Receptores de Angiotensina/biossíntese , Órgão Subfornical/metabolismo , Regulação para Cima , Privação de Água/fisiologia , Animais , Peso Corporal , Hibridização In Situ , Masculino , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Fatores de Tempo , Transcrição GênicaRESUMO
Endothelins are vasoactive peptides and are believed to act as vascular smooth muscle mitogens. Vascular injury results in medial smooth muscle migration and proliferation with the formation of a neointima. Using quantitative autoradiography, we examined the expression of endothelin receptor subtypes ET(A) and ET(B) in the rat carotid artery 2, 8, and 16 days after balloon-catheter injury. At two and eight days after balloon catheterization, ET(A) receptor expression was significantly increased in the media of the injured vessel when compared to that in the media of the intact vessel. The enhanced expression of receptors returned to normal levels by 16 days after the injury. Neointimal cells also expressed ET(A) receptors at a lower level than that expressed by the injured media 8 days after injury, and continued to express ET(A) receptors 16 days after the injury. ET(B) receptors were not detectable in the media or the neointima at any time after the injury. Our results suggest the ET(A) receptors may have a significant role in injury induced vascular smooth muscle proliferation and neointima formation.
Assuntos
Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas , Cateterismo/efeitos adversos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Receptores de Endotelina/biossíntese , Animais , Autorradiografia , Ligação Competitiva , Artérias Carótidas/patologia , Antagonistas dos Receptores de Endotelina , Endotelina-1/metabolismo , Radioisótopos do Iodo , Cinética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Músculo Liso Vascular/patologia , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A , Receptor de Endotelina BRESUMO
In this study, [125I]CGP 42112, a ligand of high affinity and selectivity for the angiotensin II AT2 receptor, was used to detect and quantify a non-angiotensin II binding site in the balloon-injured carotid artery of the rat. The amount of [125I]CGP 42112 binding was significantly enhanced in the adventitia of the injured arteries. Localization of the binding site using emulsion autoradiography and immunocytochemistry suggests that the binding sites may be expressed by macrophages in the inflamed tissue surrounding the injured artery.
Assuntos
Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas , Cateterismo/efeitos adversos , Oligopeptídeos/metabolismo , Angiotensina II/metabolismo , Animais , Autorradiografia , Artérias Carótidas/patologia , Imuno-Histoquímica , Inflamação , Radioisótopos do Iodo , Macrófagos , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Angiotensina/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) may modulate vascular permeability, chemotaxis for monocytes, and protease activity. In addition, VEGF may play a role in embryonic and tumor angiogenesis. In fetal mouse kidney, VEGF mRNA and protein expression have been demonstrated. This finding led to the hypothesis that VEGF might be involved in renal growth and development. To further elucidate the role of VEGF in human kidney, expression of VEGF and its receptors, the specific tyrosine kinase receptors, fit-1 and KDR, were studied. In fetal (6-24 gestational wk; mesonephros and metanephros) and adult kidney, VEGF mRNA and protein could be colocalized in glomerular epithelia and collecting duct cells by in situ hybridization and immunohistology. By reverse transcription-polymerase chain reaction, mRNA of three VEGF isoforms, VEGF121, VEGF165, and VEGF189, were found in fetal kidney and cortex, isolated glomeruli, and medulla of adult human kidney. KDR and flt-1 mRNA were coexpressed in endothelia of glomeruli and in peritubular capillaries in fetal and adult kidney. These data support the assumption that VEGF and its receptors may influence renal ontogenesis. We speculate that the constitutive expression of VEGF in adult kidney may be required for the function of VEGF receptor positive-fenestrated endothelia in glomeruli and postglomerular vessels. The expression of VEGF in collecting duct and of its receptors in medullary capillaries may in addition be relevant for maintaining medullary osmolality.