RESUMO
We have discovered a non-AT(1), non-AT(2) angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.
Assuntos
Neurônios/citologia , Neurônios/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , 4-Cloromercuriobenzenossulfonato/farmacologia , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Feminino , Glutationa/farmacologia , Dissulfeto de Glutationa/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos , Neurônios/efeitos dos fármacos , Prosencéfalo/citologia , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Temperatura , Ácido p-Cloromercurobenzoico/farmacologiaRESUMO
AIMS: To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. MAIN METHODS: Radioligand binding assays of (125)I-sarcosine(1), isoleucine(8) angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 microM losartan to saturate AT1 receptors, 10 microM PD123319 to saturate AT2 receptors, with or without 10 microM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. KEY FINDINGS: The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77+/-0.16 and 1.52+/-0.17 fmol/mg initial wet weight in female and male brains, respectively. The K(D) values, 1.79+/-0.09 nM for females, and 1.53+/-0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine(1), isoleucine(8) angiotensin II>angiotensin III>angiotensin II>angiotensin I'angiotensin IV>angiotensin 1-7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. SIGNIFICANCE: The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.
Assuntos
Angiotensinas/metabolismo , Química Encefálica/fisiologia , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Adulto , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Córtex Cerebral/metabolismo , Feminino , Humanos , Imidazóis/farmacologia , Ligantes , Losartan/farmacologia , Masculino , Pessoa de Meia-Idade , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Piridinas/farmacologia , Ensaio Radioligante , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores de Angiotensina/efeitos dos fármacos , Caracteres Sexuais , Ácido p-Cloromercurobenzoico/farmacologiaRESUMO
The effects of losartan on angiotensin receptors in hypertrophic rat hearts were studied. The study was prompted by inconsistent findings of either an increase or decrease in the mRNA of the AT1 receptor in the hearts of cardiac hypertrophic rats treated with losartan, and a paucity of information on the effects of losartan on functional angiotensin receptors in the heart. Losartan, administered i.p. to aortic coarcted rats, dose-dependently attenuated the cardiac hypertrophy. Significant effect was observed with a dose of 2.72 micromol/kg/day for four days. Hypertrophy was accompanied by an increase in [125I]-Sar1-Ile8-angiotensin II binding sites (due mainly to an increase in AT2 binding) and AT2 receptor protein in cardiac ventricles of aortic coarcted rats. Treatment with effective anti-hypertrophic doses of losartan dose-dependently downregulated the [125I]-Sar1-Ile8-angiotensin II binding sites, constitutive AT1 receptor protein, and the over expressed AT2 receptor protein. It was suggested that the anti-cardiac hypertrophic action of losartan resulted from its ability to suppress the expression of both the basal and enhanced cardiac angiotensin receptors. This raises the question as to whether such drastic action could form the therapeutic basis for the use of losartan in cardiac pathologies.
Assuntos
Cardiomegalia/tratamento farmacológico , Cardiomegalia/metabolismo , Losartan/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Sequência de Bases , Cardiomegalia/genética , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Masculino , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/genéticaRESUMO
The proposal that the mas oncogene is an angiotensin receptor was evaluated in Xenopus oocytes injected with human and rat mas RNA transcripts, and during transient expression of mas in several cell lines. No evidence of mas-induced angiotensin II (AII) receptors or [Ca2+]i responses was observed in Xenopus oocytes or in most of the transfected cells. However, Cos-1 cells, which showed a small endogenous [Ca2+]i response to AII, exhibited a modest but reproducible enhancement of this response after mas transfection. Such responses were inhibited by [Sar1, Ala8]AII and [Sar1, Ile8]AII, but not by [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, an antagonist reported to inhibit mas-induced responses to AII in oocytes. These findings are not compatible with the proposal that the mas oncogene is an angiotensin receptor, but suggest that expression of mas leads to increased responsiveness of the endogenous AII signaling system.
Assuntos
1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Cálcio/metabolismo , Oncogenes , Precursores de RNA/metabolismo , Receptores de Angiotensina/metabolismo , Proteínas Recombinantes , Saralasina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Antagonistas de Receptores de Angiotensina , Animais , Linhagem Celular , Microinjeções , Oócitos , Saralasina/farmacologia , Transdução de Sinais , Substância P/análogos & derivados , Substância P/farmacologia , Transfecção , XenopusRESUMO
A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-¿Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ¿Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.
Assuntos
Furões/genética , Losartan/metabolismo , Receptores de Angiotensina/genética , 1-Sarcosina-8-Isoleucina Angiotensina II/análogos & derivados , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Antagonistas de Receptores de Angiotensina , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Células COS , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , Imidazóis/farmacologia , Radioisótopos do Iodo , Losartan/farmacologia , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Treonina/genética , Treonina/metabolismoRESUMO
Chinese Hamster Ovary Cells (CHO-K1) were transiently and stably transfected to express the human angiotensin AT(1) receptor. Cell surface receptor expression was maximal 2 days after transient transfection. Their pharmacological and signalling properties differed from stably expressed receptors. Receptor reserve was significant in the transient cells but not in stable cells, explaining the higher potency of angiotensin II and the lower degree of insurmountable inhibition by candesartan in the transient cells. [Sar(1)Ile(8)]angiotensin II (sarile) is a potent angiotensin AT(1) receptor antagonist for the stable cells but is a partial agonist, producing 19% of the maximal response by angiotensin II, in transient cells. Internalization of [(3)H]angiotensin II and [(125)I]sarile (i.e., acid-resistant binding) was more pronounced in stable cells. CHO-K1 cells were also transiently transfected with the enhanced green fluorescence-AT(1) receptor gene. Confocal microscopy revealed rapid internalization induced by angiotensin II and sarile but not by candesartan. The above disparities may result from differences in receptor maturation and/or cellular surrounding.
Assuntos
Receptor Tipo 1 de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Ligação Competitiva/efeitos dos fármacos , Compostos de Bifenilo , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cinética , Ligantes , Microscopia Confocal , Ensaio Radioligante , Receptor Tipo 1 de Angiotensina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tetrazóis/metabolismo , Tetrazóis/farmacologia , Transfecção , TrítioRESUMO
Specific angiotensin II (Ang II) binding and functional responses have been studied in a number of male and female reproductive structures, but to date have not been characterized in the epididymis. Some epididymal functions, including smooth muscle contraction and regulation of fluid and electrolyte balance, are mediated by Ang II in other tissues. This study demonstrates specific, saturable (63 fmol/mg protein), high affinity (Kd, less than 1 nM) Ang II-binding sites in the epididymis. These binding sites, localized in the circumference of the epididymal tubule and most concentrated within the proximal cauda, are present throughout the caput, corpus, and remaining cauda epididymis. Ang II caused powerful expulsions of spermatozoa in segments of epididymis in situ. These results suggest Ang II involvement in epididymal function.
Assuntos
Epididimo/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Autorradiografia , Sítios de Ligação , Epididimo/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
In mammalian zona glomerulosa cells, angiotensin II (AII)-induced increases in intracellular Ca2+ ([Ca2+]i) and AII-induced aldosterone production seem to be inextricably linked. However, in avian adrenal steroidogenic (adrenocortical) cells studied thus far, inducible aldosterone production seems to be insensitive to alterations in the mobilization of cellular Ca2+. This raises the hypothesis that alternative signal transduction pathways are implemented to induce aldosterone production in avian adrenocortical cells. In the present study, this hypothesis was investigated by using isolated turkey (Meleagris gallopavo) adrenocortical cells that are known to be three times more sensitive to AII than to ACTH for aldosterone production. In isolated turkey adrenocortical cells, the mammalian AII receptor antagonist, [Sar1,Ile8]AII, was as efficacious as [Ile5]AII in stimulating aldosterone production, albeit it had about 1/150 the potency of [Ile5]AII. The actions of both analogs required extracellular K+, suggesting a voltage-sensitive event. However, a maximal aldosteronogenic concentration of [Sar1,Ile8]AII not only failed to increase [Ca2+]i but also completely blocked maximal (10(-8) M)[Ile5]AII-induced increases in [Ca2+]i when added before [Ile5]AII and partially dampened (approximately 50%) maximal [Ile5]AII-induced increases in [Ca2+]i when added after (3 min) [Ile5]AII. This blockade in [Ca2+]i elevation was surmounted by high concentrations of [Ile5]AII (> 10(-6) M). By contrast, [Sar1,Ile8]AII did not alter maximal aldosterone production induced by [Ile5]AII and vice versa, thus suggesting that the action of both analogs converged on the same aldosteronogenic pathway, and that AII-induced aldosterone production was not coupled to elevations in [Ca2+]i. Detailed homologous-heterologous ligand-binding analyses supported the presence of two AII-binding sites that were discriminated by [Sar1,Ile8]AII (dissociation constants, 4.2 +/- 1.4 and 21.9 +/- 2.2 nM; concentration distribution, approximately 40% and approximately 60%, respectively; mean +/- SE, n = 4) but not by [Ile5]AII (dissociation constant, 2.1 +/- 0.1 nM for both sites). In addition, [Sar1,Ile8]AII- and [Ile5]AII-binding sites exhibited different physicochemical and pharmacological properties. The sensitivity of [Sar1,Ile8]AII-binding sites was about twice that of [Ile5]AII-binding sites to dithiothreitol. In addition, whereas both the high- and low-affinity sites detected by [Sar1,Ile8] AII exhibited equivalent competitive sensitivities to the type-1 receptor, the nonpeptidic antagonist, losartan (DuP 753), the sensitivity of the low-affinity site was 2.7 times that of the high-affinity site to the type-2 receptor, nonpeptidic antagonist, PD123319. Taken collectively, the data suggest that in turkey adrenocortical cells, elevations in [Ca2+]i and aldosterone production are dissociable events regulated by distinct AII receptor subtypes or isomorphs.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Aldosterona/biossíntese , Angiotensina II/farmacologia , Cálcio/metabolismo , Receptores de Angiotensina/fisiologia , Perus , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Córtex Suprarrenal/metabolismo , Angiotensina II/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Masculino , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismoRESUMO
Ovarian angiotensin II (Ang II) receptors display a cyclical pattern of variation during the rat estrous cycle. Ang II receptors, estimated by the specific binding of the Ang II receptor antagonist [125I]iodo-[Sar1,Ile8] Ang II to ovarian membranes, were lowest at estrus [binding site density (Bmax) = 35 +/- 2 fmol/mg; binding site affinity (KD) = 2.0 +/- 0.2 nM] and highest at diestrus I (Bmax = 59 +/- 3 fmol/mg; KD = 1.6 +/- 0.1 nM). We have previously shown that Ang II receptors in the rat ovary predominantly exist on the granulosa cell layer of a subpopulation of follicles. Our present studies show that the Ang II receptor-containing follicles in the rat ovary are mainly atretic (approximately 80%) or show signs of early atresia (approximately 15%) during all stages of the estrous cycle. A small number of Ang II receptor-containing follicles were healthy (approximately 5%). In contrast to the Ang II receptor-containing follicles, the FSH receptor-containing follicles were predominantly healthy (greater than 90%). Follicles which contained both Ang II receptors and FSH receptors were mainly early atretic. Since Ang II receptor-containing follicles in the rat ovary were mainly atretic these studies suggest that in the rat Ang II may be a major factor in regulating the function of atretic ovarian follicles.
Assuntos
Angiotensina II/metabolismo , Estro/metabolismo , Atresia Folicular , Fase Folicular , Ovário/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Animais , Membrana Celular/metabolismo , Epitélio/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Radioisótopos do Iodo , Folículo Ovariano/anatomia & histologia , Folículo Ovariano/metabolismo , Ratos , Ratos Endogâmicos , Receptores do FSH/metabolismo , Distribuição TecidualRESUMO
The role of angiotensin II (AII) in human preadipocyte physiology has been investigated in primary cultures from human adipose tissue. Receptor binding studies indicated that human preadipocytes express a high affinity AII binding site of the AT1 subtype, as binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific. As AII has previously been demonstrated to affect the cell cycle in adrenal and cardiac cells, the effect of AII on regulation of cycle progression was examined in human preadipocytes. Stimulation of preadipocytes with AII resulted in G1 phase progression of the cell cycle, as determined by flow cytometric analysis. AII treatment was associated with induction of expression of the messenger RNA for the cell cycle regulatory protein cyclin D1 in a dose-dependent manner. Pretreatment of cells with subtype-selective AT receptor ligands before AII stimulation indicated that the cyclin response was mediated via the AT1 receptor. The identity of the cells as preadipocyte was verified by culture in a defined differentiation medium, observing both leptin message expression and triglyceride accumulation by flow cytometry. These findings indicate that AII has early, receptor-mediated effects on cell cycle progression in human preadipocytes that may contribute to differentiation to the adipocyte phenotype.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/citologia , Ciclo Celular/fisiologia , Receptores de Angiotensina/fisiologia , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Adipócitos/citologia , Adulto , Sítios de Ligação , Diferenciação Celular , Separação Celular , Células Cultivadas , Ciclina D1/biossíntese , Ciclina D1/genética , Feminino , Citometria de Fluxo , Humanos , Masculino , RNA Mensageiro/metabolismo , Receptores de Angiotensina/metabolismoRESUMO
Ovarian angiotensin I (Ang I)-converting enzyme (ACE), estimated by the specific binding of the ACE inhibitor [125I]iodo-MK-351A, is localized on multiple ovarian structures, including follicular granulosa cells, corpora lutea, terminal epithelium, and ovarian blood vessels, but total ovarian ACE does not display a cyclic pattern of variation during the rat estrous cycle. We have previously shown that ACE is localized on the granulosa cell layer of a subpopulation of rat ovarian follicles. Our present study shows that ovarian granulosa cells contain high affinity [binding site affinity (Kd), approximately 90 pM] and low capacity [binding site density (Bmax), approximately 12 fmol/2.5 X 10(5) cells] [125I]iodo-MK-351A-binding sites and convert [125I]iodo-Ang I to [125I]iodo-Ang II (greater than 85% of this conversion was inhibited by the ACE inhibitor captopril). Throughout the rat estrous cycle, 94-100% of developing follicles and 89-96% of atretic follicles contained high levels of ACE; however, ACE was either not observed or its levels were very low in preovulatory follicles. These findings indicate the presence of high levels of biologically active ACE on the surface of granulosa cells and suggest a potential role for follicular ACE in early stages of follicular maturation and atresia. Although ACE is known to process a variety of peptides found within the ovary, and these peptides may have opposing effects on follicular maturation, we attempted to define the cumulative effect of ACE inhibition on follicular maturation. Short and long term (6- and 14-day) infusions of captopril (6-day, 30.5 +/- 3.5 ova; 14-day, 28.5 +/- 7.5 ova) in immature rats, in which ovulation was induced by sequential treatments with PMSG and hCG, did not significantly affect ovulation compared with that in vehicle-infused control rats (6-day, 22.4 +/- 2.4 ova; 14-day, 20.8 +/- 3.1 ova), suggesting that ACE inhibition does not modify the follicular selection process in a way that affects ovulation. This may explain the lack of any reports of adverse effects of clinically used ACE inhibitors on ovulation.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Estro/fisiologia , Folículo Ovariano/enzimologia , Ovário/enzimologia , Ovulação/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animais , Sítios de Ligação , Captopril/farmacologia , Membrana Celular/enzimologia , Gonadotropina Coriônica/metabolismo , Dipeptídeos/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/enzimologia , Radioisótopos do Iodo , Ratos , Ratos EndogâmicosRESUMO
The present study was undertaken to investigate the role of angiotensin II (Ang II) in ovulation and ovarian steroidogenesis and prostaglandin (PG) production via the Ang II receptors in rabbit ovaries. In in vitro perfused rabbit ovaries, PD123319, a selective nonpeptide antagonist for AT2 receptors, reduced the Ang II-induced ovulation in a dose-dependent manner, whereas CV-11974, a selective nonpeptide antagonist for AT1 receptor, did not affect the Ang II-induced ovulation. Ang II also significantly stimulated the meiotic maturation of ovulated ova and follicular oocytes in the absence of gonadotropin. The addition of PD123319 at 10 (-6) M to the perfusate significantly inhibited the Ang II-induced oocyte maturation. Ang II did not stimulate the production of progesterone by perfused rabbit ovaries but significantly stimulated the production of estradiol (E2) and PGs. When PD123319 at 10(-6) M was added to the perfusate 30 min before the onset of Ang II administration, the Ang II-stimulated production of E2 and PGs was significantly blocked. Saralasin, a peptide analog of Ang II, inhibited the specific binding of [125I] iodo-[Sar1, Ile8] Ang II to rabbit ovarian membranes in a concentration-dependent manner, yielding an inhibitory constant (IC50) value of 1.58 x 10(-9) M. PD123319 and CV-11974 also inhibited the binding of [125I]iodo-[Sar1, Ile8] Ang II; however, PD123319 and CV-11974 were 15 and 40 times less potent than saralasin, respectively. Autoradiographic study revealed that an intense localization of Ang II receptors in the rabbit ovaries was present in the granulosa cell layers and the stroma of the preovulatory follicles. AT2 receptors were predominantly located in granulosa cells, whereas AT1 receptors were more concentrated in the stroma and thecal cell layers. In summary, Ang II induced ovulation and oocyte maturation and stimulated the production of E2 and PG by perfused rabbit ovary in vitro via the AT2 receptor. Thus, locally produced Ang II may be part of a novel intraovarian paracrine or autocrine control mechanism during the ovulatory process.
Assuntos
Angiotensina II/farmacologia , Oócitos/fisiologia , Ovário/efeitos dos fármacos , Indução da Ovulação , Receptores de Angiotensina/fisiologia , 1-Sarcosina-8-Isoleucina Angiotensina II/análogos & derivados , 1-Sarcosina-8-Isoleucina Angiotensina II/antagonistas & inibidores , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Autorradiografia , Benzimidazóis/farmacologia , Compostos de Bifenilo , Senescência Celular/efeitos dos fármacos , Feminino , Imidazóis/farmacologia , Piridinas/farmacologia , Coelhos , Receptores de Angiotensina/agonistas , Saralasina/farmacologia , Tetrazóis/farmacologiaRESUMO
Angiotensin II receptor binding sites in type 1 interstitial cells in the inner stripe of the outer medulla are readily labeled in vitro by the radioligand but not in vivo after systemic radioligand administration. In anesthetized rats, we investigated if reduced vascular delivery due to angiotensin II-induced renal vasoconstriction or, alternatively, prior occupancy of these sites by endogenous angiotensins modulates angiotensin II subtype 1 receptor binding to renal medullary interstitial cells in vivo using electron microscopic autoradiography. Using 125I-angiotensin II, administered systemically, as a radioligand, binding in control rats occurred predominantly in the glomeruli and proximal tubules, while only low binding was observed in the inner stripe of the outer medulla. Pretreatment of rats with unlabeled [Sar1,Ile8]angiotensin II or with the angiotensin II subtype 1 receptor antagonist losartan before receiving the radioligand completely abolished binding to all sites. Renal vasodilatation induced by sodium nitroprusside or use of the radiolabeled antagonist analogue 125I-[Sar1,Ile8]angiotensin II did not alter binding to the inner stripe. In contrast, chronic salt loading or inhibition of angiotensin-converting enzyme by perindopril significantly increased binding not only to the cortical sites but also to the sites in the inner stripe of the outer medulla. Electron microscopic autoradiographs of the inner stripe detected binding in the interstitial cells only in rats treated with chronic salt loading or perindopril. These results suggest that endogenous angiotensins may modulate binding of circulating angiotensin II to the interstitial cells in vivo, and these angiotensin II receptor-bearing cells are more likely to be more responsive to interstitial angiotensin II than to the circulating hormone.
Assuntos
Medula Renal/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/antagonistas & inibidores , Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Autorradiografia , Compostos de Bifenilo/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Medula Renal/citologia , Medula Renal/ultraestrutura , Losartan , Masculino , Microscopia Eletrônica , Ratos , Ratos Sprague-Dawley , Tetrazóis/farmacologiaRESUMO
In contrast to the abundant expression of the AT2 subtype of angiotensin II (AII) receptors during fetal development, AT2 receptor in adult life is expressed in few tissues. We now report studies on the presence and hormonal regulation of AT2 receptor in human pregnant and nonpregnant myometrium obtained from a large study population (n = 50). AT2 receptor subtypes have been characterized using self- and cross-competition curves among [125I]CGP42112A (a selective AT2 ligand), [125I](Sar1,Ile8)AII (a unselective antagonist), the corresponding unlabeled ligands, and several peptidic and nonpeptidic analogs with different affinities for the AT1 and AT2 receptor subtypes. We found that the human nonpregnant uterus expresses almost exclusively the AT2 subtype, and that [125I]CGP42112A is a selective probe to study human AT2 receptor. By using [125I]CGP42112A, we demonstrated that the density of AT2 receptor in human myometrium is dramatically affected by the hormonal milieu. Indeed, in the estrogen-dominant uterus of normal cycling women in the proliferative phase and that of perimenopausal women with anovulatory cycles, the density of binding sites was very high [1565 +/- 246 fmol/mg protein (n = 11) and 2176 +/- 429 (n = 7), respectively]. The concomitant presence of progestogens blunted the estrogen effect [term pregnancy, 61 +/- 12 fmol/mg protein (n = 5); secretive phase of the cycle, 453 +/- 154 (n = 10); combined oral contraceptive, 243 +/- 74 fmol/mg protein (n = 6)]. Very low concentrations of binding sites are also present in the sex steroid-deprived uterus of postmenopausal women (100 +/- 12 fmol/mg protein; n = 8) and the uterus of fertile women chronically treated with GnRH agonists (199 +/- 100 fmol/mg protein; n = 3). Hence, these data confirm the presence of AT2 receptors in human uterus and indicate their regulation by sex steroids.
Assuntos
Hormônios Esteroides Gonadais/farmacologia , Miométrio/metabolismo , Receptores de Angiotensina/efeitos dos fármacos , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Adulto , Idoso , Ligação Competitiva , Membrana Celular/metabolismo , Feminino , Humanos , Radioisótopos do Iodo , Menopausa/fisiologia , Ciclo Menstrual , Pessoa de Meia-Idade , Oligopeptídeos/metabolismo , Gravidez , Receptores de Angiotensina/metabolismoRESUMO
NG108-15 cells, a neurally derived clonal cell line, express various components of the renin-angiotensin system and thus serve as a model of the cellular action of angiotensin (Ang) II. NG108-15 cells contain a high-affinity binding site for Ang II, with a Kd of 1.1 nM and a Bmax of 6.5 fmol/mg protein. Ang peptides competed for 125I-Ang II binding with an order of potency of Ang II greater than Ang-(2-8) much greater than Ang-(1-7). The subtype 1 (or B)-selective Ang II receptor antagonist DuP 753 as well as [Sar1,Ile8]Ang II and [Sar1,Thr8]Ang II competed for Ang II binding with high affinity, whereas the subtype 2 (or A)-selective Ang receptor antagonist CGP 42112A was partially effective only at a 300-fold higher concentration. When NG108-15 cells were induced to differentiate by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate, the density of Ang II receptors increased dramatically, with little change in affinity (1.1 versus 4.2 nM) or competition by Ang peptides. In marked contrast to undifferentiated cells, CGP 42112A became a potent competitor (IC50, 1 nM) for the majority (90-95%) of Ang II binding, whereas DuP 753 competed for only 5-10% of the binding sites. Ang II caused a dose-dependent mobilization of cytosolic Ca2+ in undifferentiated NG108-15 cells through activation of phospholipase C and the production of inositol 1,4,5-trisphosphate. In these cells, Ca2+ mobilization was blocked by either DuP 753 or the sarcosine Ang II analogues, whereas CGP 42112A was ineffective. Ang II also mobilized intracellular Ca2+ in differentiated NG108-15 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Neurônios/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/antagonistas & inibidores , Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Ligação Competitiva , Cálcio/metabolismo , Células Híbridas , Imidazóis/metabolismo , Losartan , Oligopeptídeos/metabolismo , Transdução de Sinais , Tetrazóis/metabolismo , Células Tumorais CultivadasRESUMO
The neuronal angiotensin II (Ang II) type 1 (AT1) receptor is coupled to the Ras-Raf-1-mitogen-activated protein (MAP) kinase signal-transduction pathway (Yang H, Lu D, Yu K, Raizada MK. Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci. 1996;16:4047-4058). In this study we compared the effects of angiotensin II (Ang II) on AT1 receptor phosphorylation and the ability of the phosphorylated receptor to bind Ang II in neuronal cultures of Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) brains to further our understanding of the Ang II signaling mechanism. Ang II caused a time-dependent phosphorylation of AT1 receptors in both WKY and SHR brain neurons. The level of phosphorylation was higher in the SHR brain neurons; this finding was consistent with increased AT1 receptors in these cells. MAP kinase was involved in this phosphorylation, a conclusion supported by the following evidence: (1) exogenous MAP kinase phosphorylated the AT1 receptor; (2) PD98059, a MAP kinase kinase inhibitor, attenuated Ang II-stimulated AT1 receptor phosphorylation; and (3) MAP kinase and AT1 receptors were coimmunoprecipitated in Ang II-stimulated neurons. Finally, MAP kinase phosphorylation was associated with the loss of 125I-[Sar1-Ile8]-Ang II binding ability of the AT1 receptor in both strains of neurons. These observations show that Ang II stimulates phosphorylation of the neuronal AT1 receptor by a mechanism involving MAP kinase and that the phosphorylated neuronal AT1 receptor does not exhibit Ang II binding activity in the brains of either WKY or SHR.
Assuntos
Angiotensina II/farmacologia , Encéfalo/metabolismo , Neurônios/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/análogos & derivados , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
125I-labelled angiotensin II (A II) specifically binds to solubilized receptors extracted from rat isolated glomeruli using CHAPS (3-[3-( cholamidopropyl ) dimethylammonio ]-1-propanesulfonate). The yield of solubilization of the binding sites was 3.3%. Equilibrium was reached after 15-20 min and specific binding represented 75% of total binding. Dissociation of the hormone-receptor complex after addition of an excess of A II was very slow in the presence of Ca2+ and Mg2+. [Sar1 Ala8] A II and [Sar1 Ile8] A II were more potent as competitive inhibitors of 125I-labelled A II than A II itself and its heptapeptide. These basic features of 125I-labelled A II binding to the extracted material were similar to those observed previously with untreated glomeruli.
Assuntos
Angiotensina II/metabolismo , Ácidos Cólicos , Glomérulos Renais/análise , Receptores de Angiotensina/isolamento & purificação , Receptores de Superfície Celular/isolamento & purificação , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Animais , Ligação Competitiva , Detergentes , Masculino , Ratos , Ratos Endogâmicos , Receptores de Angiotensina/metabolismo , Saralasina/metabolismo , SolubilidadeRESUMO
High-affinity receptors for angiotensin II were identified on Xenopus laevis cardiac membranes and characterized by binding-inhibition studies with peptide and non-peptide AII antagonists. Scatchard analysis of the binding data identified a high-affinity site with Kd1 = 1.6 nM and Bmax1 = 3.7 pmol/mg protein and a low-affinity site with Kd2 = 22 nM and Bmax 2 = 9.5 pmol/mg protein. Treatment with dithiothreitol reduced the number of binding sites by greater than 70%. The rank order of potency for ALL analogs was (agent, IC50) [Sar1,Ile8]AII, 0.91 nM greater than AII, 2.0 nM greater than AI, 5.3 nM greater than [Sar1, Ala8]AII, 19 nM much greater than CGP42112A, 1.2 microM much much greater than DuP 753 approximately PD-123177, greater than 100 microM. The relative potencies of these compounds differ markedly from their activities on the two known mammalian AII receptor subtypes, AT1 and AT2. These results indicate that amphibian AII receptors are pharmacologically distinct from both the AT1 and AT2 receptors characterized in mammalian tissues.
Assuntos
Angiotensina II/metabolismo , Miocárdio/metabolismo , Receptores de Angiotensina/metabolismo , Xenopus laevis/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina I/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Ditiotreitol/farmacologia , Cinética , Oócitos/metabolismo , Saralasina/metabolismoRESUMO
Angiotensin II (Ang II) binding sites were localized in the thalamus, basal ganglia, midbrain, and pons of the human central nervous system by in vitro autoradiography, employing 125I-[Sar1, Ile8]angiotensin II as the radioligand. High-density binding occurs in the substantia nigra pars compacta, the interpeduncular nucleus and two of the raphe nuclei, the raphe magnus, and median raphe nucleus. Moderate densities occur in the caudate nucleus, putamen, bed nucleus of the stria terminalis, rostral linear nucleus, caudal linear nucleus, dorsal and paramedian raphe nuclei, locus coeruleus, and region of the subcoeruleus, oral dorsal paramedian nucleus, and A5/periolivary region. Low levels occur in the region between the subthalamic nucleus and the zona incerta, the mediodorsal thalamic nucleus, the central gray, the lateral and medial parabrachial nuclei, and the molecular layer of the cerebellum. The high density of Ang II receptor binding in the substantia nigra occurs over pigmented, presumably dopaminergic, neurons. The binding in this site, and in the striatum, is not observed in any of the other species we have studied. It displays similar pharmacological characteristics to the Ang II receptor binding site in other regions of the human brain. Overall we demonstrate a discrete pattern of Ang II receptor binding sites in the human brain, which shows a high correlation with the distribution observed in other mammalian species.
Assuntos
Angiotensina II/metabolismo , Cerebelo/metabolismo , Ponte/metabolismo , Prosencéfalo/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Idoso , Autorradiografia , Gânglios da Base/metabolismo , Sítios de Ligação , Feminino , Humanos , Masculino , Mesencéfalo , Tálamo/metabolismo , Distribuição TecidualRESUMO
Angiotensin II (Ang II) exerts a number of central actions on fluid and electrolyte homeostasis, autonomic activity, and neuroendocrine regulation. In order to evaluate likely sites where these actions are mediated, Ang II receptor binding was localized in rat brain by in vitro autoradiography with the aid of the antagonist analogue 125I-[Sar1, Ile8]Ang II. Two subtypes of Ang II receptor have been identified using recently developed peptide and nonpeptide antagonists. In the periphery, the receptor subtypes differ in distribution, second messenger coupling, and function. Brain Ang II receptor subtypes were therefore differentiated into AT-1 (type I) and AT-2 (type II) subtypes by using unlabelled nonpeptide antagonists specific for the two Ang II subtypes. AT-1 binding was determined to be that inhibited by Dup 753 (10 microM) and AT-2 binding to be that inhibited by PD 123177 (10 microM). The reducing agent dithiothreitol (DTT) decreased binding to AT-1 receptors and enhanced binding to AT-2 receptors. Many brain structures, such as the vascular organ of the lamina terminalis, subfornical organ, median preoptic nucleus, area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus, which are known to be related to the central actions of Ang II, contain exclusively AT-1 Ang II receptors. By contrast, the locus coeruleus, ventral and dorsal parts of lateral septum, superior colliculus and subthalamic nucleus, many nuclei of the thalamus, and nuclei of the inferior olive contain predominantly AT-2 Ang II receptors. The detailed binding characteristics of each subtype were determined by competition studies with a series of analogues of angiotensin and antagonists. The pharmacological specificity obtained in rat superior colliculus and the nucleus of the solitary tract agreed well with published data on AT-1 and AT-2 receptors, respectively. There was a high degree of correlation between the distribution of Ang II binding sites with published data on Ang II-immunoreactive fields and on the sites of Ang II-responsive neurons. The present study also reveals pharmacological heterogeneity of brain Ang II receptors. The subtype-specific receptor mapping described here is relevant to understanding the role of angiotensin peptides in the central nervous system and newly discovered central actions of nonpeptide Ang II receptor antagonists.