1. We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. 2. Compounds were delivered into the cytoplasm of de-endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10-5 M). 3. Intracellular administration of Ang II (incorporation range: 0.01-300 nmol mg-1) resulted in a dose-dependent contraction, insensitive to extracellular administration (10-6 M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non-selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]-Ang II (P < 0.05). 4. Intracellular administration of CV11947 or PD 123319 right shifted the dose-response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg-1 Ang II was incorporated. 5. Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P < 0.05). 6. Intracellular administration of Ang I and saralasin also induced contraction (P < 0.05). Both responses were sensitive to intracellular CV11947 (P < 0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. 7. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre-treatment with saralasin or CV11947 (P < 0.05), but not with PD 123319. Extracellular saralasin induced no contraction. 8. Intracellular Ang II induced contraction was not affected by pre-treatment with heparin filled liposomes, but completely abolished in Ca2+-free external medium. 9. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+-influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+-stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In mammals, it is well know that resumption of meiosis occurs after the preovulatory LH surge and results in germinal vesicle breakdown (GVBD), initiating the so-called oocyte maturation. However, the pathway by which this gonadotrophin acts is not completely clear. We have recently demonstrated that AngII plays an important role on the onset of ovulation in cattle, potentially acting as an intrafollicular LH mediator. We also observed that AngII prevents the inhibitory effect of follicular cells during bovine oocyte nuclear maturation in vitro. These results suggest that AngII plays a role in LH-induced resumption of meiosis in the bovine oocyte. The aim of this study was to verify the involvement of AngII in LH-induced meiosis resumption and test the hypothesis that prostaglandins E2 and F2α participates of AngII-induced meiosis resumption in bovine oocytes. In the first experiment, seven cows were superovulated with FSH and follicles larger than 12 mm in diameter were subjected to an intrafollicular injection of saralasin or saline. Follicles from the right ovary (n=17) where intrafollicular injected with saralasin (10μM) and follicles from the left ovary (n=17) were treated with saline (control group). A preovulatory LH surge was induced by im injection of a GnRH agonist (gonadorelin 100μg im) following the intrafollicular injections. Fifteen hours later, the animals were ovariectomized and the oocytes were recovered to evaluate the stage of meiotic maturation. All oocytes (n=12) were at germinal vesicle stage (GV) 15 hours after GnRH agonist injection in the saralasin group while in the control group (n=13) the oocytes were at the GVBD (30.8%) or Metaphase I (MI; 69.2%; P<0.001) stage. In other experiment, oocytes were co-cultured with follicular hemisections during 15 hours, to evaluate the role of prostaglandins mediating the effect of AngII on meiotic resumption. The inhibitory effects caused by follicular cells on oocyte nuclear maturation was prevented by adding 100pM of AngII to the culture medium (26.6% MI without AngII vs. 77.5% MI with AngII; P<0.001). However, when a nonselective ciclooxigenase (COX) inhibitor (10μM of indometacin) was present in the culture system with AngII and follicular hemisections, oocytes reached MI in a percentage (13.4%) significantly lower than without indometacin (P<0.001). Furthermore, when 1μM of PGE2 or PGF2α was added to the co-culture system with follicular cells, oocyte nuclear maturation rate followed the same pattern as the high maturation rate observed in the presence of AngII (PGE2 77.4%, PGF2α 70.0% and AngII 75.0% of MI). In conclusion, these results suggest that AngII mediates meiosis resumption induced by LH surge in bovine oocytes, which is dependent of PGE2 and PGF2α production by follicular cells. / Em mamíferos, é bem estabelecido que o reinício da meiose ocorre após o pico préovulatório de LH e resulta no rompimento da vesícula germinativa (RVG), iniciando a maturação do oócito. Entretanto, a via pela qual essa gonadotrofina atua não está completamente elucidada. Nosso grupo demonstrou que a angiotensina II (AngII) apresenta uma importante função no início da ovulação em bovinos, potencialmente atuando como um mediador intrafolicular do LH. Nós também observamos que a AngII previne o efeito inibitório das células foliculares durante a maturação nuclear in vitro de oócitos bovinos. Estes resultados sugerem que a AngII apresenta uma função importante durante o reinício da meiose induzido pelo LH em oócitos bovinos. Portanto, os objetivos deste estudo foram verificar a participação da AngII no reinício da meiose induzido pelo pico ovulatório de LH, e investigar o envolvimento das prostaglandinas E2 e F2α como mediadores da AngII para desencadear o reinício da meiose em oócitos bovinos. No primeiro experimento, sete vacas foram superovuladas com FSH e os folículos maiores que 12mm de diâmetro foram submetidos a uma injeção intrafolicular de saralasina ou NaCl 0,9%. Os folículos do ovário direito (n=17) receberam uma injeção intrafolicular de saralasina (10μM) e os do ovário esquerdo (n=17) foram injetados com NaCl 0,9% (grupo controle). Um pico de LH foi induzido pela administração IM de um agonista do GnRH (gonadorelina 100μg) imediatamente após as injeções intrafoliculares. Quinze horas após, os animais foram ovariectomizados e os oócitos foram recuperados para avaliar o estádio da maturação nuclear. Todos os oócitos do grupo saralasina (n=12) estavam no estádio de vesícula germinativa (VG) 15 horas após a administração IM de um agonista do GnRH enquanto que no grupo controle (n=13) os oócitos estavam no estádio de RVG (30,8%) ou Metáfase I (MI; 69,2%; P<0,001). Em outro experimento, oócitos foram co-cultivados com metades foliculares durante 15 horas para avaliar a participação das prostaglandinas como mediadores do efeito da AngII sobre o reinício da meiose. O efeito inibitório causado pelas células foliculares sobre a maturação nuclear do oócito foi prevenido pela adição de 100pM de AngII ao meio de cultivo (26,6% de MI sem AngII vs. 77,5% de MI com AngII; P<0,001). Entretanto, quando um inibidor não seletivo da COX (10μM de indometacina) foi adicionado ao sistema de cultivo contendo AngII e metades foliculares, os oócitos atingiram MI em uma percentagem (13,4%) significativamente mais baixa que sem indometacina (P<0,001). Além disso, quando 1μM de PGE2 ou PGF2α foram adicionados ao sistema de co-cultivo in vitro com metades foliculares, a taxa de maturação nuclear dos oócitos seguiu o mesmo padrão observado na presença de AngII (PGE2 77,4%, PGF2α 70,0% e AngII 75,0% de MI). Portanto, este estudo demonstra que o reinício da meiose em oócitos bovinos, induzido pelo pico ovulatório de LH, requer AngII, e que as prostaglandinas E2 e F2α participam dessa ação.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this work was to investigate the role of angiotensin II (Ang II) in the mechanism of ovulation in the bovine, using an in vivo model, through the injection of the Ang II receptor antagonists in mature follicles. The animals were pre-synchronized and when the follicles reached a minimum diameter of 12mm, they received the treatments and were challenged with an IM application of GnRH-analogous. The intrafolicular application of 100 μM of saralasin blocked the ovulation only before estrous; therefore, before the LH surge (14.3% e 83.3% cows ovulated in the saralasin and control group, respectively; P<0.05). Based in these results, a second experiment was carried out to determine the moment in which Ang II plays critical role in the ovulation. Saralasin blocked ovulation only when applied at 0 and 6 hours (16.7 e 42.9% in the 0 and 6 hours groups, respectively) but not at 12 hours (100%) after GnRh-analogous treatment (P<0.001). To determine which Ang II receptor is involved in the LH-induced ovulation, an intrafolicular application of losartan (AT1-Ang II receptor-antagonist), PD123,319 (AT2 antagonist), losartan+PD123,319 or saline was performed at the moment in which the cows were challenged with GnRH-analogous. The ovulation was inhibited by PD123,319 and losartan+PD123,319 application (50.0 e 33.3% on ovulation rate, respectively), but not by the application of losartan or saline solution (100% in both the groups). The results demonstrated that Ang II plays a basic role in the early mechanism of bovine ovulation via AT2 receptor subtype. / O presente trabalho teve por objetivo averiguar o papel da angiotensina II (Ang II) no mecanismo de ovulação em bovinos, utilizando um modelo in vivo através da injeção de inibidores da AngII em folículos pré-ovulatórios. Os animais foram pré-sincronizados e quando os folículos atingiram um diâmetro mínimo de 12mm, receberam os tratamentos e foram desafiados com uma aplicação IM de análogo de GnRH. A aplicação intrafolicular de 100μM de saralasina bloqueou a ovulação somente quando realizada antes do início do cio, ou seja, antes do pico de LH (14,3% e 83,3% das vacas ovularam nos grupos saralasina e controle, respectivamente; P<0,05). Baseado nesses resultados, foi delineado um segundo experimento para determinar o momento em que a Ang II desempenha papel crítico na ovulação. Quando a saralasina foi aplicada 0 e 6 horas após a aplicação do análogo do GnRH, houve um bloqueio da ovulação (16,7% e 42,9%, para os grupos 0 e 6 horas, respectivamente), mas não quando esta foi aplicada 12 horas após a aplicação de GnRH (100%; P<0,001). Para determinar qual receptor está envolvido no mecanismo de ovulação induzido por Ang II, foi realizada uma aplicação intrafolicular de losartan (inibidor dos receptores AT1 de Ang II), PD123,319 (inibidor AT2), losartan+PD123,319 ou solução fisiológica em folículos pré-ovulatórios desafiados com GnRH. A ovulação foi inibida pela aplicação de PD123,319 e losartan+PD123,319 (50% e 33,3%, respectivamente), mas não pela aplicação de losartan ou solução fisiológica (100% em ambos os grupos). Os resultados apresentados demonstram que a Ang II desempenha um papel fundamental na regulação da ovulação em bovinos via receptores AT2.

Hemos inducido en ratas wistar dos modelos de hipertensión arterial (HTA): hipertensión vasculorrenal 2 riñones-1 clip (2R-1C) e hipertensión por déficits crónico de óxido nítrico (NO). La angiotensina II (AII) participa en el desarrollo y mantenimiento de la hipertensión vasculorrenal y por déficit crónico de NO. La administración crónica de losartán, aún previniendo el desarrollo de la hipertensión por déficit crónico de NO, no confiere protección completa frente a las alteraciones hemodinámicas que dicha hipertensión conlleva. Los bloqueantes de los receptores de la AII, saralasina y losartán, poseen efectos hemodinámicos similares, aunque de distinta potencia, sobre los grupos de HTA. En condiciones normales, los efectos hemodinámicos del péptido natriurético auricular (PNA) no son modulados por los receptores de AII. La HTA potencia los efectos hemodinámicos del PNA por un efecto en el que intervienen los receptores de AII. / We have induced in Wistar rats two models of experimental hypertension: two kidney-one clip hypertension (2K-1C) and hypertension induced by chronic inhibition of nitric oxide production (L-NAME-induced hypertension). In both, angiotensin II (AII) participates in the development and maintenance of the hypertension. Chronic administration of losartan prevents the development of the L-NAME-induced hypertension, although it does not confer complete protection to the hemodynamic alterations that this hypertension causes. The AII receptor antagonists, saralasin and losartan, have similar hemodynamic effects on the hypertensive groups, although in different degrees. Moreover, the hemodynamic effects of the atrial natriuretic peptide (ANP) under normal conditions are not modulated by the AII receptor. However, hypertension increases the hemodynamic effects of the ANP by an effect partially due to the AII receptors