Influence of antihypertensive drugs on aortic and coronary effects of Ang-(1-7) in pressure-overloaded rats

This study investigated the influence of antihypertensive drugs, such as angiotensin-converting enzyme inhibitors (ACEIs), AT1 receptor blockers (ARBs), voltage-gated L-type calcium channel blockers, and mineralocorticoid receptor antagonists (MRAs), on the effects of angiotensin-(1-7) [Ang-(1-7)] on aorta and coronary arteries from pressure-overloaded rats. Pressure overload was induced by abdominal aortic banding (AB). To evaluate the role of antihypertensive drugs on the effect of Ang-(1-7), AB male Wistar rats weighing 250–300 g were treated with vehicle or low doses (5 mg·kg-1·day-1, gavage) of losartan, captopril, amlodipine, or spironolactone. Isolated aortic rings and isolated perfused hearts under constant flow were used to evaluate the effect of Ang-(1-7) in thoracic aorta and coronary arteries, respectively. Ang-(1-7) induced a significant relaxation in the aorta of sham animals, but this effect was reduced in the aortas of AB rats. Chronic treatments with losartan, captopril or amlodipine, but not with spironolactone, restored the Ang-(1-7)-induced aorta relaxation in AB rats. The coronary vasodilatation evoked by Ang-(1-7) in sham rats was blunted in hypertrophic rats. Only the treatment with losartan restored the coronary vasodilatory effect of Ang-(1-7) in AB rat hearts. These data support a beneficial vascular effect of an association of Ang-(1-7) and some antihypertensive drugs. Thus, this association may have potential as a new therapeutic strategy for cardiovascular diseases.

Angiotensin- (1-7) inhibits inflammation and oxidative stress to relieve lung injury induced by chronic intermittent hypoxia in rats

Obstructive sleep apnea is associated with inflammation and oxidative stress in lung tissues and can lead to metabolic abnormalities. We investigated the effects of angiotensin1–7 [Ang-(1–7)] on lung injury in rats induced by chronic intermittent hypoxia (CIH). We randomly assigned 32 male Sprague-Dawley rats (180–200 g) to normoxia control (NC), CIH-untreated (uCIH), Ang-(1–7)-treated normoxia control (N-A), and Ang-(1–7)-treated CIH (CIH-A) groups. Oxidative stress biomarkers were measured in lung tissues, and expression of NADPH oxidase 4 (Nox4) and Nox subunits (p22phox, and p47phox) was determined by Western blot and reverse transcription-polymerase chain reaction. Pulmonary pathological changes were more evident in the uCIH group than in the other groups. Enzyme-linked immunosorbent assays and immunohistochemical staining showed that inflammatory factor concentrations in serum and lung tissues in the uCIH group were significantly higher than those in the NC and N-A groups. Expression of inflammatory factors was significantly higher in the CIH-A group than in the NC and N-A groups, but was lower than in the uCIH group (P<0.01). Oxidative stress was markedly higher in the uCIH group than in the NC and N-A groups. Expression of Nox4 and its subunits was also increased in the uCIH group. These changes were attenuated upon Ang-(1–7) treatment. In summary, treatment with Ang-(1-7) reversed signs of CIH-induced lung injury via inhibition of inflammation and oxidative stress.

Hidrolisado proteico da semente de cupuaçu como fonte de peptídeos inibidores da enzima conversora da angiotensina I / Hydrolyzed cupuassu seed protein as a source of angiotensin I- converting enzyme inhibitory peptides

Peptídeos com ação inibitória da enzima conversora da angiotensina I (ECA) podem ser obtidos a partir de diversos alimentos e exercer efeito anti-hipertensivo. O cupuaçu (Theobroma grandiflorum S.), fruto nativo da Amazônia, possui sementes comestíveis contendo proteína de reserva similar à do cacau (Theobroma cacao L.), as quais parecem ser fonte de peptídeos inibidores da ECA. Desse modo, o objetivo deste trabalho foi investigar in vitro a ocorrência de peptídeos inibidores da ECA no hidrolisado proteico da semente de cupuaçu obtido por ação da Alcalase. O hidrolisado revelou o desaparecimento de proteínas entre 27 a 180 kDa, incluindo as globulinas, e o surgimento daquelas abaixo de 15 kDa após 2 h de hidrólise, indicando a formação de peptídeos menores. O ensaio de atividade utilizando o substrato Abz-FRK(Dnp)-P-OH revelou que o hidrolisado total promoveu 65% de inibição da ECA e esse pool peptídico foi fracionado em cinco frações (F1-F5) por cromatografia em fase reversa (RP-HPLC). Após a etapa de purificação, o monitoramento da inibição apontou, ao final, duas frações (3.2.8 e 3.4.10) com maior atividade inibitória. Oito peptídeos foram identificados por LC-MS/MS, sendo três deles já conhecidos como inibidores da ECA. Outros cinco novos peptídeos identificados (FLEK, GSGKHVSP, LDNK, MVVDKLF e MEKHS) foram sintetizados e tiveram sua ação inibitória validada por ensaios in vitro. O peptídeo GSGKHVSP apresentou a menor IC50 (3,11 µM) e Ki (0,74 µM), sendo um inibidor tipo misto quanto ao seu mecanismo de inibição revelado pelo gráfico de Lineweaver-Burk. Os resultados permitem concluir que o isolado proteico da semente de cupuaçu pode ser uma fonte para obtenção de peptídeos anti-hipertensivos, a despeito de serem necessárias investigações sobre a resistência desses peptídeos à digestão gastrointestinal e a eficácia da inibição in vivo

Peptides with angiotensin I-converting enzyme (ACE) inhibitory activity may be obtained from several foods and cause antihypertensive effect. Cupuassu (Theobroma grandiflorum S.), a native fruit from Amazon, has edible seeds with a storage protein similar to that of cocoa (Theobroma cacao L.) which seems to have incrypted ACE inhibitor peptides. Thus, the aim of this project was to investigate the in vitro formation of ACE inhibitory peptides in protein hydrolysate from cupuassu seeds using Alcalase enzyme. The hydrolysate revealed the disappearance of proteins between 27 and 181 kDa after 2h hydrolysis, including the globulin, and the increase of those below 15 kDa, indicating the formation of peptides. The ACE inhibitory activity assays using the substrate Abz-FRK(Dnp)P-OH revealed the hydrolysate had 65% ACE inhibition and the pool of peptides was fractionated into five fractions (F1-F5) by reversed phase high-performance liquid chromatography (RP-HPLC). After the purification step, two fractions (3.2.8 e 3.4.10) exhibited the highest ACE-inhibitory activity. Eight peptides had been identified by LC-MS/MS and three of them were ACE inhibitors. The other newly identified peptides (FLEK, GSGKHVSP, LDNK, MVVDKLF and MEKHS) were synthesized and in vitro assayed for ACE inhibitory activity. The peptide GSGKHVSP had the lower IC50 (3.11 µM) and Ki (0.74 µM). Lineweaver-Burk plots suggest this peptide is a mixed-type inhibitor according to the inhibition mechanism. The results indicate that protein isolate from cupuassu seeds may be a good protein source of antihypertensive peptides and further investigation is needed in order to evaluate the resistance of these peptides to gastrointestinal digestion and the inhibitory activity in vivo

Angiotensina-(1-9) disminuye el daño renal en la hipertensión arterial renina independiente / Angiotensin (1-9) decreases renal damage in hypertension with low renin

Antecedentes: Angiotensina [Ang-(1-9)] disminuye la presión arterial (PA) y el remodel amiento cardíaco en la hipertensión arterial experimental independiente de renina. No hay antecedentes sobre el efecto de Ang-(1-9) en la progresión de daño renal de ratas hipertensas por expansión de volumen (con renina baja). Objetivo: Determinar la participación de Ang-(1-9) en la progresión del daño renal en ratas hipertensas (DOCA-sal). Métodos: Se utilizaron ratas Sprague Dawley macho de 150 +/- 10 g uninefrectomizadas tratadas con DOCA (60mg/Kg/2 veces sem, im) por 4 semanas. Como controles (Sham) se usaron ratas uninefrectomizadas. Desde la 2a semana las ratas DOCA, con presión arterial sistólica (PAS) > 140 mmHg, recibieron vehículo o Ang-(1-9) [602 ng/Kg min] por 2 semanas (minibomba Alzet). Se evaluó la respuesta inflamatoria y el daño renal profibrótico por la presencia de macrófagos infiltrativos y de mio-fibroblastos intersticiales. Se determinó, además, la presión arterial sistólica (PAS), masa corporal (MC), masa del riñón derecho (MR) y masa renal relativa (MRR). Resultados: Se observó una disminución del daño renal en las ratas DOCA-sal, cuando recibieron Ang-(1-9), respecto a aquellas que no la recibieron, evidenciado por una significativa disminución de macrófagos infiltrativos y miofibroblastos en el intersticio renal. El bloqueo de los receptores Mas y AT2, no tuvieron efectos adicionales. Conclusion: En este modelo experimental, Ang-(1-9) disminuyó la hipertensión y redujo significativamente la infiltración de macrófagos y la aparición de miofibroblastos en el intersticio renal. Estos resultados son la primera evidencia de que Ang-(1-9) reduce la fibrosis túbulo-intersticial renal y el daño renal hipertensivo.

Angiotensin [Ang] - (1-9) decreases blood pressure (BP) and cardiac remodeling in renin independent hypertension. There are not studies about the effect of Ang- (1-9) in the progression of hypertensive renal damage by volume overload (low-renin). The aim of this study was to determine the effect of Ang- (1-9) on renal damage in volume overload hypertensive rats by (DOCA-salt rats). Methods: Male Sprague Dawley rats, 150 +/- 10 g, were uni-nephrectomized and treated with DOCA (60mg/Kg) 2 times per week, for 4 weeks. Uninephrec-tomized rats were used as controls (Sham). From the 2nd week on DOCA rats with systolic blood pressure (SBP)> 140 mmHg received vehicle or Ang- (1-9) [602 ng / kg min] for 2 weeks (Alzet minipump). Inflammatory and profibrotic renal damage was evaluated by the presence of infiltrating macrophages and interstitial myofibroblasts. Results: Compared to rats receiving vehicle renal damage in DOCA-salt rats decreased when they received Ang- (1-9), as evidenced by a significant decrease in infiltrating macrophages and myofibroblasts in the renal interstitium. Mas and AT2 receptor blockade had no additional effect. The SBP, body mass (BM), mass of the right kidney (MR) and relative renal mass (MRR) were also determined. Conclusion: in this experimental model, Ang-(1-9) decreased hypertension and significantly reduced macrophage infiltration and the appearance of myofibroblasts in the renal interstitium. These results are the first evidence that Ang-(1-9) reduces renal tubulointerstitial fibrosis and hypertensive renal damage.

The pressor effect of angiotensin-(1-7) in the rat rostral ventrolateral medulla involves multiple peripheral mechanisms

OBJECTIVE: In the present study, the peripheral mechanism that mediates the pressor effect of angiotensin-(1-7) in the rostral ventrolateral medulla was investigated. METHOD: Angiotensin-(1-7) (25 pmol) was bilaterally microinjected in the rostral ventrolateral medulla near the ventral surface in urethane-anesthetized male Wistar rats that were untreated or treated (intravenously) with effective doses of selective autonomic receptor antagonists (atenolol, prazosin, methyl-atropine, and hexamethonium) or a vasopressin V1 receptor antagonist [d(CH2)5 -Tyr(Me)-AVP] given alone or in combination. RESULTS: Unexpectedly, the pressor response produced by angiotensin-(1-7) (16 ± 2 mmHg, n = 12), which was not associated with significant changes in heart rate, was not significantly altered by peripheral treatment with prazosin, the vasopressin V1 receptor antagonist, hexamethonium or methyl-atropine. Similar results were obtained in experiments that tested the association of prazosin and atenolol; methyl-atropine and the vasopressin V1 antagonist or methyl-atropine and prazosin. Peripheral treatment with the combination of prazosin, atenolol and the vasopressin V1 antagonist abolished the pressor effect of glutamate; however, this treatment produced only a small decrease in the pressor effect of angiotensin-(1-7) at the rostral ventrolateral medulla. The combination of hexamethonium with the vasopressin V1 receptor antagonist or the combination of prazosin, atenolol, the vasopressin V1 receptor antagonist and methyl-atropine was effective in blocking the effect of angiotensin-(1-7) at the rostral ventrolateral medulla. CONCLUSION: These results indicate that angiotensin-(1-7) triggers a complex pressor response at the rostral ventrolateral medulla that involves an increase in sympathetic tonus, release of vasopressin and possibly the inhibition of a vasodilatory mechanism.

Efeitos da angiotensina-I e isquemia na recuperação funcional em corações isolados / Effects of angiotensin-I and ischemia on functional recovery in isolated hearts

FUNDAMENTO: A ressuscitação de parada cardíaca pode apresentar disfunção miocárdica determinada pelo tempo da isquemia, e a inibição da enzima conversora de angiotensina (ECA) pode reduzir a disfunção cardíaca durante a reperfusão. OBJETIVO: Investigar os efeitos da angiotensina-I e diferentes períodos de isquemia na recuperação funcional em corações de ratos isolados. MÉTODOS: Os corações isolados de ratos Wistar (n = 45; 250-300 g) foram submetidos a diferentes períodos de isquemia global (20, 25 ou 30 min) e reperfundidos (30 min) com o tampão Krebs-Henseleit, ou com a adição de 400 nmol/L de angiotensina-I, ou com 400 nmol/L de angiotensina-I + 100 µmol/L de captopril durante o período de reperfusão. RESULTADOS: A derivada positiva máxima de pressão (+dP/dt max) e o produto frequência-pressão foram reduzidos nos corações expostos à isquemia de 25 min (~ 73 por cento) e à isquemia de 30 min (~ 80 por cento) vs. isquemia de 20 min. A pressão diastólica final do ventrículo esquerdo (PDFVE) e a pressão de perfusão (PP) foram aumentadas nos corações expostos à isquemia de 25 min (5,5 e 1,08 vezes, respectivamente) e à isquemia de 30 min (6 e 1,10 vezes, respectivamente) vs. isquemia de 20 min. A angiotensina-I ocasionou uma diminuição no +dP/dt max e no produto frequência-pressão (~ 85-94 por cento) em todos os períodos de isquemia e um aumento na PDFVE e na PP (6,9 e 1,25 vezes, respectivamente) apenas na isquemia de 20 min. O captopril foi capaz de reverter parcial ou completamente os efeitos da angiotensina-I na recuperação funcional nas isquemias de 20 e 25 min CONCLUSÃO: Os dados sugerem que a angiotensina-II participa direta ou indiretamente no dano pós-isquêmico e que a capacidade de um inibidor da ECA atenuar esse dano depende do tempo de isquemia.

BACKGROUND: Cardiac arrest resuscitation can present myocardial dysfunction determined by ischemic time, and inhibition of the angiotensin-converting enzyme (ACE) can reduce cardiac dysfunction during reperfusion. OBJECTIVE: To investigate the effects of angiotensin-I and different periods of ischemia on functional recovery in isolated rat hearts. METHODS: Isolated hearts from Wistar rats (n=45; 250-300 g) were submitted to different periods of global ischemia (20, 25 or 30 min) and reperfused (30 min) with Krebs-Henseleit buffer alone or with the addition of 400 nmol/L angiotensin-I, or 400 nmol/L angiotensin-I + 100 mmol/L captopril along the reperfusion period. RESULTS: The maximal positive derivative of pressure (+dP/dt max) and rate-pressure product were reduced in hearts exposed to 25 min ischemia (~73 percent) and 30 min ischemia (~80 percent) vs. 20 min ischemia. Left ventricular end-diastolic pressure (LVEDP) and perfusion pressure (PP) were increased in hearts exposed to 25 min ischemia (5.5 and 1.08 fold, respectively) and 30 min ischemia (6 and 1.10 fold, respectively) vs. 20 min ischemia. Angiotensin-I caused a decrease in +dP/dt max and rate-pressure product (~85-94 percent) in all ischemic periods and an increase in LVEDP and PP (6.9 and 1.25 fold, respectively) only at 20 min ischemia. Captopril was able to partially or completely reverse the effects of angiotensin-I on functional recovery in 20 min and 25 min ischemia. CONCLUSION: These data suggest that angiotensin-II directly or indirectly participates in the post-ischemic damage, and the ability of an ACE inhibitor to attenuate this damage depends on ischemic time.

Angiotensin-(1-7) increases osmotic water permeability in isolated toad skin

Angiotensin-(1-7) (Ang-(1-7)) increased osmotic water permeability in the isolated toad skin, a tissue with functional properties similar to those of the distal mammalian nephron. Concentrations of 0.1 to 10 ÁM were effective, with a peak at 20 min. This effect was similar in magnitude to that of frog skin angiotensin II (Ang II) and oxytocin but lower than that of human Ang II and arginine-vasotocin. The AT2 angiotensin receptor antagonist PD 123319 (1.0 ÁM) fully inhibited the response to 0.1 ÁM Ang-(1-7) but had no effect on the response to Ang II at the same concentration. The specific receptor antagonist of Ang-(1-7), A-779, was ineffective in blocking the response to Ang-(1-7) and to frog skin Ang II. The AT1 receptor subtype antagonist losartan, which blocked the response to frog skin Ang II, was ineffective in blocking the response to Ang-(1-7). The present results support the view of an antidiuretic action of Ang-(1-7) in the mammalian nephron

Angiotensin-(1-7) potentiates the coronary vasodilatatory effect of bradykinin in the isolated rat heart

It has been shown that angiotensin-(1-7) (Ang-(1-7)) infusion potentiates the bradykinin (BK)-induced hypotensive response in conscious rats. The present study was conducted to identify Ang-(1-7)-BK interactions in the isolated rat heart perfused according to the Langendorff technique. Hearts were excised and perfused through the aortic stump under a constant flow with Krebs-Ringer solution and the changes in perfusion pressure and heart contractile force were recorded. Bolus injections of BK (2.5, 5, 10 and 20 ng) produced a dose-dependent hypotensive effect. Ang-(1-7) added to the perfusion solution (2 ng/ml) did not change the perfusion pressure or the contractile force but doubled the hypotensive effect of the lower doses of BK. The BK-potentiating Ang-(1-7) activity was blocked by pretreatment with indomethacin (5 mg/kg, ip) or L-NAME (30 mg/kg, ip). The Ang-(1-7) antagonist A-779 (50 ng/ml in Krebs-Ringer) completely blocked the effect of Ang-(1-7) on BK-induced vasodilation. These data suggest that the potentiation of the BK-induced vasodilation by Ang-(1-7) can be attributed to the release of nitric oxide and vasodilator prostaglandins through an Ang-(1-7) receptor-mediated mechanism.

The hyperglycemia induced by angiotensin II in rats is mediated by AT1 receptors

We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33 per cent of the initial values, P<0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 µmol/100 g body weight as a bolus, iv, plus a 30-min infusion of 0.018 µmol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P<0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 ñ 0.28 mM, angiotensin II, N = 9 vs 6.4 ñ 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.

Rol de la ECA en la citoproteccion gastrica: mecanismo dopaminérgico y *2 adrenérgico periférico / Role of ACE in the gastric cytoprotection: dopaminergic and peripheral *2 adrenergic mechanisms

En ratas Wistar, se llevaron a cabo cuatro experiencias, donde fueron estudiados los bloqueadores de la ECA: captopril, enalapril, lisinopril, ramipril y cilazapril ante la injuria del etanol absoluto en mucosa gástrica. Asimismo, se estudió el efecto del Lisinopril y la Angiotensina I ante la agresión del etanol al 20 y 95%. Por otro lado, se estudiaron drogas citoprotectoras, de conocido mecanismo de acción como el enprostil, paracetamol, ketotifeno, levamisol, diazepan, bromocriptina, dopamina y clonidina, con posterior injuria con etanol al 95%. Por fin, se estudió el rol de la ECA previo pretratamiento con Lisinopril, seguido por el tratamiento de la mucosa gástrica con las drogas citoprotectoras estudiadas y posterior injuria con etanol al 95%. Se concluyó que todas las drogas bloqueadoras de la ECA agravaron las lesiones gástricas inducidas por etanol al 20 y 95%, en forma similar a la Angiotensina I. Por otro lado, la ECA como integrante de la barrera defensiva gástrica, actuaría como regulador de la microcirculación, por un doble mecanismo, a través de las aminas vasopresoras Angiotensina I y II y por otro, activando receptores vasoactivos, como son los dopaminérgicos DA2 y los alfa2 adrenérgicos periféricos

Mecanismos de acción de angiotensina II sobre la liberación de ACTH / Mechanism of action of angiotensin II in ACTH liberation

El presente trabajo fue desarrollado, utilizando sistemas in vitro e in vivo con el objeto de comparar la actividad liberadora de ACTH (ALC) de angiotensina II (A II) con la respectiva de otros péptidos neurales con ALC intrínseca, tales como el factor liberador de ACTH (CRF), arginina-vasopresina (AVP) y ocitocina (OXY), y determinar el sitio más probable de acción de A II para inducir liberación de ACTH. Inyecciones intraperitoneales de A II en ratas macho intactas aumentaron los niveles circulantes de ACTH, aunque dicha respuesta no fue tan pronunciada como las que produjeron menores dosis molares de CRF y AVP. La inyección intravenosa (i.v.) de A II en ratas macho centralmente bloqueadas no modificó los valores plasmáticos de ACTH, cuando CRF y AVP aumentó varias veces los valores de esta hormona. Además, las inyecciones i.v. de A II en ratas hembra deficientes de AVP (Brattleboro, D.I.) indujeron respuestas semejantes a las obtenidas en ratas hembras controles (Long Evans, L. E. y Sprague-Dawley, S-D), donde se incrementaron significativamente los valores de ACTH plasmática. Las ratas D.I. hiper respondieron a la inyección i.v. de CRF si se compara con la respuesta obtenida en las ratas control apropiadas (L.E.). Los estudios in vitro sugieren que A II es capaz de liberar ACTH de células adenohipofisarias dispersas obtenidas de donantes machos adultos en el rango 10**8 a 10**6 M. OXY fue capaz de liberar ACTH a la concentración 10**8 M. Las curvas respuesta-dependientes de la concentr