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.

In der vorliegenden Untersuchung wurde der Einfluss des AT1-R -Antagonisten Valsartan auf die Nierenfunktion bei nierentransplantierten Ratten mit der Fragestellung analysiert, ob eine Langzeittherapie mit diesem Wirkstoff einen positiven Effekt auf die Nierenfunktion entfaltet und sich somit sein Einsatz gegen die Entwicklung einer chronischen Transplantatnephropathie empfiehlt. Die über den gesamten Versuchszeitraum gegenüber der allogenen Kontrollgruppe signifikant erhöhten Urinvolumina stellen allein kein Indiz für eine bessere Nierenfunktion unter Therapie mit Valsartan dar. Dieses Ergebnis ist am ehesten durch Veränderungen der glomerulären Hämodynamik post transplantationem zu erklären. Wie nunmehr in mehreren tierexperimentellen Untersuchungen und klinischen Patientenstudien nachgewiesen worden ist, zeigt sich auch in der Synopsis der eigenen Befunde ein signifikant günstigerer Verlauf des Serumkreatinins, des Serum-BUN, der Kreatinin-Clearance sowie der Proteinurie unter Blutdrucksenkung mit dem AT1-R-Antagonisten Valsartan. An einigen Zeitpunkten der Studie waren die Ergebnisse allerdings statistisch nicht signifikant. Eine positive Wirkung auf die Transplantatfunktion und auf das Langzeitüberleben der Versuchstiere ist anzunehmen, ist aber in dieser Studie nicht weiter verfolgt worden. Eine Untersuchung mit einer größeren Anzahl von Versuchstieren und über einem längeren Versuchzeitraum hin scheint sinnvoll, um signifikante Unterschiede zwischen den Kontrollgruppen und der Versuchgruppe unter Valsartan zu belegen. Die im Vergleich zu den Kontrollgruppen geringere Entwicklung des Körpergewichts hatte bei der o.g. Fragestellung keine Relevanz. Wie in zahlreichen klinischen Studien für die Progredienz des chronischen Nierenversagens seit längerem eindrucksvoll belegt ist, scheint eine pharmakologische Blockade des RAAS auch einen protektiven Effekt auf die Entstehung einer chronischen Transplantatnephropathie zu entfalten. Die eigene Untersuchung liefert hinreichend Belege für diese Vermutung. Auch wenn in einzelnen Studien über negative Auswirkungen einer Blockade des RAAS auf die Transplantatfunktion berichtet worden ist, gibt es genügend Anhaltspunkte für einen günstigeren Verlauf nach Transplantation sowohl in Tierversuchen als auch für den transplantierten Patienten. Das allmähliche Fortschreiten der chronischen Transplantatnephropathie kann damit allerdings nicht ganz aufgehalten werden. Somit bleibt trotz dieser erfolgversprechenden experimentellen Ergebnisse nach Organtransplantation durch diese neuen Therapieansätze (Immunsuppressiva, RAAS-Blockade, Plasmapherese u.a.) die chronische Transplantat-Abstoßung immer noch ein therapeutisch fortbestehendes Problem. Weitere Untersuchungen über die Zusammenhänge immunologischer sowie nicht-immunologischer Ursachen einer chronischen Transplantatnephropathie und eine Optimierung der Immunsuppression sind deshalb auch weiterhin dringend erforderlich. / In spite of the new immunosuppressive drug therapies used in renal transplants, chronic rejection continues to be a major cause of graft dysfunction after the first posttransplant year. This so called chronic allograft nephropathy is characterized by a slow but variable decrease in renal function appearing months or years after transplantation, is often accompanied or proceded by proteinuria ans hypertension and does not respond to immunosuppressive therapy. In our study we could show that posttransplant therapy with the angiotensin-II-receptor antagonist Valsartan improves chronic renal allograft nephropathy in Lewis rats. A similar effect on long-term survival of human kidney transplants can be supposed. Nevertheless further investigations have to be done in oder to understand all immunologigal and non immunological mechanisms of renal chronic rejection and to improve therapies.

The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.<p> Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme® in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.

The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.<p> Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme® in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.

The renin-angiotensin-aldosterone system (RAAS) plays an essential role in maintaining plasma volume and arterial blood pressure by regulating angiotensin II levels. Dysregulation of the RAAS can result from an underlying disorder that results in a severe and untreatable form of hypertension, known as secondary hypertension. Measurement of plasma renin activity is a commonly employed method of diagnosing secondary hypertension. Plasma renin activity is quantified by determining the amount of angiotensin I generated through the enzymatic cleavage of angiotensinogen by renin. Radioimmunoassay is routinely used to measure plasma renin activity, however there are limitations to the method. With the prevalence of hypertension on the rise, there is need for a more accurate and rapid method of assessing the RAAS for diagnostic purposes and therapeutic monitoring. Multiplexed measurement of angiotensin I and angiotensin II would provide comprehensive understanding of the RAAS by determining dysregulation in the production of either molecule. In this thesis, the relationship between endogenous angiotensin I concentrations and plasma renin activity are studied in order to examine the research hypothesis that measurement of angiotensin I concentration correlates with plasma renin activity and whether this may provide a more accurate and rapid method of screening for hypertension when multiplexed with angiotensin II. To overcome the current limitations of radioimmunoassay for measuring plasma renin activity, a mass spectrometric-based method was developed to measure angiotensin I and plasma renin activity. Evaluation of the assay against radioimmunoassay demonstrates that the assay is reproducible and provides a linear response over a diagnostically relevant concentration range. Comparison of endogenous levels of angiotensin I with normal plasma renin activity levels show a correlation in this study (R=0.74). Comparison of plasma renin activity values by radioimmunoassay and iMALDI also show correlation (R=0.98), indicating that the iMALDI assay may provide an improved method for diagnosing secondary hypertension.

INTRODUÇÃO: O infarto agudo do miocárdio (IAM) é uma doença cardiovascular que apresenta altas taxas de morbimortalidade associado à prejuízo da função cardíaca, disfunção autonômica e alteração do sistema renina angiotensina (SRA). Tem se estabelecido na literatura que a atuação do polimorfismo de genes do SRA, como o da enzima conversora da angiotensina (ECA), poderia contribuir para maior ocorrência de IAM e óbito. Dessa forma, o objetivo desse estudo foi avaliar os efeitos da variação do número de cópias do gene da ECA no infarto agudo do miocárdio. MÉTODOS: Foram utilizados camundongos machos adultos, geneticamente modificados para expressar diferentes concentrações fisiológicas da enzima conversora de angiotensina: animais com uma cópia do gene da ECA (ECA 1), que apresentam menores níveis plasmáticos de ECA; com duas cópias do gene da ECA (ECA 2), que apresentam níveis plasmáticos intermediários de ECA; com três cópias do gene da ECA (ECA 3), que apresentam maiores níveis plasmáticos de ECA. Os animais foram divididos em nove grupos (n=10): três grupos sem infarto - ECA 1, ECA 2 e ECA 3; três grupos infartados e avaliados sete dias após o infarto - ECA 1-7, ECA 2-7 e ECA 3-7; três grupos infartados e avaliados 28 dias após o infarto - ECA 1-28, ECA 2-28 e ECA 3-28. Os animais infartados foram submetidos ao IAM pela ligadura da coronária esquerda. Todos os animais passaram por avaliação de ecocardiografia, para determinar a morfometria e função cardíaca e foram canulados para registro direto da pressão arterial. A partir dos sinais de pressão arterial foram realizadas as análises da variabilidade da frequência cardíaca (VFC), da variabilidade da pressão arterial sistólica e do barorreflexo espontâneo. O tecido cardíaco dos animais infartados foi coletado para quantificação da área de infarto e de colágeno, por meio de técnicas histológicas, e o plasma foi destinado para análise do perfil inflamatório por técnica de ELISA. RESULTADOS: Quando comparados os animais não infartados, em relação aos dados ecocardiográficos, observou-se que os animais com três cópias do gene da ECA apresentaram maiores valores de relação E/A do que os animais com duas e uma cópia do gene da ECA (ECA 3: 1,80 ± 0,14 vs. ECA 2: 1,24 ± 0,16 e ECA 1: 1,08 ± 0,14). Não houve diferença entre os grupos não infartados na pressão arterial sistólica (ECA 1: 124,6 ± 4,0; ECA 2: 127,1 ± 5,62; ECA 3: 129,2 ± 3,32 mmHg) e diastólica (ECA 1: 87,90 ± 3,00; ECA 2: 86,79 ± 2,63; ECA 3: 85,08 ± 1,85 mmHg), bem como na frequência cardíaca (ECA 1: 682,7 ± 12,54; ECA 2: 654,6 ± 15,77; ECA 3: 625,6 ± 25,60 bpm). Em relação aos parâmetros autonômicos nos grupos não infartados, o componente de muito baixa frequência da VFC, em valores percentuais, foi menor no grupo ECA 1 do que no ECA 2 (ECA 1: 7,87 ± 1,98 vs. ECA 2: 18,00 ± 4,25 %) e o índice de Down Gain do barorreflexo espontâneo foi maior no grupo ECA 3 em comparação com os grupos ECA 2 e ECA 1 (ECA 3: 3,80 ± 0,50 vs. ECA 2: 2,00 ± 0,36 e ECA 1: 2,24 ± 0,30 ms/mmHg). Em relação aos dados ecocardiográficos, comparando os animais não infartados e os animais infartados avaliados sete dias após o infarto, observou-se que: a relação E/A foi maior no grupo ECA 3-7 do que nos grupos não infartados com uma e duas cópias e do que no grupo infartado com duas cópias do gene da ECA (ECA 3-7: 3,47 ± 0,50 vs. ECA 1: 1,08 ± 0,14, ECA 2: 1,24 ± 0,16 e ECA 2-7: 1,74 ± 0,25); a mudança de área fracional foi menor nos grupos infartados ECA 1-7, ECA 2-7 e ECA 3-7 em comparação com os não infartados (ECA 1-7: 30,10 ± 1,80, ECA 2-7: 31,12 ± 1,47 e ECA 3-7: 21,30 ± 1,47 vs. ECA 1: 37,14 ± 1,67, ECA 2: 37,16 ± 1,23 e ECA 3: 40,68 ± 1,28 %), e entre os infartados, foi menor no grupo ECA 3-7 do que nos grupos ECA 2-7 e ECA 1-7; a fração de encurtamento foi menor nos grupos ECA 3-7 e ECA 2-7 do que no grupo ECA 3 (ECA 3-7: 16,81 ± 1,6 e ECA 2-7: 17,25 ± 2,1 vs. ECA 3: 25,00 ± 1,2 %). Em relação aos parâmetros hemodinâmicos, não foram observadas diferenças na pressão arterial e frequência cardíaca entre os grupos sete dias após o infarto. Quanto aos parâmetros autonômicos comparando os animais não infartados e os animais infartados avaliados sete dias após o infarto, observou-se que: a modulação simpática foi maior no grupo ECA 3-7 do que nos grupos ECA 1, ECA 2 e ECA 1-7 (ECA 3-7: 14,85 ± 2,36 vs. ECA 1: 4,76 ± 2,00, ECA 2: 4,34 ± 1,40 e ECA 1-7: 5,13 ± 1,88 ms²); e o balanço simpatovagal foi maior no grupo ECA 3-7 do que no grupo ECA 1-7 (ECA 3-7: 2,22 ± 0,17 vs. ECA 1-7: 0,81 ± 0,25). Sete dias após o infarto, foi observado aumento no peso do coração no grupo ECA 3-7 em comparação aos grupos não infartados (ECA 3-7: 0,205 ± 0,01 vs. ECA 1: 0,135 ± 0,006, ECA 2: 0,146 ± 0,006 e ECA 3: 0,145 ± 0,008 g). Quando comparados os grupos infartados sete e 28 dias após o infarto do miocárdio observou-se que: a relação E/A foi menor no grupo ECA 2-28 do que o grupo ECA 3-7 (ECA 2-28: 1,75 ± 0,14 vs. ECA 3-7: 3,47 ± 0,50); a espessura do septo intraventricular na diástole foi menor no grupo ECA 1-28 em comparação ao grupo ECA 1-7 (ECA 1-28: 0,63 ± 0,03 vs. ECA 1-7: 0,87 ± 0,06 mm); não houve diferença entre os grupos na pressão arterial e na frequência cardíaca; o componente de baixa frequência da VFC em valores absolutos foi menor nos grupos ECA 1-28 e ECA 3-28 em comparação ao grupo ECA 3-7 (ECA 1-28: 4,09 ± 1,45 e ECA 3-28: 6,04 ± 1,03 vs. ECA 3-7: 14,85 ± 2,36 ms2); o índice de eficiência do barorreflexo foi maior no grupo ECA 1-28 do que nos grupos infartados avaliados sete dias após o infarto (ECA 1-28: 0,28 ± 0,03 vs. ECA 1-7: 0,15 ± 0,02, ECA 2-7: 0,17 ± 0,01 e ECA 3-7: 0,17 ± 0,01); a avaliação histológica mostrou maior deposição de colágeno no grupo ECA 2-7 em comparação ao grupo ECA 1-7 (ECA 2-7: 41,20 ± 2,87 vs. ECA 1-7: 23,09 ± 4,15 mm²) e menor deposição de colágeno no grupo ECA 1-28 do que o grupo ECA 2-7 (ECA 1-28: 22,09 ± 5,41 vs. ECA 2-7: 41,20 ± 2,87 mm²); o grupo ECA 1-28 apresentou menor área de infarto comparado ao grupo ECA 3-7 (ECA 1-28: 33,62 ± 2,40 vs. ECA 3-7: 48,90 ± 1,72 mm²). Em relação ao perfil inflamatório dos animais infartados sete e 28 dias após o infarto, os animais com uma cópia do gene da ECA apresentam maiores valores de IL-10 comparados aos animais com duas e três cópias do gene da ECA (ECA 1(7-28): 65,04 ± 5,90 vs. ECA 2(7-28) + ECA 3(7-28): 37,06 ± 6,00 pg/ml) e menores valores de TGF beta (ECA 1 (7-28): 91,44 ± 11,33 vs. ECA 2(7-28) + ECA 3(7-28): 122,0 ± 8,00 pg/ml). A curva de sobrevivência mostrou que os animais infartados com 3 cópias do gene da ECA apresentaram menor sobrevivência (41%) e os animais com 1 cópia do gene da ECA maior sobrevivência (71%) ao final do estudo. CONCLUSÃO: De maneira geral, os nossos resultados de função cardíaca, função autonômica cardiovascular, perfil inflamatório, área de infarto e curva de sobrevivência indicam que os animais com três cópias do gene da ECA apresentaram pior evolução após o infarto do miocárdio em relação aos animais com uma cópia do gene da ECA / INTRODUCTION: Acute myocardial infarction (AMI) is a cardiovascular disease with high morbidity and mortality rates associated with impairment of cardiac function, autonomic dysfunction, and altered renin angiotensin system (RAS). It has been established in the literature that the performance of the RAS gene polymorphism, such as angiotensin converting enzyme (ACE), could contribute to a higher occurrence of AMI and death. Thus, the objective of this study was to evaluate the effects of the variation of the number of copies of the ACE gene in the acute myocardial infarction. METHODS: Adult male mice, genetically modified to express different physiological concentrations of the angiotensin converting enzyme were used: animals with one copy of the ACE gene (ACE 1), which had lower plasma levels of ACE; with two copies of the ACE gene (ACE 2), which present intermediate plasma levels of ACE; with three copies of the ACE gene (ACE 3), which have higher plasma levels of ACE. The animals were divided into nine groups (n = 10): three groups without infarction - ACE 1, ACE 2 and ACE 3; three infarcted groups assessed seven days after infarction - ACE 1-7, ACE 2-7 and ACE 3-7; three infarcted groups assessed 28 days after infarction - ACE 1-28, ACE 2-28 and ACE 3-28. Infarcted animals were submitted to AMI by ligature of the left coronary artery. All animals underwent echocardiographic evaluation to determine morphometry and cardiac function and were cannulated for direct recording of blood pressure. The analysis of heart rate variability (HRV), systolic blood pressure variability and spontaneous baroreflex were performed from blood pressure signals. The cardiac tissue of the infarcted animals was collected for quantification of the infarct area and collagen by means of histological techniques, and the plasma was used for analysis of the inflammatory profile by ELISA technique. RESULTS: When compared non-infarcted animals, in relation to echocardiographic data, it was observed that animals with three copies of the ACE gene had higher values of E/A ratio than animals with two and one copies of the ACE gene (ACE 3 : 1.80 ± 0.14 vs. ACE 2: 1.24 ± 0.16 and ACE 1: 1.08 ± 0.14). There was no difference between the non-infarcted groups on systolic blood pressure (ACE 1: 124.6 ± 4.0, ACE 2: 127.1 ± 5.62, ACE 3: 129.2 ± 3.32 mmHg) and diastolic blood pressure (ACE 1: 87.90 ± 3.00, ACE 2: 86.79 ± 2.63, ACE 3: 85.08 ± 1.85 mmHg), as well as in heart rate (ACE 1: 682.7 ± 12, 54, ACE 2: 654.6 ± 15.77, ACE 3: 625.6 ± 25.60 bpm). Regarding the autonomic parameters in the non-infarcted groups, the very low frequency component of HRV, in percentage values, was lower in the ACE 1 group than in the ACE 2 (ACE 1: 7.87 ± 1.98 vs. ACE 2: 18.00 ± 4.25 %) and the Down Gain index of the spontaneous baroreflex was higher in the ACE 3 group compared to the ACE 2 and ACE 1 groups (ACE 3: 3.80 ± 0.50 vs. ACE 2: 2.00 ± 0.36 and ACE 1: 2.24 ± 0.30 ms/mmHg). Regarding the echocardiographic data, comparing non-infarcted animals and infarcted animals evaluated seven days after infarction, it was observed that: the E/A ratio was higher in the ACE group 3-7 than in the non-infarcted groups with one and two copies of ACE and higher than in the infarcted group with two copies of the ACE gene (ACE 3-7: 3.47 ± 0.50 vs. ACE 1: 1.08 ± 0.14, ACE 2: 1.24 ± 0.16 and ACE 2-7: 1.74 ± 0.25); the fractional area change was smaller in the infarcted groups ACE 1-7, ACE 2-7 and ACE 3-7 compared to non-infarcted (ACE 1-7: 30,10 ± 1,80, ACE 2-7: 31, 12 ± 1.47 and ACE 3-7: 21.30 ± 1.47 vs. ACE 1: 37.14 ± 1.67, ACE 2: 37.16 ± 1.23 and ACE 3: 40.68 ± 1.28 %), and among infarcts, was lower in the ACE group 3-7 than in the ACE groups 2-7 and ACE 1-7; the shortening fraction was lower in the ACE groups 3-7 and ACE 2-7 than in the ACE group 3 (ACE 3-7: 16.81 ± 1.6 and ACE 2-7: 17.25 ± 2.1 vs. ACE 3: 25.00 ± 1.2 %). Regarding hemodynamic parameters, no differences were observed in blood pressure and heart rate among the groups seven days after the infarction. Regarding the autonomic parameters comparing non-infarcted animals and infarcted animals evaluated seven days after infarction, it was observed that: sympathetic modulation was higher in the ACE group 3-7 than in the ACE 1, ACE 2 and ACE 1-7 groups (ACE 3-7: 14.85 ± 2.36 vs. ACE 1: 4.76 ± 2.00, ACE 2: 4.34 ± 1.40 and ACE 1-7: 5.13 ± 1.88 ms2); and the sympatovagal balance was higher in the ACE group 3-7 than in the ACE group 1-7 (ACE 3-7: 2.22 ± 0.17 vs. ACE 1-7: 0.81 ± 0.25). Seven days after infarction, an increase in heart weight was observed in the ACE group 3-7 compared to non-infarcted groups (ACE 3-7: 0.205 ± 0.01 vs. ACE 1: 0.135 ± 0.006, ACE 2: 0.146 ± 0.006 and ACE 3: 0.145 ± 0.008 g). When comparing infarcted groups 7 and 28 days after myocardial infarction, it was observed that: the E/A ratio was lower in the ACE group 2-28 than the ACE group 3-7 (ACE 2-28: 1.75 ± 0.14 vs. ACE 3-7: 3.47 ± 0.50); the thickness of the intraventricular septum in diastole was lower in the ACE group 1-28 compared to the ACE group 1-7 (ACE 1-28: 0.63 ± 0.03 vs. ACE 1-7: 0.87 ± 0.06 mm); there was no difference between groups in blood pressure and heart rate; the low frequency component of HRV in absolute values was lower in the ACE groups 1-28 and ACE 3-28 compared to the ACE group 3-7 (ACE 1-28: 4.09 ± 1.45 and ACE 3-28: 6.04 ± 1.03 vs. ACE 3-7: 14.85 ± 2.36 ms2); the baroreflex efficiency index was higher in the ACE group 1-28 than in the infarcted groups assessed seven days after the infarction (ACE 1-28: 0.28 ± 0.03 vs. ACE 1-7: 0.15 ± 0.02, ACE 2-7: 0.17 ± 0.01 and ACE 3-7: 0.17 ± 0.01); the histological evaluation showed greater collagen deposition in the ACE group 2-7 compared to the ACE group 1-7 (ACE 2-7: 41.20 ± 2.87 vs. ACE 1-7: 23.09 ± 4.15 mm²) and lower collagen deposition in the ACE group 1-28 than the ACE group 2-7 (ACE 1-28: 22.09 ± 5.41 vs. ACE 2-7: 41.20 ± 2.87 mm²); the ACE group 1-28 had a lower infarction area compared to the ACE group 3-7 (ACE 1-28: 33.62 ± 2.40 vs. ACE 3-7: 48.90 ± 1.72 mm²). In relation to the inflammatory profile of infarcted animals seven and 28 days after infarction, animals with one copy of the ACE gene had higher IL-10 valuescompared to animals with two and three copies of the ACE gene (ACE 1 (7- 28): 65.04 ± 5.90 vs. ACE 2 (7-28) + ACE 3 (7-28): 37.06 ± 6.00 pg/ml) and lower TGF beta values (ACE 1 (7 -28): 91.44 ± 11.33 vs. ACE 2 (7-28) + ACE 3 (7-28): 122.0 ± 8.00 pg/ml). The survival curve showed that animals infarcted with 3 copies of the ACE gene had lower survival (41%) and animals with 1 copy of the ACE gene had greater survival (71%) at the end of the study. CONCLUSION: Our results of cardiac function, cardiovascular autonomic function, inflammatory profile, infarct area and survival curve indicate that animals with three copies of the ACE gene presented a worse evolution after myocardial infarction in relation to the animals with a copy of the ACE gene

O sistema renina-angiotensina (SRA) circulante é um sistema endócrino que promove a produção de angiotensina (Ang) II, a qual exerce seus efeitos pela interação com receptores específicos. O conceito clássico do SRA circulante está sendo modificado, pois tem sido demonstrada a existência de sistemas locais capazes de gerar angiotensinas de forma independente do SRA circulante em vários tecidos e órgãos. Trabalhos recentes sugerem a existência de alguns componentes do SRA em tecido gengival e fibroblastos gengivais de diferentes espécies. Porém, não são encontrados na literatura achados inequívocos sobre a presença de importantes componentes do SRA, tais como renina e angiotensinogênio, no tecido gengival de rato. Portanto, os objetivos do presente trabalho foram: 1) estudar a expressão e localização de componentes do SRA no tecido gengival de rato e 2) estudar in vitro a funcionalidade do SRA local em homogenato de tecido gengival de rato quanto à formação de Ang II e outros peptídeos vasoativos a partir de precursores de Ang II. Transcrição reversa seguida de reação em cadeia da polimerase (RTPCR) foi utilizada para avaliar a expressão de RNAm. Análise imunohistoquímica foi utilizada para detecção e localização de renina no tecido gengival de rato. Um método fluorimétrico padronizado com o tripeptídeo Hipuril-Histidina-Leucina (Hip-His-Leu) foi usado para medir a atividade da ECA em homogenatos de tecido gengival de rato. A técnica de cromatografia líqüida de alto desempenho (HPLC) foi usada para analisar os produtos formados após a incubação de homogenatos de tecido gengival de rato com Ang I ou tetradecapeptídeo substrato de renina (TDP). RT-PCR revelou a expressão de RNAm para renina, angiotensinogênio, ECA e receptores de Ang II (AT1a, AT1b e AT2) em tecido gengival; em fibroblastos cultivados de tecido gengival foi observada expressão de RNAm para renina, angiotensinogênio e receptor AT1a. A técnica de imunohistoquímica demonstrou a existência de renina em vasos de tecido gengival de rato. Atividade da ECA foi detectada por meio do ensaio fluorimétrico (4,95±0,89 nmol His-Leu/g.min). Quando Ang I foi usada como substrato, análises de HPLC mostraram a formação de Ang 1-9 (0,576±0,128 nmol/mg.min), Ang II (0,066±0,008 nmol/mg.min) e Ang 1-7 (0,111±0,017 nmol/mg.min), enquanto que os mesmos peptídeos (0,139±0,031; 0,206±0,046 e 0,039±0,007 nmol/mg.min, respectivamente) e Ang I (0,973±0,139 nmol/mg.min) foram formados quando TDP foi usado como substrato. Adicionalmente, análises de HPLC revelaram a ausência de enzimas que degradam Ang II em homogenatos de tecido gengival de rato. Em conclusão, os resultados apresentados neste trabalho mostram claramente a existência de um SRA local em tecido gengival de rato, que é capaz de gerar Ang II e outros peptídeos vasoativos in vitro. Estudos adicionais são necessários para elucidar o papel deste sistema local no tecido gengival de rato. / Systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through the interaction with specific receptors. The concept of this classic circulating RAS has been modified since there is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of the circulating RAS. Recent works suggest the existence of some RAS components in the gingival tissue and cultured gingival fibroblasts of different species, but there is paucity of data in the literature regarding the unequivocal existence of crucial RAS components, such as renin and angiotensinogen, in the rat gingival tissue. Therefore, the aims of the present work were to: 1) study the expression and localization of RAS components in the rat gingival tissue and 2) evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different precursors of Ang II. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess mRNA expression. Immunohistochemical (IHC) analysis aimed to detect and localize renin in the rat gingival tissue. A standardized fluorimetric method with the tripeptide Hippuryl-Histidyl-Leucine (Hip-His-Leu) was used to measure tissue ACE activity in rat gingival tissue homogenates. High performance liquid chromatography (HLPC) was used to analyze the products formed after the incubation of rat gingival tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). RT-PCR revealed the mRNA expression for renin, angiotensinogen, ACE and Ang II receptors (AT1a, AT1b and AT2) in the rat gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen and AT1a receptor. IHC demonstrated the existence of renin in vessels of the rat gingival tissue. ACE activity was detected by the fluorimetric assay (4.95±0.89 nmol His-Leu/g.min). When Ang I was used as the substrate, HPLC analyses showed the formation of Ang 1-9 (0.576±0.128 nmol/mg.min), Ang II (0.066±0.008 nmol/mg.min) and Ang 1-7 (0.111±0.017 nmol/mg.min) whereas these same peptides (0.139±0.031; 0.206±0.046 and 0.039±0.007 nmol/mg.min, respectively) and Ang I (0.973±0.139 nmol/mg.min) were formed when TDP was the substrate. Additionally, HPLC revealed absence of Ang II degrading enzymes in rat gingival tissue homogenates. In conclusion, the results presented here clearly show the existence of a local RAS in the rat gingival tissue, which is capable of generating Ang II and other vasoactive peptides in vitro. Further studies are required to elucidate the role of this system in the rat gingival tissue.

O arroz (Oryza sativa L.), principal alimento para cerca de metade da população mundial, é consumido principalmente na forma polida. Contudo, o arroz integral vem se destacando, devido principalmente aos compostos bioativos presentes nas camadas mais externas do grão. Os benefícios à saúde são atribuídos, em parte, à sua capacidade de combater radicais livres e exercer atividades biológicas, tais como a inibição de determinadas enzimas. Neste trabalho foram analisados os teores de compostos fenólicos totais (FT), solúveis (FS) e insolúveis (FI) e avaliado o efeito do cozimento de 17 genótipos de arroz integral, sete com pericarpo pigmentado e dez genótipos não-pigmentados. Ainda foi avaliada a inibição da enzima conversora de angiotensina I (ECA) e da -amilase por esses compostos, no arroz cru e cozido. O arroz pigmentado se mostrou rico em compostos fenólicos, em média da ordem de 4200 &#181;g eq. ácido ferúlico/g, devido aos seus altos teores de FS, constituídos principalmente por antocianinas e proantocianidinas. Os FI, representados principalmente pelos ácidos fenólicos contribuíram com apenas 20% dos compostos fenólicos totais. O arroz não-pigmentado contém, em média, ao redor de 1000 &#181;g eq. ácido ferúlico/g, distribuídos quase equitativamente entre a fração solúvel e insolúvel. O cozimento do arroz provocou redução nos teores de FT e alteração na proporção entre FS e FI. Essas alterações foram mais pronunciadas no arroz com pericarpo vermelho, afetando principalmente a fração solúvel. O arroz preto, contudo, manteve a proporção entre FS e FI após o cozimento. A -amilase não foi inibida de forma significativa pelos fenólicos das amostras de arroz cozido. O arroz pigmentado inibiu mais fortemente a ECA do que o arroz não-pigmentado, levando a crer que a pigmentação seja um fator importante. No entanto, entre os diferentes genótipos pigmentados, o perfil de fenólicos parece ser o fator determinante para a maior ou menor atividade inibitória. O cozimento do arroz reduziu significativamente a inibição da ECA pelos fenólicos, fato observado principalmente nos genótipos vermelhos, devido à diminuição dos teores de FS e da capacidade inibitória dos fenólicos presentes. O arroz preto se destacou por ter o maior teor de fenólicos solúveis e a maior ação inibidora da ECA, após o cozimento. / Rice (Oryza sativa L.) sustains at present about half of the world´s population. Consumption is mainly in its milled form, but brown rice has prompted further research due to bioactive compounds present in the pericarp of the grain. Some of the positive health effects have been attributed to radical scavenging activity and other biological effects such as inhibition of certain enzymes. In this study it was analyzed the contents of total, soluble and insoluble phenolic compounds of 17 different genotypes of brown rice as well as the effect of cooking. Seven genotypes had pigmented pericarp and ten were non-pigmented. In addition, the extracts from crude and cooked rice were tested for their capacity to inhibit the angiotensin I (ACE) converting enzyme and -amylase activities. Pigmented rice genotypes were highest in phenolic compounds, with an average of about 4200 &#181;g ferulic acid eq./g, due to their high contents of soluble phenolics, mostly represented by anthocyanins and proanthocyanidins. Insoluble phenolics, represented mainly by phenolic acids, contributed with only 20% of total phenolics. Non-pigmented rice showed overall lower levels of phenolics. The mean content was about 1000 &#181;g ferulic acid eq./g, almost equally distributed between the soluble and insoluble fractions. Levels of total phenolics were significantly reduced by rice cooking and proportions between soluble and insoluble fractions were altered. These alterations were more pronounced for pigmented rice, and soluble phenolics were the most affected. However, after cooking, black rice was the only that maintained the original proportion between soluble and insoluble phenolics. Alpha-amylase was not significantly inhibited by phenolics after cooking. Pigmented rice showed a potent inhibition of ACE, much higher than of non-pigmented rice, which seems to indicate that color of the pericarp is an important factor. Nevertheless, different profiles of phenolic compounds may explain why individual pigmented genotypes with similar phenolic levels can have different ACE inhibiting capacities. Rice cooking reduced significantly the inhibition of ACE by phenolics, which feature was more pronounced in pigmented rice due to the reduction of soluble phenolics and the activity of individual phenolics. Among pigmented rice, the highest soluble phenolic content and the most potent ACE inhibition after cooking was observed for black rice turning it the most distinguished notable.

Evidências sobre a presença do sistema renina-angiotensina (SRA) na medula óssea e a possível participação da enzima conversora de angiotensina-I (ECA) na regulação hematopoética tem despertado o interesse da comunidade científica. Como a ECA também é um componente chave do sistema calicreína-cininas (SCC), é possível que elementos deste sistema, possam estar envolvidos no controle hematopoético. Assim, avaliamos a participação da ECA na regulação hematopoética de animais não modificados (WT) e nocautes dos receptores B1 de cininas (KOB1). Para isso, utilizamos técnicas de cultura de células de medula óssea, a saber: os ensaios clonogênicos em soft-ágar para granulócitos e macrófagos (CFU-GM) e o sistema de cultura líquida de longa duração (CLLD). Os resultados mostraram a presença da ECA em células das CLLD e indicaram a participação da enzima na proliferação de progenitores hematopoéticos possivelmente através do controle dos níveis de AcSDKP, pois o tratamento com o tetrapeptídeo e com captopril, reduziu significativamente o número CFU-GM in vitro e in vivo. Quando adicionado às CLLD, o AcSDKP foi capaz de aumentar significativamente a expressão do mRNA da ECA, sugerindo que seus níveis possam controlar a expressão gênica desta enzima. Em relação aos animais KOB1, os resultados mostraram maior atividade da ECA, acompanhado de aumento não significativo da expressão gênica e protéica da enzima. O tratamento das CLLD de animais WT com agonistas de receptores de cininas, não alterou a expressão gênica e a atividade da ECA. Assim, nossos dados sugerem que a ECA participa da regulação hematopoética neste modelo. No entanto, novos estudos serão necessários para a elucidação dos mecanismos envolvidos na expressão e/ou controle da atividade da ECA pelos receptores de cininas. / Evidences on the presence of the renin angiotensin system in the bone marrow and the possible participation of the angiotensin-I converting enzyme (ACE) in the hematopoietic regulation have aroused interest of the scientific community. As the ACE also is a common element of the kallikrein-kinin system (KKS), it is possible that elements of KKS, can be involved in the hematopoietic control. Thus, we evaluated the participation of the ACE on the hematopoietic regulation of wild-type (WT) and kinin receptor B1 knockout mice (KOB1). For this, we use techniques of bone marrow cell culture, to know the clonogenic assays for granulocyte-macrophage (GM-CFU) and the long term bone marrow cultures (LTBMC). The results shown the presence of the ACE in cells from LTBMC and its possible participation on hematopoietic proliferation through the control of AcSDKP levels, therefore the treatment with AcSDKP and captopril, decreased significantly the GM-CFU number in vitro and in vivo. When added to the LTBMC, the AcSDKP increase significantly the expression of ACE mRNA, suggesting that its levels could control the gene expression of this enzyme. In relation to KOB1 mice, the results shown increase of the ACE activity and not significant increase of the gene and protein expression of the enzyme. The treatment of the LTBMC of WT mice with kinins receptors agonists, did not modify the gene expression and the ACE activity. Thus, our data suggesting that ACE participate of the hematopoietic regulation in this model. However, new studies will be necessary to understand the involved mechanisms in the expression and/or control of ACE activity by kinins receptors.

Peptídeos bioativos são de grande interesse tanto para a indústria farmacêutica como para a de alimentos e são obtidos a partir da hidrólise enzimática de várias fontes protéicas, como as do leite, glúten de milho, soja, e músculos de suínos, aves e peixes. Estes peptídeos podem desempenhar atividades benéficas para a saúde, entre elas, a regulação ou inibição de enzimas, com destaque à inibição da enzima conversora da angiotensina I (ECA). Esta enzima é responsável pela clivagem de dois importantes substratos envolvidos na regulação da pressão arterial, a angiotensina I e a bradicinina. Neste trabalho foi estudada a atividade inibitória da ECA em hidrolisados dos peixes tilápia tailandesa (Oreochromis niloticus - linhagem tailandesa) e corvina (Micropogonias furnien) . Os \"minced\" destes peixes foram hidrolisados com pepsina e proteases de Streptomyces griséus por 5 horas em condições ideais de pH e temperatura. A atividade inibitória foi avaliada pela medida da atividade residual da enzima sobre substrato sintético fluorescente. Os hidrolisados com um grau de hidrólise de cerca de 34% apresentaram atividade inibitória semelhante, com valor de IC50 = 0,040 e 0,036 mg proteína/mL, respectivamente para a tilápia e a corvina. Observou-se aumento da atividade inibitória com o progresso da hidrólise, sendo que este aumento também está relacionado com a especificidade das enzimas proteolíticas. Em outro experimento observou-se que os peptídeos presentes no hidrolisado de tilápia inibiram indistintamente os domínios C e N terminais da ECA, não demonstrando especificidade. A atividade inibitória dos hidrolisados foi mantida após submetê-los à ação de enzimas proteolíticas gastrointestinais, indicando a sua provável estabilidade \"in vivo\". Da mesma forma, o minced de tilápia quando incubado sucessivamente com pepsina/tripsina/quimotripsina produziu peptídeos ativos (IC50 = O,025mg proteína/mL). Apesar do reduzido grau de hidrólise obtido neste ensaio (17%), os peptídeos liberados apresentaram atividade inibitória elevada, confirmando que a atividade não está relacionada apenas com o grau de hidrólise, mas também com a sequência de aminoácidos, liberados em função da especificidade das enzimas. Concluiu-se que o consumo destes peixes, ou seus respectivos hidrolisados poderá eventualmente, auxiliar na prevenção e no tratamento não medicamentoso da hipertensão, embora estudos \"in vivo\" sejam necessários para comprovar as suas funções biológicas. / Bioactive peptides have been highly valued by pharmaceutical industries and by food industries as well. These peptides can be released by the enzymatic hydrolysis of various protein sources such as milk, com gluten, soybean, muscles of pigs, chicken and fish and have been recognized to exhibit important health benefits. Among these, the regulation or inhibition of enzymes, like the inhibition of the angiotensin I-converting enzyme (ACE) have been focused. This enzyme is responsible for the cleavage of two important substrates, angiotensin I and bradykinin, both of them involved with the regulation of blood pressure. In this study the inhibitory activity of hydrolysates from tilapia tailandesa (Oreochromis niloficus - linhagem tailandesa) and corvina (Micropogonias furnien) was evaluated. The hydrolysates of the minced fishes were produced in a 5-hour lasting controlled process under optimal conditions of pH and temperature by the sequential action of pepsin and enzymes from Streptomyces griseus. Inhibitory activity was evaluated by measuring the residual activity of the enzyme on a synthetic fluorescent peptidic substrate. The extent of hydrolysis was about 34% and the hydrolysates of tilapia and corvine showed similar inhibitory activity (IC50 of 0.040 and 0.036 mg protein/mL), respectively. An íncrease of activity proportional to the degree of hydrolysis was observed, as well as a relationship with the specificity of the enzymes used. In another experiment it was observed that the bioactive peptides present in the hydrolysate of tilapia did not show specificity for the C- and N-terminal catalytic domains of the angiotensin I-converting enzyme. The inhibitory activity of the hydrolysates was still active afier a further hydrolysis by gastrointestinal enzymes, which seems to indicate an eventual activity in vivo. In a similar way, when the minced tilapia was consecutively incubated with pepsine, trypsin, chymotrypsin active peptides were produced with an activity of IC50 = 0.025 mg protein/mL. Despite the low extend of hydrolysis of about 17%, a high inhibitory activity was observed confirming that activity is not only related to the degree of hydrolysis but also to the sequence of aminoacids and therefore, to the specificity of the enzymes as well. It was concluded that the intake of fish or fish hydrolysates has the potential to help control or to prevent hypertension by a non-drug food-based treatment.