AT1, AT2, and AT(1-7) receptor expression in the uteroplacental unit of normotensive and hypertensive rats during early and late pregnancy.

INTRODUCTION: We investigated the expression of angiotensin receptors in early pregnancy and established whether normal pregnancy or preeclampsia alters the expression and distribution of the uteroplacental AT1R, AT2R and mas/AT1-7R at late gestation. METHODS: The percentage of each receptor subtype present in tissues from virgin rats and from normotensive and RUPP hypertensive pregnant rats was established by in vitro receptor autoradiography. Receptor mRNA levels were determined by quantitative PCR at early and late pregnancy. RESULTS: AT1R mRNA levels were up-regulated in the interimplantation (IIS) site at day 7 of gestation. AT2R mRNA levels were decreased at day 5 and 7 in the IIS but increased in the implantation site (IS) at day 5 and 7 as compared to the IIS at day 5. Mas/AT1-7R mRNA was increased in early pregnancy. In normal pregnancy and RUPP the mRNA for all angiotensin receptors was reduced in the uterus at late gestation. The AT1R accounted for the majority of binding in the uterus of virgin and the placenta of pregnant and RUPP. In RUPP pregnancy there was a significant competition with d-Ala in the placenta labyrinth. DISCUSSION AND CONCLUSION: The expression of angiotensin receptors suggests their involvement in the maintenance of early stages of pregnancy. During late gestation down-regulation of Ang receptors in the uterus may arise from feedback down-regulation by Ang II. In the placenta the levels of AT1Rs are equivalent in the RUPP model. The increased binding of mas/AT1-7R at late gestation in RUPP may represent a compensatory mechanism to reduce uteroplacental vascular resistance.

Decidualized pseudopregnant rat uterus shows marked reduction in Ang II and Ang-(1-7) levels.

UNLABELLED: Previous studies showed that angiotensin (Ang) II and Ang-(1-7) concentrations were reduced in the implantation site at day 7 of pregnancy in Sprague-Dawley rats as compared to the site immediately adjacent to it, which does not have the embryo attached, clearly showing the importance of the blastocyst in the regulation of renin-angiotensin system (RAS). OBJECTIVE: The objective of this study was to evaluate the regulation of the RAS in the decidualized uterus in the pseudopregnant rat, a model without the presence of a conceptus. METHODS: Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments; decidualization was induced by oil-injection of the right horn; the left horn served as a control. The uterine content of Ang I, Ang II, and Ang-(1-7) was examined in the decidualized and non-decidualized uteri. RESULTS: Both Ang-(1-7) and Ang II and ACE and ACE2 mRNA were significantly reduced in the decidualized horn as compared to the non-decidualized horn. Immunocytochemical characterization of Ang II, Ang-(1-7), ACE and ACE2 demonstrated that Ang-(1-7), Ang II, and ACE2 polarize to the anti-mesometrial pole with decidualization. CONCLUSION: The decidualization process elicits marked reduction in uterine Ang II and Ang-(1-7) content as compared to the non-decidualized horn. The differential immunocytochemical expression of Ang II and Ang-(1-7) with ACE2, but not ACE in the anti-mesometrial pole of the decidualized horn may favor the formation and action of Ang-(1-7) in the anti-mesometrial pole, an area which plays a role in triggering the decidualization process.

Angiotensin-(1-7) serves as an aquaretic by increasing water intake and diuresis in association with downregulation of aquaporin-1 during pregnancy in rats.

We previously demonstrated that kidney and urine levels of angiotensin-(1-7) [ANG-(1-7)] were increased in pregnancy. To explore the role of ANG-(1-7) on fluid and electrolyte homeostasis during pregnancy, we evaluated the effect of the ANG-(1-7) antagonist D-alanine-[ANG-(1-7)] (A-779) on kidney function. Virgin and pregnant rats received infusion of vehicle or A-779 (48 microg.kg(-1).h(-1)) for 8 days by osmotic minipumps. Metabolic studies were done on treatment day 7-8. Virgin and pregnant rats at day 15 and 19 were killed, and blood and kidneys were collected. Kidneys were prepared for Western blot analysis for aquaporin-1 (AQP1) and aquaporin-2. In virgin female rats, A-779 increased urine volume and decreased urinary osmolality and AQP1 with no change in water intake. In 19-day pregnant rats, A-779 significantly decreased water intake and urine volume and increased urinary osmolality and kidney AQP1 expression. Only in late gestation did A-779 treatment decrease the difference between intake and output (balance). A-779 treatment increased plasma vasopressin in late gestation but did not change vasopressin in virgins. In virgin and pregnant animals, A-779 administration had no effect on blood pressure, plasma volume, blood volume, or urinary electrolytes. These results suggest that ANG-(1-7) produces antidiuresis associated with upregulation of AQP1 in virgin rats, whereas ANG-(1-7) produces diuresis in late gestation with downregulation of AQP1. ANG-(1-7) contributes to the enhanced water intake during pregnancy, allowing maintenance of the normal volume-expanded state despite diuresis produced in part by decreased AVP and AQP1.

Temporal-spatial expression of ANG-(1-7) and angiotensin-converting enzyme 2 in the kidney of normal and hypertensive pregnant rats.

We recently demonstrated that renin-angiotensin system (RAS) overactivity during late gestation in rats is associated with increased kidney and urine levels of ANG-(1-7) and enhanced kidney immunostaining of ANG-(1-7) and angiotensin-converting enzyme 2 (ACE2). To understand the temporal-spatial changes in normal and hypertensive pregnancies, the renal distribution of ANG-(1-7) and ACE2 in association with kidney angiotensin peptides and ACE2 activity was examined in virgin, normal pregnant (NP; gestational days 5, 15, and 19) and reduced uterine perfusion pressure (RUPP at day 19) pregnant Sprague-Dawley rats. ANG-(1-7) and ACE2 immunocytochemical staining increased 1.8- and 1.9-fold and 1.7- and 1.8-fold, respectively, at days 15 and 19 of NP, compared with virgin rats. ANG-(1-7) and ANG II concentrations were increased in the kidney at 19 days of gestation. ACE2 activity measured using a fluorescent substrate was increased 1.9- and 1.9-fold in the cortex and 1.9- and 1.8-fold in the medulla at days 15 and 19 of NP. In the RUPP animals, ANG-(1-7) immunostaining and concentration were significantly decreased compared with 19-day NP rats. ACE2 activity was unchanged in the cortex and medulla of RUPP rats. In conclusion, during NP, the concurrent changes of ACE2 and ANG-(1-7) suggest that ACE2 plays an important role in regulating the renal levels of ANG-(1-7) at mid to late gestation. However, the decrease in renal ANG-(1-7) content in the absence of a concomitant decrease in ACE2 implicates the participation of other ANG-(1-7) forming or degrading enzymes during hypertensive pregnancy.

Effect of estrogen on neprilysin expression in uterus and kidney of Sprague-Dawley normotensive and heterozygous (mRen2)27-transgenic hypertensive rats.

The present study was designed to determine whether estrogen modulates the angiotensin processing enzymes in membrane homogenates obtained from uterus and kidney cortex and medulla of Sprague-Dawley (SD) and heterozygous (mRen2)27-transgenic hypertensive (Tg(+)) female rats treated with or without 17beta-estradiol (E2). We evaluated estrogen's influence on neprilysin (NEP), an endopeptidase that forms angiotensin-(1-7) [Ang-(1-7)] and on aminopeptidase (AMP), which degrades Ang-(1-7). Renal tissue from normotensive and hypertensive male rats was also evaluated. E2 up-regulated NEP mRNA in the uterus of both SD and Tg(+) and this was associated with increased NEP activity in the uterus of SD (0.31+/-0.03 nmol/min/mg versus 0.18+/-0.04 nmol/min/mg of protein, p<0.05) and Tg(+) (0.26+/-0.04 nmol/min/mg versus 0.13+/-0.02 nmol/min/mg of protein, p<0.05) female). E2 had no significant effect on NEP activity in cortex and medulla of hypertensive and normotensive female. In female animals, cortical NEP activity is two-fold higher than medullary; in males there is a four-fold higher cortical NEP activity as compared to medulla. In male animals, medullary NEP was significantly lower than females with or without E2 treatment; no gender specific effect was found in cortex. E2 treatment also caused a two-fold increase in AMP activity in the uterus and 1.6-fold decrease in kidney cortex of SD and Tg(+) female (p<0.05). Our studies indicate that NEP may be a primary candidate for increased Ang-(1-7) processing in the uterus with estrogen treatment; kidney NEP, on the other hand, showed no modulation by estrogen, suggesting that down regulation of other processing enzymes, like AMP and ACE, may come into play in the kidney with estrogen replacement. In addition, these studies showed that there is tissue-specific regulation of NEP with estrogen treatment that is strain independent.

Distribution of angiotensin-(1-7) and ACE2 in human placentas of normal and pathological pregnancies.

This work was designed to study the expression of the vasodilator peptide angiotensin-(1-7) [Ang-(1-7)] and its generating enzyme (ACE2) in the uteroplacental interface. Placentas were obtained from 11 early pregnancy failures (5 miscarriages and 6 ectopic pregnancies), 15 normotensive, and 10 preeclamptic gestations. In placental villi, the main sites of immunocytochemical expression of Ang-(1-7) and ACE2 were the syncytiotrophoblast, cytotrophoblast, endothelium and vascular smooth muscle of primary and secondary villi. Syncitial Ang-(1-7) expression in samples obtained from miscarriages and ectopic pregnancies was increased compared to normal term pregnancy [2.0 (2.0-2.25 for the 25 and 75% interquartile range) vs 1.3 (1.0-1.9), p<0.01]. In the maternal stroma, Ang-(1-7) and ACE2 were expressed in the invading and intravascular trophoblast and in decidual cells in all 3 groups. Ang-(1-7) and ACE2 staining was also found in arterial and venous endothelium and smooth muscle of the umbilical cord. The expression of Ang-(1-7) and ACE2 was similar in samples obtained from normal term or preeclamptic pregnancies, except for increased expression of ACE2 in umbilical arterial endothelium in preeclampsia [0.5 (0.5-0.8) vs 0.0 (0.0-0.0), p<0.01]. The uteroplacental location of Ang-(1-7) and ACE2 in pregnancy suggests an autocrine function of Ang-(1-7) in the vasoactive regulation that characterizes placentation and established pregnancy.

Enhanced expression of Ang-(1-7) during pregnancy

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7) [Ang-(1-7)], and of a newly cloned angiotensin converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7). These findings provide a basis for the physiological role of Ang-(1-7) and ACE2 during pregnancy.

Enhanced expression of Ang-(1-7) during pregnancy.

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7) [Ang-(1-7)], and of a newly cloned angiotensin converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7). These findings provide a basis for the physiological role of Ang-(1-7) and ACE2 during pregnancy.

Effect of angiotensin-(1-7) on reperfusion arrhythmias in isolated rat hearts

There is increasing evidence that angiotensin-(1-7) (Ang-(1-7) is an endogenous biologically active component of the renin-angiotensin system (RAS). In the present study, we investigated the effects of Ang-(1-7) on reperfusion arrhythmias in isolated rat hearts. Isolated rat hearts were perfused with two different media, i.e., Krebs-Ringer (2.52 mM CaCl2) and low-Ca2+ Krebs-Ringer (1.12 mM Cacl2) and low-Ca2+ Krebs-Ringer (1.12 mM CaCl2). In hearts perfused with Krebs-Ringer, Ang-(1-7) produced a concentration-dependent (27-210 nM) reduction in coronary flow (25 percent reduction at highest concentration), while only slight and variable changes in contaction force and heart rate were observed. Under the same conditions, angiotensin II (Ang II; 27 and 70 nM) produced a significant reduction in coronary flow (39 percent and 48 percent, respectively) associated with a significant increase in force. A decrease in heart rate was also observed. In low-Ca2+ Krebs-Ringer solution, perfusion with Ang-(1-7) or Ang II at 27 nM concentration produced similar changes in coronary flow, contraction force and heart rate. In isolated hearts perfused with normal Krebs- Ringer, Ang-(1-7) produced a significant enhancement of reperfusion arrhythmias revealed by an increase in the incidence and duration of ventricular tachycardia and ventricular fibrillation (more than 30-min duration). The faciliation of reperfusion arrhythmias by Ang-(1-7) was associated with an increase in the magnitude of the decreased force usually observed during the postischemic period. The effects of Ang-(1-7) were abolished in isolated rat hearts perfused with low-Ca2+ Krebs-Ringer. The effect of Ang II (27 nM) was similar but less pronounced than that of Ang-(1-7) at the same concentration. These results indicate that the heart is a site of action for Ang-(1-7) and suggest that this heptapeptide may be involved in the mediation of the cardiac effects of the RAS.

Utilizaçäo do AMP (2-amino-2-metil-1-propanol) em substituiçäo à DEA (Dietanolamina) na dosagem de fosfato inorgânico no soro e urina pelo método direto de NCL / The use of amp (2-amino-2-methyl-1-propanol) as a substitute of DEA (Diethanolamine) in the determination of inorganic phosphate ins erum and urine, by the MCL direct method

O emprego do AMP (2-amino-2-metil-1-propanol) é proposto como substituto da DEA (Dietanolamina) na determinaçäo de fosfato inorgânico no soro na urina, pelo método direto MCL (Mendes eta al., 1988) Este reagente é mais estável em condiçöes de armazenamento e possui alto poder de solubilizaçäo do precipitado de fosfomolibdato. A análise dos resultados demonstrou uma excelente correlaçäo linear, concluindo que o AMP preenche todos os requisitos necessários para seu emprego na determinacäo de fosfato inorgânico

Perfil hematologico e bioquimico de pacientes com tuberculose / Hematological and biochemical profile of patients carrying lung tuberculosis

Foram colhidas amostras de sangue de 30 pacientes com tuberculose pulmonar comprovada atraves do encontro do B.A.A.R. em esfregaços de escarro corados pelo Ziehl-Neelsen.. As amostras de sangue foram empregadas, atraves de metodologia adequada, para a determinaçao do perfil hematologico e bioquimico dos tuberculosos em busca de alteraçoes que pudessem ser distribuidas a infeccao. Os resultados observados revelaram uma tendencia a leucocitose (23//dos casos com global acima de 10.000/mm3) e um elevado aumento da velocidade de hemossedimentaçao (86//apresentaram elevaçao com media de 72 a 44 mm para mulheres ehomens, respectivamente). A monocitose, referida por varios autores na tuberculose ativa, nao foi observada. Quanto ao perfil bioquimico, os resultados da determinaçao de fosfatase alcalina e valores obtidos de globulinas se mostraram elevados em 46//dos casos. Esses resultados representaram dados preliminares obtidos atraves das avaliaçoes hematologicas e bioquimicas de pacientes com tuberculose,realizadas antes do emprego da terapeutica recomendada. Estes pacientes representam uma amostragem adequadamente selecionada para a primeira etapa do projeto depesquisa sobre a avaliaçao da conduta farmacoterapeutica no setor publico, com aparticipaçao integrada dos Departamentos de Farmacia Social, Produtos Farmaceuticos e Analises Clinicas e Toxicologicas da Faculdade de Farmacia da UFMG.