The G protein-coupled receptor Mas was recently described as an angiotensin-(1-7) [ANG-(1-7)] receptor. In the present study we evaluated the anatomical localization of Mas using immunofluorescence in the central nervous system of adult male Wistar rats. An abundant labeling was found in the hippocampus, amigdala, anterodorsal thalamic nucleus, cortex, and hypoglossal nucleus. More importantly, a dense ANG-(1-7) receptor Mas immunoreactivity was observed in cardiovascular-related areas of the medulla and forebrain, shown in several previous studies as sites for the action of ANG-(1-7) in the brain. A strong staining was found in the nucleus of the solitary tract, caudal and rostral ventrolateral medulla, inferior olive, parvo and magnocellular portions of the paraventricular hypothalamic nucleus, supraoptic nucleus, and lateral preoptic area. Furthermore, Mas staining was predominantly present in neurons. At the medullary sites, a specific and high-intensity binding for rhodamine-ANG-(1-7) was also shown. The specific ANG-(1-7) binding was completely displaced by the anti-Mas antibody or by the ANG-(1-7) antagonist, A-779. The data presented provide the first anatomical basis for the physiological role of ANG-(1-7)/Mas axis in the modulation of different cardiovascular functions and give new insights for clarifying the role of ANG-(1-7) in the central nervous system.

We have previously shown the presence of immunoreactive angiotensin-(1-7) [Ang-(1-7)] in rat ovary homogenate and its stimulatory effect on estradiol and progesterone production in vitro. In the current study, we investigated the presence and cellular distribution of Ang-(1-7) and the Mas receptor, the expression of Mas and angiotensin-converting enzyme 2 (ACE2) messenger RNA (mRNA), and the enzymatic activity in the rat ovary following gonadotropin stimulation in vivo. Immature female Wistar rats (25 days old) were injected subcutaneously (SC) with equine chorionic gonadotropin (eCG, 20 IU in 0.2 mL) or vehicle 48 hours before euthanasia. Tissue distributions of Ang-(1-7), Mas receptor, and ACE2 were evaluated by immunohistochemistry, along with angiotensin II (Ang II) localization, while the mRNA expression levels of Mas receptor and ACE2 were evaluated by real-time polymerase chain reaction (PCR). In addition, we determined the activity of neutral endopeptidase (NEP), prolyl endopeptidase (PEP), and ACE by fluorometric assays. After eCG treatment, we found strong immunoreactivity for Ang-(1-7) and Mas primarily in the theca-interstitial cells, while Ang II appeared in the granulosa but not in the thecal layer. Equine chorionic gonadotropin treatment increased Mas and ACE2 mRNA expression compared with control animals (3.3- and 2.1-fold increase, respectively; P < .05). Angiotensin-converting enzyme and NEP activities were lower, while PEP activity was higher in the eCG-treated rats (P < .05). These data show gonadotropin-induced changes in the ovarian expression of Ang-(1-7), Mas receptor, and ACE2. These findings suggest that the renin-angiotensin system (RAS) branch formed by ACE2/Ang-(1-7)/Mas, fully expressed in the rat ovary and regulated by gonadotropic hormones, could play a role in the ovarian physiology.

SUMMARYThe most sensitive nonradiometric routine assay for angiotensin-converting enzyme (ACE) activity uses fluorometry to detect His-Leu released from Hip-His-Leu. Our results indicate that, in contrast to human serum, rat serum and plasma contain large and variable amounts of dipeptidase activity that lead to a subestimation of the ACE activity measured in 0.1 M potassium phosphate buffer, pH 8.3, containing 0.3 M NaCI, the most commonly used assay for human serum and tissue ACE. We describe and validate an assay for 1 to 10 /xL rat and human serum or plasma using 5 mM Hip-His-Leu in 500 fiL of 0.4 M sodium borate buffer, pH 8.3, containing 0.9 M NaCI at 37°C that reduced the subestimation error to ^ 3% (rat serum) and ^ 0.1% (human serum) and increased the ACE activity twofold to threefold. The K m and V,^ are reported for rat serum ACE

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

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