Angiotensin-(1-7) mediated calcium signalling by MAS.

The G protein-coupled receptor, MAS, is the receptor of the endogenous ligand, Angiotensin (Ang)-(1-7). It is a promising drug target since the Ang-(1-7)/MAS axis is protective in the cardiovascular system. Therefore, a characterization of MAS signalling is important for developing novel therapeutics for cardiovascular diseases. In this paper, we show that Ang-(1-7) increases intracellular calcium in transiently MAS-transfected HEK293 cells. The calcium influx induced by the activation of MAS is dependent on plasma membrane Ca2+ channels, phospholipase C, and protein kinase C. Specifically, we could demonstrate that MAS employs non-selective, transient receptor potential channels (TRPs) for calcium entry.

Different reactive species modulate the hypotensive effect triggered by angiotensins at CVLM of 2K1C hypertensive rats.

Hypertension is associated with increased central activity of the renin-angiotensin system (RAS) and oxidative stress. Here, we evaluated whether reactive species and neurotransmitters could contribute to the hypotensive effect induced by angiotensin (Ang) II and Ang-(1-7) at the caudal ventrolateral medulla (CVLM) in renovascular hypertensive rats (2K1C). Therefore, we investigated the effect of Ang II, Ang-(1-7), and the Ang-(1-7) antagonist A-779 microinjected before and after CVLM microinjection of the nitric oxide (NO)-synthase inhibitor, (L-NAME), vitamin C (Vit C), bicuculline, or kynurenic acid in 2K1C and SHAM rats. Baseline values of the mean arterial pressure (MAP) in 2K1C rats were higher than in SHAM rats. CVLM microinjection of Ang II, Ang-(1-7), l-NAME, or bicuculline induced decreases in the MAP in SHAM and 2K1C rats. In addition, Vit C and A-779 produced decreases in the MAP only in 2K1C rats. Kynurenic acid increased the MAP in both SHAM and 2K1C rats. Only the Ang-(1-7) effect was increased by l-NAME and reduced by bicuculline in SHAM rats. L-NAME also reduced the A-779 effect in 2K1C rats. Only the Ang II effect was abolished by CVLM Vit C and enhanced by CVLM kynurenic acid in SHAM and 2K1C rats. Overall, the superoxide anion and glutamate participated in the hypotensive effect of Ang II, while NO and GABA participated in the hypotensive effect of Ang-(1-7) in CVLM. The higher hypotensive response of A-779 in the CVLM of 2K1C rats suggests that Ang-(1-7) contributes to renovascular hypertension.

Functional cross-talk between aldosterone and angiotensin-(1-7) in ventricular myocytes.

High serum levels of aldosterone have been linked to the development of cardiac disease. In contrast, angiotensin (Ang)-(1-7) was extensively shown to possess cardioprotective effects, including the attenuation of cardiac dysfunction induced by excessive mineralocorticoid activation in vivo, suggesting possible interactions between these 2 molecules. Here, we investigated whether there is cross-talk between aldosterone and Ang-(1-7) and its functional consequences for calcium (Ca(2+)) signaling in ventricular myocytes. Short-term effects of aldosterone on Ca(2+) transient were assessed in Fluo-4/AM-loaded myocytes. Confocal images showed that Ang-(1-7) had no effect on Ca(2+) transient parameters, whereas aldosterone increased the magnitude of the Ca(2+) transient. Quite unexpectedly, addition of Ang-(1-7) to aldosterone-treated myocytes further enhanced the amplitude of the Ca(2+) transient suggesting a synergistic effect of these molecules. Aldosterone action on Ca(2+) transient amplitude was mediated by protein kinase A, and was related to an increase in Ca(2+) current (I(Ca)) density. Both changes were not altered by Ang-(1-7). When cardiomyocytes were exposed to aldosterone, increased Ca(2+) spark rate was measured. Ang-(1-7) prevented this change. In addition, a NO synthase inhibitor restored the effect of aldosterone on Ca(2+) spark rate in Ang-(1-7)-treated myocytes and attenuated the synergistic effect of these 2 molecules on Ca(2+) transient. These results indicate that NO plays an important role in this cross-talk. Our results bring new perspectives in the understanding of how 2 prominent molecules with supposedly antagonist cardiac actions cross-talk to synergistically amplify Ca(2+) signals in cardiomyocytes.

Pharmaceutical composition of hydrochlorothiazide:ß-cyclo-dextrin: preparation by three different methods, physico-chemical characterization and in vivo diuretic activity evaluation.

Hydrochlorothiazide is a common diuretic antihypertensive drug of the thiazide family. Its poor aqueous solubility is one of the reasons for its limited bioavailability after oral administration. This work aimed at the development of a hydrochlorothiazide:ß-cyclodextrin (HTZ:ß-CD) pharmaceutical composition in order to improve water solubility and bioavailability of the drug. The HTZ:ß-CD complexes were prepared by three different methods: spray-drying, freeze-drying and fluid bed. Complexes were characterized by thermal analysis, Fourier transform-infrared (FTIR) spectroscopy, powder X-ray diffractometry, NMR (2D-ROESY), scanning electron microscopy (SEM), particle analysis and intrinsic dissolution. The findings reveal that three binary systems prepared presented better solubility results in comparison with free HTZ. Increased diuretic effect was observed to HTZ:ß-CD obtained by fluid bed in comparison to free drug in rats. Results taken together suggest that pharmacological effect of HTZ in complex was increased by solubility improvement promoted by cyclodextrin.

Angiotensin-(3-7) pressor effect at the rostral ventrolateral medulla.

Ang-(3-7) is a fragment of the renin-angiotensin system that can be derived both from Ang II or Ang-(1-7). In the present study we determined the cardiovascular effects produced by angiotensin-(3-7) [Ang-(3-7)] microinjection into the rostral ventrolateral medulla (RVLM), a key region for the control of sympathetic drive to the periphery. RVLM microinjection of Ang-(3-7) (20, 40 or 80 ng) in male Wistar rats anesthetized with urethane produced significant increases in MAP (19+/-3.8 mm Hg, n=5; 16+/-1.6 mm Hg, n=15 and 11+/-1.2 mm Hg, n=4, respectively) as compared to saline (4+/-0.7 mm Hg, n=6). These alterations were similar to that induced by Ang-(1-7) (14+/-1.3 mm Hg, 40 ng; n=12) and Ang II (17+/-2.3 mm Hg, 40 ng; n=7). Microinjection of losartan (AT(1) receptor antagonist, 100 pmol) or A779 (selective Mas receptor antagonist, 100 pmol) did not alter the pressor effect caused by Ang-(3-7). Microinjection of an Ang-(3-7) analogue, d-Ala(7)-Ang-(3-7) (100 pmol), completely abolished the pressor effect caused by Ang-(3-7). These results suggest that Ang-(3-7) may be an additional peptide of the RAS to act as neuromodulator, at least at the RVLM. Further, the Ang-(3-7) pressor effect is not mediated by the interaction with AT(1) or the Ang-(1-7), Mas, receptors.

Angiotensin-(1-7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways.

Angiotensin-(1-7) [Ang-(1-7)] causes endothelial-dependent vasodilation mediated, in part, by NO release. However, the molecular mechanisms involved in endothelial NO synthase (eNOS) activation by Ang-(1-7) remain unknown. Using Chinese hamster ovary cells stably transfected with Mas cDNA (Chinese hamster ovary-Mas), we evaluated the underlying mechanisms related to receptor Mas-mediated posttranslational eNOS activation and NO release. We further examined the Ang-(1-7) profile of eNOS activation in human aortic endothelial cells, which constitutively express the Mas receptor. Chinese hamster ovary-Mas cells and human aortic endothelial cell were stimulated with Ang-(1-7; 10(-7) mol/L; 1 to 30 minutes) in the absence or presence of A-779 (10(-6) mol/L). Additional experiments were performed in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin (10(-6) mol/L). Changes in eNOS (at Ser1177/Thr495 residues) and Akt phosphorylation were evaluated by Western blotting. NO release was measured using both the fluorochrome 2,3-diaminonaphthalene and an NO analyzer. Ang-(1-7) significantly stimulated eNOS activation (reciprocal phosphorylation/dephosphorylation at Ser1177/Thr495) and induced a sustained Akt phosphorylation (P<0.05). Concomitantly, a significant increase in NO release was observed (2-fold increase in relation to control). These effects were blocked by A-779. Wortmannin suppressed eNOS activation in both Chinese hamster ovary-Mas and human aortic endothelial cells. Our findings demonstrate that Ang-(1-7), through Mas, stimulates eNOS activation and NO production via Akt-dependent pathways. These novel data highlight the importance of the Ang-(1-7)/Mas axis as a putative regulator of endothelial function.

Angiotensin-(1-7) antagonist A-779 attenuates the potentiation of bradykinin by captopril in rats.

We evaluated the possibility that endogenous angiotensin-(1-7) [Ang-(1-7)] could participate in the potentiation of bradykinin (BK) by the angiotensin-converting enzyme inhibitor (ACEI) captopril in conscious Wistar rats. Catheters were introduced into descending aorta (through the left carotid artery) for BK injection, femoral artery for arterial pressure measurement, and both femoral veins for BK injection and vehicle or Ang-(1-7) antagonist, A-779 infusion. Infusion of vehicle or A-779 started 40 to 45 minutes after captopril administration. Sequential BK dose-response curves were made before, 10 minutes after captopril, and within 10 minutes of infusion of vehicle or A-779. To evaluate angiotensin I conversion, dose-response curves for angiotensin I and angiotensin II were made following the same protocol used for BK. Captopril treatment markedly increased the BK hypotensive effect and significantly decreased angiotensin I conversion. Infusion of A-779 did not modify the angiotensin II pressor effect or the effect of captopril on angiotensin I conversion. However, A-779 significantly reduced the potentiating effect of captopril on the hypotensive effect of BK administered intravenously or intra-arterially. These results suggest that endogenous Ang-(1-7) and/ or an Ang-(1-7)-related peptide plays an important role in the BK potentiation by ACEI through a mechanism not dependent upon inhibition of ACE hydrolytic activity.