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The angiotensin receptor type 1–Gq protein–phosphatidyl inositol phospholipase Cβ–protein kinase C pathway is involved in activation of proximal tubule Na+-ATPase activity by angiotensin(1–7) in pig kidneys
Lucienne S. Lara,
Departamento de Farmacologia Básica e Clínica, Instituto de Ciências Biomédicas
Corresponding author C. C. Neves: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-bloco G, 21941-902, Rio de Janeiro, RJ, Brazil. Email: firstname.lastname@example.org
In a previous study, we observed that angiotensin(1–7) (Ang(1–7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1–7) on Na+-ATPase activity through AT1R involves a Gq protein–phosphatidyl inositol-phospholipase Cβ(PI-PLCβ) pathway because: (1) the effect was reversed by GDPβS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPγS, a non-hydrolysable GTP analogue; (3) Ang(1–7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCβ substrate; and (4) U73122, a specific inhibitor of PI-PLCβ, abolished Ang(1–7)-induced stimulation of Na+-ATPase activity. Angiotensin(1–7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1–7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1–7) stimulates Na+-ATPase activity through the AT1R–Gq protein–PI-PLCβ–PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.
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Angiotensin(1–7) (Ang(1–7): Asp-Arg-Val-Tyr-Ile-His-Pro) plays a crucial role in the control of fluid and electrolyte balance, at least in part through modulating renal function (Santos et al. 2000; Magaldi et al. 2003). There are two processing pathways for its production: the classical hydrolysis of angiotensin I (Ang I) by neutral endopeptidase and the direct conversion of angiotensin II (Ang II) by the carboxypeptidase angiotensin-converting enzyme (ACE2; Chappel et al. 2004; Rice et al. 2004). This latter pathway is responsible for the major intrarenal formation of Ang(1–7), especially in the proximal tubule, the segment of the nephron responsible for most of the water and Na+ reabsorption (Ferrario et al. 2005; Li et al. 2005).
Although it is accepted that the kidney is the crucial organ for both production and action of Ang(1–7), many studies have addressed the complex nature of the effects this heptapeptide on renal function. Differences between species, nephron segment, sodium and water status and level of renin–angiotensin system (RAS) activation may be responsible for different results concerning the handling of renal sodium excretion (Santos et al. 2000, 2005; Simões-e-Silva et al. 2006). It is accepted that these variations suggest several Ang(1–7) binding sites, namely angiotensin receptor type 1 (AT1R), angiotensin receptor type 2 (AT2R) or Ang(1–7) specific receptor (AT(1–7)R), which are coupled to multiple intracellular signalling pathways.
In our previous study, we showed that Ang(1–7) has a biphasic effect on the ouabain-insensitive proximal tubule Na+-ATPase activity, in that lower concentrations (10−11–10−8m) stimulate and higher concentrations (10−8–10−5m) inhibit. We observed that the stimulatory effect of Ang(1–7) on the proximal tubule Na+-ATPase activity is mediated by the AT1R (Caruso-Neves et al. 2000); however, the signalling pathway involved in this process is still to be determined.
Our work aimed to investigate the molecular mechanisms involved in AT1R-stimulated proximal tubule Na+-ATPase activity by Ang(1–7). We used isolated basolateral membrane (BLM) of proximal tubule cells. In this report, we show that the effect of Ang(1–7) results from the activation of associated basolateral membrane protein kinase C (PKC) triggered by diacylglycerol (DAG) produced by a Gq protein–phosphatidyl inositol phospholipase Cβ (PI-PLCβ) pathway. This result opens new possibilities for understanding the diversity of the effects of Ang(1–7) in renal sodium excretion and, consequently, the long-term regulation of extracellular volume and blood pressure.
Adenosine triphosphate, furosemide, guanosine 5-[γ-thio]triphosphate (GTPγS), guanosine 5-[β-thio]diphosphate (GDPβS), protein kinase A (PKA) inhibitor peptide (iPKA), sodium dodecyl sulphate (SDS), ouabain, EDTA, Hepes, Tris, Ang(1–7), sodium vanadate, hydroxylamine, bovine serum albumin (BSA) and histone were purchased from Sigma Aldrich; polyclonal anti-Gqα-subunit, monoclonal anti-phosphoserine antibody, phorbol-12-myristate-13-acetate (PMA), catalytic subunit of PKC (PKC-M), 1-[6((17b-3-methoxyestra-1,3,5-(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), horseradish-peroxidase-conjugated sheep anti-rabbit and anti-mouse antibodies were purchased from Calbiochem (San Diego, CA, USA); Percoll was from GE Biosciences (Uppsala, Sweden). The AT1 receptor selective antagonist, losartan, was obtained from Medley S.A. Autoradiograph films (XO-Mat and T-MAT) were from Kodak T-Mat (Resende, Brazil). Silica gel thin-layer chromatography (TLC) plates, organic solvents and all other reagents were from Merck (Darmstadt, Germany). All other reagents were of the highest purity available. [32P]Inorganic phosphate (Pi) was obtained from the Institute of Energetic and Nuclear Research (São Paulo, SP, Brazil). All solutions were prepared with deionized glass-distilled water. [γ-32P]Adenosine triphosphate was prepared as described by Maia et al. (1993).
Preparation of isolated basolateral membranes
The basolateral membranes of the outer cortex were prepared from adult pig kidneys. The kidneys were obtained from a commercial slaughterhouse immediately after death of the animals and were maintained in a cold solution (4°C) containing 250 mm sucrose, 10 mm Hepes–Tris (pH 7.6), 2 mm EDTA and 1 mm phenylmethylsulphonic fluoride. Thin slices of outer cortex were removed using a scalpel. After dissection, slices were homogenized in the same cold solution with a Teflon–glass homogenizer. The homogenate was centrifuged at 1900g for 10 min at 4°C in a SCR20B centrifuge using an RP12-2 rotor (Hitachi). The supernatant was collected and stored at 4°C. The fraction enriched in BLM was isolated by the Percoll gradient method (Grassl & Aronson, 1986). The membrane preparation was resuspended in 250 mm sucrose at a final concentration of 5–10 mg protein ml−1 and was stored at −20°C. The Na+–K+-ATPase activity, a marker for BLM, was five- to eightfold higher than the activity found in the cortex. The same enrichment was observed for the Na+-ATPase specific activity; both were determined as described by Caruso-Neves et al. (2002). In contrast, the alkaline phosphatase and 5′-nucleotidase specific activities, markers of the luminal membrane, were only 1.2- and 0.25-fold enriched, respectively. Residual contamination with other subcellular membrane fractions was minimal. The specific activities of succinate dehydrogenase (a marker for mitochondrial contamination), acid phosphatase (a marker for lysosomal membranes) and glucose-6-phosphatase (a marker for endoplasmic reticulum) were decreased by 95, 90 and 94%, respectively, determined as described previously (Vieyra et al. 1986; Coka-Guevara et al. 1999). Protein concentration was determined by the Folin phenol method (Lowry et al. 1951) using BSA as a standard.
Measurement of Na+-ATPase activity
The assay medium for the measurement of Na+-ATPase activity was (0.1 ml): 4 mm MgCl2, 4 mm ATP (specific activity of approx. 10 MBq (nmol ATP)−1), 20 mm Hepes–Tris (pH 7.0), 90 mm NaCl and 1 mm ouabain. The ATPase activity was measured according to the method described by Grubmeyer & Penefsky (1981). The reaction was started by adding membranes to a final protein concentration of 0.3 mg ml−1, and was stopped after 10 min by adding 0.1 m HCl-activated charcoal. The [32P]Pi released was measured in an aliquot of 0.2 ml of the supernatant obtained after centrifugation of the charcoal suspension for 5 min at 1500g in a clinical centrifuge. Spontaneous hydrolysis of [γ-32P]ATP was measured simultaneously in tubes to which protein was added after the acid. The Na+-ATPase activity was calculated from the difference between the [32P]Pi released in the absence and in the presence of 2 mm furosemide, both in the presence of 1 mm ouabain (Caruso-Neves et al. 2002).
Phospholipid extraction from purified BLM
Total phospholipids were extracted from isolated BLM following the method of Horwitz & Perlman (1987), modified by Malaquias & Oliveira (1999). Basolateral membrane phospholipids were labelled by incubating them at 37°C for 4 h with 5 mm ATP (672 Bq nmol−1), 10 mm MgCl2, 20 mm Hepes–Tris (pH 7.0), 5 mm azide and 90 mm NaCl. The reaction was started by the addition of 10−9m Ang(1–7) or vehicle (10 mm HCl). At different incubation times, 1 ml aliquots were taken, the reaction was stopped with 2 ml of CHCl3:CH3OH:HCl (200:100:0.75, v/v/v), and the tubes were kept on ice for 10 min. After reaching room temperature, 0.375 ml of 0.6 n HCl was added. After centrifugation at 800g for 10 min at room temperature, the organic (lower) phase was transferred to a new tube and washed twice with 1 mLl of CHCl3:CH3OH:HCl (0.6 n; 3:48:47, v/v/v). The pH of the organic phase was neutralized with 0.2 n NH3 in methanol, and then the solvent was dried under N2 and lipids stored at −20°C.
Separation and identification of phospholipids
Lipid samples were solubilized in 20 μl of CHCl3:CH3OH:H2O (75:25:2, v/v/v) and applied to TLC silica gel plates along with phospholipid standards. The plates were precoated with 1.3% potassium oxalate solution in H2O:CH3OH (3:2, v/v/v) and allowed to dry overnight. Prior to lipid loading, the plates were activated at 110°C for 10 min. The TLC plates were developed in the following solvent mixture: CHCl3:CH3OH:CH3COCH3:CH3COOH:H2O (40:13:15:12:8, by volume) for 80 min. The plate was dried in a fume hood, and the lipid spots were visualized by exposure to I2 vapour. Autoradiography of the TLC plates was performed with X-ray film. The radiolabelled lipid spots were located, scraped from the silica into scintillation vials, and the radioactivity was quantified in a liquid scintillation counter (Packard Tri-carb model A2100TR, Ramsey, MN, USA).
Measurement of hydroxylamine-resistant protein phosphorylation in isolated BLM of renal outer cortex
To measure the incorporation of 32Pi from [γ-32P]ATP into isolated BLM, we measured the radioactivity bound to an insoluble protein fraction. The reaction was initiated with the addition of the membrane preparation (final concentration of 1.5 mg ml−1) to reaction medium containing 1 mm[γ-32P]ATP (259 Bq nmol−1), 10 mm MgCl2, 20 mm Hepes–Tris (pH 7.0), 6 mm NaCl, 1.1 m hydroxylamine and 1 mm EGTA. The reaction was stopped with 1.5 ml of an ice-cold solution (0.25 m perchloric acid, 1 mm ATP and 4 mm sodium phosphate, pH 7.0). The mixture was centrifuged for 1 h at 2000g, and the pellet resuspended in 0.15 ml of an ice-cold solution (0.1 m NaOH, 2% (w/v) Na2CO3, 2% (w/v) SDS). The radioactivity was quantified by liquid scintillation counting (Rangel et al. 2001).
Protein kinase activity assay
The protein kinase activity of isolated BLM was measured by protein kinase inhibitor-sensitive incorporation of [32P]Pi from [γ32-P]ATP (259 Bq nmol−1), using histone as substrate. The composition of the reaction medium was 4 mm MgCl2, 20 mm Hepes–Tris (pH 7.0), 1.5 mg ml−1 histone and 0.7 mg ml−1 protein. After 10 min, the reaction was stopped with 40% trichloroacetic acid (TCA) and the sample was immediately placed on ice. An aliquot (0.1 ml) was filtered through a Millipore filter (0.45 μm pore size) and washed with ice-cold 20% TCA solution and 0.1 m phosphate buffer (pH 7.0). The radioactivity was quantified by liquid-scintillation counting. The specific PKC activity was calculated from the difference between the activity in the absence and in the presence of 10−8m calphostin C. Phorbol ester (PMA; 10−12m) was used as the activator of PKC (Lara et al. 2006).
The means were compared by one-way ANOVA, taking into account the treatment of the experimental groups. The magnitudes of the differences were evaluated using the multiple comparative Bonferroni test. The data are presented as means ±s.e.m. The n values correspond to the results obtained from different basolateral membrane preparations.
The Gq–PI-PLCβ pathway is involved in the stimulatory effect of Ang(1–7) on Na+-ATPase activity
It is well known that the AT1R belongs to the G protein-coupled receptor family (de Gasparro et al. 2000). Moreover, our group showed that BLM fractions contain diverse isoforms of trimeric Gα proteins (Caruso-Neves et al. 2003; De Souza et al. 2004; Lara et al. 2006). To test the involvement of a trimeric G protein in the Ang(1–7)-induced stimulation of Na+-ATPase activity, we used GDPβS, a non-hydrolysable GDP analogue (Fig. 1A) and GTPγS, a non-hydrolysable GTP analogue (Fig. 1B). It was observed that the GDPβS (from 10−9 to 10−6m) reversed the stimulatory effect of 10−9m Ang(1–7) on the enzyme activity in a dose-dependent manner, with maximal effect obtained at 5 × 10−8m. The addition of 10−6m GDPβS alone did not change the Na+-ATPase activity. In contrast, the addition of 10−6m GTPγS increased the enzyme activity in a similar way to that observed in the presence of Ang(1–7) alone. Moreover, the increasing concentrations of GTPγS (from 10−11 to 10−6m) in the presence of Ang(1–7) did not promote any further additive effect from that of Ang(1–7) alone.
Since it is known that the AT1R could be coupled to Gq protein in proximal tubule cells (Douglas & Hopfer, 1994; Houillier et al. 1996; Rangel et al. 1999), we decided to investigate the involvement of this G protein in the stimulatory effect of Ang(1–7). A polyclonal anti-Gqα-subunit antibody was used as a specific inhibitor (Fig. 1C). The addition of different dilutions of the antibody (from 1:4800 to 1:400) completely reversed the effect of Ang(1–7), confirming the involvement of Gq protein. The addition of the polyclonal anti-Gqα-subunit antibody alone at 1:400 did not modify the enzyme activity.
One of the downstream pathways known to be activated by Gq proteins is the PI-PLCβ–PKC pathway (Rhee, 2001). To investigate involvement of PI-PLCβ in the signalling pathway triggered by Ang(1–7), the levels of phosphatidyl inositol 4,5 bisphosphate (PIP2), its specific substrate, were measured. Initially, BLM were pre-incubated with [γ-32P]ATP for 4 h. Then, labelled PIP2 was measured in the absence or in the presence of 10−9m Ang(1–7) in a course- and time-dependent manner as described in the Methods section (Fig. 2A). Angiotensin(1–7) induced a rapid decrease in PIP2 levels; the maximal effect was observed in 30 s. The complete reversal of the phenomenon was observed after 3 min of reaction. Coupling between the activation of PI-PLCβ and activation of Na+-ATPase activity by Ang(1–7) was tested using 5 × 10−8m U73122, a specific inhibitor of PI-PLCβ (Fig. 2B). It was observed that the stimulatory effect induced by Ang(1–7) on Na+-ATPase activity was completely blocked by U73122.
So far, the scenario is that a nanomolar concentration of Ang(1–7) stimulates Na+-ATPase activity through the Gq protein–PI-PLCβ pathway coupled to AT1R.
Protein kinase C mediates the stimulatory effect of Ang(1–7) on Na+-ATPase activity
In the next step, the phosphorylation of proteins induced by Ang(1–7) was investigated (Fig. 3). Protein phosphorylation was measured through the incorporation of 32Pi from [γ-32P]ATP into BLM fractions. It is well known that BLM has several ATPases belonging to the P-ATPase family (Feraille & Doucet, 2001). This protein family is characterized by formation of covalently bound acylphosphate hydroxylamine during the catalytic cycle. To avoid the formation of this phosphoenzyme intermediate (acylphosphate linkage), 1.1 m hydroxylamine was used. Angiotensin(1–7)-induced hydroxylamine-resistant protein phosphorylation was observed after 1 min and sustained for 7 min (Fig. 3A). In these conditions, the phosphoprotein (PP) level increased by 260%. In addition, this effect was completely abolished by 10−10m losartan, a specific antagonist of AT1R, and 10−8m calphostin C, a specific inhibitor of PKC (Fig. 3B). Therefore, 10−12m PMA, an activator of PKC, had a similar and not additive effect to Ang(1–7) on hydroxylamine-resistant protein phosphorylation. These data suggest that Ang(1–7) enhances the protein phosphorylation levels, possibly due to an increase of PKC activity.
To evaluate the involvement of PKC in the modulation of Na+-ATPase activity by Ang(1–7), PMA and calphostin C were used initially (Fig. 4A). The PMA (10−12m) stimulated the Na+-ATPase activity in a similar and non-additive manner to Ang(1–7), and the calphostin C blocked the stimulatory effect promoted by Ang(1–7). It is known that both PKC and PKA could be involved in AT1R-mediated modulation of renal proximal tubule sodium reabsorption (Inagami & Harris, 1993; Douglas & Hopfer, 1994; Houillier et al. 1996). However, the possible involvement of PKA in the effect of Ang(1–7) on the enzyme activity can be ruled out because the 10−8m peptide inhibitor of PKA (iPKA) did not modify the Ang(1–7)-stimulated Na+-ATPase activity (Fig. 4A).
We also tested the effect of the catalytic subunit of PKC (PKC-M) on Na+-ATPase activity (Fig. 4B). The addition of 2 × 10−9m PKC-M increased enzyme activity in a similar and non-additive manner to 10−9m Ang(1–7). In the next step, we investigated the direct effect of Ang(1–7) on PKC activity through the phosphorylation of histone (Fig. 5). Angiotensin(1–7) increased the PKC activity by 300%, which was completely abolished by 10−10m losartan and mimicked 10−12m PMA. These effects of losartan and PMA were similar to those observed on Ang(1–7)-induced hydroxylamine-resistant phosphoprotein formation (Fig. 3B).
Angiotensin(1–7) is particularly interesting because many studies have shown that it is functionally linked to the overall mechanisms that intrinsically regulate the function of the RAS (Lara et al. 2002, 2006; Ferrario et al. 2005; Santos et al. 2005). In part, the complexity of the effects of Ang(1–7) is correlated to the diversity of the receptors that mediate its different effects. In general, it is accepted that Ang(1–7) counteracts the effect of Ang II on the cardiovascular and renal systems (Santos et al. 2000; Lara et al. 2002; Grobe et al. 2007). The data obtained in the present work revealed that the stimulation of Na+-ATPase activity by a nanomolar concentration of Ang(1–7) involves the AT1R–Gq protein–PI-PLCβ–PKC pathway. This effect is similar to that described for Ang II (Rangel et al. 1999, 2001, 2005), showing, for the first time, that these compounds could have non-contradictory effects.
In spite of the controversial results, it has become clear that Ang(1–7) participates in renal handling of Na+ and water, at least in part through the modulation of sodium reabsorption in proximal tubule cells. The active sodium transporters that act as targets for Ang(1–7) during the modulation of proximal tubule sodium reabsorption still need to be determined. Some authors have postulated that Ang(1–7) modulates Na+–K+-ATPase activity, based on the observation of oxygen consumption (Handa et al. 1996). Direct evidence for the modulation of this enzyme by Ang(1–7) was obtained in Madin–Darby canine kidney cells, a distal tubule model, where inhibition of this enzyme by Ang(1–7) was shown (Lara et al. 2005). However, in isolated BLM of proximal tubules, our group showed that Ang(1–7) did not change Na+–K+-ATPase activity (Caruso-Neves et al. 2000). The lack of effect of Ang(1–7) on enzyme activity in the BLM could be explained by the requirement of cell integrity for the modulation of Na+–K+-ATPase activity (Aperia et al. 1998). The data described in the present paper, together with observations reported by our group in previous studies, indicate that Na+-ATPase is involved, at least in part, in the effect of Ang(1–7) on the proximal tubule sodium reabsorption (Garcia & Garvin, 1994; Handa et al. 1996; Caruso-Neves et al. 2000; Lara et al. 2006).
Generally, it has been shown that the effects of Ang(1–7) are mediated by the interaction with AT2R and AT(1–7)R (Mas receptor; Santos et al. 2000; Magaldi et al. 2003; Lara et al. 2006). Only a few studies have described the effects of Ang(1–7) mediated by AT1R (Caruso-Neves et al. 2000; Giani et al. 2007), but the Ang(1–7)-induced signalling pathways triggered by these receptors have not yet been determined. Here, we observed that a PI-PLCβ is activated by binding of Ang(1–7) to AT1R, and this effect is mediated by Gq protein. One concern in this regard could be the recycling of PIP2 in isolated BLM used as a substrate for PI-PLCβ. However, it has been well documented that the formation of PIP2 can occur in the plasma membrane without the need for cytoplasmic enzymes (Catz et al. 1998; Rangel et al. 2005). We measured the formation of labelled PIP2 in the isolated BLM after the addition of [γ-32P]ATP through TLC. Furthermore, other characteristics, such as the time dependence of mobilization of the PIP2 induced by Ang(1–7) (maximal 30 s) and its reversal by U7312, a specific inhibitor of PI-PLCβ, confirm the involvement of PI-PLCβ on the effect of Ang(1–7) on Na+-ATPase activity.
The PI-PLCβ catalyses the hydrolysis of PIP2 to DAG and IP3. Since we used BLM isolated from proximal tubule, the role of IP3 in altering the intracellular level of Ca2+ can be ruled out. Here, we showed that Ang(1–7) increases the activity of a resident pool of PKC in isolated BLM. Furthermore, this PKC activated by Ang(1–7) must belong to the novel PKC isoform family because it is not Ca2+ dependent and it is activated by the phorbol ester PMA, a diacylglycerol analogue. The observation that Ang(1–7)-induced phosphorylation of BLM is mimicked by PMA and reversed by calphostin C suggests that PKC increases Na+-ATPase activity through direct phosphorylation. However, we cannot rule out a possible involvement of intermediary proteins or other factors in this process.
The data obtained in the present paper, together with those obtained in previous studies, show the presence of complete signalling complexes in plasma membrane, from receptors to all intermediary elements, to final membrane targets. These findings illustrate an important means by which fine and fast modulation of renal sodium reabsorption by proximal tubule cells can occur during acute changes in sodium intake. This hypothesis is in agreement with the proposal that cellular signalling could occur in distinct components or even in microdomains of the plasma membrane, such as caveoles. Such compartmentalization is usually associated with epithelial cell types, such as kidney cells (Garcia & Garvin, 1994). Since we used only isolated basolateral membranes, the modulation of Na+-ATPase by Ang(1–7) occurs only by events of phosphorylation without the necessity to recruit enzyme from the cytosolic compartments. In this way, activation of the Gq protein–PI-PLCβ signalling pathway present in basolateral membrane could modulate ion transporters present in this membrane. This kind of regulation is important for very rapid events of regulation required in transporting epithelial cells.
In the present paper we showed that Ang(1–7) interacted with AT1R in isolated BLM of proximal tubule cells and in a previous paper we observed that Ang(1–7), in the presence of antagonist of AT1R, binds to AT2R. The interaction of Ang(1–7) with AT1R stimulates proximal tubule Na+-ATPase activity, while interaction with AT2R inhibits it. Another important factor to determine the final effect of Ang(1–7) on sodium reabsorption in the proximal tubule is the Ang II content. In a previous paper we observed that Ang(1–7), through AT(1–7)R (Mas receptor), reversed the Ang II-induced activation of Na+-ATPase mediated by AT1R. Thus, we can postulate that the final effect of Ang(1–7) on the proximal tubule sodium reabsorption is a consequence of the co-ordination of several factors that will determine the binding of Ang(1–7) to different receptors, triggering distinct cellular signalling pathways. Among these factors we can highlight the ratio between Ang II and Ang(1–7). This hypothesis could explain, at least in part, the different effects of Ang(1–7) on renal sodium excretion that have been observed by several authors.
We thank Dr Luiz Roberto Leão Ferreira for critical reading of the manuscript. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Antonio Luiz Vianna Program from Fundação José Bonifácio (FUJB).