GPR30 Expression Is Required for the Mineralocorticoid Receptor–Independent Rapid Vascular Effects of Aldosterone
It has been increasingly appreciated that steroids elicit acute vascular effects through rapid, so-called nongenomic signaling pathways. Though aldosterone, for example, has been demonstrated to mediate rapid vascular effects via both mineralocorticoid receptor–dependent and –independent pathways, the mechanism(s) of this mineralocorticoid receptor–independent effect of aldosterone is yet to be determined. For estrogen, its rapid effects have been reported to be, at least in part, mediated via the 7-transmembrane–spanning, G protein–coupled receptor GPR30. Previous studies have demonstrated common response outcomes in response to both aldosterone and estrogen on GPR30 expression, ie, activation of phosphatidylinositol 3-kinase–dependent contraction and extracellular signal-regulated kinase activation in vascular smooth muscle cells. The present studies were undertaken to test the hypothesis that the rapid response to aldosterone in smooth muscle is dependent on the availability of a GPR30-dependent signaling pathway. These findings not only reconcile differences in the literature for aldosterone response in freshly isolated versus cultured aortic smooth muscle cells but also suggest alternative therapeutic strategies for modulating aldosterone actions on the vasculature in vivo.
See Editorial Commentary, pp 370–372
Aldosterone and other vasoactive steroids are important physiological and pathophysiological regulators of cardiovascular function. The canonical mechanisms for vasoactive steroids, such as aldosterone and estrogen, have focused on regulation of transcription via activation of their “traditional” cytosolic/nuclear mineralocorticoid and estrogen receptors. However, scientists have speculated that these and other vasoactive steroids might have transcriptionally independent effects mediating their rapid changes in smooth muscle contractile reactivity and regulation of cell growth/death, based on confounding findings going back more than half a century.1,–,6
The mechanism(s) mediating rapid effects of steroids has remained a focus of controversy. Though the rapid effects of aldosterone and estrogen have been attributed to classical mineralocorticoid receptor (MR), estrogen receptor α (ERα), and ERβ receptor in some studies,5,7,8 the insensitivity of these rapid steroid actions to “classical receptor” antagonists9,–,11 has implicated additional mechanisms of action for these responses.
Recent studies have implicated GPR30 (also called GPER) as a mediator of the rapid effects of estrogens. GPR30, first characterized as an “orphan G protein–coupled receptor,”12 is expressed on the cell surface and mediates its effects via several members of the G protein family of GTP-binding proteins13,14; this receptor has been shown to contribute to the rapid effects of estrogen in several model systems.14,–,20 GPR30 is widely distributed and has been identified in both endothelial cells and vascular smooth muscle cells (VSMCs).21 However, to date, data have suggested that GPR30 is selectively activated by estradiol (E2).22
Because recent studies from our laboratory have revealed common activation of extracellular signal-regulated kinase (ERK) and of vascular contraction via phosphatidylinositol 3-kinase (PI3)-dependent kinases,13,23 we postulated that the rapid vascular actions of aldosterone might be mediated, at least in part, through a GPR30-dependent pathway. Based on this hypothesis, we examined the role of GPR30 in mediating the rapid vascular effects of aldosterone on vascular smooth muscle ERK phosphorylation and apoptosis as well as on myosin light chain (MLC) phosphorylation and contractility. Our findings are consistent with the interpretation that estrogens are not the sole hormones that mediate their rapid effects via GPR30-dependent pathways and that aldosterone can mediate its rapid vascular effects potently via both GPR30- and MR-dependent mechanisms.
VSMC Primary Cultures
Wistar-Kyoto rats (10 to 12 weeks of age; Charles River) were utilized in our studies as a source of VSMCs. They were cared for in accordance with the Canadian Council on Animal Care guidelines. The protocol for their use was approved by the Animal Use Subcommittee, University of Western Ontario. Isolation of rat aortic VSMCs was performed as described previously.24
Aortic Endothelial Cell Primary Cultures
Rat aortic endothelial cells were cultured as per the manufacturer's instructions (Cell Applications) and utilized between passages 4 and 8. See http://hypertension.ahajournals.org for further details.
Generation of Adenoviral Vectors for Expression of GPR30 and MR and for GPR30 Knockdown
The adenoviral vectors used in these studies were generated with the AdMax adenovirus vector creation kit as per the manufacturer's instructions (Microbix BioSystems Inc.). See supplemental data for further details.
Assessment of Endogenous Receptor Expression in Endothelium-Denuded Aortic Tissue, VSMCs, and Vascular Endothelial Cells
Isolation of total RNA was performed utilizing the 1-step RNA isolation reagent TRIzol (Invitrogen). Receptor mRNA expression was determined using SuperScript One Step RT-PCR kits (Invitrogen) with receptor-specific primers. See supplemental data for details.
Assessment of ERK Content and EKR1/2 Phosphorylation
The effects of aldosterone, E2, corticosterone, or the GPR30 agonist G1 on ERK1/2 phosphorylation were assessed by immunoblotting, as described previously.13 See supplemental data for details relating to each cellular preparation.
Assessment of Apoptosis by Annexin Labeling
VSMCs were cultured 24 hours before gene transfer and infected with adenoviral constructs expressing MR, GPR30, or GFP (control) for 16 hours. Infection medium was then replaced with DMEM without serum. After 48 hours of serum starvation, cells were treated with aldosterone (10 pM) for 24 hours, in the absence or presence of eplerenone or the GPR30 antagonist G15, detached with trypsin, and washed in PBS. Pooled intact cells were suspended in annexin binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2, pH 7.4) containing fluorescein isothiocyanate–conjugated annexin V (0.25 μg/mL) and propidium iodide (5 μg/mL) and incubated in the dark for 15 minutes. Annexin binding was assessed using a BD FACScalibur flow cytometer (BD Biosciences). A total of 20 000 events were analyzed for double-stained positive cells for each sample with FlowJo software (Tree Star Inc.) by a blinded observer. Data were normalized relative to the control levels of annexin-positive staining determined for each experiment (1.7±0.3% of cells, n=20).
Assessment of MLC Phosphorylation
In vascular smooth cells, phosphorylation of the 20-kDa regulatory subunit of myosin light chain (MLC20) was assessed by Western blotting using antibodies against phospho-MLC (Cell Signaling Technology) and total MLC (Santa Cruz Biotechnology) as described previously. See supplemental data for details.23
For multiple group comparisons, initial analysis by ANOVA was followed by Dunnett's multiple comparison tests. The significance of difference for between-paired groups was determined by Student's t test for paired data. P<0.05 on a 2-sided test was taken as a minimum level of significance.
The PI3 kinase inhibitor LY294002 and the GPR30-selective agonist G1 were purchased from Calbiochem-Novabiochem Corporation. The specific p38 inhibitor SB203580 and its p38 mitogen-activated protein kinase (MAPK)-inactive analog SB202474 were purchased from Calbiochem. Eplerenone was graciously provided by Pfizer Pharmaceuticals. The selective GPR30 antagonist G15 was synthesized as described recently.25 All other chemical reagents were obtained from Sigma-Aldrich Canada Ltd. Antiphospho ERK and antiphospho MLC20 antibodies were obtained from Cell Signaling Technology. The anti-ERK antibody was purchased from Upstate Cell Signaling. The anti-MLC20 antibody was obtained from Santa Cruz Biotechnology.
Aldosterone Stimulates ERK1/2 Phosphorylation in Freshly Isolated Aortic Ring Segments via Both MR- and GPR30-Dependent Mechanisms
Aldosterone stimulation of ERK phosphorylation is a rapid method for assessing aldosterone sensitivity of aortic tissue and isolated cells to this mineralocorticoid. As shown in Figure 1A, aldosterone causes a concentration-dependent and saturable increase in detectability of phospho-ERK in freshly isolated and endothelium-denuded aortic ring segments (Figure 1A). The GPR30 agonist G1 (1 μmol/L) also significantly enhanced ERK phosphorylation (151±16% of control, P<0.05, n=3) in isolated and endothelium-denuded aortic ring segments (data not shown). The aldosterone-mediated stimulation of ERK phosphorylation was partially attenuated by both the MR antagonist eplerenone as well as the GPR30-selective antagonist G15 (Figure 1B), suggesting that the effects of aldosterone might be mediated via both GPR30-dependent and MR-dependent actions in this tissue.
GPR30 Expression Diminishes After Culturing of VSMCs
Consistent with the functional studies in Figure 1, endogenous GPR30 and MR expression could be detected by reverse transcription-polymerase chain reaction (RT-PCR) in freshly isolated, endothelium-denuded aortic tissue (supplemental Figure 1A). Because GPR30-dependent aldosterone signaling could be detected in aortic rings denuded of endothelium, we tested whether cultured VSMCs might serve as a biological source for interrogation of the molecular contributors to the rapid aldosterone (mediated effects on growth regulatory mechanisms and contractility). RT-PCR analysis of cultured VSMCs revealed that GPR30 expression is significantly reduced in VSMCs compared to the freshly isolated aorta from which these cells were harvested and cultured (supplemental Figure 1), consistent with our previously reported findings.13 Though this finding was at first frustrating, we decided to use these cultured VMSCs as a heterologous background in which we could modulate relative GPR30 and MR expression via adenoviral-mediated gene transfer, to test the relative roles of these receptors, and their downstream signaling pathways, in mediating rapid aldosterone activation of signaling pathways and of contraction. We have previously successfully utilized this strategy in delineating the GPR30- versus ER-dependent mechanisms underlying the rapid effects of E2 in VSMCs.13 As shown in supplemental Figure 1B, we could express flag-tagged GPR30 (Mr 44 kDa14) and MR (Mr 107.0 kDa26) proteins in VSMCs. Although we do not have antibodies that can sensitively detect and thus quantitate the relative levels of endogenous GPR30 and MR receptors in these VSMCs, we do know that that virally transduced expression of these receptors was greater than that of their endogenous receptors based on relative RT-PCR product quantity (supplemental Figure 1C). Interestingly, gene transfer of GPR30 resulted in a reduction in MR expression as detected by RT-PCR (supplemental Figure 1C), though neither ERα nor ERβ expression was altered by GPR30 gene transfer (data not shown). Thus, VSMCs with and without gene transfer-mediated GPR30 expression provides a useful model to testing our hypothesis regarding the role of GPR30 in aldosterone-mediated responses in these cells.
Aldosterone-Mediated ERK Activation in VSMCs With Varying Levels of GPR30 Versus MR Expression
Our previous studies demonstrated that in primary cultured VSMCs, aldosterone mediated a concentration-dependent increase in ERK activation and phosphorylation with maximal effects in the picomolar concentration range.27 Similar to those previous findings in native VSMCs (and similar to those effects seen in freshly isolated tissues), in green fluorescent protein (GFP) (control)-infected VSMCs, aldosterone mediated a concentration-dependent increase in ERK phosphorylation (Figure 2A) (Emax ERK activation was 153±4% of control). However, in contrast to our findings in freshly isolated aortic rings (Figure 1A), aldosterone-mediated ERK activation in these cultured VSMCs (transduced with GFP-expressing control virus) was almost completely inhibited following pretreatment with the MR antagonist eplerenone (Figure 2A) and was not affected by pretreatment with the GPR30 antagonist G15 (Figure 3A). Thus, reduction in expression of GPR30 on VSMC culturing (supplemental Figure 1C) leads to a loss of GPR30 antagonist-sensitive signaling, emphasizing the specificity of this agent for blocking GPR30-mediated events. Similarly, the GPR30 agonist G1 (1 μmol/L) was without significant effect in GFP-infected cells (104±8% of control, P>0.1, n=5), again affirming the specificity of this agonist in evoking GPR30-mediated signaling.
Enrichment of Either MR or GPR30 in Cultured VSMC Enhanced Aldosterone-Mediated ERK Activation Compared to Findings in GFP-Infected Cells
In MR-transduced cultured VMSCs, the MR antagonist eplerenone completely suppressed aldosterone ERK activation (Figure 2B). Although robust expression of GPR30 reduces MR expression (cf supplemental Figure 1C), aldosterone-mediated ERK phosphorylation in these cells was still partially attenuated by the MR antagonist eplerenone (Figure 2C). Interestingly, following gene transfer of GPR30, aldosterone-mediated activation of ERK phosphorylation was almost completely inhibited by the GPR30 antagonist G15 (Figure 3A). The persistent inhibitory effects of eplerenone were somewhat surprising, given that in GPR30-expressing cells the actions of aldosterone appeared to be almost entirely GPR30 dependent (at least judging from the studies with G15). Thus, we determined whether eplerenone might also antagonize GPR30. To test whether eplerenone might serve as a GPR30 antagonist, we determined its effect on the ability of the GPR30 agonist G1 to activate ERK phosphorylation in GPR30-expressing cells. As illustrated in Figure 3B, both eplerenone and spironolactone partially inhibit G1-mediated responses, suggesting that both can serve as partial antagonists of GPR30.
Corticosterone-Mediated Regulation of ERK Phosphorylation Is Not GPR30 Dependent
We were appreciative of the potency of corticosteroids for the MR28 and their dominant effects on MR activation (versus aldosterone) in cells in the absence of high levels of 11 β-hydroxysteroid dehydrogenase activity.29 Therefore, we sought to determine whether corticosterone had a potency/effectiveness comparable to aldosterone in activating GPR30-dependent ERK phosphorylation. Corticosterone did stimulate ERK phosphorylation in both control and GPR30-expressing cells (supplemental Figure 2). However, over the concentration range at which aldosterone had maximal GPR30-dependent effects (1 to 1000 pM), the effects of corticosterone were not enhanced with GPR30 expression (supplemental Figure 2A), nor were they inhibited by the GPR30 antagonist G15 (supplemental Figure 2B).
GPR30-Mediated Regulation of ERK Phosphorylation in Vascular Endothelial Cells
To more definitively determine the importance of endogenous GPR30 expression on aldosterone-stimulated ERK activation, we additionally studied rat aortic vascular endothelial cells. These cells, when maintained in culture, demonstrate persistent expression of GPR30 as detected by RT-PCR (Figure 4A). Additionally, ERα and androgen receptor mRNA were detectable. Interestingly, no MR mRNA was detectable in this endothelial cell line, nor was ERβ mRNA (Figure 4A). Thus, rat aortic endothelial cells offered us the opportunity to examine the importance of endogenous GPR30 expression in mediating the effects of aldosterone on ERK activation in a model with robust GPR30 expression but attenuated MR expression.
In these endothelial cells, aldosterone increased ERK phosphorylation (Figure 4B) with a maximal effect in the 10 pM range following a 15-minute exposure, similar to that seen in VSMCs. In contrast, E2 mediated a concentration-dependent inhibition of ERK phosphorylation with a maximal effect in the low nanomolar range (Figure 4C). This inhibitory effect was similar in extent and concentration dependence to that seen for E2 in cultured VSMCs (and as we have reported recently13). In native endothelial cells, the GPR30 antagonist G15 almost completely attenuated the stimulatory effect of aldosterone on ERK activation (Figure 5A). In contrast, G15 had no significant effect on the E2-mediated inhibition of ERK phosphorylation, although the ER-specific antagonist ICI-182780 almost completely blocked the effects of E2 (Figure 5B). These findings suggest that, in this vascular endothelial cell model, aldosterone (and not E2) is important in mediating GPR30-directed effects on ERK activation.
To examine the effect of attenuation of GPR30 expression on aldosterone-mediated ERK phosphorylation, we studied endothelial cells infected with our adeno-shGPR30 construct compared to cells infected with our adeno-short hairpin (sh) GFP (control) construct. Transduction of shGPR30 mediated a dose-dependent inhibition of GPR30 expression as detected by RT-PCR (Figure 5C). With attenuation of GPR30 expression, the effect of aldosterone to stimulate ERK phosphorylation was almost completely ablated, as was the effect of the GPR30 agonist G1 (Figure 5D). In contrast, angiotensin II-mediated ERK phosphorylation was not altered. Interestingly, the effect of E2 to inhibit ERK phosphorylation was further enhanced with shGPR30 transduction. This would suggest that under native conditions, the net effect of E2 on ERK activation reflected its countervailing effects to mediate ERK inhibition via ERα (presumably) versus ERK stimulation via GPR30.
Aldosterone-Mediated Regulation of Apoptosis
Another effect on VSMCs of clinical relevance is the ability of aldosterone to increase apoptosis, measurable by changes in annexin labeling. Aldosterone mediated a concentration-dependent increase in apoptosis (Figure 6A). The pathways underlying aldosterone-accelerated apoptosis in these cells have been argued.30,31 However, our data are consistent with a MAPK kinase (MEK)-dependent pathway, since aldosterone (10 pM)-stimulated apoptosis was inhibited by the selective MEK inhibitor U0126 but not the chemically related, but MEK inactive, analog U0124 (Figure 6B) nor by the p38-MAPK inhibitor SB203580. Aldosterone-mediated apoptosis was also blocked by the PI3 kinase inhibitor LY294002 (Figure 6C), paralleling our previously reported findings demonstrating the PI3 kinase dependence of the “rapidly” mediated effects of aldosterone.23,27
It was of interest to examine the pathways involved in this apoptotic response in VMCs when we manipulated the relative GPR30 and MR expression levels by adenoviral transduction. As shown in Figure 7A, transduction with the control GFP vector, as anticipated, did not alter the aldosterone-mediated VSMC apoptotic responses when compared to control VSMCs (compare with Figure 6A) Elevated MR expression in VSMCs resulted in enhanced aldosterone-mediated apoptosis, which was completely blocked following eplerenone pretreatment (Figure 7C), whereas in GPR30-infected cells, aldosterone-mediated apoptosis was only partially inhibited by eplerenone pretreatment (Figure 7B). In contrast, G15 significantly blunted the aldosterone-mediated increase in apoptosis in GPR30-infected VSMCs although G15 had no effect in GFP-infected cells (Figure 7D).
Rapid Effects of Aldosterone in Mediating MLC Phosphorylation
MLC phosphorylation provides a downstream marker of signaling that is more closely linked to smooth muscle cell contraction. Furthermore, whereas ERK activation is a cellular function common to all vascular cell types, regulation of MLC phosphorylation is smooth muscle-specific. Thus, we also studied the mechanism of aldosterone-mediated regulation of MLC phosphorylation in (1) freshly isolated tissue with expression of endogenous GPR30, (2) primary smooth muscle cultures where GPR30 expression is attenuated, and (3) smooth muscle cells following reintroduction of GPR30 by gene transfer.
Aldosterone Can Stimulate MLC Phosphorylation in Freshly Isolated Aortic Ring Segments via a GPR30-Dependent Mechanism
In freshly isolated aortic vascular tissue, denuded of endothelium, both aldosterone (10 nmol/L for 15 minutes) and G1 (1 μmol/L for 15 minutes) stimulated MLC phosphorylation to an extent comparable to that of the alpha adrenergic agonist phenylephrine (10 μmol/L for 15 minutes) (Figure 8). The effect of aldosterone was almost completely inhibited by G15, consistent with an effect predominantly mediated by GPR30 activation.
Aldosterone Can Stimulate MLC Phosphorylation in Cultured VSMCs via Both MR- and GPR30-Dependent Mechanisms
Aldosterone mediated a dose-dependent increase in MLC phosphorylation in GFP-expressing VSMCs (Figure 9A) that was insensitive to G15 (Figure 9C). Gene transfer of either MR or GPR30 resulted in an enhancement of aldosterone-mediated MLC phosphorylation (Figure 9A), and the expected antagonists blocked this activation, depending on whether MR or GPR30 was introduced. Thus, while aldosterone-mediated MLC phosphorylation was completely abolished with eplerenone pretreatment in GFP- and MR-expressing VSMCs, the effects of aldosterone were only partially inhibited by eplerenone pretreatment in GPR30-infected VSMCs (Figure 9B). Additionally, whereas G15 had no significant effect on the aldosterone-mediated MLC phosphorylation in GFP-infected cells, G15 almost completely abolished the effects of aldosterone in GPR30-infected cells. Furthermore, the GPR30 agonist G1 only increased MLC phosphorylation in GPR30-infected cells (125±3% of control, n=6), but not in either GFP-infected (97±4% of control, n=6) or MR-infected (93±4%, n=6) VSMCs (Figure 9C).
The present studies demonstrate that aldosterone exploits both GPR30 and MR to mediate apoptosis and to activate intermediate signaling pathways, including PI3 kinase, ERK, and MLC phosphorylation. The relative contribution of GPR30 to this signaling depends on the relative concentrations of GPR30 in the target cells. Thus, although MR and GPR30 are expressed in freshly isolated VSMCs,23,32,33 GPR30 expression declines in cultured VSMCs in parallel with GPR30-dependent signaling. These findings suggest that studies examining the pathways mediating the effects of aldosterone in primary cultures of VSMCs will underestimate the role of GPR30 in mediating the effects of aldosterone in target cells in vivo. Additionally, these studies demonstrate, in this vascular endothelial cell model with persistent expression of GPR30 but no detectable MR expression, that the effects of aldosterone are completely dependent on GPR30-mediated mechanisms.
Our current findings are consonant with our previous findings regarding the directionality of GPR30-dependent effects on ERK phosphorylation and apoptosis in VSMCs.13 In freshly isolated aortic tissue, we have previously reported that E2 activation of ERK phosphorylation was due to its actions on GPR30 (ie, ERK stimulation). On primary culture and parallel attenuation of GPR30 expression in VSMCs, the predominant effect of E2 on ERK phosphorylation became inhibitory. For E2, expression of GPR30 was required for recovery of its ERK-stimulatory (and proapoptotic) actions. For aldosterone, our current findings indicate that its actions are ERK stimulatory under all conditions studied. Thus, whereas the actions of E2 on ERK are bidirectional and dependent on the differential expression of GPR30 versus ER, those of aldosterone converge in a parallel fashion on the same signaling pathways whether or not that signaling is primarily mediated by the MR or by GPR30.
We previously demonstrated that E2 activation of GPR30 results in activation of PI3 kinase–dependent ERK phosphorylation and apoptosis.13 In the current study, we observed that aldosterone mediates its effects on ERK phosphorylation, apoptosis, and MLC phosphorylation/contraction via a PI3 kinase–dependent pathway either in the presence or absence of GPR30 expression. Reintroduction of GPR30 into cultured VSMCs, and the consequent downregulation of MR expression, leads to signaling and functional changes that are almost completely attenuated by G15, a pattern that closely parallels the conditions seen in vivo with more robust endogenous GPR30 expression. Furthermore, in the absence of endogenous MR expression, as seen in endothelial cells, the effects of aldosterone were entirely GPR30 dependent. Thus, in total, our findings suggest an important role for GPR30 receptors in mediating the rapid vascular actions of aldosterone physiologically.
The relative potency of aldosterone versus E2 raises an important question regarding which ligand is more important, physiologically, in the activation of vascular GPR30-dependent pathways. The effects of E2 on ERK phosphorylation were only apparent in the high nanomolar range, well beyond its physiological concentrations, whereas the peak effects of aldosterone were notable in the lower picomolar range. Ultimately, the impact of these agonists on GPR30 would be dependent both on their relative potencies as well as their relative free concentrations. However, our data would suggest that, at equivalent free concentrations, aldosterone would be the more important physiological GPR30 agonist in VSMCs.
Our findings have also helped to clarify the relative importance of GPR30 in mediating the rapid signaling effects in vascular endothelial cells. Previous studies in ER knockout models have suggested that E2-mediated effects in endothelial cells were GPR30 independent and entirely dependent on ER expression.34 In our current studies, the effects of E2 on endothelial cell ERK phosphorylation were directionally opposite of those seen with selective GPR30 activation mediated by either aldosterone or the GPR30 agonist G1. Furthermore, with attenuation of endogenous endothelial cell GPR30 expression with shGPR30 transduction, E2 inhibitory effects were more pronounced. These findings suggest that, under ambient conditions in endothelial cells, E2 interacts with several receptors with countervailing effects on ERK activation and that GPR30 activation is only a minor contributor to the overall effect of E2. In contrast, our findings suggest that GPR30 activation is the major contributor to the action of aldosterone in these cells.
Eplerenone has been well characterized as a specific aldosterone antagonist.35 Our studies would suggest that eplerenone acts as both an MR antagonist and as a partial GPR30 antagonist. The significance of this GPR30-blocking effect in mediating the therapeutic actions of eplerenone is unclear. However, assuming that the effect of eplerenone to inhibit GPR30 activation is agonist independent and that the cardiovascular effects of eplerenone are aldosterone selective, these findings, in total, would suggest that aldosterone is the predominant mediator of GPR30 activation in vascular cells. At the least, our studies would suggest caution in assuming that an aldosterone-mediated effect antagonized by eplerenone proves that the effect was mediated solely through the MR.
The present studies provide evidence that acute effects of aldosterone on mechanisms regulating VSMC contractility and apoptosis rely on both MR- and GPR30-dependent pathways and suggest that agents targeting each of these receptors may provide differential levers to modulate aldosterone actions in different pathological states.
Sources of Funding
These studies were supported by grants-in-aid from the Heart and Stroke Foundation of Ontario (R.D.F.) and the Canadian Institutes of Health Research (R.G.). R.G. was supported by a New Investigator Award from the Heart and Stroke Foundation of Canada.
The authors have no conflicts to disclose.
We are very grateful to Dr. Lee Limbird, Vanderbilt University, for her editorial contributions in the revision of this manuscript.
- Received August 24, 2010.
- Revision received September 8, 2010.
- Accepted December 16, 2010.
- © 2011 American Heart Association, Inc.
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