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(Circulation. 2003;108:2400.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Aldosterone Regulates Vascular Reactivity

Short-Term Effects Mediated by Phosphatidylinositol 3-Kinase–Dependent Nitric Oxide Synthase Activation

Selina L. Liu, BSc; Saskia Schmuck, MD; Jozef Z. Chorazcyzewski, MSc; Robert Gros, PhD; Ross D. Feldman, MD

From the Departments of Physiology and Pharmacology (S.L.L., R.D.F.) and of Medicine (R.G., R.D.F.), University of Western Ontario, London, and the Robarts Research Institute (S.L.L., S.S., J.Z.C., R.G., R.D.F.), London, Ontario, Canada.

Correspondence to Dr Ross D. Feldman, Robarts Research Institute, PO Box 5015, 100 Perth Dr, London, Ontario, Canada N6A 5K8. E-mail feldmanr{at}lhsc.on.ca

Received September 30, 2002; de novo received March 31, 2003; revision received July 9, 2003; accepted July 10, 2003.

	Abstract

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Background— There is increasing evidence for rapid nongenomic effects of aldosterone. Therefore, we studied the immediate effects of aldosterone on vascular reactivity in rat aortic ring segments and on endothelial and vascular smooth muscle cellular responses.

Methods and Results— In endothelium-intact ring segments, aldosterone attenuated phenylephrine-mediated constriction (maximal reduction, 25±4% below control phenylephrine-mediated constriction). In contrast, in endothelium-denuded vessels, aldosterone mediated a monophasic dose-dependent enhancement of vasoconstrictor response. In endothelial cells, aldosterone caused a phosphatidylinositol 3-kinase (PI3K)–dependent increase in nitric oxide synthase activity as well as PI3K-dependent activation of extracellular signal–regulated kinase 1/2 and p70 S6 kinase.

Conclusions— Overall, these data support a novel effect of aldosterone on vascular endothelial and smooth muscle cell function. These rapid effects of aldosterone might be important in both the short- and long-term regulation of peripheral vascular resistance. Furthermore, in the setting of endothelial dysfunction, alterations in aldosterone’s short-term vascular responses might contribute to its pathophysiological effects in cardiovascular disease.

Key Words: arteries • endothelium • hormones • vasodilation

	Introduction

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The primary cardiovascular effect of aldosterone has traditionally been ascribed to regulation of electrolyte homeostasis and extracellular fluid volume by promotion of sodium retention and potassium excretion in the renal collecting duct. However, evidence for aldosterone-mediated responses beyond the collecting duct has been increasingly appreciated. Cardiac, vascular, and endothelial cells express "classic" aldosterone receptors.¹ It has been hypothesized that the correlation between increased circulating levels of aldosterone and cardiac hypertrophy² and myocardial and vascular fibrosis^3,4 is mediated by a direct effect on myocardial and vascular smooth muscle cells (VSMCs).⁵ However, the molecular mechanisms by which these might occur are unknown.

The renal effects of aldosterone have been ascribed to a genomic mechanism—binding to its intracellular receptor, followed by translocation of the steroid-receptor complex to the nucleus, where it acts as a transcriptional regulator. However, there has been increasing evidence to support the existence of short-term effects of aldosterone,⁶ including activation of Na⁺/H⁺ exchange,⁷ increased cytosolic calcium levels ([Ca²⁺]_i),⁸ and increased intracellular cAMP.⁹

The present study was undertaken to examine the rapid effects of aldosterone on vascular tone and to elucidate the signaling and regulatory mechanisms involved in the vascular effects of aldosterone. In data presented below, we describe a novel short-term effect of aldosterone to modulate vascular function via PI3K-dependent activation of endothelial nitric oxide synthase (NOS), as well as to activate mitogen-activated protein kinases (MAPKs) through a PI3-kinase dependent mechanism.

	Methods

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Materials
All drugs (unless otherwise specified) were purchased from Sigma Chemical Co.

Animal Protocol
Ten-week-old male, normotensive, Wistar rats (Charles River, Montreal, Canada) and spontaneously hypertensive rats (SHR; Harlan, Indianapolis, Ind) were used. The rats were cared for in accordance with Canadian Council on Animal Care guidelines. Indirect tail-cuff measurements of systolic blood pressure were obtained in lightly anesthetized rats, as previously described.¹⁰ Mean systolic pressures in 10-week-old Wistar rats and SHR were 90.8±2.9 mm Hg and 181.9±8.5 mm Hg, respectively.

Assessment of Vascular Reactivity in Aortic Rings
Assessment of vascular reactivity was performed in vascular ring preparations according to our previously published methods.¹⁰ In experiments with endothelium-denuded aortic rings, the endothelial layer was removed by flushing with distilled water.¹¹

Endothelial and VSMC Primary Cultures and Immunostaining
Bovine aortic endothelial cell (BAEC) primary cultures were isolated and cloned by the method of Schwartz.¹² Rat aortic VSMC primary cultures were isolated by a modification of the methods of Touyz et al.¹³

Assessment of NOS Activity
NOS activity was determined by assessment of alterations in 4,5-diaminofluorescein diacetate (DAF2-DA) fluorescence¹⁴ in BAECs in response to short-term addition of aldosterone. NO production mediated by the NO donor sodium nitroprusside (1 µmol/L) and acetylcholine (1 µmol/L) was used as a positive control. In experiments with inhibitors, BAECs were incubated with spironolactone (10 µmol/L), N^G-monomethyl-L-arginine (L-NMMA) (10 µmol/L), or LY-294002 (50 µmol/L) 5 minutes before the addition of aldosterone.

[Ca²⁺]_i Imaging
Alterations in [Ca²⁺]_i were assessed by fura-2 microspectrofluorimetry according to previously published techniques.¹⁵ ATP or aldosterone was applied locally onto a single cell by pressure ejection from micropipettes positioned 30 to 50 µm from the cell (Picospritzer II, General Valve Corp). In other experiments, [Ca²⁺]_i was measured in populations of cells in suspension loaded with indo-1, as described previously.¹⁶

Assessment of Protein Kinase A and Adenylyl Cyclase Activity
Protein kinase A (PKA) activity was assessed by ³²P phosphorylation of a synthetic substrate (Kemptide) according to our previously published techniques.¹⁷ Adenylyl cyclase activity was assessed by the conversion of [³H]ATP to [³H]cAMP by a modification of the methods described by Conklin et al.¹⁸

MAPK Assays
MAPK activity assays were performed on the basis of the methods of Ahn et al.¹⁹

PI3K Assays
PI3K assays were performed according to the methods of Record et al.²⁰ Kinase activity was assessed in BAEC lysates treated with aldosterone (1 nmol/L) for 10 minutes at room temperature. In experiments with inhibitors, BAECs were incubated with either spironolactone (10 µmol/L) or BAPTA (10 µmol/L, Molecular Probes) 5 minutes before stimulation with aldosterone.

Western Blot Analysis: Phospho-ERK and Phospho-p70 S6K Labeling
BAECs and VSMCs were treated and lysates were prepared as for the MAPK assay. Proteins were separated, transferred to a polyvinylidene difluoride membrane, and blotted with mouse anti–phospho-extracellular signal–regulated kinase (ERK1/2; Upstate Biotechnology) or rabbit anti–phospho-p70 S6K (Cell Signaling Technology). Equal protein loading was confirmed by stripping the membranes and reprobing them for total ERK1/2 (Upstate Biotechnology) and p70 S6K (Santa Cruz Biotechnology).

	Results

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Effect of Aldosterone on Vascular Reactivity
Aldosterone caused a biphasic attenuation of phenylephrine-mediated constriction in endothelium-intact aortic ring preparations of normotensive Wistar rats, to a maximal inhibition of 25±4% of control phenylephrine-mediated constriction at an aldosterone concentration of 0.01 nmol/L (Figure 1A and 1B). This effect was attenuated by the aldosterone receptor antagonist spironolactone (Figure 1C). Spironolactone at 0.1, 1.0, and 10 µmol/L had no significant effect on phenylephrine-mediated constriction (100.3±3.9%, 107.0±7.2%, and 95.8±8.5% of control, respectively; n=5, P=NS). However, higher doses of spironolactone (30 and 100 µmol/L) did significantly reduce phenylephrine-mediated constriction (30 µmol/L, 65.2±9.0% of control, n=5, P<0.05; 100 µmol/L, 27.4±9.4% of control, n=3, P<0.01).

View larger version (20K): [in this window] [in a new window]   Figure 1. A, Aldosterone attenuates phenylephrine-mediated constriction in endothelium-intact aortic ring segments. Ring segments were incubated for 2 minutes with indicated concentrations of aldosterone, followed by submaximal constriction with 100 nmol/L phenylephrine for 10 minutes. Constriction response with phenylephrine was quantified by determination of area under curve by trapezoidal method of analysis (See B). Data represent geometric mean±SEM for 10 independent experiments. *P<0.05, **P<0.01, ANOVA; Dunnett’s multiple comparison test. B, Representative tracings from control and aldosterone (ALDO)–treated ring segments. AUC indicates area under curve; DMSO, dimethyl sulfoxide; and PE, phenylephrine. C, Spironolactone blocks aldosterone inhibition of phenylephrine-mediated constriction. Ring segments were incubated for 5 minutes with 10 µmol/L spironolactone, followed by 2-minute incubation with indicated concentrations of aldosterone and then stimulation with 100 nmol/L phenylephrine. Data represent geometric mean±SEM for 9 (control) and 6 (spironolactone) independent experiments. *P<0.05 vs control, unpaired t test.

To determine the specificity of the inhibitory effect of aldosterone on phenylephrine-mediated constriction, we assessed the effects of 17ß-estradiol and hydrocortisone. Hydrocortisone caused a significant increase in phenylephrine-mediated constriction (Figure 2A). 17ß-Estradiol (at concentrations equivalent to those used in aldosterone studies) had no significant effect on phenylephrine-mediated constriction (Figure 2B).

View larger version (19K): [in this window] [in a new window]   Figure 2. Effect of other steroid hormones on phenylephrine-mediated constriction. Ring segments were incubated for 2 minutes with indicated concentrations of hydrocortisone or 17ß-estradiol, followed by constriction with 100 nmol/L phenylephrine for 10 minutes. A, Hydrocortisone enhances phenylephrine-mediated constriction. Data represent geometric mean±SEM for 5 independent experiments. *P<0.05, ANOVA; Dunnett’s multiple-comparison test. B, 17ß-Estradiol does not affect phenylephrine-mediated constriction. Data represent geometric mean±SEM for 7 independent experiments.

Endothelium Dependence of the Aldosterone Effect
The NOS inhibitor L-NMMA blocked the aldosterone-mediated attenuation of constrictor response in endothelium-intact aortic rings (Figure 3A). Furthermore, in rings denuded of endothelium, aldosterone mediated a monophasic enhancement of vasoconstrictor responses (Figure 3B). In endothelium-intact aortic ring segments from SHR (a model of vascular endothelial dysfunction),²¹ the inhibitory effect of aldosterone on phenylephrine constriction was essentially abolished compared with that in ring segments from normotensive Wistar rats (Figure 3C). In aggregate, these data support the hypothesis that the inhibitory effect of aldosterone on phenylephrine-mediated constriction is dependent on intact endothelial function—specifically, endothelial NOS activation.

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of aldosterone to attenuate vasoconstrictor response is endothelium dependent. A, L-NMMA attenuates aldosterone inhibition of phenylephrine-mediated constriction. Ring segments were incubated with 10 µmol/L L-NMMA, followed by 2-minutes incubation with indicated concentrations of aldosterone and then stimulation with 100 nmol/L phenylephrine. Data represent geometric mean±SEM for 10 (control) and 5 (L-NMMA) independent experiments. *P<0.01, **P<0.001 vs control, unpaired t test. B, Aldosterone enhances phenylephrine-mediated constriction in endothelium-denuded rings. Data represent geometric mean±SEM for 5 independent experiments. *P<0.05, **P<0.01, ANOVA; Dunnett’s multiple-comparison test. C, Aldosterone inhibition of phenylephrine-mediated constriction is decreased in ring segments from SHR. Ring segments from hypertensive SHR and normotensive Wistar rats were incubated for 2 minutes with indicated concentrations of aldosterone, followed by constriction with 100 nmol/L phenylephrine for 10 minutes. Data represent geometric mean±SEM for 9 (Wistar) and 6 (SHR) independent experiments.

Effect of Aldosterone on NOS Activity in ECs
To confirm that aldosterone does act by NOS activation, NOS activity was assessed in BAECs. Development of fluorescence was linear over 30 minutes. In BAECs, short-term aldosterone exposure increased NOS activity in a time- and dose-dependent manner (Figure 4A), with an ED₅₀ of 7 pmol/L and an E_max of 449±183% above control.

View larger version (31K): [in this window] [in a new window]   Figure 4. Aldosterone stimulates NOS activity. A, Aldosterone increases DAF2-DA fluorescence in dose-dependent manner. Data represent mean±SEM for 3 independent experiments. B, Spironolactone, LY-294002, and L-NMMA inhibit aldosterone-stimulated NOS activity. Before treatment with 100 pmol/L aldosterone, cells were incubated with spironolactone (10 µmol/L), LY-294002 (50 µmol/L), or L-NMMA (10 µmol/L). Data represent geometric mean±SEM for 4 or 5 independent experiments. *P<0.05 vs aldosterone.

At 100 pmol/L, the aldosterone-mediated increase in NOS activity was 54±13% of the increase seen with acetylcholine (1 µmol/L) stimulation (P<0.05, n=5). Pretreatment of BAECs with either spironolactone, the PI3K inhibitor LY-294002, or the NOS inhibitor L-NMMA attenuated the aldosterone-mediated increase in NOS activity (Figure 4B). Wortmannin (500 nmol/L) similarly attenuated aldosterone’s effect, whereas LY-303511 (50 µmol/L; the structurally related but PI3K-inactive analogue of LY-294002) had no significant effects (data not shown).

Assessment of Potential Mechanisms Responsible for Aldosterone-Mediated NOS Activation in ECs
NOS has been reported to be activated by a range of hormones and growth factors, including estrogen²² and insulin,²³ through a PI3K-dependent mechanism. To confirm that aldosterone acted by this mechanism, we assessed its effects on phosphatidylinositol phosphate (PIP) accumulation. Aldosterone treatment alone (1 nmol/L) increased PI3K activity to a maximum of 219±39% of control (n=4, P<0.05 vs control). Pretreatment of BAECs with either spironolactone (10 µmol/L) or BAPTA (10 µmol/L) alone did not significantly affect PI3K activity (119±39% and 85±23% of control, respectively; n=4). However, spironolactone or BAPTA pretreatment significantly reduced the aldosterone-mediated increase in PI3K activity (119±13% and 129±11% of control, respectively; n=4).

To determine whether the effect of aldosterone to increase NOS activity might also occur through increased [Ca²⁺]_i, [Ca²⁺]_i in BAECs was determined. By single-cell Ca²⁺ imaging, aldosterone (10 pmol/L to 10 nmol/L) alone did not produce any observable effect on calcium concentrations (data not shown).

NOS can also be activated through the adenylyl cyclase/PKA pathway.^24,25 Therefore, the short-term effect of aldosterone on the adenylyl cyclase/PKA pathway in ECs was assessed. Aldosterone (10 pmol/L to 10 nmol/L) had no effect on isoproterenol-, forskolin-, or cAMP-mediated PKA activity (data not shown). Additionally, aldosterone (1 nmol/L or 100 nmol/L) had no effect on basal or forskolin-mediated adenylyl cyclase activity (data not shown).

Lack of Effect of Aldosterone on [Ca²⁺]_i in VSMCs
Aldosterone did not produce any observable effect on [Ca²⁺]_i (12 cells), even though the cells were healthy and exhibited robust, reversible responses to ATP (100 µmol/L; data not shown).

Effect of Aldosterone on MAPK Activation
To determine whether short-term aldosterone exposure might regulate MAPK pathways—important in the long-term regulation of cell growth—we assessed its effects on ERK1/2 as well as p70 S6 kinase activity in both ECs and VSMCs. Aldosterone (10 pmol/L) increased ERK1/2 activity (140±22% above control) in BAECs. In addition, aldosterone increased phospho-ERK1/2 labeling in both BAECs and VSMCs (Figure 5A and 5C). Aldosterone also caused a significant increase in phospho-p70 S6K activation in BAECs and VSMCs (Figure 5B and 5D). In both cell types, labeling of phospho-ERK1/2 and phospho-p70 S6K was completely inhibited by pretreatment of the cells with the PI3K inhibitor LY-294002 (Figure 5).

View larger version (21K): [in this window] [in a new window]   Figure 5. PI3K inhibitor LY-294002 abrogates aldosterone-mediated ERK1/2 and p70 S6K phosphorylation in both BAECs and VSMCs. Primary cultured BAECs and VSMCs were incubated with 50 µmol/L LY-294002 for 30 minutes before 10 pmol/L aldosterone treatment. Quantification of ERK1/2 (A) and p70 S6K (B) phosphorylation in BAECs and ERK1/2 (C) and p70 S6K phosphorylation (D) in VSMCs. Data represent geometric mean±SEM for 3 to 13 independent experiments. *P<0.05.

	Discussion

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In this study, we have demonstrated a short-term effect of aldosterone on vascular function. Specifically, in endothelium-intact vascular ring segments, aldosterone rapidly attenuated phenylephrine-mediated constriction. The effect of aldosterone was potent, highly specific, and endothelial NOS dependent. In ECs, we demonstrated that aldosterone-mediated NOS activation was PI3K dependent. Furthermore, short-term aldosterone exposure resulted in both EC and VSMC ERK and p70 S6 kinase activation, which was also PI3K dependent. In aggregate, these data delineate a previously unappreciated pathway by which aldosterone modulates vascular reactivity.

The short-term attenuation of phenylephrine constriction by aldosterone was rapid, occurring within minutes, consistent with the time course of a "nongenomic" effect. The effect was potent, occurring at concentrations in the picomolar range (well within the physiological circulating concentrations of aldosterone). Furthermore, the effect was specific, ie, not mimicked by comparable concentrations of either hydrocortisone or estrogen. Notably, short-term hydrocortisone exposure mediated an enhancement of vasoconstrictor response—consistent with the findings of others.^26,27 17ß-Estradiol has previously been demonstrated to "nongenomically" enhance vasorelaxation and attenuate vasoconstriction,^28,29 ie, effects comparable to those of aldosterone. However, these previously reported effects of estradiol were seen at much higher concentrations (nmol/L to µmol/L) than those used in the present studies. The ability of spironolactone to block the aldosterone effects suggests that the attenuation of constriction is due to high-affinity binding of aldosterone to a "classic" mineralocorticoid receptor. This is in contrast to the effects observed by other investigators, where the rapid (nongenomic) effects of aldosterone were not blocked by spironolactone treatment.^6,8

The effect of aldosterone to attenuate vasoconstrictor responses is endothelial/NO dependent, although a minor role for vasodilatory prostanoids has not been ruled out. Three lines of evidence support this conclusion. First, the effect of aldosterone to attenuate vasoconstrictor responses was lost after endothelial denudation. In fact, in the absence of the endothelium, the effect of aldosterone was converted to a vasoconstrictor response—reminiscent of the pattern of response to acetylcholine in endothelium-intact versus denuded preparations,¹³ and similar to the effect of hydrocortisone in intact ring preparations. Second, in rings from SHR, which demonstrate endothelial dysfunction,²¹ the ability of aldosterone to attenuate vasoconstrictor responses was lost. Last, the effect of aldosterone to attenuate vasoconstriction was lost after inhibition of NOS activity by L-NMMA. Overall, these data would predict that the effects of aldosterone to increase peripheral resistance (or the effect of aldosterone antagonism to reduce peripheral resistance) will be most evident either in the settings of (1) endothelial dysfunction or (2) experimental models where any impact of endothelium-mediated vasodilation is minimized (eg, with preexisting shear stress activation).

We used cellular models of ECs and VSMCs to delineate the mechanisms underlying the short-term vascular effects of aldosterone. In ECs, we confirmed that short-term aldosterone exposure stimulated NOS activation Also, these studies suggested that NOS activation by aldosterone was, at least in part, PI3K dependent. First, the PI3K inhibitor LY-294002 inhibited the aldosterone-mediated NOS activation. Second, we demonstrated that aldosterone stimulated PI3K activation at picomolar concentrations—comparable to the concentrations at which aldosterone activated NOS. Notably, PI3K-mediated NOS activation has been implicated in the vasodilator effects of both insulin²³ and estrogen²² (at higher concentrations than those tested here). The endothelial actions of aldosterone appear to utilize a common pathway.

The effect of aldosterone on vascular reactivity and on NOS activation does not appear to be mediated by either adenylyl cyclase activation or alterations in [Ca²⁺]_i (by techniques that we have previously demonstrated to be very sensitive in detecting hormone-mediated effects³⁰). Aldosterone-mediated adenylyl cyclase activation has been reported in porcine vascular SMCs.⁹ The reason for these divergences with previous findings is not apparent but could be related to differences in the tissues studied or the higher concentrations of aldosterone used in previous studies (maximal effects of aldosterone in those studies were only evident at nmol/L to µmol/L concentrations). However, regardless of the explanation, it is unlikely that adenylyl cyclase activation accounts for the effects of aldosterone to either activate NOS or to attenuate vasoconstrictor responses via an endothelium-dependent mechanism.

Notably, there are recent reports of (1) no immediate effects of aldosterone on vascular reactivity,³¹ (2) no effect of aldosterone on norepinephrine-mediated vasoconstriction but attenuation of acetylcholine-mediated vasodilation,³² and (3) aldosterone-mediated vasoconstriction.³³ However, all of these studies were performed at concentrations that, on the basis of our studies and the biphasic nature of aldosterone-mediated attenuation of vasoconstriction response, we would have predicted to have either no effect (with intact endothelial function) or vasoconstriction (with endothelial dysfunction). The most direct interpretation of all of these studies is that (1) at physiological concentrations of aldosterone there are constitutive, nongenomic effects mediated via NOS activation and (2) dependent on vessel and species variability (as well as endothelial integrity), there are vasoconstrictor effects at pathologic concentrations.

Beyond its effects to activate NOS, we demonstrated that short-term aldosterone exposure results in activation of ERK and p70 S6 kinase, critical enzymes with critical roles in proliferative signaling cascades. The effect of aldosterone to mediate the rapid phosphorylation of p70 S6 kinase in both BAECs and VSMCs is consistent with the well-recognized activation of this enzyme via PI3K pathways.³⁴ Aldosterone-mediated ERK activation (previously demonstrated in Madin-Darby canine kidney cells³⁵) appears to be PI3K dependent, consistent with a role for PKB-mediated regulation of ERK activation.³⁶ Notably, the short-term effects of aldosterone to regulate MAPK activation might be expected to be synergistic with both (1) the short-term effects of angiotensin II to activate MAPK pathways³⁷ and (2) in a more general sense, the classic "transcriptional" effects of aldosterone. The implications of these synergies on vascular and endothelial hypertrophic mechanisms remain to be elucidated.

Beyond its effect on proliferative pathways, aldosterone-mediated ERK activation might be implicated in the effect of aldosterone to enhance short-term vasoconstrictor responses in endothelium-denuded preparations. A recent study has suggested such a role for ERK activation as a vasoconstrictor mechanism.³⁸ Whether the effect of aldosterone to enhance vasoconstrictor response occurs via ERK activation remains to be established.

In summary, we have demonstrated a complex pattern of short-term effects of aldosterone at both a functional level and in endothelial and vascular smooth muscle cells, thus reflecting a novel nongenomic pathway of vascular regulation by aldosterone. These effects could be seen as a balance among (1) endothelial and vascular smooth muscle mechanisms, (2) vasodilator and vasoconstrictor mechanisms, and ultimately, and (3) pathways that regulate vascular tone in the short term and longer-term proliferative pathways. The balance between these mechanisms might have an important role in short- and long-term vascular regulation (Figure 6). In addition, a shift in the balance of these mechanisms, ie, in the setting of a range of syndromes characterized by endothelial dysfunction (eg, hypertension and congestive heart failure), might contribute to the increasingly appreciated pathophysiological role of aldosterone in the progression of cardiovascular disease complications.

View larger version (34K): [in this window] [in a new window]   Figure 6. Schematic demonstrating possible divergence between short- and long-term effects of aldosterone (A) on ECs and VSMCs. Other abbreviations are as defined in text.

	Acknowledgments

 
Acknowledgments

These studies were supported by a grant-in-aid from the Canadian Institutes of Health Research. Dr Feldman is a Career Investigator of the Heart and Stroke Foundation of Ontario. We are grateful to Dr S.J. Dixon, Dr Stephen Sims, and Caiqiong Liu for assistance with measurement of [Ca²⁺]_i.

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