Rapid actions of aldosterone in vascular health and disease—friend or foe?

Pharmacology & Therapeutics 111 (2006) 495 – 507 www.elsevier.com/locate/pharmthera

Associate editor: F. Brunner

Rapid actions of aldosterone in vascular health and disease—friend or foe? Ole Skøtt *, Torben R. Uhrenholt, Jeppe Schjerning, Pernille B.L. Hansen, Lasse E. Rasmussen, Boye L. Jensen Department of Physiology and Pharmacology, University of Southern Denmark, DK-5000 Odense, Denmark

Abstract The mineralocorticoid receptor (MR) and the enzyme 11hhydroxysteroid dehydrogenase type 2, which confers aldosterone specificity to the MR, are present in endothelium and vascular smooth muscle. In several pathological conditions aldosterone promotes vascular damage by formation of reactive oxygen species. The effect of aldosterone on vascular function, however, is far from clear. By rapid non-genomic mechanisms aldosterone may cause calcium mobilization and vasoconstriction, or may stimulate nitric oxide formation through the PI-3 kinase/ Akt pathway and thereby counteract vasoconstriction. Vasoconstrictor, vasodilator or no effects of aldosterone have been reported from studies on human forearm blood flow. Inhibition of MR with spironolactone improves endothelial function in patients with heart failure but worsens endothelial function in type 2 diabetic patients. The aim of the present review is to reconcile some of the apparently conflicting data. A key observation is that reactive oxygen and nitrogen species serve as physiological signaling molecules at low concentrations, while they initiate pathological processes at higher concentrations. The net effect of aldosterone, which stimulates ROS production, therefore depends on the ambient level of oxidative stress. Thus, in situations with low levels of oxidative stress aldosterone may promote vasodilatation, while at higher oxidative stress (high NaCl intake, pre-existing vascular pathological conditions, high oxygen tension in vitro) aldosterone is likely to be associated with vasoconstriction and oxidative damage, and in this setting inhibition of the MR is likely to be beneficial. D 2006 Elsevier Inc. All rights reserved. Keywords: Endothelium; Kidney; Steroid; Smooth muscle Abbreviations: 11hHSD1, 11hhydroxysteroid dehydrogenase type 1; 11hHSD2, 11hhydroxysteroid dehydrogenase type 2; eNOS, endothelial nitric oxide synthase; ERK1/2, extracellular signal-regulated kinases 1 and 2; Hsp, Heat shock protein; IP3, inositol 1,4,5 triphosphate; K d, equilibrium dissociation constant; K off, dissociation rate constant; MR, mineralocorticoid receptor; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; PI-3 kinase, phosphatidylinositol 3-kinase; PKB/Akt, protein kinase B; PLC, phospholipase C; ROS, reactive oxygen species; VLDL, very low density lipoprotein.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mineralocorticoid receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. MR structure and regulation of ligand specificity and response specificity . 2.2. Origin of aldosterone that activates the mineralocorticoid receptor . . . . . 11hHydroxysteroid dehydrogenase (11hHSD2) . . . . . . . . . . . . . . . . . . MR and 11hHSD2 in the vascular wall . . . . . . . . . . . . . . . . . . . . . . Rapid vasoconstrictor actions of aldosterone . . . . . . . . . . . . . . . . . . . . Rapid vasodilator actions of aldosterone . . . . . . . . . . . . . . . . . . . . . . 6.1. Signaling cascade involved in the vasodilator effect of aldosterone . . . . Acute vascular effects of aldosterone in conscious humans . . . . . . . . . . . . Interim conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress and cellular signaling . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +45 6550 3752; fax: +45 6613 3479. E-mail address: [email protected] (O. Skøtt). 0163-7258/$ - see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.10.010

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10. 11. 12.

Aldosterone as an activator of reactive oxygen (and General effects of steroids on endothelial function . NaCl and aldosterone . . . . . . . . . . . . . . . . 12.1. Salt and human evolution. . . . . . . . . . 13. Consequences for treatment . . . . . . . . . . . . . 14. Conclusion . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Aldosterone is synthesized and released mainly from the zona glomerulosa cells in the adrenal glands. The classical target cells for aldosterone are involved in NaCl transport: the principal cells of the renal distal tubules and collecting ducts, distal colon, and sweat glands, where aldosterone binds to mineralocorticoid receptors (MR), which are translocated to the nucleus, where they initiate transcription of target genes. Consistent with this, deletion of the MR in mice leads to a lethal salt-wasting phenotype, which is salvaged by NaCl supplementation (Berger et al., 1998). More recently, it has been realized that aldosterone acts on a number of non-traditional target tissues, and initiates rapid responses that do not involve gene transcription. In normal physiological situations, aldosterone is regulated inversely with salt status. With a low salt intake plasma aldosterone concentration may increase to high levels without any negative effects on the organism. If aldosterone becomes inappropriately high for the salt status, then aldosterone becomes associated with cardiac fibrosis, left ventricular remodeling, endothelial dysfunction, vasculopathy, vascular remodeling, and renal injury (review Brown, 2005). All of these effects are alleviated by inhibition of the MR with spironolactone, K+-canrenoate or eplerenone. In several human pathological conditions, the MR seems inappropriately activated, even under conditions where plasma aldosterone concentration is not increased. There has been a dramatic increase in the interest of the role played by aldosterone in the cardiovascular system after the Randomized Aldactone Evaluation Study (RALES) which, in 1999, demonstrated that inhibition of MR led to a 30% reduction in mortality rates in patients with heart failure after myocardial infarction (Pitt et al., 1999). The aim of the present review is to delineate some aspects of aldosterone effects on vascular function based on a previous shorter overview (Uhrenholt et al., 2004), and to possibly contribute to reconciliation of apparently conflicting data regarding acute (non-genomic) effects of aldosterone. In order to appreciate the basics involved in aldosterone signaling we include short overviews of the functions of the MR and 11hhydroxysteroid dehydrogenase type 2 (11hHSD2), as well as redox signaling and oxidative stress. 2. The mineralocorticoid receptor The mineralocorticoid receptor is a steroid hormone receptor that belongs to the larger family of nuclear receptors, which comprises the steroid hormone receptors (estrogen a and

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h, progesterone, androgen, glucocorticoid and mineralocorticoid), non-steroid hormone receptors (retinoid acid, thyroid hormone, vitamin D and retinoid X), nutritional sensors (e.g. peroxisome proliferator-activated receptor), receptors with structural ligands, and a large group of orphan receptors (Benoit et al., 2004). The steroid receptor family arose in early vertebrates by a series of gene duplications of an ancestral nuclear receptor, which was probably the estrogen receptor (Baker, 2001). Initially, steroid binding specificity was low, as it still is in fishes, and physiological response specificity was achieved to a large degree by local regulation of steroid concentration, which was mediated by steroidogenic and steroid-inactivating enzymes. Glucocorticoids and aldosterone bind to the MR with high affinity, and glucocorticoids bind to glucocorticoid receptors with lower affinity. Fishes possess mineralocorticoid and glucocorticoid receptors while they generally do not possess aldosterone or the enzymes that produce it (Baker, 2004) suggesting that MR originally may have served as a high affinity glucocorticoid receptor. After the colonization of land, expression in mineralocorticoid target tissues (kidney, colon, sweat glands) of 11hHSD2 that metabolizes glucocorticoids, but not aldosterone, had the consequence that aldosterone became the main mineralocorticoid in land animals. 2.1. MR structure and regulation of ligand specificity and response specificity The human MR gene is located to chromosome 4 (q31.1), it contains 10 exons and is about 450 kb in size. The usual transcription initiation site is two base pairs into exon 2 and gives rise to a 107 kDa translation product. Recently ten putative transcription initiation sites (AUG codons) have been identified (Lu & Cidlowski, 2005) which may potentially give rise to mRNA with different stability and translational efficiency (Pascual-Le Tallec & Lombe`s, 2005). The nuclear receptors consist of a highly conserved DNA binding domain localized centrally in the receptor, a well conserved ligand-binding domain at the carboxyterminal end, and a more variable N-terminal domain (Fig. 1). Activation function domains in the N-terminal domain and in the ligand binding domain bind coactivators and corepressors that are important for transcriptional activation (review Benoit et al., 2004). The DNA binding domain binds to hormone response elements of DNA by two zinc finger domains and is highly conserved among the different steroid hormone receptors. The

O. Skøtt et al. / Pharmacology & Therapeutics 111 (2006) 495 – 507

Fig. 1. The mineralocorticoid receptor consists of three main moieties: the ligand binding domain which binds aldosterone (and glucocorticoids), the DNA binding domain, which binds to canonical hormone response elements in the DNA, and the N-terminal domain, which contains regulatory elements. Arrow indicates interdomain interactions in the mineralocorticoid receptor that are stronger in the presence of aldosterone than cortisol and thereby contributes to transactivation specificity. Adapted from Pascual-Le Tallec et al. (2005).

glucocorticoid and the mineralocorticoid receptors have 94% homology in this segment, which is consistent with the fact that after ligand binding the two receptors bind to the same glucocorticoid response element. This raises the questions as to how differential cellular responses can be obtained after activation of the glucocorticoid versus the mineralocorticoid receptor. The DNA binding domain also contains a nuclear localization signal. A naturally occurring mutation (G633R) in the DNA binding domain of the human MR is associated with a reduced dissociation rate from DNA, and to a preferential nuclear localization of the unbound receptor. The mutation is associated with a reduced transcriptional activity and leads to pseudohypoaldosteronism type 1 (Sartorato et al., 2004). The C-terminal part of the receptor forms the ligand binding domain which has a length of 251 amino acids. There is a relatively high homology (57%) between mineralocorticoid and glucocorticoid receptors in the ligand binding domain (review Rashid & Lewis, 2005). This part of the molecule contains a hydrophobic pocket that binds the different hormones. In the absence of hormone, the ligand binding domain binds chaperone proteins such as Heat shock proteins (Hsp) 90 and 70, and different immunophilins (review PascualLe Tallec & Lombe`s, 2005). After binding of the hormone, the chaperones are released from the ligand binding domain, and the nuclear localization signal is exposed. The ligand binding domain is also responsible for the formation of receptor dimers. Naturally occurring mutations in the ligand binding domain region (Q776R and L979P) lead to a decrease or a total loss of the aldosterone binding and leads to pseudohypoaldosteronism type 1 (Sartorato et al., 2004). The K d values for binding of aldosterone and glucocorticoids to the MR are similar, so in vitro, both hormones bind equally well. In spite of this, the

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binding kinetics are not identical, since the K off rate for glucocorticoids is about 5 times faster than that of aldosterone (Lombe`s et al., 1994), allowing the aldosterone-MR complex a much more active role in the transactivation process. An extra hydrogen bond between aldosterone and helix 10 of the ligand binding domain, as revealed by crystallography, may explain the tighter binding (Bledsoe et al., 2005). The N-terminal domain consists of 602 amino acids and is very variable among the different steroids. Thus, there is less than 15% homology between mineralocorticoid and glucocorticoid receptors in this segment, and in the MR there are two activation function 1 segments in comparison to only one in the glucocorticoid receptor (Pascual-Le Tallec et al., 2005). A number of cofactors are molecular partners of the N-terminal domain. Recently the human elongation factor ELL (elevennineteen lysine-rich leukemia) was identified as a coactivator of MR that increased MR-mediated transactivation by 2.5 fold. At the same time the elongation factor was a corepressor for glucocorticoid receptors and inhibited transactivation mediated by this receptor by 90% (Pascual-Le Tallec et al., 2005). Thus, this elongation factor is a candidate to provide specificity for a MR response over a glucocorticoid receptor response in cells that express both receptors. Recruitment and binding of the coactivators RNA helicase A/Creb binding protein (CBP) complexes to the AF1a region lead to potentiation of MR transcriptional activity when aldosterone is the ligand, but not when hydrocortisone is bound to the ligand binding domain of the MR (Kitagawa et al., 2002). This mechanism may help aldosterone to be more efficient than glucocorticoids in inducing gene activation after binding to the MR. The activation function segments are likely to be key determinants for MR specificity. Thus, a number of intrinsic properties of the mineralocorticoid receptor and its ability to recruit corepressors and coactivators help conferring selectivity after binding of aldosterone. This is especially important in tissues with low activity of the glucocorticoid-metabolizing enzyme 11hhydroxysteroid dehydrogenase type 2 (11hHSD2, see below). The distribution and expression in cardiovascular tissue of coactivators and corepressors that confer aldosterone-specificity to the MR and MR-response-specificity to the glucocorticoid response element is yet largely unknown but is highly relevant for identification of potential targets for intervention with MR response. 2.2. Origin of aldosterone that activates the mineralocorticoid receptor It is controversial whether all of the aldosterone that activates vascular mineralocorticoid receptor is derived from the circulation. Thus, local production of aldosterone in endothelium and vascular smooth muscle was suggested in initial reports (Takeda et al., 1994a, 1996), while this has not been confirmed in several recent studies (Ahmad et al., 2004; Jaffe & Mendelsohn, 2005). Overexpression of aldosterone synthase in cardiomyocytes leads to dysfunction of the coronary arteries, suggesting that aldosterone produced in the local tissues at least under some circumstances, may reach the blood vessels (Garnier et al., 2004).

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3. 11BHydroxysteroid dehydrogenase (11BHSD2) The circulating concentration of glucocorticoids is about 100-fold higher than that of aldosterone, depending on the level of sodium intake. With variations in sodium content in the rat feed from 3% to 0.014% the plasma corticosterone concentration varies between 180 and 480 nmol/L, while plasma aldosterone concentration is between 0.074 and 20 nmol/L, resulting in glucocorticoid to aldosterone ratios between 2400 and 24 (Bistrup et al., 2005; Nørregaard et al., 2003). The affinity for the mineralocorticoid receptor is equally high for the two hormones (K d 0.5 nM, Table 1) and specificity is maintained in the classical aldosterone target tissues by the enzyme 11hHSD2, which reduces cortisol/corticosterone to inactive 11-keto metabolites (Funder et al., 1988; Sandeep & Walker, 2001). Mice with deletion of 11hHSD2 display a renal phenotype with increased MR-activation resulting in sodium retention, hypertension (to 150 mm Hg), and potassium wasting (Kotelevtsev et al., 1999), which all can be prevented by MRantagonists. These results show that the specificity conferred to the MR by ligand-specific coactivator recruitment is not sufficient to avoid inappropriate activation of the MR when 11hHSD2 is absent from the kidney. Therefore, the presence of 11hHSD2 in mineralocorticoid sensitive tissues protects the high-affinity MR against activation by glucocorticoids. Even when 11hHSD2 is present, significant binding of glucocorticoids to the MR is likely to occur. However, in the presence of 11hHSD2 this occupancy does not lead to MR activation. Funder (2005) has suggested a hypothesis to explain this paradox: 11hHSD2 produce one NADH per molecule of glucocorticoid which is metabolized, and this NADH could contribute to inhibition of glucocorticoid – MR complexes. Based on the description of a redox-sensitive activation of the co-repressor C-terminal binding protein, which binds to the activation function 2 segment of several nuclear receptors, including the estrogen and glucocorticoid receptor (Zhang et al., 2002). Funder speculated that NADH stimulates corepressor function after binding to the MR of glucocorticoids but not of aldosterone. If true, changes in redox status of the cells could induce transcriptional activity by MR already occupied by glucocorticoids. Thus, inhibition of MR could have beneficial clinical effects in situations with local inflammation and increased oxidative stress, such as atherosclerosis—even in the absence of changes in steroid hormone concentration or Table 1 Corticosteroid binding to hMR and hGR

Aldosterone Cortisol

Human mineralocorticoid receptor

Human glucocorticoid receptor

K d (nM)

t 1/2 (min)

K d (nM)

t 1/2 (min)

0.52 T 0.03 0.49 T 0.02

140 45

14.4 T 2.1 11.7 T 0.89

5 5

Corticosteroid binding to synthetic human mineralocorticoid and glucocorticoid receptors. The equilibrium dissociation constant (K d) is similar for binding of aldosterone and cortisol to both receptors, but the half life (t 1/2) of the aldosterone – mineralocorticoid receptor complex is longer than that of the cortisol – mineralocorticoid receptor complex (from Hellal-Levy et al., 1999).

receptor expression. In addition, the hypothesis implies that glucocorticoids may act as functional inhibitors of MR activation by competing with aldosterone for binding. Although there is circumstantial support for this intriguing hypothesis, more evidence is clearly needed. 4. MR and 11BHSD2 in the vascular wall The first report on the presence of MR and 11hHSD in blood vessels came in 1982, (Kornel et al., 1982), This was followed by the demonstration of a vascular 11hHSD activity, which was considerably lower than the renal 11hHSD activity (Walker et al., 1991). The presence of active forms of both 11hHSD isoforms (types 1 and 2) in human blood vessels from the female reproductive organs (uterine arteries, chorionic vessels) was reported by Alzamora et al. (2000). Regarding the cellular localization, endothelial cells express 11hHSD2 (Brem et al., 1998) and MR (Golestaneh et al., 2001; Uhrenholt et al., 2003). Vascular smooth muscle cells also express both 11hHSD2 (Takeda et al., 1994a, 1994b) and MR, and in smooth muscle cell cultures the two are colocalized (Kornel, 1994). We demonstrated 11hHSD2 and MR mRNA and protein in renal afferent arterioles, aorta and left cardiac ventricle (Uhrenholt et al., 2003). Jaffe and Mendelsohn (2005) demonstrated MR and 11hHSD2 mRNA and protein in vascular smooth muscle freshly isolated from human coronary arteries and in immortalized vascular smooth muscle cell cultures derived there from. In addition, transfection of the smooth muscle cells with a MRresponse element coupled to a luciferase gene allowed them to show that inhibition of 11hHSD2 with glycyrrhetinic acid increased the cortisol-sensitivity of MR, thus demonstrating that 11hHSD2 is functional in the human VMSC. In contrast, 11hHSD2 in mice was reported to be confined to the endothelium and not present in smooth muscle cells, while 11hHSD1 had the opposite distribution (Christy et al., 2003). The concomitant expression of 11hHSD2 and MR in vascular smooth muscle and endothelial cells suggest that the high-affinity MR has important physiological functions as a target for aldosterone in the blood vessels. 5. Rapid vasoconstrictor actions of aldosterone In addition to the classical genomic effects, aldosterone has effects that are observed after a few minutes, and therefore are too rapid for gene transcription. These effects are insensitive to transcription inhibitors, such as actinomycin D. Two different types of non-genomic responses have been reported. One type is mediated by the classical MR and involves activation of intracellular kinases of the ERK1/2, PI-3 kinase S6 kinase (see below), while the other response has been suggested to involve a yet unidentified membrane receptor and lead to an increase of the intracellular calcium concentration and is associated with stimulation of phospholipase C, liberation of IP3 and diacyl glycerol, activation of protein kinase C, and of sodium –proton exchange (Christ et al., 1995a, 1995b; Wehling et al., 1994). Consistent with this, Arima et al. (2003) found that aldosterone (1– 10 nM) induced vasoconstriction in microperfused afferent

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arterioles by a mechanism involving activation of PLC and calcium mobilization. This vasoconstrictor response seemed not to involve the MR, because it was not inhibited by spironolactone, and it was elicited by aldosterone coupled to albumin, which does not readily cross cell membranes. Consistent with this, aldosterone increased the intracellular calcium concentration in Human Embryonic Kidney cells that did not express the MR (Grossmann et al., 2005) and in fibroblasts of MR knockout mice (Haseroth et al., 1999). The notion that the ‘‘vasoconstrictor pathway’’ is activated by aldosterone only independently of the MR is questioned by studies of Alzamora et al. (2000) and Michea et al. (2005). Alzamora et al. reported a rapid non-genomic activation of sodium proton exchange by aldosterone in human uterine and chorionic arteries. The effect was not affected by spironolactone while the MR-inhibitor RU28318 completely abolished the response. Similarly, Michea et al. (2005) reported that aldosterone (10 nM) enhanced the vasoconstrictor response to phenylephrine in large mesenteric vessels and caused a vasoconstrictor response and a slow increase in intracellular calcium concentration and pH in mesenteric resistance arteries. The aldosterone response was significantly reduced by inhibition of protein kinase C and by eplerenone, suggesting that the classical MR was involved. Considerations of the binding properties of the different inhibitors to the classical MR, led Lo¨sel et al. (2003) to suggest that eplerenone and the RU compound simply inhibits the yet unidentified nonclassical aldosterone receptor. If the non-genomic vasoconstrictor action is mediated by a non-classical MR, then there may be a clinical advantage in using eplerenone which may inhibit both the classical and non-classical MR instead of spironolactone, which inhibits only the classical MR. Aldosterone led to an increase in intracellular pH which was interpreted to be dependent on activation of the sodium – proton exchanger type 1, because it was blocked by 100 AM

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amiloride. Furthermore, 100 AM amiloride blocked aldosterone-mediated vasoconstriction. Recent studies based on atomic force microscopy showed that aldosterone mediated endothelial swelling which involved activation of the epithelial Na+ channel (Oberleithner et al., 2004). Because amiloride inhibits the epithelial Na+ channel, the ability of amiloride to inhibit aldosterone-mediated vasoconstriction may have several explanations. Whatever the basis for the effect of amiloride, treatment of heart failure patients with amiloride does not improve endothelial function (Farquharson & Struthers, 2002). Thus, there is experimental evidence that aldosterone in a number of circumstances can stimulate an increase in intracellular calcium concentration and cause vasoconstriction by a mechanism which involves PKC, and which may be independent of the classical MR (Fig. 2). 6. Rapid vasodilator actions of aldosterone Based on the prior reports on aldosterone-induced vasoconstriction we tested the effect of aldosterone in microperfused rabbit renal afferent arterioles (inner diameter 15– 18 Am) (Uhrenholt et al., 2003), and were surprised to find that aldosterone in concentrations from 10 9 to 10 5 M did not change vascular diameter. To the contrary, aldosterone counteracted vasoconstriction induced by exposure to 100 mM K+. The response was rapid with significant inhibition of vasoconstriction after 5 min, and maximal response after 20 min, after which time the effect declined. Consistent with a non-genomic effect, inhibition of transcription with actinomycin D did not inhibit the response. The effect was fully reversible, and it was significant already at subpicomolar concentrations of aldosterone, and it was blocked by spironolactone, but not by blockade of the glucocorticoid receptor with mifepristone (Uhrenholt et al., 2003). The findings in the small rabbit renal resistance arterioles were confirmed simultaneously in rat

Fig. 2. Cellular pathways induced by aldosterone. In endothelial cells aldosterone stimulates NO production via the classical mineralocorticoid receptor and the PI-3 kinase pathway. NO diffuses to the smooth muscle where it counteracts vasoconstriction. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), and the ribosomal S6-kinase (p70S6K) are also activated via the MR, but are not involved in NO-activation. In vascular smooth muscle aldosterone mobilises calcium via a mechanism which may not involve the classical MR. The intracellular signaling cascade involves activation of the PLC, IP3, diacylglycerol (DAG), and protein kinase C (PKC) signaling systems to promote an increase in intracellular calcium concentration and, via activation of sodium – proton exchanger type 1 (NHE1), alkalinisation of the cytosol. If not inhibited by NO from the endothelium, activation of this pathway will lead to vasoconstriction. Thus, in conditions with endothelial dysfunction, aldosterone is likely to cause vasoconstriction.

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aortic rings by Liu et al. (2003a), who reported that aldosterone counteracted vasoconstriction induced by phenylephrine. The effect was observed after 2 min, and it was significant at picomolar concentrations of aldosterone, while at supraphysiological concentrations, 100 nM, there was no effect (Liu et al., 2003a). Also in this case, the rapid non-genomic effect of aldosterone was blocked by spironolactone (Liu et al., 2003a). In conclusion, aldosterone counteracts vasoconstriction within 2– 5 min by a mechanism, which is mediated by the MR, and which does not depend on transcription. 6.1. Signaling cascade involved in the vasodilator effect of aldosterone Steroid hormones and growth factors may mediate vasodilatation by a signal sequence that involves activation of the PI-3 kinase, activation of PKB/Akt, phosphorylation of the endothelial NO synthase (eNOS), and release of NO (Bucci et al., 2002, Fig. 2). The PI-3 kinase is composed of a regulatory (p85a) and a catalytic (p110a) subunit, which is present in the vascular tree (Uhrenholt et al., 2003). Aldosterone doubled PI-3 kinase activity in endothelial cells through the MR (Liu et al., 2003a), and inhibition of the kinase with LY294002 abolished the vasodilator effect of aldosterone in renal afferent arterioles and in aorta (Liu et al., 2003a; Uhrenholt et al., 2003). Aldosterone enhanced vascular NO production as measured by DAF fluorescence, and its effect on vasoconstriction was abolished by inhibition of eNOS with l-NAME in aorta and arterioles (Liu et al., 2003a; Uhrenholt et al., 2003). Furthermore, a NO-donor, sodium nitroprusside, mimicked the effect of aldosterone, and inhibition of the soluble guanylate cyclase, which is a main target of NO, abolished the aldosterone effect (Uhrenholt et al., 2003). In endothelium-denuded rings, aldosterone enhanced phenylephrine-induced vasoconstriction at the same low concentrations that counteracted vasoconstriction in the endothelium-intact rings (Liu et al., 2003a). This finding demonstrates the importance of a functioning endothelium for the vasodilator response. Further evidence for the importance of a functioning endothelium was the observation of Liu et al. (2003a) that the vasodilator effect of aldosterone was absent in spontaneously hypertensive rats that are notorious for endothelial dysfunction. In addition to the PI-3 kinase, aldosterone also activates the extracellular regulated kinase ERK1/2 and the p70 S6kinase (Liu et al., 2003a). This is consistent with the findings of Grossmann et al. (2005) who transfected Chinese Hamster Ovary and Human Embryonic Kidney (HEK) cells with human mineralocorticoid receptors and reported that aldosterone induced ERK1/2 phosphorylation within 5 min in both cell types. The response to aldosterone was inhibited by spironolactone, by inhibitors of MAPK kinase and Src kinase, while the response was unaffected by PKC inhibition. The ERK1/2 and p70 S6kinase seem not to be involved in the vasodilatory response (Liu et al., 2003a). These studies allow the conclusion that aldosterone counteracts vasoconstriction by an endothelium-dependent mecha-

nism, which involves activation of PI-3 kinase, eNOS and release of NO. Activation of eNOS is an intricate process, where calciumindependent stimulation of eNOS involves phosphorylation of Ser 1179 (1177 in rodents). This site is an important target for the PKB/Akt mediated stimulation of eNOS, and activation of the PI-3 kinase—PKB/Akt pathway is involved in many different stimuli leading to calcium-independent stimulation of NO production (steroid hormones, tyrosine kinase receptor activating hormones including growth factors and insulin). Also, protein kinase A, AMP-activated protein kinase and Ca2+/calmodulin-dependent protein kinase II have been implicated in eNOS phosphorylation at the Ser 1179 site. On the other hand, dephosphorylation of Thr497 on eNOS seems important for the ability of the eNOS to yield NO. If Thr497 is phosphorylated, eNOS activity is increased, but at the same time its function is shifted to produce superoxide, and Thr497 may thereby serve as a molecular switch that determines whether eNOS generates NO or superoxide (Lin et al., 2003). Heat shock protein 90 (Hsp90) is a molecular chaperone that may participate in aldosterone-activation of eNOS in several ways. Hsp90 is involved in the complex formation of eNOS with PKB/Akt, which is important for calcium-independent activation of eNOS (Balligand, 2002), and two Hsp90 molecules are released from the ligand-binding domain of the MR after binding of aldosterone. It is tempting to speculate that the liberation of Hsp90 from the receptor complex may assist in activating the eNOS. The observation that inhibition of Hsp90 with geldanamycin abolishes the effect of aldosterone is consistent with an important role of Hsp90 in aldosterone signaling (Uhrenholt et al., 2003). In conclusion, aldosterone counteracts vasoconstriction by a mechanism that involves activation of the MR, stimulation of PI-3 kinase, Akt, eNOS and release of NO from the endothelium. When active, the mechanism obviously overrides the direct ability of aldosterone to increase vascular calcium concentration. 7. Acute vascular effects of aldosterone in conscious humans The picture in studies on humans is as diverse as the animal studies described above. Schmidt et al. (2003) infused aldosterone (1.4 nmol/min = 500 ng/min) into the forearm of healthy humans and observed a vasodilatation, which became significant after 4 min. Other investigators using this paradigm observed rapid vasoconstriction after infusion of aldosterone at 2.5 pmol/min (= 0.9 ng/min) (Romagni et al., 2003) or observed no effect after infusion of 10, 50 or 100 ng/min (= 28, 140, and 280 pmol/min)(Gunaruwan et al., 2002). Over 40 years ago Klein & Henk (1963) injected aldosterone (0.5 mg Aldocorten) into human patients with gastrointestinal diseases. The authors describe their findings as follows: ‘‘Aldosterone led to a rapid increase of low stroke volumes and cardiac outputs simultaneously with an increase in blood pressure, blood pressure amplitude, and a fall in the peripheral resistance and pulse wave velocity (the changes

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were maximal 5 minutes after the injection)’’ (originally in German). The 38.9% fall in peripheral resistance was maximal 5 min after the bolus injection of aldosterone, consistent with a rapid, non-genomic vasodilatory effect of aldosterone. In recent years the paper has mostly been cited to demonstrate the opposite. Schmidt et al. (1999) reported that bolus injection of 0.5 mg aldosterone into healthy young volunteers was associated with a higher systemic vascular resistance 6 and 30 min after injection when compared to controls or injection of 0.05 mg aldosterone. However, the systemic vascular resistance was higher in the 0.5 mg group already before injection of aldosterone. If this is taken into consideration there was no acute effect of aldosterone. By contrast, bolus injection of 1 mg aldosterone into 59 years old patients with suspected coronary artery disease was associated with a significant transient 5 – 6% increase in systemic vascular resistance after 3 min, which had disappeared after 10 min (Wehling et al., 1998). The latter study suggests that in patients with a high likelihood of endothelial dysfunction, aldosterone acts as a vasoconstrictor. The effect of inhibition of mineralocorticoid receptors on human endothelial function varies depending on disease. Treatment of type 2 diabetic patients for 1 month with spironolactone worsened endothelial function measured as acetylcholine-induced increase in forearm blood flow and heart rate variability (Davies et al., 2004), while the same group, using the same paradigm, could show that spironolactone improved endothelial vasodilator dysfunction and NO bioactivity in patients with chronic heart failure (Farquharson & Struthers, 2000). In hypertensive patients a high plasma aldosterone is associated with reduced flow-mediated vasodilatation (Nishizaka et al., 2004). 8. Interim conclusion Two in vitro studies from different laboratories (Liu et al., 2003a; Uhrenholt et al., 2003) show that aldosterone counteracts vasoconstriction by activating eNOS phosphorylation by a PI-3 kinase dependent pathway, while two other studies, also from different laboratories (Arima et al., 2003; Michea et al., 2005), arrive at the conclusion that aldosterone induce vasoconstriction by a mechanism involving PKC and increase in intracellular calcium. When looking at studies of the human forearm, the situation is similar: infusion of aldosterone may lead to vasodilatation, vasoconstriction or no effect, and inhibition of mineralocorticoid receptors with spironolactone improves endothelial function in patients with chronic heart failure (Farquharson & Struthers, 2000) and worsens endothelial function in type 2 diabetic patients (Davies et al., 2004). Since both the vasodilatory and the vasoconstrictor effects of aldosterone have been independently confirmed, it is likely that the experimental observations are correct and that the explanation for the paradox may be that seemingly identical experiments are, in fact, different. We may thus use the paradox to get more insight into the complex aldosterone signaling mechanisms.

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9. Oxidative stress and cellular signaling In recent years, it has become clear that one of the most important pathways by which aldosterone exerts its negative effects on the cardiovascular system is through production of reactive oxygen species (ROS). We, therefore, make a little digression into the mechanisms of oxidative stress. ROS are produced by several cellular pathways: in unstressed cells a significant source is NADPH oxidase which is a membranebound flavocytochrome that uses flavin and heme groups to shuttle electrons from NADPH to oxygen, yielding superoxide (Liu et al., 2003b). Xanthine oxido reductase is a major source of superoxide in stressed conditions such as heart failure and ischemia/reperfusion injury. Xanthine oxido reductase expression is stimulated by glucocorticoids, at least in epithelial cells, but the effect of aldosterone is unknown (review Berry & Hare, 2004). In ischemia/reperfusion xanthine oxido reductase converts nitrite into NO in the hypoxic and acidic environment of the ischemic area and thereby reduces infarct sizes (Duranski et al., 2005). In a normal cell the mitochondria are probably net scavengers of ROS (Andreyev et al., 2005), but in situations with cellular stress, the mitochondrial cytochromes become significant sources of ROS. Endothelial NOS usually produces NO, but after uncoupling, (e.g. by lack of tetrahydrobiopterin) it also becomes a source of superoxide. Hemoglobin in the blood may also deliver ROS to the vasculature. When superoxide is formed at a relatively slow rate (basal rate in endothelial cells: 19 nM min 1 mg protein 1 (Ushio-Fukai et al., 2002); basal rate in vascular smooth muscle 14 nM min 1 mg protein 1 (Dikalov et al., 1998)) it is converted by superoxide dismutase to H2O2, which may be further converted by catalase. The intracellular concentration of superoxide dismutase is high in most cells (4– 10 Amol/L in brain and liver (Beckman & Koppenol, 1996)) and the reaction rate for formation of H2O2 (and O2) from superoxide catalysed by superoxide dismutase is 2  109 M 1 s 1. Therefore, the capacity for superoxide breakdown is very high and a doubling of superoxide production rate such as observed after stimulation of endothelial cells and vascular smooth muscle (Dikalov et al., 1998; Ushio-Fukai et al., 2002) is likely to increase the H2O2 formation, and not necessarily the free superoxide concentration. However, macrophages and neutrophils may increase their superoxide production rate 10 and 100 fold, to about 200 and 20 000 nM min 1 mg protein 1, respectively (Dikalov et al., 2002; Wyche et al., 2004). In inflammatory conditions (e.g. atherosclerosis) the scavenging capacity of superoxide dismutase may therefore be overwhelmed (Fig. 3). Superoxide may also react with NO to form peroxynitrite that has physiological functions at low concentrations (10 – 50 AM, Adachi et al., 2004). The reaction rate for formation of peroxynitrite from NO and superoxide is high (about 6.7  109 M 1 s 1). However the usual NO concentration is low: 5 nmol/L is enough to activate the guanylate cyclase (Beckman & Koppenol, 1996) although endothelial cells can make 10- to 40-fold more than that (Malinski & Taha, 1992). Thus, at low NO production rate H2O2 production mediated by superoxide dismutase is the major pathway for removal of superoxide. Only

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Fig. 3. Upper panel: In the unstressed situation with normal endothelial function, production of NO (5 – 40 nmol/L) leads to vasodilation. The low production rate of superoxide (OU2 ) leads to concentrations of H2O2 (<100 Amol/L) and peroxynitrite (ONOO ) (10 – 40 Amol/L) that have vasodilatory properties. SOD: superoxide dismutase. Addition of aldosterone is likely mainly to cause vasodilation. Lower panel: With increased oxidative stress the superoxide production is increased. This may lead to concentrations of superoxide (OU2 ), hydrogen peroxide (H2O2), and peroxynitrite (ONOO ) that cause oxidative damage and vasoconstriction. Uncoupling of eNOS from NO production to superoxide production and scavenging of NO by peroxynitrite formation both reduce bioavailability of NO. Further stimulation of superoxide production by aldosterone is likely to increase the oxidative damage and vasoconstriction.

when the concentration of NO approaches the micromolar range of superoxide dismutase, peroxynitrite will be formed in large amounts (Beckman & Koppenol, 1996). When larger amounts of superoxide are formed, oxidative damage will prevail. Two major systems that act as scavengers of oxygen radicals are glutathione and the thioredoxin system. Thus, in unstressed cells reactive oxygen and nitrogen species take part in normal cellular signaling. H2O2 participates in flowmediated dilation in human coronary resistance arteries (Liu et al., 2003b). Oxidative stress stimulates PI-3 kinase, and, thereby, eNOS activation by inhibition of phosphatases (protein tyrosine-, lipid-, and serine/threonine-phosphatases) that inactivate the PI3 kinase. The lipid phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is probably the most important physiological regulator of PI-3 kinase. Exposure to H2O2 at moderate concentrations (100 AM) inactivates the phosphatases and increases PI-3 kinase activity (Barthel & Klotz, 2005; Tanaka et al., 2005). At higher concentrations of H2O2 the Akt eNOS pathway is not activated (Tanaka et al., 2005). Similar dose-dependent effects are observed with reactive nitrogen species. Together with glutathione the peroxynitrite induces reversible S-glutathiolation and activation of sarcoplasmic reticulum Ca2+ ATPase, thereby reducing cellular calcium concentration and causing vasodilatation (Adachi et al., 2004). At higher levels of oxidative stress irreversible thiol oxidation leads to irreversible modification of the Ca2+

ATPase; and peroxynitrite no longer affects the calcium uptake. Hare and Stamler (2005) suggested that ROS may affect cellular signaling by changing the set points for initiation of phosphorylation and nitrosylation signaling (review Hare & Stamler, 2005), thereby implicating a profound effect of redox status on cellular signaling. In conclusion, at low levels of oxidative stress, reactive oxygen species and reactive nitrogen species are important vasodilators and cellular signaling molecules, while at higher levels of oxidative stress the same molecules contribute to oxidative damage (Fig. 3). 10. Aldosterone as an activator of reactive oxygen (and nitrogen) species Aldosterone stimulates NADPH oxidase in blood vessels (Callera et al., 2005) and in macrophages (Keidar et al., 2004). The stimulation occurs via c-src (Callera et al., 2005). In the collecting duct cell line M-1, the ability of aldosterone to stimulate c-src is rapid (Braun et al., 2004). Treatment of mice with mineralocorticoid (DOCA) and a high salt diet causes hypertension and stimulates NADPH oxidase to produce ROS (superoxide and peroxynitrite) that oxidize tetrahydrobiopterin and thereby uncouples eNOS to further produce superoxide, thereby initiating a vicious cycle of increasing ROS formation (Landmesser et al., 2003). Glucocorticoids down-regulate the

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expression of GTP cyclohydrolase, which is the rate limiting enzyme involved in tetrahydrobiopterin formation (Mitchell et al., 2003a, 2003b, 2004), and a similar effect has been ascribed to aldosterone, but yet only reported in abstract form (Mitchell et al., 2003a, 2003b, abstract). Thus, aldosterone stimulates ROS production by activation of the NADPH oxidase, and by uncoupling of eNOS. In view of the Janus face of reactive oxygen species (serving as vasodilators at low concentrations, and the opposite at higher concentrations) it is to be expected that hormones that stimulate formation of ROS, such as aldosterone, have diverse effect depending on the pre-existing redox status of the cells. Thus, in situations with increased levels of oxidative stress, a further stimulation of ROS formation by aldosterone is more likely to lead to pathological damage. In classical in vitro studies the experimental solutions were equilibrated with 95% or 100% oxygen to assure sufficient oxygenation of the tissue. However, many investigators have now abandoned the high oxygen tensions because of a concern that the high oxygen tension may damage cellular processes. Proteome analysis of cells incubated with 95% compared to 21% oxygen demonstrated significant changes in expression levels of proteins involved in cytoskeletal structure, glycolysis, redox status, signal transduction, transcription, and protein folding (Vorum et al., 2004). The regulation by NO of the ryanodine receptor/ calcium release channel (RyR1) of skeletal muscle sarcoplasmic reticulum is disrupted after incubation with 95% O2 (Eu et al., 2003). Microvascular endothelial cells may have a greater ROS production than macrovascular endothelium (review Li & Shah, 2004) meaning that the microvasculature may be more sensitive to additional oxidative stress induced by experimental conditions. These observations may resolve the apparently contradicting reports on rapid effects of aldosterone on the microvasculature. Uhrenholt et al. (2003) equilibrated their solutions with 21% oxygen and reported that aldosterone counteracted vasoconstriction in rabbit afferent arterioles. Michea et al. (2005) and Arima et al. (2003, 2004) used solutions equilibrated with 95% oxygen, and reported that aldosterone induced vasoconstriction in small mesenteric arteries and rabbit afferent arterioles. We suggest that differences in ambient redox status may contribute to the observed differences in microvascular reactivity to aldosterone. 11. General effects of steroids on endothelial function A dual effect on vascular function is not limited to aldosterone, but has been observed also with other steroid hormones, such as glucocorticoids and estrogen. High dose glucocorticoids (dexamethasone) increase cerebral blood flow and reduce cerebral infarct size. The effect is mediated by a non-transcriptional mechanism initiated via the glucocorticoid receptor, activation of PI-3 kinase, PKB/Akt and phosphorylation of eNOS (Limbourg et al., 2003). Activation of the same pathway reduces heart infarct size in mice (HafeziMoghadam et al., 2002). In contrast, long-term dexamethasone treatment suppresses endothelium-dependent vasodilatation in

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awake mice by down-regulation of eNOS expression, and of the l-arginine transporter, as well as by an increase in the production of ROS (Scha¨fer et al., 2005). Rapid non-genomic activation of PI-3 kinase-PKB/Akt pathway and stimulation of NO signaling after activation of estrogen a receptors has been reported in a number of different vascular tissues (Simoncini et al., 2002; Stirone et al., 2005). It has also, however, recently become clear that estrogen stimulates ROS generation—most likely H2O2 from mitochondria (Felty et al., 2005), and estrogen down-regulates the ability of MCF-7 cells to metabolize peroxide by decreasing catalase activity and reduce cellular glutathione levels (Mobley & Brueggemeier, 2004). The ability to phosphorylate and stimulate eNOS by phosphorylation at Ser 1179 through a non-genomic mechanism seems to be a universal characteristic of nuclear receptors. Thus, stimulation of NO production has not only been described for all steroid hormones, but also for non-steroid receptors (vitamin D receptors), and for nutritional sensors, such as peroxisome proliferator-activated receptor g (Hwang et al., 2005). Because all steroid hormone receptors may mediate activation of eNOS through interaction with PI-3 kinase this ability is likely to be linked to a well-conserved sequence of the nuclear receptors. Consistent with this, mutations in the well conserved DNA binding domain of estrogen receptora led to a loss of the abilities to bind PI-3 kinase and to activate eNOS (Chambliss et al., 2005), while mutations in the more variable ligand-binding domain had no effect on eNOS activation. The genomic effect on the redox status by activation of nuclear receptors is highly variable and has many different targets. The relative contribution in vivo of the many potential activators of eNOS is unknown. 12. NaCl and aldosterone Ingestion of a low-sodium diet is associated with increased plasma aldosterone concentration, and this combination does not promote pathologic processes in the vasculature. However, infusion of aldosterone into normal rats on a high NaCl diet leads to hypertension and development of severe vascular inflammation and fibrosis, which is attenuated by inhibition of the MR (Rocha et al., 2002). The combined effect of aldosterone and the high salt diet on redox status may explain this finding because a high salt intake by itself is associated with reduced endothelial NO production and increased superoxide production. Thus, Lenda et al. (2000) compared skeletal muscle arterioles from rats fed a 0.45% or a 7% salt diet for 4– 5 weeks and observed a blunted vasodilator response to acetylcholine and a higher oxidant activity in the high-salt rats. The vasodilator response returned to normal after scavenging oxygen radicals with superoxide dismutase and catalase. Zhu et al. (2004) showed that methacholine increased aortic NO release from rats on a 0.4% NaCl diet while it led to superoxide production in rats on a 4% NaCl diet. Scavenging superoxide with the superoxide dismutase mimetic tempol restored NO release in the high-salt group. Taken together, these studies show that a high sodium diet impairs endothelium-dependent

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relaxation via reduced NO levels and increased superoxide production. Thus, with a high sodium intake, oxidant stress is already relatively high, and a further addition of aldosterone may drive the oxidant stress into the pathological level. Oxidative and nitrosative stress also is centrally involved in the pathway leading from activation of the angiotensin – aldosterone system to the proinflammatory phenotype which precedes tissue invasion of macrophages and fibroblasts that eventually lead to vascular fibrosis (Kuwahara et al., 2004; Weber, 2004). 12.1. Salt and human evolution In view of the many negative effects of aldosterone on cardiovascular function, it could be asked why aldosterone has not been purged during evolution? One of the answers to this question is probably that the higher oxidative stress associated with a high sodium intake only has become part of human life after the transition from a hunter/gatherer lifestyle to agricultural living during the last 10 000 years, which in an evolutionary context is a short period (Meneton et al., 2005). In the preceding millions of years, sodium intake in land animals was usually low, and consequently efficient sodiumsparing mechanisms were highly advantageous. Furthermore, conditions with endothelial dysfunction, where the negative effects of aldosterone on vascular function are likely to prevail, are mainly characteristics of mature human life and are therefore not likely to exert a significant evolutionary pressure. 13. Consequences for treatment The multifactorial origin of endothelial dysfunction and oxidative stress suggest that these pathological conditions may be more successfully treated by combinations of several different treatment strategies. As summarized by Struthers (2004) five pharmacological treatments (statins, aspirin, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and MR blockers) have been demonstrated in large trials to improve endothelial dysfunction and reduce cardiovascular events. In addition, non-pharmacological intervention such as salt restriction is a cornerstone of heart failure treatment. Dietary supplementation of antioxidants is not helpful for cardiovascular prevention. One of several reasons is that ROS negatively regulate hepatic VLDL secretion, and that inhibition by antioxidants increases VLDL secretion and blunts the VLDL-lowering effect of poly-unsaturated fatty acids (Williams & Fisher, 2005). In keeping with this, long-term vitamin E supplementation to patients with vascular disease or diabetes mellitus does not prevent major cardiovascular events and may, to the contrary, increase the risk for heart failure (Lonn et al., 2005). 14. Conclusion The redox status of vascular cells depends on the combined effects of many pathways leading to production of reactive oxygen and nitrogen species and of systems that scavenge

these reactive molecules. At low physiological concentrations ROS and reactive nitrogen species are vasodilators and have antithrombotic and antigrowth effects, while at higher formation rates or with reduced scavenging capacity, the concentrations rise into the pathological range, where the aggressive molecules initiate molecular modifications that reduce NO bioavailability, and cause endothelial dysfunction and vasoconstriction and become prothrombotic. The biological effect of aldosterone should be seen in this context. At low ambient oxidative stress, aldosterone promotes NO production and vasodilatation, while in situations with increased oxidative stress and endothelial dysfunction (heart failure, hypertension, high oxygen tension, and high salt status) the further production of superoxide initiated by aldosterone is likely to have detrimental effects on vascular function, and inhibition of MR is likely to be beneficial for cardiovascular function. If a non-classical MR is involved in mediating the negative effects of aldosterone on the cardiovascular system, then there might be an advantage in using newer open-ring inhibitors of MR, such as eplerenone, which also inhibits the responses ascribed to activation of these yet unidentified receptors. Acknowledgments Work from the author’s laboratory was supported by grants from the Danish Medical Research Council (9601829, 9902742 and 9903058), the Novo Nordisk Foundation, the Danish Heart Foundation (02-1-2-28A-22983, 01-201-61A-22939, 99223622743, 01123022896, and 02-1-2-33A-22982), the Danish Medical Association Research Foundation, and Kønig Petersen’s Foundation. References Adachi, T., Weisbrod, R. M., Pimentel, D. R., Ying, J., Sharov, V. S., & Scho¨neich, C., et al. (2004). S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide. Nat Med 10, 1200 – 1207. Ahmad, N., Romero, D. G., Gomez-Sanchez, E. P., & Gomez-Sanchez, C. E. (2004). Do human vascular endothelial cells produce aldosterone? Endocrinology 145, 3626 – 3629. Alzamora, R., Michea, L., & Marusic, E. T. (2000). Role of 11betahydroxysteroid dehydrogenase in nongenomic aldosterone effects in human arteries. Hypertension 35, 1099 – 1104. Andreyev, A. Y., Kushnareva, Y. E., & Starkov, A. A. (2005). Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 70, 200 – 214. Arima, S., Kohagura, K., Xu, H. L., Sugawara, A., Abe, T., & Satoh, F., et al. (2003). Nongenomic vascular action of aldosterone in the glomerular microcirculation. J Am Soc Nephrol 14, 2255 – 2263. Arima, S., Kohagura, K., Xu, H. L., Sugawara, A., Uruno, A., & Satoh, F., et al. (2004). Endothelium-derived nitric oxide modulates vascular action of aldosterone in renal arteriole. Hypertension 43, 352 – 357. Baker, M. E. (2001). Adrenal and sex steroid receptor evolution: environmental implications. J Mol Endocrinol 26, 119 – 125. Baker, M. E. (2004). Co-evolution of steroidogenic and steroid-inactivating enzymes and adrenal and sex steroid receptors. Mol Cell Endocrinol 27, 55 – 62. Balligand, J. L. (2002). Heat shock protein 90 in endothelial nitric oxide synthase signaling: following the lead(er)? Circ Res 90, 838 – 841. Barthel, A., & Klotz, L. O. (2005). Phosphoinositide 3-kinase signaling in the cellular response to oxidative stress. Biol Chem 386, 207 – 216.

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