Nongenomic actions of aldosterone: physiological or pathophysiological role?

The actions of aldosterone are usually divided into persistent genomic mediated by the classical mineralocorticoid receptor versus acute nongenomic actions. Rapid, nongenomic effects of aldosterone have been shown in a variety of tissues, although the physiological relevance of these nongenomic actions remains to be established. There is now growing evidence that both the nongenomic and genomic actions of aldosterone, are mediated via the same classical mineralocorticoid receptor, and there is cross talk between the nongenomic and classical actions of steroid hormones. Activation of tissue-specific, second messenger pathways may contribute to integration of nongenomic and classical actions of aldosterone. Further studies are required to determine the physiological or pathophysiological role of these nongenomic actions of aldosterone and whether they might amplify pathophysiological effects of aldosterone.     

Rapid actions of aldosterone: lymphocytes, vascular smooth muscle and endothelial cells.

The genomic theory of steroid action has been the unquestioned dogma for the explanation of steroid effects over the past four decades. Despite early observations on rapid steroid effects being clearly incompatible with this theory, only recently has nongenomic steroid action been recognized more widely and led to a critical reappraisal of unsolved questions about this dogma. Evidence for nongenomic steroid effects come from all fields of steroid research now, and mechanisms of agonist action are studied with regard to membrane receptors and second messengers involved. A prominent example of a receptor/effector-cascade for nongenomic steroid effects has been described for rapid aldosterone effects in various cell types, including lymphocytes, cultured vascular smooth muscle, and endothelial cells involving nonclassical membrane receptors with a high affinity for aldosterone, but not for cortisol, and phosphoinositide turnover. As another important second messenger, [Ca2+]i is consistently increased by aldosterone within 1-2 min. In vascular smooth muscle cells, calcium is released from perinuclear stores, while in endothelial cells a predominant increase of subplasmalemmal calcium is seen. Effects are half maximal at physiological concentrations of free aldosterone (0.1 nmol/L), while cortisol is inactive up to 0.1 micromol/L; the classical mineralocorticoid antagonist canrenone is ineffective in blocking the action of aldosterone. The data show that intracellular signaling for nongenomic aldosterone effects also involves calcium, but pathways of cell activation may vary between different cell types. Future research will have to target the cloning of the first membrane receptor for steroids, and the evaluation of the clinical relevance of these rapid steroid effects.     

Rapid nongenomic effects of aldosterone in mineralocorticoid-receptor-knockout mice

In addition to genomic effects of aldosterone, rapid nongenomic effects of steroids have been reported in various tissues that were clearly incompatible with a genomic action of aldosterone. Rapid effects of aldosterone involve second messengers such as calcium and cAMP. Specific high affinity binding sites for aldosterone have been characterized in membranes for different cells, which probably transmit those rapid steroid effects. To date, it is unclear if these binding sites are modified classical mineralocorticoid receptors (MR) or if they represent an unrelated receptor protein. The aim of the present study was to investigate whether rapid aldosterone action still occurs in the absence of the classical MR. For this purpose we used the model of MR knockout mice. Rapid effects were analyzed in skin cells, measuring intracellular calcium and cAMP levels after stimulation with aldosterone. We found that rapid effects are not only present in MR knockout mice, but that the effects are even larger than in wild-type mice cells. The results of the present study demonstrate that the classic MR is dispensable for rapid aldosterone action. The study, thus, proves that a receptor different from the classic intracellular receptor is involved in rapid aldosterone signaling.     

Nongenomic effects of mineralocorticoid receptor activation in the cardiovascular system

Fifteen years ago Wehling and colleagues showed unequivocal rapid effects of aldosterone, neither mimicked by cortisol nor blocked by spironolactone, and postulated that these nongenomic effects are mediated via a membrane receptor distinct from the classical mineralocorticoid receptor (MR). Several recent studies have challenged this view. Alzamora et al. showed 11beta-hydroxysteroid denydrogenase 1 and 2 (11betaHSD1, 11betaHSD2) expression in human vascular smooth muscle cells, and that aldosterone rapidly raises intracellular pH via sodium-hydrogen exchange; cortisol is without effect and spironolactone does not block the aldosterone response. When, however, 11betaHSD activity is blocked by carbenoxolone, cortisol shows agonist effects indistinguishable from aldosterone; in addition, the effect of both aldosterone and cortisol is blocked by the open E-ring, water soluble MR antagonist RU28318. In rabbit cardiomyocytes, aldosterone increases intracellular [Na+] by activating Na+/K+/2Cl- cotransport, with secondary effects on Na+/K+ pump activity. Pump current rises approximately 10-fold within 15', is unaffected by actinomycin D or the MR antagonist canrenone, and not elevated by cortisol. Pump current is, however, completely blocked by the open E-ring, water soluble MR antagonist K+ canrenoate and stoichometrically by cortisol. PKCepsilon agonist peptides (but not PKCalpha, PKCdelta or scrambled PKCepsilon peptides) mimic the effect of aldosterone, and PKCepsilon antagonist peptides block the effect. Very recently, cortisol has been shown to mimic the effect of aldosterone when cardiomyocyte redox state is altered by the installation of oxidized glutathione (GSSG) via the pipet, paralleling the effect of carbenoxolone on vascular smooth cells and suggesting possible pathophysiologic roles for an always glucocorticoid occupied MR.     

EF domains are sufficient for nongenomic mineralocorticoid receptor actions

The mineralocorticoid receptor (MR) is important for salt homeostasis and reno-cardiovascular pathophysiology. Signaling mechanisms include, besides classical genomic pathways, nongenomic pathways with putative pathophysiological relevance involving the mitogen-activated protein kinases ERK1/2. We determined the MR domains required for nongenomic signaling and their potential to elicit pathophysiological effects in cultured cells under defined conditions. The expression of full-length human MR or truncated MR consisting of the domains CDEF (MR CDEF), DEF (MR DEF), or EF (MR EF) renders cells responsive for the MR ligand aldosterone with respect to nongenomic ERK1/2 phosphorylation, whereas only full-length MR and MR CDEF conferred genomic responsiveness. ERK1/2 phosphorylation depends on the EGF receptor and cSRC kinase. MR EF expression is sufficient to evoke the aldosterone-induced increase of collagen III levels, similar to full-length MR expression. Our data suggest that nongenomic MR signaling is mediated by the EF domains and present the first proof of principle showing that nongenomic signaling can be sufficient for some pathophysiological effects. The minimum amino acid motif required for nongenomic MR signaling and its importance in various effects have yet to be determined.     

Aldosterone stimulates vacuolar H(+)-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

Urinary acidification in the collecting duct is mediated by the activity of H(+)-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H(+)-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H(+)-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific G(αq) inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca(2+) with BAPTA, and blockade of protein kinase C prevented the stimulation of H(+)-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H(+)-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H(+)-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H(+)-ATPase activity. Thus, the nongenomic modulation of H(+)-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a G(αq) protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H(+)-ATPase activity and contribute to final urinary acidification.     

Nongenomic actions of aldosterone and progesterone revisited

After almost 30 years of research, the existence of nongenomic steroid actions is no longer disputed. Yet, there is still a debate on the nature of receptors involved, and answers to the inherent questions are important for translational activities. In the case of aldosterone, the existence of receptors different from the classic mineralocorticoid receptors (MR) had been postulated 25 years ago as the pharmacology of about 50% of rapid actions of aldosterone reported so far is incompatible with MR involvement (insensitivity to classic MR antagonists). Candidates proposed as alternatives to MR were protein kinase C, sodium-potassium ATPase or aberrant forms of MR, none of which supported convincing evidence to represent 'the aldosterone membrane receptor'. Early in 2011, data on GPR30 showed its involvement in rapid aldosterone action, and major pharmacological aspects of this action are compatible with the landmark deviations from MR pharmacology mentioned above. GPR30, therefore, may be a receptor candidate for nongenomic aldosterone action. Similarly, a variety of promising candidates mediating rapid progesterone action has been described, including progesterone receptor membrane component 1 (PGRMC1) which seems to be associated with tumor proliferation, and membrane progesterone receptor (mPR) originally identified in fish with potential linkage to reproductive processes. So far, no candidate was unanimously convincing. In 2010, two independent groups reported that CatSper, a calcium channel, is a strong receptor candidate for the rapid action of progesterone on sperm fertilization. With these novel receptors cloned, translational activities ultimately leading to new drugs for cardiovascular protection (in the case of aldosterone) or fertilization benefits (for progesterone) are much more promising.     

Oxidative refolding of lysozyme in trifluoroethanol (TFE) and ethylene glycol: interfering role of preexisting alpha-helical structure and intermolecular hydrophobic interactions

We investigated the effect of aldosterone on Src kinase. In the kidney cell line, M-1 aldosterone leads to a >2-fold transient activation of Src kinase seen as early as 2 min after aldosterone administration. Maximal Src kinase activation was measured at an aldosterone concentration of 1 nM. In parallel to activation, autophosphorylation at Tyr-416 of Src kinase increased. Src kinase activation was blocked by spironolactone. Aldosterone led to increased association of Src with HSP84. Furthermore, rapamycin blocked aldosterone-induced Src activation. We conclude that Src activation by aldosterone is mediated through the mineralocorticoid receptor and HSP84.     

Aldosterone upregulates vascular endothelial growth factor expression in mouse cortical collecting duct epithelial cells through classic mineralocorticoid receptor

Accumulating evidence shows that aldosterone plays an important role in the pathogenesis of renal fibrosis but its mechanism has not been completely defined. Recently, exogenous administration of aldosterone significantly alleviated ischemic states in a model of femoral artery ligated rats, accompanied by an obvious enhancement of VEGF upregulation. We hypothesized that aldosterone may also regulate the expression of VEGF in the kidney. To confirm this, cultured cortical collecting duct epithelial cells (M-1 cell line) were incubated with aldosterone and control media, respectively. The pathway by which aldosterone regulates VEGF expression was tested by the administration of spironolactone, a specific mineralocorticoid receptor (MR) antagonist. VEGF expression was detected by immunofluorescence staining, ELISA, Western blot and RT-PCR. Aldosterone induced an elevation of VEGF excretion in a time- and dose-dependent manner. Western blotting showed a 1.4-fold elevation in cytosolic VEGF expression following aldosterone (10(-8) M) incubation for 48 h (p<0.01). After aldosterone (10(-7) M) incubation for 48 h, the mRNA level of VEGF164 and VEGF120 showed 1.8- and 1.7-fold increases, respectively (p<0.01). This upregulation was almost completely blocked by spironolactone as shown both by mRNA levels and cytosolic protein levels. In addition, the mRNA of aldosterone receptor was detected in M-1 cells. We demonstrated for the first time that aldosterone induced VEGF expression in M-1 cells, an effect mediated by classic mineralocorticoid receptor. This finding provides experimental evidence for the local non-hemodynamic action of aldosterone.     

Nongenomic effects of aldosterone: cellular aspects and clinical implications

Nongenomic action of aldosterone has been observed in many cell types which often are different from the classic target tissues for mineralocorticoid action, such as vascular cells. As judged from their time scale and insensitivity toward inhibitors of protein synthesis, effects are not mediated by the classic mineralocorticoid receptor pathway. Here we summarize studies on rapid in vitro aldosterone effects, e.g. ion fluxes, and second messengers involved therein. Furthermore, several clinical studies on in vivo aldosterone action have shown rapid effects on cardiovascular parameters, among them baroreflex and vascular resistance. Taken together with the beneficial effect of aldosterone antagonism in heart failure patients that was demonstrated in the Randomized Aldactone Evaluation Study (RALES), aldosterone may be an equally important factor of the renin-angiotensin-aldosterone system in cardiovascular pathogenesis.     

Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions

The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inhibition of MAPK kinase (MEK) kinase but not of protein kinase C prevented the rapid action of aldosterone and also reduced aldosterone-induced transactivation, most probably due to impaired nuclear-cytoplasmic shuttling of MR. Cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extend due to Ca2+ influx, whereas dexamethasone had virtually no effect. Spironolactone did not prevent the Ca2+ response. We conclude that some nongenotropic effects of aldosterone are MR dependent and others are MR independent (e.g. Ca2+), indicating a higher degree of complexity of rapid aldosterone signaling. According to this model, we have to distinguish three aldosterone signaling pathways: 1) genomic via MR, 2) nongenotropic via MR, and 3) nongenotropic MR independent.     

Rapid effects of aldosterone in primary cultures of cardiomyocytes – do they suggest the existence of a membrane-bound receptor?

Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone?+?spironolactone?+?BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca²⁺. Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.