Rapid Actions Of Aldosterone Research Articles

FRI126 Autoreactive T Cells To Angiotensin II Type 1 Receptor In Salt-dependent Hypertension Due To Primary Aldosteronism

Abstract Disclosure: R. Scarpa: None. M. Piazza: None. B. Caroccia: None. S. Carraro: None. F. Vianello: None. F. Cinetto: None. C. Agostini: None. T.M. Seccia: None. G. Rossi: None. Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but the pathogenic role of this acquired immunity is still unknown. Primary Aldosteronism, the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary (‘essential’) hypertension and by high titer of autoantibodies against the type 1 angiotensin receptor (AT1R). As T cells are broadly recognised as HMOD-mediators, we aim to investigate: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of know aldosterone receptors on T lymphocytes. The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number by droplet digital PCR and immunoblotting from healthy donors (HD) subjects and PA patients. Peripheral blood mononuclear cells (PBMCs) from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFNɣ release with flow cytometry. PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR>MR>>GPER. Aldosterone significantly increased IFNɣ release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-γ production by CD8+ T cells by acting via the MR, as it was prevented (p < 0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p < 0.003), but also by G36 (p < 0.02), and mimicked by G1. These results showed the presence of AT1R-autoreactive T cells in patients with PA. Moreover, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs. Presentation: Friday, June 16, 2023

AUTOREACTIVE T CELLS TO ANGIOTENSIN II TYPE 1 RECEPTOR IN SALT-DEPENDENT HYPERTENSION DUE TO PRIMARY ALDOSTERONISM

Objective: Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but its pathogenic role is still unknown. Primary Aldosteronism (PA), the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary hypertension and by high titer of autoantibodies against the angiotensin type-1 receptor (AT1R). As T cells are broadly recognised as HMOD-mediators we investigated: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of aldosterone receptors on T lymphocytes. Design and Methods: The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number and immunoblotting from healthy donors and PA patients. PBMCs from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFN? release with flow cytometry. Results: PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR>MR>>GPER. Aldosterone significantly increased IFN? release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-? production by CD8+ T cells by acting via the MR, as it was prevented (p<0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p<0.003), but also by G36 (p<0.02), and mimicked by G1. Conclusion: These results showed the presence of AT1R-autoreactive T cells in patients with PA and demonstrates that, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs.

Aldosterone rapidly activates p-PKC delta and GPR30 but suppresses p-PKC epsilon protein levels in rat kidney.

Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.

Rapid Action of Aldosterone on Protein Levels of Sodium-Hydrogen Exchangers and Protein Kinase C Beta Isoforms in Rat Kidney.

Previous in vitro studies demonstrated that aldosterone rapidly activates sodium-hydrogen exchangers 1 and 3 (NHE 1 and 3). In vitro investigations revealed that protein kinase C (PKC) regulates NHE properties. We previously demonstrated that aldosterone rapidly enhances PKCα protein abundance in the rat kidney. There are no reports of renal PKCβ (I and II) protein levels related to the regulation by aldosterone. There are also no in vivo data regarding the rapid effects of aldosterone on renal protein levels of NHE (1 and 3) and PKCβ (I and II), simultaneously. In the current study, rats received normal saline solution or aldosterone (150 μg/kg BW, i.p.). After 30 minutes, abundance and immunoreactivity of these proteins were determined by Western blot analysis and immunohistochemistry, respectively. Aldosterone increased NHE1 and NHE3 protein abundance to 152% and 134%, respectively (P < 0.05). PKCβI protein level was enhanced by 30%, whereas PKCβII declined slightly. Aldosterone increased NHE protein expression mostly in the medulla. PKCβI immunostaining intensity was increased in the glomeruli, renal vasculature, and thin limb of the loop of Henle, while PKCβII was reduced. This is the first in vivo study to simultaneously demonstrate that aldosterone rapidly elevates PKCβI and NHE (1 and 3) protein abundance in the rat kidney. Aldosterone-induced NHE (1 and 3) protein levels may be related to PKCβI activation.

P.2.f.025 - Escitalopram versus duloxetine in acute treatment of major depressive disorder: a meta-analysis and systematic review

Steroid hormones like aldosterone have been conclusively shown to elicit both late genomic and rapid, nongenomically initiated responses. Aldosterone was among the first for which rapid, clinically relevant effects were even shown in humans. Yet, after over 30 years of research, the nature of receptors involved in rapid actions of aldosterone is still unclear. Such effects may be assigned to the classical, intracellular steroid receptors, in this case mineralocorticoid receptors (MR, class IIa action Mannheim classification). They typically disappear in knockout models and are blocked by MR-antagonists such as spironolactone, as shown for several cellular and physiological, e.g. renal or cardiovascular effects. In contrast, there is also consistent evidence suggesting type IIb effects involving structurally different receptors (“membrane receptors”) being insensitive to classic antagonists and persistent in knockout models; IIb effects have lately even been confirmed by atomic force detection of surface receptors which bind aldosterone but not spironolactone. Type IIa and b may coexist in the same cell with IIa often augmenting early IIb effects. So far cloning of IIb receptors was unsuccessful; therefore results on G-protein coupled estrogen receptor 1 (GPER1) being potentially involved in rapid aldosterone action raised considerable interest. Surprisingly, GPER1 does not bind aldosterone. Though under these circumstances GPER1 should not yet be considered as IIb-receptor, it might be an intermediary signaling enhancer of mineralocorticoid action as shown for epithelial growth factor receptors reconciling those results. We still seem to be left without IIb-receptors whose identification would however be highly desirable and essential for clinical translation.

Rapid Action of Aldosterone on Protein Expressions of Protein Kinase C Alpha and Alpha1 Sodium Potassium Adenosine Triphosphatase in Rat Kidney

Previous in vitro studies showed that aldosterone rapidly stimulates protein kinase C alpha (PKC α) that could activate alpha (α) 1 isoform of Na, K-ATPase and then enhances its activity. There are, however, no in vivo data that demonstrate the rapid effects of aldosterone on renal protein expressions of PKC α and α1-Na, K-ATPase simultaneously. The present study further investigates the expression of these proteins. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 μg/kg BW). After 30 minutes, abundances and localizations of PKC α and α1-Na, K-ATPase proteins were determined by Western blot analysis and immunohistochemistry, respectively. Aldosterone administration significantly increased plasma aldosterone levels from 1,251.95 ± 13.83 to 6,521.78 ± 209.92 pmol/L. By Western blot analysis, aldosterone enhanced renal protein abundances of PKC α (homogenate samples) and α1-Na, K-ATPase (plasma membrane) approximately 50% and 30%, respectively (P<0.05). From immunohistochemistry examination in sham group, the protein expression of PKC α was prominent in the medulla. Aldosterone stimulated the expression both in the cortex and medulla with the translocation from the basolateral to luminal side of the proximal convoluted tubule (PCT). As for α1-Na, K-ATPase protein expression, the sham rats showed a strong immunostaining in the distal convoluted tubules, collecting ducts, and thick ascending limbs. Aldosterone elevated the expression in the PCT and medullary collecting duct (MCD). This in vivo study is the first to demonstrate simultaneously that aldosterone rapidly elevates PKC α and α1-Na, K-ATPase protein abundances in rat kidney. Both immunoreactivities were stimulated in the cortex and medulla. The greater affected areas were noted for PKC α expression, whereas the alterations of α1-Na, K-ATPase were observed only in the PT and MCD. The stimulation of Na, K-ATPase protein expression by aldosterone, per se, may occur through PKC activation.

Biphasic Action of Aldosterone on Akt Signaling in Cardiomyocytes

Both aldosterone and Akt signaling play pivotal roles in the pathogenesis of heart failure. However, little is known about the correlation between them. We herein investigated whether aldosterone interacts with Akt signaling in a coordinated manner in cardiomyocytes. Neonatal rat cardiomyocytes were stimulated with aldosterone for either a short (10-min) or long (24-h) time. The phosphorylation of Akt and its downstream effector, GSK3β, were transiently increased after short-term stimulation, which was blocked by either PI3K or Na(+)/H(+) exchanger inhibitors, but not by the mineralocorticoid receptor antagonist, eplerenone. Long-term stimulation also significantly increased Akt-GSK3β phosphorylation and this effect was reduced by eplerenone. Thus, these results suggest that aldosterone activates Akt signaling via a biphasic reaction that occurs through different cascades. To understand the significance of the rapid action of aldosterone, cardiomyocytes were exposed to hydrogen peroxide for from 10 to 60 min. A short-term aldosterone stimulation (for up to 30 min) significantly protected cardiomyocytes from oxidative stress-induced cellular damage. Eplerenone did not abrogate this beneficial effect, while a PI3K inhibitor did. Therefore, during the early phase, aldosterone has favorable effects on cardiomyocytes, partly by acute activation of a mineralocorticoid receptor-independent cascade through the Na(+)/H(+) exchanger, PI3K, and Akt. In contrast, its persistent activity produces pathological effects partly by chronic Akt activation in a mineralocorticoid receptor-dependent manner.

Is GPR30 the Membrane Aldosterone Receptor Postulated 20 Years Ago?

HomeHypertensionVol. 57, No. 5Is GPR30 the Membrane Aldosterone Receptor Postulated 20 Years Ago? Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBIs GPR30 the Membrane Aldosterone Receptor Postulated 20 Years Ago? Alexandra Wendler and Martin Wehling Alexandra WendlerAlexandra Wendler Search for more papers by this author and Martin WehlingMartin Wehling Search for more papers by this author Originally published21 Mar 2011https://doi.org/10.1161/HYPERTENSIONAHA.111.170977Hypertension. 2011;57:e16Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2011: Previous Version 1 To the Editor:Gros et al1 describe the involvement of GPR30 in rapid actions of aldosterone. The hypothesis that a structure other than the mineralocorticoid receptor (MR) may mediate rapid actions of aldosterone was postulated many years ago. Wehling et al2 showed in 1991 that the rapid effect of aldosterone on the sodium-proton exchanger in human mononuclear leukocytes and vascular smooth muscle cells was independent of classic aldosterone antagonists like canrenoate or canrenone.3 Grossmann et al4 showed almost 15 years later that cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extent, and spironolactone did not block this effect, pointing to MR-independent mechanisms. Most recently, aldosterone binding sites were detected at the membrane of human endothelial cells using atomic force microscopy. Aldosterone binding to these sites was spironolactone and dexamethasone insensitive.5 These examples are just a few of those pointing to MR-independent rapid aldosterone actions, and this interpretation is mainly based on their insensitivity to MR antagonists.The functions of GPR30, especially with respect to estrogen actions, are a matter of ongoing debate, and the new findings on its involvement in rapid aldosterone actions add an important facet to this discussion.Gros et al1 postulate that eplerenone and spironolactone are partial antagonists of GPR30. This needs further clarification. Analyzing Figure 2 of the article by Gros et al1 in detail, it is tempting to assume that, in vascular smooth muscle cells which have been transfected with the control construct (green fluorescent protein), extracellular signal–regulated kinase phosphorylation by aldosterone reflects basic MR expression, because it is completely inhibited by eplerenone. When MR is overexpressed in those cells, the effect of aldosterone is roughly doubled, but it remains completely (=100%) eplerenone sensitive. In GPR30-transfected cells, extracellular signal–regulated kinase phosphorylation by aldosterone is also roughly doubled. However, the added effect is not eplerenone sensitive, because eplerenone decreases extracellular signal–regulated kinase phosphorylation only by exactly the same amount seen in cells transfected with the control construct (green fluorescent protein). Therefore, the effect of eplerenone in the GPR30-transfected cells seems to be fully explained by the inhibition of the assumed endogenous MR activity. This is underlined by numerically identical results in Figure 7A through 7C of the article by Gros et al,1 which show identical relations for apoptosis as readout or by Figure 9B in the article by Gros et al1 for myosin light chain phosphorylation as readout. Figure 3 in the article by Gros et al1 demonstrates an incomplete inhibition by eplerenone of G1-stimulated responses, as seen in Figure 5A of this article for endothelial cells and aldosterone as an agonist. This interpretation is only flawed by more complete effects of G15 on aldosterone responses in GPR30-transfected cells (Figure 9C of the article by Gros et al1), which have to remain unexplained. If correct, these interpretations underscore the fact that eplerenone and spironolactone are not antagonists for the rapid action of aldosterone mediated through GPR30.This interpretation would identify GPR30 as an almost perfect candidate for the membrane aldosterone receptor, which we characterized mainly by its landmark property of resistance to classic MR antagonists more than 2 decades ago. Further studies need to validate GPR30 as an aldosterone receptor and to corroborate our interpretation of the impressive data by Gros et al.1Alexandra WendlerMartin Wehling University of Heidelberg Clinical Pharmacology Mannheim Mannheim, GermanyDisclosuresM.W. received consulting and lecture fees from Pfizer, Roche, Novartis, Lilly, PAION, MorphoSys, 4D-Biomedical, and Daiichi-Sankyo. He is a former employee of AstraZeneca (2004–2006).FootnotesLetters to the Editor will be published, if suitable, as space permits. They should not exceed 1000 words (typed double-spaced) in length and may be subject to editing or abridgment.

Mechanisms Underlying Rapid Aldosterone Effects in the Kidney

The steroid hormone aldosterone is a key regulator of electrolyte transport in the kidney and contributes to both homeostatic whole-body electrolyte balance and the development of renal and cardiovascular pathologies. Aldosterone exerts its action principally through the mineralocorticoid receptor (MR), which acts as a ligand-dependent transcription factor in target tissues. Aldosterone also stimulates the activation of protein kinases and secondary messenger signaling cascades that act independently on specific molecular targets in the cell membrane and also modulate the transcriptional action of aldosterone through MR. This review describes current knowledge regarding the mechanisms and targets of rapid aldosterone action in the nephron and how aldosterone integrates these responses into the regulation of renal physiology.

Nitric Oxide-mediated Dilation of Arterioles to Intraluminal Administration of Aldosterone

The nature of the rapid action of aldosterone on blood vessels, whether endothelium-dependent dilation or smooth muscle-dependent constriction is predominant, is still in dispute. In this study, we administered aldosterone intraluminally or extraluminally to isolated mesenteric and cerebral arterioles of male Wistar rats. Extraluminal administration of aldosterone (10(-11) or 10(-7) M) elicited a transient vasodilatation. The peak response appeared at approximately 5 minutes. In contrast, intraluminal administration of aldosterone elicited a greater and sustained dilation. When aldosterone (10(-12)-10(-7) M) was administered extraluminally in a cumulative manner, dose-dependent vasodilator responses were elicited, except a reduced dilation was observed to 10(-7) M aldosterone. The dilations were significantly inhibited by spironolactone (10(-7) M), a mineralocorticoid receptor antagonist or Nomega-nitro-l-arginine methyl ester (3 x 10(-4) M), a NO synthesis inhibitor. In endothelium-denuded vessels, extraluminal aldosterone induced a dose-dependent vasoconstrictor response. Scavenging superoxide with Tempol (10(-4) M) sustained the extraluminal aldosterone (10(-11) or 10(-7) M)-induced dilation, whereas inhibition of NO synthesis or removal of the endothelium abolished intraluminal aldosterone-induced dilation. Dilation to 10(-7) M aldosterone was significantly enhanced after inhibition of NAD(P)H-oxidase with apocynin (10(-5) M). Furthermore, in the presence of endothelial dysfunction, induced by chronic inhibition of NO synthesis, intraluminal administration of aldosterone failed to dilate the arterioles. We conclude that in physiological conditions, acute elevation of aldosterone will evoke mainly an endothelium-dependent NO-mediated dilation.

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.

Aldosterone inhibits HCO absorption via a nongenomic pathway in medullary thick ascending limb.

Rapid actions of aldosterone that are independent of transcription and translation have been described in a variety of cells; however, whether nongenomic pathways mediate aldosterone-induced regulation of renal tubule transport has not been determined. We report here that aldosterone induces rapid (<3.5 min) inhibition of HCO absorption in the medullary thick ascending limb (MTAL) of the rat. This inhibition is observed over the physiological range of hormone concentrations (IC(50) approximately 0.6 nM) and is not affected by pretreatment with actinomycin D (12.5 microg/ml), cycloheximide (40 microg/ml), or spironolactone (10 microM). The glucocorticoids dexamethasone, cortisol, and corticosterone (1 or 500 nM) did not affect HCO absorption in the absence or presence of carbenoxolone. Thus the specificity of rapid aldosterone action is not dependent on 11beta-hydroxysteroid dehydrogenase activity. The inhibition by aldosterone is additive to inhibition by angiotensin II and vasopressin, indicating that these factors regulate MTAL transport through distinct pathways. These results demonstrate that aldosterone inhibits HCO absorption in the MTAL via a pathway that is rapid, highly selective, independent of transcription and protein synthesis, and not mediated through the classic mineralocorticoid receptor. The results establish a role for nongenomic pathways in mediating aldosterone-induced regulation of transepithelial transport in the mammalian kidney. The novel action of aldosterone to inhibit luminal acidification in the MTAL may play a role in enabling the kidney to regulate acid-base balance independently of Na(+) balance and extracellular fluid volume.

Rapid actions of aldosterone on cells from renal epithelium: the possible role of EGF-receptor signaling.

It has been suggested that steroids interact with peptide hormones in part by rapid, potentially non-genomic, mechanisms. The peptide hormone epidermal growth factor (EGF) regulates cell proliferation and ion transport using ERK1/2 as downstream signal. Furthermore, the EGF-receptor (EGF-R) is involved in signaling by G-protein-coupled receptors, growth hormone and cytokines via transactivation. We show that aldosterone modulates Na(+)/H(+)-exchange in renal collecting duct-derived Madin-Darby canine kidney (MDCK) cells via ERK1/2 in a similar way as compared to growth factors. Furthermore, we tested the hypothesis that aldosterone uses the EGF-R as heterologous signal transducer in MDCK cells. Aldosterone induces a rapid increase of ERK1/2 phosphorylation and cytosolic Ca(2+)-concentration of similar extend as compared to EGF. Furthermore, aldosterone stimulates EGF-R Tyr-phosphorylation. Inhibition of EGF-R kinase abolished aldosterone-induced signaling. Aldosterone-induced Ca(2+)-influx seems to be mediated by the activation of ERK1/2, whereas ERK1/2 activation does not depend on Ca(2+)-influx. Our data show that aldosterone uses the EGF-R-ERK1/2 signaling cascade to elicit its rapid effects in MDCK cells.

Aldosterone.

Aldosterone plays a pivotal role in electrolyte and fluid homeostasis and thus control of blood pressure. The "classical" view of aldosterone action is that it targets epithelia of the distal colon and renal nephron to stimulate Na(+) (re)absorption and K(+) secretion. In these cells, aldosterone binds steroid receptors, promoting translocation to the nucleus, where they modulate gene expression with the induced proteins stimulating transport. This "genomic" action is dependent on transcription and translation and has a latency of 0.5-1.0 h. Recently, more rapid actions of aldosterone that are independent of transcription and translation have been described. These "nongenomic" actions are mediated by a distinct receptor that is insensitive to inhibitors of the classical mineralocorticoid receptor, such as spironolactone. The present review describes advances in our understanding of the classical model of aldosterone action as well as those that broaden this model to encompass nongenomic actions, nonepithelial targets, production of aldosterone outside of the adrenal gland, novel mechanisms of specificity, and novel mechanisms for mediating genomic actions.

Non-genomic actions of aldosterone: role in hypertension.

There is universal acceptance of the existence of rapid, non-genomic effects of aldosterone, although their physiological relevance and potential importance in hypertension are not yet clear. What has emerged over the year under review is that at least some of such rapid non-genomic effects of aldosterone may be mediated by the activation of the classical intracellular mineralocorticoid receptor, rather than a putative membrane receptor. The post-receptor mechanisms of rapid aldosterone action appear variously to involve protein kinase C, calcium, cyclic adenosine 3', 5'-monophosphate and inositol 1, 4, 5-triphosphate, with downstream effects on a variety of ion pumps and channels.

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.

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, [Ca 2+] 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 μmol/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.