Aldosterone Regulation Research Articles

Sensing tension: epithelial sodium channel/acid-sensing ion channel proteins in cardiovascular homeostasis.

The epithelial sodium (Na+) channel (ENaC) plays a critical role in blood pressure regulation by controlling renal salt and water reabsorption. Channel overactivity can lead to severe hypertension and underactivity to salt wasting and hypotension.1 In addition to their role in salt/water homeostasis, recent studies suggest that ENaC proteins, and their relatives, the acid-sensing ion channel (ASIC) proteins, may play more ubiquitous roles in cardiovascular regulation than considered previously. Recent evidence suggests that ENaC/ASIC proteins may act as mechanosensors and chemosensors in the cardiovascular system. ENaC/ASIC proteins are expressed in mechanosensing and chemosensing tissues, such as vascular smooth muscle cells (VSMCs), carotid body glomus cells, and sensory neurons innervating arterial baroreceptors, heart, and skeletal muscle. Disruption of the ENaC/ASIC channels alters myogenic constriction, arterial chemoreceptor and baroreceptor responses, and acid-induced responses in heart and skeletal muscle. This brief review summarizes the evidence supporting a role for ENaC and ASIC proteins in diverse systems of cardiovascular mechanosensing and chemosensing. Together, these studies suggest that ENaC/ASIC proteins contribute to cardiovascular homeostasis by mediating neural and local regulatory mechanisms. ENaC and ASIC proteins are members of a protein family termed the degenerin (DEG)/ENaC/ASIC family. Members of this family are expressed in a wide range of species (nematode Caenorhabditis elegans , Drosophila , and mammals) and participate in diverse biological functions, including neurodegeneration, acid sensation, taste, learning and memory, proprioception, Na+/water transport, and mechanosensation. All of the members of the DEG/ENaC/ASIC family share a highly conserved structure: intracellular N and C termini and 2 membrane-spanning domains separated by a large extracellular domain. Most DEG/ENaC/ASIC proteins form amiloride sensitive, nonvoltage, gated cation channels.1,2 ### C elegans DEGs Members were first identified in the nematode, where a chemically induced mutation caused a subset of neurons to swell and lyse. This phenotype led to the first …

A Lifetime of Aldosterone Excess: Long-Term Consequences of Altered Regulation of Aldosterone Production for Cardiovascular Function

Up to 15% of patients with essential hypertension have inappropriate regulation of aldosterone; although only a minority have distinct adrenal tumors, recent evidence shows that mineralocorticoid receptor activation contributes to the age-related blood pressure rise and illustrates the importance of aldosterone in determining cardiovascular risk. Aldosterone also has a major role in progression and outcome of ischemic heart disease. These data highlight the need to understand better the regulation of aldosterone synthesis and its action. Aldosterone effects are mediated mainly through classical nuclear receptors that alter gene transcription. In classic epithelial target tissues, signaling mechanisms are relatively well defined. However, aldosterone has major effects in nonepithelial tissues that include increased synthesis of proinflammatory molecules and reactive oxygen species; it remains unclear how these effects are controlled and how receptor specificity is maintained. Variation in aldosterone production reflects interaction of genetic and environmental factors. Although the environmental factors are well understood, the genetic control of aldosterone synthesis is still the subject of debate. Aldosterone synthase (encoded by the CYP11B2 gene) controls conversion of deoxycorticosterone to aldosterone. Polymorphic variation in CYP11B2 is associated with increased risk of hypertension, but the molecular mechanism that accounts for this is not known. Altered 11beta-hydroxylase efficiency (conversion of deoxycortisol to cortisol) as a consequence of variation in the neighboring gene (CYP11B1) may be important in contributing to altered control of aldosterone synthesis, so that the risk of hypertension may reflect a digenic effect, a concept that is discussed further. There is evidence that a long-term increase in aldosterone production from early life is determined by an interaction of genetic and environmental factors, leading to the eventual phenotypes of aldosterone-associated hypertension and cardiovascular damage in middle age and beyond. The importance of aldosterone has generated interest in its therapeutic modulation. Disadvantages associated with spironolactone (altered libido, gynecomastia) have led to a search for alternative mineralocorticoid receptor antagonists. Of these, eplerenone has been shown to reduce cardiovascular risk after myocardial infarction. The benefits and disadvantages of this therapeutic approach are discussed.

Phenotypic consequences of variation across the aldosterone synthase and 11‐beta hydroxylase locus in a hypertensive cohort: data from the MRC BRIGHT Study

Aldosterone is an important cardiovascular hormone; 15% of hypertensive subjects have alteration in aldosterone regulation, defined by a raised ratio of aldosterone to renin (ARR). Studies of the aldosterone synthase gene (CYP11B2) have focused on a single nucleotide polymorphism in the 5'promoter region (-344 C/T). In normotensive subjects, the T allele associates with raised levels of the 11-deoxysteroids, deoxycorticosterone and 11-deoxycortisol which are substrates for 11beta-hydroxylase, encoded by the adjacent and homologous gene, CYP11B1. We have speculated that this altered 11beta-hydroxylase efficiency leads to increased ACTH drive to the adrenal gland to maintain cortisol production and reported herein the association between the -344 C/T single nucleotide polymorphism (SNP) and adrenal steroid production in subjects with essential hypertension. The CYP11B2-344 C/T polymorphism was genotyped and urinary excretion of adrenal steroid metabolites was measured (by GCMS) in 511 unrelated hypertensives from the Medical Research Council (MRC) British Genetics of Hypertension (BRIGHT) study. Thirty-five per cent of subjects were homozygous for the -344T allele whilst 16% were CC homozygotes. There was no difference in cortisol excretion rate between the two genotype groups but the index of adrenal 11beta-hydroxylation (ratio of tetrahydrodeoxycortisol/total cortisol) was significantly higher in the TT group (P < 0.005) than in the CC group. Excretion rates of the major urinary metabolite of aldosterone (tetrahydroaldosterone) correlated strongly with the ACTH-regulated steroids, cortisol (r = 0.437, P < 0.0001) and total androgen metabolites (r = 0.4, P < 0.0001) in TT but not CC subjects. Hypertensives homozygous for the -344 T allele of CYP11B2 demonstrate altered 11beta-hydroxylase efficiency (CYP11B1); this is consistent with the hypothesis of a genetically determined increase in adrenal ACTH drive in these subjects. The correlation between excretion of aldosterone and cortisol metabolites and suggests that, in TT subjects, ACTH exerts an important common regulatory influence on adrenal corticosteroid production in subjects with hypertension.

Response to Polycystic Ovary Syndrome: Implications of Corticotropin in the Regulation of Blood Pressure, Aldosterone, and Androgen Secretion

We thank Armanini et al1 for their interests and comments on our work2 regarding the association between hyperandrogenemia and elevated systolic and diastolic blood pressures in young women with polycystic ovary syndrome. Armanini et al1 propose that the chronic corticotropin drive could activate aldosterone synthesis and …

Polycystic Ovary Syndrome: Implications of Corticotropin in the Regulation of Blood Pressure, Aldosterone, and Androgen Secretion

To the Editor: In a recent issue of Hypertension , Chen et al1 have described a correlation between androgen levels and blood pressure in young women with polycystic ovary syndrome (PCOS), and this correlation was independent from age, insulin resistance, obesity, and dyslipidemia. We would like to discuss other factors involved in the interpretation of these data. High adrenal androgen levels in women with PCOS can also be related to an increase of corticotropin (ACTH). The major question is why …

Early Aldosterone-Regulated Genes in Cardiomyocytes: Clues to Cardiac Remodeling?

Recent clinical studies demonstrated beneficial effects of mineralocorticoid receptor (MR) antagonists in patients with heart failure and other cardiovascular diseases. However, the underlying molecular mechanisms are poorly understood, and the genes that mediate direct effects of aldosterone in the cardiovascular system are yet to be identified. The goal of this study was to identify genes that are directly regulated by aldosterone in cardiomyocytes and thus potentially play a role in initiating MR-mediated effects in the heart. We generated clonal cell lines of cardiomyocytes (H9C2 cells) stably expressing the MR. Using these cell lines and Affymetrix microarrays, we determined the effects of physiological concentrations of aldosterone on the gene expression profile. In two independent microarrays we identified 48 genes that were induced more than 1.5-fold (27 known genes and 21 expressed sequence tags) and five (three known genes and two expressed sequence tags) that were suppressed by a 2-h aldosterone treatment. We focused on eight genes that have a potential function in cardiovascular regulation and verified their aldosterone regulation using quantitative RT-PCR. These include genes related to extracellular matrix regulation (tenascin-X, ADAMTS1, PAI-1, UPAR, and hyaluronic acid synthase-2), signaling, and regulation of vascular tone (RGS2, adrenomedullin) and inflammation (orosomucoid). Protein synthesis inhibitors did not prevent aldosterone induction of these genes. We conclude that in cardiomyocytes aldosterone rapidly and directly regulates the expression of several genes that are involved in cardiac remodeling and regulation of blood pressure and thus might be mediators of the physiological and pathophysiological effects of aldosterone on the cardiovascular system.

Reactive Oxygen Species–Dependent Hypertension in Dopamine D 2 Receptor–Deficient Mice

Dysfunction of D2-like receptors has been reported in essential hypertension. Disruption of D2R in mice (D2-/-) results in high blood pressure, and several D2R polymorphisms are associated with decreased D2R expression. Because D2R agonists have antioxidant activity, we hypothesized that increased blood pressure in D2-/- is related to increased oxidative stress. D2-/- mice had increased urinary excretion of 8-isoprostane, a parameter of oxidative stress; increased activity of reduced nicotinamide-adenine dinucleotide phosphate oxidase in renal cortex; increased expression of the reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits Nox1, Nox2, and Nox4; and decreased expression of the antioxidant enzyme heme-oxygenase-2 in the kidneys, suggesting that regulation of reactive oxygen species (ROS) production by D2R involves both pro-oxidant and antioxidant systems. Apocynin, a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, or hemin, an inducer of heme oxigenase-1, normalized the blood pressure in D2-/- mice. Because D2Rs in the adrenal gland are implicated in aldosterone regulation, we evaluated whether alterations in aldosterone secretion contribute to ROS production in this model. Urinary aldosterone was increased in D2-/- mice and its response to a high-sodium diet was impaired. Spirolactone normalized the blood pressure in D2-/- mice and the renal expression of Nox1 and Nox4, indicating that the increased blood pressure and ROS production are, in part, mediated by impaired aldosterone regulation. However, spironolactone did not normalize the excretion of 8-isoprostane and had no effect on expression of Nox2 or heme-oxygenase-2. Our results show that the D2R is involved in the regulation of ROS production and that, by direct and indirect mechanisms, altered D2R function may result in ROS-dependent hypertension.

Teaching aldosterone regulation and basic scientific principles using a classic paper by Dr. James O. Davis and colleagues

Classroom discussion of scientific articles can be an effective means of teaching scientific principles and methodology to both undergraduate and graduate science students. The availability of classic papers from the American Physiological Society Legacy Project has made it possible to access articles dating back to the early portions of the 20th century. In this article, we discuss a classic paper from the laboratory of Dr. James O. Davis on the regulation of aldosterone synthesis from the adrenal zona glomerulosa cell. Dr. Davis has conducted much of the seminal research investigating the renin-angiotensin system and the regulation of aldosterone release by angiotensin II. In addition to a characterization of the effects of ACTH on aldosterone regulation, this study is useful for discussing the basic principles of negative feedback pathways of the hypothalamic-pituitary axis. This study also provides examples of early bioassay techniques for the detection of angiotensin II and of the importance of quantitative measurements when investigating physiological responses. Three figures and one table are reproduced from the original article along with a series of discussion questions designed to facilitate discovery learning.

Type 2 Pseudohypoaldosteronism: New Insights Into Renal Potassium, Sodium, and Chloride Handling

Type 2 Pseudohypoaldosteronism: New Insights Into Renal Potassium, Sodium, and Chloride Handling

Aldosterone and the Pathogenesis of Hypertension

Hypertension remains a major public health problem, affecting up to 20% of the adult population in Western societies. Despite progress in treatment, the rates of blood pressure control remain suboptimal. Hypertension is a heterogeneous disorder, and in the majority of cases, with so-called essential hypertension, no clear single identifiable cause is found. Syndromes of excessive mineralocorticoid production or activity are among the important causes of secondary hypertension. Aldosterone is the principal mineralocorticoid in humans, and primary aldosterone excess, when associated with an aldosterone secreting adenoma (Conns tumor), is amenable to surgical cure. Classically, patient with Conns tumor present with spontaneous hypokalemia and have a relative excess of aldosterone production with suppression of plasma levels of renin (a proxy for angiotensin II, the major trophic substance regulating aldosterone secretion). This combination of a high aldosterone and a low renin is however more commonly associated with nodular hyperplasia of the adrenal glands, a condition not improved by surgery and variably responsive to the effects of mineralocorticoid antagonists such as spironolactone. Although primary aldosteronism was previously considered to be rare, recent studies have reported prevalence rates of up to 20% among hypertensive patients. This reflects the increasing use of the plasma aldosterone concentration to renin activity ratio (ARR), rather than spontaneous hypokalemia, as a screening tool for aldosteronism. Many patients with high ARR have normokalemia and, although renin activity is low, the level of aldosterone is usually within the normal range. This group of patients may thus include those who were previously classified as having low-renin essential hypertension. Recent data suggest that disturbances in aldosterone metabolism and regulation may not be uncommon in patients with essential hypertension. Thus, relatively high serum aldosterone levels within the reference range in normotensive individuals are associated with a substantially increased risk of developing hypertension, highlighting the potential role for aldosterone in the etiology of essential hypertension. The present review addresses the physiology of aldosterone action and its role in the pathogenesis of hypertension.

Nongenomic Renal Effects of Aldosterone

There is increasing evidence for a dependency on aldosterone in the development of hypertension.1 How aldosterone influences the physiology that determines blood pressure or for that matter promotes the pathophysiology of high blood pressure turns out to be less than straightforward. There is the genomic pathway that begins with the binding of aldosterone to the classical mineralocorticoid receptor (MR) and ends with sodium reabsorption at the distal nephron. But aldosterone can convey nongenomic influences as well, actions that have received less attention, possibly in part because they seem counterintuitive to our genomic view of biology. Adding to the complexity of aldosterone’s actions is the fact that there are also nonepithelial (nondistal nephron) targets of aldosterone, such as the heart, the vasculature, the kidney, and other sites. In this issue of Hypertension , Schmidt et al2 describe a rapid (thus nongenomic) effect of administered aldosterone to increase resistance in renal vasculature (a nonepithelial target) in healthy human subjects. The findings follow on the heels of a rather large body of work on the genomic actions of aldosterone. Aldosterone’s nongenomic effects were first recognized more than a decade before an aldosterone-inducible gene was identified in the late 1990s.3 A nongenomic action characteristically occurs almost immediately, usually within several minutes,4 before sufficient time has elapsed for gene …

Aldosterone Regulation of the Cardiac Sodium‐Calcium Exchanger

The objective of this study was to determine whether the protein expression and functional activity of the cardiac sodium-calcium exchanger was subject to regulation by activation of mineralocorticoid receptors. The studies were carried out in cultured adult rat ventricular myocytes. Protein expression was evaluated by Western blotting, using a monoclonal antibody to the NCX1 protein (R3F1, Swant, Switzerland). Functional activity of the sodium-calcium exchanger was evaluated by first inducing reverse-mode exchanger activity and then using fluorescent calcium indicators to measure changes in cytosolic calcium. Aldosterone strongly inhibited exchanger protein expression in a dose-dependent manner (0.1–10 nM). This effect depended on mineralocorticoid activation, as it could be blocked by spironolactone. Cytosolic calcium measurements confirmed that the decreased protein expression seen with 10 nM aldosterone was accompanied by a significant inhibition of sodium-calcium exchanger functional activity. This work was supported by NHLBI R01HL056910 (RWH).

Aldosterone-induced abnormal regulation of ENaC and SGK1 in Dahl salt-sensitive rat

Aldosterone plays a crucial role in controlling mineral balance in our body. The mechanism of aldosterone has been reported to elevate renal Na + reabsorption by stimulating expression of epithelial Na + channel (ENaC) and also activate an ENaC-regulating protein kinase, serum and glucocorticoid-regulated kinase 1 (SGK1). However, it is unknown whether aldosterone shows its stimulatory action on ENaC and SGK1 under an abnormal, salt-sensitive hypertensive condition. To clarify this point, we studied how aldosterone regulates expression of ENaC and SGK1 in Dahl salt-sensitive (DS) rat that shows hypertension with high salt diet. RNA and protein were extracted from the kidney 6 h after application of aldosterone (1.5 mg/kg body weight) subcutaneously injected into adrenalectomized DS and Dahl salt-resistant (DR) rats. Aldosterone decreased mRNA expression of β- and γ-ENaC in DS rat unlike DR rat, while aldosterone increased α-ENaC mRNA expression in DS rat similar to DR rat. Further, we found that aldosterone elevated SGK1 expression in DR rat, but not in DS rat. These observations indicate that ENaC and SGK1 are abnormally regulated by aldosterone in salt-sensitive hypertensive rats, suggesting that disturbance of the aldosterone regulation would be one of factors causing salt-sensitive hypertension.

A Novel Role for Glucocorticoid-induced Leucine Zipper Protein in Epithelial Sodium Channel-mediated Sodium Transport

The steroid hormone aldosterone stimulates sodium (Na+) transport in tight epithelia by altering the expression of target genes that regulate the activity and trafficking of the epithelial sodium channel (ENaC). We performed microarray analysis to identify aldosterone-regulated transcripts in mammalian kidney epithelial cells (mpkC-CD(c14)). One target, glucocorticoid-induced leucine zipper protein (GILZ), was previously identified by serial analysis of gene expression (SAGE); however, its function in epithelial ion transport was unknown. Here we show that GILZ expression is rapidly stimulated by aldosterone in mpkCCD(c14) and that GILZ, in turn, strongly stimulates ENaC-mediated Na+ transport by inhibiting extracellular signal-regulated kinase (ERK) signaling. In Xenopus oocytes with activated ERK, heterologous GILZ expression consistently inhibited phospho-ERK expression and markedly stimulated ENaC-mediated Na+ current, in a manner similar to that of U0126 (a pharmacologic inhibitor of ERK signaling). In mpkCCD(c14) cells, GILZ transfection similarly consistently inhibited phospho-ERK expression and stimulated transepithelial Na+ transport. Furthermore, aldosterone treatment of mpkCCD(c14) cells suppressed phospho-ERK levels with a time course that paralleled their increase of Na+ transport. Finally, GILZ expression markedly increased cell surface ENaC expression in epidermal growth factor-treated mammalian kidney epithelial cells, HEK 293. These observations suggest a novel link between GILZ and regulation of epithelial sodium transport through modulation of ERK signaling and could represent an important pathway for mediating aldosterone actions in health and disease.

The Utility of Nesiritide for Hemodialysis-Dependent Patients with Acutely Decompensated Heart Failure As a Bridge to Outpatient Dialysis

Study Objectives: Acutely decompensated heart failure is a common ED presentation in patients with renal dysfunction and are particularly challenging to manage due to limited response to standard therapies. B-type natriuretic peptide (BNP) is released as a result of left ventricular strain and aids by decreasing wall stress by relaxation of the cardiac myocyte, arterial vasodilatation, anti-fibrotic properties and down regulation of aldosterone, endothelium and norepinephrine function. No studies have investigated the use of nesiritide in the ADHF ED population with chronic renal dysfunction. The study objective is to evaluate the role of nesiritide in ADHF ED patients with hemodialysis-dependent renal failure as a bridge to dialysis.

Seasonal changes of water, electrolytes and aldosterone levels in blood serum, brain and kidney of the Egyptian cobra “Naja haje haje”

1. The levels of water, Na, K, Ca and Mg in blood serum, brain and kidney and aldosterone level in blood of Naja haje haje were studied during the different phases of the annual cycle. 2. The water content in the tissues studied displayed only minor changes as the animals passed from one phase to the other. 3. A significant increase in Na was recorded in the brain during the different phases indicating a depressed sodium pump, whereas the blood Na level showed a significant decrease during hibernation. 4. K increased in blood serum, brain and kidney during hibernation, while a nonsignificant decrease was found in blood serum during arousal. The brain may act as a potassium reservoir. 5. An increase in Ca and Mg concentration was recorded in blood serum, brain and kidney during prehibernation and hibernation. The data suggested a homeostatic function in the transport and metabolism of these cations. 6. Aldosterone exhibited a highly significant decrease especially during hibernation. The aldosterone regulation of ionic composition is discussed. 7. Na/K and Ca/Mg ratios in the brain may explain the decreased excitability during winter torpor.

Glucocorticoid metabolism and Na+ transport in chicken intestine

The role of aldosterone in regulation of electrogenic Na+ transport is well established, though mineralocorticoid receptors bind glucocorticoids with similar binding affinity as aldosterone and plasma concentration of aldosterone is much lower than glucocorticoids. In mammals, the aldosterone specificity is conferred on the low-selective mineralocorticoid receptors by glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase (11HSD) that converts cortisol or corticosterone into metabolites (cortisone, 11-dehydrocorticosterone) with lower affinity for these receptors. The present study examined the chicken intestine, whether changes in 11HSD activity are able to modulate the effect of corticosterone on Na+ transport, and how the metabolism of this hormone is distributed within the intestinal wall. This study shows that not only aldosterone, but also corticosterone (B), was able to increase the electrogenic Na+ transport in chicken caecum in vitro. The effect of corticosterone was higher in the presence of carbenoxolone, an inhibitor of steroid dehydrogenases, and was comparable to the effect of aldosterone. The metabolism of B in the intestine was studied; results showed oxidation of this steroid to 11-dehydrocorticosterone (A) and reduction to 11-dehydro-20beta-dihydrocorticosterone (20diA) as the main metabolic products at low nanomolar concentration of the substrate. In contrast, 20beta-dihydrocorticosterone and 20diA were the major products at micromolar concentration of B. Progesterone was converted to 20beta-dihydroprogesterone. The metabolism of corticosterone was localized predominantly in the intestinal mucosa (enterocytes). In conclusion, the oxidation at position C11 and reduction at position C20 suggest that both 11HSD and 20beta-hydroxysteroid dehydrogenase (20HSD) operate in the chicken intestine and that the mucosa of avian intestine possesses a partly different system of modulation of corticosteroid signals than mammals. This system seems to protect the aldosterone target tissue against excessive concentration of corticosterone and progesterone.

The Renin-Aldosterone Response to Stimulation and Suppression During Normal Pregnancy

Objective. During normal pregnancy, studies have shown increased activity of the renin-angiotensin-aldosterone system (RAAS) and a dissociation of plasma renin activity (PRA) and aldosterone (Aldo) evidenced by a greater increase in Aldo relative to PRA. The aims of this study were to examine the RAAS response to stimulation by upright posture and suppression by saline infusion and to investigate the PRA-Aldo dissociation under these two conditions. Methods. We studied 24 healthy normotensive women (mean ± standard error of mean, ages 29 ± 1 yrs) in sodium (Na) balance in the second and third trimesters and postpartum. Subjects underwent a 24-hour urine collection which was analyzed for Na, norepinephrine (NE), epinephrine (Epi), and dopamine (DA); a posture study with analysis of blood pressure (BP), PRA, Aldo, NE, Epi, DA, and cortisol; and a 0.9% NaCl infusion study (500 mL/hr for 3 hrs) with analysis of BP, PRA, Aldo, cortisol, and digitalis-like factor (DLF). Analyses included paired t tests to compare posture and saline responses, repeated measures to compare across periods, and percent change to evaluate the PRA-Aldo dissociation. Results. During pregnancy, PRA, Aldo, BP, catecholamines, and cortisol levels were significantly greater in upright than left lateral decubitus (LLD) posture, and the percent change in Aldo was significantly greater than the percent change in PRA. During pregnancy in response to saline infusion, BP did not change; the PRA and Aldo significantly decreased; the percent change in Aldo was significantly greater than the percent change in PRA in the second trimester; and serum DLF and cortisol levels significantly decreased. Conclusions. In longitudinally studied normal pregnancy, PRA and Aldo levels were dissociated at baseline, with stimulation and, to a lesserdegree, with suppression. Norepinephrine, adrenocorticotrophic hormone, and DLF may contribute to this dissociation, and clarification of these interactions may provide insight into the regulation of aldosterone during normal and hypertensive pregnancy.

Aldosterone regulation for 18 years in a case of primary aldosteronism

Aldosterone regulation for 18 years in a case of primary aldosteronism

Isoform specificity of human Na(+), K(+)-ATPase localization and aldosterone regulation in mouse kidney cells.

Short-term aldosterone coordinately regulates the cell-surface expression of luminal epithelial sodium channels (ENaC) and of basolateral Na(+) pumps (Na(+), K(+)-ATPase alpha1-beta1) in aldosterone-sensitive distal nephron (ASDN) cells. To address the question of whether the subcellular localization of the Na(+), K(+)-ATPase and its regulation by aldosterone depend on subunit isoform-specific structures, we expressed the cardiotonic steroid-sensitive human alpha isoforms 1-3 by retroviral transduction in mouse collecting duct mpkCCD(c14) cells. Each of the three exogenous human isoforms could be detected by Western blotting. Immunofluorescence indicated that the exogenous alpha1 subunit to a large extent localizes to the basolateral membrane or close to it, whereas much of the alpha2 subunit remains intracellular. An ouabain-sensitive current carried by exogenous pumps could be detected in apically amphotericin B-permeabilized epithelia expressing human alpha1 and alpha2 subunits, but not the alpha3 subunit. This current displayed a higher apparent Na(+) affinity in pumps containing human alpha2 subunits (10 mM) than in pumps containing human alpha1 (33.2 mM) or endogenous (cardiotonic steroid-resistant) mouse alpha1 subunits (mean: 16.3 mM). A very low mRNA level of the Na(+), K(+)-ATPase gamma subunit (FXYD2) in mpkCCD(c14) cells suggested that this ancillary gene product is not responsible for the relatively low apparent Na(+) affinity measured for a1 subunit-containing pumps. Aldosterone increased the pump current carried by endogenous pumps and by pumps containing the human alpha1 subunit. In contrast, the current carried by pumps with a human alpha2 subunit was not stimulated by the same treatment. In summary, quantitative basolateral localization of the Na(+), K(+)-ATPase and its responsiveness to aldosterone require alpha1 subunit-specific sequences that differentiate this isoform from the alpha2 and alpha3 subunit isoforms.