This Article Abstract Full Text (PDF) Methods Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mihailidou, A. S. Articles by Rasmussen, H. H. Search for Related Content PubMed PubMed Citation Articles by Mihailidou, A. S. Articles by Rasmussen, H. H. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB OUABAIN POTASSIUM SODIUM SPIRONOLACTONE Related Collections Gene regulation Ion channels/membrane transport

(Circulation Research. 2000;86:37.)
© 2000 American Heart Association, Inc.

Cellular Biology

Hyperaldosteronemia in Rabbits Inhibits the Cardiac Sarcolemmal Na⁺-K⁺ Pump

Anastasia S. Mihailidou, Henning Bundgaard, Mahidi Mardini, Peter S. Hansen, Keld Kjeldsen, Helge H. Rasmussen

From the Department of Cardiology, Royal North Shore Hospital (A.S.M., M.M., P.S.H., H.H.R.) and The University of Sydney (M.M., P.S.H., H.H.R.), Sydney, Australia, and Department of Medicine (H.B., K.K.), The Heart Centre, Rigshospitalet, National University Hospital, Copenhagen, Denmark.

Correspondence to Professor H.H. Rasmussen, Department of Cardiology, Royal North Shore Hospital, Pacific Highway, St. Leonards, Sydney, NSW, Australia 2065. E-mail helger{at}mail.med.usyd.edu.au

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Aldosterone upregulates the Na⁺-K⁺ pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na⁺-K⁺ pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na⁺-K⁺ pump current, arising from the 3:2 Na⁺:K⁺ exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na⁺ in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na⁺. Aldosterone had no effect on myocardial Na⁺-K⁺ pump concentration evaluated by vanadate-facilitated [³H]ouabain binding or by K⁺-dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na⁺ activity. The aldosterone-induced decrease in pump current and increased intracellular Na⁺ were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na⁺ affinity of the Na⁺-K⁺ pump, whereas it has no effect on maximal pump capacity.

Key Words: cardiac • mineralocorticoid receptor • spironolactone • ouabain binding • sodium

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is widely accepted that aldosterone increases abundance and activity of the Na⁺-K⁺-pump in kidney^{1 2 3 4} and colon.⁵ These organs are considered classical targets for aldosterone. Because the hormone can also bind with high affinity in the heart,⁶ effects on the cardiac sarcolemmal Na⁺-K⁺ pump have been examined. In vitro exposure of cultured rat cardiac myocytes to physiologically relevant nanomolar concentrations of aldosterone increases the abundance of mRNA for the catalytic ₁ subunit of the Na⁺-K⁺ pump, and when myocytes are exposed to micromolar concentrations, an increase in expression of the corresponding protein isoform can be demonstrated.⁷

The effects of aldosterone have also been examined in vivo. In one study,⁸ rats were given aldosterone via osmotic minipumps at a dose that caused a 5-fold increase in serum levels. There was no change in mRNA levels for Na⁺-K⁺ pump subunits in the heart after 1, 3, or 15 days of treatment, and the authors concluded that the myocardial Na⁺-K⁺ pump is not regulated by aldosterone. In another study,⁹ guinea pigs were given aldosterone for 90 days via osmotic minipumps at a dose that produced a 2-fold increase in serum levels. Northern and Western blot analysis showed that aldosterone induced a substantial increase in mRNA and protein levels of the ₂ subunit, whereas there was no effect on the ₁ subunit. The authors suggested that the increase in the ₂ isoform would cause an increase in pump activity and, hence, a decrease in the intracellular Na⁺ concentration. However, neither pump activity nor intracellular Na⁺ levels were measured.

If Na⁺-K⁺ pump gene expression is assumed to reflect activity, the previous studies suggest that aldosterone either has no effect⁸ or that it induces an increase in myocardial Na⁺-K⁺ pump activity.^{7 9} However, pump function was not directly examined in any of these studies. In the present study, the effect of aldosterone on myocardial Na⁺-K⁺ pump function was examined. Aldosterone was administered to rabbits via implanted osmotic minipumps to achieve increases in plasma levels similar to those encountered in human hyperaldosteronemia. We measured Na⁺-K⁺ pump current (I_p) in isolated ventricular myocytes using the whole-cell patch-clamp technique. Treatment with aldosterone induced a decrease in I_p measured when the intracellular Na⁺ concentration was set near physiological levels. However, there was no effect of treatment when Na⁺ was at a level expected to nearly saturate intracellular pump sites, suggesting that aldosterone has no effect on maximal pump capacity. This conclusion was supported by the absence of an effect of aldosterone on vanadate-facilitated [³H]ouabain binding capacity in intact samples and K⁺-dependent paranitrophenylphosphatase (pNPPase) activity in crude myocardial homogenates. Taken together, our results indicate that hyperaldosteronemia induces a functionally significant decrease in myocardial Na⁺-K⁺ pump activity but has no effect on the number of pump units.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
A total of 71 male New Zealand White rabbits weighing 2.5 to 3.0 kg were used. They were maintained on standard chow and had free access to tap water. In vivo interventions were followed by in vitro studies of cells or tissues. To administer aldosterone or spironolactone, we implanted osmotic minipumps (Alza) subcutaneously in the interscapular region under a general anesthetic of 2% halothane with 2 parts nitrous oxide and 1 part oxygen. Aldosterone and spironolactone were dissolved in a stock solution of ethanol and diluted in 0.9% sterile saline. Control rabbits were infused with the ethanol vehicle only. When treatment protocols were completed, we anesthetized rabbits with intramuscular ketamine (50 mg kg^-1) and xylazine hydrochloride (20 mg/kg) and excised the heart. Experimental protocols were approved by the institutional ethics committee at Royal North Shore Hospital, Sydney, and at Rigshospitalet, Copenhagen, and were conducted in accord with Danish Ministry of Justice legislation.

Single myocytes from either ventricle were isolated and voltage clamped with wide-tipped (4- to 5-µm) patch pipettes with resistances of 0.9 to 1.1 M, as described previously.¹⁰ For measurement of I_p, myocytes were superfused with Ca²⁺-containing modified Tyrode’s solution, as described previously.¹⁰ This solution was used while the whole-cell configuration was established and the membrane capacitance was measured. The superfusate was then changed to one that was identical except that it was nominally Ca²⁺-free and contained 0.2 mmol/L CdCl₂ and 2 mmol/L BaCl₂. I_p was identified in myocytes voltage clamped at -40 mV as the shift in holding current induced by 100 µmol/L ouabain. Currents are normalized for membrane capacitance. Details of the experimental setup and of the experimental protocols used to measure membrane capacitance and I_p have been described previously.¹¹

Vanadate-facilitated [³H]ouabain binding to intact left ventricular samples of 2 to 4 mg (wet weight) was performed as described in detail for rat skeletal muscle.¹² K⁺-dependent pNPPase activity was determined in crude homogenates (10 mg tissue/mL), as described previously for rat myocardium.¹³ Tissue K⁺ content was measured in samples of 25 mg wet weight by flame photometry using lithium as an internal standard. All measurements were made in duplicate. Details have been described.¹⁴ Intracellular Na⁺ activity (aⁱ_Na) was measured in intact isolated right ventricular papillary muscles with Na⁺-sensitive microelectrodes as described previously.^{10 11}

Reagents and Chemicals
Aldosterone, spironolactone, ouabain, dihydroouabain (DHO), and pNPP were purchased from Sigma. Tetramethyl-ammonium chloride was of purum grade and was purchased from Fluka. [³H]ouabain was from Amersham International. Vanadate was purchased from Merck. Chemicals used for the K⁺-dependent pNPPase activity, [³H]ouabain binding, and tissue K⁺ content experiments were purchased from Bie and Berntsen. All other chemicals were purchased from BDH. All chemicals were of analytical grade.

Statistical Analysis
Results are expressed as mean±SE. Statistical comparisons were made by both paired and unpaired Student t test and 1-way ANOVA followed by a Tukey test. Differences were regarded as statistically significant when P<0.05.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A previous study on the renal effects of aldosterone in rabbits used a dose of 50 µg/kg body weight per day for 7 days.⁴ To determine an appropriate dose that would produce a clinically relevant increase in plasma aldosterone levels in our rabbits, we administered aldosterone in doses of 30, 50, or 100 µg/kg body weight per day via osmotic minipumps for 7 days. Blood was collected for plasma concentrations of aldosterone at the time of implantation of minipumps and immediately before the rabbits were euthanized. The relationship between the infused dose and levels of plasma aldosterone is shown in Figure 1. The dose of 50 µg/kg body weight per day used in the previous study⁴ induced an increase in aldosterone levels similar to that seen in hyperaldosteronemic states and was therefore adopted for our study. Unless specified otherwise, this dose infused for 7 days was used throughout the study.

View larger version (11K): [in this window] [in a new window]   Figure 1. Aldosterone dose and achieved plasma levels. Plasma levels of aldosterone (Ald) before infusion of aldosterone are indicated by solid bars, and plasma levels after a 7-day period of infusion in doses of 30, 50, and 100 µg/kg body weight per day are indicated by open bars. N indicates number of animals in each group. *Significant increase in plasma levels.

Effect of Aldosterone on Serum and Tissue K⁺
Infusion of 50 µg/kg body weight per day aldosterone for 7 days produced a decrease in serum K⁺ from 4.8±0.1 to 3.6±0.2 mmol/L. The difference was statistically significant. To examine whether there was an associated depletion of tissue K⁺, we measured myocardial and skeletal muscle K⁺ content in control rabbits and in rabbits given aldosterone. The K⁺ contents are shown in Figure 2. There was no significant difference between control and aldosterone-treated rabbits. Because K⁺ depletion is known to cause a decrease in the abundance of Na⁺-K⁺ pumps in skeletal muscle,¹⁵ we also measured vanadate-facilitated [³H]ouabain binding. There were no significant changes in aldosterone-treated rabbits as compared with controls in the soleus muscle (202±14 versus 225±7 pmol/g wet weight) or in the extensor digitorum longum (EDL) muscle (140±8 versus 136±8 pmol/g wet weight). Similarly, treatment with aldosterone had no effect on K⁺-dependent pNPPase activity in soleus and EDL muscles (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 2. Aldosterone and tissue K+ content. The K+ content in the myocardium (myocard), soleus, and EDL muscles is shown. Open bars indicate K+ content in 6 control rabbits; solid bars, content in 6 rabbits infused with aldosterone for 7 days with 50 µg/kg body weight per day.

Effect of Aldosterone on I_p
We isolated myocytes from rabbits infused with ethanol vehicle and from the rabbits used in the series of experiments in which an appropriate dose of aldosterone was determined. We measured I_p in the myocytes using a Na⁺ concentration in the pipette solution ([Na]_pip) of 10 mmol/L. Figure 3 shows representative recordings of membrane currents during measurement of I_p in myocytes from a control rabbit and an aldosterone-treated rabbit. The mean I_p for myocytes from control rabbits and rabbits given 30, 50, or 100 µg/kg body weight per day is summarized in Figure 4. Mean I_p of myocytes from rabbits given 50 or 100 µg/kg body weight per day was significantly lower than mean I_p of myocytes from controls. A plateau in aldosterone-induced pump inhibition was reached with a dose of 50 µg/kg body weight per day.

View larger version (11K): [in this window] [in a new window]   Figure 3. Representative records of holding currents (Ih). A, Ih of a myocyte from a control rabbit. B, Ih of a myocyte from a rabbit treated with aldosterone 50 µg/kg body weight per day. Cells were of similar size, with membrane capacitances of 157 and 154 pF, respectively. Ih before and during superfusion of ouabain was considered stable when it varied by <2 pA over a period of 50 seconds. To determine Ip, we sampled Ih 5 times with an electronic cursor at 10-second intervals before and during superfusion of ouabain. Ip for each cell was defined as difference in mean values of the sampled currents.

View larger version (14K): [in this window] [in a new window]   Figure 4. Aldosterone (Ald) dose and Ip. Bars indicate mean Ip in myocytes isolated from control rabbits and rabbits administered aldosterone in a dose of 30, 50, or 100 µg/kg body weight per day for 7 days. Numbers in parentheses indicate numbers of myocytes studied; N, number of animals in each group. *Significant difference between Ips of myocytes from aldosterone-treated rabbits and myocytes from control rabbits.

To determine the effect of duration of treatment with aldosterone on I_p, we infused rabbits with aldosterone for 3 or 14 days. Infusion of aldosterone for 3 days caused an increase in plasma aldosterone levels similar to the levels achieved after 7 days (see Figure 1). In contrast, infusion for 14 days caused a marked increase to 8±2 nmol/L. Myocytes were isolated and I_p was measured using a [Na]_pip of 10 mmol/L. Mean levels of I_p are shown in Figure 5, with mean levels of I_p of myocytes isolated from rabbits given aldosterone for 7 days. The aldosterone-induced decrease in I_p at 7 days was sustained for 14 days.

View larger version (16K): [in this window] [in a new window]   Figure 5. Duration of aldosterone (Ald) treatment and Ip. Bars indicate mean Ip of myocytes from rabbits treated with aldosterone in a dose of 50 µg/kg body weight per day for 3, 7, or 14 days. Data for myocytes from control rabbits and rabbits given aldosterone for 7 days are also included in Figure 4. *Significant difference between Ip of myocytes from aldosterone-treated rabbits and Ip of myocytes from controls.

Effect of Aldosterone on Na⁺-K⁺ Pump Concentration
The results shown in Figures 4 and 5 were obtained with a [Na]_pip similar to physiological levels of intracellular Na⁺. To examine whether aldosterone affects I_p when intracellular Na⁺ is at a level expected to nearly saturate binding sites, I_p was measured using a [Na]_pip of 80 mmol/L in 12 myocytes from 5 rabbits infused with aldosterone and in 16 myocytes from 5 control rabbits. The mean I_p of myocytes from rabbits treated with aldosterone was 1.61±0.06 pA/pF and mean I_p of myocytes from control rabbits was 1.77±0.04 pA/pF. There was no significant difference (P=0.06). These results do not definitively rule out an effect of aldosterone on the number of sarcolemmal Na⁺-K⁺ pump units. We used 2 additional methods to determine the effect of aldosterone on the abundance of pump units, vanadate-facilitated [³H]ouabain and measurement of K-dependent pNPPase activity. We measured vanadate facilitated [³H]ouabain binding in intact myocardial samples isolated from 6 rabbits given aldosterone and from 6 control rabbits. The mean [³H]ouabain binding site concentration in the myocardium from rabbits treated with aldosterone was 669±21 pmol/g wet weight, whereas the mean concentration in control rabbits was 642±29 pmol/g wet weight. There was no significant difference. We measured K-dependent pNPPase activity in crude homogenates of myocardium. The mean activity in myocardium isolated from 6 rabbits treated with aldosterone was 0.97±0.05 µmol min^-1/g wet weight, whereas the mean activity in 6 control rabbits was 0.91±0.09 µmol min^-1/g wet weight. There was no significant difference between the groups.

Effect of Mineralocorticoid Receptor Blockade
To examine whether the classical mineralocorticoid receptor is involved in the effect of aldosterone on I_p, we used the mineralocorticoid receptor blocker spironolactone, comparing rabbits given aldosterone alone, those given spironolactone alone, and those given both aldosterone and spironolactone. Spironolactone was administered via osmotic minipumps in a dose of 200 µg/kg body weight per day. All rabbits were treated for 7 days. The mean serum K⁺ levels for the 3 groups are shown in Figure 6A. During the 7-day treatment period, serum K⁺ decreased by a similar amount in rabbits given combined aldosterone and spironolactone and rabbits given aldosterone alone. We conclude that spironolactone in the dose we used had no effect on serum K⁺.

View larger version (19K): [in this window] [in a new window]   Figure 6. A, Spironolactone treatment and serum K+. Serum K+ levels measured before treatment are indicated by solid bars, and serum K+ levels after a 7-day treatment of rabbits with aldosterone (Ald, 50 µg/kg body weight per day), spironolactone (SP, 200 µg/kg body weight per day), or a combination of aldosterone and spironolactone (Ald+SP) are indicated by open bars. N indicates number of rabbits in each group. *Significant difference between levels before and after treatment. B, Spironolactone treatment and Ip. Bars indicate mean Ip of myocytes from the 3 groups of rabbits referred to in panel A. Numbers in parentheses indicate numbers of myocytes studied. Asterisks indicate a significant difference in mean Ip between groups. Data for myocytes from rabbits treated with aldosterone alone are also shown in Figures 4 and 5 and are included here to facilitate comparison.

We compared I_p of myocytes from rabbits given aldosterone alone, those given spironolactone alone, and those given aldosterone and spironolactone in combination. I_p was measured using a [Na]_pip of 10 mmol/L. Mean I_p values of myocytes from each of the 3 groups of rabbits are shown in Figure 6B. Mean I_p of myocytes from rabbits given spironolactone alone was similar to mean I_p shown in Figures 4 and 5 of myocytes from untreated control rabbits (0.34±0.02 versus 0.35±0.02 pA/pF). Spironolactone completely prevented the aldosterone-induced decrease in mean I_p. We conclude that the effect of hyperaldosteronemia on myocardial Na⁺-K⁺ pump function is mediated by the classical mineralocorticoid receptor.

Effect of Aldosterone and Spironolactone on aⁱ_Na
Because treatment with aldosterone results in a decrease in I_p when [Na]_pip is near physiological intracellular levels, one would expect that treatment induces an increase in intracellular Na⁺ level. To examine this, we measured aⁱ_Na in single intact papillary muscles isolated from 7 rabbits infused with aldosterone for 7 days and from 7 control rabbits. The mean aⁱ_Na in papillary muscles from aldosterone-treated rabbits was 10.4±0.9 mmol/L, whereas the mean aⁱ_Na in control papillary muscles was 7.0±0.4 mmol/L. Aldosterone induced a significant increase in aⁱ_Na. We also measured aⁱ_Na in papillary muscles from a third group of 4 rabbits treated with both aldosterone and spironolactone. Spironolactone prevented the aldosterone-induced increase in aⁱ_Na (7.2±1.1 mmol/L). The effects of aldosterone and spironolactone on aⁱ_Na are summarized in Figure 7A.

View larger version (19K): [in this window] [in a new window]   Figure 7. A, Effect of aldosterone and spironolactone on intracellular sodium activity. aiNa was measured in intact papillary muscles isolated from control rabbits, rabbits infused with aldosterone alone (Ald), and rabbits infused with both aldosterone and spironolactone (Ald+SP). Numbers in parentheses indicate number of rabbits. *Significant difference between aiNa in papillary muscles from rabbits given aldosterone alone and control rabbits or rabbits given both aldosterone and spironolactone. B, Effect of aldosterone on rate of increase in aiNa during Na+-K+ pump blockade. •, Mean increase in aiNa after onset of Na+-K+ pump blockade with 500 µmol/L DHO in papillary muscles from 5 control rabbits; , aiNa in papillary muscles from 6 rabbits treated with aldosterone. There was no significant difference between mean levels of aiNa in papillary muscles from the 2 groups of rabbits according to a repeated-measures ANOVA.

We have previously found that acute exposure of isolated papillary muscles to aldosterone in vitro enhances influx of Na⁺ via the Na⁺/K⁺/2Cl^– cotransporter. We next examined whether enhanced Na⁺ influx via the Na⁺/K⁺/2Cl^– cotransporter contributed to the increase in steady-state aⁱ_Na observed in hyperaldosteronemic rabbits (Figure 7A). Aldosterone-induced Na⁺ influx can be detected as an increase in the rate of rise of aⁱ_Na on Na⁺-K⁺ pump blockade with the fast-acting cardiac steroid DHO.¹⁰ We isolated single papillary muscles from 6 rabbits treated with aldosterone and from 5 control rabbits. We then recorded the rate of rise of aⁱ_Na on superfusion with 500 µmol/L DHO. Figure 7B summarizes the time course of the recorded changes in aⁱ_Na. There was no significant difference between the rate of rise in aⁱ_Na in papillary muscles from rabbits treated with aldosterone and in papillary muscles from controls. This suggests that the aldosterone-induced increase in steady-state aⁱ_Na is due to a reduction in extrusion of Na⁺ via the Na⁺-K⁺ pump rather than to an increase in Na⁺ influx.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Aldosterone on Na⁺-K⁺ Pump Function
Two previous studies have examined the effect of aldosterone on activity of the Na⁺-K⁺ pump in the heart. Hegyvary¹⁶ reported that aldosterone administered to guinea pigs in vivo induces an increase in the activity of isolated myocardial Na⁺-K⁺ ATPase, and Ikeda et al⁷ reported that exposure of isolated intact cardiac myocytes to aldosterone in vitro induces an increase in activity of the Na⁺-K⁺ pump. These findings are difficult to reconcile with the aldosterone-induced decrease in sarcolemmal Na⁺-K⁺ pump function demonstrated in the present study. It is important to consider details of the experimental evidence supporting such conflicting conclusions.

Hegyvary¹⁶ gave guinea pigs twice daily intraperitoneal injections of aldosterone for 2 weeks in a weight-adjusted dose 3-fold higher than the daily dose in our study. The serum aldosterone levels achieved were not reported. However, given the intermittent mode of administration, it is likely that high peak levels were reached, possibly activating glucocorticoid as well as mineralocorticoid receptors. Because glucocorticoids induce an upregulation of the Na⁺-K⁺ pump,¹⁷ such an increase in Na⁺-K⁺ ATPase activity should not necessarily be taken to indicate a physiologically relevant effect mediated by the mineralocorticoid receptor.

Ikeda et al⁷ exposed cardiac myocytes to aldosterone for 72 hours and studied gene expression and function of the Na⁺-K⁺ pump. Aldosterone was reported to cause an increase in Na⁺ influx. A decrease in myocyte Na⁺ content to levels below those of control myocytes after withdrawal of aldosterone was taken to indicate "unmasking" of functionally significant aldosterone-induced upregulation of the Na⁺-K⁺ pump. The accuracy of this indirect approach is critically dependent on assumptions that are difficult to verify with certainty. In addition, it should be noted that, whereas physiologically relevant concentrations of aldosterone were used in some of the studies on gene expression, a concentration of 1 µmol/L was used in studies on function of the Na⁺-K⁺ pump. At such concentrations, aldosterone is expected to have nonspecific effects.

We used the whole-cell patch clamp technique to control membrane voltage and the concentrations of intracellular and extracellular ligands during measurement of pump function in our study. Aldosterone induced a decrease in electrogenic Na⁺-K⁺ pump current measured when [Na]_pip was near physiological intracellular levels. The conclusion that aldosterone, as administered in our study, induces pump inhibition was strongly supported by the demonstration of an aldosterone-induced increase in free cytosolic Na⁺ activity in intact papillary muscles. A role of the mineralocorticoid receptor was indicated by the reversal of aldosterone-induced changes in pump current and aⁱ_Na by spironolactone.

Effect of Aldosterone on Myocardial Na⁺-K⁺ Pump Concentration
Aldosterone had no effect on I_p measured using a [Na]_pip expected to nearly saturate intracellular pump sites. Because I_p measured under similar conditions in oocytes accurately reflects the number of pump sites determined with the [³H]ouabain binding technique,¹⁸ aldosterone appears to have no effect on the number of myocardial pump sites. Measurements of vanadate-facilitated [³H]ouabain binding to intact myocardial samples in the present study support this conclusion. Because isoforms relatively insensitive to ouabain might not be detected by our measurements of I_p and [³H]ouabain binding, we measured K⁺-dependent pNPPase activity, an index independent of the sensitivity of Na⁺-K⁺ pump isoforms to ouabain. Taken together, our findings indicate that aldosterone had no effect on the concentration of myocardial Na⁺-K⁺ pump sites. The demonstrated decrease in pump function is consistent with an aldosterone-induced decrease in the apparent affinity of the pump for intracellular Na⁺.

Metabolic Effects of Hyperaldosteronemia and the Na⁺-K⁺ Pump
Because hyperaldosteronemia can be associated with K⁺ depletion and because K⁺ depletion has been reported to affect the Na⁺-K⁺ pump in skeletal^{15 17 19 20} and cardiac muscle,²¹ the possibility that K⁺ deficiency accounts for the decrease in I_p in our study should be considered. However, the decrease in serum K⁺ in our study was considerably less than the decrease usually associated with downregulation of the pump. Because skeletal muscles contain 75% of the total body K⁺ content,²² a major effect of aldosterone on K⁺ balance should also be reflected in the skeletal muscle K⁺ content. We did not find an aldosterone-induced reduction in K⁺ contents of skeletal muscle or myocardium. An effect of aldosterone on K⁺ balance is associated with a decrease in the abundance of Na⁺-K⁺ pump units. There was no such decrease in skeletal or cardiac muscle in our study. It is also important to note that spironolactone completely abolished the effect of aldosterone on I_p without having any effect on the decrease in serum K⁺ that developed during treatment with aldosterone (see Figure 6). Finally, it should be noted that K⁺ depletion in rabbits increases rather than decreases electrogenic pump activity in cardiac myocytes.²³ Therefore, it is likely that the aldosterone-induced decrease in I_p in our study is independent of an effect of aldosterone on K⁺ balance.

Because hyperaldosteronemia can be associated with a decrease in levels of thyroid hormone⁹ and because hypothyroidism can reduce Na⁺-K⁺ pump function in rabbit heart,²⁴ the possibility that hypothyroidism accounts for the decrease in pump function in the present study should also be considered. However, given that thyroid hormone regulates synthesis of Na⁺-K⁺ pumps,²⁵ the absence of an effect of aldosterone on the abundance of Na⁺-K⁺ pump units suggests that the aldosterone-induced decrease in I_p is not related to thyroid function. To obtain independent support for this, we measured levels of triiodothyronine and thyroxine in 6 rabbits before and after treatment with aldosterone. There was no detectable effect on thyroid function by aldosterone treatment (data not shown).

Clinical Implications of Aldosterone-Induced Na⁺-K⁺ Pump Inhibition
Serum levels of aldosterone in patients with congestive heart failure are 3-fold higher than levels in patients without heart failure. Both clinical²⁶ and experimental^{27 28} evidence suggests that chronically elevated aldosterone levels have an adverse effect on the heart. High intracellular Na⁺ levels in the myocardium of patients with heart failure²⁹ may at least in part be related to aldosterone-induced Na⁺-K⁺ pump inhibition. Because of the steep, nonlinear dependence of intracellular Ca²⁺ on the transmembrane Na⁺ concentration gradient,³⁰ this is expected to cause a large increase in intracellular Ca²⁺. Cellular overload of Na⁺ and Ca²⁺ is believed to be important in the pathogenesis of cardiac arrhythmias,³¹ a common complication of congestive heart failure. Aldosterone-induced Na⁺-K⁺ pump inhibition may also contribute to cardiac remodeling in heart failure, because pump inhibition can cause activation of key growth-related genes in cardiac myocytes³² and contribute to myocyte hypertrophy.^{33 34} The present study suggests that aldosterone receptor antagonists offer a rational therapeutic approach, a notion supported by recent reports of clinical benefits of such drugs.^{35 36}

	Acknowledgments

 
This study was supported by the North Shore Heart Research Foundation, the Danish Heart Foundation, and a grant from G.D. Searle/Monsato. We thank Judy Monaghan for performing the aldosterone assays.

Received July 8, 1999; accepted October 20, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Jørrgensen PL. The role of aldosterone in the regulation of (Na⁺ + K⁺)-ATPase in rat kidney. J Steroid Biochem. 1972;3:181–191.[Medline] [Order article via Infotrieve]

2. Verrey F, Kraehenbuhl JP, Rossier BC. Aldosterone induces a rapid increase in the rate of Na, K-ATPase gene transcription in cultured kidney cells. Mol Endocrinol. 1989;3:1369–1376.[Abstract/Free Full Text]

3. Blot-Chabaud M, Jaisser F, Bonvalet J-P, Farman N. Effect of cell sodium on Na⁺/K⁺-ATPase-dependent sodium efflux in cortical collecting tubule of rabbits under different aldosterone status. Biochim Biophys Acta. 1990;1022:126–128.[Medline] [Order article via Infotrieve]

4. Welling PA, Caplan M, Sutters M, Giebisch G. Aldosterone-mediated Na/K-ATPase expression is ₁ isoform specific in the renal cortical collecting duct. J Biol Chem. 1993;268:23469–23476.[Abstract/Free Full Text]

5. Wiener H, Nielsen JM, Klaerke DA, Jørrgensen PL. Aldosterone and thyroid hormone modulation of 1-, ß1-mRNA, and Na, K-pump sites in rabbit distal colon epithelium: evidence for a novel mechanism of escape from the effect of hyperaldosteronemia. J Membr Biol. 1993;133:203–211.[Medline] [Order article via Infotrieve]

6. Pearce P, Funder JW. High affinity aldosterone binding sites (type I receptors) in rat heart. Clin Exp Pharmacol Physiol. 1987;14:859–866.[Medline] [Order article via Infotrieve]

7. Ikeda U, Hyman R, Smith TW, Medford RM. Aldosterone-mediated regulation of Na⁺, K⁺-ATPase gene expression in adult and neonatal rat cardiocytes. J Biol Chem. 1991;266:12058–12066.[Abstract/Free Full Text]

8. Robert V, Silvestre J-S, Charlemagne D, Sabri A, Trouve P, Wassef M, Swynghedauw B, Delcayre C. Biological determinants of aldosterone-induced cardiac fibrosis in rats. Hypertension. 1995;26:971–978.[Abstract/Free Full Text]

9. Ramirez-Gil JF, Trouve P, Mougenot N, Carayon A, Lechat P, Charlemagne D. Modifications of myocardial Na⁺, K⁺-ATPase and Na⁺/Ca²⁺ exchanger in aldosterone/salt-induced hypertension in guinea pigs. Cardiovasc Res. 1998;38:451–462.[Medline] [Order article via Infotrieve]

10. Mihailidou AS, Buhagiar KA, Rasmussen HH. Na⁺ influx and Na⁺-K⁺ pump activation during short-term exposure of cardiac myocytes to aldosterone. Am J Physiol. 1998;274:C175–C181.[Abstract/Free Full Text]

11. Whalley DW, Hool LC, Ten Eick RE, Rasmussen HH. Effect of osmotic swelling and shrinkage on Na⁺-K⁺ pump activity in mammalian cardiac myocytes. Am J Physiol. 1993;265:C1201–C1210.[Abstract/Free Full Text]

12. Kjedsen K. Complete quantification of the total concentration of rat skeletal-muscle Na⁺ + K⁺-dependent ATPase by measurement of [³H]ouabain binding. Biochem J. 1986;240:725–730.[Medline] [Order article via Infotrieve]

13. Larsen JS, Kjeldsen K. Quantification in crude homogenates of rat myocardial Na⁺, K⁺- and Ca²⁺-ATPase by K⁺- and Ca²⁺-dependent pNPPase: age-dependent changes. Basic Res Cardiol. 1995;90:323–331.[Medline] [Order article via Infotrieve]

14. Dørrup I, Skajaa K, Clausen T. A simple and rapid method for the determination of the concentrations of magnesium, sodium, potassium and sodium, potassium pumps in human skeletal muscle. Clin Sci. 1988;74:241–248.[Medline] [Order article via Infotrieve]

15. Nørrgard A, Kjeldsen K, Clausen T. Potassium depletion decreases the number of ³H-ouabain binding sites and the active Na-K transport in skeletal muscle. Nature. 1981;293:739–741.[Medline] [Order article via Infotrieve]

16. Hegyvary C. Effect of aldosterone and methylprednisolone on cardiac NaK-ATPase. Experientia. 1977;33:1280–1281.[Medline] [Order article via Infotrieve]

17. Dørrup I, Clausen T. Effects of adrenal steroids on the concentration of Na⁺-K⁺ pumps in rat skeletal muscle. J Endocrinol. 1997;152:49–57.[Abstract/Free Full Text]

18. Jaunin P, Horisberger J-D, Richter K, Good PJ, Rossier BC, Geering K. Processing, intracellular transport and functional expression of endogenous and exogenous -ß3 Na, K-ATPase complexes in Xenopus oocytes. J Biol Chem. 1992;267:577–585.[Abstract/Free Full Text]

19. Hsu Y-M, Guidotti G. Effects of hypokalemia on the properties and expression of the (Na⁺, K⁺)-ATPase of rat skeletal muscle. J Biol Chem. 1991;266:427–433.[Abstract/Free Full Text]

20. Thompson CB, McDonough AA. Skeletal muscle Na, K-ATPase and ß subunit protein levels respond to hypokalemic challenge with isoform and muscle type specificity. J Biol Chem. 1996;271:32653–32658.[Abstract/Free Full Text]

21. Nørrgaard A, Kjeldsen K, Hansen O. K⁺-dependent 3-O-methylfluorescein phosphatase activity in crude homogenate of rodent heart ventricle: effect of K⁺ depletion and changes in thyroid status. Eur J Pharmacol. 1985;113:373–382.[Medline] [Order article via Infotrieve]

22. DeFronzo RA, Bia M, Smith D. Clinical disorders of hyperkalemia. Annu Rev Med. 1982;33:521–554.[Medline] [Order article via Infotrieve]

23. Shattock MJ, Matsuura H, Ward JPT. Sodium pump current measured in cardiac ventricular myocytes isolated from control and potassium depleted rabbits. Cardiovasc Res. 1994;28:1854–1862.[Abstract/Free Full Text]

24. Doohan MM, Gray DF, Hool LC, Robinson BG, Rasmussen HH. Thyroid status and regulation of intracellular sodium in rabbit heart. Am J Physiol. 1997;272:H1589–H1597.[Abstract/Free Full Text]

25. Hensley CB, Azuma KK, Tang M-J, McDonough A. Thyroid hormone induction of rat myocardial Na⁺-K⁺-ATPase: ₁-, ₂-, and ß₁-mRNA and -protein levels at steady state. Am J Physiol. 1992;262:C484–C492.[Abstract/Free Full Text]

26. Swan JW, Anker SD, Walton C, Godsland IF, Clark A, Leyva F, Stevenson JC, Coats AJS. Insulin resistance in chronic heart failure: relation to severity and etiology of heart failure. J Am Coll Cardiol. 1997;30:527–532.[Abstract]

27. Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT. Remodeling of the rat right and left ventricles in experimental hypertension. Circulation. 1990;67:1355–1364.

28. Young M, Head G, Funder J. Determinants of cardiac fibrosis in experimental hypermineralocorticoid states. Am J Physiol. 1995;269:E657–E662.[Abstract/Free Full Text]

29. Maier LS, Pieske B. Frequency-dependent changes in intracellular Na⁺-concentration in isolated human myocardium. Circulation. 1997;96(suppl I):I-178. Abstract.

30. Sheu SS, Fozzard HA. Transmembrane Na⁺ and Ca²⁺ electrochemical gradients in cardiac muscle and their relationship to force development. J Gen Physiol. 1982;80:325–351.[Abstract/Free Full Text]

31. Noble D. Ionic mechanisms determining the timing of ventricular repolarization: significance for cardiac arrhythmias. Ann N Y Acad Sci. 1992;644:1–22.

32. Komentiani P, Li J, Gnudi L, Kahn BB, Askari A, Xie Z. Multiple signal transduction pathways link Na⁺/K⁺-ATPase to growth-related genes in cardiac myocytes. J Biol Chem. 1998;24:15249–15256.

33. Kent RL, Hoober K, Cooper G IV. Load responsiveness of protein synthesis in adult mammalian myocardium: role of cardiac deformation linked to sodium influx. Circ Res. 1989;64:74–85.[Abstract/Free Full Text]

34. Huang L, Kometiani P, Xie Z. Differential regulation of Na/K-ATPase -subunit isoform gene expressions in cardiac myocytes by ouabain and other hypertrophic stimuli. J Mol Cell Cardiol. 1997;29:3157–3167.[Medline] [Order article via Infotrieve]

35. Ramires FJA, Mansur A, Coelho O, Maranhao M, Mady C, Ramires JAF. Spironolactone and ventricular arrhythmias in patients with heart failure. Circulation. 1998; 98(suppl I):I-300. Abstract.

36. Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez, Palensky J, Wittes J. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med. 1999;341:709–717.[Abstract/Free Full Text]

This article has been cited by other articles:

				V. McEneaney, B. J. Harvey, and W. Thomas Aldosterone Regulates Rapid Trafficking of Epithelial Sodium Channel Subunits in Renal Cortical Collecting Duct Cells via Protein Kinase D Activation Mol. Endocrinol., April 1, 2008; 22(4): 881 - 892. [Abstract] [Full Text] [PDF]

				W. C De Mello Beneficial Effect of Eplerenone on Cardiac Remodelling and Electrical Properties of the Failing Heart Journal of Renin-Angiotensin-Aldosterone System, March 1, 2006; 7(1): 40 - 46. [Abstract] [PDF]

				M. Yamamuro, M. Yoshimura, M. Nakayama, K. Abe, M. Shono, S. Suzuki, T. Sakamoto, Y. Saito, K. Nakao, H. Yasue, et al. Direct Effects of Aldosterone on Cardiomyocytes in the Presence of Normal and Elevated Extracellular Sodium Endocrinology, March 1, 2006; 147(3): 1314 - 1321. [Abstract] [Full Text] [PDF]

				R. Perrier, S. Richard, Y. Sainte-Marie, B. C. Rossier, F. Jaisser, E. Hummler, and J.-P. Benitah A direct relationship between plasma aldosterone and cardiac L-type Ca2+ current in mice J. Physiol., November 15, 2005; 569(1): 153 - 162. [Abstract] [Full Text] [PDF]

				J. W. Funder The Nongenomic Actions of Aldosterone Endocr. Rev., May 1, 2005; 26(3): 313 - 321. [Abstract] [Full Text] [PDF]

				A. S. Mihailidou Nongenomic Cardiovascular Actions of Aldosterone: A Receptor for All Seasons? Endocrinology, March 1, 2005; 146(3): 971 - 972. [Full Text] [PDF]

				A. S. Mihailidou, M. Mardini, and J. W. Funder Rapid, Nongenomic Effects of Aldosterone in the Heart Mediated by {epsilon} Protein Kinase C Endocrinology, February 1, 2004; 145(2): 773 - 780. [Abstract] [Full Text] [PDF]

				S. Arima, K. Kohagura, H.-L. Xu, A. Sugawara, A. Uruno, F. Satoh, K. Takeuchi, and S. Ito Endothelium-Derived Nitric Oxide Modulates Vascular Action of Aldosterone in Renal Arteriole Hypertension, February 1, 2004; 43(2): 352 - 357. [Abstract] [Full Text] [PDF]

				S. Arima, K. Kohagura, H.-L. Xu, A. Sugawara, T. Abe, F. Satoh, K. Takeuchi, and S. Ito Nongenomic Vascular Action of Aldosterone in the Glomerular Microcirculation J. Am. Soc. Nephrol., September 1, 2003; 14(9): 2255 - 2263. [Abstract] [Full Text] [PDF]

				P. C. White Aldosterone: Direct Effects on and Production by the Heart J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2376 - 2383. [Full Text] [PDF]

				A. Cittadini, M. G. Monti, J. Isgaard, C. Casaburi, H. Stromer, A. Di Gianni, R. Serpico, L. Saldamarco, M. Vanasia, and L. Sacca Aldosterone receptor blockade improves left ventricular remodeling and increases ventricular fibrillation threshold in experimental heart failure Cardiovasc Res, June 1, 2003; 58(3): 555 - 564. [Abstract] [Full Text] [PDF]

				R. Alzamora, E. T. Marusic, M. Gonzalez, and L. Michea Nongenomic Effect of Aldosterone on Na+,K+-Adenosine Triphosphatase in Arterial Vessels Endocrinology, April 1, 2003; 144(4): 1266 - 1272. [Abstract] [Full Text] [PDF]

				R. H.G Schwinger, H. Bundgaard, J. Muller-Ehmsen, and K. Kjeldsen The Na, K-ATPase in the failing human heart Cardiovasc Res, March 15, 2003; 57(4): 913 - 920. [Abstract] [Full Text] [PDF]

				K Kjeldsen, A Norgaard, and M Gheorghiade Myocardial Na,K-ATPase: the molecular basis for the hemodynamic effect of digoxin therapy in congestive heart failure Cardiovasc Res, September 1, 2002; 55(4): 710 - 713. [Abstract] [Full Text] [PDF]

				G. Crambert, M. Fuzesi, H. Garty, S. Karlish, and K. Geering Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties PNAS, August 20, 2002; 99(17): 11476 - 11481. [Abstract] [Full Text] [PDF]

				A. S. Mihailidou, M. Mardini, J. W. Funder, and M. Raison Mineralocorticoid and Angiotensin Receptor Antagonism During Hyperaldosteronemia Hypertension, August 1, 2002; 40(2): 124 - 129. [Abstract] [Full Text] [PDF]

				H. G. Glitsch Electrophysiology of the Sodium-Potassium-ATPase in Cardiac Cells Physiol Rev, October 1, 2001; 81(4): 1791 - 1826. [Abstract] [Full Text] [PDF]

				M. Mardini, A. S. Mihailidou, A. Wong, and H. H. Rasmussen Cyclosporine and FK506 Differentially Regulate the Sarcolemmal Na+-K+ Pump J. Pharmacol. Exp. Ther., April 12, 2001; 297(2): 804 - 810. [Abstract] [Full Text]

				O. M. Sejersted and G. Sjogaard Dynamics and Consequences of Potassium Shifts in Skeletal Muscle and Heart During Exercise Physiol Rev, October 1, 2000; 80(4): 1411 - 1481. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Methods Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mihailidou, A. S. Articles by Rasmussen, H. H. Search for Related Content PubMed PubMed Citation Articles by Mihailidou, A. S. Articles by Rasmussen, H. H. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB OUABAIN POTASSIUM SODIUM SPIRONOLACTONE Related Collections Gene regulation Ion channels/membrane transport