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Endocrinology Vol. 145, No. 2 773-780
Copyright © 2004 by The Endocrine Society

Rapid, Nongenomic Effects of Aldosterone in the Heart Mediated by {epsilon} Protein Kinase C

Anastasia S. Mihailidou, Mahidi Mardini and John W. Funder

Department of Cardiology (A.S.M., M.M.), Royal North Shore Hospital, Sydney 2065, Australia; Faculty of Medicine, University of Sydney 2006 (A.S.M., M.M.); Department of Cardiology (M.M.), Westmead Hospital, Sydney 2145, Australia; and Prince Henry’s Institute of Medical Research (J.W.F.), Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Dr. Anastasia Susie Mihailidou, Department of Cardiology, Royal North Shore Hospital, Pacific Highway, St. Leonards, Sydney, New South Wales, Australia 2065. E-mail: amihaili{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aldosterone elevates Na+/K+/2Cl- cotransporter activity in rabbit cardiomyocytes within 15 min, an effect blocked by K-canrenoate and thus putatively mineralocorticoid receptor mediated. Increased cotransporter activity raises intracellular [Na+] sufficient to produce a secondary increase in Na+-K+ pump activity; when this increase in intracellular [Na+] is prevented, a rapid effect of aldosterone to lower pump activity is seen. Addition of transcription inhibitor actinomycin D did not change basal or aldosterone-induced lowered pump activity, indicating a direct, nongenomic action of aldosterone. We examined a possible role for protein kinase C (PKC) in the rapid nongenomic effects of aldosterone. Single ventricular myocytes and pipette solutions containing 10 mM intracellular [Na+] were used in patch clamp studies to measure Na+-K+ pump activity. Aldosterone lowered pump current, an effect abolished by {epsilon} PKC ({epsilon}PKC) inhibition but neither {alpha}PKC nor scrambled {epsilon}PKC; addition of {epsilon}PKC activator peptide mimicked the rapid aldosterone effect. In rabbits chronically infused with aldosterone, the lowered pump current in cardiomyocytes was acutely (<=15 min) restored by {epsilon}PKC inhibition. These studies show that rapid effects of aldosterone on Na+-K+ pump activity are nongenomic and specifically {epsilon}PKC mediated; in addition, such effects may be prolonged (7 d) and long-lived (~4 h isolated cardiomyocyte preparation time). The rapid, prolonged, long-lived effects can be rapidly (<=15 min) reversed by {epsilon}PKC blockade, suggesting a hitherto unrecognized complexity of aldosterone action in the heart and perhaps by extension other tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW WIDELY recognized that aldosterone has rapid nongenomic effects in a variety of tissues, including vascular smooth muscle cells (1, 2) and cardiac myocytes (3). In the latter tissue, we have previously shown that aldosterone acutely activates the Na+/K+/2Cl- cotransporter to enhance Na+ influx and stimulate the Na+-K+ pump. In preliminary experiments we recently examined the direct effect of aldosterone on pump activity. When Na+ influx in response to aldosterone is blocked by the addition of the cotransporter inhibitor bumetanide, a direct inhibitory effect of aldosterone on pump activity can be shown (3A ). We have also recently reported that chronic treatment of rabbits with aldosterone similarly induces a decrease in cardiac Na+-K+ pump function (4).

Phosphorylation has been reported to either activate or inhibit cotransporter and pump activity, with studies showing that both cotransporter (see Ref.5 for review) and pump (see Ref.6 for review) are phosphorylated by mechanisms involving protein kinase C (PKC) in a variety of systems. Isozymes of PKC differ in their involvement in many cellular functions, and isozyme-specific stimulation or inhibition of PKC may be important therapeutic targets (7, 8). In noncardiac tissue, {delta}PKC has been shown to regulate cotransporter activity during {alpha}-adrenergic stimulation (9, 10), whereas there are no reports of specific PKC isozyme regulation of the cardiac Na+/K+/2Cl- cotransporter. Previous studies from this laboratory have shown that {epsilon}PKC regulates cardiac sarcolemmal Na+-K+ pump activity during captopril treatment (11). Similarly, a regulatory role for {epsilon}- and {delta}PKC in the dopaminergic stimulation of pump activity in the lung has recently been reported (12), confirming that PKC isozymes may mediate distinct functions in different cells.

Aldosterone has been reported to activate PKC in leukocytes (13), gut (14, 15), and a renal cell line (16) and decrease basal and phorbol-12-myristate-13-acetate-stimulated PKC activity in rat neonatal cardiomyocytes (17). Isozyme-specific activation has been explored in only one study (13) in vascular smooth muscle cells in which aldosterone (100 nM) stimulated translocation of {alpha}PKC. The aims of the present study were therefore to examine whether the effects of aldosterone on Na+/K+/2Cl- cotransporter and Na+-K+ pump activity are mediated by PKC and if so to determine the isoform(s) involved by the use of isoform-specific peptide blockers and activators. Second, because both the rapid direct in vitro effect and the chronic (7 d) in vivo effect of aldosterone to reduce cardiac myocyte Na+-K+ pump activity are similar, we have explored these effects in terms of PKC dependence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
A total of 169 male New Zealand White rabbits (2.5–3.0 kg, age 12–14 wk) were used for the study. For the in vitro studies, control rabbits receiving normal rabbit chow and water were used, and for the in vivo experiments, rabbits received either ethanol-vehicle alone (control) or aldosterone (50 µg/kg body weight·d) for 7 d by implanted osmotic minipumps, as previously described (4). In a subset of rabbits, {alpha}-D-tocopherol, the more active and natural form of vitamin E (500 IU) or vitamin C (500 mg) were given as once-daily oral doses and coadministered with aldosterone. The doses selected are comparable with those used in previous studies (18, 19). The Joint Royal North Shore Hospital/University of Technology of Sydney Animal Care and Ethics Committee approved the experimental protocols.

Isolation of single ventricular myocytes
Rabbits were anesthetized with im ketamine (50 mg/kg) and xylazine hydrochloride (20 mg/kg) and the heart excised after deep anesthesia was achieved. Single myocytes from either ventricle were isolated by modification of previously described methods (3). Briefly, hearts were rapidly removed and rinsed in ice-cold (2–4 C) Ca2+-free Krebs-Henseleit buffer (KHB) solution containing (in mM) 130 NaCl, 2.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 20.0 taurine, 25.0 NaHCO3, and 11.1 glucose, gassed with 95% O2-5% CO2 to achieve a pH of 7.4 at 35 C. The aorta was secured onto a glass cannula of a Langendorff perfusion column. Ice-cold Ca2+-free KHB solution was retrogradely perfused through the aorta and coronary arteries for 2–3 min in a nonrecirculating manner at a constant rate (20–30 ml/min). The perfusate was then switched to warm (35 C) KHB supplemented with 5 µM Ca2+ for an additional 5 min and then changed to oxygenated Ca2+-free KHB supplemented with collagenase (1 mg/ml; type II, Worthington Biochemical, Freehold, NJ) and hyaluronidase (0.8 mg/ml, Sigma Chemicals, St. Louis, MO). The digestion procedure was similar to that previously described (3) except that the there was no BSA in the Ca2+-free KHB used to mince the ventricular tissue. After the third wash, the supernatant was aspirated, and the cellular pellet resuspended in Ca2+-supplemented KHB. Myocytes were left to settle for 10 min at room temperature (22 C) and were then stored in modified Tyrode solution containing gentamicin (16 mg/liter).

Measurement of Na+-K+ pump current
We used the whole-cell patch clamp technique to measure Na+-K+ pump current (Ip), arising from the 3Na+:2K+ exchange. This technique allows control of the concentration of the pump’s intracellular ligands, provided the compositions of superfusates and pipette filling solutions are designed to block nonpump-mediated ion fluxes. Myocytes were voltage clamped with wide-tipped (tip diameter 4–5 µm) patch pipettes with initial resistances of 0.9–1.1 M{Omega} and series resistance of 1.0–2.0 M{Omega}. The pipette filling solution contained (in mM): 70 K glutamate, 1 KH2PO4, 5 HEPES, 5 ethylene glycol-bis (ß-aminoethyl ether)-EGTA, 2 MgATP, 10 Na glutamate, and 80 tetramethyl-ammonium chloride (TMA-Cl). The solution was titrated with 1 M KOH to pH 7.20 ± 0.01 at 22 C. In a series of experiments, we included PKC isozyme-specific inhibiting and activating peptides in pipette filling solutions. The peptides for {alpha}PKC as well as the novel {epsilon}PKC were unmodified. The inhibitory peptide for {delta}PKC was conjugated to Antennapedia, amino acids 43–58 [RQIKIWFQRRMKKWK] via an N-terminal Cys-Cys bond (20). Peptides were added to pipette solutions on the day of the experiment to achieve a final concentration of 100 nM.

Myocytes were superfused with modified Tyrode’s solution which contained (in mM): 140 NaCl, 5.6 KCl, 2.16 CaCl2, 0.44 NaH2PO4, 10 glucose, 1.0 MgCl2, and 10 HEPES. The solution was titrated with 1 M NaOH to pH of 7.40 ± 0.01 at 35 C. This solution was used while the whole-cell configuration was established and the membrane capacitance measured. The superfusate was then changed to one that was identical except that it was nominally Ca2+ free and contained 0.2 mmol/liter CdCl2 and 2 mmol/liter BaCl2; in a subset of experiments, it also contained 1 µM bumetanide to block Na+/K+/2Cl- cotransport. All solutions were warmed to 35 C. Ip was identified in myocytes voltage clamped at -40 mV as the shift in holding current induced by 100 µmol/liter ouabain. Reported currents are normalized for membrane capacitance and thus cell size. Membrane capacitance was determined by pClamp version 7.0 software (Axon Instruments, Foster City, CA).

Measurement of Na+/K+/2Cl- cotransporter activity
The pipette solution used to identify Na+/K+/2Cl- cotransport activity was identical with that used for Na+-K+ pump studies except that it was Na+ free, with the concentration of TMA-Cl increased from 80 to 90 mM to maintain osmotic balance. The superfusates were identical with those used for Na+-K+ pump studies. When Na+-free pipette solutions are used, ouabain-induced shifts in membrane holding currents are almost eliminated because the intracellular concentration of Na+ is well controlled by the perfusing pipette solution (21). In contrast, when Na+/K+/2Cl- cotransport is activated (in the absence of bumetanide), the intracellular Na+ concentration cannot be controlled, and transmembrane Na+ influx causes an increase in intracellular Na+ concentration as indicated by a large increase in ouabain-sensitive Na+-K+ pump current. This bumetanide-sensitive increase can thus be used to specify aldosterone-induced activation of the Na+/K+/2Cl- cotransporter (3).

Reagents and chemicals
Aldosterone, bumetanide, potassium canrenoate, ouabain, and actinomycin D were purchased from Sigma. TMA-Cl was purum grade from Sigma-Aldrich (Sydney, Australia), and other chemicals analytical grade from BDH Laboratory Supplies (Melbourne, Australia). Staurosporine and bisindolylmaleimide I were purchased from Calbiochem-Novabiochem (Victoria, Australia). PKC isozyme specific-inhibiting and -activating peptides were purchased from the Stanford Protein and Nucleic Acid Facility (Stanford University, Palo Alto, CA).

Statistical analysis
Results are expressed as means ± SEM. Statistical comparisons were by unpaired t test, and one-way ANOVA followed by Tukey’s test, with statistical significance set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Na+/K+/2Cl- cotransporter studies
In our previous studies, we showed cotransporter activity was stimulated when cardiac myocytes were exposed to 10 nM aldosterone for 20–60 min (3). In the present study, we show that aldosterone regulates cotransporter activity within 13–22 min of exposure. Figure 1Go shows that when patch pipette-filling solutions are Na+ free, there is only a small ouabain-sensitive pump current in control myocytes, indicating that intracellular [Na+] is well controlled by the perfusing pipette solution. In contrast, brief exposure to 10 nM aldosterone, measured under Ca2+-free conditions, activates cotransporter activity, which causes an increase in intracellular [Na+], indicated by a large increase in ouabain-sensitive pump current. This effect appears to be mediated via the classical mineralocorticoid receptor, in that addition of 1 µM K+-canrenoate to the superfusate completely blocked the aldosterone-induced increased cotransporter activity over the same time period (Fig. 1Go).



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  		FIG. 1. Mineralocorticoid receptor blockade and PKC inhibition during aldosterone-induced increased Na+/K+/2Cl- cotransporter activity. Pipette solutions were Na+ free, and experimental conditions were designed to reduce nonpump transsarcolemmal Na+ fluxes. Na+-K+ Ip recorded when using Na+-free patch pipette filling solutions gives an indication of the degree of control of intracellular Na+ that can be achieved. Bars indicate mean Ip of myocytes superfused with control solutions (control) or solutions containing 10 nM aldosterone (Ald) alone or aldosterone combined with 1 µM K-canrenoate (K-can) for 13–22 min. In separate experiments, aldosterone-superfused myoyctes were exposed to 10 nM staurosporine ([Stauro]pip) or 0.5 µM bisindolylmaleimide ([Bis I]pip) added to pipette solutions. Numbers in parentheses indicate the number of myocytes studied. Asterisk indicates significant difference between Ip of myocytes exposed to aldosterone and Ip of control myocytes.

 
Aldosterone-induced increased cotransporter activity is also reversed by addition of the relatively nonspecific kinase blocker staurosporine or the more selective blocker bisindolylmaleimide I (Fig. 1Go), suggesting that PKC is involved in mediating regulation of cotransport activity by aldosterone. To exclude a nonspecific effect of the PKC inhibitors, we measured Ip in the absence of aldosterone in an independent series of control experiments. Myocytes were voltage clamped at -40 mV with patch pipettes containing 10 mM Na+ and either 10 nM staurosporine or 0.5 µM bisindolylmaleimide I. Mean Ip was 0.29 ± 0.03 pA/pF (n = 5) and 0.32 ± 0.02 pA/pF (n = 5), respectively, indistinguishable from the mean Ip of 0.32 ± 0.01 pA/pF (n = 6) measured under identical experimental conditions except that the myocytes were not exposed to PKC inhibitors.

Although these pharmacological inhibitors identify a PKC-mediated mechanism, both are isozyme-nonselective PKC inhibitors. We have therefore examined the effect of blocking peptides for the classical PKC {alpha}-isozyme as well as the novel {epsilon}- and {delta}-isozymes in terms of the acute effect of aldosterone on cardiac myocyte cotransporter activity. Our experimental conditions would favor a role for the novel, Ca2+-insensitive {epsilon}- and/or {delta}-isozymes, in that myocytes are exposed to nominally Ca2+-free superfusates and pipette solutions contained EGTA; however, aldosterone-induced translocation of the classical PKC {alpha}-isozyme (13) as well as increases in intracellular Ca2+ (22, 23, 24) has been reported. Therefore, we used a blocking peptide for the classical {alpha}-isozyme that has been reported to inhibit translocation and function of classical PKC isozymes (25). Figure 2Go shows the effect of blockade of {alpha}-, {epsilon}-, and {delta}-PKC on aldosterone- induced increased cotransport activity. We patch-clamped myocytes using Na+-free pipette filling solutions that included 100 nM {epsilon}PKC blocking peptide [{epsilon}V1–2, amino acids 14–21 (EAVSLKPT), {epsilon}PKC] (26). After the whole-cell configuration was achieved, myocytes were exposed to aldosterone and Ip measured. The {epsilon}PKC blocking peptide reversed the acute (13–16 min) and therefore presumably nongenomic effect of aldosterone on cotransporter activity. In contrast, the {alpha}PKC blocking peptide (100 nM, SLNPQWNET, modeled on ß peptide, ßC2–4) (27) and {delta}PKC blocking peptide [100 nM, {delta}V1–1, amino acids 8–17 (SFNSYELGSL), {delta}PKC, 20] showed no equivalent effect.



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  		FIG. 2. Blockade of {alpha}-, {epsilon}-, and {delta}-PKC during aldosterone-induced increased Na+/K+/2Cl- cotransporter activity. Pipette solutions were Na+ free, and experimental conditions were designed to reduce nonpump transsarcolemmal Na+ fluxes so that activity of the Na+-K+ pump was used to indicate the presence of intracellular Na+. Myocytes were superfused with solutions containing 10 nM aldosterone (Ald) for 10–15 min and 100 nM specific inhibitor peptides for {alpha}- ({alpha}PKCinh), {epsilon}- ({epsilon}V1–2), and {delta}- ({delta}V1–1) PKC were included in pipette solutions. Numbers in parentheses indicate the number of myocytes studied. Asterisks indicate significant difference between mean Ip of control myocytes and Ip of aldosterone-superfused myocytes (ANOVA).

 
To examine whether the effect of aldosterone on cotransporter activity could be reproduced by directly activating {epsilon}PKC, we patch-clamped myocytes using Na+-free pipette solutions that contained 100 nM of the {epsilon}PKC selective agonist peptide [{psi}{epsilon}RACK, HDAPIGYD, {epsilon}PKC (85–92)], (28). As shown in Fig. 3Go, myocytes exposed to {epsilon}PKC agonist peptide, {psi}{epsilon}RACK, in the absence of aldosterone showed Ip values significantly higher than those of control myocytes. Further evidence that this effect is via cotransporter activation is shown by the action of bumetanide. In myocytes patch-clamped with pipettes containing {psi}{epsilon}RACK, in the presence of bumetanide (Fig. 3Go), the {epsilon}PKC agonist-induced activation of cotransporter activity was completely abolished. When myocytes were patch-clamped with pipette solutions containing {psi}{epsilon}RACK and simultaneously exposed to aldosterone, no synergistic effect was seen. The effect in the presence of both {psi}{epsilon}RACK and aldosterone was modestly but significantly lower than that seen in myocytes exposed to {psi}{epsilon}RACK alone (Fig. 3Go).



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  		FIG. 3. Activation of {epsilon}PKC and cotransporter activity. Pipette solutions were Na+ free, and experimental conditions were designed to reduce nonpump transsarcolemmal Na+ fluxes, and activity of the Na+-K+ pump was used to indicate the presence of intracellular Na+. Myocytes were exposed to 100 nM specific activating peptide for {epsilon}PKC ({psi}{epsilon}RACK) included in pipette solutions alone or during superfusion with solutions containing 1 µM bumetanide (Bumet) or 10 nM aldosterone (Ald) for 10–15 min. Numbers in parentheses indicate the number of myocytes studied. Asterisks indicate significant difference between mean Ip of control myocytes and Ip of myocytes exposed to {psi}{epsilon}RACK (ANOVA).

 
Na+-K+ pump: acute effects
The studies shown in Fig. 4Go provide evidence that the acute direct effect of aldosterone on cardiac myocyte Na+-K+ pump activity is also mediated by PKC and specifically by the PKC {epsilon}-isozyme. When pipette solutions contain 10 mM [Na+], control values of pump current are 0.32 ± 0.01, and bumetanide alone has no effect on pump activity. With the Na+/K+/2Cl- cotransporter blocked by bumetanide; however, aldosterone rapidly (10–15 min) lowers pump activity, evidence for its modest, direct negative regulatory effect. To confirm that this effect was nongenomic, the transcription inhibitor, actinomycin D (1 µM) was included in pipette solutions and perfused into cells for 5 min after the whole-cell configuration had been established; cardiomyocytes were then superfused with or without 10 nM aldosterone and pump activity recorded. Actinomycin D did not affect basal pump activity (mean Ip 0.33 ± 0.01 pA/pF, n = 5) or reverse the negative effect of aldosterone (0.17 ± 0.02 pA/pF, n = 5), confirming the nongenomic mechanism of action of aldosterone in regulating pump activity.



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  		FIG. 4. Acute effect of aldosterone on Na+-K+ pump activity. Pipette solutions contained 10 mM Na+ and superfusates contained 1 µM bumetanide to block cotransport. Myocytes were superfused with control solutions or solutions containing 10 nM aldosterone (Ald) for 10–15 min; actinomycin D (Act-D, 1 µM) or 100 nM specific inhibitor peptides for {alpha}PKC ({alpha}PKCinh), {epsilon}PKC ({epsilon}V1–2), and {delta}PKC ({delta}V1–1) were included in pipette solutions. Numbers in parentheses indicate the number of myocytes studied. An asterisk indicates significant differences (ANOVA).

 
That this effect can be mimicked by the {epsilon}PKC agonist, {psi}{epsilon}RACK, is also shown in Fig. 4Go, with no significant further lowering in the presence of both aldosterone and {psi}{epsilon}RACK. In addition, we explored the specificity of the aldosterone-induced response by studies with a {epsilon}PKC inhibitory peptide, {epsilon}V1–2. This peptide, as shown in Fig. 4Go, has no effect when administered alone in the presence of bumetanide; it does, however, completely block the negative regulatory effect of aldosterone on pump activity. To exclude the possibility of a nonspecific effect of the {epsilon}PKC blocking peptide in abolishing the aldosterone-induced decrease in Ip, we patch-clamped myocytes using pipette solution containing 100 nM scrambled {epsilon}PKC blocking peptide (LSETKPAV) and exposed them to aldosterone before measuring Ip. Mean levels of Ip were similar to levels of Ip for myocytes exposed to aldosterone alone in the presence of bumetanide, further evidence that the acute direct inhibitory effect of aldosterone on cardiac myocyte pump activity, like its stimulatory effect on cotransporter function, is mediated by {epsilon}PKC.

Na+-K+ pump: chronic effects
In our previous studies, rabbits infused with aldosterone at a rate of 50 µg/kg body weight·d showed plasma levels of 1.5–2 nM, approximately three times the normal levels for rabbits on the customary low sodium laboratory diet. After 7 d of such infusion, no residual effects on the Na+/K+/2Cl- cotransporter can be seen on the basis of ion-sensitive microelectrode studies (4). In contrast, the inhibitory effect of aldosterone on the Na+-K+ pump appears to persist (4). It is well established that PKC regulates pump activity (see Ref.6 for review) with stimulation, inhibition, and absence of effect reported for different tissues. In the heart, previous studies from this laboratory have shown an PKC-induced decrease in pump activity (11), and it appears likely that this chronic effect of aldosterone might also involve PKC.

Before the specific PKC peptides were available, we used the combined antioxidant/PKC inhibitor, vitamin E, to examine whether the chronic effect of aldosterone on pump activity is PKC mediated. Vitamin E, but not other antioxidants, has marked effects on preventing activation of PKC (29). As shown in Fig. 5Go, the inhibitory effect of aldosterone on pump activity was abrogated by the combined antioxidant/PKC inhibitor vitamin E. When vitamin C, an antioxidant without PKC inhibitory activity, was used as an antioxidant control, there was no reversal of the aldosterone effect.



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  		FIG. 5. Chronic effect of aldosterone on Na+-K+ pump activity. Pipette solutions contained 10 mM Na+. Bars indicate mean Ip of myocytes isolated from control rabbits and rabbits treated with either aldosterone alone (Ald) or in combination with vitamin E (500 IU, Ald+Vit E) or vitamin C (500 mg, Ald+Vit C) for 7 d. Rabbit number in each group is indicated by N and cell number by parentheses. Asterisks indicate significant difference in mean Ip in cells from control and treated rabbits.

 
In subsequent studies, as shown in Fig. 6Go, we used the specific PKC peptides to further explore the direct inhibitory effect of aldosterone on the Na+-K+ pump. Myocytes prepared (over a time course of ~4 h) from chronically (7 d) aldosterone-infused rabbits show the characteristic response to aldosterone, a substantial decrease in activity, compared with cardiac myocytes from noninfused rabbits. If, however, myocytes from aldosterone-infused rabbits are exposed to the {epsilon}PKC blocking peptide, {epsilon}V1–2, in the pipette for 15 min, complete reversal of the effect of chronic aldosterone exposure is seen. As shown in Fig. 6Go, these data are selective in that the effect of the {epsilon}PKC blocking peptide, {epsilon}V1–2, is not reproduced by the {alpha}PKC blocking peptide or by the scrambled {epsilon}PKC blocking peptide. Taken together, these data show that the acute and chronic effects of aldosterone to lower pump activity are both PKC dependent, specifically via the PKC{epsilon} isoform and that such a chronic effect can be rapidly reversed by {epsilon}PKC antagonism.



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  		FIG. 6. Aldosterone-induced pump inhibition and {epsilon}PKC. Bars indicate mean Ip from myocytes isolated from control (control) or aldosterone-treated rabbits (Ald). Pipette filling solutions contained 10 mM Na+ and were either peptide free or included 100 nM inhibitory peptides for {epsilon}PKC ({epsilon}V1–2), scrambled {epsilon}PKC ({epsilon}V1–2-scr), or {alpha}PKC. Rabbit number in each group is indicated by N and cell number by parentheses. An asterisk indicates a significant difference, compared with mean Ip of myocytes isolated from rabbits not treated with aldosterone.

 
We previously reported that aldosterone induces a decrease in Na+-K+ pump activity by decreasing the Na+-K+ pump’s apparent affinity for intracellular Na+ rather than a change in maximal pump capacity (4). Previous reports suggest that sodium binds to the pump at three intracellular sites, two near the cytosolic surface and the third inside the membrane dielectric. Na+ binding at the cytosolic sites competes with K+, and because of the location of these sites, this binding is independent of membrane voltage. In contrast, binding at the third site is highly selective for Na+ and voltage dependent (30, 31, 32). To determine whether there is an effect of chronic aldosterone infusion at the cytosolic binding sites, we measured the current-voltage (Ip-Vm) relationship for myocytes from rabbits infused with aldosterone and controls. Details of pipette solutions and superfusates designed to eliminate time- and voltage-dependent nonpump currents, and the voltage clamp protocol, have been reported previously from this laboratory (33). Figure 7Go shows a summary of Ip-Vm relationships of myocytes from control- and aldosterone-treated rabbits. To facilitate comparison of slopes, the Ip-Vm relationships have been normalized to the Ip recorded at 0 mV (34). There was no significant difference between the curves (two-way ANOVA), evidence for an effect of aldosterone at the cytosolic sites rather than inside the membrane dielectric site.



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  		FIG. 7. Summary of Ip-Vm relationships for seven cardiac myocytes from control rabbits (open circles) and nine cardiac myocytes from rabbits treated with aldosterone (filled circles). To facilitate comparison of slopes, the Ip-Vm relationships have been normalized to the Ip recorded at 0 mV.

 
To confirm that aldosterone regulates the sensitivity of the pump for Na+ near the cytoplasmic surface, we examined the K+/Na+ antagonism relationship (6). Details of the composition of solutions and protocols have been previously described by studies from this laboratory, which demonstrated [K]pip-dependent changes in sarcolemmal pump activity by in vivo treatment with captopril (35) or cyclosporine (36). We measured Ip with K+-free pipette solutions in myocytes isolated from rabbits infused with aldosterone and found a mean Ip of 0.90 ± 0.07 pA/pF (n = 10), similar to the level in myocytes isolated from control rabbits (0.91 ± 0.07 pA/pF, n = 9). However, when we used a [K+]pip of 70 mM, the mean Ip in myocytes isolated from aldosterone-infused rabbits (0.21 ± 0.07 pA/pF, n = 10) was significantly lower than that from myocytes isolated from control rabbits (0.32 ± 0.02 pA/pF, n = 6). These results similarly suggest that aldosterone regulates the sensitivity of the pump for Na+ at sites near the cytoplasmic surface by a mechanism of action involving an increased antagonistic effect of K+ on Na+ binding.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies show that both the Na+/K+/2Cl- cotransporter and the Na+-K+ pump, important determinants of intracellular ionic status in the cardiomyocyte, are acutely regulated by aldosterone. Second, they show that aldosterone-induced up-regulation of cotransporter activity and direct down-regulation effect on pump activity are both mediated by {epsilon}PKC, in that the effect of aldosterone can be mimicked by {epsilon}PKC agonists and blocked by {epsilon}PKC antagonists. Third, the studies on in vivo administration of aldosterone for 7 d, followed by patch clamp studies after a 4-h period of myocyte isolation and equilibration, provide strong evidence for possible chronic nongenomic effects of aldosterone, again mediated via {epsilon}PKC.

The acute nongenomic effects of aldosterone have been very clearly shown for a variety of tissues in a series of studies from the Wehling laboratory (see Ref.37 for review). In such studies the effects of aldosterone can generally be mimicked by 9{alpha}-fluorocortisol and deoxycorticosterone, classical mineralocorticoid agonists, but not by cortisol; in addition and in contrast to genomic effects of aldosterone on in vivo renal Na+ handling, for example, they are not acutely blocked by spironolactone. On this basis these rapid, nongenomic effects of aldosterone have been ascribed to a membrane receptor, distinct from the classical mineralocorticoid receptor involved, for example, in renal Na+ retention.

Despite more than a decade’s search for an alternative receptor for aldosterone, this has not been rewarding, and recently evidence has been presented that clearly implicates the classical mineralocorticoid receptor in at least some rapid nongenomic effects of aldosterone (2). Human vascular smooth muscle cells (VSMCs) express both 11ß-hydroxysteroid dehydrogenase (11ßHSD)1 and 11ßHSD2, the enzyme that acts to metabolize cortisol to receptor-inactive cortisone. In studies on human VSMCs, Alzamora et al. (2) confirmed the rapid effects of aldosterone on Na+/H+ exchanger activity, shown by elevation of intracellular pH and blocked by amiloride or its derivative, 5-(N-ethyl-N-isopropyl)amiloride. The effects of aldosterone were neither mimicked by cortisol nor blocked by spironolactone, consistent with an action via a nonclassical membrane receptor. When, however, the authors added carbenoxolone, to block the enzymatic action of 11ßHSD, cortisol equivalently elevated intracellular pH; second, although spironolactone was ineffective, the acute effects of aldosterone were completely blocked by the water soluble, open E-ring mineralocorticoid receptor antagonist RU28318.

Although cardiac myocytes do not express substantial levels of 11ßHSD2 [cardiac levels are less than 1% of those in kidney, in which expression is confined to distal elements (38)], we have sufficient evidence for a working hypothesis of a similar rapid nongenomic aldosterone effect via classical mineralocorticoid receptors in our studies. This includes the lack of effect of actinomycin D in blocking the rapid effect of aldosterone on pump activity and the effect of spironolactone in chronic, in vivo studies, which blocks the effect of coadministered aldosterone on pump activity (4). In contrast, preliminary studies show that acutely, spironolactone is inactive in blocking the rapid effects of aldosterone, similar to the study by Alzamora et al. (2); the open-ring, water-soluble mineralocorticoid antagonist potassium canrenoate, however, completely blocked the rapid aldosterone effect in the present study and previously (3).

The present studies thus begin to map the pathway of action of aldosterone activating classical mineralocorticoid receptors to produce rapid effects on cotransporter and pump activity. In each instance the action of {epsilon}PKC appears crucially involved; whether either or both cotransporter or pump are direct substrates for {epsilon}PKC or whether additional steps are interposed is not addressed by the current studies. There are, however, previous reports that PKC directly regulates Na+-K+ pump activity (39, 40, 41, 42), which tend to support the former hypothesis; in addition, a volume-sensitive, staurosporine-inhibitable kinase has been reported to directly regulate Na+/K+/2Cl- cotransporter activity (43).

Na+-K+ pump activity is the major determinant of cytosolic levels of Na+, and sustained aldosterone-induced pump inhibition will interfere with a variety of cell functions by changing the Na+ and K+ gradients across the plasma membrane. The energy derived from activation of the Na+-K+ pump drives secondary active transmembrane transport of other ions and a variety of organic compounds important for the regulation of cell volume, metabolism, and excitation-contraction coupling. PKC-mediated reduction in pump activity has been reported to involve either internalization of Na+-K+ pump units (40) or changes in the apparent affinity of the pump for Na+ (6). The results of the present study favor the latter mechanism, that the sustained aldosterone-induced reduction in pump activity mediated by {epsilon}PKC involves changes in the apparent affinity of the pump for Na+.

The pathophysiological effects of aldosterone on the heart are crucially salt dependent. Experimentally, rats infused with aldosterone but on a low-salt diet do not show the cardiac hypertrophy and fibrosis seen in animals similarly infused but with 0.9% NaCl solution to drink (44). Relatively small (4%) increments in ambient [Na+] in vitro have been shown to produce very much amplified effects on protein synthesis in VSMCs and cardiac myofibroblasts from neonatal rats, of the order of 50% increases (45). It remains unclear how the elevated Na+ intake synergizes with an inappropriate level of aldosterone to produce the pathophysiological effects it does. Aldosterone-induced pump inhibition might amplify these pathophysiological effects, in that increased intracellular Na+ in cardiac myocytes has been shown to induce translocation of {epsilon}PKC (46). In addition, overexpression of {epsilon}PKC (47) or the novel {epsilon}PKC-specific translocation enhancer peptide {psi}{epsilon}RACK (8) produces hypertrophy in transgenic mice, and activation of {epsilon}PKC has been shown to induce activation of transcription factors activator protein-1 and nuclear factor-{kappa}B in adult rabbit cardiac myocytes (48).

Perhaps the most challenging of the present findings is that the rapid (within 15 min and thus presumably nongenomic) effect of aldosterone on the pump is maintained over 7 d and persists in the absence of aldosterone over the time of cell preparation. In addition, this rapid, maintained, and persistent effect can in turn be rapidly reversed by {epsilon}PKC antagonist administration. As previously noted, these effects of aldosterone have been shown to be blocked by the classical mineralocorticoid receptor antagonist spironolactone in vivo, thus strongly suggesting an aldosterone effect via the classical mineralocorticoid receptor. This provides additional evidence for maintained or chronic nongenomic effects: to date, aldosterone effects have generally been considered as acute nongenomic vs. more persistent genomic actions, a distinction that may need to be refined in light of the recent findings.

Second, and perhaps equally important, the prompt reversal by the PKC antagonist of the chronic effects of aldosterone suggests that the default position, in mechanistic terms, is that of the system once stimulated being on, given the 4-h washout period between killing and addition of the PKC antagonist. Although the processes of preparing isolated myocytes and equilibration may inevitably introduce artifact, the positive result found with the addition of the {epsilon}PKC antagonist is most easily reconciled with a maintained nongenomic effect of aldosterone over the period of isolation, despite the clearly rapid washout of bound and tissue-free aldosterone of adrenal origin under such conditions. Although there have been a series of reports on cardiac biosynthesis of aldosterone, this has varied between species and strains (49) and appears much more likely in heart failure than in other circumstances (50). It remains a formal but distant possibility to explain the apparently persistent action of aldosterone in these circumstances.

In conclusion, the directionality and time course of the cardiac nongenomic effects of aldosterone need to be considered. These include rapid but not continuing activation of the Na+/K+/2Cl- cotransporter and rapid and persistent direct inhibition of Na+-K+ pump activity, in both instances {epsilon}PKC mediated. The way in which these effects of aldosterone, acute and chronic, contribute to cardiac pathophysiology, and the extent to which the consequent changes in intracellular [Na+] are involved in cardiac hypertrophy and fibrosis, remain to be determined.


   Footnotes
 
This study was supported by the North Shore Heart Research Foundation and a grant from Pharmacia.

Abbreviations: 11ßHSD, 11ß-Hydroxysteroid dehydrogenase; Ip, pump current; Ip-Vm, current-voltage; KHB, Krebs-Henseleit buffer; PKC, protein kinase C; TMA-Cl, tetramethyl-ammonium chloride; VSMC, vascular smooth muscle cell.

Received August 29, 2003.

Accepted for publication October 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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