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Laboratory of Cellular and Molecular Physiology, Faculty of Medicine, University of Los Andes, Las Condes 6782468, Santiago, Chile
Address all correspondence and requests for reprints to: Luis Michea, M.D., Ph.D., Laboratory of Cellular and Molecular Physiology, Faculty of Medicine, University of Los Andes, San Carlos Apoquindo 2200, Las Condes 6782468, Santiago, Chile. E-mail: lmichea{at}uandes.cl.
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Abstract |
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Introduction |
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Na+,K+-ATPase regulation in cardiovascular muscle is particularly critical as an indirect regulator of contractility, and several studies have been carried out on the long-term effects of aldosterone on the Na+-K+ pump (11, 16, 17, 18, 19). For example, we have shown that rat vascular Na+,K+-ATPase activity and its 
2-subunit were reduced to half the control values after adrenalectomy (11). Deoxycorticosterone administration restored activity and subunit expression levels, suggesting mineralocorticoid regulation of the Na+ pump (11). Whether aldosterone has rapid (nongenomic) effects on Na+,K+-ATPase in cardiovascular tissue is less certain. Rapid activation of the cardiac Na+ pump and Na+,K+-2Cl- cotransporter by aldosterone has been observed in voltage-clamped ventricular myocytes (20). Recently, it has been reported that hyperaldosteronemia in rabbits inhibits the cardiac sarcolemmal Na+-K+ pump (21).
We and others have described rapid effects of aldosterone on the Na+-H+ exchanger activity of fetal and adult human arteries (22), vascular smooth muscle cells, and human mononuclear leukocytes (23, 24). Inhibitors of transcription or translation do not affect rapid aldosterone action, demonstrating that aldosterone has nongenomic effects mediated by additional signal transduction pathways. Short-term aldosterone effects also include intracellular Ca2+ increase, inositol trisphosphate turnover and protein kinase C (PKC) activation (24, 25, 26). To date, there is no evidence of a direct rapid effect of aldosterone on the Na+,K+-ATPase in blood vessels.
We investigated whether aldosterone could have a rapid action on vascular Na+,K+-ATPase. An effect on the Na+-K+ pump, with no latency, is of interest because it would imply a new nongenomic action of aldosterone affecting ionic homeostasis in vascular tissue. Therefore, we examined early effects of aldosterone on Na+,K+-ATPase function of aortic rings by measuring ouabain-sensitive 86Rb/K uptake.
We show here that aldosterone has a rapid effect on the Na+-K+ pump in rat arteries: an early inhibition of ouabain-sensitive 86Rb/K uptake, followed by a return to control values after 120 min of continuous stimulation. The inhibitory effect of aldosterone on the Na pump is blocked by PKC inhibitors and occurs through a mechanism involving the microtubule system. Removal of catalytic units from the plasma membrane by short-term aldosterone action could account for the inhibition of the Na pump. Interestingly, eplerenone, a new mineralocorticoid antagonist, blocked the nongenomic aldosterone effect on Na+,K+-ATPase of the vascular tissue.
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Materials and Methods |
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Drug treatments
The effect of aldosterone (10 nM) on ouabain-sensitive 86Rb/K uptake was measured in the presence of actinomycin D (5 µM), cycloheximide (20 µM), monensin (2.5 µM), bisindolylmaleimide I (120 nM), rapamycin (15 µM), or colchicine (100 µM) as indicated in the figures. Drugs were prepared as stock solutions and diluted in KRB to the desired concentration before use. Aldosterone was dissolved in methanol (final concentration, 0.01%). Actinomycin D was prepared as a 800-µM solution in water and was diluted to 5 µM in KRB. Cycloheximide was dissolved in methanol (final concentration in KRB, 0.09%). Monensin was dissolved in ethanol (final concentration in KRB, 0.005%). Bisindolylmaleimide I (bisindole I) was dissolved in dimethylsulfoxide (final concentration in KRB, 0.005%). Rapamycin was dissolved in dimethylsulfoxide (final concentration in KRB, 0.01%). Colchine was dissolved in water. Identical aliquots of solvents used for the drugs were added to the control and hormone-treated paired samples in each experiment. Eplerenone was provided by Amersham Pharmacia Biotech (Piscataway, NJ). Bisindolylmaleimide I was obtained from Calbiochem (La Jolla, CA), and the other reagents were purchased from Sigma-Aldrich (St. Louis, MO). 86RbCl was obtained from the Chilean Commission of Nuclear Energy.
Na+,K+-ATPase function in aortic rings
Na pump activity was determined by measuring ouabain-sensitive 86Rb/K uptake in aortic rings as described previously (27). Briefly, after the aorta was removed and cleaned, rings of 4 mm were incubated in separate vials for 30 min at 37 C in 2 ml KRB constantly gassed with 95% O2-5% CO2. Thereafter, the aortic rings were preincubated for 30 min in KRB in the presence or absence of 1 mM ouabain. Finally, the rings were incubated for different periods of time in the presence of 10 nM aldosterone and/or the respective drug, as indicated in Results. The activity of the Na pump was measured during the last 10 min of hormone action in KRB containing 86Rb (0.1 µCi/ml). The reaction was stopped by transferring the rings into ice-cold KRB; the tissues were then quickly washed in cold buffer and gently blotted. The radioactivity of the samples was measured by Cerenkov radiation in a liquid scintillation counter in the presence of 0.1% Tween 20 (28).
Plasma membrane protein biotinylation and Western blotting of biotinylated Na pump catalytic subunits
Thoracic aorta segments were carefully excised, washed in KRB (37 C, 95% O2-5% CO2), and treated for various periods (10–120 min) with 10 nM aldosterone or vehicle in KRB, as described for the Na+,K+-ATPase function experiments. Plasma membrane proteins were covalently labeled with EZ-link sulfo-NHS-biotin (0.5 mg/ml in KRB, 5 ml solution/two thoracic aortas; Pierce Chemical Co., Rockford, IL) by quick immersion of aortic rings in KRB at 4 C with or without aldosterone for 2 h on a roller system. After labeling, the rings were washed three times to remove unreacted sulfo-NHS-biotin with washing solution [120 mM KCl, 1.2 mM MgCl2, 2.5 mM EGTA, and 50 mM Tris-HCl (pH 7.4); 2 min each time in 4 C, ice-cold buffer].
To prepare crude membrane fractions, tissues were homogenized (four strokes, 12 sec at maximum speed each, in a Polytron, Kinematica, Luzern, Switzerland) in ice-cold buffer containing 20 mM sucrose, 2.5 mM MgCl2, 1.2 mM EGTA, 50 mM Tris-HCl (pH 7.4), and Complete Mini protease inhibitor cocktail minitablets (one tablet per 10 ml; Roche, Indianapolis, IN). The homogenate was centrifuged at 3,000 x g for 10 min (4 C), and the supernatant was centrifuged at 150,000 x g for 90 min (4 C). Crude membrane fractions were carefully solubilized in the presence of solubilization buffer [120 mM NaCl, 2.5 mM MgCl2, 1.2 mM EGTA, 2 mM EDTA, 0.5% Triton X-100, 0.5% Tween 20, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), and Complete protease inhibitor cocktail (one minitablet per 10 ml; Roche)]. The protein concentration of solubilized membranes was measured (bicinchoninic acid method; Pierce Chemical Co.), and biotinylated proteins were precipitated in the presence of TetraLink tetrameric avidin resin (200 µg solubilized protein/100 µl beads; Promega Corp.), suspended in 1 ml solubilization buffer, and incubated overnight at 4 C in a roller system.
To elute the biotinylated proteins, the beads were decanted, washed four times in solubilization buffer, resuspended in Laemmli buffer, and warmed to 37 C for 3 min. Beads were decanted by centrifugation, and eluted proteins were processed for SDS-PAGE in 10% polyacrylamide gel. Gels were stained with Coomassie Blue and used for densitometry to check for protein abundance in each sample. Equal protein amounts were loaded on new SDS-PAGE gels and processed for Western blotting, as previously described (11). Briefly, after blotting, the polyvinylidene difluoride membranes were blocked with 5% nonfat milk in Tris-buffered saline (20 mM Tris/HCl and 137 mM NaCl) plus 0.1% Tween 20. Separate membranes were incubated with mouse monoclonal anti-1 subunit (provided by Dr. M. J. Caplan) and rabbit polyclonal anti-2-subunit (provided by Dr. T. A. Presley). Blots were developed using the enhanced chemiluminescent method (Chemiluminescence Reagent Plus, Perkin-Elmer, Boston, MA) with horseradish peroxidase-linked antibodies. Films were placed in contact with the membranes in cassettes containing intensifying screens, and four or five plates with different exposure times were used to avoid film saturation. The signal intensity present in each lane was quantified by computer scanning densitometric analysis, comparing the intensity of the aldosterone-treated sample with that of the control vehicle-treated sample. Results are expressed as the relative band intensity compared with that of the control paired sample.
In additional experiments total catalytic subunit abundance was studied. In these experiments aortic segments were incubated in the presence of 10 nM aldosterone or vehicle as described above, and crude membrane fractions were prepared as described previously (11). Equal amounts of proteins were analyzed by SDS-PAGE and Western blot with the anti-1 or anti-2 catalytic subunit antibodies. Catalytic subunit abundance in each parallel sample was also analyzed by computer scanning densitometry.
Statistical analysis
For analysis of the ouabain-sensitive 86Rb/K uptake time course and biotinylation and Western blot studies in the presence or absence of aldosterone, t test was used. For analysis of the effects of drugs, the mean Na+/K+-ATPase activities for the four groups were compared after incubation at 20 min. For each set of experiments, one-way ANOVA was conducted on the raw data, followed by planned comparisons between group means. The = 0.05 level of significance was used for the planned comparisons between the means. The least significant differences method was used for planned comparisons between groups. Data were analyzed using PROC TTEST in the SAS statistical software package (SAS PC, version 6.12, SAS Institute, Inc., Cary, NC). Data are reported as the mean ± SEM.
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Results |
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). Exposure to aldosterone of rat aortic rings revealed a rapid inhibitory effect on ouabain-sensitive 86Rb/K uptake. Maximal inhibition (57.0 ± 2.3% of control values; P < 0.05; n = 8) was observed as early as 20 min of hormone action. However, the aldosterone-induced decrease in Na+,K+-ATPase activity was not sustained. After 120 min of continuous hormone presence in the incubation medium, the activity of the Na+-K+ pump returned to initial basal levels.
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). Cycloheximide or actinomycin D alone had no effect on ouabain-sensitive 86Rb/K uptake or short-term aldosterone inhibition of sodium pump.
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).
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, eplerenone completely blocked the rapid inhibitory aldosterone effect on ouabain-sensitive 86Rb/K uptake. Eplerenone alone (2 µM) had no effect on basal Na+,K+-ATPase activity.
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). Rapamycin alone had no effect on basal ouabain-sensitive 86Rb/K uptake. These results are similar to those observed by Tumlin et al. (30), where rapamycin also inhibited the rapid induction of calcineruin activity by aldosterone.
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Role of microtubules in aldosterone-induced Na+,K+-ATPase inhibition
An intact microtubular system is needed to observe a decrease in pump function by endocytosis of Na pump subunits in epithelia. To determine whether the microtubular system is involved in the short-term effect of aldosterone on Na+,K+-ATPase function, aortic rings were incubated in the presence of colchicine (100 µM). This alkaloid binds to the tubular system and prevents polymerization. As shown in Fig. 5, colchicine completely suppressed the rapid aldosterone inhibition of ouabain-sensitive 86Rb/K uptake. No significant effect of colchicine was observed, however, on basal Na pump activity.
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1 and 2 catalytic isoforms. As shown in Fig. 6, aldosterone induced a 40% decrease in both 1 and 2 isoform abundance in plasma membrane after 10 and 20 min of hormone action (Fig. 6, A–C; P < 0.05; n = 3–4). The membrane protein abundance of both isoforms returned to control values at 120 min (Fig. 6A).
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1 and 2 catalytic isoforms by measuring the effect of colchicine. For this purpose, aortic rings were pretreated with 100 µM colchicine for 20 min, previous to the action of aldosterone (10 nM, 20 min). As shown in Fig. 6C
, colchicine suppressed the aldosterone-mediated decrease in plasma membrane abundance of catalytic subunits.
To analyze possible changes in the total abundance of catalytic subunits (plasma membrane plus intracellular pools), a Western blot of crude membrane fractions obtained from rings incubated with or without aldosterone was performed. We observed no change in total catalytic subunit abundance (Fig. 6D). These results indicate that the observed reduction in catalytic cell surface abundance is not due to a general decrease in total 1 and 2 secondary to increased protein degradation.
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Discussion |
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Inhibitors of transcription or translation did not affect the short-term inhibitory effect of aldosterone on Na+,K+-ATPase. Therefore, this response is considered to be a nongenomic effect. As nongenomic steroid effects become more widely studied, it has been suggested that nongenomic aldosterone effects may be transmitted by a putative membrane receptor and/or by the classic intracellular MR (22, 31, 32, 33). Previously, we demonstrated that rapid Na+-H+ exchanger activation in human vascular tissue by aldosterone was not sensitive to spironolactone. However, aldosterone activation of Na+/H+ exchanger was completely inhibited by the mineralocorticoid antagonist RU28318 (22). Furthermore, cortisol mimicked aldosterone activation of the Na+/H+ exchanger when the enzyme 11ß-hydroxysteroid dehydrogenase, which confers cytosolic MR specificity, was blocked (22). In the present study it was found that the new, highly selective MR antagonist, eplerenone, was able to block the rapid action of aldosterone in vascular Na+,K+-ATPase. The present results suggest that the nongenomic effect of aldosterone on the Na+ pump involves activation of the cytosolic MR. Alternatively, eplerenone could be able to bind to the putative new MR. In addition, it is possible that not all of the nongenomic effects of aldosterone on vascular tissue are mediated by the same mechanism.
It is known that the steroid hormone receptors constitute a heterodimeric 8–9S complex of proteins, which includes the hormone-binding receptor and several other proteins, such as 90-, 70-, and 56-kDa HSP (34, 35). Rapamycin, an immunosuppressive drug that stabilizes the HSP-steroid hormone receptor complex, completely blocked the rapid inhibitory action of aldosterone on the Na+-K+ pump (29, 30). The exact role of HSPs is unknown, but recent studies indicate that 90-kDa HSP facilitates the anchoring of steroid receptors on the cytoskeleton, regulates its subcellular localization, and maintains the hormone-binding receptor in a high affinity conformation (36, 37). Aldosterone activates kidney tubule calcineurin, a nongenomic hormone action that depends on the release of HSPs (30). Rapamycin blocked aldosterone-induced stimulation of calcineurin, whereas 90- or 70-kDa HSP increased calcineurin activity via a transcription-independent path in permeabilized rat cortical ducts (38). Although our results with rapamycin could be interpreted as an argument in favor of MR activation to elicit the nongenomic effect of aldosterone on the Na pump, it is not possible to rule out the eventual interaction of the drug with other cellular targets.
It is known that the intracellular Na concentration is a primary determinant of Na-K pump activity, as the Km values of the enzyme are close to the intracellular Na+ concentration. In the present study we found that the short-term aldosterone-induced inhibition of ouabain-sensitive 86Rb/K uptake was not suppressed by monensin, a Na ionophore that allows Na+ entry from the extracellular space. In contrast with these results, studies performed in voltage-clamped ventricular myocytes have shown increased Na+ influx and Na pump activation during short-term exposure to aldosterone (20). These researchers conclude that in vitro exposure of cardiac myocytes to aldosterone activates the Na+,K+,2Cl- cotransporter to enhance Na+ influx and stimulate the Na pump. Tissue and/or species differences may exist, as in Mihailidou’s work (20) no activation of the Na/H exchanger was detected. Ebata et al. (39) have shown that aldosterone and corticosterone can stimulate Na+ uptake in rat vascular smooth muscle cells by genomic and nongenomic mechanisms activating the Na/H exchanger. More recently, we (22) demonstrated nongenomic Na/H exchanger activation caused by physiological concentrations of aldosterone in human vascular tissue, which should result in a rapid Na influx. Taken together, these observations indicate that the inhibitory effect of aldosterone on the Na pump is not the consequence of an eventual decrease in the intracellular Na concentration.
There are several possibilities to explain the initial inhibitory action on the sodium pump. Microtubules regulate the internalization of Na+-K+ pumps from the plasma membrane, and this could account for the diminished ouabain-sensitive 86Rb/K uptake in aldosterone-treated aorta. Chibalin et al. (40) have shown that inhibition of Na+,K+-ATPase activity by dopamine in renal proximal tubule cells is associated with removal of Na+,K+-ATPase from the plasma membrane secondary to an increased endocytosis of - and ß-subunits. This effect requires PKC activation and a dynamic actin microtubule cytoskeleton (41). Interestingly, the results of the present study indicate that the microtubular system is required to observe the nongenomic inhibition of Na pump function by aldosterone. Also, the biotinylation experiments demonstrated a rapid decrease in the abundance of catalytic subunits present in the plasma membrane after aldosterone addition. These observations, the microtubule involvement, reduced cell surface abundance of catalytic subunits, and no change in the total cell content of 1- and 2-subunits, are consistent with increased endocytosis of the Na pump after short-term aldosterone stimulus.
Previous studies of aldosterone action on blood cells and vascular smooth muscle cells have shown that intracellular signaling for nongenomic aldosterone action involves changes in intracellular Ca2+ and PKC activity (23, 24). More recently, using electrophysiological techniques, Mihailidou (26) demonstrated that inhibition of the Na pump by chronic hyperaldosteronemia in rabbit cardiac myocytes involved PKC
activation. In the present study we found that bisindole I, an inhibitor of PKC, -ß, -
, -
, and - isoforms, blocked the nongenomic aldosterone action in rat aorta. Our results indicate that PKC could mediate this new nongenomic action of aldosterone and are consistent with the view of a key role for PKC in the induction of Na+,K+-ATPase endocytosis from the plasma membrane (42). The PKC-mediated action of dopamine in proximal tubule cells involves PKC and -ß, indicating that although PKC activation could be a general mechanism for the control of Na pump function (43), different isoforms of PKC have a tissue-specific and/or hormone-specific role in mediating Na pump regulation by endocytosis and trafficking. For example, dopamine-induced exocytosis is dependent on activation of PKC and - (44).
In sharp contrast to the effect of short-term exposure to aldosterone, it is known that long-term exposure stimulates Na+-K+ pump activity. Previous studies carried out in our laboratory and others proved that mineralocorticoids regulate the expression of Na+-K+ pump subunits in rat vascular tissue (11) and also in vascular smooth muscle cells (8, 19). However, chronic treatment with aldosterone induced a decrease in the pump current of rabbit myocardiocytes, suggesting tissue-specific effects (21).
Taken together, these data favor the view of a double effect of aldosterone on vascular Na+,K+-ATPase: a nongenomic inhibitory action on the ouabain-sensitive 86Rb/K uptake, followed by a late genomic effect on the de novo synthesis of pumps and their activity. Nongenomic effects were blocked by eplerenone, a new MR antagonist.
The rapid aldosterone action in vascular smooth muscle cells, human arteries, and cardiac myocytes suggests that aldosterone action plays a role in the regulation of cardiovascular function. Na+-K+ pump inhibition may also contribute to vascular remodeling. Several studies have shown that aldosterone can mediate vascular fibrosis of cardiac arterioles and large arteries (12, 13, 45). Furthermore, it has been shown that a nontoxic concentration of ouabain that causes partial inhibition of Na+,K+-ATPase and induces an increase in intracellular Ca2+ led to transcriptional regulation of several early growth-related genes in cardiac myocytes (46, 47). It is tempting to speculate that in addition to the putative roles that the rapid aldosterone action could have in vascular contractility, these effects could be part of the early transduction signal machinery that need to be activated to observe genomic effects in vascular tissue.
The present study provides evidence suggesting that aldosterone directly modulates vascular function by genomic and nongenomic mechanisms. Rapid aldosterone action on the cardiovascular system could form part of a fine-tuning mechanism by which arterial contractility may be regulated up to certain limits by modulating the ionic homeostasis of the vascular smooth muscle cell. The fact that aldosterone also regulates vascular function by nongenomic effects adds a level of complexity to its action in the cardiovascular system.
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Acknowledgments |
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Footnotes |
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Abbreviations: ATPase, Adenosine triphosphatase; HSP, heat shock protein; KRB, Krebs-Ringer buffer; MR, mineralocorticoid receptor; PKC, protein kinase C.
Received September 9, 2002.
Accepted for publication December 23, 2002.
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contributes to regulation of sarcolemmal Na+-K+ pump. Am J Physiol 50:C1059–C1063
-subunit constitutes a triggering signal for Na+, K+-ATPase endocytosis. J Biol Chem 273:8814–8819 and -. Mol Biol Cell 13:1381–1389This article has been cited by other articles:
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) and 
) plasma membrane abundance in the presence of aldosterone (10 nM). Results are expressed as the relative abundance compared with that in the control sample treated with vehicle. Points represent the mean ± SEM of three or four independent experiments. *, P < 0.05. B, Representative Western blot of