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Am J Physiol Heart Circ Physiol 288: H477-H485, 2005. First published September 30, 2004; doi:10.1152/ajpheart.00083.2004
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The {alpha}2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro

Iva Dostanic,1 Richard J. Paul,2 John N. Lorenz,2 Steven Theriault,4 James W. Van Huysse,3,4 and Jerry B. Lingrel1

Departments of 1Molecular Genetics, Biochemistry, and Microbiology and 2Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio; and 3Hypertension Unit, University of Ottawa Heart Institute, and 4Department of Medicine and Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada

Submitted 29 January 2004 ; accepted in final form 28 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the {alpha}2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the {alpha}2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive {alpha}2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the {alpha}2-isoform of Na-K-ATPase. As the {alpha}1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in {alpha}2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the {alpha}2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.

vascular tone; blood pressure; cardiac glycosides; serum level

CARDIAC GLYCOSIDES REPRESENT a class of drugs used to treat congestive heart failure and have been implicated in the development of hypertension in rats (10, 15, 16, 26, 31). In addition, endogenous ouabain-like substances occur in mammals and are associated with development and maintenance of hypertension (5, 27). Plasma levels of endogenous ouabain are high in several animal hypertension models, as well as in human essential hypertension (1, 5, 6, 17, 18, 28). However, the role of cardiac glycosides in the regulation of blood pressure remains controversial, as some reports demonstrate that chronic administration of ouabain does not always result in hypertension (14, 19, 35).

The mechanism of how ouabain affects blood pressure is uncertain. Increased sympathetic activity, alternation in baroreceptor function, as well as increased myocardial contraction and vascular resistance have all been proposed to play a role in the development and maintenance of ouabain-induced hypertension (2, 3, 7–9, 11, 24, 30, 32).

Although the Na-K-ATPase is the only known receptor for cardiac glycosides, at present it is unknown whether ouabain-induced hypertension is linked to the inhibition of this enzyme. Furthermore, as there are four {alpha}-isoforms ({alpha}1, {alpha}2, {alpha}3, {alpha}4) of Na-K-ATPase, each exhibiting a unique tissue distribution, it is important to define which specific {alpha}-isoform mediates the ouabain effect on blood pressure.

The major objective of the present study was to determine whether the {alpha}2-isoform of Na-K-ATPase mediates ouabain-induced hypertension. We analyzed the effect of ouabain on blood pressure in wild-type mice, where the {alpha}2-isoform is sensitive to ouabain, and on our genetically engineered mice expressing a cardiac glycoside-insensitive {alpha}2-isoform of the Na-K-ATPase. In both of these mice, the {alpha}1-isoform is resistant to ouabain and thereby is not affected by low levels of ouabain. The knock-in mice have normal baseline cardiovascular function, which makes them an excellent model for analysis of ouabain-induced hypertension (4). If hypertension is not observed in these gene-targeted mice following the administration of ouabain, this would demonstrate that the {alpha}2-isoform mediates the ouabain-induced increase in systolic blood pressure (SBP). However, if ouabain-induced hypertension still occurs in the targeted animals, it would indicate that this effect is mediated through another {alpha}-isoform or some other receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice and genotype analysis. Development of mice expressing the ouabain-insensitive {alpha}2-isoform of Na-K-ATPase by gene targeting was described previously (4). The use of mice in all of the experiments was approved by the University of Cincinnati Animal Care and Use Committee.

Western blot analysis. Western blot analysis was performed via standard methods, as previously described (4). Whole tissue preparations of aorta were analyzed for the expression of the {alpha}2-isoform using the {alpha}2-isoform-specific rabbit Mc HERED antibody (gift of Dr. Alicia McDonough). The expression of the total amount of Na-K-ATPase was analyzed by using KETTY antibody (gift of Dr. Jack Kyte) that detects the COOH-terminal region of all of the {alpha}-isoforms of Na-K-ATPase.

Prolonged administration of saline and ouabain. Mice were administered saline or ouabain (Calbiochem) daily by intraperitoneal injection in the early morning for either 6 days or 2 or 5 wk. Tail-cuff measurements of SBP were always obtained 10 h following administration (late afternoon).

Tail-cuff blood pressure measurements. Tail-cuff measurements were obtained by using a Visitech System (Apex, NC) computerized apparatus. For each measurement, 10 preliminary blood pressure measurements were obtained to acclimate the mice to the apparatus, and these were followed by 10 recorded SBP and heart rate (HR) measurements. There was no time delay between measurements. The blood pressure waveform was carefully observed for movement artifacts during both preliminary and measurement cycles. Data were accepted if SBP was identified by the computer in at least 5 of the 10 measurements and was >80 or <200 mmHg. Recordings not meeting these criteria were discarded. These were <1% of the recordings.

Serum ouabain-like immunoreactivity. Serum preparation and ELISA experiments were performed as previously described with modifications (33, 38a). Briefly, after serum samples (400–500 µl) were deproteinized with an equal volume of 0.1% trifluoroacetic acid, ouabain-like substances were collected by using Sep-Pak Vac C-18 cartridges according to the manufacturer’s instructions (Waters, Milford, MA) and eluted by using 3 ml of 25% acetonitrile. The samples were dried under vacuum and resuspended in 250 µl of PBS containing 0.05% Tween-20. Two days before the assay, microplate wells were coated with 200 µl of an ovalbumin-ouabain conjugate (0.6 µg/ml). The day before the assay, wells were washed (all washes consisted of 400 µl PBS-Tween 20, five times) and were then blocked with 300 µl of 4% normal rabbit serum (Sigma). At the time of the assay, wells were washed and 100 µl of sample (or ouabain standard) and 100 µl (0.002 mg/ml) of primary antibody (Digibind, GlaxoSmithKline) were added (22). After incubation and washing were completed, 200 µl (0.002 mg/ml) of horseradish peroxidase-conjugated secondary antibody [F(ab')2-specific anti-sheep IgG secondary antibody (313-036-047, Jackson ImmunoResearch, West Grove, PA)] were added per well. After final washing was completed, the amount of bound secondary antibody was detected colorimetrically by reaction with 3,3',5,5'-tetramethylbenzidine (Sigma). The reaction was stopped with 50 µl of 2 M H2SO4, and optical densities were measured at 450 nm. Because Digibind cross-reacts with endogenous marinobufagenin (albeit with a 20-fold lower affinity than ouabain), the activity in this assay is referred to as ouabain-like immunoreactivity.

Cardiovascular measurements in anesthetized mice. Cardiovascular measurements in anesthetized animals were performed as previously described (4). Values for HR, mean arterial pressure (MAP), SBP, and left ventricular blood pressure (LVBP) were measured before and 5 min after intravenous infusion of 300 µg/kg ouabain. The 300 µg/kg dose of ouabain was accomplished by a single 10-µl bolus intravenous infusion. The maximum rate of cardiac contractility (dP/dtmax) and the rate of cardiac contractility at 40 mmHg (dP/dt40) before and after infusion of ouabain were calculated from the first derivative of the pressure waveforms.

Calculation for determining the levels of ouabain in extracellular fluid. As an estimate of the acting concentration of ouabain in blood serum following intravenous infusion of 300 µg/kg of ouabain, we calculated the amount of ouabain in extracellular fluid (ECF). Approximate levels of ouabain in ECF were determined as previously described (4). Briefly, we assumed that metabolism and excretion of ouabain were negligible 5 min following intravenous infusion when the measurements were obtained. The 300 µg/kg of ouabain correspond to 0.412 nmol/g body wt. Because ECF corresponds to ~25% of total body weight, ouabain infusions of 0.412 nmol/g body wt correspond to 1.6 nM ouabain in ECF.

Contractile analysis of blood vessels. Analysis of contractile properties of blood vessels was performed as previously described (13). Studies were completed in both intact and endothelium-denuded aortas. Before the beginning of the experiment, each aortic segment was challenged once with 50 mM KCl and three times with 1 µM phenylephrine (PE) to ensure reproducible forces. The PE concentration-isometric force relationships were generated for each intact and endothelium-denuded aorta before beginning the saline or ouabain incubation. PE contraction-isometric force relationship was generated following 1-h incubation with saline and different concentrations of ouabain, including 0.1, 1, and 10 µM. Experiments in portal vein were carried out at optimal tension based on adjusting this blood vessel to a point at which maximum peak-to-peak oscillations were observed. Force measurements were obtained by using a Harvard Apparatus differential capacitor force transducer (South Natick, MA) connected to a Biopac MP100 data-acquisition system that allowed measurements and calculations of contractile parameters, including the frequency of spontaneous contraction and tension-time integral. For analysis of the long-term effect of ouabain on mechanical properties of aortas and portal veins, vessels were incubated for 12 h with saline or 1 µM ouabain during which time measurements were obtained.

Statistical analysis. Student’s t-test was used to compare the experimental group of mice to the corresponding control mice. Two-way ANOVA for time and treatment with repeated measurements was performed, and a post hoc test of Fisher’s least significant difference was obtained.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ouabain-induced hypertension in wild-type restrained mice. Previous studies demonstrated that ouabain has a pressor effect in rats. Daily administration of 30 µg·kg–1·day–1 of ouabain induced hypertension during the first week of treatment, and this hypertension persisted throughout the 5 wk of ouabain administration (10, 15, 16, 31, 38). However, it was unknown whether prolonged administration of ouabain affects blood pressure in mice. Thus we first determined the effect of daily administration of ouabain on SBP in wild-type ({alpha}2W/W) mice, which express the ouabain-sensitive {alpha}2-isoform of Na-K-ATPase. The {alpha}1-isoform is naturally resistant to ouabain in rodents and thereby could not be inhibited by the doses used in our experiment. Consistent with previous studies done in rats, the tail-cuff method of measurement was used to obtain the values for SBP (10, 15, 16, 31, 38). In contrast to rats, daily administration of 30 µg·kg–1·day–1 of ouabain had no effect on SBP in wild-type mice (data not shown). Daily administration of 100 µg·kg–1·day–1 of ouabain also had no effect (data not shown), and the level of ouabain in blood serum in these mice (0.87 ± 0.07 nM) did not differ from that in saline-treated animals (0.75 ± 0.08 nM). Administration of 300 µg·kg–1·day–1 of ouabain for 6 days increased the level of ouabain in blood serum to 3.3 ± 0.09 nM and SBP without altering HR in wild-type mice (Fig. 1). On the first day of ouabain administration, the SBP of the wild-type mice increased significantly compared with those administered saline. A gradual increase in SBP occurred during the next 3 days and reached a maximum at the fourth day of treatment. Although administration of 300 µg·kg–1·day–1 of ouabain slightly increased HR in wild-type mice, this increase was statistically nonsignificant (Fig. 1B).



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  		Fig. 1. Systolic blood pressure (SBP) and heart rate (HR) measurements in saline and ouabain-treated wild-type mice. A: tail-cuff measurements of SBP in wild-type mice injected with saline (n = 7) and wild-type mice injected with 300 µg·kg–1·day–1 of ouabain (n = 6) were measured once a day for 6 days. B: tail-cuff measurements of HR in wild-type mice injected with saline (n = 7) and wild-type mice injected with 300 µg·kg–1·day–1 of ouabain (n = 6) were measured once a day for 6 days. bpm, Beats/min. Values are averages ± SE. *P < 0.01 compared with wild-type mice injected with saline.

 
Absence of ouabain-induced hypertension in {alpha}2R/R mice. To determine whether Na-K-ATPase, specifically the high-affinity {alpha}2-isoform of Na-K-ATPase, mediates ouabain-induced hypertension, we analyzed the effect of prolonged administration of 300 µg·kg–1·day–1 of ouabain for 2 wk on SBP (Fig. 2A) and HR (data not shown) in {alpha}2W/W and targeted ({alpha}2R/R) mice. The {alpha}2 gene of {alpha}2R/R animals has been modified so that it is relatively resistant to ouabain (4). Before the treatment was started, there was no significant difference in baseline SBP or HR between wild-type and targeted animals. There was also no significant difference in SBP or HR between saline-administered wild-type and targeted animals. Ouabain had no significant effect on HR in mice from either genotype (data not shown). However, ouabain treatment resulted in a significant difference in SBP between {alpha}2W/W and {alpha}2R/R mice. Although ouabain induced hypertension in wild-type mice, it had no effect on SBP in {alpha}2R/R mice.



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  		Fig. 2. SBP and ouabain-like immunoreactivity in serum from {alpha}2W/W and {alpha}2R/R following administration of saline and ouabain. Tail-cuff SBP was measured before daily intraperitoneal injections with saline and 300 µg·kg–1·day–1 of ouabain (day 0 of treatment) to obtain baseline pressures. Thereafter, SBPs of saline-administered {alpha}2W/W mice (n = 5), saline-administered {alpha}2R/R mice (n = 5), ouabain-administered {alpha}2W/W mice (n = 19), and ouabain-administered {alpha}2R/R mice (n = 20) were obtained for 14 consecutive days of treatment. A: daily plot of mean SBP ± SE for each group. *P < 0.001 compared with ouabain-administered wild-type animals. #P < 0.001 compared with the saline-administered wild-type animals. B: level of ouabain-like immunoreactivity in serum from saline-administered {alpha}2W/W mice (n = 10), saline-administered {alpha}2R/R mice (n = 10), ouabain-administered {alpha}2W/W mice (n = 13), and ouabain-administered {alpha}2R/R mice (n = 13) at the 14th day of daily treatment. Values are averages ± SE. #P < 0.001 compared with the saline-administered control animals.

 
After the 2 wk of treatment, serum ouabain-like reactivity was essentially the same in ouabain-treated wild-type and targeted animals and was significantly higher than in the corresponding saline control groups (Fig. 2B). The level of ouabain immunoreactivity in serum from both wild-type and targeted mice treated with ouabain was within the range shown previously to be sufficient enough to induce hypertension in rats (16). It is important to point out that the level of ouabain following 14 days of treatment is slightly higher than that obtained following 6 days of administration. Because the half-life of ouabain in serum is between 18 and 22 h, it is reasonable to assume that, with each day of administration, the concentration of ouabain in blood serum will be slightly increased.

To ensure that targeted animals do not exhibit a delayed development of hypertension, we analyzed the effect of 300 µg·kg–1·day–1 of ouabain on SBP throughout 5 wk of daily treatment. Whereas ouabain-induced hypertension persisted in wild-type mice, ouabain had no effect on SBP in targeted animals (Fig. 3). Throughout the 5-wk period, SBP in targeted animals treated with saline was not significantly different from that in saline-administrated wild-type mice.



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  		Fig. 3. Tail-cuff measurements of SBP in {alpha}2W/W and {alpha}2R/R mice following administration of saline and ouabain for 5 wk. Tail-cuff SBP in saline-administered {alpha}2W/W mice (n = 5), saline-administered {alpha}2R/R mice (n = 5), ouabain-administered {alpha}2W/W mice (n = 10), and ouabain-administered {alpha}2R/R mice (n = 10) was measured once a week during 5 wk of treatment. Values are average ± SE SBP. *P < 0.001 compared with saline-administered wild-type animals.

 
Acute effect of ouabain on cardiovascular function in anesthetized {alpha}2W/W and {alpha}2R/R mice. We further determined whether acute administration of ouabain affects blood pressure in anesthetized mice and whether this effect is mediated by the {alpha}2-isoform of Na-K-ATPase. We analyzed MAP, SBP, LVBP, HR, dP/dtmax, and dP/dt40 in anesthetized {alpha}2W/W and {alpha}2R/R mice before and after intravenous infusion of ouabain (Table 1). Before treatment with ouabain (values for 0 µg/kg), there was a slight, but insignificant, difference in HR, MAP, dP/dtmax, and dP/dt40 between the two genotypes. The small differences in anesthetized animals could possibly be due to variability among this group of animals caused by their mixed background of Black Swiss and 129/SvJ strains. Furthermore, variability in the depth of anesthesia between animals could possibly account for slight differences in basal hemodynamics. The intravenous infusion of 300 µg/kg of ouabain increased significantly the MAP, SBP, and LVBP in wild-type mice without changing HR. Along with the increase in blood pressure, intravenous infusion of 300 µg/kg of ouabain significantly increased cardiac contractility. In contrast to wild-type mice, intravenous infusion of 300 µg/kg of ouabain had no marked effect on blood pressure, HR, and cardiac contractility in anesthetized {alpha}2R/R mice.


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  		Table 1. Hemodynamics before and after bolus intravenous infusion of ouabain in anesthetized {alpha}2W/W and {alpha}2R/R mice

 
We also calculated the approximate levels of ouabain in ECF following its administration by intravenous infusion, as described previously (4). Administration of 300 µg/kg of ouabain should have resulted in 1.6 nM ouabain in ECF, if ouabain was distributed equally throughout the ECF. In wild-type mice, this concentration of ouabain would inhibit only the ouabain-sensitive {alpha}2- and {alpha}3-isoforms. The {alpha}1-isoform is naturally resistant to ouabain in mice and therefore would not be inhibited by this low concentration of ouabain. As we have established that metabolism and extrusion of ouabain in targeted animals are indistinguishable from that in the wild-type mice, it is reasonable to assume that the ouabain levels in ECF of ouabain-administered targeted animals will be essentially the same as in wild-type animals. As in targeted animals, both the {alpha}1- and {alpha}2-isoforms are resistant to ouabain; it is highly unlikely that either will be inhibited by 1.6 nM ouabain. This level of ouabain would only affect the remaining ouabain-sensitive {alpha}3-isoform in targeted mice. As ouabain had no effect on cardiovascular hemodynamics in targeted animals, this clearly indicates that only the {alpha}2-isoform mediated the pressor effect of ouabain observed in wild-type anesthetized mice.

It is important to note that the approximate concentration of ouabain in ECF is threefold smaller than the concentration of ouabain in blood serum obtained following prolonged administration. This is expected as the half-life of ouabain in serum is between 18 and 22 h. It is reasonable to assume that, with each day of administration, the concentration of ouabain in blood serum will be slightly increased. Thus it is expected that the level of ouabain in serum will be greater following prolonged daily administration than following one acute injection.

The effect of ouabain on the PE response of intact and endothelium-denuded {alpha}2W/W and {alpha}2R/R aortas. Because increased vascular resistance is one of the proposed mechanisms for the ouabain-induced hypertension and as the {alpha}2-isoform is expressed in rodent vasculature, we analyzed the role of this isoform in the ouabain-induced increase in vascular reactivity (2, 3, 24, 30). We determined the effect of 0.1, 1, and 10 µM ouabain on the PE response of intact and endothelium-denuded aortas from {alpha}2W/W and {alpha}2R/R mice. As the expression of the {alpha}2-isoform is normal in aortas of the {alpha}2R/R animals (Fig. 4A), any differences in aortic contractile parameters between the two genotypes following ouabain treatment will only be accounted for by the difference in the ouabain sensitivity of the {alpha}2-isoform of Na-K-ATPase.



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  		Fig. 4. Characterization of intact and endothelium-denuded aortic rings from {alpha}2W/W and {alpha}2R/R mice. A: Western blot analysis of intact aorta from wild-type and targeted animals. The expression of the {alpha}2-isoform was analyzed. Kidney is used as a negative control and brain as a positive control for the expression of this {alpha}-isoform. The expression of the total amount of Na-K-ATPase was analyzed by using KETTY antibody that detects the COOH-terminal region of all of the {alpha}-isoforms of Na-K-ATPase. B: phenylephrine (PE) concentration-isometric force relationship of intact (n = 6 for each genotype) and endothelium-denuded (n = 6 for each genotype) aortic segments from {alpha}2W/W and {alpha}2R/R mice. C: PE-induced force per area of intact (n = 6 for each genotype) and endothelium-denuded (n = 6 for each genotype) aortas from {alpha}2W/W and {alpha}2R/R mice. Values are averages ± SE.

 
Before the beginning of the saline or ouabain treatment, there was no significant difference in the PE isometric force relationship of intact and endothelium-denuded aortic rings between the two genotypes (Fig. 4, B and C). As expected, the endothelium-denuded aortic rings had enhanced PE sensitivity compared with intact aortas, indicating that the removal of endothelium did not damage smooth muscle tissue. After incubation with saline, there was also no significant difference in PE sensitivity of intact or endothelium-denuded aortas between the two genotypes (Fig. 5, A and C). Whereas treatment with 0.1 µM ouabain had no significant effect on vascular tone of intact and endothelium-denuded aortas from both genotypes (data not shown), the incubation with 1 and 10 µM ouabain did. The 1-h incubation with 1 µM ouabain increased isometric force of only intact aorta from wild-type mice. However, this increase occurred in response to low doses of PE (Fig. 5A). This indicates that an increase in vascular tone was negligible compared with the maximal force of contraction induced by high concentrations of PE. It is well established that the presence of endothelium inhibits ouabain-induced increase in vascular tone, which is possibly due to enhanced secretion of endothelial vasodilating factors (20, 24–26). Thus the presence of endothelial cells could prevent a large increase in vascular tone of intact aortas. In common with these studies, the removal of endothelium enhanced the effect of ouabain on the vascular tone of only wild-type aorta, resulting in a significant difference in PE sensitivity between endothelium-denuded {alpha}2W/W and {alpha}2R/R aortas (Fig. 5C). The PE concentration yielding 50% of maximum contraction, half-maximal effective dose, was significantly different between the two genotypes.



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  		Fig. 5. Effect of ouabain on the PE sensitivity of intact and endothelium-denuded aortas from {alpha}2W/W and {alpha}2R/R mice. The PE concentration-isometric force relationship of intact (A) and endothelium-denuded (C) aortic segments from {alpha}2W/W and {alpha}2R/R mice following a 1-h treatment with saline (n = 6 for each genotype) and 1 µM ouabain (n = 6 for each genotype) is shown. The PE-induced force per area of intact (B) and endothelium-denuded (D) aortas from {alpha}2W/W and {alpha}2R/R mice following a 1-h treatment with saline (n = 6 for each genotype) and 1 µM ouabain (n = 6 for each genotype) is shown. Values are averages ± SE. *P < 0.05 relative to ouabain-treated wild-type aorta.

 
The removal of endothelium also enhanced the ouabain-induced increase in force per area of contraction in wild-type aorta. Treatment with 1 µM of ouabain had no significant effect on the PE-induced force of contraction of intact aortas from either genotype (Fig. 5B). There was a small decrease in the force of contraction in ouabain-treated aortic rings compared with their corresponding saline control. Previous studies demonstrated that ouabain increases the amount of released endothelial vasodilating agent nitric oxide (39). This possibly could account for the slight decrease in force of contraction in ouabain-treated intact aortas. After removal of endothelium, ouabain resulted in a significant increase in the force of contraction in wild-type aorta (Fig. 5D). No effect of ouabain was detected in targeted endothelium-denuded aortas.

In contrast to this differential effect, treatment with 10 µM ouabain increased the PE sensitivity of intact and endothelium-denuded aortas from both genotypes (data not shown). However, as this concentration of ouabain affects both the modified {alpha}2-isoform and low-affinity {alpha}1-isoform in targeted aortas, the differential effect of these two {alpha}-isoforms in vascular contractility cannot be determined.

Unexpectedly, the 1-h treatment with saline decreased total force per area contraction of the endothelium-denuded aortas compared with their baseline and saline-treated intact aortic segments. As removal of endothelium did not affect the PE isometric force relationship before the beginning of the saline and ouabain treatment (Fig. 4, B and C), this decrease could result from the time-induced dissipation in basal tone of endothelium-denuded aortic rings. However, 1 and 10 µM of ouabain prevented this decrease in the total force per area of contraction and resulted in higher force per area of contraction compared with that in the saline controls.

The long-term effect of ouabain on contraction of portal vein and intact and endothelium-denuded aorta from {alpha}2W/W and {alpha}2R/R mice. We also analyzed the role of the {alpha}2-isoform of Na-K-ATPase in the long-term effect of ouabain on vascular contractility in vitro. The mechanical properties of portal veins (phasic smooth muscle) and intact aortas (tonic smooth muscle) from {alpha}2W/W and {alpha}2R/R mice were determined during 12 h of incubation with either saline or 1 µM of ouabain. Tracings of the spontaneous mechanical activity profiles of isometrically mounted {alpha}2W/W and {alpha}2R/R portal veins are shown in Fig. 6A. Within 5 h of exposure to ouabain, a marked increase in mechanical force was apparent in the {alpha}2W/W portal veins, whereas no increase was observed in {alpha}2R/R veins. The mechanical force of {alpha}2R/R portal vein slowly decreased in amplitude over time. The mechanical force before and after ouabain administration, as determined by the tension-time integral, is shown in Fig. 6B. In the absence of ouabain, there was no significant difference in the mechanical force of portal veins between the two genotypes. After ouabain incubation, the mechanical force significantly increased in {alpha}2W/W portal veins and decreased slightly in portal veins from {alpha}2R/R mice.



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  		Fig. 6. Contractile activity of isolated portal veins following administration of 1 µM ouabain. A: representative tracings of {alpha}2W/W (top) and {alpha}2R/R (bottom) portal veins showing isometric force vs. time for spontaneous contractions. Ouabain treatment is indicated by the arrow and leads to a clear increase in force in {alpha}2W/W but not in {alpha}2R/R portal veins. Scales represent 40 min of time and 1 mN of force. B: tension-time integrals for {alpha}2W/W and {alpha}2R/R portal veins (n = 5 for each genotype) in absence and presence of 1 µM ouabain. *P < 0.05 relative to ouabain-treated wild-type portal vein. #P < 0.05 relative to baseline of wild-type portal vein.

 
Figure 7A shows the force per area-time integral for the {alpha}2W/W and {alpha}2R/R aortas during incubation with either saline or 1 µM ouabain. There was no significant difference in force per area between the {alpha}2W/W and {alpha}2R/R aortas at their baselines. There was also no significant difference in force per area values between saline-treated intact aortas from {alpha}2W/W and {alpha}2R/R mice. The basal tone of the saline-treated aortas from both genotypes slightly decreased over time. After ouabain treatment, there was a significant difference in the force of contraction between intact aortas from {alpha}2W/W and {alpha}2R/R mice. The basal tone of the {alpha}2W/W aorta spontaneously increased after 6 h of ouabain administration and reached its maximum after 12 h. Ouabain incubation did not affect basal tone of the {alpha}2R/R aortas, and it was not different from saline-treated aortas. Interestingly, removal of endothelium abolished the effect of ouabain on wild-type aorta and had no effect on aorta from the targeted animals (Fig. 7B).



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  		Fig. 7. Effect of long-term ouabain treatment on basal tone of intact and endothelium-denuded {alpha}2W/W and {alpha}2R/R aortas. A: force per area-time relationship for intact aortic segments from {alpha}2W/W (n = 6) and {alpha}2R/R (n = 6) mice treated with saline and 1 µM ouabain. B: force per area-time relationship for endothelium-denuded aortic segments from {alpha}2W/W (n = 6) and {alpha}2R/R (n = 6) mice treated with 1 µM ouabain. *P < 0.05 relative to ouabain-treated wild-type aorta.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It is not known whether cardiac glycosides affect blood pressure in certain species. Although ouabain was shown to induce hypertension in rats (10, 15, 16, 26, 31), it was unknown whether this cardiac glycoside has the same effect in mice. In the present study, we demonstrated that both prolonged and acute administration of ouabain increases blood pressure in these animals. However, in contrast to rats, a 10-fold higher dose of ouabain was necessary to induce hypertension in mice. Nevertheless, the level of ouabain in the serum of hypertensive mice was similar to that in hypertensive rats (16), indicating that there may be differences in the rate of extrusion and metabolism of ouabain between the two species (16). Thus it is reasonable to assume that administration of low concentrations of ouabain did not result in serum levels that are sufficient to induce hypertension in mice. Consistent with the previous studies in rats, the acute pressor effect of ouabain was observed in anesthetized mice (25). It occurred following intravenous administration of the same dose of ouabain that induced hypertension in wild-type mice following prolonged administration. Thus the present study demonstrates that ouabain increases blood pressure in mice.

In addition to the uncertainty of whether ouabain has pressor effects, it was unknown by what mechanisms this cardiac glycoside affects blood pressure. Although the Na-K-ATPase is the only known receptor for cardiac glycosides, it has not been directly demonstrated that this enzyme plays a role in ouabain-induced hypertension. Furthermore, as there are four different {alpha}-isoforms of Na-K-ATPase, each with different tissue distribution and affinity for cardiac glycosides, it is unknown whether a specific {alpha}-isoform mediates ouabain-induced hypertension.

As the ubiquitously expressed {alpha}1-isoform is naturally resistant to ouabain in rodents, it is highly unlikely that it plays a role in ouabain-induced hypertension. In contrast, the {alpha}2- and {alpha}3-isoforms have a high affinity for cardiac glycosides and thereby could mediate the pressor effects of ouabain. In addition, both of these {alpha}-isoforms are expressed in tissues that are known to be involved in regulating blood pressure. The {alpha}2-isoform is expressed in brain, vasculature, heart, and skeletal muscle. The {alpha}3-isoform is expressed in both the central and peripheral nervous systems.

To determine whether the Na-K-ATPase, and specifically the {alpha}2-isoform of Na-K-ATPase, plays a role in ouabain-induced hypertension, we used a unique approach of analyzing whether the inability of this {alpha}-isoform to bind ouabain affects the development and maintenance of hypertension. This was achieved by utilizing genetically engineered mice expressing the ouabain-resistant {alpha}2-isoform of Na-K-ATPase. The fact that ouabain had no effect on blood pressure in these animals clearly demonstrates that the {alpha}2-isoform of Na-K-ATPase mediates the ouabain-induced hypertension. The indistinguishable levels of ouabain in serum from ouabain-treated wild-type and targeted animals indicate that metabolism and extrusion of ouabain are normal in the targeted mice. The present study also demonstrates that the {alpha}2-isoform mediates the acute pressor effects of ouabain in anesthetized mice, as acute administration of ouabain had no significant effect on blood pressure in these targeted animals.

Our finding that the {alpha}2-isoform of Na-K-ATPase mediates both chronic and acute pressor effects of ouabain is an important step in understanding the mechanism by which ouabain affects blood pressure. In addition, this study implies that the {alpha}2-isoform of Na-K-ATPase is involved in regulating blood pressure. This is consistent with previous reports indicating that the {alpha}2-isoform of Na-K-ATPase contributes to the regulation of HR and blood pressure in healthy subjects (23). However, the mechanism by which the {alpha}2-isoform regulates blood pressure remains to be determined. As the {alpha}2-isoform is expressed in brain, heart, and vasculature, its inhibition in one or all of these tissues could play a role in development and maintenance of ouabain-induced hypertension (26, 29, 36). Thus understanding the functional role of the {alpha}2-isoform in each of these tissues and its involvement in regulating blood pressure is crucial.

In the present study, we demonstrate that the {alpha}2-isoform of Na-K-ATPase plays a role in the regulation of vascular contractility and thereby in the regulation of the ouabain-induced increase in vascular reactivity in vitro. The increase in vascular contractility in response to ouabain has been observed previously and has been suggested to play a role in ouabain-induced hypertension in rats (3, 12, 24, 30). It has been shown that acute treatment with ouabain increases vascular tone of isolated rat aortic rings (3, 24, 30). We also demonstrate that the same effect occurs in isolated mouse aorta and that it is mediated by the {alpha}2-isoform of Na-K-ATPase. Consistent with previous studies performed in rats, the present study demonstrates that prolonged incubation with ouabain increased vascular contraction of isolated mouse aortic rings and portal vein (12). These long-term effects of ouabain were absent in aortic and portal vein segments from targeted mice and thus are also mediated by the {alpha}2-isoform of Na-K-ATPase.

An interesting property of the long-term effect of ouabain is its dependence on the presence of the endothelium. The removal of the endothelium abolished the ouabain-induced increase in basal tone of aorta, suggesting that the endothelium facilitates the enhancement of basal tone by ouabain. This is in contrast to the inhibitory effect of the endothelium during the short-term treatment of aorta with ouabain. However, our results are consistent with previous reports demonstrating the positive endothelial modulation of the ouabain-induced vasoconstriction in rats (20, 21). In addition, a recent study demonstrated that ouabain enhanced the production of endothelin-1, which is a potent vasoconstriction agent, in cultured human endothelial cells (27). Thus it needs to be determined whether enhanced endothelin-1 production causes this ouabain-induced, endothelium-dependent increase in wild-type aortic basal tone.

In summary, we have demonstrated that chronic ouabain-induced hypertension occurs in conscious restrained mice and is mediated by the {alpha}2-isoform of Na-K-ATPase. The present study also demonstrates that the {alpha}2-isoform mediates the acute effect of ouabain on blood pressure in anesthetized mice. These studies are important as ouabain-induced hypertension is suggested to be a model for salt-dependent hypertension in which elevated blood pressure is mediated by endogenous ouabain. However, whether these compounds function through the {alpha}2-isoform is yet to be determined. In addition, the biological role of the {alpha}2-isoform in ouabain-induced increase in vascular contractility suggests that the increase in vascular resistance may play a role in hypertension induced by ouabain. However, this does not eliminate the possibility that the increased sympathetic activity also plays a role in ouabain-induced hypertension to the same extent as proposed previously (7–11). As the {alpha}2-isoform is expressed in the central nervous system, its role in the regulation of ouabain-induced sympathetic activity should be analyzed in the future.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants R01 HL-28573 (J. B. Lingrel), R01 HL-66062 (J. B. Lingrel), and R01 DK-57552 (J. N. Lorenz) and by Heart and Stroke Foundation of Ontario Grant NA-5102 (J. W. Van Huysse).


   ACKNOWLEDGMENTS
 
We thank Michelle L. Nieman and Peggy Bowman for expert technical assistance. We thank Maureen Luehrmann for expert animal husbandry. We also thank Dr. Robert C. Krueger for animal genotyping.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. B. Lingrel, Dept. of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, Univ. of Cincinnati, Cincinnati, OH 45267 (E-mail: jerry.lingrel{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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