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ACTH-induced hypertension is dependent on the ouabain-binding site of the ₂-Na⁺-K⁺-ATPase subunit

¹Department of Molecular and Cellular Physiology and ²Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 20 February 2008 ; accepted in final form 8 May 2008

	ABSTRACT

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ACTH-induced-hypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabain-resistant ₂-Na⁺-K⁺-ATPase subunits (2^R/R mice). Since tail-cuff measurements involve restraint stress, the present study used telemetry to distinguish between an effect of ACTH on resting BP vs. an ACTH-enhanced stress response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant 2^R/R mice vs. wild-type mice (ouabain-sensitive ₂-Na⁺-K⁺-ATPase, 2^S/S mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in 2^S/S (104.0 ± 2.6 to 117.7 ± 3.0 mmHg) but not in 2^R/R mice (108.2 ± 3.2 to 111.5 ± 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of ₂-Na⁺-K⁺-ATPase on vascular smooth muscle by endogenous cardiotonic steroids, we measured BP and regional blood flow. Results suggest a differential sensitivity of renal, mesenteric, and cerebral circulations to ACTH and that the response depends on the ouabain sensitivity of the ₂-Na⁺-K⁺-ATPase. Baseline cardiac performance was elevated in 2^S/S but not 2^R/R mice. Overall, the data establish that the ₂-Na⁺-K⁺-ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations, presumably caused by elevated levels of circulating cardiotonic steroids.

cardiac glycosides; telemetry; blood flow; vascular resistance; hemodynamics

There are four major -isoforms of the Na⁺-K⁺-ATPase (NKA), and in most species all four are extremely sensitive to ouabain (5). In mice and rats, however, the ₁-NKA has low affinity for, and is resistant to, the inhibitory effects of ouabain and other cardiac glycosides. To explore the physiological role of the ouabain-binding site, mice have been generated with targeted mutation of the ₂-NKA such that it, like the ₁-isoform, is also resistant to ouabain. Using these mice, we recently reported that ACTH treatment for 5 days significantly increased systolic blood pressure, measured by tail cuff, in the ₂ ouabain-sensitive wild-type mice (2^S/S) but not in ouabain-resistant ₂-NKA mutant mice (2^R/R). These data suggested that ACTH-induced cardiovascular effects are mediated through the actions of an endogenous substance acting on the ₂-NKA ouabain-binding site (6). Since the tail-cuff method and attendant restraint were employed in this study, there are two potential interpretations of these earlier findings: first, that the primary effect of ACTH treatment was to elevate resting blood pressure, and second, that ACTH only serves to augment the cardiovascular response to restraint stress. Indeed, this latter possibility that ACTH-induced hypertension is not a true hypertension has been previously argued in a study using rats (8). If this were the case, then perhaps ACTH-induced increases in the stress response would be related to the release of an endogenous cardiotonic steroid on the imposition of acute stress. Thus an important goal of this study was to distinguish between these two possible interpretations, and we hypothesized that ACTH-administration does not elevate resting blood pressure in 2^R/R mice.

ACTH-induced hypertension has been associated with alterations in a variety of cardiovascular variables including cardiac output, total peripheral vascular resistance, endothelial NO production, and sympathetic activity (26, 27, 29). For example, it has been shown in rats that angiotensin-converting enzyme inhibition prevented the ACTH-induced rise in renal vascular resistance and the development of hypertension (26). Moreover, in this same study, β-adrenergic blockade prevented ACTH-induced increases in cardiac output but only partially prevented the increase in blood pressure. It is therefore reasonable to hypothesize that ACTH-induced changes in blood pressure are related to an increase in vascular and cardiac muscle reactivity, which in turn may be dependent on the actions of an endogenous glycoside interacting with the ₂-NKA on vascular smooth muscle and, perhaps, on cardiac muscle. A second goal of the present study, then, was to explore the potential role of altered vascular and cardiac contractility in ACTH-induced hypertension in mice with a ouabain-sensitive vs. ouabain-resistant ₂-NKA.

	METHODS

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Animals. Animals were obtained from an established colony at the University of Cincinnati. The development of mice expressing the ouabain-resistant ₂-isoform of the Na⁺-K⁺-ATPase by gene targeting was described previously (2). Homozygous mutant (2^R/R) and wild-type (2^S/S) offspring were generated from heterozygous matings, and all mice were maintained on a mixed 129SvJ and Black Swiss background. For all experiments, animals were paired for age, weight, and sex. Genotypes were determined by PCR analysis of DNA from tail biopsies, as described previously (2). All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati in accordance with established guidelines.

Telemetric measurement of blood pressure. Continuous ambulatory blood pressure recordings were made in 2^S/S and 2^R/R mice as described previously using the TA11PA-C10 pressure transmitter (Data Sciences International, St. Paul, MN) (19). Transmitters were implanted under isoflurane anesthesia using carotid artery cannulation and subcutaneous transmitter placement. After implantation, mice were allowed to recover for at least 7 days before data collection. The mice were synchronized to a 12:12-h light-dark schedule with lights on at 7:00 AM. After 3–5 days of baseline recording, mice were administered saline vehicle or ACTH every 8 h by subcutaneous injection for 5 days (0.5 µg/g body wt of fragment 1–24; Sigma). Blood pressure was monitored continuously in 1-min episodes at 5-min intervals using a PowerLab system and Chart software (ADInstruments, Colorado Springs, CO). To evaluate the participation of the renin-angiotensin system (RAS) in maintaining blood pressure, we injected mice with losartan (10 µg/g body wt ip) and monitored blood pressure continuously for 60 min. This was performed once before administration of ACTH in each mouse and repeated after 5 days of ACTH treatment. On a different day before ACTH treatment, we also evaluated the contribution of sympathetic tone to blood pressure maintenance by injecting hexamethonium (40 µg/g body wt ip) to induce ganglionic blockade. To evaluate the specificity of hexamethonium for ganglionic blockade vs. neuromuscular blockade of the diaphragm, we administered escalating doses of hexamethonium with and without artificial ventilation and while monitoring blood pressure and ventilation with a spirometer (ADInstruments).

Regional blood flow and vascular resistance. 2^S/S and 2^R/R mice, treated with vehicle or ACTH as described, were anesthetized with ketamine (50 µg/g body wt ip) and thiobutabarbital (Inactin; 100 µg/g boy wt ip) and instrumented as previously described (18). After tracheostomy and cannulation of the femoral artery and vein, mice were provided a maintenance infusion of 2.5% BSA in PBS at 0.15 µl·min^–1·g body wt^–1 to maintain euvolemia. For carotid flow measurements, the left common carotid artery, just proximal to the internal/external bifurcation, was isolated and fitted with a 0.5-PSB perivascular flow probe connected to a dual-channel TS420 flowmeter (Transonic Systems, Ithaca, NY). The probe window was filled with acoustical gel, and the probe body was embedded in 4% agar to secure its position. For renal and mesenteric blood flow, a flank incision was made and the renal and/or superior mesenteric artery was carefully isolated and fitted with a 0.5-PSB flow probe, which was held in position with a micromanipulator. Since flow could only be measured in two arteries at a time, the probe configuration was varied systematically from experiment to experiment (i.e., carotid and renal, carotid and mesenteric, renal and mesenteric). Continuous traces of regional vascular resistance were derived by dividing the mean arterial pressure signal by the mean blood flow signal. After completion of the surgical procedure and a 30-min recovery period, dose-dependent responses to phenylephrine (PE; 10, 40, and 80 ng·min·g body wt ^–1 iv) were tested. Individual doses were infused until a plateau was achieved (1–2 min), with a 3- to 5-min recovery period between doses.

Vascular smooth muscle reactivity. Analyses of contractile properties of vascular smooth muscle were performed in intact thoracic aortas as previously described (11). Briefly, aortic rings from vehicle- and ACTH-treated 2^S/S and 2^R/R mice were gently dissected and mounted in a myograph chamber. After the experiment, the aortas were gently blotted and weighed, and dimensions were measured. Aortic wall thickness (t_w) was estimated from the equation t_w = blot weight/(1.05 x length x circumference), and the cross-sectional area (CSA) for force normalization was calculated as CSA = 2(t x length). The bath solution contained (in mmol/l) 118 NaCl, 4.73 KCl, 1.2 MgCl₂, 0.026 EDTA, 1.2 KH₂PO₄, 2.5 CaCl₂, and 5.5 glucose, buffered with 25 NaH₂CO₃; pH when bubbled with 95%O₂-5% CO₂ was 7.4 at 37°C. For force measurement, rings were mounted on 100-µm stainless steel wires and attached to a force transducer (South Natick, MA). Resting tension on each aorta was set to 25 mN, the estimated in vivo tension calculated for a pressure of 100 mmHg and a CSA of 0.42 mm². Before the start of the experiment, each aortic segment was challenged once with 50 mM KCl and three times with 1 µM PE to ensure reproducible forces. Cumulative PE concentration-isometric force relationships were generated for each aorta. EC₅₀ was determined using a logistic nonlinear curve-fitting routine (OriginLab, Northampton, MA).

Left ventricular function. Left ventricular (LV) function was evaluated in closed-chest anesthetized mice as described previously (18). Since we have previously reported that LV function is nearly identical between vehicle-treated 2^S/S and 2^R/R mice (2), only ACTH-treated mice were evaluated in the current study. Mice were anesthetized with ketamine and Inactin as before and were instrumented with femoral artery and vein catheters and with a micromanometer (model SPR-671; Millar Instruments, Houston, TX) in the LV via the right carotid artery. Arterial and LV pressure and the change in LV pressure over time (dP/dt) signals were recorded at 2,000 Hz and analyzed using a PowerLab system. Hemodynamic measurements were taken at the basal state and after the administration of graded doses of dobutamine (1, 2, 4, 8, 16, and 32 ng·min^–1·g body wt^–1) to evaluate contractile reserve and β-adrenergic responsiveness. Maximum dP/dt (dP/dt_max) and dP/dt at 40 mmHg of developed pressure (dP/dt₄₀) were calculated from the first derivative of the pressure waveforms.

Adrenal cortical steroid levels. 2^S/S and 2^R/R mice were treated for 5 days with either saline vehicle or ACTH as described above. After anesthesia was administered, blood was withdrawn by aortic puncture and centrifuged, and the serum was stored at –20°C for later analysis. Aldosterone was measured by enzyme immunoassay (EIA) using a kit from Diagnostic Systems Laboratories (Webster, TX), and corticosterone was measured by EIA using a kit from Immunodiagnostic Systems (Fountain Hills, AZ).

Data analysis. Statistical analysis was performed by analysis of variance (ANOVA) using either a single-factor within-subjects design or a two- or three-factor mixed design with repeated measures on the last factor. Individual contrasts were used to compare group effects and interactions when needed, and Tukey's post hoc test was used to compare individual means where appropriate (SigmaStat version 3.5; Point Richmond, CA). Data are means ± SE, and differences were regarded as significant at P < 0.05.

	RESULTS

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Telemetric blood pressure. Baseline blood pressure was found to be not different between 2^S/S and ^R/R mutant mice, as shown in Fig. 1. Both groups showed normal diurnal variations in blood pressure and heart rate (not shown) over the 3 days of control recording. In response to treatment with ACTH over a 5-day period, 2^S/S mice demonstrated a substantial and significant increase in blood pressure from a control level of 104.0 ± 2.6 mmHg to a final sustained level of 117.7 ± 3.0 mmHg (n = 8), as shown in Fig. 2, left. In contrast, blood pressure did not significantly change in response to ACTH in 2^R/R mutant mice (108.2 ± 3.2 to 111.5 ± 4.0, n = 6). Examination of daytime vs. nighttime blood pressure (Fig. 2, right) suggests that the ACTH -induced hypertension persisted during both sleep and active periods in wild-type mice. Although there appeared to be a transient mild elevation in blood pressure in 2^R/R mice during the light cycle, perhaps related to the stress of the three-times-daily injections, this effect did not persist and there was no significant elevation of pressure through days 4 and 5 of ACTH treatment. Before ACTH administration, administration of losartan resulted in equivalent decreases in blood pressure in 2^S/S (111 ± 2 to 96 ± 3 mmHg, n = 8) and 2^R/R mice (104 ± 4 to 90 ± 4 mmHg, n = 7; ANOVA: genotype effect, P = 0.11; treatment effect, P < 0.001; interaction, P = 0.82). After 5 days of ACTH treatment, the hypotensive response to losartan was not altered in either group (120 ± 5 to 107 ± 4 mmHg in 2^S/S, n = 6, vs. 101 ± 4 to 90 ± 4 mmHg in 2^R/R, n = 6; ANOVA: genotype effect, P < 0.001; treatment effect, P = 0.032; interaction, P = 0.79), suggesting that the systemic RAS was not an important contributor to the development of hypertension in the ACTH-treated wild-type mice. Hexamethonium injection (40 µg/g body wt) did not significantly alter blood pressure in the 2^S/S mice (114 ± 2 to 108 ± 4 mmHg) but, surprisingly, caused rapid death in 2^R/R mice. Since the specificity of hexamethonium for cholinergic receptors of autonomic ganglia vs. motor end plate is, in our experience, not particularly high in mice, we evaluated blood pressure and ventilatory responses in anesthetized mice. We found that administration of hexamethonium at doses >30 µg/g body wt in 2^R/R mice caused immediate but modest decreases in blood pressure (12 ± 2 mmHg, n = 3), followed by complete respiratory arrest, cardiovascular collapse, and death. Moreover, we found that artificial ventilation prevented the cardiovascular collapse and that mice tolerated doses of hexamethonium up to 80 µg/g body wt with only small decreases in blood pressure. When subsequently removed from the ventilator, mice were unable to resume spontaneous breathing.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Twenty-four-hour averages of mean arterial pressure (MAP) measured by telemetry for mice expressing ouabain-sensitive (2S/S wild type) and ouabain-resistant 2-Na+-K+-ATPase subunits (2R/R mutant) during the control period 3 days before treatment with ACTH. Data represent the mean blood pressure from each 1-min episode, recorded at 5-min intervals.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Telemetric MAP measurements before and during administration of ACTH in 2S/S and 2R/R mice. Left: data are shown as 12-h means and are calculated as the mean pressure between 9:00 AM and 5:00 PM (light period) and 9:00 PM and 5:00 AM (dark period). Right: changes in (delta) MAP in response to ACTH during the light (top) and dark period (bottom). *P < 0.05 compared with control period within the same genotype. BW, body weight.

View larger version (18K): [in this window] [in a new window]   Fig. 3. MAP values from anesthetized vehicle- and ACTH-treated 2S/S and 2R/R mice instrumented with perivascular flow probes on the renal, mesenteric, or carotid arteries. Values represent the average from a 20- to 30-s recording taken under resting conditions and at the peak of the response during graded infusions of phenylephrine. Inset shows the percent change in MAP relative to baseline to highlight interactions. ANOVA results: *P < 0.05, significant genotype effect for vehicle- vs. ACTH-treated 2S/S mice. P < 0.05, significant genotype x dose interaction for vehicle- vs. ACTH-treated 2R/R mice. P < 0.05, significant genotype x dose interaction for ACTH-treated 2R/R vs. 2S/S mice.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Renal, mesenteric, and carotid blood flow under resting conditions in ACTH- and vehicle-treated 2S/S and 2R/R mice. In each experiment, flow probes were placed on 2 of the 3 arteries, and values reflect subsets of the data shown in Fig. 3. *P < 0.05 compared with vehicle-treated mice in the same group. P < 0.05 compared with 2S/S mice with the same treatment.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Regional vascular resistance (VR) from anesthetized mice instrumented with perivascular flow probes on the renal (top), mesenteric (middle), or carotid arteries (bottom). In each experiment, flow probes were placed on 2 of the 3 arteries, and values reflect subsets of the data shown in Fig. 3. ANOVA results: *P < 0.05, significant genotype x dose interaction for 2S/S vs. 2R/R mice with the same treatment. P < 0.05, significant genotype x dose interaction for ACTH vs. vehicle within the same genotype. P < 0.05, significant genotype effect for ACTH 2S/S vs. vehicle 2S/S mice.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Force development in response to graded doses of phenylephrine in isolated intact aortas from vehicle- and ACTH-treated 2S/S and 2R/R mice. *P < 0.05 for vehicle- vs. ACTH-treated 2S/S at corresponding doses.

View larger version (20K): [in this window] [in a new window]   Fig. 7. MAP, left ventricular systolic pressure (LVPsys), maximum LV dP/dt (dP/dtmax), and LV dP/dt at 40 mmHg of developed pressure (dP/dt40) in anesthetized ACTH-treated 2S/S and 2R/R mice instrumented with a high-fidelity transducer in the left ventricle. Measurements were made at baseline and during graded doses of dobutamine. *P < 0.05 compared with corresponding value in 2R/R.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Serum aldosterone and corticosterone concentrations in vehicle- and ACTH-treated mice. There were no differences in serum aldosterone levels, and corticosterone levels increased to the same degree in both genotypes. *P < 0.01 compared with vehicle-treated mice.

	DISCUSSION

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The underlying nature of ACTH-induced hypertension has been investigated thoroughly over the last several decades, particularly through the work of Whitworth et al. (29), and although there appear to be clear hallmarks regarding mechanisms of hypertension, a consolidated hypothesis has yet to emerge. Importantly, it is likely that there are substantial species differences in the mechanisms underlying the elevation in blood pressure associated with increased levels of serum ACTH. One important observation is that in all species tested, the rise in blood pressure can be prevented by prior adrenalectomy and is therefore dependent to a substantial degree on adrenally produced steroids. In humans and rats, for example, administration of the primary glucocorticoid (cortisol and corticosterone, respectively) can in large part reproduce the hypertensive state (30). We found that corticosterone increased dramatically in response to ACTH treatment in both ouabain-sensitive and -resistant mice and that the magnitude of this response was not different between the genotypes. Likewise, aldosterone concentration was not different between groups either before or after ACTH and, in fact, did not respond at all to ACTH treatment. Therefore, the difference in the blood pressure response cannot be attributed to altered secretion of these adrenal cortical steroids. On the other hand, endogenous cardiotonic steroids, presumably derived from the adrenal gland as well, also have been implicated in the development of ACTH hypertension (7, 13, 14). In our previous study (6), we demonstrated that ACTH treatment increased plasma levels of endogenous cardiac glycosides in both 2^S/S and 2^R/R mice and that the ACTH-induced hypertension in the 2^S/S mice could be blocked by concomitant treatment with Digibind. In light of these previous findings, and since we have repeatedly found that ACTH-induced hypertension in mice is dependent on the sensitivity of the ₂-NKA to ouabain, it seems reasonable to postulate that endogenous cardiotonic steroids are a necessary mediator of the hypertensive response. Such a role of endogenous cardiotonic steroids does not negate an important role for other adrenal steroids, such as corticosterone, but rather suggests that a common and necessary component is the binding of a ligand to the ₂-NKA subunit. Since the ₂-NKA isoform is widely expressed throughout the mouse cardiovascular system, potential candidates for the downstream targets and mechanism(s) of action are numerous.

As an initial attempt to identify potential targets mediating the elevated blood pressure, we used the telemetered mice to evaluate whether the RAS was activated differentially by ACTH in the two genotypes. Losartan was administered to block AT₁ receptors, and the degree of blood pressure decline was evaluated as an index of the prevailing level of RAS activation. Before ACTH treatment, losartan treatment decreased pressure in the wild-type and mutant mice by 15 and 14 mmHg, respectively. After ACTH treatment, losartan decreased pressure by 13 and 11 mmHg, respectively. Thus the RAS appears to contribute to blood pressure maintenance equally in both genotypes, both before and after administration of ACTH, and does not appear to play a role in the development of hypertension in the wild-type mice, a finding that is consistent with earlier studies (23, 24, 28).

To likewise explore the potential role of the sympathetic nervous system in the development of hypertension, we also attempted to perform a similar experiment using the ganglionic blocker hexamethonium. To our surprise, hexamethonium at a dose of 40 µg/g body wt resulted in only a modest decrease in blood pressure in vehicle-treated 2^S/S mice (5–10 mmHg), whereas this same dose resulted in apparent cardiovascular collapse and abrupt death in all of the 2^R/R mice. Although this result at first suggested that blood pressure was maintained in these mice through elevated sympathetic drive, subsequent experiments showed that this dose of hexamethonium was causing skeletal muscle paralysis and cessation of breathing in the mutant mice. When anesthetized mice were artificially ventilated, the applied dose of hexamethonium did not cause cardiovascular collapse and, in fact, the decline in blood pressure was minimal and not different between wild-type and mutant mice. The reason for the altered sensitivity of cholinergic receptors at the motor end plate in 2^R/R mice is unknown, but functional interactions between nicotinic receptors and the ₂-NKA have been described (10).

Conventional paradigms propose that the stimulatory effects of cardiac glycosides on cardiac and vascular smooth muscle are related to decreases in Ca²⁺ extrusion by the Na⁺/Ca²⁺ exchanger secondary to increases in intracellular Na⁺ concentration (5, 9). Accordingly, we hypothesized that ACTH-stimulated increases in endogenous cardiotonic steroids mediate the hypertension that develops in these animals by blocking the ₂-NKA and that mice with a resistant ₂-isoform are therefore resistant to the development of hypertension. We evaluated vascular function, both in situ and ex vivo, and found that blood pressure and vascular resistance responses to PE were not significantly augmented in the ACTH-treated wild-type mice that developed hypertension. In fact, vascular responsiveness in both mesenteric and renal beds was paradoxically elevated in the ACTH-treated mutant mice that were resistant to hypertension, and blood flows were correspondingly low throughout the dose-response range. In the cerebral vasculature, there was a modest elevation in resistance that reached statistical significance in the hypertensive 2^S/S mice but not in the 2^R/R mice. In isolated aortas, responsiveness to PE was actually decreased by ACTH treatment in 2^S/S mice but not in the 2^R/R mutants. Vascular responses to ACTH treatment are therefore complex, and the possibility remains that subtle differences in vascular tone contribute to elevated blood pressure in ACTH-treated wild-type mice. It is further possible that differences in skeletal muscle blood flow, which were not evaluated in this study, may underlie the differences in blood pressure between the two groups. Since skeletal muscle blood flow is dramatically lowered by anesthesia (25), it would likely be necessary to evaluate this possibility in conscious mice.

There have been several prior studies evaluating hemodynamics in ACTH-induced hypertension. Notably, a study in rats reported that ACTH-induced hypertension was associated with increases in cardiac output and renal vascular resistance (RVR) but not in other vascular beds or in overall peripheral resistance, suggesting that the increase in RVR may specifically contribute to the increased blood pressure (27). Although we saw no such increase in renal blood flow in either wild-type or mutant mice, a renal basis for the differences in blood pressure remains a distinct possibility. It is possible, for example, that ACTH-induced elevations in endogenous cardiotonic steroids cause a shift of the pressure natriuresis relationship through a direct effect on the ₂-NKA in the kidney. However, since renal tubular cells express only the ₁-subunit, this effect would likely involve alterations in renal microvascular behavior.

Since previous investigations had consistently reported elevations in cardiac output in the early phases of ACTH-dependent hypertension (22, 26, 27), we sought to determine whether there were any differences in cardiac performance that might directly underlie the differences in blood pressure following ACTH treatment. Our hypothesis predicts that elevations in endogenous cardiotonic steroids following ACTH would enhance contractility in the 2^S/S wild-type mice but not in the 2^R/R mutants. We have previously shown that basal and β-adrenergic-stimulated cardiac contractility is comparable between wild-type and mutant mice and that ouabain administration increases contractility in the 2^S/S but not 2^R/R mice (2). In the present study we found that baseline cardiac contractility was elevated in ACTH-treated 2^S/S vs. 2^R/R mice. The combined data are consistent with the notion that cardiac output may be elevated to some degree in the hypertensive 2^S/S mice and that this increase in output is accommodated by elevated cardiac contractile function.

In summary, the data presented provide important confirmation that mice with a ouabain-resistant ₂-isoform of the Na⁺-K⁺-ATPase are resistant to the development of ACTH-induced hypertension. Use of telemetry confirms that differences in blood pressure between ACTH-treated 2^S/S and 2^R/R mice persist in the absence of the restraint stress associated with tail-cuff measurements. Vascular tone and reactivity appear to be relatively similar between 2^S/S and 2^R/R mice following ACTH treatment, with only small differences in responses between the genotypes. Cardiac function was incrementally elevated in the 2^S/S but not 2^R/R mice. Given that the ₂-NKA is expressed in a wide variety of tissues and cell types relevant to cardiovascular function, it is possible that the difference in phenotype between the wild-type and mutant mice is the result of a combination of subtle differences in cardiac, vascular, endothelial, and neuronal function.

	GRANTS

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This work was supported by National Institutes of Health Grants DK-57552 (to J. N. Lorenz), HL-66062, and HL-28573 (to J. B. Lingrel).

	ACKNOWLEDGMENTS

 
We thank Michelle Nieman for additional technical expertise.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. N. Lorenz, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (e-mail: john.lorenz{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.

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