Estradiol Attenuates Angiotensin-Induced ... - Semantic Scholar

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 12 Printed in U.S.A.

Estradiol Attenuates Angiotensin-Induced Aldosterone Secretion in Ovariectomized Rats* DARREN M. ROESCH†, YING TIAN, WEI ZHENG, MIN SHI, JOSEPH G. VERBALIS, AND KATHRYN SANDBERG Divisions of Endocrinology and Metabolism and Nephrology and Hypertension, Department of Medicine, Georgetown University, Washington, D.C. 20007 ABSTRACT Estrogen replacement therapy significantly reduces the risk of cardiovascular disease in postmenopausal women. Previous studies indicate that estradiol (E2) decreases angiotensin II (AT) receptor density in the adrenal and pituitary in NaCl-loaded rats. We used an in vivo model that eliminates the potentially confounding influence of ACTH to determine whether the E2-induced decrease in adrenal AT receptor expression affects aldosterone responses to angiotensin II (Ang II). Female rats were ovariectomized, treated with oil (OVX) or E2 (OVX⫹E2; 10 ␮g, sc) for 14 days, and fed a NaCl-deficient diet for the last 7 days to maximize adrenal AT receptor expression and responsiveness. On days 12–14 rats were treated with dexamethasone (DEX; 25 ␮g, ip, every 12 h) to suppress plasma ACTH. On day 14 aldosterone secretion was measured after a 30-min infusion of Ang II (330 ng/min). Ang II infusion increased the peak plasma aldosterone levels to a lesser degree in the OVX⫹E2 than in the OVX rats (OVX, 1870 ⫾ 290 pg/ml; OVX⫹E2, 1010 ⫾ 86 pg/ml; P ⬍ 0.05). Ang

A

S WOMEN ADVANCE in age from the premenopausal to the postmenopausal years, the risk of cardiovascular disease (CVD) increases (1). Evidence suggests that an age-related decline in cardiovascular function contributes to the increased risk for CVD; mean arterial pressure is increased and cardiac output is decreased in postmenopausal women (2). Although few studies have studied the effect of ovarian senescence on the activity of the renin-angiotensinaldosterone system (RAAS), an age-related decline in PRA that results in decreased plasma aldosterone concentrations in aged humans and rats (3) appears to accompany the decline in cardiovascular function. Although some studies suggest that adrenal responsiveness to angiotensin II (Ang II) is also reduced in the aged (4 – 6), numerous other studies suggest that the age-associated decrease in activity of the RAAS is partially compensated for by increased Ang II receptor (AT) expression and increased tissue responsiveness to Ang II (3, 7–10). Estrogen replacement therapy (ERT) decreases mean arterial pressure, increases cardiac output, and significantly reduces the risk of CVD in postmenopausal women (11). Although 25–50% of the cardiovascular benefits of ERT ther-

Received May 19, 2000. Address all correspondence and requests for reprints to: Dr. Darren M. Roesch, 350 Building D, 4000 Reservoir Road NW, Washington, D.C. 20007. E-mail: [email protected]. * This work was supported by NIH Grants HL-57502 (to K.S.), AG16902 (to K.S.), and DK-38094 (to J.G.V.). † Supported by a National Research Service Award (HL-10419).

II-induced ACTH and aldosterone secretion was also studied in rats that were not treated with DEX. In the absence of DEX, the peak plasma aldosterone response was also significantly decreased (OVX, 5360 ⫾ 1200 pg/ml; OVX⫹E2, 2960 ⫾ 570 pg/ml; P ⬍ 0.05). However, E2 also reduced the plasma ACTH response to Ang II (P ⬍ 0.05; OVX, 220 ⫾ 29 pg/ml; OVX⫹E2, 160 ⫾ 20 pg/ml), suggesting that reduced pituitary ACTH responsiveness to Ang II contributes to the effect of E2 on Ang II-induced aldosterone secretion. Adrenal AT1 binding studies confirmed that E2 significantly reduces adrenal AT1 receptor expression in both the presence and absence of DEX in NaCl-deprived rats. These results indicate that E2-induced decreases in pituitary and adrenal AT1 receptor expression are associated with attenuated pituitary ACTH and adrenal aldosterone responses to Ang II and suggest that estrogen replacement therapy may modulate Ang IIstimulated aldosterone secretion as part of its well known cardioprotective actions. (Endocrinology 141: 4629 – 4636, 2000)

apy have been attributed to the effects of estrogens on the lipid profile (11), most of the protective effects of ERT have not been adequately explained. Some studies have suggested that differential regulation of the RAAS by estrogen contributes to the cardiovascular benefits of ERT (12, 13). Recent studies suggest that the plasma renin concentration is decreased in postmenopausal women receiving ERT (12, 13), and ERT decreases angiotensin-converting enzyme (ACE) activity in monkeys (14) and ACE messenger RNA (mRNA) levels in rats (15). Despite these concurrent changes in the regulation of angiotensinogen, renin concentration, ACE expression and activity, PRA (13), plasma Ang II (14), and plasma aldosterone (12, 13) concentrations do not change significantly during ERT. However, ERT has been shown to decrease vascular smooth muscle type 1 AT receptor (AT1) mRNA expression (16) and attenuate vascular (17) and renal (18) responses to Ang II. Together, these findings suggest that reduced target tissue responsiveness to Ang II may constitute a significant component of the cardiovascular benefit of ERT. Surprisingly, the effect of ERT on the responsiveness of the adrenal to Ang II has not been studied. We have recently observed that estradiol (E2) reduces maximal binding to AT1 receptors in the adrenal and pituitary of ovariectomized (OVX) rats (19). This finding is consistent with previous reports of E2-induced decreases in total AT binding in the adrenal and pituitary (20, 21) and suggests that E2 may attenuate Ang II-stimulated aldosterone secretion by decreasing the sensitivity of the adrenal and pituitary to Ang II. As Ang II infusions are known to stimulate ACTH release (22,

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ESTRADIOL ATTENUATES ANGIOTENSIN-INDUCED ALDOSTERONE SECRETION

23), and ACTH has also been shown to increase aldosterone production in humans (24), rats (25), dogs (26), and sheep (27), it is impossible to study the effect of exogenous Ang II on aldosterone secretion without considering the contribution of Ang II-stimulated ACTH secretion. Before the late 1970s, the study of Ang II-induced aldosterone secretion was also hindered by the fact that when rats are fed standard laboratory chows that are typically high in sodium, the adrenal is minimally responsive to exogenous Ang II (28). Since then, dietary sodium deprivation has been shown to increase both the expression of adrenal AT receptors and the adrenal aldosterone response to Ang II (28). In this study we used a protocol that combines dietary sodium deprivation and dexamethasone (DEX) suppression of ACTH to produce 1) a high magnitude of Ang II-induced aldosterone secretion, 2) a return of plasma aldosterone levels to baseline after Ang II stimulation, and 3) an absence of Ang II-stimulated ACTH secretion. Using this improved protocol, we tested the effect of E2 on ACTH-independent adrenal responsiveness to Ang II in OVX rats. To assess whether the E2-induced decrease in pituitary AT1 receptor expression affects Ang II-induced ACTH secretion and contributes to decreased Ang II-induced aldosterone secretion, we also measured the effect of E2 on Ang II-induced aldosterone secretion in OVX rats that were not treated with DEX. Materials and Methods Animals and maintenance Male and female Sprague Dawley rats, weighing 240 –300 g, were individually housed in a temperature-controlled room with a regular light-dark cycle. Rats were allowed to acclimate to the facility for 5–7 days before beginning any study and were provided standard chow and tap water ad libitum. When female rats were studied, these animals were OVX under methoxyflurane anesthesia 14 days before any studies. All procedures were approved by the Georgetown University animal care and use committee.

Jugular vein catheterization Silicone rubber catheters (id, 0.02 in.; od, 0.037 in.; Sil-Med Corp., Taunton, MA) were prepared by allowing a ball of SILASTIC brand medical adhesive (Dow Corning Corp., Midland, MI) to dry around the perimeter 3.1 cm from one end of a 12.5-cm-long piece of tubing. Under methoxyflurane anesthesia (Schering Plough Animal Health Corp., Union, NJ), the short end of a catheter filled with heparinized (100 U/ml), sterile 0.15-m NaCl solution was inserted into the right jugular vein. The catheter was anchored to the pectoral muscle by tying 4 – 0 surgical silk around the adhesive ball, and a 15-gauge trocar (Becton Dickinson and Co., Sparks, MD) was used to exteriorize the free end of the catheter in the dorsal scapular region. The incision was closed with 9-mm wound clips (Becton Dickinson and Co., Franklin Lakes, NJ), and the catheter was plugged with a 1-cm piece of 21-gauge stainless steel wire (Small Parts, Inc., Miami Lakes, FL).

Ang II infusion Ang II was dissolved in sterile 0.15 m NaCl and infused using Razel syringe pumps at the rates specified in the infusion protocols (Razel Scientific Instruments, Inc., Stamford, CT).

Blood sampling Each jugular catheter was connected to a 100-cm long piece of polyethylene tubing (id, 0.76 mm; od, 1.22 mm; PE-60, Becton Dickinson and Co.) filled with heparinized (50 U/ml) saline using a 1-cm-long piece of 21-gauge stainless steel hypodermic tubing (Small Parts, Inc.). The dead

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space in this catheter was approximately 0.5 ml. The catheter was routed to the exterior of the animal’s cage, and a three-way stopcock (VWR Scientific, Bridgeport, NJ) was connected to the end of the catheter with a 21-gauge blunt needle. The rats were allowed to recover from the stress of handling for at least 30 min before beginning a study. Blood samples were withdrawn slowly (over 1–2 min), and a volume of heparinized saline (50 U/ml) equal to the sample volume was infused after each sample. Food, water, and saline were not provided during the bloodsampling experiments. Blood samples were collected in 3-ml Vacutainer blood collection tubes (Becton Dickinson and Co.) containing EDTA or sodium heparin.

Effect of DEX on the dose response of Ang II-induced aldosterone secretion in NaCl-deprived rats Twelve male rats were fed a NaCl-deficient diet (TD 95087, Harlan Teklad, Madison, WI) for 7 days. On day 5, jugular vein catheters were inserted, and on day 7, aldosterone secretion was studied after graded 30-min infusions of Ang II at rates of 1.7, 3.3, 33, and 330 ng/min. As several investigators have found that Ang II infusions increase plasma ACTH (22, 23), four of the rats were pretreated with an ip injection of dexamethasone sodium phosphate (DEX; 100 ␮g) 3 h before beginning the study. The following pattern was repeated until all four infusions were completed: a baseline sample was taken (0.5 ml), a dose of Ang II was infused over 30 min, a sample was taken 30 min after beginning the infusion (0.5 ml), another sample was taken 30 min after the end of the infusion (0.5 ml), and the next infusion was begun immediately thereafter.

Effects of E2 on PRA, aldosterone, plasma Na⫹, plasma K⫹, osmolality, hematocrit, and plasma protein in OVX, NaCldeprived rats OVX rats were treated daily with sc injections of peanut oil (0.2 ml; n ⫽ 12) or E2 (10 mg in 0.2 ml peanut oil; n ⫽ 12) for 14 days. On days 10 –14, the rats were fed TD 95087. On day 12, catheters were inserted into the right jugular veins of half of the animals [6 OVX and 6 OVX and E2 treated (OVX⫹E2)]. On day 14, an iv blood sample (2.5 ml) was collected from these animals in tubes containing heparin for measurement of aldosterone, plasma Na⫹, plasma K⫹, osmolality, hematocrit, and plasma protein. The other 12 animals (6 OVX and 6 OVX⫹E2) were killed by decapitation on day 14, and a sample of trunk blood was collected in tubes containing EDTA for measurement of PRA.

Effect of E2 on ACTH-independent and ACTH-dependent Ang II-induced aldosterone secretion in OVX, NaCldeprived rats To determine the effect of E2 on ACTH-independent Ang II-induced aldosterone secretion, OVX rats were treated with sc injections of peanut oil (0.2 ml; n ⫽ 17) or E2 (10 ␮g; n ⫽ 17) for 14 days. All rats were fed TD 95087 on days 8 –14. On day 11, silicone catheters were inserted into the right jugular vein. On days 12–14, injections of DEX (25 ␮g in 1 ml sterile isotonic saline, injected ip) were given every 12 h at lights-on and lights-off for a total of five injections. This DEX injection protocol has been shown to inhibit ACTH responses to potent ACTH-releasing stimuli (29 –31). On day 14, aldosterone secretion was studied after a 30-min infusion of Ang II (330 ng/min) in conscious ambulatory animals. To determine whether the observed effects of the Ang II infusion on aldosterone secretion were mediated via the AT1 receptor subtype, some of the OVX (n ⫽ 4; OVX⫹L) and some of the OVX⫹E2 (n ⫽ 6; OVX⫹E2⫹L) rats were given a 2.5-mg iv bolus of the AT1 selective antagonist losartan (L; dissolved in 1 ml sterile, isotonic saline) 30 min before beginning the infusion of Ang II (control rats were given an iv bolus of 1 ml sterile isotonic NaCl 30 min before beginning the infusion). In a recent study this dose and route of losartan administration were shown to eliminate the pressor response to an iv infusion of Ang II (32). Plasma samples were collected at baseline and 30 (1.2 ml), 60 (0.7 ml), and 120 (0.9 ml) min after the beginning of the Ang II infusion for measurement of aldosterone and ACTH. Each sample volume was immediately replaced with an equal volume of sterile isotonic NaCl. Blood samples were collected in 3-ml Vacutainer blood collection tubes (Becton


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Dickinson and Co.) containing EDTA (for ACTH) or sodium heparin (for aldosterone). As plasma ACTH normally varies between 15– 45 pg/ml during the course of the day in female rats (33), animals that attained baseline ACTH levels greater than 45 pg/ml and stimulated ACTH levels greater than 100 pg/ml were excluded from subsequent analysis. To determine the effect of E2 on ACTH-dependent Ang II-induced aldosterone secretion, 10 female rats were OVX and treated with sc injections of peanut oil (0.2 ml; n ⫽ 5) or E2 (10 ␮g; n ⫽ 5) for 14 days. All rats were fed TD 95087 on days 8 –14, and jugular catheters were inserted on day 11. On day 14, plasma samples were collected as described above for measurement of aldosterone and ACTH after a 30-min infusion of Ang II (330 ng/min).

adrenal membrane preparation made from each animal (three OVX, three OVX⫹E2, three OVX⫹DEX, and three OVX⫹E2⫹DEX rats), and all determinations were performed in triplicate. Data from each animal were fit to the equation y ⫽ x(Bmax)/(Kd ⫹ x), where Bmax is the total number of receptors, and Kd is the equilibrium dissociation constant) using nonlinear regression and the program PRISM (GraphPad Software, Inc., San Diego, CA). The relationship between bound and bound/ free was plotted (34), and lines were computer generated (slope ⫽ ⫺1/Kd; y-intercept ⫽ ⫺slope ⫻ Bmax) using the average values for Bmax and Kd obtained by nonlinear regression.

Blood analyses

The integrated (s) aldosterone response in each animal was calculated using the program PRISM. Integrated aldosterone responses, AT1 receptor binding values, and basal values for PRA, aldosterone, plasma Na⫹, plasma K⫹, osmolality, hematocrit, and protein were compared using an unpaired t test. To analyze the effects of both treatment (DEX or E2) and time on plasma aldosterone and ACTH levels, values were analyzed by two-way ANOVA corrected for repeated measures, and individual means were compared using the Student-Newman-Keuls post-hoc test. Data are expressed as the mean ⫾ sem.

Hematocrits were measured using a microhematocrit centrifuge and a microcapillary reader. Blood samples were stored on ice until centrifugation at 3000 rpm in a refrigerated centrifuge. Plasma was stored at ⫺20 C until assayed. Plasma Na⫹ and K⫹ were measured with ionselective electrodes using a Synchron Elise Electrolyte System (Beckman Coulter, Inc., Brea, CA). Plasma osmolality was measured using Advanced Osmometer model 3D3 (Advanced Instruments, Inc., Norwood, MA). Plasma proteins were determined using a clinical refractometer (type 5711–2020, Schuco, Carle Place, NY). PRA was measured by RIA (GammaCoat Plasma Renin Activity, DiaSorin, Inc., Stillwater, MN). Plasma aldosterone was measured by RIA (Coat-A-Count Aldosterone, Diagnostic Products, Los Angeles, CA). Using 200 ␮l plasma, this assay detects plasma levels ranging from 25–1200 pg/ml. Stimulated samples were diluted in the supplied zero calibrator. Plasma ACTH was measured by RIA (ACTH Double Antibody RIA, DiaSorin, Inc.). When 100 ␮l plasma are assayed, this method detects plasma ACTH concentrations ranging from 20 –500 pg/ml. When plasma aldosterone or ACTH levels were below the limit of assay detection, the values were reported as the limit of detection.

Effect of E2 on adrenal AT1 receptor expression in OVX, NaCl-deprived rats To determine whether E2 reduces AT1 receptors in the adrenals of NaCl-deprived rats, OVX rats were treated with oil (n ⫽ 6) or E2 (n ⫽ 6) for 14 days as described in the protocol above. On days 8 –14 the rats were fed TD 95087. On days 12–14, three OVX and three OVX⫹E2 rats were treated with five ip injections of DEX as described above. On day 14, all rats were killed, and adrenals were removed for radioligand binding analysis. Adrenals were homogenized by Polytron in 10 vol homogenization buffer (50 mm Tris-HCl and 1 mm EDTA, pH 7.4) supplemented with 10 ␮g/ml leupeptin, 0.2 U/ml aprotinin, 10 ␮g/ml antipain, and 0.1 mm phenylmethylsufonylfluoride at 4 C. The homogenate was filtered through four-ply gauze and centrifuged at 48,000 ⫻ g for 15 min. The pellet was resuspended in homogenization buffer and centrifuged at 48,000 ⫻ g for 15 min. The second pellet was resuspended in binding buffer (10 mm Na2HPO4, 100 mm NaCl, and 5 mm EDTA, pH 7.4). Protein concentrations of adrenal membranes were determined by the Bradford method using BSA as the standard (Bio-Rad Laboratories, Inc., Richmond, CA). Adrenal membranes (5–10 ␮g/tube) were sonicated for 5 sec with a Sonifer cell disrupter immediately before being incubated with increasing concentrations (0.05– 4 nm) of 125I-[Sar1,Ile8]Ang II (Peptide Radioiodination Center, Pullman, WA) for 2 h at room temperature in 0.3 ml binding buffer supplemented with 0.1% BSA. Bound tracer was rapidly separated from unbound tracer by washing filters four times with icecold PBS using a Brandel vacuum harvester (model M-24, Gaithersburg, MD). Radioactivity was measured in a ␥-counter (Cobra, Packard, Downers Grove, IL). Preincubation of glass-fiber filters with 10% BSA overnight was used to reduce nonspecific absorption of the radioligand to the filters. To determine changes in AT1 receptor expression, membranes were incubated the presence of increasing concentrations of 125 I-[Sar1,Ile8]Ang II and 10 ␮m PD-123,319 (AT2 receptor antagonist). For each curve, nonspecific binding was determined at each concentration of 125I-[Sar1,Ile8]Ang II in the presence of 200 nm Ang II and 10 ␮m PD-123,319. Specific binding was defined as the total radioligand bound minus nonspecific binding. All binding analyses were conducted on the

Statistical analyses

Results Effect of DEX on the dose-response of Ang II-induced aldosterone secretion in NaCl-deprived rats

Basal plasma aldosterone was 526 ⫾ 93 pg/ml in control, NaCl-deprived rats (n ⫽ 8). Plasma aldosterone was not significantly increased from baseline after 30-min infusions of Ang II at rates of 1.7 and 3.3 ng/min (Fig. 1). However, 30-min infusions of Ang II at rates of 33 and 330 ng/min significantly (P ⬍ 0.05) increased plasma aldosterone levels in control, NaCl-deprived rats (1950 ⫾ 77 and 3170 ⫾ 340 pg/ml, respectively; Fig. 1). DEX significantly reduced basal aldosterone concentrations in NaCl-deprived rats to near the assay detection limit (163 ⫾ 63 pg/ml; n ⫽ 4; Fig. 1). Plasma aldosterone levels were not significantly increased from baseline in DEX-treated rats after 30-min infusions of Ang II

FIG. 1. Aldosterone secretion in response to 30-min graded infusions of Ang II. Male rats (n ⫽ 12) were NaCl deprived for 7 days before beginning the study. Four of the rats were treated with an ip injection of DEX (100 ␮g) 3 h before beginning the study. The box indicates the timing and rate of each Ang II infusion. Blood samples (0.5 ml each) were taken every 30 min during the 4-h study. E, Control, NaCldeprived male rats (n ⫽ 8). F, DEX-treated, NaCl-deprived male rats (n ⫽ 4). Data are expressed as the mean ⫾ SEM. *, P ⬍ 0.05 compared with basal levels. ‡, P ⬍ 0.05 compared with control, NaCl-deprived rats.

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at rates of 1.7, 3.3, and 33 ng/min (Fig. 1). In comparison, a 30-min infusion of Ang II at a rate of 330 ng/min significantly (P ⬍ 0.05) increased plasma aldosterone levels above baseline levels in DEX-treated animals (1240 ⫾ 140 pg/ml). In addition, the stimulated values (at 2.5 and 3.5 h) were significantly (P ⬍ 0.05) lower in DEX-treated rats than in control, NaCl-deprived rats (Fig. 1). Effect of E2 on basal values of PRA, aldosterone, plasma Na⫹, plasma K⫹, osmolality, hematocrit, and plasma protein in OVX, NaCl-deprived rats

E2 did not significantly alter basal PRA or plasma aldosterone concentrations in NaCl-deprived, OVX rats (Table 1). However, E2 significantly (P ⬍ 0.05) increased plasma Na⫹ and protein and decreased plasma K⫹ (Table 1). Effect of E2 on ACTH-independent aldosterone secretion in OVX, NaCl-deprived rats

FIG. 2. Aldosterone secretion in response to a 30-min infusion of Ang II (330 ng/min) in OVX, NaCl-deprived, DEX-treated rats. OVX (n ⫽ 8; E), OVX⫹L (n ⫽ 3; F), OVX⫹E2 (n ⫽ 7; ƒ), OVX⫹E2⫹L (n ⫽ 3; ). The period of Ang II infusion is indicated with a box. Data are expressed as the mean ⫾ SEM. *, P ⬍ 0.05 compared with basal levels. ‡, P ⬍ 0.05 compared with OVX rats.

In DEX-treated rats, basal plasma aldosterone levels were near the limit of detection of the assay, and there was no significant effect of treatment on the basal aldosterone concentration (Fig. 2). In OVX (n ⫽ 8) rats, plasma aldosterone increased to 1870 ⫾ 290 pg/ml by the end of the infusion of Ang II at 330 ng/min (P ⬍ 0.05 compared with baseline), and levels returned toward baseline by 60 and 120 min (Fig. 2). In E2-treated rats (n ⫽ 7), plasma aldosterone only increased to 1010 ⫾ 86 pg/ml after the end of the Ang II infusion (P ⬍ 0.05 compared with baseline; Fig. 2). This level of stimulation was significantly (P ⬍ 0.05) less than the level of stimulation attained in OVX rats at the same time point. Plasma aldosterone did not significantly change over time in OVX (OVX⫹L; n ⫽ 3) and OVX⫹E2 (OVX⫹E2⫹L; n ⫽ 3) rats pretreated with an iv bolus of the AT1-selective antagonist losartan (Fig. 2), and plasma ACTH did not change significantly over time in any treatment group (Table 2).

tions increased to 220 ⫾ 29 pg/ml after the end of the Ang II infusion, and in E2-treated rats, the peak ACTH response was significantly decreased (P ⬍ 0.05; 160 ⫾ 20 pg/ml; Fig. 3B).

Effect of E2 on ACTH-dependent aldosterone secretion in OVX, NaCl-deprived rats

Effect of E2 on adrenal AT1 receptor expression in OVX, NaCl-deprived rats

Basal aldosterone levels were not significantly different between OVX (527 ⫾ 180 pg/ml) and E2-treated (398 ⫾ 73 pg/ml) rats (Fig. 3A). In OVX rats plasma aldosterone concentrations increased to 5360 ⫾ 1200 pg/ml after the end of the Ang II infusion, and in E2-treated rats, the peak aldosterone response was significantly decreased (P ⬍ 0.05; 2960 ⫾ 570 pg/ml; Fig. 3A). In OVX rats, plasma ACTH concentra-

When the relationship between free radioligand concentration and specific AT1 receptor binding in each individual adrenal obtained from OVX, NaCl-deprived rats was plotted and fit by nonlinear regression to the equation y ⫽ x(Bmax)/ (Kd ⫹ x), the r2 values for the nonlinear regressions ranged from 0.87– 0.99 (representative plots and nonlinear regressions are shown in Fig. 4A). The average values of the nonlinear regression analyses indicate that in OVX, NaCl-deprived rats, E2 significantly (P ⬍ 0.05) decreased adrenal AT1 receptor number (Bmax) from 345 ⫾ 4 to 190 ⫾ 10 fmol/mg protein, and E2 did not significantly change the Kd for AT1 binding (OVX, 0.08 ⫾ 0.02; OVX⫹E2, 0.19 ⫾ 0.09). A Scatchard plot of the data showing the relationship between bound and bound/free for all rats analyzed is presented in Fig. 4B. The lines shown in Fig. 4B were generated from the average Bmax and Kd values obtained by nonlinear regression. In DEX-treated rats, the r2 values for the nonlinear regressions ranged from 0.86 – 0.99 (representative plots and nonlinear regressions are shown in Fig. 5A). The average values of the nonlinear regressions indicate that in OVX, NaCl-deprived, DEX-treated rats, E2 also significantly (P ⬍

TABLE 1. Effect of E2 on basal values for PRA, aldosterone, Na⫹, K⫹, osmolality, hematocrit, and protein in NaCl-deprived, OVX rats

PRA (ng/ml 䡠 h) Aldosterone (pg/ml) Na⫹ (mmol/liter) K⫹ (mmol/liter) Osmolality (mosmol/KgH2O) Hematocrit (%) Plasma protein (g/100 ml)

OVX

OVX ⫹ E2

97 ⫾ 13 1300 ⫾ 400 136.0 ⫾ 0.5 6.2 ⫾ 0.3 283 ⫾ 3

97 ⫾ 18 1100 ⫾ 240 139.0 ⫾ 0.5a 4.6 ⫾ 0.2a 286 ⫾ 2

37 ⫾ 1 5.3 ⫾ 0.2

34 ⫾ 2 6.6 ⫾ 0.2a

Note, these rats were not treated with DEX. a P ⬍ 0.05 compared to OVX.

TABLE 2. Plasma ACTH levels after a 30-min infusion of Ang II (333 ng/min) in OVX, NaCl-deprived, DEX-treated rats pretreated with an iv bolus of saline or losartan (2.5 mg) Time (min)

Baseline ⫹30 ⫹60 ⫹120

ACTH (pg/ml) OVX

OVX ⫹ L

OVX ⫹ E2

OVX ⫹ E2 ⫹ L

28.9 ⫾ 3.4 32.8 ⫾ 2.6 41.8 ⫾ 9.3 31.6 ⫾ 3.8

36.1 ⫾ 3.2 37.3 ⫾ 2.6 35.9 ⫾ 3.5 38.1 ⫾ 6.8

30.3 ⫾ 1.3 35.3 ⫾ 1.4 35.2 ⫾ 3.2 30.0 ⫾ 1.6

34.0 ⫾ 3.5 33.8 ⫾ 4.7 32.0 ⫾ 4.7 33.8 ⫾ 2.6


FIG. 3. Plasma aldosterone (top) and ACTH (bottom) responses to a 30-min infusion of Ang II (330 ng/min) in OVX, NaCl-deprived rats. Note that these rats were not treated with DEX. Data are expressed as the mean ⫾ SEM. E, OVX; ƒ, OVX⫹E2. *, P ⬍ 0.05 compared with basal levels. ‡, P ⬍ 0.05 compared with OVX rats.

0.05) decreased the AT1 Bmax from 450 ⫾ 5 to 317 ⫾ 38 fmol/mg protein, and E2 did not significantly change the Kd for AT1 binding (OVX⫹DEX, 0.12 ⫾ 0.01 nm; OVX⫹E2⫹DEX, 0.14 ⫾ 0.01 nm). A Scatchard plot of the data showing the relationship between bound and bound/free for all rats analyzed and the lines generated from the average Bmax and Kd values obtained by nonlinear regression is presented in Fig. 5B. Summary

In the absence of DEX, E2 significantly (P ⬍ 0.05) reduced adrenal AT1 Bmax by 45% (Fig. 6A), and significantly (P ⬍ 0.05) reduced the integrated (concentration ⫻ time) aldosterone response to Ang II by a similar magnitude (47%, Fig. 6B). Likewise, in DEX-treated rats, E2 significantly (P ⬍ 0.05) reduced adrenal AT1 Bmax by 30% (Fig. 6A) and the integrated aldosterone response (Fig. 6B) by 23%. Discussion

Previous studies have shown that E2 reduces adrenal AT receptor expression in OVX rats that are NaCl loaded (20, 21). In this study we demonstrate that E2 also reduces the expression of adrenal AT1 receptors by approximately 30% both in rats that are NaCl deprived and in rats that are NaCl deprived and DEX treated. By feeding rats a NaCl-deficient diet and treating them with DEX, we were able to show that

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FIG. 4. Adrenal AT1 receptor binding in OVX, NaCl-deprived rats. Note that these rats were not treated with DEX. A, Adrenal membranes from OVX (E and —-) and OVX⫹E2 (ƒ and - - -) animals were incubated with increasing concentrations of 125I-[Sar1,Ile8]Ang II (0.05– 4 nM). The data shown are representative of nonlinear regression analyses of saturation plots from three experiments performed in duplicate. B, Scatchard plots of adrenal AT1 receptor binding in OVX (E and —-) and OVX⫹E2 (ƒ and - - -) rats. The relationship between bound and bound/free is plotted for each of the OVX⫹DEX (E) and OVX⫹E2 (ƒ) rats analyzed (n ⫽ 3/group). The lines were generated from the average Bmax and Kd values obtained by nonlinear regression analysis using the program PRISM (—-, OVX; 䡠䡠䡠, OVX⫹E2).

at the time points when E2 reduces adrenal AT1 receptor expression, E2 also attenuates the plasma aldosterone response to an infusion of Ang II, independently of plasma ACTH. We also found that E2 attenuates Ang II-induced aldosterone secretion in NaCl-deprived rats that are not treated with DEX. However, E2 also significantly decreased the plasma ACTH response to Ang II in these rats, most likely as a result of decreased AT1 receptor binding in the pituitary (19) and in the subfornical organ of the brain (35). Taken together, these findings suggest that E2 attenuates Ang IIstimulated aldosterone secretion in NaCl-deprived rats by reducing both pituitary ACTH and adrenal aldosterone responses to Ang II. The AT1 receptor subtype predominates in adrenal glomerulosa cells (36) and in the pituitary (37), and AT1-selective antagonists completely abolish Ang II-stimulated aldosterone secretion in vivo (38). As Ang II-induced aldosterone secretion was completely blocked by pretreating the OVX, NaCl deprived, DEX-treated rats used in this study with the AT1-selective antagonist losartan, the E2-induced decrease in

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FIG. 5. Adrenal AT1 receptor binding in OVX, NaCl-deprived, DEXtreated rats. A, Adrenal membranes from OVX⫹DEX (E and —-) and OVX⫹E2⫹DEX (ƒ and - - -) animals were incubated with increasing concentrations of 125I-[Sar1,Ile8]Ang II (0.05– 4 nM). The data shown are representative of nonlinear regression analyses of saturation plots from three experiments performed in duplicate. B, Scatchard plots of adrenal AT1 receptor binding in OVX⫹DEX (E and —-) and OVX⫹E2⫹DEX (ƒ and - - -) rats. The relationship between bound and bound/free is plotted for each of the OVX⫹DEX (E) and OVX⫹E2⫹DEX (ƒ) rats analyzed (n ⫽ 3/group). The lines were generated from the average Bmax and Kd values obtained by nonlinear regression analysis using the program PRISM (—-, OVX⫹DEX; 䡠䡠䡠, OVX⫹E2⫹DEX).

the plasma aldosterone response to an Ang II infusion is probably attributable to decreased adrenal AT1 expression. However, the mechanism by which E2 reduces adrenal AT1 expression is currently unknown. An estrogen response element (ERE) has been identified in the 5⬘-flanking region of the AT1 receptor gene (39), suggesting that E2 may directly regulate adrenal AT1 receptor transcription. In addition, this laboratory has recently discovered cytosolic proteins (BP) that bind to the 5⬘-leader sequence (5⬘LS) of the AT1 receptor mRNA (40). We observed that the magnitude of 5⬘LS-BP complex formation inversely correlates with tissue-specific E2-induced changes in maximal AT1 binding. Furthermore, in vitro translation of the AT1 receptor mRNA is decreased when these studies are conducted in the presence of cytosolic extracts obtained from a tissue rich in 5⬘LS-BP activity. Thus, we proposed that E2 may also indirectly modulate AT1 receptor translation by modifying the expression and/or activity of 5⬘LS-BP (19). It is interesting to note that despite equally high PRA and

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FIG. 6. Summary of the effect of E2 on maximal adrenal AT1 binding (A) and Ang II-induced aldosterone secretion (B) in NaCl-deprived, OVX rats treated with (⫹DEX) or without (⫺DEX) DEX. Data are expressed as the mean ⫾ SEM. 䡺, OVX; f, OVX⫹E2. *, P ⬍ 0.05 compared with OVX.

aldosterone levels in both OVX and OVX⫹E2 rats, plasma Na⫹ was significantly increased, and plasma K⫹ was significantly decreased in E2-treated rats. One interpretation of this finding is that E2 enhances the efficacy of the high circulating concentrations of aldosterone. The sodium-retaining effects of estrogens have been known for many years (41). Two recent observations support the hypothesis that estrogens actually enhance chronic mineralocorticoid activity, at least in the kidney: 1) a study of postmenopausal women indicated that ERT increased renal sodium reabsorption after a hypertonic saline infusion without altering plasma aldosterone levels (42); and 2) E2 increased the expression of a mineralocorticoid receptor (MR)-regulated protein, the thiazide-sensitive NaCl cotransporter, in the distal convoluted tubule (43, 44). Although a few studies have investigated the effects of estrogens on MR expression in the brain (45), the results of these studies have been contradictory. Moreover, there are no known reports of the effects of estrogens on MR expression in the kidney. Thus, it is possible that ERT alters renal responsiveness to aldosterone by modifying MR expression, and that this effect leads to increased plasma Na⫹ and decreased plasma K⫹. Although the mechanism by which E2 alters plasma Na⫹ and K⫹ remains to be determined, this effect of E2 on plasma K⫹ may contribute to the effects of E2 on pituitary and adrenal AT1 receptor expression. In the rat, adrenal AT receptor density directly correlates with dietary potassium intake (46, 47), and adrenal responsiveness to Ang II is increased by a


high K⫹ diet (48, 49). Moreover, when rats are fed potassiumdeficient diets, PRA and Ang II concentrations are increased, whereas plasma aldosterone concentrations remain unchanged (46). This constellation of low potassium-induced changes in RAAS activity closely corresponds to the increases in plasma angiotensinogen, PRA, and Ang II concentration that have been associated with the physiological effects of E2 (50) and suggests that E2-induced changes in potassium homeostasis play an important role in modulating adrenal sensitivity to Ang II. We did not observe a significant effect of E2 on basal aldosterone levels in rats that were not treated with DEX. Similarly, other studies have not reported a significant effect of ERT on plasma aldosterone concentrations (12, 13). It is possible that an existing effect of E2 on basal aldosterone concentrations is obscured by individual animal variations and/or environmental stress-induced variations in plasma ACTH. We also did not observe a significant effect of E2 on basal aldosterone levels in the NaCl-deprived, OVX rats that were treated with DEX. However, DEX decreased basal plasma aldosterone levels in the NaCl-deprived rats that were used in this study to near the assay detection limit, so any effect E2 may have had on basal aldosterone concentrations in DEX-treated rats would not have been detected by our assay. The mechanism by which DEX, given as a single injection 3 h before sampling or as a series of five injections over the course of 3 days, decreased basal aldosterone levels in NaCl-deprived rats to near the assay detection limit is unclear. In the past, DEX inhibition of ACTH has been used to reveal the circadian relationship between PRA and aldosterone independently of variation in plasma ACTH (51), suggesting that PRA and aldosterone secretion are not inhibited by DEX. However, others have shown that the natural glucocorticoid, cortisol, attenuates nitroprusside-stimulated PRA in adult ewes (52), suggesting that the synthetic glucocorticoid, DEX, may contribute to inhibition of basal aldosterone levels in NaCl-deprived rats by decreasing PRA. Further study is needed to determine the mechanism by which DEX decreases basal aldosterone concentrations in NaCl-deprived rats and to clarify the potential effects of E2 on basal aldosterone concentration. Although the physiological relevance of the E2-induced decrease in adrenal AT1 receptor expression and Ang IIinduced aldosterone secretion remains to be determined, it is reasonable to consider the possibility that an E2-induced decrease in acute adrenal aldosterone responses may contribute to the cardiovascular benefits of ERT. Recent studies suggest that aldosterone can have rapid effects on cardiovascular function. The hypertensive effects of mineralocorticoids are mediated at least in part via actions in the brain. Intracerebroventricular (icv) infusions or injections of mineralocorticoids increase blood pressure (53, 54), and icv MR antagonists block the hypertensive effects of mineralocorticoids (55, 56) and decrease blood pressure when administered alone (54, 57). A recent study demonstrated that icv administration of a MR antagonist affects blood pressure and renal excretion of water and electrolytes as early as 30 min after icv injection (57). As these studies have found rapid effects of centrally administered adrenal steroids on cardiovascular function, and steroids are known to rapidly diffuse

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across the blood-brain barrier (58), we propose that by modifying relatively acute changes in plasma aldosterone concentration, E2 may exert significant effects on the central regulation of cardiovascular function. It is also well known that when laboratory animals are treated chronically with mineralocorticoids, such as deoxycorticosterone acetate, and diets high in sodium, they develop hypertension (59). This model of hypertension is characterized by increased vascular responsiveness to Ang II, increased vascular resistance, and decreased baroreflex sensitivity; all of these factors are thought to be risk factors for myocardial infarction and stroke (59). The recently completed Randomized Aldactone Evaluation Study supports the likelihood that aldosterone has potential deleterious cardiovascular effects. Blockade of aldosterone receptors with spironolactone substantially reduced the risk of both morbidity and mortality among patients with severe heart failure who were also receiving standard ACE inhibition therapy (60). Therefore, if an E2-induced decrease in acute adrenal aldosterone responses also contributes to decreased chronic mineralocorticoid effects, this could clearly be cardioprotective. In summary, our results demonstrate that in DEX-treated rats, E2 attenuates Ang II-induced secretion independently of plasma ACTH. This finding indicates that E2 simultaneously reduces adrenal AT1 receptor expression and adrenal responsiveness to Ang II. In both DEX-treated and control rats, the magnitude of the E2-induced decrease in the integrated aldosterone response to Ang II correlated closely with the measured decrease in adrenal AT1 binding. In addition, we have shown that in rats that are NaCl deprived but not treated with DEX, E2 also attenuates Ang II-induced ACTH secretion. This finding suggests that E2 attenuates adrenal aldosterone responses to surges in plasma Ang II in NaCldeprived rats partly by decreasing pituitary responsiveness to Ang II. Taken together, these results suggest that by decreasing the expression of brain, pituitary, and adrenal AT1 receptors, E2 attenuates acute adrenal aldosterone responses to Ang II. We propose that this effect may contribute to the cardioprotective effects of ERT. References 1. National High Blood Pressure Education Program Working Group 1994 National High Blood Pressure Education Program Working Group Report on Hypertension in the Elderly. Hypertension 23:275–285 2. Hunt BE, Davy KP, Jones PP, DeSouza CA, Van Pelt RE, Tanaka H, Seals DR 1997 Systemic hemodynamic determinants of blood pressure in women: age, physical activity, and hormone replacement. Am J Physiol 273:H777–H785 3. Belmin J, Levy BI, Michel JB 1994 Changes in the renin-angiotensin-aldosterone axis in later life. Drugs Aging 5:391– 400 4. Rakotondrazafy J, Brudieux R 1993 Age-related change in plasma aldosterone response to exogenous angiotensin II in the rat. Horm Res 39:156 –160 5. Takeda R, Morimoto S, Uchida K, Miyamori I, Hashiba T 1980 Effect of age on plasma aldosterone response to exogenous angiotensin II in normotensive subjects. Acta Endocrinol (Copenh) 94:552–558 6. Kau MM, Chen JJ, Wang SW, Cho WL, Wang PS 1999 Age-related impairment of aldosterone secretion in zona glomerulosa cells of ovariectomized rats. J Invest Med 47:425– 432 7. Heymes C, Silvestre JS, Llorens-Cortes C, Chevalier B, Marotte F, Levy BI, Swynghedauw B, Samuel JL 1998 Cardiac senescence is associated with enhanced expression of angiotensin II receptor subtypes. Endocrinology 139:2579 –2587 8. Duggan J, Kilfeather S, O’Brien E, O’Malley K, Nussberger J 1992 Effects of aging and hypertension on plasma angiotensin II and platelet angiotensin II receptor density. Am J Hypertens 5:687– 693 9. Tank JE, Vora JP, Houghton DC, Anderson S 1994 Altered renal vascular responses in the aging rat kidney. Am J Physiol 266:F942–F948

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