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Endocrinology Vol. 147, No. 7 3539-3546
Copyright © 2006 by The Endocrine Society

Angiotensin II Inhibition Reduces Stress Sensitivity of Hypothalamo-Pituitary-Adrenal Axis in Spontaneously Hypertensive Rats

Walter Raasch, Christian Wittmershaus, Andreas Dendorfer, Inga Voges, Friedrich Pahlke, Christoph Dodt, Peter Dominiak and Olaf Jöhren

Institutes of Experimental and Clinical Pharmacology and Toxicology (W.R., C.W., A.D., I.V., P.D., O.J.) and Medical Biometry and Statistics (F.P.) and the Medical Clinic I (C.D.), University Clinic of Schleswig-Holstein, Campus Lübeck, 23538 Lübeck, Germany

Address all correspondence and requests for reprints to: Walter Raasch, Ph.D, Institute of Experimental and Clinical Pharmacology and Toxicology, University Clinic of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail: raasch{at}medinf.mu-luebeck.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II type 1 (AT1) receptors are expressed within organs of the hypothalamo-pituitary-adrenal (HPA) axis and seem to be important for its stress responsiveness. Secretion of CRH, ACTH, and corticosterone (CORT) is increased by stimulation of AT1 receptors. In the present study, we tested whether a blockade of the angiotensin II system attenuates the HPA axis reactivity in spontaneously hypertensive rats. Spontaneously hypertensive rats were treated with candesartan (2 mg/kg), ramipril (1 mg/kg), or mibefradil (12 mg/kg) for 5 wk. In addition to baseline levels, CORT and ACTH responses to injection of CRH (100 µg/kg) were monitored over 4 h. mRNA of CRH, proopiomelanocortin, AT1A, AT1B, and AT2 receptors was quantified by real-time PCR. All treatments induced equivalent reductions of blood pressure and had no effect on baseline levels of CORT and ACTH. However, both candesartan and ramipril significantly reduced CRH-stimulated plasma levels of ACTH (–26 and –15%) and CORT (–36 and –18%) and lowered hypothalamic CRH mRNA (–25 and –29%). Mibefradil did not affect any of these parameters. Gene expression of AT1A, AT1B, and AT2 receptors within the HPA axis was not altered by any drug. We show for the first time that antihypertensive treatment by inhibition of AT1 receptors or angiotensin-converting enzyme attenuates HPA axis reactivity independently of blood pressure reduction. This action is solely evident after CRH stimulation but not under baseline conditions. Both a reduced pituitary sensitivity to CRH and a down-regulation of hypothalamic CRH expression have the potential to reduce HPA axis activity during chronic AT1 blockade or angiotensin-converting enzyme inhibition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (ANG) is the main active peptide of the renin-angiotensin-aldosterone system (RAAS) and stimulates two distinct receptor subtypes, namely type 1 (AT1) and type 2 (AT2) receptors. The significance of AT1 receptors in the regulation of cardiovascular functions, fluid homeostasis, and hormone release is well known, and several AT1 blockers are established in the treatment of cardiovascular diseases (1). Two pharmacologically similar AT1 receptor isoforms, AT1A and AT1B, have been identified in rodents, which are encoded by two distinct genes and are expressed and regulated differentially (2). Both isoforms of the AT1 receptor as well as the AT2 receptor are present in organs of the hypothalamo-pituitary-adrenal (HPA) axis, the major system of endocrine stress (3, 4, 5, 6, 7). Because ANG receptors are known to be regulated within the HPA axis by restraint stress (8, 9, 10, 11), it was concluded that ANG itself may modulate the reactivity of the HPA axis. Indeed, ANG has been shown to influence synthesis and secretion of CRH and ACTH as well as corticosterone (the major glucocorticoid in the rat) (12, 13, 14). In pituitary cells, stimulation of ANG receptors increases the CRH-induced ACTH secretion (15, 16). In the hypothalamus, AT1 receptors activate the HPA axis by modulation of gene expression; CRH was up-regulated in an AT1-dependent manner during immobilization stress (13), and hypothalamic AT1A receptor mRNA was reduced after chronic AT1 receptor blockade (17).

Activation of the HPA axis by ANG may contribute to the pathogenesis, and in particular to the metabolic derangements, of genetic hypertension. Spontaneously hypertensive rats (SHR) respond more sensitively to ANG regarding release of ACTH and corticosterone, and this has been related to a higher expression of AT1A receptors in the pituitary gland when compared with normotensive WKY rats, whereas AT1B receptors were decreased in pituitary and adrenal glands (18). A similar coincidence between hypertension and enhanced stress sensitivity of the HPA axis has also been demonstrated in hypertensive patients (19). Thus, it is important to know whether antihypertensive drugs differ in their ability to attenuate the stress sensitivity of the HPA axis and whether treatments targeting the RAAS show such beneficial potential independently of blood pressure reduction.

In the present study, we determined reactivity of the HPA axis in terms of ACTH and corticosterone levels after CRH injection and plasma glucose profiles in SHR that were long-term treated with the AT1 blocker candesartan or the angiotensin-converting enzyme (ACE) inhibitor ramipril. Additional rats were treated with the T-type calcium antagonist mibefradil to control for the effects of blood pressure reduction, because mibefradil was shown to reduce blood pressure equieffectively to candesartan and ramipril in previous studies (20, 21). Chronic alteration in hypothalamus and pituitary glands were assessed by measurements of the expression of CRH, proopiomelanocortin (POMC), and AT1A and AT1B receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and chemicals
Ramipril, candesartan-cilexetil, enalaprilat, and mibefradil were generous gifts from Astra-Zeneca (Wedel, Germany) and Hoffmann-La Roche (Grenzach-Wyhlen, Germany), respectively. For drug administration, substances were suspended in distilled water by using gum arabic (10% wt/vol) and were kept at 4 C for not more than 1 wk.

Animals
Adult male SHR (Charles River, Sulzfeld, Germany) were used. All rats were 9 wk old at the beginning of the study, and the groups did not differ in body weight before drug treatment (275.0 ± 1.4 g). The study was conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The animals were kept at room temperature with a 12-h light (0200–1400 h), 12-h dark (1400–0200 h) cycle. They received a standard diet and water ad libitum. Rats were habituated to research assistants and vice versa 3 wk before drug treatment was initiated.

Study protocol
SHR (n = 10 each for dosage, each drug) were daily treated by gavage for 5 wk with either candesartan-cilexetil (2 mg/kg body weight per day), ramipril (1 mg/kg body weight per day) or mibefradil (12 mg/kg body weight per day). Controls were given an identical volume of gum arabic suspension (10% wt/vol; 1 µl/1 g body weight). At d 29 of treatment, chronic polyethylene catheters were inserted during pentobarbitone anesthesia (75 mg/kg) into the right femoral vein and artery. Catheters were tunneled under the back skin, exteriorized in the region of the cervical vertebra, and fixed at the skin. For habituation toward isolation, rats were housed individually into cages (height x width x length, 20 x 22 x 25 cm) 3 d before the CRH test was performed until the end of the study.

Blood pressure and heart rate were monitored via arterial catheters 1 d after catheterization between 0900 and 1000 h. Values were recorded for 5 min and expressed as means within this time period. Afterward, blood (1 ml) was withdrawn from the femoral artery to obtain serum for the determination of baseline ACTH, corticosterone, aldosterone, glucose, and insulin (Fig. 1Go).


Figure 1
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  		FIG. 1. Schedule of the study protocol.

 
One day later, CRH tests were performed. During CRH tests, rats had free access to water and food. Three hours before starting the tests, arterial catheters were extended by approximately 4 cm to avoid stress reactions during blood withdrawal. CRH (100 µg/kg, iv) was injected 3 h before the light cycle. During CRH tests, the lights at working benches were kept as dim as possible to maintain the dark period. Before CRH injections and every 15 min within a 4-h time period, 50 µl blood was withdrawn. To avoid hemorrhage-induced alterations, platelets and erythrocytes were reconstituted and returned to each animal. One day after CRH tests, rats were killed and organs were removed for biochemical analysis. For the determination of ANG, blood (2 ml) was collected into an inhibitor solution containing 12.1 mM EDTA and 20 µM bestatin (final concentration).

Biochemical analysis
Plasma concentrations of ACTH, corticosterone, aldosterone (MP Biomedicals, Heidelberg, Germany), ANG (IBL, Hamburg, Germany) or insulin (Linco, St. Charles, MO) were determined by RIAs using commercial kits. Assays were adapted to the small sample size and performed accordingto the manufacturer’s instructions (ACTH, 100 µl; corticosterone, 25 µl of a 1:20 dilution; insulin, 50 µl; ANG, 250 µl). After determining corticosterone, plasma samples of CRH test were pooled for measuring ACTH and insulin because of limited sample volumes, covering the time periods 45–90, 135–160, and 195–240 min with respect to ACTH and covering the time periods 0–60, 75–120, 135–180, and 195–240 min with respect to insulin. Blood glucose was determined using glucose sensors based on an amperometric measurement after enzymatic glucose oxidation (Ascensia ELITE XL; Bayer, Leverkusen, Germany).

ACE activity was measured in serum of trunk blood using ABz-Gly-p-nitro-Phe-Pro-OH and a fluorometric quantification after HPLC separation (21).

Quantification of mRNA of CRH, POMC, AT1A, AT1B, and AT2 receptors in organs of the HPA axis
Hypothalamus was prepared from whole brain in a freezer by taking care that tissue did not defrost. Total RNA was isolated. Isolation of genomic DNA was avoided by thorough treatment with DNase I. cDNA was synthesized using standard kits RNeasy Kit (QIAGEN, Hilden, Germany) and Reverse Transcription System (Promega, Mannheim, Germany). Quantitative measurements of mRNA were performed by kinetic PCR with the cycle threshold method using SYBR green I as a fluorescent dye on the GeneAmp 7000 sequence detection system (PerkinElmer Applied Biosystems, Weiterstadt, Germany) by using cDNA-specific primers (CRH sense, 5'-AAA GGG GAA AGG CAA AGA AA-3'; CRH antisense, 5'- GTT TAG GGG CGC TCT CTT CT-3'; POMC sense, 5'- GAA GGT GTA CCC CAA TGT CG-3'; POMC antisense, 5'- CTT CTC GGA GGT CAT GAA GC-3'). If possible, primers across exon-exon junctions were designed. Primers for AT1A, AT1B, and AT2 receptors and ß-actin were published previously (21). All primers were obtained from Live Technologies GmbH (Karlsruhe, Germany). Contamination with genomic DNA was monitored by omitting the reverse transcriptase, and no-template controls served as negative control (22).

Statistics
Data shown are expressed as means ± SEM. Values were excluded from statistical analysis according to Grubb’s outlier test (www.graphpad.com/quickcalcs/Grubbs1.cfm). To quantify the overall effect of CRH toward corticosterone or ACTH release, the area under the curves (AUC) were calculated for each individual animal on the basis of their delta-values. Statistical analysis was performed by one- or two-way ANOVA, followed by appropriate post hoc tests (Bonferroni's multiple comparison test). Wilcoxon signed rank test was used when variances differed between groups. Student’s t test was executed for the statistical analysis of only two groups. Differences were considered to be statistically significant at an error level of P < 0.05. A nonparametric bootstrap procedure with 1 million replicates was applied to investigate differences in mRNA levels of CRH, POMC, and AT receptors between the different treatment groups in various organs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Basal parameters
Body weight was not affected by different treatment regimes (controls, 313 ± 2 g). Mean arterial blood pressure was reduced by at least 30 mm Hg by all substances, with candesartan being slightly more effective (P < 0.05; Fig. 2AGo). Heart rate was not affected by candesartan or ramipril but was decreased by mibefradil (Fig. 2BGo), which has to be attributed to a suppression of sinoatrial node activity (23). Left ventricular weight was diminished only after treatment with candesartan (15%) or ramipril (9%; Fig. 2CGo). Specific influences of ramipril or candesartan on ACE and AT1 receptors, respectively, were demonstrated by a halved plasma ACE activity after ramipril and a reduced aldosterone plasma concentration after candesartan (Table 1Go). ANG levels were increased by candesartan but not by ramipril or mibefradil treatments (Table 1Go). Baseline concentrations of glucose and insulin were not influenced by candesartan or by ramipril. However, plasma glucose was significantly increased by mibefradil, which may be attributed to a reduction in insulin (Table 1Go).


Figure 2
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  		FIG. 2. Effects of candesartan-cilexitil (CAN), ramipril (RAM), mibefradil (MIB), and vehicle (CON) in SHR after 5 wk of treatment on mean arterial pressure (MAP) (A), heart rate (HR) (B), and left ventricular weight (LVW) (C). Results are shown as means ± SEM (n = 9–10); *, P < 0.05 vs. controls; {dagger}, P < 0.05 vs. MIB.

 

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  		TABLE 1. Baseline endocrinological and metabolic parameters of SHR after pretreatment (5 wk) with candesaratan-cilexetil (CAN), ramipril (RAM), or mibefradil (MIB) but before CRH tests

 
HPA axis reactivity
The first day after catheter insertion, plasma concentrations of corticosterone and ACTH were not specifically altered by candesartan, ramipril, or mibefradil (Table 1Go). The second day after catheter insertion directly before the CRH injection, corticosterone levels tended to be decreased (25%) in all treatment groups (Fig. 3Go), indicating recovery from surgical stress. Consequent to CRH injections, ACTH and corticosterone increased in controls by factors of 3.6 and 2.3, respectively (Figs. 3Go and 4Go), and corticosterone remained elevated throughout the observation period. In contrast, ACTH levels returned to baseline values. The maximal ACTH increase in response to CRH was observed in pooled samples covering the time period 45–90 min after injections (Fig. 4AGo). Peak corticosterone levels were obtained after 78 ± 10 min (Fig. 3Go). Maximal ACTH release was significantly blunted by candesartan (–26%) and ramipril (–15%) but not by mibefradil (–4%). The reduced ACTH response after candesartan treatment was also evident in the 135- to 180-min fraction (–25%; Fig. 4AGo). The candesartan- and ramipril-mediated reduction in ACTH release is also evident when focused on the integral between concentration and time (Fig. 4BGo). In parallel to ACTH, corticosterone release in response to CRH was diminished by candesartan or ramipril but not by mibefradil (Fig. 3AGo), as reflected by the AUC (Fig. 3BGo) and the maximal increases of plasma concentrations (controls, 327 ± 28 ng/ml; candesartan, 209 ± 18 ng/ml, P < 0.05; ramipril, 269 ± 24 ng/ml, P < 0.05; mibefradil, 332 ± 21 ng/ml, P > 0.05). Time points of peak levels (Tmax) were not influenced (Fig. 3BGo).


Figure 3
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  		FIG. 3. A, Corticosterone plasma concentrations in response to CRH (100 µg/kg) in rats pretreated with candesartan-cilexetil ({triangleup}), ramipril ({square}), mibefradil ({circ}), or vehicle (bullet); B, to statistically compare corticosterone release, AUC were calculated for various corticosterone plasma time curves. Results are shown as means ± SEM (n = 9–10); *, P < 0.05 vs. controls. CAN, Candesartin-cilexetil; CON, vehicle control; MIB, mibefradil; RAM, ramipril.

 

Figure 4
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  		FIG. 4. A, ACTH plasma concentrations in response to CRH (100 µg/kg) in rats pretreated with candesartan-cilexetil ({triangleup}), ramipril ({square}), mibefradil ({circ}), or vehicle (bullet); B, for statistical comparison of ACTH release dependent on pretreatment regime, AUC were calculated for various ACTH plasma time curves. Results are shown as means ± SEM (n = 9–10); *, P < 0.05 vs. controls. CAN, Candesartin-cilexetil; CON, vehicle control; MIB, mibefradil; RAM, ramipril.

 
Whereas AT1 blockade did not influence baseline glucose and insulin, these parameters were differentially regulated within the CRH test; plasma glucose increased after CRH injections and returned to baseline levels within 4 h. Candesartan attenuated this increase in glucose (AUC for controls was 35.7 ± 1.0 g/dl·min and for candesartan was 32.6 ± 0.7 g/dl·min; P < 0.05; Fig 5AGo) and enhanced the insulin release to CRH (Fig. 5BGo).


Figure 5
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  		FIG. 5. Plasma concentrations of glucose (A) and insulin (B) in response to CRH (100 µg/kg) in rats pretreated with candesartan-cilexetil ({triangleup}) or vehicle (bullet). Results are shown as means ± SEM (n = 9–10); *, P < 0.05 vs. controls.

 
Hypothalamic CRH mRNA levels were reduced by candesartan and ramipril by approximately 25% (Fig. 6AGo). This pattern was paralleled by corticosterone, because corticosterone was attenuated in trunk blood compared with controls (108 ± 13) by candesartan (74 ± 9; P < 0.05 vs. controls) and ramipril (66 ± 9; P < 0.05 vs. controls), but not by mibefradil (81 ± 12). Overall, corticosterone levels 1 d after CRH test were lower than before. In contrast, pituitary POMC mRNA was not affected. Mibefradil influences neither CRH mRNA nor POMC mRNA (Fig. 6BGo).


Figure 6
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  		FIG. 6. mRNA steady-state levels of CRH in hypothalamus (A) and POMC in pituitary (B) of SHR pretreated for 5 wk with vehicle (CON), candesartan-cilexitil (CAN), ramipril (RAM), or mibefradil (MIB). Rats were killed 1 d after CRH test. Results are shown as means ± SEM (n = 9–10); *, P < 0.05 vs. controls.

 
Expression of ANG receptors
A specific distribution of AT1A, AT1B, and AT2 receptor mRNA was found in different tissues of the HPA axis. (Fig. 7Go). Although distinct differences in mRNA levels of AT1A, AT1B, and AT2 receptors were detected in hypothalami and pituitary glands, expression of AT receptor subtypes was equal in the adrenals. The highest AT1B receptor mRNA levels were found in pituitary glands. Conversely, the expression of AT1A receptor in hypothalami was approximately 40-fold higher compared with AT1B receptors. In pituitary glands, AT1A mRNA was approximately three and four times increased, compared with the hypothalami or adrenals. Treatment with candesartan or ramipril did not effect mRNA levels of each receptor subtype in any organ of the HPA axis. (Fig. 7Go). A slight down-regulation in AT1A mRNA was detected in hypothalami of mibefradil-treated rats. However, the relevancy of this finding is not clear and remains for future investigations, because no functional correlate could be observed in our study.


Figure 7
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  		FIG. 7. mRNA steady-state concentrations of AT1A, AT1B, and AT2 receptors in hypothalami (HYP) (A–C), pituitaries (PIT) (D–;F), and adrenals (ADR) (G–I) of SHR pretreated with vehicle (CON), candesartan-cilexetil (CAN), ramipril (RAM), and mibefradil (MIB). Results are shown as means ± SEM (n = 9–10); *, P < 0.05 vs. controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates for the first time that the CRH-induced ACTH and corticosterone release is reduced after long-term oral treatment with the AT1 blocker candesartan or the ACE inhibitor ramipril in parallel to the antihypertensive effects in SHR. In contrast, the T-type calcium blocker mibefradil did not regulate secretion of stress hormones despite its antihypertensive influence. As shown previously, blood pressure reduction was almost similar to candesartan and ramipril but was not accompanied by regression of left ventricular growth (20, 21). Biochemical findings reflect the different targets of candesartan and ramipril within the RAAS. Blockade of AT1 receptors increased ANG and decreased aldosterone in plasma. Although ramipril reduced ACE activity in plasma, levels of ANG and aldosterone were equal to controls in our study, a result that is somehow in contrast to the expectation based on the underlying mechanism and the common literature (for review see Ref. 24). However, an ANG escape and a subsequent aldosterone breakthrough consequent to a therapeutic ACE inhibition was previously observed in experimental and human studies (25, 26, 27, 28), which might result from the loss of the feedback inhibition of the renin activity (29). These phenomena may account for the unchanged levels in our study. Although the blood pressure reduction after mibefradil was not equally effective as that of candesartan or ramipril, after all, the distinct blood pressure reduction by mibefradil of 30 mm Hg did not produce a consequent decrease of the HPA axis reactivity. The ineffectiveness of mibefradil on HPA axis reactivity indicates that an enhanced stress sensitivity does not develop as a phenomenon secondary to hypertension and is not normalized by general antihypertensive treatment. Accordingly, the attenuation of HPA axis reactivity by RAAS inhibitors is based on mechanisms independent from blood pressure regulation.

In hypertensive patients a chronic elevation of plasma cortisol has been observed, and its relevance for the pathogenesis of hypertension has been suggested (30), a finding that is contradictory to other findings (31, 32). However, there is clear evidence that the HPA axis is sensitized in hypertension, because hypertensive in contrast to normotensive patients show an exaggerated cortisol response to CRH stimulation (33) as well as larger cortisol elevations during stress (19, 34), which was similarly observed as a property of the SHR model (18). Accordingly, suppression of HPA axis reactivity must be discussed for its possible contribution to the antihypertensive actions of RAAS inhibitors. However, in the present study, candesartan and ramipril did not influence the baseline levels of ACTH and corticosterone in SHR, suggesting that an attenuation of HPA axis reactivity is not an essential mechanism for antihypertensive activity. Whether an influence of AT1 antagonists on plasma glucocorticoids can be effective to prevent the development of hypertension remains to be investigated.

Previous studies have documented impressively the specific role of ANG for the reactivity of the HPA axis, and expression of ANG receptors has been demonstrated to be regulated by stress within the HPA axis (8, 9, 11). ANG itself has been shown to stimulate ACTH or corticosterone secretion in isolated pituitary and adrenal cells, and ip or intracerebroventricular (icv) application of ANG in rats enhances the stimulatory effects of CRH through AT1 receptors (17, 35, 36, 37). However, controversial findings have been demonstrated for ACE inhibitors or AT1 blockers regarding their effects on ACTH release. We have observed in this study that CRH-induced secretion of ACTH and corticosterone is reduced by candesartan and ramipril (Fig. 3Go and 4Go), a finding that is in line with various studies showing 1) that candesartan reduces the corticosterone release stimulated by ANG in rats (17), 2) that captopril after icv administration attenuates the ACTH release in response to hemorrhage (38), and 3) that losartan reduces ACTH responses to hypoglycemia in volunteers (39). On the other hand, there is evidence that central ANG is not required for the secretory response of the hypothalamus and pituitary gland to stress because ACTH responses to ether, shaking, or immobilization stress in rats are unchanged by central AT1 antagonists or ACE inhibitors (13, 40, 41).

Differences in the sensitivity of the HPA axis to ANG may be ascribed to pathophysiological conditions of the different models used. It is conspicuous that studies showing no influence of AT1 or ACE inhibition on HPA axis reactivity were performed in normotensive rats (13, 40, 41). Because hypertensive patients exhibit larger cortisol elevations during stress compared with normotensive patients (19, 34) and because the increase in endocrine stress response was paralleled by an increase of AT1A receptors in pituitary glands of SHR (18), hypertension may be a prerequisite for an effective attenuation of the HPA axis reactivity after AT1 blockade. This experimental activity of AT1 blockade reflects an enhanced ANG sensitivity of the HPA axis in hypertensive patients that may be relevant for the development of high blood pressure. The potential of ANG to provoke such actions may be enhanced in hypertension because ANG levels were found to be increased in this condition (42, 43). The specific ability of RAAS inhibitors to reduce HPA axis reactivity in the course of an antihypertensive treatment is emphasized in the present study by the lack of effect of mibefradil.

In our study, the attenuation of HPA axis reactivity by RAAS blockade was observed only under the condition of simulated endocrine stress. This is in line with previous findings that describe ANG-facilitating actions on ACTH release in response to CRH stimulation (12, 14, 15, 16, 44, 45, 46). This mechanism seems to be of clinical relevance, because baseline cortisol salivary levels were similar between normotensive and hypertensive patients but increased only in hypertensive patients after various stress conditions (19). In contrast to our results, in one study, baseline corticosterone and ACTH were found to be diminished by AT1 blockade in an experimental study using SHR (17). However, baseline corticosterone in that study was 2- to 5-fold higher compared with our values and thus rather similar to maximal plasma concentrations after CRH stimulation in controls. This indicates that these animals have had stress as even conceded by the authors themselves (17). Thus, it is less astonishing but rather fitting to our results that such stress-induced corticosterone levels are reduced by AT1 blockade.

There is consensus that both CRH-R1 receptors and AT1A receptors increase ACTH release in response to CRH and ANG, respectively (47). Because AT1 receptors have been identified in the hypothalamus and pituitary and adrenal glands, several options have to be considered regarding the predominant site of action of RAAS blockade. We propose that the pituitary AT1A receptors are of high functional relevance for the exaggerated endocrine stress responses to ANG in SHR (18). AT1A receptors were found to be colocalized with CRH-R1 receptors in pituitary cells (13). Thus, these cells constitute the primary targets of CRH when it is physiologically supplied to the pituitary glands by hypothalamic neurons. Activation of CRH response to stress (48) is simulated in a physiological manner by systemic administration in the CRH test. The peripheral localization of pituitary glands provides sufficient access for ramipril or candesartan after chronic treatment to block local ACE or AT1 receptors in pituitary cells.

AT1 receptors are also present in adrenal glands and may modulate the release of glucocorticoids in response to stress. In view of reduced ACTH levels during RAAS inhibition, it seems unlikely that a direct influence of these drugs at the adrenal level would contribute to the observed reduction in corticosterone release. It has even been suggested that stimulation by ANG would have a suppressive effect on the corticosterone secretion of isolated adrenals in response to ACTH (49).

AT1 receptors are also expressed in the hypothalamus, and their significance for the regulation of HPA axis reactivity has been intensively investigated (13, 47). Circulating ANG enhances ACTH release indirectly through a central stimulation of CRH secretion. The hypothesis that this reaction is provoked by direct stimulation of the circumventricular organ was supported by the observation that an increase in ACTH to iv ANG could be prevented by pentobarbital and morphine, which inhibited release of CRH, and by CRH removal with specific antibodies (12, 37, 50). However, indirect ANG effects on ACTH release occur after administration of high doses and under circumstances that produce a concomitant increase in CRH release (45, 47). Because our study protocol did not include the application of ANG, this mechanism is unlikely to contribute to the acute ACTH release induced by exogenous CRH.

To investigate a possible chronic influence of RAAS inhibition on the HPA axis at the hypothalamic/pituitary level, we determined the expression of ANG receptors, CRH, and POMC. Because the expression of ANG receptors was not altered within tissues of the HPA axis by RAAS blockade, it is concluded that global adaptive changes in response to the chronic treatment did not occur. With regard to candesartan and ramipril, we found no differences in the mRNA levels except a down-regulation of CRH in the hypothalamus. In the context of previous findings that icv ANG stimulated the expression of CRH in the hypothalamus (8, 14) and icv injection of losartan decreased CRH mRNA after acute immobilization stress (13), our study supports the notion that hypothalamic CRH expression is under the control of ANG. Suppression of CRH expression would be supposed to reduce baseline stress hormones in rats during chronic RAAS inhibition. This was not reflected by alterations of plasma ACTH and corticosterone in our study but may become more evident under conditions of chronic stress. Indeed, Armando et al. (10) have observed that the increase in pituitary ACTH and plasma corticosterone levels induced by immobilization stress was attenuated by concomitant AT1 blockade.

Stress is thought to induce or maintain cardiovascular diseases and diabetes (51, 52, 53). In addition, AT1 blockade was proved to be beneficial in the treatment of cardiovascular diseases and in the prevention of diabetes. The presented evidence that chronic blockade of AT1 receptors reduces HPA axis reactivity in hypertension suggests a new mechanism influencing glucose metabolism. A first clue to a therapeutic reduction of plasma glucose during endocrine stress could already be obtained in this study, because glucose was in fact reduced and insulin enhanced after AT1 blockade when the HPA axis was stimulated by CRH but not under baseline conditions. The identification of this endocrine mechanism allows for future clinical development of preventive antidiabetic therapy.


   Footnotes
 
This work was supported by the Dean of the Medical Faculty of the University Clinic of Schleswig-Holstein, Campus Lübeck, and by the supply of test substances from the companies Astra-Zeneca (Wedel, Germany) and Hoffmann-La Roche (Grenzach-Wyhlen, Germany).

W.R., C.W., A.D., I.V., F.P., C.D., P.D., and O.J. have nothing to declare.

First Published Online March 30, 2006

Abbreviations: ACE, Angiotensin-converting enzyme; ANG, angiotensin II; AUC, area under the curve; HPA, hypothalamo-pituitary-adrenal; icv, intracerebroventricular; POMC, proopiomelanocortin; RAAS, renin-angiotensin-aldosterone system.

Received February 15, 2006.

Accepted for publication March 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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