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Endocrinology Vol. 142, No. 9 3880-3889
Copyright © 2001 by The Endocrine Society

ARTICLES

Peripheral Administration of an Angiotensin II AT1 Receptor Antagonist Decreases the Hypothalamic-Pituitary-Adrenal Response to Isolation Stress

Ines Armando, Andrea Carranza, Yasuaki Nishimura, Kwang-Lae Hoe, Marta Barontini, José A. Terrón, Alicia Falcón-Neri, Takeshi Ito, Augusto V. Juorio and Juan M. Saavedra

Centro de Investigaciones Endocrinologicas, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Buenos Aires (I.A., A.C., M.B.), Buenos Aires 1013, Argentina; and Section on Pharmacology, National Institute of Mental Health, National Institutes of Health (I.A., Y.N., K.-L.H., J.A.T., A.F.-N., T.I., A.V.J., J.M.S.), Bethesda, Maryland 20814

Address all correspondence and requests for reprints to: Dr. Ines Armando, Section on Pharmacology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20814. E-mail: saavedrj{at}irp.nimh.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II, which stimulates AT1 receptors, is a brain and peripheral stress hormone. We pretreated rats with the AT1 receptor antagonist candesartan for 13 d via sc-implanted osmotic minipumps, followed by 24-h isolation in individual metabolic cages. We measured angiotensin II receptor-type binding and mRNAs and tyrosine hydroxylase mRNA by quantitative autoradiography and in situ hybridization, catecholamines by HPLC, and hormones by RIA. Isolation increased AT1 receptor binding in hypothalamic paraventricular nucleus as well as anterior pituitary ACTH, and decreased posterior pituitary AVP. Isolation stress also increased AT1 receptor binding and AT1B mRNA in zona glomerulosa and AT2 binding in adrenal medulla, adrenal catecholamines, tyrosine hydroxylase mRNA, aldosterone, and corticosterone. Candesartan blocked AT1 binding in paraventricular nucleus and adrenal gland; prevented the isolation-induced alterations in pituitary ACTH and AVP and in adrenal corticosterone, aldosterone, and catecholamines; abolished the increase in AT2 binding in adrenal medulla; and substantially decreased urinary AVP, corticosterone, aldosterone, and catecholamines during isolation. Peripheral pretreatment with an AT1 receptor antagonist blocks brain and peripheral AT1 receptors and inhibits the hypothalamic-pituitary-adrenal response to stress, suggesting a physiological role for peripheral and brain AT1 receptors during stress and a possible beneficial effect of AT1 antagonism in stress-related disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (Ang II) regulates fluid and electrolyte homeostasis, hormone secretion, and the autonomic nervous system through receptors present within the brain and many peripheral tissues (1, 2, 3). In addition, Ang II may influence other brain functions, including the central regulation of peripheral sympathetic activity and hormonal secretion from the anterior and posterior pituitary gland (2, 3). In particular, Ang II was implicated in the response of the hypothalamic-pituitary-adrenal axis to stress. Stress increases the production of circulating Ang II (4) and the expression of Ang II receptors in brain areas such as the hypothalamic paraventricular nucleus (PVN) that are crucial for the central control of the stress reaction (2, 3, 5, 6). Ang II stimulates CRH formation in the PVN during stress (7, 8). Released into the hypothalamic portal system, CRH increases pituitary ACTH release. In turn, increased adrenal corticoids regulate the expression of Ang II receptors in the PVN (6, 9). In the anterior pituitary, locally synthesized and circulating Ang II enhances the secretion of ACTH during stress (2). In addition, Ang II has direct effects on adrenal function, stimulating aldosterone secretion from the zona glomerulosa and catecholamine release from the medulla (10, 11, 12). Ang II also stimulates vasopressin (AVP) formation and release in the PVN (2). Released from the median eminence, AVP is taken through the portal circulation to participate with CRH and enhance ACTH production (13).

There are, in mammals, two types of Ang II-binding sites, the AT1 and AT2 types, cloned and characterized pharmacologically (14, 15). The AT1 and AT2 types have a seven-transmembrane region structural topology typical of the G protein-linked superfamily of receptors, but only about 30% amino acid sequence homology (14). The AT1 site is the physiologically active Ang II receptor, mediating most, if not all, of the well known functions of Ang II (15) and is the receptor subtype proposed to mediate the regulation of the stress reaction by Ang II (2, 5, 16). In rodents, there are two AT1 receptor subtypes, AT1A and AT1B, encoded by two distinct genes and pharmacologically indistinguishable, sharing 95% amino acid sequence identity in their open reading frame (ORF), but only 60% nucleotide sequence homology in their 5'- and 3'-untranslated regions (UTRs) (14). The AT2 site is highly expressed in peripheral fetal tissues, immature brain, and rodent adrenal medulla and zona glomerulosa (3, 17, 18, 19, 20). This site has been proposed to be involved in the regulation of growth and to cross-talk with the AT1 receptor (3, 21, 22). However, the experimental evidence is equivocal, and its physiological function has not yet been clarified (3, 21).

In adult male rats only AT1A receptors are present in brain areas involved in the response to stress (16, 18, 23). In the anterior pituitary of adult rats, AT1B receptors are the predominant isoform (23, 24, 25). Both AT1A and AT1B receptors, in addition to AT2, are expressed in the adrenal zona glomerulosa (26). Only AT1A and AT2 receptors are expressed in the adrenal medulla; the AT2 receptor is the predominant Ang II receptor subtype (17, 18, 19, 20).

Based on the above evidence, it was reasonable to postulate that AT1 receptor blockade could inhibit the hormonal and sympathetic responses to stress. However, initial reports of the effects of AT1 antagonists in stress were equivocal. When administered peripherally, the AT1 antagonists losartan (27, 28) and irbesartan (28) did not modify (27) or only partially antagonized (28) the effects of Ang II on the brain, probably due to their limited access to brain AT1 receptors (27, 28). When the AT1 receptor inhibitor losartan was administered acutely into the brain, it partially inhibited the stress-mediated catecholamine release, but did not decrease the ACTH response to stress (29). The apparent lack of specificity of changes in AT1 receptor expression after different stressors (5), and the lack of effect of intracerebral administration of Ang II in AVP mRNA levels (7) suggested that Ang II, although important to modulate PVN function, might not be a major factor in the regulation of the acute hypothalamic-pituitary-adrenal response to stress (5, 7, 29).

To further clarify the role of Ang II AT1 receptors in stress, we selected the AT1 antagonist candesartan, an insurmountable antagonist that, when administered peripherally, readily crosses the blood-brain barrier, inhibiting not only peripheral, but also central AT1 receptors (30). After peripheral administration of candesartan, we studied a more clinically relevant form of stress, the stress of isolation. We asked the question of how pretreatment with a peripheral and central Ang II AT1 receptor antagonist modulated the hypothalamo-pituitary-adrenal response to isolation stress. We found that pretreatment with candesartan almost completely abolished the hormonal and sympathoadrenal responses to stress, suggesting that simultaneous antagonism of peripheral and brain AT1 receptors could represent an advantage in the control of the stress reaction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and preparation of tissues
Wistar male rats inbred at the Centro de Investigaciones Endocrinologicas, CONICET (Buenos Aires, Argentina), or purchased from Taconic Farms, Inc. (Germantown, NY), were kept at 22 C, six animals per each 390-in.2 cage, under a 12-h dark, 12-h light cycle with lights on at 0700 h and were given free access to normal rat diet and tap water. The NIMH (U.S.) and CONICET (Argentina) animal care and use committees approved all procedures.

At the time of the experiment, when the rats were 10 wk old and weighed between 250 and 300 g. They were anesthetized with ketamine (100 mg/kg, ip) and xylocine (10 mg/kg), and Alzet osmotic minipumps (Alza Corp., Palo Alto, CA) were implanted sc. Three groups of six animals (treated) received minipumps containing candesartan (Astra GmBH, Wedel, Germany) dissolved in 1 mol/liter sodium carbonate and further diluted in isotonic saline, to be delivered at rates of 0.1, 0.5 or 1.0 mg/kg·d, respectively, and these animals were later subjected to isolation stress. An additional group of six animals received osmotic minipumps filled with vehicle, at a final pH of 7.5–8.0, and these animals were also subjected to isolation stress. Control animals (referred as grouped rats) were implanted with minipumps filled with vehicle and remained grouped, six animals per cage and undisturbed, until killed as described below.

After minipump implantation the rats were kept in their cages in groups of six under standard conditions for 13 d. On d 13, the animals were individually housed in standard, 50-in.2 plastic metabolic cages (Nalgene, Rochester, NY) that were located in the same animal room. Regular rat food and water were provided ad libitum throughout the experiment. A 24-h urine production was collected in plastic containers and stored at -20 C until use. At the end of the experiment, on d 14, the animals were killed by decapitation and the brain, pituitary gland, and adrenal glands were removed, frozen in isopentane at -30 C on dry ice, and stored at -80 C until used. For determination of ACTH and AVP, anterior and posterior pituitary glands were homogenized in 0.1 N acetic acid. For determination of catecholamines, aldosterone and corticosterone, adrenal glands were homogenized in 0.3 N perchloric acid. The clear supernatant obtained after centrifugation was stored frozen until assayed.

Tissue hormone determinations were performed in grouped, nonstressed rats, and the results were compared with those found in isolated rats treated with the AT1 antagonist or vehicle. Thus, comparisons were made between tissue levels of catecholamines and/or hormones in isolated vs. grouped animals and of the effects of AT1 receptor blockade during isolation. On the other hand, determination of urinary catecholamines and hormones was performed only in rats isolated in their metabolic cages, and the results represent only the effects of the AT1 antagonist on the hormone and catecholamine secretion during isolation. We did not measure urinary excretion of catecholamines or hormones in grouped control rats. Therefore, we do not present urinary excretion data comparing grouped and isolated rats.

Hormone determinations
Urine, pituitary, and adrenal hormones were determined by RIA using commercially available kits [ICN Biomedicals, Inc. (Costa Mesa, CA), for ACTH, aldosterone and corticosterone; Diagostics Systems Laboratories, Inc. (Webster, TX), for urinary AVP, and Phoenix Pharmaceuticals, Inc. (Belmont, CA), for pituitary AVP].

Catecholamine determinations in urine and adrenal glands
Catechols in 20-µl aliquots of the 24-h urine collection or in aliquots of adrenal supernatants were determined by reverse phase HPLC with electrochemical detection (31). Sample catechols were partially purified by batch alumina extraction, separated by HPLC using a 4.6 x 250-mm Zorbax Rx C18 column (DuPont Merck Pharmaceutical Co., Wilmington, DE) and quantified amperometrically by the current produced upon exposure of the column effluent to oxidizing and then reducing potentials in series using a triple electrode system (ESA, Bedford, MA). Recovery through the alumina extraction step averaged 70–80% for epinephrine (E) and norepinephrine (NE) and 45–55% for dihydroxyphenylglycol (DHPG). Catechol concentrations in each sample were corrected for recovery of an internal standard, dihydroxybenzylamine. DHPG levels were further corrected for differences in recovery of the internal standard and of this catechol in a mixture of external standards. The limit of detection was about 15 pg/volume assayed for each catechol.

Autoradiography of Ang II receptor subtypes
Consecutive coronal brain sections, 16 µm thick, and consecutive sections from adrenal gland, 16 µm thick, were cut in a cryostat, thaw-mounted on gelatinized slides, dehydrated overnight in a dessicator at 4 C, and kept at -80 C until used. Binding experiments were performed as described previously (32). Sections were preincubated for 15 min in 10 mM sodium phosphate buffer, pH 7.4, containing 120 mM NaCl, 5 mM EDTA, 0.005% bacitracin, and 0.2% BSA. After preincubation, slides were transferred to fresh buffer containing 0.5 nM [125I]Sar1-Ang II (Peninsula Laboratories, Inc., Belmont, CA), iodinated by NEN Life Science Products (Boston, MA) to a specific activity of 2200 Ci/mmol to determine total binding. Nonspecific binding was determined by incubation of consecutive sections as described above in the presence of 5 µM unlabeled Ang II. The binding of [125I]Sar1-Ang II to AT1 receptors was determined in consecutive sections incubated with 0.5 nM [125I]Sar1-Ang II in the presence of 10 µM of the AT1 receptor antagonist losartan (DuPont Merck Pharmaceutical Co.). Losartan has been shown previously to completely displace binding from AT1 receptors at this concentration (32). AT1 receptor binding was the binding selectively displaced by losartan in our experiments. The binding of [125I]Sar1-Ang II to AT2 receptors was determined in consecutive sections incubated with 0.5 nM [125I]Sar1-Ang II in the presence of 1 µM of the selective AT2 ligand PD 123319 (Parke-Davis, Ann Arbor, MI). PD 123319 has been shown previously to completely displace binding from AT2 receptors at this concentration (32). AT2 receptor binding was the binding selectively displaced by PD 123319 in our experiments.

After incubation for 2 h at 22 C, sections were washed four times, 1 min each time, in ice-cold 50 mM Tris-HCl buffer, pH 7.4, rinsed in ice-cold water, and dried under a stream of cold air. Sections were exposed to Hyperfilm-3H (Amersham Pharmacia Biotech, Piscataway, NJ). The optical densities of autoradiograms were determined by computerized microdensitometry using the Image 1.61 program (NIMH, Bethesda, MD), quantified by comparison with 125I-labeled microscales (Amersham Pharmacia Biotech) or 14C-labeled microscales (American Radiolabeled Chemicals, Inc., St. Louis, MO) and transformed to corresponding values of femtomoles per mg protein (33). Brain regions were identified by comparison with toluidine blue staining of consecutive brain sections.

In situ hybridization of Ang II receptor subtype mRNAs
To obtain rat AT1A, AT1B, and AT2 receptor-specific riboprobes, partial fragments of full-length cDNA were subcloned into the polylinker site of the pBluescript KS+ vector (Stratagene, La Jolla, CA). The rat (r) AT1A cDNA was restricted with HaeIII (34). The restriction fragment of 368 bp [from nucleotides (nt) 1317–1684] was prepared and ligated into the EcoRV site of the vector. The fragment corresponded to the 3'UTR of the gene with a 35-bp ORF region that did not show any identity with rAT1B cDNA ORF. The rAT1B cDNA was restricted with HindIII and EcoRI (35). The restriction fragment of 398 bp (from nt 1832–2229) was prepared and ligated into the HindIII-EcoRI site of the vector. The fragment corresponded to the 3'UTR of the gene. The rAT2 cDNA was restricted with XbaI and BglII (36) and the fragment of 375 bp (from nt 1478–1852) was prepared and ligated into the XbaI-BglII site of the vector. The fragment corresponded to the 3'UTR of the gene. The subclones, rAT1A-S2, rAT1B-S1, and rAT2, were confirmed by DNA sequencing.

For in vitro transcription of the 35S-labeled antisense and sense (as a control) riboprobes, the subclones were linearized with HindIII or EcoRI for the rAT1A-S2 and rAT1B-S1 or with XbaI or EcoRI for the rAT2, and then they were treated with T3 or T7 RNA polymerase, respectively. In vitro transcription was performed using the RNA labeling kit (Amersham Pharmacia Biotech) as previously described (37). The quality of riboprobes was monitored with liquid scintillation counting and controlled by a preexperiment using adrenal gland as a positive control.

To perform in situ hybridization, sections were fixed in 4% paraformaldehyde for 10 min, acetylated for 10 min in 0.1 M triethanolamine HCl, pH 8.0, containing 0.25% acetic anhydride, dehydrated in alcohols, and air-dried. Each section was covered with 150 µl hybridization buffer containing 50% formamide, 0.3 M NaCl, 2 mM EDTA, 20 mM Tris (pH 8.0), 1 x Denhardt’s solution, 10% dextran sulfate, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast tRNA, 150 mM dithiothreitol, 0.1% SDS, and 40,000 cpm/µl sense or antisense probe. Sections were hybridized overnight at 54 C, treated with 40 µg/ml ribonuclease A (Sigma, St. Louis, MO) for 30 min, and washed in sodium chloride/sodium citrate with increasing stringency. After a final wash in 0.1 x SSC (standard saline citrate) at 65 C for 60 min, sections were dehydrated through alcohols and exposed to Hyperfilm-3H (Amersham Pharmacia Biotech) along with 14C-labeled microscales (American Radiolabeled Chemicals, Inc.) for 7 d. Films were developed as described above. The intensities of hybridization signals were quantified as nanocuries per g tissue equivalent by measuring optical film densities using the NIH Image 1.61 program after calibration with the 14C-labeled microscales (38). Nonspecific hybridization was analyzed using sense (control) probes.

In situ hybridization of tyrosine hydroxylase (TH) mRNA
One antisense (TH-AS) of 48-mer for the rat TH cDNA sequence localized in nt 1562–1609 (39) was synthesized (Lofstrand Laboratories Ltd., Gaithersburg, MD). Labeling was performed with a 3'-end labeling kit (Amersham Pharmacia Biotech) using terminal deoxynucleotidyl transferase to a specific activity of 3–4 x 108 dpm/µg. Each reaction was performed with 70 pmol oligonucleotides in the presence of 70 µCi [{alpha}-35S]ATP (Amersham Pharmacia Biotech). The labeled oligonucleotides were separated from unincorporated nucleotides using MicroSpin G-25 columns (Amersham Pharmacia Biotech). In situ hybridization of rat adrenal sections and posthybridization washings were performed as previously described (38). In situ hybridization was performed in consecutive adrenal sections, one incubated with the TH-AS oligonucleotide and another with excess unlabeled TH-AS probe (157 pmol/ml). After the washing, sections were dehydrated in alcohols containing 0.3 M ammonium acetate, air-dried, and exposed to Hyperfilm-3H (Amersham Pharmacia Biotech). The films were developed and quantified as described above by comparison with 14C-labeled standards (American Radiolabeled Chemicals, Inc.).

Statistics
Data are the mean ± SEM. One-way ANOVA followed by post-hoc analysis using the Newman-Keuls multiple comparison test was used to assess the significance of differences among groups. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Brain Ang II AT1 receptors
In the PVN, isolation stress produced a significant (P < 0.03) increase in AT1 receptor number, from 12 ± 3 fmol/mg protein in control grouped rats to 25 ± 3 fmol/mg protein in isolated animals (Fig. 1Go). In the subfornical organ, AT1 receptor binding showed a tendency to increase during isolation, from 50 ± 7 fmol/mg protein in control grouped rats to 61 ± 10 fmol/mg protein in isolated animals, but the change was not statistically significant.



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  		Figure 1. Autoradiography of Ang II AT1 receptor binding in the paraventricular nucleus. A, Binding to AT1 receptors in the PVN of control grouped rats. The section was incubated, as described in the text, in the presence of 0.5 nM [125I]Sar1-Ang II and 1 µM PD 123319 to reveal binding to AT1 receptors. B, Binding to AT1 receptors in the PVN from a rat isolated for 24 h, incubated as described above. C, Binding to AT1 receptors in the PVN from a rat isolated for 24 h and treated with candesartan (1.0 mg/kg·d) for 13 d before the isolation, incubated as described above. Note the marked increase in AT1 binding in the PVN during isolation and the marked decrease in binding during isolation after candesartan pretreatment. Scale bar, 1 mm.

 
We studied the effect of pretreatment with 1.0 mg/kg·d candesartan, as described above, on AT1 receptor binding in the PVN after isolation stress. Predictably, AT1 blockade eliminated the increase in AT1 binding in the PVN produced by isolation (Fig. 1Go). Binding after pretreatment with candesartan and isolation was 2 ± 1 fmol/mg protein, a 90% decrease from values obtained in the PVN of isolated rats treated with vehicle (P < 0.01) and an 80% decrease in binding compared with that found in control grouped rats (P < 0.01). Pretreatment with 0.1 mg/kg·d candesartan significantly decreased AT1 binding in the subfornical organ, to 15 ± 3 fmol/mg protein compared with control grouped animals or isolated vehicle-treated rats (P < 0.05).

Adrenal Ang II receptor subtypes
In the zona glomerulosa, isolation stress produced a significant increase in AT1 receptor binding and a selective increase in AT1B receptor mRNA, without changes in AT1A receptor mRNA (Fig. 2Go). Binding to AT2 receptors and AT2 mRNA showed a tendency to increase in the zona glomerulosa after isolation, although the difference did not reach statistical significance (Fig. 2Go). In the adrenal medulla, there was no significant change in AT1 receptor binding after isolation (Fig. 3Go). The expression of AT1A receptor mRNA was lower during isolation compared with that in grouped controls, but the result was not statistically significant (Fig. 3Go). Conversely, isolation stress produced a large significant increase in AT2 receptor binding (Fig. 3Go).



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  		Figure 2. Ang II receptor subtype mRNA and binding in the adrenal zona glomerulosa. Results are the mean ± SEM of groups of six rats, measured individually. *, P < 0.05 vs. control grouped rats.

 


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  		Figure 3. Ang II receptor subtype mRNA and binding in the adrenal medulla. Results are the mean ± SEM of groups of six rats, measured individually. *, P < 0.05 vs. control grouped rats.

 
In the zona glomerulosa, administration of an AT1 antagonist altered the expression of Ang II receptor type binding in opposite directions. Candesartan abolished the increased AT1 expression produced by isolation stress (Fig. 2Go). In addition, AT1 receptor binding was strongly reduced to amounts much lower than those found in control nontreated grouped rats, even at low doses of the AT1 antagonist (Fig. 2Go). Candesartan also reduced the increased expression of AT1B mRNA produced by isolation stress, although a significant reduction, to levels not different from those in control grouped rats, was found only at the highest dose of the AT1 antagonist (Fig. 2Go). AT1A mRNA expression was also reduced by candesartan pretreatment, but the decrease was significant when compared with control grouped or vehicle-treated isolated rats only when the highest dose of the AT1 receptor antagonist was administered (Fig. 2Go).

Conversely, AT2 binding in the zona glomerulosa, which showed a tendency toward an increase during isolation (Fig. 2Go), increased even further after pretreatment with the AT1 antagonist (Fig. 2Go). In addition, the highest dose of the AT1 antagonist significantly increased the expression of AT2 mRNA during isolation (Fig. 2Go).

In the adrenal medulla, pretreatment with the AT1 receptor antagonist decreased AT1 binding to levels markedly lower than those observed in grouped controls or vehicle-treated isolated rats (Fig. 3Go). Pretreatment with candesartan did not significantly affect AT1A mRNA expression during isolation (Fig. 3Go).

Pretreatment with the AT1 receptor antagonist eliminated the stress-related increase in AT2 binding in the adrenal medulla (Fig. 3Go), and the highest dose of candesartan reduced AT2 binding in the adrenal medulla even further to levels lower than those observed in grouped rats (Fig. 3Go). Conversely, the highest dose of the AT1 antagonist enhanced the expression of AT2 mRNA in the adrenal medulla to levels significantly higher than those in control grouped or vehicle-treated isolated rats (Fig. 3Go).

Pituitary hormones
Isolation produced significant elevations in the pituitary ACTH content (Fig. 4Go) and a significant reduction in the posterior pituitary AVP content (Fig. 5Go). Pretreatment with the AT1 receptor blocker completely eliminated the increase in pituitary ACTH produced by isolation stress, and this effect occurred even at the lowest dose of candesartan used (Fig. 4Go). Similarly, candesartan pretreatment reversed the decrease in pituitary AVP concentrations, dose-dependently restoring the AVP content to concentrations not different from those in grouped control rats (Fig. 5Go).



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  		Figure 4. Anterior pituitary ACTH and adrenal and urinary corticosterone. Results are the mean ± SEM of groups of six rats, measured individually. *, P < 0.05 vs. control grouped rats.

 


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  		Figure 5. Posterior pituitary and urinary vasopressin. Results are the mean ± SEM of groups of six rats, measured individually. *, P < 0.05 vs. control grouped rats.

 
Adrenal hormones
After isolation, the adrenal corticosterone content was markedly elevated compared with that in grouped animals (Fig. 4Go). The adrenal aldosterone content was also significantly increased after isolation compared with that in grouped control rats (Fig. 6Go). In the adrenal gland, AT1 receptor blockade abolished the increase in adrenal corticosterone (Fig. 4Go) and aldosterone (Fig. 6Go) content produced by isolation.



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  		Figure 6. Adrenal and urinary aldosterone. Results are the mean ± SEM of groups of six rats, measured individually. *, P < 0.05 vs. control grouped rats.

 
Urinary hormones
We measured urinary hormones only in groups subjected to isolation. Pretreatment with the AT1 receptor antagonist significantly reduced the 24-h urinary corticosterone excretion (Fig. 4Go). Significant reductions in urinary corticosterone occurred after candesartan treatment at doses of 0.1 and 0.5 mg/kg·d (Fig. 4Go). Treatment with 1.0 mg/kg·d candesartan reduced urinary corticosterone by 17%, but the change was not statistically significant. The urinary excretion of aldosterone was also significantly reduced after pretreatment with the AT1 receptor antagonist at a dose of 0.5 and 1.0 mg/kg·d (Fig. 6Go).

Treatment with the AT1 receptor antagonist significantly decreased the 24-h urinary elimination of AVP, an effect that occurred even when the lowest doses of the antagonist were used (Fig. 5Go).

Adrenal TH mRNA and catecholamines
Isolation produced significant elevations in TH mRNA in the adrenal medulla (Fig. 7Go) and in adrenal E and NE contents (Fig. 7Go). Blockade of AT1 receptors abolished the increase in TH mRNA produced by isolation (Fig. 7Go). At the highest doses of candesartan, expression of TH mRNA showed a tendency to decrease to levels even less than those present in control grouped rats (Fig. 7Go). Similarly, candesartan pretreatment eliminated the increase in adrenal E and NE concentrations produced by isolation, decreasing concentrations of both catecholamines to levels not significantly different from those in control grouped rats (Fig. 7Go).



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  		Figure 7. Adrenal medulla TH mRNA, adrenal catecholamine content, and urinary catecholamine and DHPG excretion. Results are the mean ± SEM of groups of six rats, measured individually. *, P < 0.05 vs. control grouped rats.

 
Urinary catecholamines
We determined urinary excretion of catecholamines only in animals subjected to isolation. Pretreatment with the AT1 receptor antagonist significantly and dose-dependently reduced the urinary excretion of E and NE and that of the NE metabolite DHPG during isolation (Fig. 7Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies using a well characterized model of stress in rodents, immobilization or restraint, indicated that peripheral and brain Ang II plays a role in the response to stress (2). We chose a more clinically relevant model of individual isolation in unfamiliar metabolic cages because it represents, for rodents, a significant emotional stress resulting from the restriction from freely regulating their exposure to novel surroundings and their access to familiar territory (40).

We found that a relatively short (24-h) period of isolation enhanced Ang II AT1 receptor expression in the PVN to an extent similar to the increase in AT1 receptors that occurs during repeated immobilization stress (2, 5, 6), increased pituitary ACTH, decreased pituitary AVP, and increased adrenal corticosterone, aldosterone, catecholamines, and TH mRNA. Increased pituitary ACTH content, in association with increased adrenal corticosterone and aldosterone, can be best interpreted as evidence of increased ACTH formation in association with increased ACTH release during stress and the consequent stimulation of adrenal hormone synthesis. The increased AT1 receptor expression in the PVN in association with the increased pituitary ACTH and adrenal corticosterone and aldosterone contents supports the hypothesis of a role for AT1 receptors in the regulation of the enhanced hypothalamic-pituitary-adrenal response to stress and indicates that isolation produces a classical stress response.

Increased AVP release into the circulation, such as that produced by water deprivation, results in a depletion of AVP stores, leading to decreased posterior pituitary AVP content (41). In some stress models, AVP secretion from the pituitary to the circulation also increases (42). We interpret the decreased AVP content during isolation stress as indirect evidence of increased release, probably in response to increased AT1 receptor stimulation (2).

The increase in adrenomedullary mRNA for TH, the rate-limiting enzyme in catecholamine biosynthesis, in parallel with increased E and NE concentrations, indicated increased catecholamine synthesis during isolation, in agreement with previous studies demonstrating that isolation produces increased sensitivity, as determined by increased E and corticosterone, to other forms of stress (43, 44).

There were associated alterations in adrenal Ang II receptor type expression during isolation. In the zona glomerulosa, higher AT1 receptor binding and AT1B mRNA during stress indicated receptor up-regulation, and this correlates with increased aldosterone content. Our results support the hypothesis that in the rat, stimulation of aldosterone synthesis by Ang II is probably mediated through AT1B receptor stimulation (45, 46).

In rat adrenal medulla, AT1 receptors, although few in number, mediate catecholamine secretion by Ang II (47). However, in the medulla of isolated rats, only AT2 receptor binding was increased, and this correlated with the increase in adrenomedullary catecholamines. Our findings suggest that rat adrenomedullary AT2 receptor expression may play a still undetermined role in catecholamine synthesis and release during isolation (26, 48).

We asked whether antagonism of AT1 receptors by blocking the physiological effects of Ang II (2) could significantly affect the hormonal and sympathoadrenal response to isolation stress. Because candesartan readily crosses the blood-brain barrier (30), peripheral administration of the AT1 antagonist almost completely blocked AT1 receptor binding, not only in peripheral tissues such as anterior pituitary and adrenal gland, but also in the brain. Candesartan treatment effectively blocked peripheral and brain AT1 binding after isolation in a manner similar to what was previously observed in control grouped rats (30).

Chronic AT1 receptor blockade totally abolished the increase in pituitary ACTH and adrenal corticosterone during isolation stress, probably as a result of combined AT1 receptor blockade in the pituitary and adrenal glands and in the parvocellular region of the PVN (30). Moreover, during isolation, AT1 receptor blockade abolished the decrease in the posterior pituitary content of AVP. These findings can be interpreted as the result of decreased AVP release from the posterior pituitary gland during blockade of AT1 receptors in magnocellular neurons (2).

In the adrenal zona glomerulosa, chronic administration of the AT1 antagonist predictably produced a marked decrease in AT1 binding to levels even lower than those in control rats, a finding that correlated with a significant decrease in adrenal aldosterone to levels not different from those in control nonstressed rats and with a significant reduction in the urinary excretion of aldosterone during isolation. These results indicate that increased AT1 receptor stimulation, in addition to ACTH (10), is an important factor in the increase in aldosterone formation and release during acute stress. AT1 blockade also suppressed the increase in AT1B receptor mRNA that occurs during isolation, indicating that receptor blockade was not compensated by receptor transcription.

Conversely, AT1 receptor blockade increased AT2 receptor expression in the zona glomerulosa. This finding is intriguing because AT2 receptor stimulation is not involved in aldosterone synthesis or release (45). In many systems, AT1 and AT2 receptors have been proposed to exert opposite actions, and the receptor type expression is sometimes inversely correlated (49). It is possible that alterations in AT2 receptor expression could be dependent on AT1 receptor blockade and a result of cross-talk between AT1 and AT2 receptors (22, 49). However, such a reciprocal influence on receptor type expression is not universal, but, rather, is tissue specific. For example, in AT2 gene-deficient mice, the expression of AT1 receptors is increased in the kidney glomeruli, but not in the inner stripe of the outer medulla (49), and in our study, binding to AT2 receptors increased in the zona glomerulosa, but decreased in the adrenal medulla after AT1 receptor blockade.

Pretreatment with the AT1 receptor antagonist decreased the adrenomedullary catecholamine response to stress to levels not different from those in control grouped rats. These changes paralleled the decrease in AT1 receptor binding expression in the adrenal medulla, supporting the hypothesis that adrenomedullary AT1 receptor stimulation is associated with increased catecholamine formation and release. Blockade of catecholamine secretion is probably the result, in addition to AT1 receptor antagonism in the adrenal medulla, of AT1 receptor blockade in brain areas related to the control of peripheral sympathetic activity, i.e. the PVN and nucleus of the solitary tract (30).

AT2 receptor expression in the adrenal medulla also decreased after AT1 blockade, to levels not different from those in control grouped animals. At high candesartan concentrations, however, AT2 receptor expression during isolation actually decreased to levels lower than control values, with a concomitant increase in AT2 receptor mRNA expression. The mechanism and significance of these changes have not been clarified, but suggest a still undetermined role of AT2 receptors during isolation stress and perhaps additional cross-talk between AT1 and AT2 receptors in the adrenal medulla (3, 22).

Pretreatment with the AT1 receptor antagonist significantly decreased the urinary excretion of catecholamines, corticosterone, aldosterone, and AVP during isolation. Because we did not measure catecholamine and hormone urinary secretion in control, grouped animals, we cannot establish with certainty that isolation increases the urinary excretion of catecholamines, corticosterone, aldosterone, or AVP. Nevertheless, the alterations in tissue hormone and catecholamine levels during isolation are indicative of a classical stress reaction, suggesting stimulation of the hypothalamic-pituitary-adrenal axis leading to increased hormone and catecholamine release. It is logical to speculate that increased catecholamine and hormone secretion during stress could result in their increased urinary excretion. The prevention of the alterations in tissue hormone and catecholamines by pretreatment with the AT1 antagonist parallels the decrease in urinary excretion as a result of the treatment. On the basis of these results, it is reasonable to conclude that peripheral and central AT1 receptor blockade inhibits the hormonal and sympathoadrenal responses to stress by preventing the stress-induced increase in pituitary and adrenal hormones, an effect indirectly reflected in their decreased urinary excretion during isolation as a result of the treatment.

In conclusion, our results demonstrate that Ang II AT1 receptor blockade is sufficient to completely abolish the hypothalamic-pituitary-adrenal response to isolation stress, strongly supporting a role for Ang II as a major stress hormone. If a blockade of pathologically enhanced responses to stress has beneficial effects, antagonism of AT1 receptors could have a place in the therapy of stress-related disorders. It appears that a dual blockade of the peripheral and central AT1 receptors, as reported here, may have advantages compared with selective peripheral or central blockade (27, 28, 29). In addition, compounds that readily and insurmountably block brain as well as peripheral AT1 receptors inhibit both the peripheral and central components of the stress response and avoid the rebound AT1 receptor stimulation that can occur when surmountable antagonists are used (50). Our results suggest that centrally acting, insurmountable AT1 antagonists could be therapeutically advantageous not only in hypertension, but also for the treatment of stress-related disorders.


   Acknowledgments
 
We thank Dr. Bernd Friedrich, Astra GmBH, for his generous supply of candesartan.


   Footnotes
 
This work was supported by a grant from Astra GmBH, Germany (to J.A.T.).

Abbreviations: Ang II, Angiotensin II; DHPG, dihydroxyphenylglycol; E, epinephrine; NE, norepinephrine; nt, nucleotides; ORF, open reading frame; PVN, paraventricular nucleus; r, rat; TH, tyrosine hydroxylase; TH-AS, tyrosine hydroxylase antisense UTR, untranslated region.

Received March 23, 2001.

Accepted for publication May 11, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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