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. Author manuscript; available in PMC: 2009 Sep 11.
Published in final edited form as: Stress. 2008;11(6):457–466. doi: 10.1080/10253890801892040

Angiotensin II AT1 receptor blockade selectively enhances brain AT2 receptor expression, and abolishes the cold-restraint stress-induced increase in tyrosine hydroxylase mRNA in the locus coeruleus of spontaneously hypertensive rats

C BREGONZIO 1, A SELTZER 1, I ARMANDO 1, J PAVEL 1, J M SAAVEDRA 1
PMCID: PMC2742314  NIHMSID: NIHMS104513  PMID: 18609298

Abstract

Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated orally for 14 days with the AT1 receptor antagonist candesartan before submission to 2 h of cold-restraint stress. In non-treated rats, stress decreased AT1 receptor binding in the median eminence and basolateral amygdala, increased AT2 receptor binding in the medial subnucleus of the inferior olive, decreased AT2 binding in the ventrolateral thalamic nucleus and increased tyrosine hydroxylase mRNA level in the locus coeruleus. In non-stressed rats, AT1 receptor blockade reduced AT1 receptor binding in all areas studied and enhanced AT2 receptor binding in the medial subnucleus of the inferior olive. Candesartan pretreatment produced a similar decrease in brain AT1 binding after stress, and prevented the stress-induced AT2 receptor binding decrease in the ventrolateral thalamic nucleus. In the locus coeruleus and adrenal medulla, AT1 blockade abolished the stress-induced increase in tyrosine hydroxylase mRNA level. Our results demonstrate that oral administration of candesartan effectively blocked brain AT1 receptors, selectively increased central AT2 receptor expression and prevented the stress-induced central stimulation of tyrosine hydroxylase transcription. The present results support a role of brain AT1 and AT2 receptors in the regulation of the stress response, and the hypothesis that AT1 receptor antagonists may be considered as potential therapeutic compounds in stress related disorders in addition to their anti-hypertensive properties.

Keywords: Angiotensin II receptors, brain, central sympathetic system, locus coeruleus, renin angiotensin system, stress

Introduction

Brain angiotensin II (Ang II), through AT1 receptor stimulation, is a multitasking peptide with established important roles in hormone formation and release, the control of central sympathetic system activity and stress (Saavedra 2005). Lines of evidence supporting the hypothesis of a major role of brain Ang II in stress include stress-induced increases in circulating and brain Ang II levels (Yang et al. 1993, 1996), high AT1 receptor expression in all areas involved in the stimulation of the hypothalamic-pituitary-adrenal axis (HPA) activity, including the hypothalamic paraventricular nucleus (PVN), the median eminence (ME) and the subfornical organ (SFO) (Tsutsumi and Saavedra 1991b), and a stress-induced increase in AT1 receptor expression in the parvocellular PVN, where cell bodies forming the corticotropin-releasing hormone (CRH) are located (Castrén and Saavedra 1988; Aguilera et al. 1995a). AT1 receptors from the PVN are transported to the ME through axons co-expressing CRH (Oldfield et al. 2001). Stimulation of PVN AT1 receptors by Ang II increases CRH formation and release followed by enhanced ACTH release (Ganong and Murakami 1989; Aguilera et al. 1995b). In turn, increased levels of adrenal glucocorticoids regulate the expression of Ang II receptors in the PVN (Castrén and Saavedra 1989; Aguilera et al. 1995a) through stimulation of glucocorticoid response elements (GREs) in the AT1 receptor promoter (Guo and Inagami 1994).

Antagonism of brain AT1 receptors decreases CRH production and release in the PVN, explaining the blockade of the hormonal response to isolation stress by this class of compounds (Armando et al. 2001, 2007). Sustained inhibition of peripheral and brain AT1 receptors by peripheral administration of the AT1 receptor antagonist candesartan prevents not only the hormonal, but also the sympathoadrenal response to isolation stress (Armando et al. 2001). In addition, candesartan pretreatment prevents the activation of the brain sympathetic system during isolation (Saavedra et al. 2006), the isolation-induced cere-brocortical alterations in CRH1 receptors and the GABAA complex, reducing anxiety (Saavedra et al. 2006). These results suggest that the effect of AT1 receptor antagonists may not be limited to their action in the hypothalamus. In addition to hypothalamic areas, AT1 receptors are expressed in brain areas regulating the response of the limbic system to stress, such as the basolateral amygdaloid nucleus (Tsutsumi and Saavedra 1991a). This, and the anxiolytic and cortical effects of AT1 receptor blockade, suggest a role for Ang II in the regulation not only of the autonomic and hormonal, but also the behavioral response to stress (Shekhar et al. 2003).

The brain also expresses another Ang II receptor type, the AT2 receptors (Tsutsumi and Saavedra 1991a) located, in the adult rat brain, in areas related to sensory and motor function and behavior, such as the inferior olivary complex and thalamic nuclei (Tsutsumi and Saavedra 1991a). In the rat, there are high numbers of AT2 receptors in the locus coeruleus (Tsutsumi and Saavedra 1991a), the major site for catecholamine synthesis projecting to the forebrain (Sawchenko and Swanson 1981). A role for brain AT2 receptors in the regulation of central catecholamine formation and stress is further supported by the decrease in AT2 receptor mRNA in the locus coeruleus and inferior olive of rats submitted to chronic cold stress (Peng and Phillips 2001) and by the increased stress response (Watanabe et al. 1999), HPA axis stimulation and AT1 receptor expression (Armando et al. 2002) in AT2 gene-disrupted (AT2- /-) mice.

We have earlier reported that candesartan pretreatment prevented a stress-induced disorder, the development of cold-restraint induced gastric ulcers in spontaneously hypertensive rats (SHRs) (Bregonzio et al. 2003). In SHR, both the brain Ang II and sympathetic systems are hyperactive (Phillips and Kimura 1988; Palmer and Printz 1999). In addition, and perhaps as a consequence of the central Ang II and sympathetic hyperactivity, SHR are hypersensitive to a variety of stressors, including immobilization (McMurtry and Wexler 1981). To further clarify, the central mechanisms involved in the therapeutic effect of candesartan, and the role of Ang II AT1 and AT2 receptors in the regulation of the central response to stress, we studied these receptors in brain areas proposed to be involved in the hypothalamic and limbic response to cold-restraint stress in SHR.

Materials and methods

Animals

Adult, 8-week-old male spontaneously hypertensive rats (SHRs) weighing 200-250 g were purchased from Taconic Farms, Germantown, NY, USA, housed at 22°C, under a 12-h dark, 12-h light cycle (lights on at 07:00 h) and given free access to normal rat diet and tap water. The National Institute of Mental Health Animal Care and Use Committee approved all procedures. All efforts were made to minimize the number of animals used and their suffering. Animals were killed by decapitation without anesthesia to prevent biochemical changes as a result of the anesthetic procedures (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 80-23, revised 1996).

Groups of 12 animals received oral candesartan-cilexetil (TCV 116, from ASTRA, Mölndal, Sweden), 10 mg/kg per day for 14 days, or vehicle, dissolved in their drinking water. Candesartan-cilexetil was first dissolved as a 1 mg/ml stock solution in polyethylene glycol (PGE) 400/ethanol/Cremophor EL (Sigma Chemicals)/water (10/5/2/83%) adjusted to pH9 with 0.2 M Na2CO3. The stock solution was diluted in water to a final concentration equal to or less than 1/0.5/0.2% PEG/ethanol/Cremophor EL.

Systolic blood pressures were measured by the tail-cuff procedure in control, non-stressed animals on the day before administration of candesartan and after 14 days of administration.

Stress protocol

The rats were fasted overnight with free access to water, on the night before the experiment. Fasting overnight was required in this model to prevent gastric protection by stomach contents (Tanaka et al. 2007). On the day of the experiment, the group of 12 rats treated with candesartan and the group of 12 rats treated with vehicle were randomly divided into two groups of six rats each. Two of the groups, one previously treated with candesartan and one group treated with vehicle, were submitted to stress for 2 h between 9:00 AM and 12:00 noon, by placing them in standard adjustable plastic restraining devices (IITC Inc., Woodland Hills, CA, USA, model 82) maintained at 4°C. Two other groups, one treated with candesartan and the another one treated with vehicle, were not submitted to stress and kept in their home cages. Immediately after the end of the restraining period, the rats were killed by decapitation. The other two groups of rats were treated with vehicle or candesartan and served as controls. They were fasted but not submitted to cold-restraint stress.

Tissue preparation

The brain and adrenal glands were immediately removed, frozen in isopentane at -30°C on dry ice, and stored at -80°C until assayed. Consecutive, 16 μm thick coronal brain sections and adrenal sections were cut at -20°C in a cryostat. For anatomical localization of Ang II receptor binding or TH mRNA, sections were stained with Toluidine Blue, and brain regions were identified and designated according to a rat brain atlas (Paxinos and Watson 1986). Each rat was evaluated independently, and four sections per brain region were studied for each animal and for each procedure.

Quantitative autoradiography of angiotensin II AT1 and AT2 receptors

Coronal brain sections, 16 μm-thick, were cut in a cryostat at -20°C, thaw-mounted on poly-1-lysine-coated slides (Labscientific Inc., Livingston, NJ, USA), dried overnight in a desiccator at 4°C, and stored at -80°C until use. Sections were labeled in vitro with 0.5 nM of [125I]sarcosine1-Ang II ([125I]Sar1-Ang II, Peninsula Laboratories, Belmont, CA, USA; iodinated by the Peptide Radioiodination Service Center, School of Pharmacy, University of Mississippi, Mississippi, MS, USA, to a specific activity of 2176 Ci/mmol). Sections were pre-incubated for 15 min at 22°C in 10 mM sodium phosphate buffer, pH 7.4, containing 120 mM NaCl, 5 mM Na2EDTA, 0.005% bacitracin (Sigma Chemical, St. Louis, MO, USA), and 0.2% proteinase-free bovine serum albumin (Sigma Chemical), followed by incubation for 120 min in fresh buffer containing 0.5 nM of [125I]Sar1-Ang II. We determined total binding by incubating the sections as described above (Tsutsumi and Saavedra 1991a). Non-specific binding was determined in consecutive sections incubated as above in the presence of 1 μM unlabeled Ang II (Peninsula), and was defined as the binding remaining in the presence of excess unlabeled agonist. To determine selective binding to the Ang II AT1 and AT2 receptors, we incubated consecutive sections with 0.5 nM of [125I]Sar1-Ang II in the presence of the selective AT1 receptor antagonist losartan (10 μM; DuPont-Merck, Wilmington, DE, USA) or the selective AT2 receptor antagonist PD 123319 (Sigma Chemical), respectively, to give maximum specific displacement. The number of AT1 and AT2 receptors was defined as the binding displaced by the AT1 and AT2 receptor antagonists, respectively (Tsutsumi and Saavedra 1991a).

After incubation, slides were rinsed four consecutive times, for 1 min each, in fresh ice-cold 50 mM Tris-(hydroxymethyl)aminomethane) HCl buffer, pH 7.6, dipped in ice-cold distilled water, and dried under air. Sections were exposed to Kodak Biomax MR film (Eastman Kodak Company, Rochester, NY, USA) together with 14C-labeled microscales (American Radiolabeled Chemicals, St. Louis, MO, USA). Films were developed in ice-cold GBX developer (Eastman Kodak) for 4 min, fixed in Kodak GBX fixer for 4 min at 22°C, and rinsed in water for 15 min. Optical densities of autoradiograms generated by incubation with the 125I-labeled ligands were quantified by computerized densitometry using the Image 1.6 Program (National Institute of Mental Health, Bethesda, MD, USA) after calibration with 14C-labeled standards as described (Tsutsumi and Saavedra 1991a). The films were exposed for different times to obtain film images within the linear portion of the standard curve and the optical densities were converted to corresponding values of fmol per mg protein (Nazarali et al. 1989). Because, we used single ligand concentrations below saturation, we could not determine whether the changes described represent alterations in receptor number or receptor affinity.

In situ hybridization of tyrosine hydroxylase mRNA

For in situ hybridization, sections at the level of the locus coeruleus consecutive to those used for autoradiography were thaw-mounted on silanated glass slides (Digene Diagnostics, Beltsville, MD, USA) and stored at -80°C. Sections from the adrenal gland were collected as above.

An antisense oligonucleotide probe corresponding to 48 nucleotides of the rat tyrosine hydroxylase (TH) cDNA sequence (nt 1562-1609) was synthesized by Lofstrand Labs Ltd (Gaithersburg, MD, USA) (Grima et al. 1985). We labeled the probe to a specific activity of 3 to 4 × 108 dpm/μg with a 3′-end labeling kit (Amersham) that used terminal deoxynucleotidyl transferase. Each reaction was performed with 70 pmol of probe in the presence of 70 μCi of [α-35S]ATP (SJ 1334) (Amersham). The labeled probes were separated from unincorporated nucleotides using MicroSpin G-25 columns (Amersham). In situ hybridization of rat brain and adrenal sections and post-hybridization washings were performed as described (Wisden and Morris 1994). In situ hybridization was performed in consecutive sections, one with the TH antisense (AS) probe and another with added excess unlabeled TH-AS probe (157 pmol/ml). After the washing, sections were dehydrated in alcohols containing 0.3 M ammonium acetate, airdried and exposed to Hyperfilm-3H (Amersham, Arlington Heights, IL, USA) for 14 days. Films were developed in D-19 developer (Eastman Kodak, Rochester, NY, USA) for 4 min at 0°C and fixed in Kodak rapid fixer for 4 min at 22°C. The intensities of hybridization signals were quantified as nCi/g tissue equivalent (Wisden and Morris 1994) by measuring optical film densities using the NIH Image 1.61 program after calibration with the [14C] micro-scales.

Statistical analysis

Data from TH mRNA and Ang II receptor binding were analyzed by one-way ANOVA and Newman-Keuls post tests. Blood pressure data were analyzed by t-test. A value of P < 0.05 was considered significant.

Results

Effects of candesartan on blood pressure

Oral administration of candesartan reduced the systolic blood pressure in SHR from 190 ± 6 to 114 ± 5mm Hg (n = 6, P < 0.01).

Autoradiographic localization of angiotensin II receptor types

We detected AT1 binding in the SFO, PVN, ME, piriform cortex, basolateral amygdaloid nucleus, median preoptic nucleus, area postrema and the nucleus of the solitary tract (Figures 1-3). AT2 binding was located in the nucleus of the lateral olfactory tract, the mediodorsal and ventrolateral thalamic nuclei, the inferior olivary complex (dorsal, medial subnucleus A and B, and medial nuclei) and in the locus coeruleus (Figures 4 and 5). In each of the brain areas studied, we could detect only one receptor type, and not the other. This selective expression was maintained in stressed rats treated with vehicle and in control or stressed rats treated with the AT1 receptor antagonist (Figures 1-5).

Figure 1.

Figure 1

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Effects of stress and AT1 receptor blockade on AT1 receptor binding in the subfornical organ and paraventricular nucleus. AT1 receptor binding, as defined in “Materials and methods,” is visualized in the autoradiograms of sections incubated with [125I]Sar1-AngII in the presence of the AT2 receptor selective antagonist PD123319. The autoradiographs show one representative individual of each group. SFO, subfornical organ and PVN, paraventricular nucleus. Columns are means ± SEM obtained from six rats, measured individually. *P < 0.05. Bars are 3 mm.

Figure 3.

Figure 3

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Effects of stress and AT1 receptor blockade on AT1 receptor binding in the basolateral amygdaloid nucleus, median preoptic nucleus, area postrema and nucleus of the solitary tract. Columns are means ± SEM obtained from six rats, measured individually. *P < 0.05.

Figure 4.

Figure 4

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Effects of stress and AT1 receptor blockade on AT2 receptor binding in the locus coeruleus, the nucleus of the lateral olfactory tract, and in the mediodorsal and ventrolateral nuclei of the thalamus. Columns are means ± SEM obtained from six rats, measured individually. *P < 0.05.

Figure 5.

Figure 5

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Effects of stress and AT1 receptor blockade on AT2 receptor binding in the inferior olive. IO A + B, inferior olive, medial subnucleus A and B; IOM, inferior olive, medial nucleus; and IOD, inferior olive, dorsal nucleus. AT2 receptor binding, as defined in Materials and methods is visualized in the autoradiograms of sections incubated with [125I]Sar1-Ang II and displaced with the AT1 receptor-selective ligand losartan. Autoradiographs show one representative individual of each group. Columns are means ± SEM obtained from six rats, measured individually. *P < 0.05. Bar is 1 mm.

Effects of oral administration of an AT1 receptor antagonist on brain AT1 and AT2 receptor expression

Long-term oral treatment with the AT1 receptor antagonist inhibited binding to AT1 receptors in all brain areas studied, either outside (SFO, ME, and area postrema) or inside (median preoptic nucleus, PVN, basolateral amygdaloid nucleus, piriform cortex and nucleus of the solitary tract) the blood brain barrier (Figures 1-3).

No significant changes in AT2 binding occurred in the nucleus of the lateral olfactory tract, the mediodorsal or ventrolateral thalamic nuclei, or the locus coeruleus after long-term AT1 receptor blockade (Figure 4). Conversely, after pretreatment with candesartan, the number of AT2 receptors was significantly increased in the medial subnucleus A and B of the inferior olivary complex (Figure 5).

Effect of cold-restraint stress on brain AT1 and AT2 receptor expression

Cold restraint stress did not alter AT1 receptor number in the median preoptic nucleus, SFO, PVN, piriform cortex, or nucleus of the solitary tract (Figures 1-3). However, stress produced a significant increase in AT1 receptor binding in the area postrema, and a decrease in AT1 binding in the median eminence and the basolateral amygdaloid nucleus (Figures 2 and 3).

Figure 2.

Figure 2

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Effects of stress and AT1 receptor blockade on AT1 receptor binding in the median eminence and piriform cortex. Columns are means ± SEM obtained from six rats, measured individually. *P < 0.05.

After stress, we found significant increases in AT2 receptor binding only in the medial subnucleus A and B of the inferior olivary complex (Figure 5). Stress did not affect AT2 receptor binding in the nucleus of the locus coeruleus, the lateral olfactory tract, or the mediodorsal thalamic nucleus (Figure 4). Conversely, stress produced a significant decrease in AT2 receptor binding localized exclusively to the ventrolateral thalamic nucleus (Figure 4).

Effect of pretreatment with the AT1 receptor antagonist on AT1 and AT2 receptor expression after cold-restraint

Pretreatment with the AT1 receptor antagonist inhibited AT1 receptor binding after cold-restraint in all brain areas studied to the same extent as the inhibition observed in non-stressed rats (Figures 1-3).

AT2 receptor binding in the medial subnucleus A and B of the inferior olive was still significantly greater after AT1 blockade followed by cold-restraint (Figure 5). Similarly, AT1 receptor antagonism did not modify the AT2 receptor expression in the locus coeruleus, the nucleus of the lateral olfactory tract, and the mediodorsal thalamic nucleus after stress (Figure 4). On the other hand, the significant stress-induced reduction in AT2 receptor binding in the ventrolateral thalamic nuclei was no longer present (Figure 4).

Effect of cold restraint and AT1 receptor blockade on tyrosine hydroxylase mRNA expression in the locus coeruleus

Cold restraint produced a significant increase in TH mRNA expression in the locus coeruleus. Oral pretreatment with the AT1 receptor antagonist did not influence the expression of TH mRNA in control rats, but completely abolished the increased TH mRNA expression in the locus coeruleus that was produced by cold-restraint (Figure 6)

Figure 6.

Figure 6

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Effect of stress and AT1 receptor blockade on tyrosine hydroxylase mRNA in the locus coeruleus and in the adrenal medulla. Tyrosine hydroxylase was measured by quantitative in situ hybridization as described in Materials and methods. Candesartan treatment prevents the stress-induced increase in tyrosine hydroxylase mRNA expression in locus coeruleus, but not in the adrenal medulla. Columns are means ± SEM obtained from six rats, measured individually. *P < 0.05.

Effect of cold restraint and AT1 receptor blockade on adrenal tyrosine hydroxylase mRNA

After cold-restraint there was a significant increase in adrenomedullary TH mRNA. Oral pretreatment with the AT1 receptor antagonist did not modify the expression of TH mRNA in non-stressed rats, but significantly reduced the stress-induced increase in the expression of adrenomedullary TH mRNA (Figure 6).

Discussion

Effects of candesartan treatment and cold-restraint stress on Ang II AT1 receptors

Orally administered candesartan, at doses that decreased systolic blood pressure in SHR to normotensive levels, significantly blocked brain AT1 receptors situated outside or inside the blood brain barrier and effectively prevented the effects of centrally administered Ang II in SHR (Seltzer et al. 2004). The present results confirm our previous observations, demonstrating that orally administered candesartan may be used to study the activity and the role of brain Ang II in a manner similar to that of candesartan administered subcutaneously at an equivalent dose (Nishimura et al. 2000).

Acute cold-restraint stress produced profound and selective alterations in AT1 receptor expression. Only repeated, but not acute immobilization produces alterations in AT1 receptor expression (Leong et al. 2002). The decrease in AT1 receptor binding in the ME after acute cold-restraint, a more complex stress than immobilization alone, may be due to fast receptor internalization following enhanced Ang II binding as a consequence of the stress-induced increase in circulating and hypothalamic levels of the peptide (Yang et al. 1993). Receptor binding, however, was enhanced in another circumventricular organ, the area postrema. These changes are not likely to result from alterations in receptor synthesis, because of the short time of exposure to stress in our model.

Alterations in AT1 receptor binding during stress are not limited to hypothalamic structures. Of particular interest is the decrease in AT1 receptor binding in the basolateral amygdaloid nucleus. The basolateral amygdala has been implicated in the emotional response to stress (McIntyre et al. 2003; Sah et al. 2003). The presence of AT1 receptors in the basolateral amygdala has been previously recognized (Tsutsumi and Saavedra 1991a). Our results indicate that AT1 receptors in the basolateral amygdala are involved in the acute response to a major stress, and are in agreement with the recent report (Shekhar et al. 2003) of an Ang II-mediated activation of the basolateral amygdala during stress. We postulate that enhanced stimulation by Ang II may increase receptor internalization, resulting in the observed decrease in receptor binding.

In most brain areas, the decrease in AT1 receptor binding produced by candesartan pretreatment was not changed when candesartan-treated rats were submitted to cold-restraint. These observations indicate that brain AT1 receptor blockade was effective during the stress procedure.

Effects of candesartan treatment and cold-restraint stress on Ang II AT2 receptors

The AT1 antagonist increased AT2 binding in the inferior olivary complex and especially in the medial subnuclei A and B in non-stressed rats, confirming previous observations (Seltzer et al. 2004). In our study, candesartan did not increase AT2 receptor binding in the locus coeruleus or in the thalamic nuclei. We had previously observed increased AT2 binding in the locus coeruleus after four weeks of candesartan administration to non-fasted SHR (Seltzer et al. 2004). Differences in treatment conditions could best explain the discrepancy between the two studies.

We conclude that brain AT1 receptor antagonism has selective and profound influences on brain AT2 receptor expression, and that AT1 receptor activity regulates AT2 receptor number, at least in some selected brain areas. In turn, AT2 receptor activity regulates AT1 receptor number, as demonstrated by the increased AT1 receptor expression in the PVN of AT2- /- mice (Armando et al. 2002). The reciprocal interaction between AT1 and AT2 receptor expression supports the hypothesis of AT1/AT2 receptor interaction, or intracellular “cross-talk” (Gelband et al. 1997). However, we have not detected co-localization of AT1 and AT2 receptors in any brain area studied. If same cell AT1/AT2 colocalization exists in the brain, it is the exception rather than the norm (Tsutsumi and Saavedra 1991a). The cross-talk between brain AT1 and AT2 receptors is therefore due to intercellular rather than intracellular interactions, and its precise mechanisms remain to be elucidated.

We report major increases in AT2 receptor expression in the medial subnucleus A and B of the inferior olive after stress. Changes in AT2 receptor binding are probably not the result of increased receptor synthesis, because of the short period of stress, but they may represent increased receptor affinity or decreased receptor agonist occupancy. These results indicate that AT2 receptors in the inferior olive may be important for the regulation of the stress reaction by brain Ang II. Ang II, by stimulation of AT2 receptors, increases neuronal firing in the inferior olive (Ambuhl et al. 1992). The role of the inferior olive during the stress reaction has not been clarified, although it is well-known that this system controls sensory information and responds readily to sensory inputs that are not anticipated (Devor 2002).

In the ventrolateral thalamic nucleus, AT2 receptor expression substantially decreased after stress. Thalamic structures are among the few areas of the rat brain expressing substantial numbers of AT2 receptors in adult animals and this may signal a role in processing of sensory information in adults (Tsutsumi and Saavedra 1991a). The present results indicate an active role of AT2 receptors in the ventrolateral thalamic nucleus in the processing of information during stress. The mechanism of the fast decrease in binding may represent increased receptor agonist occupancy, because the AT2 receptors do not internalize after agonist binding (De Gasparo and Siragy 1999), and the timing of the changes is too short to be the result of alterations in receptor turnover.

Alterations in AT2 receptor binding in the inferior olivary complex did not change when candesartan-treated rats were submitted to cold-restraint. Conversely, the decrease in AT2 receptor binding in the ventrolateral thalamic nucleus observed during stress was abolished in rats pretreated with candesartan. The mechanism and significance of this finding remains an open question.

Effects of candesartan treatment and cold-restraint stress on tyrosine hydroxylase mRNA in the locus coeruleus

Candesartan completely prevented the stress-induced increase in TH mRNA expression in the locus coeruleus, a hallmark of the central response to stress and an indication of increased catecholamine synthesis and central sympathetic activity (Rusnak et al. 1998). This indicates that AT1 receptor antagonism abolishes the increased TH transcription not only during the relatively mild stress of isolation (Saavedra et al. 2006), but also during a major challenge such as cold-restraint as reported here. These findings indicate that the control of central catecholamine formation by brain Ang II (Saavedra and Benicky 2007) has important physiological correlates.

While the locus coeruleus of the rat expresses only AT2 receptors (Tsutsumi and Saavedra 1991a), the locus coeruleus of the mouse expresses only AT1 receptors, indicating that receptor type expression in this area is species-dependent (Häuser et al. 1998). There is evidence for a localization of Ang II (Fuxe et al. 1988) and Ang II receptors (Rowe et al. 1990) in catecholaminergic neurons in the locus coeruleus. However, Ang II does not affect norepinephrine release from this region (Huang et al. 1987). Instead, Ang II depresses the depolarizing effect of glutamate and excitatory postsynaptic potentials in the locus coeruleus through AT2 receptor stimulation (Xiong and Marshall 1994). We did not detect alterations in AT2 receptor expression in the locus coeruleus during stress or after AT1 receptor blockade. Others have reported increased AT2 receptor mRNA in the locus coeruleus of Wistar rats after acute or chronic immobilization or air-jet stress (Dumont et al. 1999) and decreased AT2 receptor mRNA expression after chronic cold stress (Peng and Phillips 2001). We conclude that the effect of AT1 receptor antagonists in the expression of TH mRNA in the locus coeruleus may not be direct. Indirect effects may include inhibition of brain stem AT1 receptors located in the nucleus of the solitary tract and the area postrema, as reported here, along with the blood pressure decrease and changes in the baroreflex that follow AT1 receptor antagonism (Hasser et al. 2000) and inhibition of the CRH mediation of the stress-induced stimulation of neuronal activity in this area (Porter 2000; Makino et al. 2002; Zeng et al. 2003).

Decreased TH transcription in the locus coeruleus may be causally related to the increase in AT2 receptor expression in the inferior olive, since inferior olivary neurons containing AT2 receptors (Tsutsumi and Saavedra 1991a) receive norepinephrine afferents from the locus coeruleus (Kobayashi et al. 1974). The regulation of AT2 receptor expression by alterations in central catecholamine formation is a novel hypothesis in need of further support.

In addition to the central alterations described above, oral pretreatment with the AT1 receptor blocker partially reduces the well-known stress-induced increase in adrenomedullary TH transcription (Kvetnansky et al. 1996). This supports our previous observations in rats submitted to isolation or cold-restraint stress and pretreated with candesartan administered subcutaneously (Armando et al. 2001; Bregonzio et al. 2003). These results are in agreement with recent findings of reduction in central and peripheral sympathetic nerve activity in neurogenic hypertensive rats by the AT1 receptor antagonist losartan (Ye et al. 2002).

In conclusion, our observations support a role for hypothalamic and limbic system AT1 receptors and brain stem AT2 receptors in the control of the central response to stress. The present findings demonstrate that AT1 receptor antagonists with dual peripheral and central effects inhibit the stress-induced increase in central and peripheral sympathetic activity. The anti-stress properties of candesartan are shared by other AT1 receptor antagonists such as losartan (Vinícius et al. 2007). These observations indicate that AT1 receptor blockers may be considered as effective anti-stress agents.

Acknowledgements

This study was supported by the Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Department of Health and Human Services, USA.

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