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Endocrinology Vol. 144, No. 5 2092-2101
Copyright © 2003 by The Endocrine Society

Angiotensin II AT1 and AT2 Receptors Contribute to Maintain Basal Adrenomedullary Norepinephrine Synthesis and Tyrosine Hydroxylase Transcription

Miroslava Jezova, Ines Armando, Claudia Bregonzio, Zu-Xi Yu, Sujuan Qian1, Victor J. Ferrans1, Hans Imboden and Juan M. Saavedra

Section on Pharmacology (M.J., I.A., C.B., J.M.S.), Division of Intramural Research Programs, National Institute of Mental Health, and Pathology Section (Z.-X.Y., S.Q., V.J.F.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and Institute of Cell Biology (H.I.), University of Bern, CH-3012 Bern, Switzerland

Address all correspondence and requests for reprints to: Miroslava Jezova, Section on Pharmacology, Division of Intramural Research Programs, National Institute of Mental Health, 10 Center Drive, Building 10, Room 2D-57, Bethesda, Maryland 20892. E-mail: armandoi{at}intra.nimh.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (Ang II) AT1 receptors have been proposed to mediate the Ang II-dependent and the stress-stimulated adrenomedullary catecholamine synthesis and release. However, in this tissue, most of the Ang II receptors are of the AT2 type. We asked the question whether AT1 and AT2 receptors regulate basal catecholamine synthesis. Long-term AT1 receptor blockade decreased adrenomedullary AT1 receptor binding, AT2 receptor binding and AT2 receptor protein, rat tyrosine hydroxylase (TH) mRNA, norepinephrine (NE) content, Fos-related antigen 2 (Fra-2) protein, phosphorylated cAMP response element binding protein (pCREB), and ERK2. Long-term AT2 receptor blockade decreased AT2 receptor binding, TH mRNA, NE content and Fra-2 protein, although not affecting AT1 receptor binding or receptor protein, pCREB or ERK2. Angiotensin II colocalized with AT1 and AT2 receptors in ganglion cell bodies. AT2 receptors were clearly localized to many, but not all, chromaffin cells. Our data support the hypothesis of an AT1/AT2 receptor cross-talk in the adrenomedullary ganglion cells, and a role for both receptor types on the selective regulation of basal NE, but not epinephrine formation, and in the regulation of basal TH transcription. Whereas AT1 and AT2 receptors involve the Fos-related antigen Fra-2, AT1 receptor transcriptional effects include pCREB and ERK2, indicating common as well as different regulatory mechanisms for each receptor type.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (Ang II) participates in the regulation of fluid and electrolyte homeostasis, hormone secretion from the anterior and the posterior pituitary gland, and in the central control of the autonomic nervous system, through receptors present within the brain and many peripheral tissues (1, 2, 3). The AT1 site is the physiologically active Ang II receptor, mediating most, if not all, the well-known functions of Ang II (4). The AT2 site is highly expressed in peripheral fetal tissues, in the immature brain and, in adult rodents, in the adrenal medulla (3). This site has been proposed to be involved in the regulation of growth and to cross-talk with the AT1 receptor (3, 5, 6). The physiological function of the AT2 receptors has not yet been clarified, and the experimental evidence is equivocal (3, 5).

Ang II participates in the regulation of the stress reaction at all levels of the hypothalamic-pituitary-adrenal axis. Stress increases the production of circulating Ang II (7) and the expression of Ang II AT1 receptors in brain areas crucial for the central control of the stress reaction such as the hypothalamic paraventricular nucleus (8, 9), in the anterior pituitary gland, and in the adrenal medulla (10). These findings indicate that the Ang II AT1 receptor mediates the regulation of the stress reaction by Ang II (9).

In the adrenal medulla, Ang II releases catecholamines by a direct action (11), mediated either by circulating Ang II or by the intrinsic adrenal renin angiotensin system (11, 12, 13). Both AT1 and AT2 Ang II receptors are expressed in the adrenal medulla. In the rat, AT2 receptors predominate, AT1 receptors representing only 5–10% of the total number of Ang II receptors (14). It appears that AT1 receptor stimulation is most important as a regulatory factor for adrenomedullary catecholamine synthesis and release. First, AT1 blockade is sufficient to inhibit in vivo adrenal catecholamine release by Ang II (15). Second, pretreatment with an insurmountable AT1 antagonist almost completely abolished the hormonal and sympathoadrenal response to the stress of isolation in unfamiliar metabolic cages (16). However, isolation stress also produced a substantial increase in adrenomedullary AT2 receptor binding, abolished by pretreatment with the AT1 receptor antagonist (16). This indicated a possible role of AT2 receptors in the adrenomedullary response to stress.

In cultured bovine adrenomedullary cells, Ang II increases catecholamine biosynthesis, producing acute activation of rat tyrosine hydroxylase (TH) and increased expression of the genes coding for this enzyme (17). Regulation of TH activity involves short-term posttranscriptional mechanisms (18), whereas long-term regulation involves transcriptional activation of the TH gene (19) by multiple factors and promoter elements. The participation of these mechanisms in the proposed Ang II-mediated regulation of TH transcription has not been fully characterized.

We wished to clarify the physiological role of Ang II and its receptor types, under basal conditions, on the molecular mechanisms of regulation of TH transcription in the adrenal medulla, including the family of proteins binding to the activator protein (AP)-1 site and to the cAMP response element (CRE; Refs.20, 21, 22). To this end, we studied the effects of long-term and selective blockade of Ang II AT1 or AT2 receptors under basal conditions, on receptor expression, adrenomedullary catecholamine levels and TH mRNA, and analyzed some of the factors proposed in the regulation of TH transcription. Because AT1 antagonists are used in the therapy of chronic hypertension, we performed long-term studies to clarify the adrenomedullary response to prolonged drug administration. In addition, we localized the expression of Ang II and Ang II receptor types by immunocytochemistry in adrenomedullary ganglion and chromaffin cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and preparation of tissues
Wistar Hanover male rats (8 wk old) were purchased from Taconic Farms, Inc. (Germantown, NY), were kept at 22 C 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 National Institute of Mental Health (NIMH) Animal Care and Use Committees approved all procedures.

Rats were anesthetized with pentobarbital (30 mg/kg) and Alzet osmotic minipumps (Alza Scientific Products, Palo Alto, CA) were implanted subcutaneously. Groups of ten animals (treated) received minipumps containing vehicle, the AT1 receptor antagonist candesartan (Astra USA, Inc., Wedel, Germany) dissolved in 1 mol/liter sodium carbonate and further diluted in isotonic saline, at a final pH of 7.5–8.0, to be delivered at a rate of 1.0 mg/kg·d, or the AT2 receptor antagonist PD 123319 (Sigma-Aldrich Corp., St. Louis, MO; 1 mg/kg·d), respectively. While candesartan is an insurmountable antagonist with long-term effects (23), the half-life of PD 123319 is short (24). For this reason, doses and routes of administration of the receptor antagonists were selected to provide continuous receptor blockade and to obtain approximately equal degrees of inhibition for each receptor type. After minipump implantation the rats were kept in their cages in groups of three to four under standard conditions with regular rat food and water ad libitum for 14 d. At the end of the experiment the animals were killed by decapitation and adrenal glands were removed, frozen in isopentane at -30 C on dry ice, and stored at -80 C until used.

Autoradiography of Ang II receptor subtypes
Consecutive sections from adrenal gland, 16 µm-thick, were cut in a cryostat at -20 C and thaw-mounted on gelatinized slides, dehydrated overnight in a desiccator at 4 C, and kept at -80 C until used. Binding experiments were performed as described earlier (23). 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] Sarcosine1-Ang II ([125I] Sar1-Ang II; Peninsula Laboratories, Inc., Belmont, CA) and iodinated by the Peptide Radioiodination Service Center, Washington State University (Pullman, WA) to a specific activity of 2176 Ci/mmol to determine total binding. Nonspecific binding was determined by incubation of consecutive sections as above in the presence of 5 x 10-6 M 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 10-5 M of the AT1 receptor antagonist losartan (DuPont Merck, Wilmington, DE). Losartan has been shown previously to completely displace binding from AT1 receptors at this concentration (25). 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 of [125I] Sar1-Ang II in the presence of 1 µM of the selective AT2 ligand PD 123319. PD 123319 has been shown previously to completely displace binding from AT2 receptors at this concentration (25). 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, 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 Kodak Biomax MR (Eastman Kodak Co., Rochester, NY). Optical densities of autoradiograms were determined by computerized microdensitometry using the Image 1.61 program (NIMH, Bethesda, MD), quantified by comparison with [14C] microscales (American Radiolabeled Chemicals, Inc., St. Louis, MO) and transformed to corresponding values of fmol/mg protein (25, 26).

In situ hybridization of tyrosine hydroxylase mRNA
We used one antisense (TH-AS) of 48-oligomer for TH cDNA sequence localized in nucleotides 1562–1609 (27 ; Lofstrand Labs Ltd., Gaithersburg, MD). Labeling was performed with a 3'-end labeling kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) using terminal deoxynucleotidyl transferase to a specific activity of 3–4 x 108 dpm/µg. Each reaction was performed with 70 pmol of oligonucleotides in the presence of 70 µCi of [{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 described (28). 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 Kodak Biomax MR. The films were developed and quantified as described above by comparison with [14C] standards (American Radiolabeled Chemicals, Inc.).

Western blot analysis
After dissection of the adrenal cortex and medulla, tissues were homogenized on ice in buffer containing 10 mM Tris (pH 7.4), 1% sodium dodecyl sulfate, and protease inhibitors (Protease Inhibitor Cocktail, Roche, Mannheim, Germany) and the homogenate was centrifuged at 1800 x g for 5 min at 4 C. Protein concentration was determined by the Bradford procedure (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins were fractionated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). After blocking with 5% nonfat dried milk (Bio-Rad Laboratories, Inc., Hercules, CA) in Tris-buffered saline (100 mM Tris-Cl, pH 7.5; and 100 mM NaCl) containing 0.1% Tween 20 (TBS-T) for 1 h, the membranes were incubated with appropriate amounts of the primary antibody [anti-Fra-2 (Fos-related antigen 2], anti-AT1 and AT2 receptor rabbit polyclonal antibody, anti-pCREB [phosphorylated CRE binding protein (CREB); Ser-133] goat polyclonal antibody and antiactin monoclonal antibody, all from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-phospho-p44/42 MAPK, control p44/42 MAPK antibody both from Cell Signaling Technology Inc. (Beverly, MA) in TBS-T containing 5% dried milk at 4 C overnight. After washing the membranes with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated donkey antirabbit, sheep antimouse IgG (Amersham Pharmacia Biotech) or donkey antigoat IgG (Santa Cruz Biotechnology, Inc.) for 1.5 h at a dilution of 1:2000 in the same buffer. The proteins were visualized with Kodak X-OMAT film using a chemiluminescence system (ECL, Amersham Pharmacia Biotech). The amount of each protein was quantified using a Microsoft Corp.-based image processing system Scion Image software (Scion Corp., Frederick, MD) using the actin protein as a control.

Catecholamine determinations in adrenal glands
The content of adrenal norepinephrine (NE) and epinephrine (E) was determined by reverse-phase HPLC with electrochemical detection in aliquots of adrenal supernatants (29). Sample catechols were partially purified by batch alumina extraction, separated by HPLC using a 4.6 x 250 mm Zorbax RxC18 column (DuPont 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 E and NE. Catechol concentrations in each sample were corrected for recovery of an internal standard, dihydroxybenzylamine. The limit of detection was about 15 pg/vol assayed for each catechol.

Colocalization studies
Dual immunofluorescent labeling was employed for simultaneous demonstration of two antigens. Briefly, frozen section of adrenal gland were air dried, fixed with cold acetone for 5 min, and washed with PBS. The sections were then incubated overnight with primary antibodies followed by secondary antibodies conjugated with fluorescein isothiocyanate or Texas red (Vector Laboratories, Burlingame, CA). Nuclei were stained with 2,6'-diamidino-4-phenyl-indole (Vector Laboratories). For AT1 receptor immunohistochemistry, we used a monoclonal antibody against the third internal loop of the human AT1 receptor, amino acids 229–246 (no. 4H2; Ref. 30). The specificity of the antibody was validated by dot blot assay, by Western blot analysis of whole adrenal protein, and by immunohistochemistry in sections of the rat adrenal gland. The AT1 receptor antibody reacted with both the AT1A and AT1B peptides (amino acids 229–246), and there was no cross-reactivity against the AT2 peptide (amino acids 314–330). In addition, the AT1 receptor antibody detected a prominent band with an apparent molecular mass of 73 kDa, consistent with that of the AT1 receptor, and detected the presence of AT1 receptors in the rat adrenal cortex and medulla (30). For AT2 receptor immunohistochemistry, we used a goat polyclonal antibody against the carboxy terminus of the AT2 receptors (no. C-18 from Santa Cruz Biotechnology, Inc.). For Ang II immunohistochemistry, we used an Ang II mouse monoclonal antibody (4B3) validated by dot-blot assay (30). In experiments for colocalization of Ang II receptor types and Ang II we used a rabbit polyclonal antibody raised against amino acids 306–359 of the AT1 receptor from Santa Cruz Biotechnology, Inc., and antibodies against the AT2 receptor and Ang II as described above. Control procedures (omission of the primary antibody and substitution of this antibody for normal IgG of the same animal species) gave negative results. Three-color fluorescence imaging was accomplished using a laser scanning confocal fluorescence microscope (TCS-4D, Leica Corp., Heidelberg, Germany).

Statistical analysis
Data are the mean ± SEM. We used Student’s t test to assess the significance of differences between groups. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of AT1 and AT2 receptor blockade on the expression of Ang II receptor types
Administration of an AT1 antagonist (1.0 mg/kg·d) for 14 d significantly decreased AT1 receptor binding in the adrenal medulla (about 36%) without significantly affecting the expression of AT1 receptor protein (Fig. 1Go). The treatment also significantly reduced AT2 receptor binding (about 16%; Fig. 2Go). The reduction in AT2 binding was associated with a 27% reduction in the expression of AT2 receptor protein (Fig. 2Go).



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  		Figure 1. Expression of Ang II AT1 receptor binding and receptor protein after AT1 or AT2 receptor blockade. AT1 receptor binding was examined by quantitative autoradiography as described in Materials and Methods. AT1 receptor protein levels were examined by Western blot. Results are presented as the mean ± SEM of groups of six rats, measured individually, and normalized to the corresponding control. *, P < 0.05 vs. vehicle-treated rats.

 


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  		Figure 2. Expression of Ang II AT2 receptor binding and protein after AT1 or AT2 receptor blockade. AT2 receptor binding was examined by quantitative autoradiography as described in Materials and Methods. AT2 receptor protein levels were examined by Western blot. Results are presented as the mean ± SEM of groups of six rats, measured individually, and normalized to the corresponding control. *, P < 0.05 vs. vehicle-treated rats.

 
Administration of the AT2 receptor antagonist PD 123319 for 14 d at the concentration of 1.0 mg/kg·d significantly (P < 0.05) reduced AT2 receptor binding (about 33%) with no effect on expression of AT2 receptor protein (Fig. 2Go). Treatment with PD 123319 also failed to affect AT1 receptor binding and expression of AT1 receptor protein (Fig. 1Go).

Effect of AT1 and AT2 receptor blockade on TH mRNA and adrenomedullary catecholamines
Treatment with the AT1 receptor antagonist significantly decreased adrenal medulla TH mRNA (about 35%) and the levels of adrenal NE (about 32%; Fig. 3Go, A and B). However, the AT1 receptor blocker did not affect adrenal E content (Fig. 3AGo).



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  		Figure 3. A, TH mRNA and adrenal catecholamines after AT1 or AT2 receptor blockade. Results are presented as the mean ± SEM of groups of six rats, measured individually. *, P < 0.05 vs. vehicle-treated rats. B, Adrenal TH mRNA after AT1 or AT2 receptor blockade. Figures are images obtained from sections of adrenal gland after in situ hybridization, as described in Materials and Methods. Scale bar, 1 mm.

 
Inhibition of AT2 receptors with PD 123319 exerted effects very similar to those of AT1 receptor blockade, namely a significant decrease in adrenal medulla TH mRNA (about 20%) and in the adrenal NE content (about 23%) without affecting the adrenal E level (Fig. 3Go, A and B).

Effect of AT1 and AT2 receptor blockade on expression of transcription factors
AT1 receptor antagonism significantly decreased the levels of Fra-2 protein (about 26%) in adrenal medulla and pCREB (about 35%) and ERK2 (p-ERK2; phospho-p42 MAPK; about 17.5%) with no effect on the phosphorylated form of ERK1 (p-ERK1; phospho-p44 MAPK; Fig. 4Go).



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  		Figure 4. Levels of Fra-2 protein and pCREB, ERK1 (pERK1, phospho-p44 MAPK), and ERK2 (pERK2, phospho-p42 MAPK) after AT1 blockade in adrenal medulla. Results are presented as the mean ± SEM of groups of six rats, measured individually, and normalized to the corresponding control. *, P < 0.05 vs. vehicle-treated grouped rats.

 
Blockade of AT2 receptors with PD 123319 significantly decreased adrenal medulla Fra-2 protein (about 38%; Fig. 5Go). Conversely, PD 123319 treatment did not change the phosphorylation of CREB or ERK1/2 protein (Fig 5Go).



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  		Figure 5. Levels of Fra-2 protein and pCREB, ERK1 (pERK1, phospho-p44 MAPK) and ERK2 (pERK2, phospho-p42 MAPK) after AT2 blockade in adrenal medulla. Results are presented as the mean ± SEM of groups of six rats, measured individually and normalized to the corresponding control. *, P < 0.05 vs. vehicle-treated grouped rats.

 
Immunohistochemical localization of Ang II and AT1 and AT2 receptors in the adrenal medulla
We report the localization of Ang II, AT1, and AT2 receptors, as determined by immunocytochemistry, in ganglion cells and chromaffin cells of the adrenal medulla. Adrenomedullary ganglion cells are very low in numbers, localized in clusters and more abundant toward the periphery of the adrenal medulla. The number of chromaffin cell is much larger than that of ganglion cells, and they are present throughout the adrenal medulla. The distribution of AT1, AT2 and Ang II immunocytochemistry was not homogenous, with AT1 and Ang II predominant in ganglion cells and AT2 predominant in chromaffin cells. We did not attempt to precisely quantify the number of positive cells.

Both Ang II and AT1 receptors were colocalized, with high intensity of immunofluorescence signals, in many, but not all ganglion cells (Fig. 6Go). Immunofluorescence for AT2 receptors was also high but was localized to fewer ganglion cells (Fig. 6Go). In ganglion cells, whereas both Ang II and AT1 receptors were expressed at the surface and within the cytoplasm (Fig. 6Go), we could localize AT2 receptors only at the cellular surface (Fig. 6Go). We did not find any ganglion cell containing Ang II without the concomitant expression of AT1 receptors. AT2 receptors were colocalized with AT1 receptors in about half of the ganglion cells expressing AT1 receptors (Fig. 6Go).



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  		Figure 6. Immunohistochemical localization of Ang II, AT1 receptors and AT2 receptors in the rat adrenal medulla. Upper row, Selective cellular localization of AT1 and AT2 receptor types. Left, AT1 receptors (red); middle, AT2 receptors (green); right, superimposed pictures of AT1/AT2 receptors (orange-yellow). Nuclei were counterstained with 2,6'-diamidino-4-phenyl-indole (blue). Asterisks point to a group of chromaffin cells expressing AT2 receptors but negative for AT1 receptors. Arrows point to ganglion cell bodies expressing AT1 receptors, but negative for AT2 receptors. Arrowheads point to AT1 and AT2 receptors colocalized to cell bodies of ganglion cells. Middle row, Cellular colocalization of AT1 and AT2 receptor types. Left, AT1 receptors (red); middle, AT2 receptors (green); right, AT1/AT2 receptors (orange-yellow). Arrows point to localization of AT1 receptors in the cell surface and in the cytoplasm, and AT2 receptors on the surface of a ganglion cell body. Lower row, Cellular colocalization of AT1 receptors and Ang II. Left, AT1 receptors (red); middle, Ang II (green); right, AT1/Ang II (orange-yellow). Arrowheads point to the colocalization of AT1 receptors and Ang II in ganglion cell bodies. Note that both AT1 receptors and Ang II appear to be located at the surface and in the cytoplasm of the ganglion cell body. Bars, 20 µm.

 
Immunocytochemical data differed in chromaffin cells. Few chromaffin cells were positive for Ang II or AT1 receptors, and the immunocytochemical images detected were of low intensity (Fig. 6Go). Conversely, many, but not all, chromaffin cells were intensely positive for AT2 receptors (Fig. 6Go). There were many chromaffin cells expressing AT2 receptors but not AT1 receptors (Fig. 6Go), and there were no clearly identifiable chromaffin cells where colocalization of Ang II and AT1 receptors, Ang II and AT2 receptors, or AT1 and AT2 receptors could be demonstrated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our in vivo studies demonstrate that both Ang II AT1 and AT2 receptors participate in the long-term maintenance of basal levels of TH transcription and NE synthesis in the rat adrenal medulla, and that the basal activation of Ang II receptors is physiologically important in this organ.

Our results are important for a number of reasons. First, our observations indicate that adrenomedullary AT1 receptors, in addition to the demonstrated participation in the Ang II-mediated (15, 31) and stress-mediated (16) in vivo catecholamine release, are important in the maintenance of catecholamine production, because their long-term selective inhibition is sufficient to significantly reduce TH mRNA and NE content in this organ. These findings are remarkable because in the adrenal medulla, one of the tissues expressing highest numbers of Ang II receptors in the adult rat (32), AT1 receptors represent not more than 10% of the total Ang II receptors (14, 33).

Second, our results are consistent with the hypothesis of a role of AT2 receptors in the synthesis and release of adrenal catecholamines. Previous contradictory results were probably dependent on assay conditions and the species studied, with reports of the AT2 receptor agonist CGP 42112 enhancing basal catecholamine release (34) or decreasing Ang-II induced catecholamine release without affecting their basal output (31). We have found evidence that long-term selective, although partial, AT2 blockade is sufficient to significantly impair the basal expression of TH mRNA and to decrease the adrenal NE content. No inhibition of AT1 receptors occurred after administration of PD 123319, confirming the selectivity of this compound, both in vitro (25) and in vivo (24).

It is clear that a cross-talk between AT1 and AT2 receptors occurs in the adrenal medulla. In this tissue, AT1 and AT2 receptors may act in a synergistic manner and may not be mutually opposing as originally postulated (6, 35). Additional support for our hypothesis is based on the recent observations of increased adrenomedullary AT1 and AT2 receptors and catecholamine synthesis during isolation stress, and by the finding that AT1 receptor antagonism abolished not only the stress-induced increase in catecholamine synthesis but also that of AT2 receptor expression (16). The increased AT1 receptor expression in the adrenal gland of AT2 receptor gene-deficient mice (36) could then be explained as an attempt to maintain an adequate basal catecholamine metabolism.

Our data contradict previous studies in cultured chromaffin cells that suggested counter-regulatory roles for AT1 and AT2 receptors in the catecholamine synthesis (37, 38). Such a contradiction could be due to the failure of cultured chromaffin cell systems to replicate the complex cellular interaction and the differential cellular localization of the Ang II receptor types that occurs in vivo.

AT1 and AT2 receptor interactions are complex and may not always be synergistic. Absence of AT2 receptor expression results in increased AT1 receptors in the paraventricular nucleus, a change associated with increased vulnerability to stress (39). AT1 receptor expression is also increased in the kidney of AT2 receptor gene deficient mice (40). However, in this organ, whereas AT1 receptor stimulation increases growth and vasoconstriction, increased AT2 receptor activation has been linked to inhibition of growth and vasodilation (41).

The third finding of interest is that AT1 receptors are not only predominantly present in adrenomedullary ganglion neurons (42), but they are colocalized in these cells with their natural agonist, indicating a possible role as presynaptic regulators of Ang II actions and release. These catecholamine and peptide-containing cells represent an intraadrenal system of ganglion neurons (42), part of the intrinsic innervation of the adrenal gland (43). Our observations highlight the importance of adrenomedullary ganglion neurons in the regulation of catecholamine synthesis by adrenal chromaffin cells (44) and suggest that ganglion neurons could be one of the sites of activity of local adrenomedullary renin angiotensin system (45).

The question remains as to how low numbers of AT1 receptors, mainly present in the low abundance adrenomedullary ganglion neurons, have such a major impact on basal catecholamine production. It appears that at least part of the effect of AT1 receptors on catecholamine synthesis may be mediated through regulation of AT2 receptor expression. Several of our findings support this hypothesis. Long-term treatment with the AT1 receptor antagonist not only blocks AT1 receptor binding, but decreases AT2 receptor binding and protein expression as well. In addition, long-term selective inhibition of AT2 receptors, although not complete, produces significant decreases in adrenomedullary TH mRNA and NE content, similar to those found after selective AT1 blockade. These results, and the occurrence of clear colocalization of AT1 and AT2 receptors in ganglion neurons, support the hypothesis of a cross-talk between both Ang II receptor types.

In addition to their expression in ganglion neurons, AT2 receptors appear to be expressed in larger numbers in many chromaffin cells, where most of the adrenomedullary catecholamine synthesis occurs. Because most of adrenomedullary AT2 receptors are expressed in chromaffin cells, it is likely that the decreased AT2 receptor expression after AT1 receptor blockade may occur not only in the ganglion neurons, but also and predominantly in the chromaffin cells. We did not attempt to perform a quantitative immunocytochemical study in individual cells because of poor reliability of the methods for such purpose.

We have found immunocytochemical signals for AT1 receptors close to the surface of a few chromaffin cells, which are located either on the chromaffin cell membranes or in terminals of ganglion neurons. Thus, regulation of AT2 receptor expression by AT1 receptors could occur by receptor cross-talk within chromaffin cells or by regulation of AT2 receptors in chromaffin cells by changes in AT1-mediated ganglion neuron activity. In the adrenal medulla, the synergistic interaction between the Ang II receptor types is probably not only the result of intracellular mechanisms in cells coexpressing both receptor types but also occurs through intercellular interactions between cells expressing only AT1 or AT2 receptors. In addition, AT1 receptor blockade very significant increases in Ang II synthesis (4), and increased Ang II could affect the expression of AT2 receptors.

Although local mechanisms in the adrenal medulla appear to be most important, it is also possible that brain mechanisms could contribute to the effects of AT1 blockade in adrenal catecholamine synthesis because the AT1 receptor antagonist used here blocks not only peripheral but also central AT1 receptors (23).

The present results appear to indicate that AT1 and AT2 regulation of basal catecholamine synthesis is restricted to that of NE, with no influence on basal E formation, suggesting that under basal conditions E containing chromaffin cells may be less sensitive to the effects of Ang II. This finding is at odds with the clear control of both NE and E formation and release by AT1 receptors during stress (16) and with the dual inhibition by AT1 and AT2 receptors of insulin-induced E release (46). However, our findings are not surprising. Although TH is the rate-limiting enzyme for all catecholamines, different sets of chromaffin cells form NE only or NE and E, where NE may only act as E metabolic precursor, and NE and E formation and release can be regulated by selective and different mechanisms (46). On the other hand, adrenomedullary E stores are larger than those of NE, and regulation of E formation by Ang II receptors under basal conditions could occur even in the absence of significant alterations in its tissue content.

Fourth, our findings shed some light on the mechanisms of transcriptional regulation of adrenomedullary TH by AT1 and AT2 receptors. It is known that a CRE-like site is involved in the Ang II stimulation of TH (20). CREB may interact with the CRE or CRE-like elements in the TH promoter region (21) or may promote de novo synthesis of other CRE-regulated genes (c-fos and fos-family members) that could interact with the AP-1 binding sites in the TH promoter region. Different members of the AP-1 family may be involved in a stimuli and region-specific manner. Fra-2 is a mediator of stress response in the adrenal medulla after repeated immobilization stress (22). We found that the decrease of adrenal NE and TH mRNA after long-term AT1 receptor blockade was accompanied by significant decreases in Fra-2 and pCREB protein levels. In vitro, Ang II produced an AT1 receptor dependent phosphorylation of CREB at serine 133 (47) and stimulation of Ang II receptors was associated with increased levels of cAMP (48). The parallel decrease in pCREB immunoreactivity and TH mRNA in adrenal medulla after pretreatment with the AT1 receptor blocker suggests a role for cAMP and CREB acting via cAMP response element in the TH promoter. Alternatively, pCREB may serve as a signal to mediate the transcription of fra-2 (49), which may further regulate the TH expression by binding to the AP-1 region in the TH promoter. In addition, Fra-2 expression may be also autoregulated in response to the phosphorylated status of its gene product (49). ERK2 phosphorylates several serine and threonine residues located in the COOH-terminal region of Fra-2. Phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an active one. The heterodimer formed between phosphorylated Fra-2 and c-jun has very high transcriptional activity, which would induce expression of a wide range of genes through AP-1 binding site, including the one present in the fra-2 promoter (50). Indeed, after AT1 blockade, we found a significant decrease in phosphorylated ERK2 (pERK2) that parallels the decrease in Fra-2 immunoreactive protein in adrenal medulla, indicating that this pathway may be involved in the regulation of Fra-2 protein expression and hence transcription of TH gene by Ang II.

Decreased TH transcription after AT2 blockade is associated with substantial decreases in the expression of the Fra-2 protein in a manner similar to that which occurs after AT1 receptor blockade. However, AT2 blockade does not affect pCREB or pERK1/2, indicating a pathway that appears to be different to that involved in the effect of the AT1 blockers.

In conclusion, we have demonstrated a synergistic effect of AT1 and AT2 receptors, through mechanisms not entirely similar, in the transcriptional regulation of NE synthesis and a clear colocalization of both receptor types in adrenomedullary ganglion neurons. A schematic representation of some of the possible sites and mechanisms presented here is outline in Fig. 7Go. Our results indicate complex mechanisms for the adrenomedullary control of catecholamine synthesis and release by Ang II.



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  		Figure 7. Interactions between Ang II, AT1, and AT2 receptors in catecholamine synthesis regulation in adrenomedullary cells. The figure represents some possible mechanisms for regulation of adrenomedullary catecholamine synthesis by Ang II and its receptors types. Ang II could reach the adrenal medulla through the bloodstream, or be locally synthesized in ganglion cells and perhaps some chromaffin cells. Ganglion cell Ang II could be released to stimulate AT1 and AT2 receptors in some chromaffin cells, or only AT2 receptors in other chromaffin cells. Presynaptic AT1 and AT2 receptors could regulate Ang II release from ganglion cells, modulating its access to postsynaptic receptors. In some chromaffin cells, both AT1 and AT2 receptor stimulation could increase phosphorylation of Fra-2, leading to stimulation of TH mRNA production. Additionally, AT1 receptor stimulation could increase ERK2 and CREB phosphorylation, additional mechanisms of possible importance in the transcriptional regulation of TH. In other chromaffin cells, expressing only AT2 receptors, possible direct or indirect effects of AT2 stimulation on Fra-2 phosphorylation could modulate regulation of TH transcription.

 


   Acknowledgments
 
The authors wish to thank Dr. Gustavo Baiardi for his generous help in the preparation of the figures.


   Footnotes
 
1 The authors S.Q. and V.J.F. are deceased. Back

Abbreviations: Ang II, Angiotensin II; AP, activator protein; CRE, cAMP response element; CREB, CRE binding protein; E, epinephrine; Fra-2, Fos-related antigen-2; NE, norepinephrine; pCREB, phosphorylated CREB; pERK2, phosphorylated ERK2; [125I] Sar1-Ang II[, [125I] sarcosine1-Ang II; TH, rat tyrosine hydroxylase; TH-AS, TH antisense.

Received November 8, 2002.

Accepted for publication January 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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