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Influence of Dietary Sodium Restriction on Angiotensin II Receptors in Rat Adrenals¹

Department of Biochemistry (J.G.L., L.D.), and Department of Anatomy and Cell Biology (N.B., D.M.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4, and Department of Obstetrics-Gynecology (I.M.B.), University of Wisconsin, Madison, Wisconsin 53715

Address all correspondence and requests for reprints to: Jean-Guy LeHoux, Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. E-mail: j.lehoux{at}courrier.usherb.ca

	Abstract

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We studied the distribution of angiotensin II (AII) receptors type 1 (AT₁) and type 2 (AT₂) and the effects of a low sodium intake on these two subtypes of receptors in male rat adrenals. Binding studies on adrenal slices, on cell membranes and on cell suspensions were performed using [¹²⁵I]AII and specific analogs for AT₁ (Losartan) and AT₂ (PD 123319) receptors. The distribution of AT₁ was also studied by immunofluorescence. Complementary approaches were necessary to reach our goal. Indeed, by autoradiography on adrenal slices, [¹²⁵I]AII was shown to bind to the zona glomerulosa (ZG) and to the medulla (M). When coincubated with [¹²⁵I]AII, PD 123319 displaced [¹²⁵I]AII from the medulla and from the ZG, indicating the presence of AT₂ receptors in both zones. Losartan partially displaced [¹²⁵I]AII from the ZG, indicating the presence of AT₁ receptors in that zone. Furthermore, the labeling intensity of the medulla (AT₂ receptors) was much stronger in adrenal sections from rats kept on a low sodium regimen than from controls. Immunofluorescence microscopy revealed that AT₁ receptors were located mainly in the ZG of control rats. After sodium restriction, AT₁ receptors appeared to be uniformly distributed within an enlarged ZG; furthermore AT₁ receptor-positive cells were found to a limited degree in the zona fasciculata and possibly in the zona reticularis, and a greater number of these positive cells appeared in these zones under sodium restriction. Cell suspensions from rats fed a low sodium diet showed a 2.7- and 2.1-fold increase in total AII receptors in adrenal ZG and ZFR + M cells when compared with controls. Based on Losartan displacement, we calculated that [¹²⁵I]AII bound to AT₁ and to AT₂ receptors was increased in both ZG and ZFR + M cell preparations under sodium restriction. Results of binding studies on cell membranes were also indicative of an increasing effect of sodium restriction on AT₁ and AT₂ receptors binding capacity. Furthermore, Northern blotting analysis revealed 3.0- and 2.5-fold increases in the level of AT₁ receptor mRNA in the ZG and the ZFR + M of rats fed a low sodium diet as compared with those fed a normal diet. The low sodium intake resulted in a weaker increase (1.5-fold) in the level of AT₂ receptor messenger RNA in the ZG, with no changes in the ZFR + M preparations. In conclusion, in this study complementary approaches were needed to determine the localization of AT₁ and AT₂ receptors in the rat adrenal, and to show the increasing effects of a low sodium regimen on the adrenal level of these receptors. Immunofluorescence studies revealed AT₁ receptors mainly in the ZG and also in some cells of the inner adrenal cortex zones; in adrenals of rats kept on a low sodium diet the ZG was markedly enlarged, and an increased number of immunoreactive cells with AT₁ receptors were observed throughout that zone; also more immunoreactive cells were present in the inner zones of the adrenal cortex. Furthermore in the adrenals of rats kept on a low sodium diet, we observed: 1) an increased number of AT₁ and AT₂ receptors in cell suspensions from the ZG, and in cell suspensions of the ZFR + M; 2) an increased level of AT₁ and AT₂ receptor mRNAs in the ZG; 3) an increased level of AT₁ receptor mRNA, with no changes in the AT₂ mRNA level in the ZFR + M. These results suggest a role for AT₁ as well as for AT₂ receptors in controlling adrenal function and differentiation under normal as well as under physiological stimulation of AII production following sodium restriction.

	Introduction

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ANGIOTENSIN II (AII) is well known to be regulator of mineralocorticoid formation in the adrenal cortex. Due to the recent development of specific AII analogs such as Losartan and PD123319, it is well documented that AII acts through two types of receptors in the adrenal, namely type 1 (AT₁) and type 2 (AT₂) (1). The function of AT₁ receptors as mediators controlling aldosterone formation has been amply studied, whereas much less is known on the function of the AT₂ receptors in the adrenal (2). Recent reports, however, lead us to believe that AT₂ receptors could be involved in the induction of differentiation. Indeed, the disappearance of AT₂ receptor messenger RNA (mRNA) in the kidney coincides with the completion of nephrogenesis (3), suggesting that AII could act through this receptor as a differentiation factor. Also, in the ovary, granulosa cells of atresic follicles, which possess high levels of AT₂ receptors, are known to be the site of apoptosis (4, 5). It was reported that AII participates in the process of apoptosis (6) in the PC12W cell line, which possesses only AT₂ receptors. Furthermore, the reexpression of AT₂ receptors after a vascular wound (7) suggests also that these receptors are involved in the pathophysiology of proliferation characterizing conditions. Our recent results showed a correlation between the appearance of AT₁ receptors, the reduction of AT₂ receptors, and an increased aldosterone secretion into the medium of cultured human fetal adrenals (8).

In rats, a low sodium intake resulted in increases in plasma AII levels, in the thickness of the adrenal cortex zona glomerulosa (ZG) (9), in cytochrome P450 aldosterone synthase activity, and aldosterone secretion (10).

In this current study using the rat model, we evaluated the effects of a low sodium intake on AT₁ and AT₂ receptors in the adrenal. Autoradiography showed that AT₂ receptors were present in the ZG and in the medulla (M), whereas AT₁ receptors were located in the ZG. Using an anti-AT₁ receptor antibody and immunofluorescence microscopy, we found that AT₁ receptors were localized only in the ZG, and that a low sodium regimen induced an enlargement of the ZG where AT₁ receptors appeared to be evenly distributed. In the latter situation, AT₁ receptors were also observed in some cells of the zona fasciculata (ZF) and possibly in the zona reticularis (ZR). When compared with controls, an increased capacity to bind AII was found in the ZG as well as in the zonae fasciculata and reticularis (ZFR)+ the medulla of cell suspensions from rats kept on a low sodium regimen, both AT₁ and AT₂ binding sites being increased under low sodium restriction. These results suggest that both AT₁ and AT₂ receptors may play crucial roles in the physiology of adrenals under normal and stimulating conditions of AII production by sodium restriction.

	Materials and Methods

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Animals
Adult male Long Evans rats were purchased from Charles River Canada, Inc. (St.-Constant, Québec, Canada). Purina rat chow and tap water were available ad libitum. Other groups of rats were fed a sodium-deficient diet (<0.01 mEq Na⁺/g; ICN Biochemicals, Cleveland, OH) with demineralized water to drink as previously described (10). The animals were decapitated between 0800 and 1000 h, in accordance with the ethical standards of the institutional review committee. Losartan was a gift from Dupont-Merck (Wilmington, DE), and PD 123319 was given by Parke-Davis (Ann-Arbor, MI).

Autoradiography
Frozen tissues were mounted in chucks and sliced into 10-µm sections using a cryostat at -20 C. These sections were thaw-mounted on gelatin-coated slides, placed in a desiccator for 18 h at 4 C, and processed for autoradiography. Serials adrenal sections were preincubated for 15 min at 20 C in 50 mM Tris-HCl, pH 7.4, containing 3 mM MgCl₂, BSA 1 mg/ml, LTI 0.05 mg/ml, bacitracin 0.5 mg/ml, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM EDTA, followed by a 45-min incubation with 200,000 dpm (0.4 nM) [¹²⁵I]AII with or without 1 µM AII to check for nonspecific binding (11). All receptors type 1 and type 2 were evaluated on serials sections incubated with 1 µM of the antagonist Losartan, thereby revealing AT₂ binding sites, or with 1 µM of the AT₂ antagonist PD 123319, to demonstrate AT₁ binding sites. After incubation, slides were washed three times for 30 sec in ice-cold HBS-glucose buffer [containing (nM) NaCl, 130; KCl, 3.5; CaCl₂, 1.8; MgCl₂, 0.5; NaHCO₃, 2.5; HEPES, 5.0, supplemented with sucrose 1g/liter and BSA 0.5% pH 7.4] and dried under cool air. Autoradiographic images were obtained after exposure of slides to Kodak X-Omat-RP film in x-ray casettes for 6–10 days.

Adrenal cell membrane preparations
ZG was separated from the rest of the gland comprising ZFR + M and the two parts were homogenized separately in ice-cold 20 mM Tris-HCl buffer, pH 7.4 (containing 1 mM EDTA, aprotinin 0.3 U/ml, phenanthroline 100 µg/ml. The homogenates were centrifuged for 5 min at 700 x g (4 C) and the supernatant for 40 min at 40,000 x g to obtain the membrane preparation. This membrane preparation was suspended in 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA, 6.5 mM MgCl₂, 125 mM NaCl, BSA 2%, aprotinin 0.3 U/ml and phenanthroline 100 µg/ml.

Cell suspensions
ZG were separated from the rest of the gland (12) and cells from the two parts were obtained by collagenase digestion in Eagle’s MEM containing penicillin 100 U/ml and streptomycin 100 mg/ml.

Binding studies
Human AII (Asp¹AII) was iodinated by the iodogen method and purified by HPLC (13). Monoiodinated AII was obtained as a homogeneous fraction with a specific activity of about 1200 Ci/mmol. Binding studies on membrane preparations were performed as follows: in polypropylene tubes were added (50 µg for ZG and 150 µg for ZFR + M) of membrane protein, 50,000 dpm, (0.1 nM) of [¹²⁵I]AII, with increasing concentrations of AII or AII analogs, or 1 µM AII for nonspecific binding determination, in a total volume of 200 µl. Incubations were performed for 90 min at 22 C, and incubation media were filtered through Whatman GF/C filters which were rinsed three times and the radioactivity determined in a Beckman gamma counter. The percentage of each type of receptor was estimated after substracting a nonspecific binding value obtained by incubating [¹²⁵I]AII in the presence of 1 µM of AII. Additional characterization of each type of receptor was made by preincubating membranes 10 min at 37 C with 1 µM Losartan or 1 µM PD 123319 followed by an incubation in the presence of [¹²⁵I]AII and increasing concentrations of the other analog. A total of 1 µM Losartan or PD 123319 was sufficient to saturate their specific receptors without affecting the alternate subtype.

Binding studies on adrenal cell suspensions
Cells (3-4 x 10⁴) isolated from ZG and from ZFR + M were suspended in HBS, glucose 0.1%, BSA 0.1% for 90 min at 22 C with 50,000 dpm (0.1 nM) of [¹²⁵I]AII in the presence of increasing amounts of AII, or Losartan or PD 123319. Cell bound radioactivity was separated by filtration through Whatman GF/C filters that were rinsed three times and the radioactivity evaluated in a Beckman counter.

RNA extraction from tissues and Northern blotting analysis
Total RNA from rat adrenal ZG and ZFR + M was extracted using the Tri-Reagent protocol (Molecular Research Center, Cincinnati, OH). RNA (15 µg) samples were denatured with glyoxal (14), and then fractionated by electrophoresis on a DEPC-treated 1% agarose gel in 0.01 M phosphate buffer, pH 7.0. The fractionated RNA was transferred to positively charged nylon membranes (Boehringer Mannheim, Mannheim, Germany), which were then hybridized for 16 h at 42 C with a [³²P]labeled bovine adrenal AT₁ cDNA probe (15) and a hamster AT₂ receptor cDNA probe (nt 521 to nt 1192) of the coding sequence, respectively.

Western blotting analysis
Homogenates of rat tissues were analyzed by immunoblotting as previously described (15), using a rabbit polyclonal anti-human AT₁ receptor. The antibody used was the affinity-purified rabbit polyclonal antibody AT₁ (306) lot F205 (Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA) raised against amino acids 306–359 of the angiotensin II AT₁ receptor of human origin. Immunoreactive proteins were detected using ECL light emitting reagents (Amersham International plc., Little Chalfont, Buckinghamshire, UK).

Immunofluorescence
For the localization of AT₁ receptors with the antihuman AT₁ receptor antibody, adrenal glands were excised from three different animals of each experimental group. The glands were fixed in buffered neutral formalin 10% solution for 24 h. The fixed adrenals were dehydrated in graded alcohols, cleared in toluene, and embedded in paraffin. Five to 7-µm thick sections were prepared according to the usual histologic procedure. Sections were deparaffinized, hydrated to water, and treated with NH₄Cl in 50 mM PBS (20 min) to block aldehydes. After two washes, tissue sections were incubated for 2 h at room temperature with the first antibody (diluted 1/100) and then washed twice. They were next incubated for 30 min with the second fluorescein-conjugated goat antirabbit IgG (Boehringer Mannheim), diluted 1/50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine (16). Adrenal sections were studied using a Reichert Polyvar 2 microscope equipped for epifluorescence.

	Results

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Figure 1 shows [¹²⁵I]AII binding in sections of rat adrenal glands. [¹²⁵I]AII was bound to the ZG and to the medulla (panel A) and sections exhibited AII labeling with low nonspecific binding in the presence of 10^-6 M AII (panel D). Pharmacological studies, using the specific AT₁ receptor antagonist Losartan, and the specific AT₂ receptor antagonist PD 123319, revealed the presence of both types of receptors in the ZG, and AT₂ receptors in the medulla. This is well shown for the low sodium diet group; indeed, 10^-6 M Losartan as well as 10^-6 M PD 123319 only partially displaced bound [¹²⁵I]AII in the ZG. In the control group, PD 123319 seemed to displace all bound [¹²⁵I]AII. However, the presence of AT₁ receptors in the ZG of control rats was established by immunofluorescence (see next section). In the medulla, the addition of 10^-6 M Losartan in the incubation medium, did not seem to change total binding, although this possibility cannot be completely ruled out, whereas coincubation with PD 123319 appeared to abolish it. By simple inspection, the medulla labeling intensity was stronger, nine times out of twelve, in adrenal sections from rat kept on a low sodium regimen (n = 12 rats) than from controls (n = 12 rats).

View larger version (118K): [in this window] [in a new window]   Figure 1. Autoradiography of [125I]AII binding to rat adrenal gland. Serial frozen sections (20 µm) of rat adrenal gland were incubated for total binding for 30 min at room temperature with 200,000 dpm (0.4 nM) of [125I]AII alone, or in the presence of 10-6 M Losartan, or 10-6 M PD 123319, or 10-6 M unlabeled AII for nonspecific binding. Bar, 1 mm.

View larger version (68K): [in this window] [in a new window]   Figure 2. Immunofluorescence localization of AT1 receptors in rat adrenal glands. Indirect immunofluorescence micrographs of paraffin sections of rat adrenals at 0 (0d) and 11 (11d) days on a low sodium intake. In control adrenals (0d), the specific fluorescein-conjugated antibody detected AT1 receptors in the zona glomerulosa (ZG). The thickness of the ZG was markedly increased under sodium restriction (11d) compared with controls, and the fluorescent signal was detected all over the whole enlarged ZG. In addition, a signal was also observed in some cells in the zona fasciculata and possibly in the zona reticularis in glands of sodium restricted animals. Data shown are representative of experiments performed on three control and three sodium restricted rats. Bar, 100 µm.

Results of binding studies on membranes are shown in Figs. 3 to 6; data obtained are only indicative of a possible enhancement of the AII binding capacity of AT₁ and AT₂ receptors by sodium restriction. Indeed, binding studies on membrane preparations revealed the presence of AII receptors in both adrenal ZG and ZFR + M, as shown by ligand analysis of the displacement of [¹²⁵I]AII by unlabeled AII (Figs. 3 and 4). Indeed, displacement (panel C) saturation (panel A) and Scatchard (panels B and D) curves show specific AII binding for all membrane preparations tested. In the ZG, maximal binding (B_max) as determined by saturation curves (Fig. 3A) was 716 fmol and 760 fmol/mg protein for preparations from control (n = 5) and from sodium restricted (n = 6) animals (statistically not significant: NS). Figure 4A shows that in the ZFR+M, B_max was 57 fmol and 77 fmol/mg protein for preparations from control (n = 3) and from sodium restricted (n = 3) animals (NS). Scatchard analysis showed the presence of two binding sites in ZG as well as in ZFR + M. For control ZG (n = 5), Kd₁ = 1.21 nM, n₁ = 422 fmol/mg; K_d2 = 16.5 nM, n₂ = 436 fmol/mg protein (Fig. 3, panel B). For low sodium ZG (n = 6), K_d1 = 0.71 nM, n₁ = 318 fmol/mg; K_d2 = 17.2 nM, n₂ = 624 fmol/mg protein (Fig. 3D). For control ZFR + M (n = 3), K_d1 = 0.23 nM, n₁ = 11.6 fmol/mg; K_d2 = 12.4 nM, n₂ = 61.3 fmol/mg protein (Fig. 4B). For low sodium (n = 3) ZFR + M, K_d1 = 0.40 nM, n₁ = 14.6 fmol/mg; K_d2 = 12.4 nM, n₂ = 82.7 fmol/mg protein (Fig. 4D).

View larger version (23K): [in this window] [in a new window]   Figure 3. Specific binding of [125I]AII to membranes from rat adrenal zona glomerulosa. Membranes (50 µg protein per assay) were incubated for 45 min at 30 C with 0.2 nM [125I]AII (100,000 dpm) in the presence of increasing concentrations of AII. The value obtained with the assay containing 10-6 M AII was taken to be nonspecific binding, and this value was subtracted from other experimental values. A, Saturation curves; C, displacement curves, B and D, Scatchard curves. (): control; (): rats on a low sodium regimen.

View larger version (24K): [in this window] [in a new window]   Figure 4. Specific binding of [125I]AII to membranes from rat adrenal zonae fasciculata and reticularis + the medulla. Membranes (150 µg protein per assay) were incubated for 45 min at 30 C with 0.2 nM [125I]AII (100,000 dpm) in the presence of increasing concentrations of AII. A, Saturation curves; C, displacement curves; B and D, Scatchard curves. (): control; (): rats on a low sodium regimen.

View larger version (24K): [in this window] [in a new window]   Figure 5. Pharmacological properties of [125I]AII binding to rat adrenal membranes. Membranes, 50 µg protein per assay for the zona glomerulosa (A) and 150 µg protein per assay for the zona fasciculata-reticularis + the medulla (B), were. incubated for 45 min at 30 C with 0.2 nM [125I]AII (100,000 dpm) in the presence of increasing concentrations of AII () or Losartan () or PD 123319 (X).

View larger version (21K): [in this window] [in a new window]   Figure 6. Pharmacological properties of [125I]AII binding to rat adrenal membranes. Membranes, 50 µg protein per assay for the zona glomerulosa (A) and 150 µg protein per assay for the zonae fasciculata and reticularis + the medulla (B), were preincubated at 22 C for 10 min with 10-6 M Losartan () or 10-6 M PD 123319 (). The media were then incubated at 22 C for 22 min with 0.2 nM [125I]AII (100,000 dpm) in the presence of increasing concentrations of the other analog. Results are the mean ± SEM, n = 3.

Membrane receptors were saturated with 10^-6 M of either Losartan or PD 123319 and in the presence of increasing concentrations of either PD 123319 or Losartan. Displacement curves (n = 3) obtained with these two analogs confirm the presence of the two AII receptor subtypes in the ZG (Fig. 6A) and also in the ZFR + M (Fig. 6B). In addition these results show that 10^-6 M of Losartan or of PD 129319 was sufficient to saturate AT₁ and AT₂ receptors in both ZG and ZFR + M.

Membrane preparations were thus incubated with [¹²⁵I]AII without or with 10^-6 M Losartan in order to accurately establish the percentage of AT₁ receptors in the various preparations. Results obtained showed that in the ZG, AT₁ receptors accounted for 77.9 ± 3.0% and 86.3 ± 2.5% (mean ± SEM, n = 8, p < 0.005) for control and sodium restricted animals, respectively. In the ZFR + M, AT₁ receptors accounted for 53.0 ± 5.2% and 71.7 ± 2.3% (n = 7, P < 0.005) for control and sodium restricted animals. Although interesting, results from binding studies on rat adrenal membrane preparations are only indicative of changes produced by a low sodium diet on the binding capacity of AT₁ and AT₂ receptors. Furthermore, as membrane preparations may not contain all the AII binding sites, and because some AII binding sites may be altered or destroyed during the preparation of membranes, binding studies were performed on intact cells.

Cell suspensions
In fact, binding studies on intact cells gave much more convincing results. Figure 7 shows saturation curves; compared with controls, with cell suspensions from rats fed a low sodium diet, a 270 ± 22% increase (mean ± SEM, n = 3, P = 0.008) and 210 ± 31% increase (n = 3, P > 0.05) in total AII receptors were obtained for adrenal ZG and ZFR + M preparations, respectively. These results indicate that the low sodium intake induced an increased binding of AII by adrenal cells. Losartan and PD 123319 were able to partially displace bound [¹²⁵I]AII to ZG (Fig. 8, A and B) as well as to ZFR + M adrenal cells (Fig. 8, C and D). Cells were incubated with [¹²⁵I]AII with or without 10^-6 M Losartan in order to establish the percentage of AT₁ receptors. Results obtained from three different experiments showed that in the ZG cells from control and sodium restricted animals, Losartan displaced (mean ± SEM, n = 3) 62.7 ± 12.6% and 67.4 ± 4.7% of bound [¹²⁵I]AII. Losartan displaced 71.7 ± 15.4% and 56.0 ± 12.7% of bound [¹²⁵I]AII in the ZFR + M cells for control and sodium restricted animals. Based on total bound [¹²⁵I]AII and on Losartan displacement percentage, we calculated that AT₁ and AT₂ receptors increased in both ZG and ZFR + M.

View larger version (18K): [in this window] [in a new window]   Figure 7. Specific binding of AII to cell suspension from rat adrenal zona glomerulosa and from zona fasciculata-reticularis + the medulla, saturation curves: 3–4 x 104 cells isolated from ZG and from ZFR + M were incubated for 90 min at 22 C with 50,000 dpm (0.1 nM) of [125I]AII in the presence of increasing amounts of AII. Results shown are representative of three experiments performed in duplicate. (): control ZG; (): low sodium ZG; (): control ZFR + M; (X: low sodium ZFR + M.

View larger version (30K): [in this window] [in a new window]   Figure 8. Pharmacological properties of [125I]AII binding to rat adrenal cell suspensions. 3–4 x 104 cells in suspension were incubated for 90 min at 22 C with 50,000 dpm (0.1 nM) of [125I]AII in the presence of increasing amounts of AII () or Losartan () or PD 129319 (). A, Control ZG; B, low sodium ZG; C, control ZFR + M; D, low sodium ZFR + M. Results shown are representative of three experiments performed in duplicate.

View larger version (81K): [in this window] [in a new window]   Figure 9. Effects of a low sodium intake for 11 days on the levels of AII receptors mRNA in rat adrenal zona glomerulosa (ZG) and zona fasciculata-reticularis + the medulla (ZFR + M). Three groups of four rats were used for control and for low Na+ diet, respectively. Northern blotting analysis was performed on 15 µg total RNA. The blots were analyzed using 32P-labeled AT1 (A) and AT2 (B); the blots were also analyzed with an 18S cDNA probe for standardization of quantities of RNA. C, Control; Low Na+, low sodium.

	Discussion

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Binding studies on membrane preparations also showed the presence of types 1 and 2 receptors in the ZG. Using Losartan to displace [¹²⁵I]AII on AT₁ receptors, we found in control rats that the AT₁ receptor population accounted for 78% of all AII receptors. This is not surprising because more than 80% of AII receptors in the rat adrenal cortex ZG were reported to be type 1 (17, 21, 22, 23, 24). The percentage of AT₁ receptors that we found in the ZG is also in agreement with the 80% recently reported by Kakiki and Horie (25), who used two specific antagonists of angiotensin II receptor type 1, Losartan and E4177, in their study. AT₁ and AT₂ receptors were also found in membrane preparations of the ZFR + M of control animals. This suggests either that angiotensin receptors type 1 are present in the rat adrenal ZFR or that contamination from the ZG occurred when the glands were decapsulated. Indeed, the separation of the ZG from the rest of the rat adrenal cannot be performed without a certain percentage of contamination, and this could explain why the two angiotensin receptor subtypes were found when membrane preparations from decapsulated glands were tested for the presence of AT₁ and AT₂ receptors. Based on microscopic visualization and counting ZG cells, which are round and smaller than inner zone cells, we concluded that contamination during mechanical separation of the ZG from decapsulated gland cells, and vice versa, was about 5%; the contamination of adrenal preparations from regular vs. low sodium diet rats seemed to be similar ruling, out the possibility that an increase in the number of ZG cells would cause an apparent increase in AT₁ receptor density in the ZFR + M preparations. Saturation curve analysis revealed the presence of slightly more AII receptors in both ZG and ZFR + M preparations from rats fed a low sodium diet than in those from control rats. Calculated as per mg protein, 760 fmol vs. 716 fmol were found in the ZG, and 77 fmol vs. 57 fmol in the ZFR + M, respectively, for low sodium and control animals. These increases, however, were not statistically different. Scatchard analyses also confirmed a greater number of receptors in the adrenals of sodium restricted animals than in controls. Indeed, the sum of n₁ + n₂ was superior in the former group compared with the latter. Two K_ds were calculated from Scatchard curves indicating that AII receptors were present in two different forms in the ZG as well as in the ZFR + M preparations. In agreement with our binding studies, Aguilera (21) found that the number of AT₁ and AT₂ receptors of the ZG significantly increased in membrane preparations from rats kept on a low sodium regimen for 6 days.

As membrane preparations do not necessarily contain all the AII binding sites, and on the other hand, as homogenization may alter certain populations of AII binding sites (21), further binding studies were performed on intact adrenal cells. Much more convincing results were obtained using cell suspensions, and clear differences were found in the number of AII binding sites between cells from control and from sodium restricted animals with 270% and 210% increase for adrenal ZG and ZFR + M preparations from the sodium restricted group. These results indicate that the low sodium intake induced an increased capacity of adrenal cells to bind AII. Our results on ZG cell suspensions are in agreement with previous reports which indicated an increase in the number of AII-binding sites in the adrenal ZG cells of rats fed a low sodium regimen (21, 26). Furthermore, AT₁ as well as AT₂ receptors increased for ZG and ZFR + M cells from the sodium restricted group. These results are also in agreement with our binding results on tissue slices (see Fig. 1). Moreover, adrenal membrane preparations appear less appropriate than intact cells for quantitative studies of AII receptors because of potential alteration of receptors that may occurred during preparation.

Northern blotting analysis showed that AT₁ receptor mRNA was present in the ZG of control rats, and after 3 days of film exposure, a small quantity of AT₁ mRNA was also revealed in the ZFR + M. Compared with controls, the low sodium intake provoked a significant 300% and 250% increase in the AT₁ receptor mRNA level in the ZG and ZFR + M. We have not studied whether the observed increases in the level of AT₁ and AT₂ receptor mRNA are controlled by induction of transcription or by post-transcription stabilization. Our results which show that the level of AT₁ receptor mRNA is increased in the ZG by sodium restriction are in agreement with our previous report (15). However, in that previous work (15) the levels of AT₁ receptor mRNA, although increased in the ZFR + M preparations following sodium restriction, was not statistically different from controls. This apparent discrepancy can be tentatively explained by the fact that in the previous work (15) rats were kept on a low sodium diet for 7 days vs. 11 days in the current study; perhaps a long sodium restriction period is needed before being able to observe a statistically significant increase with the detection technique used. Furthermore, as mentioned previously, we cannot, at present, rule out the possibility that the presence of AT₁ receptor mRNA in the ZFR + M is due or not to contamination from the ZG. However, by in situ hybridization (27), whereas AT_1A mRNA was detected with the highest intensity in the ZG of rat adrenals, a weaker signal was also detected in the ZFR and some clusters of cells in the medulla were labeled with the AT_1A probe. AT_1B mRNA was detected only in the ZG. These results are in agreement with our data on the presence of AT₁ receptors in the ZG as well as in the ZFR + M. Our Northern analyses are also in agreement with the results of Llorens-Cortes et al. (28) who found that adrenal AT_1A and AT_1B mRNA levels were increased following sodium depletion. Furthermore, in a previous report (15) we had shown that the level of AT₁ receptors was mediated by AII itself, which is also in agreement with the findings of Imai and Inagami (29) who reported that a continuous infusion of AII increased the level of AT₁ receptor mRNA in the adrenal and with those of Hauger et al. (30) who reported an increase of bound AII in adrenal ZG membrane preparations from rats receiving an infusion of AII for 36 h. Taken together these results clearly demonstrate that a low sodium diet acts through AII to increase the thickness of the adrenal ZG, the levels of AT₁ receptors and of cytochrome P450 aldosterone synthase (10); the net result being the increase in aldosterone secretion. AT₁ receptor protein and mRNA were also found to be up-regulated in tissue other than the adrenal, namely the aortic ring incubated in the presence of corticosteroids (31). However, a different situation appears to take place in bovine adrenal cells (BAC) in culture, since both AII receptor subtypes 1 and 2 were reported to be down-regulated by AII (32). These discrepancies between the above-mentioned results in vivo and in vitro, may be tentatively explained by the fact that BAC are from the zonae fasciculata reticularis which could react differently than zona glomerulosa cells to stimulus by AII. Also, BAC might well have lost some of their properties during culture.

Using immunofluorescence studies, AT₁ receptors were localized in the ZG of control rats. Under sodium restriction for 11 days, however, the ZG was considerably enlarged and an even distribution of the immunofluorescent antibody signal was observed throughout the ZG. At first sight it was difficult to determine if the immunofluorescent signal per area unit was increased in the experimental animals, but because the ZG from sodium restricted rats was much larger than that from controls, we can effectively conclude that there were more AT₁ receptors in the ZG of rats kept on a low sodium diet than in controls. AT₁ receptor-positive cells were found to a limited degree in the zona fasciculata and possibly in the zona reticularis, and a greater number of these cells appeared in these zones under sodium restriction. Using an in situ immunocytochemical technique, McEwan et al. (33) have localized AT₁ receptors within the rat adrenal gland; in agreement with our results, they found AT₁ receptors in the ZG but also in the zona reticularis. Also in agreement with our results (data not shown) they found no evidence of AT₁ receptors in the medulla. We do not know at present the physiological significance of the presence of these immunoreactive cells in the inner zones of the adrenal cortex. There is no indication that the few cells seen to be positive for AT₁ receptors the zona fasciculata are steroidogenic or belong to other cell types. Moreover in the rat, it was reported that AII does not stimulate corticosterone production in the zonae fasciculata reticularis (17). Furthermore, the increase of the number of AT₁ receptors in membrane preparations of the ZFR + M that we found in animals fed a low sodium diet, could be explained by the presence of such cells in the ZF and possibly in the ZR.

A specific rabbit antihuman AT₁ receptor antibody was used for AT₁ receptor immunolocalization. The specificity of the anti-AT₁ receptor antibody has been previously validated on the ovine adrenal gland, which is known to express AT₁ receptors in the zonae fasciculata reticularis and in the zona glomerulosa (34, 35). These authors demonstrated the specificity of the antisera to the subcapsular zona glomerulosa cells together with a reduced level of staining in zonae fasciculata and reticularis. No staining could be observed in the outer capsule nor adrenal medullary cells, consistent with the previously reported lack of AII-binding in the medulla of human, monkey, and cow adrenals (36, 37). The specificity of the antisera was also established by Western analysis on homogenates from ovine liver, kidney, and adrenal cortex, all of which are classic target tissues known to express AT₁ receptors. A major band was stained at 54 kDa in all tissues tested. In sheep, no staining was found in the adrenal medulla homogenate which was used as control. Using the same above mentioned rabbit antihuman AT₁ receptor antibody, Western studies on rat adrenal zona glomerulosa, zonae fasciculata reticularis, and medulla homogenates revealed the presence of specific protein bands at about 56 kDa. Furthermore the antisera also recognized protein bands at 56 kDa for the liver and the kidney, two tissues known to express AT₁ receptors in rats (results not shown). Using another AT₁ antisera, namely Blanka-1 (38), we previously reported an apparent molecular mass of about 56 kDa for the rat adrenal AT₁ receptor protein (15). This molecular mass appears to be identical with the value we found in the present study using the Santa Cruz AT₁ (306) antibody. By Western analysis, we previously showed an increase in the level of AT₁ receptors in the ZG of rats fed a low sodium diet (15).

In agreement with our Northern blotting analysis and with our binding studies on slices as well as on membrane and cell preparations, the presence of AT₂ receptor mRNA was observed in rat adrenal by in situ hybridization (39); using this technique, expression of AT₂ receptor mRNA was detected in the ZG, and no labeling was seen in the ZFR. In the medulla, the labeling was heterogeneous as if only some cords of cells were positive. In our study, the low sodium intake resulted in a small but significant increase in AT₂ receptor mRNA in the ZG; these results are in agreement with increased AT₂ binding sites that we found in ZG membrane and cell preparations and with Aguilera’s report (21), indicating that sodium restriction increased the level of AT₂ receptors in the rat adrenal ZG. In our study, however, no increase in AT₂ receptor mRNA level was found in the ZFR + M preparations of rats fed a low sodium diet for 11 days, so contrasting with the increase found for AT₂ receptor binding sites. The discordance between Northern and binding studies could be related to differences in the turnover rate of the mRNA and the protein. A similar explanation was given by Shanmugam et al. (39) to explain that in the fetal and the newborn rat adrenal ZG, a strong hybridization signal for AT₂ receptor mRNA was observed, whereas the presence of AT₂ binding sites was not found.

Noteworthy is the fact that a high level of AT₂ receptors persists in adult rat adrenal ZG and medulla in comparison to a diminution in other tissues where they were highly expressed in fetal life. Furthermore, it is known that the adrenal cortex is the site of much apoptosis in rats (40) and humans (41), and apoptotic cells were found in the ZG and also in the ZFR (40); the maximal apoptotic rate was situated in the ZG (40). Because adrenal dead cells must be replaced in order to maintain the integrity of the gland, differentiation must be a permanent process in the adrenal cortex. A proper balance between the effects of AII via AT₁ and AT₂ might well be necessary to maintain this integrity. Thus, one can speculate on a putative role of AT₂ receptors in the differentiation and/or apoptotic processes. Indeed, as already mentioned in the introduction, it was reported that AII participates in the process of apoptosis (6) in the PC12W cell line that possesses only AT₂ receptors. Furthermore, the reexpression of AT₂ receptors after a vascular wound (7) suggests also that these receptors are involved in the pathophysiology of cellular proliferation. In the rat kidney, the disappearance of AT₂ mRNA was synchronous with the completion of nephrogenesis suggesting that AII could act through this receptor as a differentiation/growth factor via the AT₂ receptor (3). In the rat adrenal, we observed an enlargement of the adrenal ZG under sodium restriction; in agreement with this, Rebuffat et al. (42) recently reported an increase in the number and volume of parenchymal cells in the adrenal ZG when rats were fed a low sodium diet, demonstrating that cell differentiation occurred in that zone under AII stimulus. McEwan et al. (33) have studied the effects of a low sodium diet on cell proliferation in the rat adrenal using an infusion of bromodeoxyuridine. They found that a low sodium diet increased proliferation 3-fold and caused hypertrophy of the ZG. These results indicate that the increased ZG thickening is due to mitogenesis. Huang et al. (43) reported that AT₁ and AT₂ receptors have opposing actions on mitogen-activated protein kinases in rat neonatal neurons and in comparison we speculate that in the rat adrenal the increase in AT₂ receptors in the ZG upon sodium restriction might well counterbalance the proliferative action of AII through AT₁ receptors to maintain the integrity of the adrenal cortex.

The function of the high level of AT₂ receptor expression in the medulla is also unclear; however, in contrast to the high level of AT₂ receptors found in the rat adrenal medulla, these receptors were reported to be absent in human phaeochromocytoma (23), an uncontrolled situation of cell proliferation. It was also reported that AII induces an increase in adrenal epinephrine secretion but this seems to be mediated by AT₁ and not by AT₂ receptors (44). There again, one might speculate that the increased AT₂ binding in the medulla might be involved to counterbalance the action of AII on AT₁ receptors; a putative role could thus be played by AT₂ receptors in the differentiation and/or apoptotic processes.

In conclusion, although the exact function(s) of AT₂ receptors in both the ZG and the medulla still remains unclear, our results suggest a role for AT₁ and AT₂ receptors in controlling adrenal function, differentiation, and apoptosis, and this under normal conditions as well as under stimulation of AII production by a physiological stimulus such as sodium restriction.

	Acknowledgments

 
We thank Dr. Gaétan Guillemette for his generous gift of [¹²⁵I]AII. The generous gift of Losartan by Dupont-Merck and that of PD 123319 by Parke-Davis are also acknowledged. We also thank Dr. Dennis Shapcott for reviewing this article and Dr. Nicole Gallo-Payet for her precious advice.

	Footnotes

 
¹ Supported by a grant from the Medical Research Council of Canada (MT 10983) and the Heart and Stroke Foundation of Canada.

Received July 14, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF 1991 Nonpeptide angiotensin II receptor antagonists. Trends Physiol Sci Rev 12:55–61
2. Nahmias C, Strosberg AD 1995 The angiotensin AT₂ receptor: searching for signal-transduction pathways and physiological function. Trends Pharmacol Sci 16:223–225[CrossRef][Medline]
3. Shanmugam S, Llorens-Cortes C, Clauser E, Corvol P, Gasc JM 1995 Expression of angiotensin II AT₂ receptor mRNA during development of rat kidney and adrenal gland. Am J Physiol 268:F922–F930
4. Pucell AG, Hodges JC, Sen I, Bumpus FM, Hussain A 1991 Biochemical properties of the ovarian granulosa cell type 2-angiotensin II receptor. Endocrinology 128:1947–1959[Abstract]
5. Tilly JL, Hsueh AJW 1993 Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J Cell Physiol 154:519–526[CrossRef][Medline]
6. Yamada T, Horiuchi M, Dzau V 1996 Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci USA 93:156–160[Abstract/Free Full Text]
7. Nio Y, Matsubara H, Marasuwa S, Kanasaki M, Inada M 1995 Regulation of gene transcription of angiotensin II receptor subtypes in myocardial infarction. J Clin Invest 95:651–657
8. Breault L, LeHoux JG, Gallo-Payet N 1996 The angiotensin AT₂ receptor is present in the human fetal adrenal gland throughout the second trimester of gestation. J Clin Endocrinol Metab 81:3914–3922[Abstract/Free Full Text]
9. Mitani F, Suzuki H, Hata J, Ogishima T, Shimada H, Ishimura Y 1994 A novel cell layer without corticosteroid-synthesizing enzymes in rat adrenal cortex: histochemical detection and possible physiological role. Endocrinology 135:431–438[Abstract]
10. Tremblay A, Parker KL, LeHoux JG 1992 Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11ß-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production. Endocrinology 130:3152–3158[Abstract]
11. Millan MA, Jacobowitz DM, Aguilera G, Catt KL 1991 Differential distribution of AT₁ and AT₂ angiotensin II receptor subtypes in rat brain during development. Proc Natl Acad Sci USA 88:11440–11444[Abstract/Free Full Text]
12. LeHoux JG, Ducharme L 1992 The differential regulation of aldosterone output in hamster adrenal by angiotensin_II and adrenocorticotropin. J Steroid Biochem Mol Biol 41:809–814[CrossRef][Medline]
13. Boulay G, Gallo-Payet N, Guillemette G 1990 Implication of phospholipase C in the steroidogenic action of angiotensin II. Eur J Pharmacol 189:267–275[CrossRef][Medline]
14. Thomas PS 1983 Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. Methods Enzymol 100:255–266[Medline]
15. LeHoux JG, Bird IM, Rainey EW, Tremblay A, Ducharme L 1994 Both low sodium and high potassium intake increase the level of adrenal angiotensin-II receptor type 1, but not that of adrenocorticotropin receptor. Endocrinology 134:776–782[Abstract]
16. Calvert R, Millane G, Beaulieu JF 1994 Immunolocalization of a mesenchymal antigen specific to the gastrointestinal tract. Anat Record 240:358–366[CrossRef][Medline]
17. Catt KJ, Mendelsohn FAC, Millan MA, Aguilera G 1984 The role of angiotensin II receptors in vascular regulation. J Cardiovasc Pharmacol 6:S-575–S586
18. Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia DV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWM 1991 Identification of angiotensin II receptor subtypes. Biochem Biophys Res Comm 165:196–203
19. Israel A, Niwa M, Plunkett LM, Saavedra JM 1985 High-affinity angiotensin receptors in rat adrenal medulla. Regul Pept 11:237–243[CrossRef][Medline]
20. Himeno A, Nazarali AJ, Saavedra JM 1988 Quantitative in vitro autoradiographic characterization of [¹²⁵I]angiotensin III binding sites in rat adrenal gland. Regul Pept 23:127–133[CrossRef][Medline]
21. Aguilera G 1992 Role of angiotensin receptor subtypes on the regulation of aldosterone secretion in the adrenal glomerulosa zone in the rat. Mol Cell Endocrinol 90:53–60[CrossRef][Medline]
22. Whitebread SE, Taylor V, Bottary SP, Kamber B, de Gasparo M 1991 Radioidonated CGP 42112A: a novel high affinity and highly selective ligand for the characterization of angiotensin AT₂ receptor. Biochem Biophys Res Commun 181:1365–1371[CrossRef][Medline]
23. Giacchetti G, Opocher G, Sarzani R, Rappelli A, Mantero F 1996 Angiotensin II and the adrenal. Clin Exp Pharmacol Physiol [Suppl 3] 13:S119–S124
24. Balla T, Baukal AJ, Eng S, Catt KJ 1991 Angiotensin II receptor subtypes and biological response in the adrenal cortex and medulla. Mol Pharmacol 40:401–406[Abstract]
25. Kakiki M, Horie T 1996 Localization of a novel non-peptide angiotensin II type 1 receptor antagonist, E4177, in rat adrenal glomerulosa. Biol Pharm Bull 19:1357–1361[Medline]
26. Aguilera G, Hauger RL, Catt KJ 1978 Control of aldosterone secretion during sodium restriction: adrenal receptor regulation and increased adrenal sensitivity to angiotensin II. Proc Natl Acad Sci USA 75:975–979[Abstract/Free Full Text]
27. Gasc JM, Shanmugam S, Sibony M, Corvol P 1994 Tissue-specific expression of type 1 angiotensin II receptor subtypes. An in situ hybridization study. Hypertension 24:531–537[Abstract/Free Full Text]
28. Llorens-Cortes C, Greenberg B, Huang H, Corvol P 1994 Tissular expression and regulation of type 1 angiotensin II receptor subtypes by quantitative reverse transcriptase-polymerase chain reaction analysis. Hypertension 24:538–548[Abstract/Free Full Text]
29. Imai N, Inagami T 1992 Regulation of the expression of the rat angiotensin II receptor mRNA. Biochem Biophys Res Commun 182:1094–1099[CrossRef][Medline]
30. Hauber RL, Aguilera G, Catt KJ 1978 Angiotensin II regulates its receptor sites in the adrenal glomerulosa zone. Nature 271:176–178[CrossRef][Medline]
31. Ullian ME, Walsh LG, Morinelli TA 1996 Potentiation of angiotensin II action by corticosteroids in vascular tissue. Cardiovasc Res 32:266–273[Abstract/Free Full Text]
32. Ouali R, Berthelon MC, Bégeot M 1997 Angiotensin II receptor subtypes AT₁ and AT₂ are down-regulated by angiotensin II through AT₁ receptor by different mechanisms. Endocrinology 138:725–733[Abstract/Free Full Text]
33. McEwan PE, Vinson GP, Kenyon CJ 1996 AT₁ receptors and cell proliferation in rat adrenals: effects of angiotensin infusion, low sodium diet and losartan. Endocr Res 22:369–371[Medline]
34. Bird IM, Zheng J, Cale JM, Magness RR 1997 Pregnancy induces an increase in angiotensin II type-1 receptor expression in uterine but not systemic artery endothelium. Endocrinology 138:490–498[Abstract/Free Full Text]
35. Bird IM, Zheng J, Corbin CJ, Magness RR, Conley AJ 1996 Immunohistochemical analysis of AT1 receptor versus P450c17 and 3ßHSD expression in ovine adrenals. Endocr Res 22:349–353[Medline]
36. Maurer R, Reubi JC 1986 Distribution and coregulation of three peptide receptors in adrenals. Eur J Pharmacol 125:241–247[CrossRef][Medline]
37. Gonzales-Garcia C, Keiser HR 1990 Angiotensin II and angiotensin converting enzyme binding in human adrenal gland and pheochromocytomas. J Hypertens 8:433–441[CrossRef][Medline]
38. Zelezna B, Richards EM, Tang W, Lu D, Sumners C, Raizada MK 1992 Characterization of polyclonal anti-peptide antibody to the angiotensin II type 1 (AT₁) receptor. Biochem Biophys Res Commun 183:781–788[CrossRef][Medline]
39. Shanmugam S, Lenkei ZG, Gasc JM, Corvol P, Llorens-Cortes C 1995 Ontogeny of angiotensinII type 2 (AT₂) receptor mRNA in the rat. Kidney Int 47:1095–1100[Medline]
40. Wolkersdörfer GW, Marx C, Brown JW, Scherbaum AW, Bornstein SR 1996 Evaluation of apoptotic parameters in normal and neoplastic human adrenal. Endocr Res 22:411–419[Medline]
41. Didenko VV, Wang X, Yang L, Hornsby PJ 1996 Expression of p21^{WAF1/CIP1/SDI1} and p53 in apoptotic cells in the adrenal cortex and induction by ischemia/reperfusion injury. J Clin Invest 97:1723–1731[Medline]
42. Rebuffat P, Rocco S, Andreis PG, Neri G, Nowak KW, Peters J, Opocher G, Mazzocchi G, Mantero F, Nussdorfer GG 1995 Morphology and function of the adrenal zona glomerulosa of transgenic rats TGR [mREN2] 27:effects of prolonged sodium restriction. J Steroid Biochem Mol Biol 54:155–162[CrossRef][Medline]
43. Huang XC, Richards EM, Summers C 1996 Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type e receptors. J Biol Chem 271:15635–15641[Abstract/Free Full Text]
44. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith, RD 1993 Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 45:205–251[Medline]

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