| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
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 |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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).
|
, 0 days). In adrenal sections of sodium
restricted animals, immunofluorescence was mainly located in the ZG,
which was much enlarged compared with controls. Immunofluorescence was
also observed in some cells dispersed in the zona
fasciculata and possibly in the zona reticularis, and a
greater number of AT1 receptor-positive cells appeared in
these zones under sodium restriction (Fig. 2, 0 days, 11 days).
Background staining was low and control tissues incubated only with the
second antibody were negative (results not shown).
|
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 (Bmax) 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, Bmax 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; Kd2 = 16.5 nM,
n2 = 436 fmol/mg protein (Fig. 3, panel B). For low sodium
ZG (n = 6), Kd1 = 0.71 nM, n1
= 318 fmol/mg; Kd2 = 17.2 nM, n2 =
624 fmol/mg protein (Fig. 3D). For control ZFR + M (n = 3),
Kd1 = 0.23 nM, n1 = 11.6 fmol/mg;
Kd2 = 12.4 nM, n2 = 61.3 fmol/mg
protein (Fig. 4B). For low sodium (n = 3) ZFR + M,
Kd1 = 0.40 nM, n1 = 14.6 fmol/mg;
Kd2 = 12.4 nM, n2 = 82.7 fmol/mg
protein (Fig. 4D).
|
|
|
|
shows displacement curves for
[125I]AII binding to ZG (panel A), and to ZFR + M
(panel B) membrane preparations from control rats. In the ZG, Losartan
displaced the majority of bound [125I]AII, indicating
that AII receptors were mainly of type 1 in that zone. In the ZFR
+ M fractions AT1 and AT2 were more evenly
distributed than in the ZG.
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.
|
|
, shows
that AT1 receptor mRNA is present in both preparations,
with a greater intensity in the ZG compared to the ZFR + M.
However, mRNA levels were significantly increased in both preparations
with a 3.0 ± 0.16-fold (mean ± SEM, n = 3,
P = 0.001) and 2.5 ± 0.04-fold increase (n =
3, P < 0.001) in the ZG and the ZFR + M. The low
sodium intake resulted in a small but significant in-crease
(1.5 ± 0.079-fold, n = 3, P = 0.039) in the
level of AT2 receptor mRNA in the ZG, (panel B) whereas no
significant changes occurred in the AT2 mRNA levels of the
ZFR + M preparations. An 18S cDNA probe was used for data
standardization.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 Kds 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 AT1A 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 AT1A probe. AT1B 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 AT1A and AT1B 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 |
|---|
|
Footnotes |
|---|
Received July 14, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. Hoeflich and M. Bielohuby Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2 J. Mol. Endocrinol., March 1, 2009; 42(3): 191 - 203. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H Brennan, A. Chittka, S. Barker, and G. P Vinson Eph receptors and zonation in the rat adrenal cortex J. Endocrinol., July 1, 2008; 198(1): 185 - 191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Otis, S. Campbell, M. D. Payet, and N. Gallo-Payet Angiotensin II Stimulates Protein Synthesis and Inhibits Proliferation in Primary Cultures of Rat Adrenal Glomerulosa Cells Endocrinology, February 1, 2005; 146(2): 633 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bedard, B. Sicotte, J. St-Louis, and M. Brochu Modulation of body fluids and angiotensin II receptors in a rat model of intra-uterine growth restriction J. Physiol., February 1, 2005; 562(3): 937 - 950. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Aguilar, M. Lo, B. Claustrat, J. M. Saez, J. Sassard, and J. Y. Li Hypersensitivity of the Adrenal Cortex to Trophic and Secretory Effects of Angiotensin II in Lyon Genetically-Hypertensive Rats Hypertension, January 1, 2004; 43(1): 87 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gambaryan, E. Butt, K. Marcus, M. Glazova, A. Palmetshofer, G. Guillon, and A. Smolenski cGMP-dependent Protein Kinase Type II Regulates Basal Level of Aldosterone Production by Zona Glomerulosa Cells without Increasing Expression of the Steroidogenic Acute Regulatory Protein Gene J. Biol. Chem., August 8, 2003; 278(32): 29640 - 29648. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Peters, S. Clausmeyer, P. Teubner, N. Obermüller, B. Kränzlin, N. Gretz, T. Inagami, and J. Peters Changes of AT2 Receptor Levels in the Rat Adrenal Cortex and Medulla Induced by Bilateral Nephrectomy and Its Modulation by Circulating ANG II J. Histochem. Cytochem., May 1, 2001; 49(5): 649 - 656. [Abstract] [Full Text]
|
|
|
|
|
|
K. Held Hales, T. Diemer, S. Ginde, B. K. Shankar, M. Roberts, H. B. Bosmann, and D. B. Hales Diametric Effects of Bacterial Endotoxin Lipopolysaccharide on Adrenal and Leydig Cell Steroidogenic Acute Regulatory Protein Endocrinology, November 1, 2000; 141(11): 4000 - 4012. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Chamoux Involvement of the Angiotensin II Type 2 Receptor in Apoptosis during Human Fetal Adrenal Gland Development J. Clin. Endocrinol. Metab., December 1, 1999; 84(12): 4722 - 4730. [Abstract] [Full Text]
|
|
|
|
 |
|
 W. C. Engeland and B. K. Levay-Young Changes in the glomerulosa cell phenotype during adrenal regeneration in rats Am J Physiol Regulatory Integrative Comp Physiol, May 1, 1999; 276(5): R1374 - R1382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hubert, J.-M. Gasc, S. Berger, G. Schütz, and P. Corvol Effects of Mineralocorticoid Receptor Gene Disruption on the Components of the Renin-Angiotensin System in 8-Day-Old Mice Mol. Endocrinol., February 1, 1999; 13(2): 297 - 306. [Abstract] [Full Text]
|
|
|
|
 |
|
 P. E. McEwan, G. P. Vinson, and C. J. Kenyon Control of adrenal cell proliferation by AT1 receptors in response to angiotensin II and low-sodium diet Am J Physiol Endocrinol Metab, February 1, 1999; 276(2): E303 - E309. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
): control; (
): rats on a low sodium regimen.
) or PD 123319 (X).
