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Department of Pharmacology (J.P., B.P., C.M.-G.), University of Heidelberg, D-69120 Heidelberg, Germany; and Medical Research Center (N.O., A.W., B.K., N.G.), D-68167 Mannheim, Germany
Address all correspondence and requests for reprints to: Dr. Jörg Peters, Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany. E-mail: peters{at}novsrv1.pio1.uni-heidelberg.de
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Abstract |
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Because the effects of losartan or ANG II on aldosterone production involved a latency period of at least 30 h after NX and were associated with a modulation or recruitment of renin-producing cells, we suggest that the intraadrenal renin-angiotensin system operates via regulation of cell differentiation on a long-term scale, rather than or additionally to its short-term effects on aldosterone synthase activity.
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Introduction |
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Observations by Aguilera et al. (4) and Campbell et al. (5) demonstrate that circulating ANG II does not contribute considerably to tissue ANG II. Instead, the majority of ANG II is formed within the tissues themselves from plasma-derived or locally produced ANG I. Within the adrenal cortex, all genes of the RAS are expressed, and the corresponding proteins have been found in many species (for review, see Ref. 6). Renin has been specifically localized to the zona glomerulosa cell (7), the only cell type known to be capable of aldosterone synthesis. Interestingly, within these cells, renin has not only been found within cytoplasmatic secretory vesicles but also within mitochondria (8, 9). This suggests that in the adrenal gland, a RAS exists intracellularly, which may regulate cell function by modes of action different to the circulating system.
The adrenal expression of renin seems to be under control of the circulating RAS. This is assumed for the following reasons: During mouse embryonic development, the highest expression of the renin gene is not found in the kidney but actually within the adrenal gland (10). After birth, adrenal renin expression strongly decreases and, henceforth, the kidney represents usually the major renin-expressing site. When rats are nephrectomized, renin content within the outer adrenal cortex increases markedly (11). The absence of circulating renin and the concomitant decrease of plasma ANG II concentrations seem to contribute significantly to the increase of intraadrenal renin levels (12).
Although the mechanisms of action of the intraadrenal RAS are far from clear, several observations support the idea that such a system is of physiological and pathophysiological relevance. For example, in contrast to plasma renin, intra-adrenal renin levels not only correlate well with aldosterone production dependent on serum sodium but also dependent on serum potassium levels. High levels of aldosterone are associated with high levels of adrenal renin but with low levels of plasma renin (11). Under these circumstances, an intraadrenal RAS may regulate aldosterone production separately from, or additionally to, the circulating system.
Support for the hypothesis that the intraadrenal RAS has functional consequences in vivo was derived from experiments with a transgenic rat model of hypertension. These rats have integrated into their genome the mouse ren-2 gene, which is expressed highly within the adrenal cortex (13). At 4–8 weeks of age, these rats are characterized by prominently increased intraadrenal levels of renin (14) and enhanced concentrations of aldosterone and other steroids in plasma and urine (13, 15), but they exhibit low plasma renin concentrations (PRCs). Nevertheless, evidence for this hypothesis is not conclusive yet, because the circulating RAS is still present in this model and the level of plasma prorenin, the inactive precursor of renin, is markedly elevated (13, 16).
To clearly distinguish between intraadrenal and circulating RAS, it was desired to examine the effects of inhibitors of the system in the absence of circulating renin and angiotensins. We have therefore investigated the effect of losartan on aldosterone production in nephrectomized rats on a normal sodium balance, where we could ascertain that renin and prorenin were absent from the circulation and plasma angiotensin levels were markedly reduced. We were also particularly interested in characterizing the interactions between the circulating and the local intraadrenal RAS in more detail. Taking advantage of the nephrectomy (NX) model, we compared the direct effect of resupplemented ANG II on aldosterone production with its indirect effects via inhibiting the intraadrenal RAS. Surprisingly, we observed that the latter was the stronger, resulting in suppression of aldosterone production by ANG II in anephric rats.
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Materials and Methods |
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All animals received chronic indwelling catheters into the femoral artery and vein under ketamine/xylazine anesthesia (75 mg/kg and 6 mg/kg, respectively) and were further allocated to 1 of 6 groups of 6–8 rats each. Each animal received an osmotic minipump (model 2ML1, Charles River Laboratories, Inc., Sulzfeld, Germany), which supplied 10 µl/h at a constant rate. The pumps were implanted sc for application of physiological saline in groups 1 and 2, or ANG II (Sigma Chemical Co., Deisenhofen, Germany) in groups 5 and 6, or ip for application of losartan (MSD Sharp & Dohme GmbH, Haar, Germany) in groups 3 and 4. The calculated infusion rate was 10 µg/kg·min for losartan and 200 ng/kg·min for ANG II. Further, the animals of groups 1, 3, and 5 underwent bilateral NX, whereas the animals of groups 2, 4, and 6 were sham-operated. All groups received hemodialysis on each day after surgery. Rats were hemodialyzed by using a hemophan-mini-dialyzer (courtesy of Dr. Meißner, AKZO, Wuppertal, Germany) with 230 fibers and a surface area of 0.01 m2. Hemodialysis sessions were performed each day for 3 h with an arterial blood flow rate of 1 ml/min and a countercurrent dialysate flow rate of 900 ml/h (17, 18). Plasma samples were obtained before surgery and subsequently every 10–20 h, in conscious rats, via catheter.
Tissue preparation
Rats were anesthetized with ketamine/xylazine (75 mg/kg and 6
mg/kg, respectively). Fixation of the adrenal glands was performed by
retrograde perfusion from the abdominal aorta with 2% freshly prepared
paraformaldehyde in PBS for 90 sec at a pressure of 220 mm Hg and for
90 sec at a pressure of 170 mm Hg, followed by perfusion with 18%
sucrose in PBS, adjusted to 800 mosmol/kg for another 3 min at the same
pressure level.
For in situ hybridization, the adrenal glands were removed, quartered, mounted quickly onto small pieces of styrofoam, and then snap-frozen in liquid nitrogen-cooled isopentane. All tissues were stored at -80 C. For quantitative determination of renin and of angiotensin binding sites within the adrenal cortex, one adrenal gland was removed before perfusion, trimmed of fat, quickly dissected into capsular and decapsular portion, and snap-frozen in liquid nitrogen. The capsular portion, consisting mainly of glomerulosa cells, was later homogenized in 0.1 M N-Tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid (TES), containing 2 mM EDTA and 10-4 M phenylmethanesulfonylfluoride (PMSF). Membranes were precipitated at 50,000 x g for 30 min, quickly frozen, and stored at -70 C. In the supernatant protein, as well as renin, concentrations were determined. Membranes were redissolved in solubilization buffer (20 mM Tris-acetate, pH 7.5, containing 1 mM EDTA and 1 mM 1,4-dithiothreitol) for binding studies immediately before analysis.
Determination of components of the RAS and plasma steroid
levels
Active and inactive renin concentrations were determined as
described previously (14). To activate inactive renin, 10 µl plasma
was incubated for 10 min on ice with 10 µl trypsin (for plasma, 400
U/ml; for tissue extracts, 50 U/ml, dissolved in TES-buffer (0.1
M TES, pH 7.2; 0.01% neomycin; 10 mM EDTA).
The reaction was terminated by the addition of 5 µl egg white trypsin
inhibitor (1600 U/ml and 200 U/ml, respectively, in TES-buffer) and
then incubated with lyophilized renin substrate isolated from
nephrectomized rat plasma (final concentration: 80 mg/ml, 0.11%
2,3-dimercapto-1-propanol, 1.15 mg/ml 8-hydroxy-chinolin in
TES-buffer). The reaction was terminated with RIA-buffer (0.1
M Tris-acetate, pH 7.4): 1) immediately before the
incubation; and 2) 1 h after incubation with substrate. Samples
were then boiled for 10 min and centrifuged, and the concentration of
ANG I generated was measured by RIA (19). Active renin concentration
was determined in parallel but with the addition of 10 µl of
TES-buffer instead of trypsin, and this value was subtracted from the
concentration obtained after trypsin activation to determine the
content of inactive renin (prorenin). For PRC, the samples were
incubated directly with substrate. To further test the specificity of
the results, aliquots of plasma and tissue extracts were incubated with
and without the addition of the specific renin inhibitor CH732 (20),
10-6 M, a gracious gift from Dr. M. Szelke,
Ferring Research Institute, Southampton, UK.
Angiotensinogen was measured by incubating samples with pig renin, as previously described (21), followed by ANG I RIA. For plasma ANG I and ANG II determinations, it was crucial to prevent the conversion of ANG I to ANG II, as well as the degradation of angiotensins by peptidases during blood collection. This was accomplished by collecting the blood quickly into a precooled inhibitor cocktail containing 5 mM EDTA, 0.1 µM PMSF, 4 µg/ml enalaprilat, and 400 µg/ml 1,10-phenantrolin (final concentrations). Plasma samples were then processed by Sep-Pak-elution (Waters GmbH, Eschborn, Germany). The eluates were lyophilized, and the dry residues were dissolved in 0.1 M Tris acetate buffer, pH 7.4, containing 0.1% BSA. Samples were then analyzed by RIA for ANG I or ANG II, respectively (19, 22).
Protein content of samples was determined by the commercial kit from Bio-Rad (Bio-Rad Laboratories, Inc. GmbH, Munich, Germany).
Plasma aldosterone and corticosterone were measured by RIA, after extraction and chromatography, as described previously (15, 23, 24).
Statistical analysis
Data were given as means ± SEM. Differences
between groups were evaluated by ANOVA, followed by the post
hoc test of Bonferoni. Only P-values less than 0.05
were accepted to indicate significant differences.
Angiotensin receptor binding studies
Each 30 µg of capsular membranes were incubated in assay
buffer (50 mM Tris-Cl (pH 7.4), 100 mM NaCl, 10
mM MgCl2, 0.1 mg/ml bacitracin, 0.1% BSA, and
10-4 M PMSF), together with 100,000 cpm
iodinated ANG II (6,667 cpm/fmol) and various amounts of unlabeled ANG
II (10-11–10-7 M; Bachem,
Heidelberg, Germany) in a total vol of 1 ml for 90 min at 25 C. The
reaction was stopped by the addition of 8 ml precooled 0.9% NaCl. To
separate bound from free angiotensin, the contents of the tubes were
filtered through GF/C membranes (Whatman Scientific Ltd.,
Maidstone, UK), presoaked in 0.9% NaCl-0.1% mg/ml BSA. The
membranes were washed twice with each 8 ml of 0.9% NaCl. Binding data
are reported as specific binding of ANG II after subtraction of
nonspecific binding (i.e. binding observed in the presence
of 10-6 M unlabeled ANG II).
In situ hybridization
Digoxigenin-11-uridine 5'-triphosphate-labeled sense and
antisense riboprobes were prepared from a full-length rat renin
complementary DNA, according to the manufacturer’s protocol
(Boehringer Mannheim, Mannheim, Germany). The riboprobes were
then partially hydrolyzed to estimated fragment lengths of 250 bases,
to facilitate tissue penetration.
In situ hybridization was carried out as described previously (25). Cryostat sections (5- to 7-µm thick) were transferred onto silane-coated glass slides. Sections were postfixed in 4% paraformaldehyde (in PBS, pH 7.4) for 20 min, rinsed three times in PBS, and washed in diethyl pyrocarbonate-treated, bidestilled water for 10 min. To improve permeabilization, a mild deproteinization step was performed by immersing slides in 0.1 M HCl for 10 min, followed by two short rinses (5 min each) in PBS.
To reduce background, slides were acetylated for 20 min in 0.1 M triethanolamine, pH 8.0, containing 0.25% acetic anhydride. After rinsing in PBS, slides were stepwise dehydrated (5 min each in 70%, 80%, and 95% ethanol) and air-dried. Sections were incubated with prehybridization solution (50% deionized formamide, 50 mM Tris-HCl (pH 7.6), 25 mM EDTA (pH 8.0), 20 mM NaCl, 0.25 mg/ml transfer RNA from yeast, 2.5 x Denhardt’s solution) at 46 C for 2 h, followed by incubation for 16 h at 42 C in a moist chamber with 25 µl hybridization mixture (final concentrations: 50% deionized formamide; 20 mM Tris-HCl (pH 7.6); 1 mM EDTA (pH 8.0); 0.33 M NaCl; 0.2 M 1,4-dithiothreitol; 0.5 mg/ml transfer RNA; 0.1 mg/ml sonicated, denatured DNA from fish sperm; 1 x Denhardt’s solution; 10% dextran sulfate; and 5–10 ng/µl denatured riboprobe), covered with a siliconized coverslip.
Slices were washed once in 2 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0) at room temperature for 20 min, three times (for 1 h each) at 49 C in 50% formamide containing 1 x SSC, 0.5 x SSC, and 0.1 x SSC, respectively, and then rinsed at room temperature in 0.5 x SSC for 15 min, in 0.2 x SSC for 10 min, and equilibrated twice (for 5 min) in buffer I (100 mM Tris-HCl, 150 mM NaCl, pH 7.4).
Incubation of slices with alkaline-phosphatase-coupled antidigoxigenin antibody and color reactions were performed according to the manufacturer’s protocol (Boehringer Mannheim).
The specificity of the obtained in situ hybridization signal was verified by parallel incubation with antisense and sense riboprobes on alternate sections. Throughout all experiments, sense probes did not produce any detectable signal.
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Results |
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). Renin
levels were increased by ANG II, as well, in sham operated rats, which
remains to be explained. Effects of hemodialysis or lack of feedback
inhibition because of ligand-mediated AT1-receptor
down-regulation may be suggested.
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).
Plasma ANG I concentrations were prominently decreased by NX in all
NX-groups to about 10% of pre-NX values (Table 1). Plasma ANG II
levels decreased in nephrectomized/NaCl- and
nephrectomized/losartan-treated rats, as well. It remained, however,
still considerably above the detection limit of the assay, with levels
of about 30% of its basal values. Administration of ANG II increased
its plasma levels in sham-operated and nephrectomized rats by about
10-fold, as intended.
Losartan decreased (whereas administration of ANG II increased)
angiotensinogen production significantly (Table 1). These observations
confirm reports concerning the stimulatory effect of ANG II on hepatic
angiotensinogen production (26). Plasma angiotensinogen concentrations
rose prominently after NX. We did not, however, observe any inhibitory
effect of losartan on the NX-induced increase in plasma angiotensinogen
levels (Table 1). This contradicts previous assumptions that the
NX-induced increase in angiotensinogen production is a result of any
transitory increases in circulating ANG II (27).
Effect on plasma aldosterone concentrations (PAC)
Losartan decreased (and ANG II increased) mean values of PAC in
sham-operated rats. Changes remained small, however, and thus did not
become significant, given the numbers of rats analyzed (Table 1 and
Fig. 1; sham/NaCl: from 18 ± 3
ng/100 ml to 11 ± 3; sham/Losartan: from 13 ± 3 to 8
± 4; sham/ANG II: from 12 ± 2 to 27 ± 12; each n = 6,
not significant).
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and Fig. 1: PAC, 55 h after NX in
NX/NaCl: 465 ± 90 ng/100 ml; n = 8; P <
0.01). Corticosterone levels, on the other hand, increased only
slightly in all six groups after operation (not shown) and were not
different among the groups at the end of the experiment (Table 1).
Despite the fact that, in nephrectomized rats, circulating renin was
eliminated and ANG I was reduced, losartan markedly suppressed the rise
of aldosterone production in nephrectomized rats (Table 1 and Fig. 1:
PAC, 55 h after NX in NX/losartan: 199 ± 26 ng/100 ml;
n = 7 vs. 465 ± 90 in NX/NaCl; P
< 0.01). Unexpectedly, ANG II, as well, inhibited the rise in
aldosterone levels by NX, which was apparent at 30 h and 55 h
(Table 1 and Fig. 1: PAC, 55 h after NX in NX/ANG II: 176 ±
35 ng/100 ml; n = 6; P < 0.01). The differences
remained significant, when analyzing the change of PAC in relation to
their basal levels (55 ± 13-fold in NX/NaCl vs.
19.2 ± 3-fold in NX/losartan or 16.7 ± 2-fold in NX/ANG II;
P < 0.02 for NX/NaCl vs. Nx/losartan and
P < 0.0002 for NX/NaCl vs. NX/ANG II).
The effects described here cannot be attributed to any decreases of potassium levels, because those were not different between NX/NaCl and NX/ANG II (6.2 ± 0.6 vs. 6.3 ± 0.7 at 30 h after NX and 5.7 ± 0.6 vs. 5.9 ± 0.6 mmol/liter at 55 h after NX; not significant), and they were even higher in NX/losartan (6.9 ± 0.5 at 30 h after NX and 7.7 ± 0.6 at 55 h after NX).
Effect on adrenal renin expression
The expression of the renin gene within the adrenal cortex is
subject to regulation by circulating ANG II, as we could demonstrate by
in situ hybridization. In rats with an intact circulating
RAS, renin messenger RNA (mRNA) was found within a few adrenocortical
cells only (Fig. 2a) and was undetectable
in ANG II-treated rats (Fig. 2e).
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). However, renin gene expression was inhomogeneously distributed and
still seemed to be restricted to certain cells or groups of cells. The
NX-induced rise of renin mRNA levels was strongly reduced by ANG II
(Fig. 2f), indicating that ANG II has an inhibitory effect on adrenal
renin gene expression.
If the inhibitory effect of ANG II were mediated by interactions with
the AT1-receptor, losartan should increase renin mRNA
levels. In sham-operated rats, however, we did not observe any change
in adrenal renin mRNA levels by losartan (Fig. 2, a vs. c).
The proposed effect of losartan may have been prevented by competition
at the AT1 receptor with circulating ANG II, which
prominently increased by losartan. In agreement with this, significant
effects of losartan on PAC were also absent. Moreover, in anephric
rats, which lack the possibility to respond to angiotensin receptor
inhibition with increased renin levels in plasma, renin mRNA levels
seemed to rise, indeed, by losartan (NX/NaCl, Fig. 2b vs.
NX/losartan, Fig. 2d).
In support of the data on renin mRNA in the adrenal cortex, enzymatic
activities of renin at that site correlate well with renin mRNA levels
in anephric rats but not with renin mRNA levels in sham-operated rats.
Adrenocortical renin concentrations were increased by NX (Fig. 3: Sham/NaCl: 11 ± 2 ng ANG
I/mg·h vs. NX/NaCl: 111 ± 16; P <
0.05), and this increase was partially abolished by supplementation
with ANG II (71 ± 16 ng ANG I/mg/h). Losartan, on the other hand,
increased adrenal renin content in anephric rats even further (172
± 30 ng ANG I/mg·h). Thus, the inhibitory effect of ANG II on renin
expression is likely to be mediated via the
AT1-receptor.
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40 ± 6 ng ANG I/mg·h and 45 ± 7, respectively,
vs. 11 ± 1 in NaCl-treated rats). This observation
rather paralleled the increase of circulating renin levels and,
therefore, may be the result of renin uptake from the circulation.
Effect of NX and ANG II on angiotensin binding to capsular
membranes
Because it has been shown that bilateral NX increases the number
of angiotensin binding sites and that ANG II dose-dependently prevents
this increase and even reduces the number of binding sites below
control levels (28), we needed to confirm that binding sites were still
available for ANG II to exert its effects in our experiment
(e.g. increasing aldosterone and decreasing renin mRNA).
Binding studies were therefore performed on capsular membranes from
sodium chloride- and ANG II-perfused rats with and without NX. Adrenal
capsular membranes of the sham-operated, ANG II-treated rat contained
less binding sites than membranes of the sham-operated sodium
chloride-treated control (Fig. 4;
open rhombs vs. open rectangles). In contrast,
membranes of nephrectomized rats (NX/NaCl) contained markedly more
binding sites than those of controls in the two cases investigated
(open circles). The number of binding sites of capsular
membranes from the two nephrectomized, ANG II-infused rats were within
the range of nephrectomized sodium chloride-infused rats (open
triangles). Because of the small number investigated, it is not
possible to draw any final conclusions. However, observations of the
cases presented here demonstrate that, even in NX/ANG II-treated rats,
there are enough binding sites available for ANG II to exert its
effects (e.g. the known stimulatory effect on aldosterone
production and its inhibitory effect on renin mRNA levels observed in
this study). Nevertheless, aldosterone levels were prominently higher
in NX/NaCl, compared with NX/ANG II (in the particular cases, where
binding studies were performed, PACs were: 845 and 457 ng/100 ml in
NX/NaCl vs. 275 and 147 ng/100 ml in NX/ANG II). Thus, a
down-regulation of angiotensin binding sites, as a cause for the lower
PAC in NX/ANG II (compared with NX/NaCl) could not be demonstrated.
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Discussion |
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In contrast, we have investigated the model of nephrectomized hemodialyzed rats on a normal sodium balance, ascertaining that renin and prorenin were absent from the circulation. The study shows that a functionally active local RAS indeed exists within the adrenal cortex, which is under control of circulating angiotensins.
These suggestions are based on the following observations: First, bilateral NX eliminates circulating renin but stimulates expression of renin within the adrenal cortex. Second, bilateral NX prominently increases plasma aldosterone, and this rise is inhibited by both losartan and ANG II. Third, the inhibition by ANG II is associated with a prominent reduction of the NX-induced increase of adrenocortical renin mRNA and renin content.
The effect of NX on the circulating RAS
We have previously shown that bilateral NX eliminates renin and
prorenin from the circulation (8). The study presented here confirms
the finding and demonstrates that neither ANG II nor losartan prevents
the disappearance of renin after NX. The experimental setting thus
allows us to analyze and differentiate ANG II-dependent from ANG
II-independent effects of NX on adrenal renin expression and
steroidogenesis. The model of nephrectomized hemodialyzed rats further
allows us to analyze the effects of ANG II and losartan on adrenal
function independently of their effects on the kidney (e.g.
angiotensin-dependent renal renin secretion and electrolyte handling).
Moreover, the decrease of PRC, to undetectable levels, allowed us to
investigate effects of losartan on adrenal function independently of
circulating renin.
ANG II, however, did not disappear from the circulation, indicating that angiotensins are produced locally within tissues, like the adrenal gland, even in the absence of the kidney. This finding confirms previous observations and the concept of Aguilera et al. (4), further strengthened by Campbell et al. (5), that tissue angiotensins are derived from local production rather than from the circulation. The fact, that ANG II is still present within the circulation after bilateral NX, however, unfortunately continues to prevent an unequivocal discrimination between the roles of intraadrenally generated and circulating ANG II in vivo.
The regulation of adrenocortical renin expression
Applying the in situ hybridization technique, we
confirmed the presence of renin mRNA within the adrenal cortex of rats.
In contrast to Deschepper et al. (7), who found prominent
staining for renin mRNA all over the zona glomerulosa, the observations
presented here show that renin gene expression is restricted to a few
cells only. Our data, however, are well in agreement with the low
levels of renin mRNA detected by other methods of highest sensitivity
(30).
Bilateral NX led to a marked increase of the number of renin-expressing cells and their renin mRNA content. Even after bilateral NX, however, renin mRNA is not distributed homogeneously but seems to be restricted to clusters of cells. This suggests that different populations of cells are present in the outer adrenal cortex. In terms of renin expression, NX may therefore be associated with selective stimulation, or even proliferation, of those cells expressing renin rather than with increasing renin expression within all glomerulosa cells.
Interestingly, replacement of ANG II, by chronical sc administration of the peptide, abolished the increase in adrenal renin mRNA markedly. The facts that, first, reduction of angiotensin levels by NX is associated with increased adrenal renin expression; second, that losartan increased renin mRNA even further, after NX; and third, that ANG II inhibits the rise of renin expression all demonstrate that adrenal renin expression is inhibited by circulating ANG II via the AT1 receptor. In agreement with this, such an inhibitory effect of ANG II on renin expression is already known for the kidney (31).
Because Baba et al. (12) already demonstrated that the rise of adrenal renin by NX is the result of several factors, including elevated plasma potassium concentrations, it was not surprising to observe that, in our experiment, the NX-induced rise of adrenal renin levels cannot be completely abolished by ANG II.
Effect of losartan and ANG II on the NX-induced rise of
PACs
Bilateral NX prominently increases PAC. Part of this effect
clearly depends on the binding of ANG II to the
AT1-receptor, because losartan reduces the NX-induced rise
of PAC. The source of ANG II is likely to be the adrenal tissue itself;
however, a role of circulating ANG II cannot be completely excluded,
because ANG II was not absent from the circulation.
Unexpectedly, although ANG II is known to stimulate aldosterone production on a short-term scale, even after NX (32), chronical infusions of ANG II in nephrectomized rats inhibited the rise of PAC. This became apparent at 30 h and 55 h in our experiment. The effect observed here rather represents a long-term change of adrenal function, because it was absent at 10 h after NX. Because the long-term effect is associated with lower adrenal renin mRNA, we propose that adrenal renin expression plays a major role in the regulation of aldosterone production in this context.
The intraadrenal RAS and its effects
Various models have been proposed to describe the mode of action
of the intraadrenal RAS (33). Adrenal cells may secrete renin in a
regulated fashion, similarly to the renin-producing cells of the
kidney. Secreted renin then leads to interstitial generation of
angiotensins, which subsequently stimulate angiotensin receptors
located at the surface of adrenocortical cells. This model is supported
by the demonstration of prorenin release from adrenocortical cells (34)
and of a regulated secretion of active renin from adrenal cells in a
transgenic rat model expressing the mouse ren-2d
renin gene (13, 14).
The expression of the components of the RAS within adrenocortical cells may additionally lead to intracellular generation of angiotensins, exerting their effects independently of the known angiotensin receptors at the cell surface (33). The findings presented here are in agreement with such a concept: Although the local production of ANG II is probably increased in anephric rats, because of the increased adrenal renin expression, the intraadrenal angiotensin content, representing the sum of local synthesis and uptake from the circulation, has been shown to be lower in nephrectomized than in sham-operated rats (5). Nevertheless, under these conditions, the rise of aldosterone production is still mediated considerably by ANG II, because losartan inhibits aldosterone production, even in the absence of the circulating RAS.
Because the elevation of intraadrenal ANG II levels, by infusion of the peptide in anephric rats, inhibits renin expression, we conclude that enough angiotensin receptors are present for ANG II to exert its effects. This has been confirmed by binding studies on adrenal capsular membranes from each two of the nephrectomized rats with and without ANG II. Besides inhibiting renin expression, as shown here, exogenously administered ANG II should, in accordance with the literature, certainly stimulate aldosterone production.
In opposition to this assumption, however, ANG II inhibits the rise of aldosterone production. The inhibitory effect of circulating ANG II via the local RAS therefore seems to exceed the known direct stimulatory effect of ANG II at the cell surface on aldosterone production. The intraadrenal RAS in NX/NaCl would thus be able to increase aldosterone production with a markedly higher efficiency than that of ANG II given systemically (in NX/ANG II).
Although several possibilities are still conceivable to explain this higher efficiency, including as-yet-unknown factors present in vivo or differential regulation of receptors, it is intriguing to speculate that the higher efficiency of the local system is caused by its intracellular site of action, as has been suggested before (33). Such a site would be accessible to intracellularly generated (but not to circulating) ANG II. Accordingly, losartan-sensitive AT1 receptors at the cell surface stimulate aldosterone release and inhibit renin gene expression, whereas an additional intracellular binding site stimulates exclusively aldosterone production with higher efficiency than the cell surface receptor, without affecting renin mRNA levels. This site would be sensitive to exogenously applied losartan, because this drug likely penetrates cell membranes freely, because of its hydrophobicity. The hypothesis could be tested by infusing a nonmembrane-permeable angiotensin antagonist, which should be unable to inhibit the intracellular system. Because essential steps of aldosterone biosynthesis are performed by mitochondria, these organelles may be the intracellular target for an intraadrenal RAS. The presence of renin and ACE within mitochondria has already been shown (8, 9). Angiotensin binding sites have been reported to be present at mitochondria (35), but further data are needed to demonstrate mitochondrial angiotensin receptors, in support of the intracellular model.
In summary, we here demonstrate the existence of a functionally active local RAS within the adrenal cortex and regulation of aldosterone production by this system. We further show that the system is physiologically suppressed by circulating ANG II and that the increased adrenal renin expression contributes to the high aldosterone levels observed in rats after NX, as has been proposed by Baba et al. (12). Whether the intraadrenal RAS regulates enzymatic steps in aldosterone synthesis, or rather, cell proliferation and cell differentiation, remains to be investigated. The fact that the response to losartan or ANG II after NX was present after a lag period of more than 30 h suggests that at least part of the effect is caused by proliferation or activation of certain cells.
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Acknowledgments |
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Footnotes |
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Received February 11, 1998.
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 J Manolopoulou, M Bielohuby, S J Caton, C E Gomez-Sanchez, I Renner-Mueller, E Wolf, U D Lichtenauer, F Beuschlein, A Hoeflich, and M Bidlingmaier A highly sensitive immunofluorometric assay for the measurement of aldosterone in small sample volumes: validation in mouse serum J. Endocrinol., February 1, 2008; 196(2): 215 - 224. [Abstract] [Full Text] [PDF]
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 B. Peters, P. Teubner, S. Clausmeyer, T. Puschner, C. Maser-Gluth, H.-J. Wrede, B. Kranzlin, and J. Peters StAR expression and the long-term aldosterone response to high-potassium diet in Wistar-Kyoto and spontaneously hypertensive rats Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E16 - E23. [Abstract] [Full Text] [PDF]
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 P. B Persson Renin: origin, secretion and synthesis J. Physiol., November 1, 2003; 552(3): 667 - 671. [Abstract] [Full Text] [PDF]
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 R. N. Re Intracellular Renin and the Nature of Intracrine Enzymes Hypertension, August 1, 2003; 42(2): 117 - 122. [Abstract] [Full Text] [PDF]
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R. N. Re Implications of intracrine hormone action for physiology and medicine Am J Physiol Heart Circ Physiol, March 1, 2003; 284(3): H751 - H757. [Full Text] [PDF]
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 C. Dechow, C. Morath, J. Peters, I. Lehrke, R. Waldherr, V. Haxsen, E. Ritz, and J. Wagner Effects of all-trans retinoic acid on renin-angiotensin system in rats with experimental nephritis Am J Physiol Renal Physiol, November 1, 2001; 281(5): F909 - F919. [Abstract] [Full Text] [PDF]
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 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]
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 S. Clausmeyer, A. Reinecke, R. Farrenkopf, T. Unger, and J. Peters Tissue-Specific Expression of a Rat Renin Transcript Lacking the Coding Sequence for the Prefragment and Its Stimulation by Myocardial Infarction Endocrinology, August 1, 2000; 141(8): 2963 - 2970. [Abstract] [Full Text] [PDF]
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 S. Clausmeyer, R. Sturzebecher, and J. Peters An Alternative Transcript of the Rat Renin Gene Can Result in a Truncated Prorenin That Is Transported Into Adrenal Mitochondria Circ. Res., February 19, 1999; 84(3): 337 - 344. [Abstract] [Full Text] [PDF]
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 S. Kantachuvesiri, S. Fleming, J. Peters, B. Peters, G. Brooker, A. G. Lammie, I. McGrath, Y. Kotelevtsev, and J. J. Mullins Controlled Hypertension, a Transgenic Toggle Switch Reveals Differential Mechanisms Underlying Vascular Disease J. Biol. Chem., September 21, 2001; 276(39): 36727 - 36733. [Abstract] [Full Text] [PDF]
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J. L. Cook, Z. Zhang, and R. N. Re In Vitro Evidence for an Intracellular Site of Angiotensin Action Circ. Res., December 7, 2001; 89(12): 1138 - 1146. [Abstract] [Full Text] [PDF]
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