Role of adrenal renin-angiotensin system in the control of
aldosterone secretion in sodium-restricted rats
Giuseppina
Mazzocchi1,
Ludwik K.
Malendowicz2,
Anna
Markowska2,
Giovanna
Albertin1, and
Gastone G.
Nussdorfer1
1 Section of Anatomy, Department of Human
Anatomy and Physiology, University of Padua, I-35121 Padua, Italy;
and 2 Department of Histology and Embryology,
School of Medicine, PL-60781 Poznan, Poland
 |
ABSTRACT |
This study
examined the effect of the pharmacological manipulation of adrenal
renin-angiotensin system (RAS) on aldosterone secretion from in situ
perfused adrenals of rats kept on a normal diet and sodium restricted
for 14 days. Neither the angiotensin-converting enzyme inhibitor
captopril nor the nonselective angiotensin II receptor antagonist
saralasin and the AT1 receptor-selective antagonist losartan affected basal aldosterone output in normally fed rats. In
contrast, they concentration dependently decreased aldosterone secretion in sodium-restricted animals, with maximal effective concentration ranging from 10
7 to
10
6 M. Captopril
(10
6 M), saralasin
(10
6 M), and losartan
(10
7 M) counteracted aldosterone
response to 10 mM K+ in sodium-restricted rats but not in
normally fed animals. Collectively, these findings provide evidence
that adrenal RAS plays a role in the regulation of aldosterone
secretion, but only under conditions of prolonged stimulation of zona
glomerulosa probably leading to overexpression of adrenal RAS.
in situ adrenal perfusion
 |
INTRODUCTION |
THE PRESENCE OF A COMPLETE renin-angiotensin system
(RAS) in the adrenal gland, probably involved in the local paracrine
regulation of aldosterone secretion, is well known (for review, see
Refs. 12, 13, 19). However, the direct in vivo demonstration that adrenal RAS plays a physiological role in the control of zona glomerulosa secretory activity is not yet available.
Therefore, we decided to address this issue by pharmacologically
manipulating adrenal RAS in in situ-perfused left rat adrenals. In
fact, this experimental model allows for the delivery of the chemicals
directly to the adrenal gland without any possible interference with
the systemic mechanisms regulating mineralocorticoid secretion.
 |
MATERIALS AND METHODS |
Reagents.
The nonselective angiotensin II (ANG II) antagonist
[Sar1,Val5,Ala8]-ANG II
(saralasin) was obtained from Peninsula Laboratories (St. Helens, UK),
the angiotensin-converting enzyme (ACE) inhibitor captopril (Capoten)
was obtained from Squibb (Milan, Italy), and the AT1
receptor antagonist losartan (DuP753) was obtained from Merck Sharp & Dohme (Rome, Italy). Human serum albumin (HSA) was from Sigma Chemical
(St. Louis, MO), and Medium 199 was from DIFCO (Detroit, MI). The
sodium-deprived diet (<0.01 meq Na+/g) was purchased from
Dr. Piccioni Laboratory (Milan, Italy), and RIA kits for aldosterone
and corticosterone were from IRE Sorin (Vercelli, Italy) and
Eurogenetix (Milan, Italy), respectively.
Animal treatment.
Adult male Wistar rats (260 ± 30 g body wt) were purchased from
Charles River (Como, Italy). A group of rats was sodium restricted (sodium-deprived diet and demineralized water as drinking fluid) for 14 days, and another group was maintained on a standard diet and tap water.
In situ adrenal perfusion.
Sodium-deprived and normally fed rats were anesthetized with
pentobarbital sodium, and the left adrenal gland was perfused in situ,
as previously detailed (11). Perfusion medium was introduced via a
cannula inserted in the celiac artery into an isolated segment of aorta
from which the adrenal arteries arise. After flowing through the
adrenal gland, medium was collected by a cannula inserted in the renal
vein. Perfusion medium (tissue culture Medium 199, modified by dilution
with KCl-free Krebs-Ringer bicarbonate to give a final K+
concentration of 3.9 mM and containing 0.2% glucose and 5 mg/ml HSA)
was gassed with 95% air-5% CO2, maintained at 37°C,
and delivered by peristaltic pump at a constant rate of 2 ml/10 min for
90 min. Perfusion pressure was monitored by a pressure transducer
inserted in the arterial cannula and was found to average 30 ± 3 mmHg. After an initial equilibration period of 30 min, three
10-min samples were collected, and then the perfusion medium was
substituted with one in which the chemicals to be tested were dissolved
to the required concentration, and three more 10-min samples were collected. Two experiments were performed. In the first experiment, captopril, saralasin, or losartan was added to the perfusion medium in
concentration ranging from 10
9 to
10
4 M. In the second experiment,
perfusion medium contained 10
6 M
captopril, 10
6 M saralasin, or
10
7 M losartan, and 10 mM K+
was added after the first three sample collections. This concentration of K+ is the maximally effective one in eliciting
aldosterone secretion (6).
Hormone assays.
Aldosterone and corticosterone were extracted from perfusion media and
purified by HPLC (15). Their concentrations were measured by RIA with
the following commercial kits: ALDOCTK2 (sensitivity, 5 pg/ml; intra-
and interassay variations, 5.8 and 7.5%, respectively), and CTRX-RIA
(sensitivity, 50 pg/ml; intra- and interassay variations, 6.6 and 8.2, respectively).
Statistics.
For each rat, the rate of hormone output was calculated as the average
of the three 10-min collection periods before (control value) and after
addition of the chemicals to the perfusion medium (experimental value).
In the first experiment, for each experimental point five rats were
perfused, control and experimental values were averaged, and their
statistical significance was assessed. The data were graphically
expressed as the means ± SE of the percent change from the group
control value. Baseline (control) values from sodium-restricted and
normally fed rats were averaged and expressed as picomoles per 10 minutes. In the second experiment, for each experimental point six rats
were perfused, and data were expressed as the means ± SE of the
average secretion rate (pmol/10 min) before and after 10 mM
K+ addition. The statistical comparison of the results was
done by ANOVA, followed by Duncan's multiple range test. A value of P < 0.05 was considered significant.
 |
RESULTS |
Pilot experiments showed that, during the 60 min of sample collection,
the basal rate of hormone production remained satisfactorily constant
in both normally fed and sodium-restricted rats (Fig. 1). Sodium restriction raised aldosterone
output by perfused rat adrenal (~80%), without affecting
corticosterone release (Fig. 2).

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Fig. 1.
Representative experiment showing that after initial period of 30 min,
aldosterone secretory rate (left panel) becomes satisfactorily
constant over the next 60 min of perfusion in both normally fed
(bottom line) and sodium-restricted rats (top line).
After 90 min of perfusion, rate of secretion starts to decline,
probably because of functional alteration of the gland. The same
behavior is shown by corticosterone secretion rate in both normally fed
(right panel) and sodium-restricted rats (not shown).
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Fig. 2.
Effects of sodium restriction on basal aldosterone (left panel)
and corticosterone (right panel) output by in situ perfused rat
adrenals. Bars are means ± SE; n = 72. * P < 0.01 from controls.
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Captopril, saralasin, or losartan did not evoke significant changes in
aldosterone production by perfused adrenal of normally fed rats (Fig.
3). In contrast, they
markedly lowered aldosterone output in sodium-restricted animals in a
concentration-dependent manner with a maximally effective concentration
ranging from 10
7 to
10
6 M (Fig. 3). Corticosterone
production was not affected in either group of rats (data not shown).

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Fig. 3.
Effect of captopril (A), saralasin (B), and losartan
(C) on basal aldosterone output by in situ perfused adrenals of
normally fed and sodium-restricted rats. Values are percentage changes
(means ± SE; n = 5) from respective baseline value (B).
+ P < 0.05 and * P < 0.01 from
baseline.
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K+ (10 mM) increased aldosterone production in both
normally fed and sodium-restricted rats (three- and fivefold rise,
respectively, Fig. 4). The presence of
captopril (10
6 M), saralasin
(10
6 M), or losartan
(10
7 M) in the perfusion medium did not
significantly affect either the basal rate of aldosterone production or
aldosterone response to 10 mM K+ in normally fed animals
(Fig. 4A). Conversely, in sodium-restricted rats, they lowered
the basal rate of aldosterone secretion (by 48-62%) and
aldosterone response to 10 mM K+ (from a five- to about a
threefold rise, Fig. 4B).

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Fig. 4.
Effects of captopril (10 6 M), saralasin
(10 6 M), and losartan
(10 7 M) on 10 mM
K+-stimulated aldosterone output by in situ perfused
adrenals of normally fed (A) and sodium-restricted (B)
rats. Aldosterone responses to 10 mM K+ in absence of
renin-angiotensin system inhibitors are also shown. Bars are means ± SE; n = 6. * P < 0.01 from respective control
value; A P < 0.01 from the baseline control
value.
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 |
DISCUSSION |
Many lines of in vitro evidence (obtained by the use of capsular zona
glomerulosa strips, adrenal slices, or zona glomerulosa cell cultures)
suggest that adrenal RAS controls aldosterone secretion. ACE inhibitors
were found to lower either basal (20) or K+- and
ACTH-stimulated aldosterone output by rat zona glomerulosa (2, 14, 17,
18, 21) and basal aldosterone yield by cultured bovine (9) and human
adrenal tissue (5), as well as by K+-stimulated aldosterone
release by human adrenocortical NCI-H295 cell line (8). A selective
AT1 receptor antagonist was reported to block both basal
and agonist-stimulated aldosterone secretion from cultured bovine zona
glomerulosa cells (7) and K+-enhanced aldosterone
production from the NCI-H295 cell line (8).
Our study, although partly confirming these observations, casts serious
doubts on the possibility that adrenal RAS in vivo plays a major role
in the regulation of aldosterone secretion under basal conditions. In
fact, both ACE inhibition by captopril and ANG II receptor blockade by
saralasin or losartan were ineffective on basal or
K+-stimulated aldosterone secretion from in situ perfused
adrenals in rats kept on a normal diet. This discrepancy stresses that marked differences occur in the adrenal cortex physiology between in
vivo and in vitro conditions. In fact, when the structural integrity of
the entire adrenal gland is preserved, several complex paracrine
interactions between cortex and medulla are operative (for review, see
Ref. 16), which conceivably may obscure under basal conditions the
stimulatory effect of adrenal RAS on the zona glomerulosa secretory activity.
Conversely, our investigation strongly suggests that adrenal RAS may be
involved in enhancing aldosterone secretion under pathophysiological
conditions leading to prolonged stimulation of zona glomerulosa, such
as those elicited by sodium intake restriction. Sodium restriction was
found to increase adrenal renin mRNA and protein (1, 3). Hence, our
observations could suggest that, only when overexpressed, adrenal RAS
plays a role in enhancing aldosterone secretion. This contention
appears to be in keeping with the fact that the transgenic rat strain
TGR(mREN2)27, which overexpresses the murine
Ren-2d gene in adrenal glands, secretes
elevated amounts of aldosterone (for review, see Refs. 4, 10). Further
studies are underway to see whether captopril and the ANG II receptor
antagonists alter aldosterone output by in situ perfused adrenals of
TGR(mREN2)27 rats kept on a normal diet, as well as those of animals
with prolonged K+ intake.
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FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. G. Nussdorfer, Dept. of Anatomy, University of Padua, Via Gabelli
65, I-35121 Padua, Italy (E-mail:
ggnanat{at}ipdunidx.unipd.it).
Received 16 September 1999; accepted in final form 12 January
2000.
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