Effects of Angiotensin II on Sodium Potassium Pumps,
Endogenous Ouabain, and Aldosterone in Bovine
Zona Glomerulosa Cells
Jui R. Shah, James Laredo, Bruce P. Hamilton, John M. Hamlyn
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Abstract—Angiotensin (Ang) II stimulates secretions of aldosterone and an endogenous ouabain-like steroid (EO) from
bovine adrenal zona glomerulosa (BAG) cells. The BAG cell sodium pump, a possible target of EO, affects aldosterone
secretion although little is known about this pump. Here, we describe the effects of Ang II on the characteristics of this
transporter and steroid secretions. Under serum-free conditions, 3H-ouabain bound to a single class of sites on BAG
cells. Binding of label was time and concentration dependent, was sensitive to extracellular potassium ions, and was
displaced by ouabain and digoxin with EC50 of '218 and '232 nmol/L, respectively. Sodium pump–mediated 86Rb
uptake was inhibited by ouabain (EC50 '301 nmol/L). Ang II dose dependently augmented secretions of EO and
aldosterone, increased ouabain-sensitive 86Rb uptake and 3H-ouabain binding, and increased the affinity for 3H-ouabain
binding (Kd, from 205 to 80 nmol/L) with no change in the maximal number of sodium pumps (5.453106) per cell.
Losartan blocked all effects of Ang II except EO secretion, which was inhibited by PD123319. We conclude that BAG
cells express sodium pumps in high density and bind ouabain to a single class of low-affinity sites. The characteristics
of the sodium pumps protect BAG cells from EO autotoxicity but may exclude them from mediating feedback inhibition
of EO secretion. The effects of Ang II on sodium pump activity, ouabain binding affinity, and aldosterone secretion are
mediated via Ang II type 1 receptors, whereas Ang II type 2 receptors augment EO secretion. The role of the Ang
II–mediated increase in the ouabain sensitivity of BAG cell sodium pumps in the secretions of aldosterone and EO
remains to be elucidated. (Hypertension. 1999;33[part II]:373-377.)
Key Words: cardiac glycosides n adrenal n Na,K-ATPase n secretion n cell culture
S
teroid hormones of adrenocortical origin are important
regulators of cardiovascular, renal, and neural function.
In addition to the classic steroid hormones, recent evidence
has suggested that the adrenal cortex may secrete $1 counterparts to the cardenolide ouabain.1–5 Endogenous “ouabain”
(EO) is a specific, high-affinity, reversible inhibitor of sodium pumps.2,3,5 Moreover, the classic secretagogues, angiotensin (Ang) II and adrenocorticotropic hormone, raise EO
secretion from primary adrenocortical cultures.6
Previously, we showed that Ang II stimulates aldosterone
and EO secretions from bovine adrenal zona glomerulosa
(BAG) cells via distinct receptor subtypes.7 Moreover, inhibition of sodium pumps in adrenal cells may augment
aldosterone secretion.8 The apparent links between Ang II,
EO, sodium pumps, and aldosterone secretion led us to
characterize the sodium pump of BAG cells and investigate
the effects of Ang II on the pump in concert with measurements of steroid secretion.
Methods
Adrenocortical Cell Culture
Bovine adrenal glands were obtained from a local slaughterhouse
and transported to the laboratory on ice in phosphate-buffered
solution. Adrenocortical cells were dispersed and cultured according
to established methods. Full details are given elsewhere.7,9,10 Cells
were plated at '4 million (for secretion experiments) or 1 million
(for binding studies) per well. All experiments were performed with
at least triplicate cell cultures.
Steroid Secretion Experiments
and Radioimmunoassays
Details are given elsewhere.10 After incubation, the secretion media
was collected and assayed for aldosterone and EO. They were rinsed
with buffer and dissolved in 1% sodium dodecyl sulfate (SDS) for
assay of total cell protein (BCA) and estimation of cell-associated
radioactivity when appropriate. Aldosterone was measured with a
commercially available radioimmunoassay (RIA) kit (Diagnostic
Products Corp). The RIA has no cross-reactivity to ouabain or EO.
Intra-assay and interassay coefficients of variation were 6.8% and
8.2%, respectively. EO was measured by RIA by use of extracted
Received September 18, 1998; first decision October 19, 1998; revision accepted November 6, 1998.
From the Departments of Physiology (J.R.S., J.M.H., J.L.) and Medicine (B.P.H.), University of Maryland, and Veterans Administration Medical
Center (B.P.H.), Baltimore, Md.
Correspondence to Jui R. Shah, PhD, Department of Physiology, School of Medicine, University of Maryland at Baltimore, 655 W Baltimore St,
Baltimore, MD 21201. E-mail [email protected]
Dr Laredo is now at the Department of Surgery, Beth Israel Hospital, Harvard University, 330 Brookline Ave, Boston, MA 02215.
© 1999 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
373
374
Adrenocortical Sodium Pump
samples and a polyclonal rabbit antiouabain serum (R8) described
elsewhere.11,12 The assay is highly cross-reactive with EO1 but
exhibits no significant cross-reactivity (,0.01%) with cortisol,
aldosterone, adrenocorticotropic hormone, and Ang II. Typical
intra-assay and interassay coefficients of variation were 6.9% and
13.6%, respectively.
3
H-Ouabain Binding Studies
After removal of the culture medium, the cells were washed twice
with buffer (containing in mmol/L NaCl 145, KCl 5, CaCl2 2, MgCl2
1, glucose 10, HEPES 10-NaOH, pH 7.4). Buffer containing 3Houabain (10 to 300 nmol/L) either alone or with pharmacological
reagents was added to the cells and incubated at 37°C in 5% CO2 for
4 or 8 hours. For nonspecific binding, 30 mmol/L cold ouabain was
present in the buffer, and specific binding was taken as the difference
between total and nonspecific binding. Competition curves for
3
H-ouabain binding were constructed by use of 10 nmol/L
3
H-ouabain in the presence of increasing amounts of cold ouabain. At
the end of the experiment, cells were rinsed with buffer and
dissolved in 1% SDS. Total cell protein (BCA) and cell-associated
radioactivity were determined.
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86
Rb Uptake Studies
The culture medium was removed, and the cells were washed twice
with a secretion buffer containing 2 mmol/L RbCl. Secretion buffer
fortified with 86RbCl (0.11 mmol/L) and various pharmacological
agents was incubated with cells at 37°C in 5% CO2 for 4 hours. The
medium was aspirated, and cells were rinsed 3 times in 2 mL
ice-cold buffer with 2 mmol/L RbCl and mixed with 1 mL of 1%
SDS for assay of total cell protein and cell-associated 86Rb (gamma
counter). Pump activity was the difference between uptake in the
absence and presence of 0.1 mmol/L ouabain. Pump fluxes are
expressed as nanomoles of Rb plus potassium per milligram per 4
hours.
Materials
PD123319-ditrifluoroacetate (DTFA) was a gift of Dr David Taylor
(Warner-Lambert, Ann Arbor, Mich). DTFA was removed by
high-performance liquid chromatography before use. Losartan (DuP
753) was a gift of Dr Ronald Smith (Du Pont Merck). Collagenase
type I and DNase type I were from Worthington Biochemicals.
Percoll was from Pharmacia. Fetal bovine serum and Dulbecco’s
modified Eagle’s medium were from GIBCO-BRL. 3H-ouabain was
from New England Nuclear. Other chemicals were from Sigma
Chemical Co.
Figure 1. Time, concentration, and potassium dependence of
3
H-ouabain binding to BAG cells. Top, Time course for binding
of 50 nmol/L 3H-ouabain alone (total) or also containing
30 mmol/L cold ouabain (nonspecific). Calculated specific binding (specific) is shown. Each point is mean6SEM of $3 culture
wells. Middle, Concentration dependence of 3H-ouabain binding
to cells. Total, nonspecific, and calculated specific binding are
shown. BAG cells were incubated for 8 hours with increasing
concentrations of 3H-ouabain. Bottom, Effect of extracellular
potassium concentration on specific binding of 10 and 100
nmol/L 3H-ouabain. Points are mean6SEM of $3 culture wells.
Where SEM values are not shown, they are smaller than symbol
size.
Statistical Analyses
Evaluations of significance were performed with ANOVA and
Fisher’s specific test. Results were expressed as mean6SEM, and
P,0.05 was used to indicate significance unless otherwise noted.
Results
Characterization of BAG Cell Sodium Pumps
Figure 1 shows the time course, concentration dependence,
and potassium sensitivity of 3H-ouabain binding to BAG
cells. In the time course and concentration experiments,
specific binding was .90% of the total. Specific binding
increased fairly linearly with time. The concentration dependence of specific binding was unexpectedly linear, showing
no clear evidence of saturation even at 300 nmol/L. These
results suggested that the sodium pumps in these cells had
fairly low affinity for ouabain. In addition, specific ouabain
binding was reduced dose dependently by extracellular potassium over the range of 0.01 to 10 mmol/L. The doseresponse curve for potassium ions was shifted '7.5-fold
rightward when binding was performed in 100 instead of 10
nmol/L 3H-ouabain.
To explore the binding affinity of the sodium pumps for
cardiac glycosides, the cells were coincubated with increasing
doses of cold cardiac glycosides in the presence of tracer
amounts (10 nmol/L) of 3H-ouabain. As shown in Figure 2,
binding of 3H-ouabain was displaced in a concentrationdependent manner by both ouabain and digoxin with IC50 of
'218 and '232 nmol/L, respectively. In each case, the Hill
coefficients were nearly unity, indicating the interaction of
ouabain and digoxin with a single class of 3H-ouabain binding
sites.
Rb1 ions substitute for K1 in stimulating ATP hydrolysis
and sodium pump–mediated transport into cells. Preliminary
experiments showed that BAG cells accumulated 86Rb and
that the ouabain-sensitive component comprised .80% of the
uptake over 4 hours (data not shown).
Shah et al
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Figure 2. Effects of ouabain, digoxin, and Ang II (AII in figure)
on 3H-ouabain binding to BAG cells. Top, Cells were incubated
with 10 nmol/L 3H-ouabain and indicated concentrations of cold
ligand (F, ouabain; f, digoxin). Curves were fitted iteratively to
4-parameter equation (Hamlyn et al1). Calculated EC50 for
ouabain and digoxin was 218 and 232 nmol/L, respectively.
SEM values are smaller than symbol size. Bottom, Cells were
incubated 4 hours with 50 nmol/L 3H-ouabain in presence of
various doses of Ang II. Shown is total, nonspecific, and calculated specific binding. All points are mean6SEM of $3 separate
cultures.
Effects of Ang II Receptors on Ouabain Binding
and Sodium Pump Activity
The effect of Ang II on the binding of 3H-ouabain to BAG
cells is shown in Figure 2. Ang II dose dependently increased
the specific binding of 3H-ouabain to BAG cells from a basal
of '6 to .14 fmol/mg at 100 nmol/L Ang II. The EC50 for
Ang II stimulation of 3H-ouabain binding was '1 nmol/L.
This value is similar to the EC50 for the stimulation of
corticosteroid secretions,7 suggesting that the same receptor
may be involved in both effects.
Further experiments in which the concentration of
3
H-ouabain was systematically varied showed that the effect
of Ang II increased the binding of label at all concentrations
tested (Figure 3). Moreover, Scatchard analysis (Figure 3,
inset) showed that the binding of 3H-ouabain to BAG cells
under basal conditions was well fit to a single class of binding
sites with an apparent Kd of 205 nmol/L, and a Bmax of 35.6
fmol/mg protein. In the presence of 10 nmol/L Ang II, a single
class of binding sites was again detected whose dissociation
constant and maximal binding were 79 nmol/L and 35.9
fmol/mg, respectively. Thus, Ang II increased the sensitivity
of the sodium pumps to ouabain '3-fold without changing
their number.
Figure 4 shows the effects of specific blockade of Ang II
type 1 and 2 receptors on 3H-ouabain binding and sodium
pump activity (as estimated by ouabain-sensitive 86Rb uptake). Ang II augmented 3H-ouabain binding and increased
sodium pump activity. In parallel incubations, Ang II stimulated (P,0.05) the secretions of aldosterone (basal,
0.8260.08 pmol z mg21 z 2 h21; Ang II, 5.6860.57 pmol z
mg21 z 2 h21) and EO (basal, 0.6460.04 pmol z mg21 z 2 h21;
January 1999 Part II
375
Figure 3. Concentration dependence of specific 3H-ouabain
binding to BAG cells and effect of Ang II. Cells were incubated
for 4 hours with various concentrations of 3H-ouabain as indicated in presence and absence of 10 nmol/L Ang II. Data are
means of 4 separate wells. Typical errors were ,3%. Inset,
Scatchard plot of data fit by iterative regression. Kd was
205614 and '7969 nmol/L for control and Ang II, respectively.
Bmax was 35.6 and 35.9 fmol/mg protein for control and Ang II,
respectively.
Ang II, 1.560.09 pmol z mg21 z 2 h21). Except for EO
secretion, all effects of Ang II were blocked by losartan. In
contrast, PD123319 specifically inhibited Ang II–stimulated
EO secretion. Neither losartan nor PD123319 affected the
measured parameters in the absence of Ang II (not shown).
Figure 4. Role of Ang II (AII in figure) type 1 and 2 receptors in
Ang II–stimulated ouabain-sensitive 86Rb1K uptake and
3
H-ouabain binding. Top, Cells were incubated with 86RbCl
(0.11 mmol/L) in buffer alone (basal) or containing Ang II (10
nmol/L), Ang II1losartan (Los, 10 mmol/L), or Ang II1PD123319
(2 mmol/L) with or without 0.1 mmol/L cold ouabain for 4 hours.
(Data with antagonists alone were not different from basal and
are not shown.) Data are mean6SEM of ouabain-sensitive
86
Rb1K flux from 3 culture wells. Bottom, Data from cells incubated 4 hours with 50 nmol/L 3H-ouabain. Similar conditions
as top.
376
Adrenocortical Sodium Pump
Discussion
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The present investigation was prompted by several observations. First, BAG cells were shown to secrete aldosterone and
EO.7,9,10 Second, EO was known to be a high-affinity and
specific inhibitor of the sodium potassium pump.2 Third,
many reports indicated that ouabain and other sodium pump
inhibitors may affect aldosterone secretion.8,13–19 Fourth, part
of the effect of Ang II on aldosterone secretion was suggested
to involve inhibition of adrenocortical sodium pumps.8 Fifth,
the kinetics of EO secretion implied autoinhibition, perhaps
mediated by sodium pumps.6 Sixth, the characteristics of the
sodium pumps in primary cell cultures of BAG cells used by
our laboratory and others were not known. Collectively, these
observations suggested that the secretion and subsequent
binding of EO to adrenocortical sodium pumps might have
multiple consequences. Accordingly, we investigated the
sodium pump of the BAG to evaluate its properties and
possible roles in the Ang II–stimulated secretions of EO and
aldosterone. Measurements of aldosterone secretion were
used for correlative and comparative purposes and to demonstrate the functional integrity of this secretory system.
Under basal conditions, both the time and concentration
dependence of 3H-ouabain binding were pseudolinear (Figure
1). This is consistent with the use of ligand concentrations
that are significantly less than the receptor affinity. More than
90% of the total binding was specific. The concentration
dependence of specific binding showed no evidence of
saturation even at 300 nmol/L. Changes in potassium ion
concentration within the physiological range reduce the
affinity for cardiac glycoside binding to the sodium pump.
The marked potassium ion sensitivity of the interaction of
3
H-ouabain with BAG cells and the weakened effect of
potassium at the highest concentration of 3H-ouabain used
show that specific binding is mediated exclusively by sodium
pumps. Further analysis of the relationship showed that the
competition between labeled and cold ouabain occurred at a
single class of binding sites whose apparent affinity for
ouabain and digoxin was surprisingly low (Figure 2). These
results, consistent with the temporal and concentration data in
Figure 1, have other implications. We suggested previously
that feedback inhibition of EO secretion may be mediated by
autoreceptors.6 The present results suggest that these autoreceptors are not sodium pumps. For example, the rate of EO
secretion falls to '10% of the initial rate as the concentration
of secreted EO rises into the range of 0.2 to 0.5 nmol/L.6 On
the basis of a binding affinity for ouabain of '200 nmol/L
and with equivalent potency assumed, an EO concentration of
0.5 nmol/L would bind and inhibit #0.25% of the bulk
sodium pumps. Another possibility is that a small number of
specialized pumps are involved. In some cell types, small
numbers of sodium pumps with high ouabain affinity are
clustered in functional microdomains over portions of the
sarcoplasmic reticulum. In vascular smooth muscle cells,
nanomolar concentrations of ouabain interact specifically
with these pumps to affect stored calcium, while leaving other
sodium pumps involved in the control of bulk cytosolic
sodium unaffected.20 If such specialized sodium pumps or
other types of ouabain receptors were present in BAG cells,
they would comprise ,5% of the specific binding sites
measured and would escape detection by our methods.
Another factor of relevance to BAG cell function is the
high density (5.453106) of sodium pumps per cell estimated
from Scatchard analysis (Figure 3). This is reminiscent of
tissues such as nerve, heart, and kidney in which the high
copy number typically is associated with large inward sodium
fluxes and high rates of aerobic metabolism. Moreover, it is
consistent with other reports that have noted high Na,KATPase activity in glomerulosa tissue.8,21
What is the possible significance of high pump turnover,
low affinity, and high pump densities? In other systems in
which a ligand may be present continuously, rapid receptor
turnover can serve as a means to alter signaling. The
concentration of EO leaving the dog adrenal is '0.75
nmol/L, roughly 4- to 5-fold higher than that entering the
gland.12 Moreover, the local intra-adrenal concentrations of
EO, in the vicinity of the secretory cells especially during
Ang II stimulation, may be 10-fold higher than those measured in the mixed venous effluent. Even under these conditions, our calculations suggest that #12% to 15% of the
available pumps could be occupied by EO. This level of
pump blockade would be expected to cause physiologically
relevant increases in cell sodium and stored calcium that
would augment signaling by Ang II and other stimulants
without toxicity. The affinity of the adrenocortical sodium
pump for cardiac glycosides in cows, coupled with a high
pump density, is poised for physiological regulation by EO
while forestalling the potential for autotoxicity in the adrenocortical environment.
Previous reports of the effects of Ang II on sodium pump
activity in adrenocortical cells are varied. In rat glomerulosa
cells and bovine adrenal cells, Ang II was thought to inhibit
sodium pumps.8,16 Exposure of cells to Ang II does not
appear to affect the Na,K-ATPase activity in isolated membranes.21,22 Consistent with this impression, our results
showed that Ang II, via Ang II type 1 receptors, increased
both the affinity of BAG sodium pumps for ouabain and their
transport activity (Figure 4) without upregulation or downregulation of surface membrane pump number (Figure 3).
The increase in pump activity in response to Ang II probably
originates from an increase in cell sodium,23 which is known
to enhance the formation of a phosphorylated intermediate of
the pump1 whose affinity for ouabain is heightened
simultaneously.
The increased EO secretion occurring at a time when the
ouabain affinity of the BAG cell sodium pump is increased,
along with the demand for heightened active transport, seems
paradoxical (Figures 3 and 4). One possibility is that intraadrenal EO might augment Ang II–stimulated aldosterone
secretion. In rat adrenal cells, aldosterone secretion was
increased during inhibition of sodium pumps by both Ang II
and ouabain.8 In other species, some studies reported inhibition,13–15,17 stimulation,24 or transient stimulation followed by
inhibition17,18 of aldosterone secretion in response to ouabain.
Studies that observed inhibition of aldosterone secretion have
in general used pharmacological concentrations of ouabain
that would inhibit .50% of the available sodium pumps.
These conditions are often lethal because the activity of the
Shah et al
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residual pumps cannot increase sufficiently to normalize
outward sodium flux and cell potassium.
Two recent sets of observations suggest that aldosterone
secretion can be augmented by concentrations of ouabain that
could inhibit no more than a small portion of the bulk sodium
pumps. In the rat, continuous infusions of ouabain for 5
weeks resulted in circulating plasma levels ranging from '1
to 5 nmol/L. Under these conditions, plasma aldosterone was
dose dependently elevated, whereas plasma renin activity was
unchanged.19 Thus, low concentrations of ouabain approaching the physiological range of EO provide a strong in vivo
stimulus to aldosterone secretion in the rat. A similar result,
albeit with pharmacological concentrations of ouabain, has
been observed with rat BAG cells in vitro.25 In addition,
Tamura et al26 have shown that ouabain at 10 nmol/L
increased basal aldosterone secretion 2- to 3-fold from bovine
adrenocortical cells in culture and enhanced Ang II–stimulated secretion. Assuming that their cells exhibited sodium
pumps whose characteristics were similar to those we observed, our calculations suggest that the binding of ouabain to
#15% of the available sodium pumps is sufficient to augment
aldosterone secretion significantly.
Elevated circulating levels of EO and aldosterone have
been described in '50% of hypertensive patients with surgically confirmed Conn’s syndrome.27 These observations,
along with those mentioned above and the accumulating
evidence that the secretions of EO and aldosterone are
regulated differently,7,10 raise the possibility that the hyperaldosteronism in some patients with Conn’s syndrome may
be secondary to a primary overproduction of EO. New classes
of agents that block ouabain and EO binding may distinguish
among these patients.28
Acknowledgment
This work was supported by the American Heart Association (Drs
Shah and Hamlyn) and in part by the National Institutes of Health.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
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Effects of Angiotensin ΙΙ on Sodium Potassium Pumps, Endogenous Ouabain, and
Aldosterone in Bovine Zona Glomerulosa Cells
Jui R. Shah, James Laredo, Bruce P. Hamilton and John M. Hamlyn
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Hypertension. 1999;33:373-377
doi: 10.1161/01.HYP.33.1.373
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