Vol. 280, Issue 1, C119-C125, January 2001
Effects of extracellular calcium and potassium on the sodium
pump of rat adrenal glomerulosa cells
Douglas R.
Yingst1,
Joanne
Davis2, and
Rick
Schiebinger2
Departments of 1 Physiology and 2 Internal Medicine,
Wayne State University School of Medicine and the John D. Dingell
Veterans Medical Center, Detroit, Michigan 48201
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ABSTRACT |
Because the activity of the
sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we
determined how extracellular potassium (Ko) and calcium
affect sodium pump activity in rat adrenal glomerulosa cells. Sodium
pump activity was measured as ouabain-sensitive 86Rb uptake
in freshly dispersed cells containing 20 mM sodium as measured with
sodium-binding benzofluran isophthalate. Increasing Ko from
4 to 10 mM in the presence of 1.8 mM extracellular calcium (Cao) stimulated sodium pump activity up to 165% and
increased intracellular free calcium as measured with fura 2. Increasing Ko from 4 to 10 mM in the absence of
Cao stimulated the sodium pump ~30% and did not increase
intracellular free calcium concentration ([Ca2+]i). In some experiments, addition of
1.8 mM Cao in the presence of 4 mM Ko increased
[Ca2+]i above the levels observed in the
absence of Cao and stimulated the sodium pump up to 100%.
Ca-dependent stimulation of the sodium pump by Ko and
Cao was inhibited by isradipine (10 µM), a blocker of L-
and T-type calcium channels, by compound 48/80 (40 µg/ml) and
calmidizolium (10 µM), which inhibits calmodulin (CaM), and by KN-62
(10 µM), which blocks some forms of Ca/CaM kinase II (CaMKII).
Staurosporine (1 µM), which effectively blocks most forms of protein
kinase C, had no effect. In the presence of A-23187, a calcium
ionophore, the addition of 0.1 mM Cao increased
[Ca2+]i to the level observed in the presence
of 10 mM Ko and 1.8 mM Cao and stimulated the
sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM
Cao was not reduced by isradipine but was blocked by KN-62.
Thus, under the conditions that Ko stimulates aldosterone
secretion, it stimulates the sodium pump by two mechanisms: direct
binding to the pump and by increasing calcium influx, which is
dependent on Cao. The resulting increase in
[Ca2+]i may stimulate the sodium pump by
activating CaM and/or CaMKII.
ouabain; signaling; Na-K-ATPase; calmodulin; calcium- and
calmodulin-dependent kinase II; aldosterone
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INTRODUCTION |
INCREASING EXTRACELLULAR
POTASSIUM (Ko) has been thought to stimulate
aldosterone secretion by depolarizing the membrane potential, which
opens voltage-dependent calcium channels and leads to an influx of
extracellular calcium (Cao; see Refs. 5,
6, 9, 26, 29). The
depolarizing effect of Ko is in large part due to a
decrease in the equilibrium potential for potassium (27). However, increasing Ko over the range that stimulates
aldosterone secretion should also stimulate the sodium pump by
increasing the amount of Ko that binds to the pump
(14). Because sodium pump activity is ultimately
responsible for most of the resting membrane potential, the ability of
Ko to regulate aldosterone secretion could in part be due
to its effect on sodium pump activity. In fact, it has been clearly
shown that changes in sodium pump activity can both stimulate and
inhibit aldosterone secretion, depending on the species studied and the
experimental conditions (4, 16, 31, 35).
The addition of physiological concentrations of Cao
stimulates the sodium pump even at normal levels of Ko
(16). The mechanism of this stimulation is not yet known
but could be due to an increase in intracellular free calcium
(Cai2+), which in some cells has been shown to activate
sodium pump activity (1, 23, 24, 34). However, if this is
the case, then one also might expect Ko to stimulate the
sodium pump by increasing calcium influx through voltage-dependent
calcium channels. Such an effect has not yet been reported.
Thus, in this study, we have examined how Cao and
Ko affect the activity of the sodium pump under conditions
that they stimulate the secretion of aldosterone. These studies will
help determine how aldosterone secretion may be affected by changes in
the activity of the sodium pump and will help define mechanisms that
regulate sodium pump activity during cell activation.
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MATERIALS AND METHODS |
A-23187, KN-62, calmidazolium, compound 48/80,
gramicidin A, and monensin were purchased from Calbiochem (San Diego,
CA). 86Rb was purchased from NEN Research Products (Boston,
MA). Isradipine was a gift from Sandoz Pharmaceuticals (East Hanover, NJ).
A stock solution of 500 µM monensin was prepared by adding monensin
to medium 199 in 0.5% DMSO at 37°C while vortexing. The solution was
maintained at 37°C and alternately was shaken and vortexed until the
monensin was dissolved. KN-62 and isradipine were dissolved in DMSO.
Adrenal cell preparation.
Adrenal capsules, containing the zona glomerulosa, were removed from
female Sprague-Dawley rats weighing 200-224 g and collagenase dispersed as previously described (7) in medium 199 (GIBCO, Grand Island, NY) containing modified Earle's salts (130 mM
NaCl, 4.0 mM KCl, 1.8 mM CaCl2, and 0.8 mM
MgCl2), 10 mM HEPES, sodium salt (pH 7.4), 0.2% BSA, and
no bicarbonate. After dispersion, the cells were incubated for 2 h
to allow for recovery. Cells were continuously gassed with 100%
O2.
In experiments where Cao was either 0 or 1.8 mM (see Figs.
3, 6, and 8), cells were prepared as usual except that they were divided into two equal groups after dispersion. One group was subsequently washed two times and resuspended in medium 199 without added calcium, and the other was suspended in normal medium 199 containing 1.8 mM CaCl2. Soon thereafter, the cells were
added to the assay, and the experiments were performed as usual. In the
experiments shown in Fig. 7, cells were washed two times in medium 199 without calcium and then were suspended in medium 199 containing 20 µM EGTA and either no added CaCl2 (0 µM free
Ca2+) or 120 µM CaCl2 (100 µM free
Ca2+). They were then immediately used to measure sodium
pump activity.
Sodium pump activity.
Sodium pump activity was measured as the difference in the uptake of
86Rb, a congener for potassium, in the presence and absence
of ouabain, a specific inhibitor of the sodium pump. Measurements were
carried out at 37°C over a 5-min period in medium 199 as previously
described (16). Quadruplicate samples were run in the
presence and absence of 1 mM ouabain. Experiments were performed in a
total volume of 150 µl containing 100,000 cells and from 1 to 2.5 µCi of 86Rb, depending on the experiment. Measurements
were carried out in a 96-well plate (cat. no. MADPN6550; Millipore)
gently agitated at constant intervals and maintained at 37°C on top
of a heated sand bath. Cells were added to medium 199, except where
noted, followed shortly thereafter by the addition of ouabain or an
equal volume of buffer (time 0). Monensin, a sodium
ionophore, was added at a final concentration of 10 µM at 20 min;
86Rb was added at 30 min. Fluxes were terminated 5 min
later by rapid filtration of the cells using a Millipore Multiscreen
Assay System followed by six washes (~300 µl/wash) with ice-cold
medium 199. The membranes (containing the washed cells) were then
punched out and separately placed in 0.5 ml of 1% SDS to which
scintillation fluid was then added. The filter blank in this assay was
<0.015% of the total counts.
KN-62 and isradipine were added to the assay 15 and 30 min before
86Rb, respectively. A-23187 was added 5 min before
86Rb. In the experiments when Ko was increased
from 4 to 10 mM, a small volume of concentrated KCl was added to the
normal medium 5 min before the addition of 86Rb. In
experiments where sodium pump activity was assayed at both 4 and 10 mM
Ko, ouabain-sensitive 86Rb uptake at 10 mM
Ko was multiplied by 2.5 to compensate for the difference
in the ratio of 86Rb to Ko.
Measurement of intracellular sodium.
Glomerulosa cells were prepared as described above and then were
incubated with shaking under oxygen at room temperature for 45 min in
medium 199 (minus phenol red) containing 5 µM of the acetoxymethyl ester of sodium-binding benzofluran
isophthalate (SBFI) and 0.03% pluronic following previously
developed procedures (17). The cells were then washed two
times by centrifugation and resuspended in the same medium without SBFI
and pluronic.
To calibrate the SBFI as a function of sodium, cells were loaded with
SBFI as described above. Cells from one preparation were then divided
into five equal groups and centrifuged, and each group was resuspended
separately in one of five solutions, each containing different
concentrations of sodium. Cells were then centrifuged again and
resuspended in the same solution. The five solutions contained 0, 10, 20, 30, and 40 mM NaCl and the appropriate concentration of choline
chloride so that the sum of the NaCl and choline chloride equaled 82 mM. In addition, all of the solutions contained a final concentration
of 50 mM KCl, 1.8 mM CaCl2, 1 mM MgSO4, 1 mM
KH2PO4, 5 mM glucose, 2 mM
L-glutamine, 10 mM HEPES, and 4 µM gramicidin. The
gramicidin was included to equilibrate sodium and potassium
concentrations across the plasma membrane (17). The
fluorescence of each set of cells was then measured as shown in Fig.
1A, and the concentration of sodium was calculated as
previously described (33).
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Fig. 1.
A: effect of extracellular sodium on the fluorescence of
sodium-binding benzofluran isophthalate (SBFI) in adrenal glomerulosa
cells in the presence of gramicidin to equilibrate sodium and
potassium. B: fluorescence of SBFI in adrenal glomerulosa
cells suspended in normal medium 199 used to measure sodium pump
activity. The signal shown in B is the mean of 4 measurements made on 4 separate preparations of cells.
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Measurement of
[Ca2+]i.
Glomerulosa cells were prepared as described above and then were
incubated with shaking under oxygen at 37°C for 45 min in medium 199 (minus phenol red) containing 3 µM of the acetoxymethyl ester of fura
2. The cells were then centrifuged and resuspended in the same medium
without fura 2 and were incubated with shaking under oxygen at 37°C
for 30 min. The cells were then centrifuged and resuspended in medium
199 made to contain no added CaCl2. The cells were then
centrifuged again. The next step depended on the type of experiment
that was performed. For the experiment shown in Fig. 4A, the
cells were resuspended in medium 199 containing 1.8 mM
CaCl2 and 10 µM monensin. Immediately thereafter, the
cells were put in a cuvette, and the change in fluorescence was
recorded as a function of time. For the experiment shown in Fig.
4B, the cells were suspended in medium 199 containing no
added CaCl2. The suspension of cells was then added to a
cuvette, and the measurement of fluorescence began. As the fluorescence
was measured, small volumes of concentrated KCl, EGTA, A-23187, and
CaCl2 were added to the cuvette as shown (Fig. 4B). The
concentration of [Ca2+]i was calculated as
previously described (15).
Measurements of fluorescence.
Fluorescence was measured using a dual excitation monochrometer
spectrofluormeter (SPEX Fluorolog 1680; Spex Industries, Edison, NJ).
The measurements were carried out at 37°C in a stirred cuvette containing 200,000 cells/ml. The fluorescence of the SBFI was excited
at 340 and 385 nm and was measured at 500 nm (17); the fluorescence of fura 2 was excited at 340 and 380 nm and was measured at 505 nm (15).
Statistics.
Each data point is the mean and SE of quadruplicate samples of an
individual experiment. Each experiment is representative of at least
three similar experiments. The data were analyzed by one-way ANOVA.
Groups were compared using a Bonferroni multiple-comparisons test.
Values of P 
0.05 were considered statistically significant.
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RESULTS |
Intracellular sodium.
To measure the concentration of intracellular sodium in adrenal
glomerulosa cells, the response of incorporated SBFI was first measured
as a function of sodium (Fig.
1A). This calibration
indicated that the initial sodium concentration in freshly dispersed
cells suspended in medium 199 was 20 ± 3.5 (SD) mM sodium (Fig.
1B). This value is close to the value of 23.9 ± 1.8 mM
measured by others in freshly dispersed cells and much less than the
value of 48.5 ± 5.5 mM in cells activated by ANG II
(33). Cultured glomerulosa cells have a lower sodium
concentration of 9.2 ± 3.5 mM, perhaps due to recovery from
deleterious treatment during enzymatic dispersion (33).
Adding monensin to freshly dispersed cells only modestly increased the
intracellular sodium (Fig. 1B). Thus, under these
experimental conditions, intracellular sodium was well within the
physiological range for adrenal glomerulosa cells, even in the presence
of monensin.
Sodium pump assay conditions.
Sodium pump activity was measured in freshly dispersed cells in the
presence of monensin, as previously described (16). Monensin was added to assure that the sodium pump activity did not
change during the assay due to a reduction in intracellular sodium,
which is a primary pump substrate. Under these conditions, the uptake
of 86Rb was linear for at least the first 6 min in the presence and absence of ouabain (Fig.
2A). Therefore, the
ouabain-sensitive uptake, which is a measure of sodium pump activity,
was also linear during this period (Fig. 2B). In most of the
experiments, the ouabain-sensitive 86Rb uptake accounted
for >85% of the total uptake; in all experiments, it accounted for at
least 70% of the total influx.
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Fig. 2.
A: uptake of 86Rb in adrenal glomerulosa
cells in the presence and absence of 1 mM ouabain as a function of time
after 86Rb was added to the cell suspension. B:
ouabain-sensitive difference in the uptake of 86Rb as
measured in A. Solid lines are the least-squares fit to the
data at the 3- and 6-min time points extrapolated to time 0.
cpm, Counts/min.
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Effects of Ko and Cao on sodium pump
activity and [Ca2+]i.
In the absence of Cao, increasing Ko from 4 to
10 mM stimulated the sodium pump 30% (Fig.
3). This is the degree of activation one
would expect from increased binding of Ko to its
extracellular binding site on Na-K-ATPase (20). In the
presence of 1.8 mM Cao, increasing Ko from 4 to
10 mM stimulated sodium pump activity 165% (Fig. 3).
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Fig. 3.
Effect of increasing extracellular potassium
(Ko) on the sodium pump in the presence and absence of
extracellular calcium (Cao). [Ca2+
]o and [K+]o, extracellular
concentration of calcium and potassium, respectively.
*P0.05 relative to corresponding control.
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Increasing Ko from 4 to 10 mM under these experimental
conditions also increased [Ca2+]i (Fig.
4A). Under similar conditions,
others have found that elevating Ko increases
[Ca2+]i from 200 to 600 nM (28).
Thus Ko may stimulate the sodium pump more in the presence
of Cao because it increases
[Ca2+]i, which stimulates the sodium pump in
some cells (1, 23, 24, 34).
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Fig. 4.
Change in [Ca2+]i as a function of time.
A: cells were suspended in normal medium 199 containing 1.8 mM Cao at time 0. Ko was increased
from 4 to 10 mM as indicated. B: cells prepared at the same
time as those used in A were suspended in normal medium 199 containing no added Cao. Ko, EGTA, A-23187, and
Cao were added at the indicated final concentrations. Cells
used in both of these experiments were washed in medium 199 containing
no added Cao just before the above measurements.
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Adrenal glomerulosa cells contain both L- and T-type calcium channels,
which are both blocked by isradipine, a 1,4-dihdropyridine (2). Thus, to test if the increased activation of
Ko observed in the presence of Cao could be due
to the ability of Ko to increase [Ca2+]i through voltage-dependent calcium
channels, we tested the effect of Ko in the presence and
absence of isradipine. For these experiments, we chose a concentration
of isradipine that fully inhibits ouabain-induced aldosterone secretion
(35). Isradipine significantly reduced the ability of
elevated Ko to stimulate the sodium pump in the presence of
Cao (Fig. 5A). In
fact, in the presence of isradipine, increasing Ko from 4 to 10 mM stimulated the sodium pump 36% (Fig. 5A), an
amount similar to that observed in the absence of Cao (Fig.
3). These results suggest that Ko could have stimulated the
sodium pump in a Ca-dependent fashion by increasing Ca influx through
voltage-gated calcium channels.
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Fig. 5.
Effect of increasing Ko on the sodium pump in the
presence of Cao. A: with and without isradipine
(10 µM), a calcium channel blocker. B: with and without
KN-62 (10 µM), an inhibitor of calmodulin-dependent kinase II
(CaMKII). *P 0.05 relative to corresponding control.
#P 0.05 relative to each other.
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Increasing Cao from 0 to 1.8 mM stimulated sodium pump
activity up to twofold at 4 mM Ko in some preparations of
adrenal glomerulosa cells (Fig. 6). Lower
levels of stimulation were sometimes seen in other experiments (data
not shown). In preparations of cells where Cao stimulated
activity, the effect was blocked by isradipine (Fig. 6A),
suggesting that Cao stimulated the sodium pump by
increasing [Ca2+]i through voltage-dependent
calcium channels.
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Fig. 6.
Effect of Cao on sodium pump activity. A:
with and without isradipine (10 µM). B: with and
without KN-62 (10 µM). *P 0.05 relative to
corresponding control.
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To determine if the addition of 1.8 mM Cao in the presence
of medium containing 4 mM Ko could stimulate the sodium
pump by increasing [Ca2+]i, the concentration
of [Ca2+]i was measured in the presence and
absence of 1.8 mM Cao. Cells previously loaded with fura 2 and washed in the absence of Cao were suspended at
time 0 in normal medium 199 containing 4 mM Ko
and 1.8 mM Cao (Fig. 4A).
[Ca2+]i immediately began to increase and
then reached a steady state ~5 min later (Fig. 4A). At
this point, [Ca2+]i was 140 nM, considerably
above the level of 70 nM observed when the cells were suspended at 4 mM
Ko in the absence of Cao (Fig. 4B).
Thus cells suspended in normal media containing 1.8 mM Cao
and 4 mM Ko have higher levels of
[Ca2+]i than cells suspended in the same
medium containing no added calcium. These measurements are consistent
with the idea that the addition of Cao could
stimulate the sodium pump by increasing [Ca2+]i in cells suspended in medium 199 containing normal levels of 4 mM K and 1.8 mM Ca. Others have shown
that freshly dispersed glomerulosa cells suspended in solutions
containing normal concentrations of Ko and Cao
have resting levels of ~200 nM [Ca2+]i
(28). Given the results that correlate sodium pump
stimulation with small increases in [Ca2+]i
above resting levels, relatively small differences in resting [Ca2+]i could account for our observation
that the addition of 1.8 mM Cao stimulates the sodium pump
more in some preparations than in others.
Effects of pharmacological agents.
To help determine the mechanism by which increasing
[Ca2+]i might stimulate the sodium pump, we
tested the effects of pharmacological agents known to mediate the
effects of Cai2+ in adrenal glomerulosa cells.
Ca-dependent stimulation of the sodium pump by both Ko and
Cao was blocked by compound 48/80 (40 µg/ml; see Ref.
13) and calmidizolium (10 µM; see Ref. 21), which inhibit calmodulin (CaM; data not shown). These results prompted
us to test the effects of drugs that inhibit more specific CaM-dependent proteins. We found that KN-62, which inhibits
Ca/CaM-dependent kinase II (CaMKII; see Ref. 32), blocked
the Ca-dependent stimulation of the sodium pump by both Ko
(Fig. 5B) and Cao (Fig. 6B). These data suggest that the Ca-dependent stimulation of the sodium pump could
be mediated by a CaM-dependent mechanism, perhaps by a form of CaMKII.
Because KN-62 can inhibit calcium influx through voltage-dependent Ca
channels (22), one explanation for the ability of KN-62
and the other CaM antagonists to inhibit Ca-dependent stimulation of
the sodium pump is by limiting calcium influx. To test this hypothesis,
we measured the effect of Cao in the presence of A-23187, a
calcium ionophore that would increase [Ca2+]i
independent of calcium channels. In designing these experiments, we
first determined how much Cao had to be added in the
presence of 10 µM A-23187 to achieve the same
[Ca2+]i observed in the presence of 10 mM
Ko and 1.8 mM Cao. For instance, in the
experiment shown in Fig. 4B, adding 140 µM Cao
(in the presence of 10 µM EGTA) gave the same
[Ca2+]i as was observed in the presence of 10 mM Ko and 1.8 mM Cao (Fig. 4A). In
other experiments, we found that under the same conditions we had to
add as little as 100 µM Cao (data not shown). Using this
information, we tested the effect of KN-62 on the ability of
Cao to stimulate the sodium pump in the presence of
A-23187. Increasing Cao from 0 to 100 µM in the presence
of A-23187 stimulated the sodium pump over twofold (Fig.
7A). This Ca-dependent
stimulation was completely inhibited by KN-62 (Fig. 7A)
without blocking the influx of calcium (data not shown). These data
suggest that KN-62 blocks the Ca-dependent stimulation of the sodium
pump independent of its ability to inhibit calcium influx through
voltage-dependent calcium channels.
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Fig. 7.
Effect of Cao and KN-62 (10 µM) on sodium pump
activity in the presence of A-23187 (10 µM). A: experiment
done in the absence of isradipine. B: experiment done in the
presence of isradipine (10 µM). *P 0.05 relative to
corresponding control.
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Finally, to test if isradipine could be blocking Ca-dependent
stimulation of the sodium pump by inhibiting protein kinase C (8,
18), we tested if isradipine would block Ca-dependent stimulation of the sodium pump when Ca influx was mediated by A-23187.
The results show that isradipine had no effect on the ability of 100 µM Cao to stimulate the sodium pump in the presence of
A-23187, which is when calcium could enter the cells independent of
endogenous calcium channels (Fig. 7B). Likewise, isradipine had no effect on the ability of KN-62 to block the Ca-dependent stimulation seen in the presence of A-23187 (Fig. 7, A vs.
B). Furthermore, staurosporine, which at least partially
inhibits all known forms of protein kinase C (19, 25), had
no effect on the ability of Cao to stimulate the sodium
pump (Fig. 8). Thus it is unlikely that
isradipine was blocking the ability of calcium to stimulate the
sodium pump by inhibiting protein kinase C.
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Fig. 8.
Effect of Cao on sodium pump activity in the
presence and absence of staurosporine (1 µM). *P 0.05 relative to corresponding control.
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DISCUSSION |
Our results show for the first time how sodium pump activity is
affected during Ko-induced aldosterone secretion. This is significant, because the activity of the sodium pump has been repeatedly shown to alter agonist-stimulated aldosterone secretion. We
have shown that increasing Ko and Cao stimulate
the sodium pump and increase [Ca2+]i in the
range that stimulates aldosterone secretion. From our measurements, we
deduce that increasing [Ca2+]i from ~70 to
~300 nM stimulated the sodium pump approximately twofold.
Ko also stimulated the sodium pump ~30% in the absence of Cao, probably due to increased binding of Ko
to the sodium pump. Thus increasing Ko over the range that
stimulates aldosterone secretion activates the sodium pump by two
mechanisms: one that is Ca-dependent and the other that is not. It is
not yet known how the stimulation of the sodium pump by Ko
and [Ca2+]i affects the membrane potential
and alters the secretion of aldosterone. Nevertheless, it is
interesting to note that both of the physiological agents that
stimulate aldosterone secretion alter sodium pump activity: ANG II
inhibits (16) and Ko stimulates. Perhaps these
opposing effects may one day help explain the apparently conflicting
reports that inhibiting sodium pump activity can both stimulate and
inhibit aldosterone secretion, depending on the preparation and the
experimental conditions (4, 16, 31, 35).
Stimulation of the sodium pump by
Cai2+.
Our results confirm that Cao stimulates the sodium pump in
rat glomerulosa cells (16). Hajnoczky et al.
(16), however, concluded that Cao did not
stimulate by increasing [Ca2+]i because the
calcium ionophore ionomycin failed to stimulate sodium pump activity.
However, they added ionomycin in the presence of 1.2 mM
CaCl2 (16), which may have increased
[Ca2+]i beyond its stimulatory range. In
contrast, we observed stimulation when A-23187 was added at much lower
concentrations of Cao (Fig. 4B).
Mechanism of stimulation.
Because the stimulatory effects of Cao, Ko, and
Cai2+ were blocked with 48/80 and calmidazolium, which
inhibit CaM, and by KN-62, which inhibits CaMKII, we suggest that the
stimulatory effects of [Ca2+]i could be
mediated by CaMKII or a related kinase. CaMKII is present in bovine
glomerulosa cells and is activated by Cai2+ under
conditions that stimulate sodium pump activity (10). In
principle, Ca-CaM or Ca-CaMKII could enhance sodium pump activity directly via changes in phosphorylation or by altering how the pump
interacts with other proteins. For example, in guinea pig ventricular
myocytes, the relative level of [Ca2+]i
determines if stimulation of protein kinase A by isoproterenol either
inhibits or stimulates sodium pump activity (11, 12).
Ca-dependent stimulation of the sodium pump in adrenal glomerulosa
cells could be due to an increase in the affinity for sodium, as
previously observed in the rat proximal tubules (1),
because the measured concentration of intracellular sodium under our
experimental conditions (Fig. 1) was close to the concentration that
half-maximally stimulates the 
1-isoform of
Na-K- ATPase (3, 30). In this case, we have assumed that
1 is the predominant isoform in these cells based on the
concentration of ouabain required to inhibit activity
(16). It is unlikely that calcium is stimulating the sodium pump by increasing its affinity for Ko, as observed
in HeLa cells (24), because both 4 and 10 mM
Ko are well above the half-maximal constant for
Ko to bind to the sodium pump (20). In other
cells, changes in physiological levels of
[Ca2+]i can either stimulate or inhibit
sodium pump activity, depending on the relative expression of
regulatory proteins (23, 24, 34).
In conclusion, increasing Cao and/or Ko in the
presence of Cao stimulated the sodium pump by increasing
Cai2+. The mechanism by which Cai2+
stimulated the sodium pump may involve CaM and either CaMKII or a
related kinase. The concentrations of Ko and
Cai2+ that stimulated the sodium pump are in the same
range that stimulate aldosterone secretion. Therefore, it is likely
that the sodium pump is stimulated by Cai2+ during
Ko-stimulated aldosterone secretion. How Ca-dependent stimulation of the sodium pump alters aldosterone secretion is not yet
known but could involve changes in the membrane potential.
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ACKNOWLEDGEMENTS |
This work was supported by a grant from the American Heart
Association of Michigan, National Heart, Lung, and Blood Institute Grant HL-48885, Veterans Association Research Funds, and by the Vascular Biology Program of the Department of Internal Medicine at
Wayne State University.
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FOOTNOTES |
Address for reprint requests and other correspondence: D. R. Yingst, Dept. of Physiology, Wayne State Univ. School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201 (E-mail:
dyingst{at}med.wayne.edu).
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. Section 1734 solely to indicate this fact.
Received 21 May 1999; accepted in final form 29 August 2000.
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