Nongenomic regulation of ENaC by aldosterone
Zhen-Hong
Zhou and
James K.
Bubien
Department of Physiology & Biophysics, University of Alabama at
Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
Aldosterone is
involved in salt and water homeostasis. The main effect is thought to
involve genomic mechanisms. However, the existence of plasma membrane
steroid receptors has been postulated. We used whole cell patch clamp
to test the hypothesis that epithelial sodium channels (ENaC) expressed
by renal collecting duct principal cells can be regulated
nongenomically by aldosterone. In freshly isolated principal cells from
rabbit, aldosterone (100 nM) rapidly (<2 min) increased ENaC sodium
current specifically. The aldosterone-activated current was completely
inhibited by amiloride. Aldosterone also activated ENaC in cells
treated with the mineralocorticoid receptor blocker spiranolactone.
Nongenomic activation was inhibited by inclusion of
S-adenosyl-L-homocysteine in the pipette
solution, which inhibits methylation reactions. Also, the nongenomic
activation required 2 mM ATP supplementation in the pipette solution.
Aldosterone did not activate any ENaC current in whole cell clamped rat
collecting duct principal cells. These functional studies are
consistent with aldosterone membrane binding studies, suggesting the
presence of a plasma membrane steroid receptor that affects cellular
processes by mechanisms unrelated to altered gene expression.
epithelial sodium channels; principal cells
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INTRODUCTION |
THE MINERALOCORTICOID
HORMONE ALDOSTERONE has the ability to increase the reabsorption
of salt and water. A part of this process appears to involve the
stimulation of amiloride-sensitive sodium channels in the cortical
collecting duct of the kidney. The classic mechanism for this
regulation is that aldosterone activates a cytosolic mineralocorticoid
receptor (20), which in turn has genomic effects resulting
in increased transcription of the genes that produce serum
glucocorticoid-regulated kinase and
Na+-K+-ATPase and epithelial sodium channel
(ENaC) subunits (1, 8, 10, 15, 29). A compelling reason
for suspecting a genomic mechanism of regulation is that in vitro, the
effects of aldosterone are not acute but, rather, take as long as
4 h to develop and can be blocked by inhibitors of transcription
and translation (7, 23, 26, 34). It is important to
consider that the vast majority of experiments to elucidate the
mechanism of action of aldosterone have been carried out on model
systems such as rat tissue and cells and A6 epithelial cells derived
from Xenopus laevis. It is possible that the mechanism of
action of aldosterone in other species, including humans, may be more
complex than in these model systems.
Recent evidence suggests that in species other than the rat,
aldosterone may produce acute effects at picomolar concentrations. Half-maximal effects have been observed at a concentration of 100 pM
(13, 14). For example, rapid aldosterone-mediated effects on cellular processes in human smooth muscle cells and human
lymphocytes have been observed, in the presence of spiranolactone, to
inhibit the mineralocorticoid receptor (9, 32). These
effects have also been observed in the presence of actinomycin D,
precluding the possibility of genomic effects (13, 14).
The direct demonstration of these aldosterone-induced effects led us to
the hypothesis that acute aldosterone-mediated ENaC activation by
nongenomic mechanisms may be present in renal principal cells. To test
this hypothesis, we used whole cell patch-clamp analysis of freshly isolated renal principal cells from rat and rabbit kidneys. Identical experiments were also carried out on lymphocytes from humans, dogs,
rats, mice, and rabbits.
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METHODS |
Principal cell preparation.
Collecting ducts were manually dissected from sagittal slices obtained
from 50-g Sprague-Dawley rats and 0.5-kg New Zealand White rabbits. The
dissected collecting ducts were suspended in RPMI 1640 supplemented
with 1.5 mg/ml collagenase A (Boehringer Mannheim, Mannheim, Germany).
The collecting ducts were enzymatically digested for 1.5 h to
isolate individual cells. The digested cells were washed in serum-free
RPMI and placed in a perfusion chamber mounted on the stage of an
inverted microscope. Once the whole cell configuration was established,
the capacitances were balanced (rabbit principal cell average
capacitance = 12.1 ± 0.76, n = 37; rat
principal cell capacitance = 10.9 ± 0.68, n = 29), and initial current measurements were
made in unsupplemented RPMI ([Na+] = 133 mM;
[K+] = 5.3 mM; [Cl
] = 108.3 mM). The bath
solution was changed (by perfusion of the entire bath chamber) to RPMI
supplemented with aldosterone (100 nM), vasopressin (250 nM), amiloride
(2 µM), spiranolactone (1 µM), or various combinations of these
compounds. To block the cytosolic mineralocorticoid receptor, we
suspended some cells in RPMI supplemented with spiranolactone for
1 h before whole cell clamping was performed.
Preparation of lymphocytes.
Human and canine lymphocytes were isolated from peripheral blood
samples by differential centrifugation over Ficoll-Paque (Pharmacia
Biotech, Uppsala, Sweden). The cells were washed and resuspended in
serum-free RPMI. Subsequently, suspended cells were placed in the
perfusion chamber. Whole cell patch clamp and testing of aldosterone
were carried out with techniques and protocols identical to those
described for principal cells. Rabbit, rat, and mouse lymphocytes were
obtained by mincing sections of spleen and manually freeing the cells
by agitation. The cell suspensions were then centrifuged over
Ficoll-Paque and treated identically to the lymphocytes obtained from
peripheral blood. All procedures were carried out under the guidelines
for animal use and with Institutional Board Approval for Human Use.
Whole cell patch clamp.
Micropipettes were constructed using a Narashigi pp-83 two-stage
micropipette puller. The tips of these pipettes had an inner diameter
of ~0.3-0.5 µm and an outer diameter of 0.7-0.9 µm.
When filled with an electrolyte solution containing (in mM) 100 K-gluconate, 30 KCl, 10 NaCl, 20 HEPES, 0.5 EGTA, and 4 ATP as well as
<10 nM free Ca2+, pH 7.2, the electrical resistance of the
tip was 1-3 M
. The bath solution was serum-free RPMI-1640 cell
culture medium. The solutions accurately approximate the ionic
gradients across the cell membrane in vivo. Pipettes were mounted in a
holder and connected to the head stage of an Axon 200A patch-clamp
amplifier affixed to a three-dimensional micromanipulator system
attached to the microscope. The pipettes were abutted to the cells, and
slight suction was applied. Seal resistance was continuously monitored (model 300 Nicolet oscilloscope) by using 0.1-mV electrical pulses from
an electrical pulse generator. After seals were formed with resistances
in excess of 1 G
, another suction pulse was applied to form the
whole cell configuration by rupturing the membrane within the seal but
leaving the seal intact. Successful completion of this procedure
produced a sudden increase in capacitance with no change in seal
resistance. The magnitude of the capacitance increase is a direct
function of the membrane available to be voltage clamped (i.e., the
membrane area, and hence cell size). Typically, this capacitance was
between 5 and 10 pF for activated peripheral blood lymphocytes.
Previous measurements of transmembrane voltage showed that once the
whole cell configuration was obtained, the pipette solution and the
cellular interior equilibrated within 30 s. The cells were then
held at a membrane potential of
60 mV and clamped sequentially for
0.8 s each to membrane potentials of
160,
140,
120,
100,
80,
60,
40,
20, 0, 20, and 40 mV, returning to the holding potential of
60 mV for 0.8 s between each test voltage. This procedure provided voltages sufficient to measure inward sodium (at
more hyperpolarized potentials) and outward potassium (at more
depolarized potentials) currents. The currents were recorded digitally
and filed in real time. The entire procedure was performed with a DOS
Pentium computer modified for analog-to-digital (A/D) signals with
pCLAMP 6 software, with an A/D interface controlled by pCLAMP (Axon
Instruments, Sunnyvale, CA).
Single-channel patch clamp.
For inside-out patch recordings, the pipette and bath solution were 140 mM Na-gluconate and 20 mM HEPES, pH 7.4. For outside-out patches, the
solution was supplemented with 5 mM vanadate and 0.1 mM fluoride to
inhibit lipid peroxidation and reduce channel rundown. Single-channel
activity was filtered at 50 Hz and recorded on videotape. For amplitude
histogram analysis, the taped records were digitized (Fetchex, pCLAMP),
measured (Fetchan, pCLAMP), and analyzed (pSTAT, pCLAMP) by computer.
The current-voltage (I-V) relation was constructed from the
average currents obtained at a variety of positive and negative
voltages applied to patches from six different cells.
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RESULTS |
Nongenomic ENaC activation in rabbit principal cells.
Enzymatic dispersion of collecting ducts resulted in two
morphologically distinct cell types, large oval cells and smaller round
cells. The large oval cells have been shown previously to be principal
cells, whereas the smaller round cells were most likely intercalated
cells (2). Only the large round cells were whole cell
clamped. These cells were exposed to 100 nM aldosterone. Positive
identification of principal cells was determined by positive response
either to aldosterone or, if the cells failed to respond to
aldosterone, to vasopressin. No cells failed both tests. Upon entering
the bath, the aldosterone induced a specific activation of the inward
currents at hyperpolarized membrane potential clamp voltages. The
activated currents were subsequently inhibited completely with 2 µM
amiloride (in the continued presence of aldosterone), confirming that
the activated currents were amiloride sensitive. A typical experiment
is shown in Fig. 1. Figure
2 shows the average I-V
relations for the current activated by aldosterone and for the current
inhibited by amiloride after aldosterone activation. These
I-V relations show inward current up to +40 mV, the most positive membrane potential tested. This I-V relation is
expected for a highly selective sodium conductance since, under our
experimental conditions, the equilibrium potential for sodium was +67
mV. Thus the activated current is carried primarily by sodium. In a
separate set of experiments, rabbit principal cells were resuspended in RPMI supplemented with 1 µM spiranolactone for 1 h before use for whole cell patch clamp. Figure 3
shows that spiranolactone had no effect on the inward currents.
However, even with mineralocorticoid receptor inhibition, 100 nM
aldosterone specifically activated the inward currents. These
experiments indicate that the acute activation of ENaC by aldosterone
did not utilize the mineralocorticoid receptor and was therefore
nongenomic. Aldosterone also acutely activates ENaC current in human
lymphocytes. These cells were used to confirm that the effect of
increased inward current was not caused by translation of ENaC. To
inhibit protein translation, we incubated human lymphocytes in 10 µg/ml cyclohexamide (33) and subsequently whole cell
clamped them. In cyclohexamide-treated cells, aldosterone acutely
activated ENaC current within seconds of exposure to the cells (Fig.
4), supporting the hypothesis that acute
ENaC activation by aldosterone did not involve genomic mechanisms.

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Fig. 1.
Whole cell current records from a freshly isolated rabbit
renal principal cell. Top: basal current record. The
increased inward currents in response to aldosterone
(middle) were completely inhibited by amiloride in the
continued presence of aldosterone (bottom). Typically,
activation of the inward sodium currents occurred within 1 min after
aldosterone entered the bath.
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Fig. 2.
Average current-voltage relations for the whole cell
current activated by aldosterone and inhibited by amiloride. These
current-voltage relations go to zero current at an equilibrium
potential for sodium that is determined by the sodium gradient across
the membrane, indicating the high sodium selectivity of the activated
and inhibited current.
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Fig. 3.
Preincubation of rabbit principal cells with the
mineralocorticoid receptor inhibitor spiranolactone failed completely
to inhibit the aldosterone-induced activation of the inward sodium
currents. Thus the acute epithelial sodium channel (ENaC) activation
observed in these experiments was independent of the mineralocorticoid
receptor.
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Fig. 4.
Whole cell currents obtained from a human lymphocyte pretreated
with 10 µg/ml cyclohexamide for 90 min to inhibit protein
transcription. These records show that inhibition of protein
transcription has no effect on the acute activation of inward current
by 100 nM aldosterone. This finding supports the hypothesis that the
acute activation of these currents does not involve genomic
mechanisms.
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Aldosterone does not acutely activate ENaC in rat principal cells.
In contrast to the findings in the rabbit principal cells, aldosterone
had no acute effect on principal cells isolated from the collecting
ducts of rats. Figure 5 shows typical
records from a whole cell- clamped rat principal cell, where procedures
used were identical to those used on the rabbit principal cells.
Because aldosterone had no effect, the expression of ENaC and the cell type (principal cell) were confirmed in every experiment by subsequent acute, specific activation of the amiloride-sensitive inward currents by 250 nM vasopressin (Fig. 5, bottom). The same protocol
was carried out on rabbit principal cells (Fig.
6). Once ENaC was activated by
aldosterone, subsequent stimulation with vasopressin did not activate
the inward currents any further, i.e., there was no synergistic effect
of the two ENaC agonists, implying that they activated the same set of
channels. For most experiments, vasopressin was added after aldosterone
was washed out of the bath. During short-term experiments (<10 min),
currents did not inactivate upon washout of aldosterone. Thus, to test
for synergy, vasopressin was superfused after aldosterone washout. Some
experiments were performed with both ENaC agonists present (not shown),
with identical results.

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Fig. 5.
Current records from a whole cell-clamped rat renal principal cell.
In 6 of 6 rat principal cell whole cell preparations, aldosterone
failed completely to activate any current. However, in each cell,
vasopressin activated the inward sodium currents, and these currents
were completely inhibited by amiloride.
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Fig. 6.
In
rabbit principal cells, aldosterone activated the inward current. When
activation was followed by vasopressin, no additional current was
activated. All of the activated current was inhibited by amiloride.
Thus there was no apparent synergy between these sodium channel
agonists, suggesting the same channels could be activated independently
by either aldosterone or vasopressin.
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Figure 7 shows the average current
activated by these ENaC agonists measured at the equilibrium potential
for potassium. At this potential (
80 mV) there can be no potassium
current; thus all of the current activated by aldosterone or
vasopressin must be sodium current. In the absence of aldosterone, or
if aldosterone activation was inhibited, vasopressin activated the
inward currents. Comparison of the average currents resulting from each
treatment was assessed statistically with an unpaired Student's
t-test. The mean current levels showed that for rat
principal cells, aldosterone did not significantly alter the current,
but vasopressin increased it significantly, while amiloride restored
the current to the basal level. For the rabbit principal cells,
aldosterone significantly increased the current, vasopressin had no
additional effect, and amiloride returned the conductance to the basal
level. Also, the basal conductance and the activated conductance in rat
and rabbit principal cells were not significantly different between the
species.

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Fig. 7.
Average whole cell current at the equilibrium potential
for potassium (EK; 80 mV) in rabbit and rat
cells. At this potential there can be no potassium current; thus the
current activated by each ENaC agonist can only be carried by sodium.
Also, the current recorded at 80 mV is within the physiological
range. Values represent means from 6 different cells that received all
of the treatments, i.e., aldosterone (Aldo), vasopressin (Vaso), and
amiloride (Amil). Current amplitudes are reported in pA.
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Nongenomic ENaC activation utilizes methylation.
We next tested the hypothesis that aldosterone-mediated nongenomic
activation of ENaC utilizes methylation rather than protein kinase
A-mediated phosphorylation to induce channel activation. For these
experiments, pipette solutions were supplemented with 100 µM
S-adenosyl-L-homocysteine (SAH). Once the whole
cell configuration was formed, the cells were given 5 min to ensure
that the SAH equilibrated with the cytosol. The compound is an
end-product inhibitor of methyl esterification and has been shown
previously to inhibit Na+ transport (23). When
SAH was included in the pipette solutions, acute ENaC activation by
aldosterone was completely prevented in rabbit principal cells.
However, in the same cells, vasopressin activated the ENaC
Na+ currents normally (Fig.
8). The average sodium conductance in these cells after each treatment is shown in Fig.
9. This finding implies that the
vasopressin response was independent of methyl esterification.

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Fig. 8.
Whole cell currents showing that inclusion of
S-adenosyl-L-homocysteine (SAH; 100 µM) in the
pipette solution completely prevents ENaC activation by aldosterone in
rabbit principal cells. However, in the same cell, vasopressin produced
ENaC activation. SAH inhibits methylation. Thus these experiments
suggest that methylation contributes to aldosterone-mediated nongenomic
activation of ENaC. The cells were allowed to equilibrate with the
pipette solution for 5 min before the cells were exposed to aldosterone
and the voltage-clamp protocols were run.
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Fig. 9.
There was no change in the average inward sodium
conductance in response to aldosterone when SAH was included in the
pipette solution (NSD, no significant difference; n = no. of cell preparations). However, in the same cells, vasopressin
induced a significant specific inward conductance increase
(P < 0.05). Conductance was calculated as the chord
between the average current measured between a clamp potential of 140
mV and the reversal potential for each cell.
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Nongenomic aldosterone-mediated ENaC activation requires ATP.
The present experiments extend those obtained from intact rabbit
cortical collecting ducts and help to resolve an apparent contradiction. A considerable delay (2-4 h) was observed when the
in vitro effect of aldosterone was measured using intact collecting duct segments (21, 26, 34). In the present study,
aldosterone-stimulated ENaC activation was observed within seconds. The
only obvious difference between the cited studies and the present study
was that we were able to control the cytosolic ATP concentration, whereas in intact collecting duct segments, the cellular ATP is controlled by cellular metabolism. Once these segments are dissected, it is possible that ATP becomes depleted. It has been shown previously that reduced ATP induces a loss of structural support for the plasma
membrane (11) and reduces phosphorylation in renal
epithelia (19). Thus we hypothesized that the difference
between the two preparations might be a difference in cytosolic ATP.
With a conventional whole cell configuration, the pipette solution is
contiguous with the cytosol and, in our experiments, routinely
supplemented with 4 mM ATP. With intact collecting ducts, it may not be
possible to maintain such a high level of cytosolic ATP concentration. If the thermodynamics of aldosterone-mediated signal transduction require high levels of ATP, these reactions may be lost if there is
even a slight reduction of ATP. To test this hypothesis, we examined
nongenomic ENaC activation by aldosterone, using four concentrations of
ATP in the pipette solutions (0, 0.5, 1, and 2 mM ATP). We found that
in the complete absence of ATP in the pipette solutions, aldosterone
failed completely to activate ENaC. The same result was observed when
the pipette solution contained 0.5 mM ATP. With 1 mM ATP, we observed
incomplete and intermittent ENaC activation by aldosterone. Full ENaC
activation was observed in rabbit principal cells only when the pipette
solutions were supplemented with a minimum of 2 mM ATP. Figure
10 shows representative whole cell
currents for three principal cells with different concentrations of ATP
in the pipette solutions. Responses of similar magnitude were obtained
when the pipette solutions contained 4 mM ATP (Figs. 1, 3, and 5),
implying that a saturating concentration of ATP for this signal
transduction was ~2 mM.

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Fig. 10.
Whole cell records from 3 rabbit principal cells showing that
aldosterone fails to activate the inward sodium current in the absence
of ATP. These titrations of the pipette ATP concentration suggest that
2 mM ATP is required for full activation of ENaC by aldosterone.
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To test the hypothesis that ATP hydrolysis was required in this signal
transduction pathway, we supplemented the pipette solution with 10 mM
5'-adenylylimidodiphosphate (AMP-PNP, a nonhydrolyzable ATP analog).
With AMP-PNP in the pipette solution, aldosterone (100 nM) failed to
activate any inward current (Fig. 11).
To confirm that the aldosterone maintained its potency, we whole cell
clamped an additional three cells using a pipette solution supplemented with 4 mM ATP, and the cells were treated with the same 100 nM aldosterone solution. With 4 mM ATP in the pipette solution, inward currents were activated within seconds. These findings indicate that
hydrolysis, and not simple occupation of ATP binding sites, is required
for aldosterone stimulated activation of ENaC.

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Fig. 11.
Whole cell records showing that
when the pipette solution was supplemented with 10 mM
5'-adenylylimidodiphosphate (AMP-PNP, a nonhydrolyzable ATP
analog), aldosterone failed completely to activate acutely any inward
current. Subsequent control experiments with an ATP-supplemented
pipette solution were performed to ensure the potency of the
aldosterone solution and to eliminate the possibility that impotent
aldosterone resulted in the inability to activate the currents.
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Aldosterone-activated lymphocyte sodium currents.
We have previously demonstrated that lymphocytes express an
amiloride-sensitive sodium conductance that is abnormal in lymphocytes from individuals with Liddle's disease (4, 5, 17).
Lymphocytes can be easily isolated from a small blood sample from any
mammalian species. Aldosterone has been shown to bind to the plasma
membrane of lymphocytes with high affinity
(Ks = 0.1 nM) (13, 14, 32). Also, since aldosterone failed to activate any sodium current in rat
principal cells, lymphocytes provided a second cell type to confirm
this negative observation. For these reasons we tested the hypothesis
that aldosterone was able to activate specifically lymphocyte
amiloride-sensitive sodium currents in peripheral blood lymphocytes
from five species (human, dog, rabbit, rat, and mouse). Lymphocytes
from each of these species were whole cell clamped under conditions
identical to those used to whole cell clamp renal principal cells.
After the basal current levels were established, the cells were
superfused with 100 nM aldosterone. Subsequently, the cells were
superfused with 40 µM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic
monophosphate (8-CPT-cAMP). Figure 12
shows the results obtained from lymphocytes of each species. We found
that 100 nM aldosterone activated specifically the inward sodium
conductance in lymphocytes from humans, dogs, and rabbits. These
currents were inhibited completely by 2 µM amiloride (not shown), the
same as when they are activated by 8-CPT-cAMP, cholera toxin, or
pertussis toxin (3-6). However, just as was found in
rat principal cells, aldosterone failed completely to activate any
currents in rat and mouse lymphocytes. This finding was not due to the
lack of sodium channel expression, because 8-CPT-cAMP activated sodium currents in each rat and mouse whole cell-clamped lymphocyte. This
observation confirmed the negative effect of aldosterone on rat
principal cells. In human, dog, and rabbit lymphocytes, after sodium
current activation by aldosterone, 8-CPT-cAMP inhibited the activated
currents. This finding was similar to findings previously reported showing that pertussis toxin and cholera toxin each activated the sodium conductance alone but that, when one was followed by the
other, the conductance was inhibited (5).

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Fig. 12.
Whole cell clamps identical to those performed on renal
principal cells were performed on lymphocytes from 5 species. These
records show that aldosterone activated the inward sodium currents in
lymphocytes from humans, rabbits, and dogs but failed to activate these
currents in lymphocytes from rats and mice. 8-CPT-cAMP,
8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate.
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Aldosterone modifies single-channel characteristics.
The "ragged" kinetics of the whole cell currents in principal cells
and lymphocytes has generated some concern about the identity of the
channels that are activated by aldosterone. To address this concern, we
examined single-channel characteristics of the sodium conductors
expressed by human lymphocytes. We have recently demonstrated the
expression of human ENaC (hENaC) mRNA by RT-PCR and direct sequencing
of the PCR products by human lymphocytes. To determine the
single-channel conductance, we formed inside-out patches (Fig.
13, top) were formed in
symmetrical Na-gluconate solutions (140 mM). The patches were
clamped to potentials ranging from
80 to +80 mV. The current
amplitudes were determined by either direct measurement of individual
openings or amplitude histogram analysis (pCLAMP) (Fig. 13,
middle right). From the slope of the I-V relation
(Fig. 13, middle left), it was found that the single-channel
conductance was 8.4 ± 1.1 pS. This conductance is the same as the
single-channel conductance reported for hENaC (
,
,
-subunits)
expressed in Xenopus oocytes (16, 28).

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Fig. 13.
Top: records showing single-channel activity in an
inside-out patch from an untreated lymphocyte plasma membrane.
Middle left: average currents from similar records observed
in patches from 6 cells were used to estimate the slope conductance
(G) from the current-voltage relation. I/O, inside-out.
Middle right: amplitude histograms constructed from
all-points histograms were used to confirm the accuracy of measurements
made on individual channel transitions. Bottom: records
obtained from an outside-out patch showing that a submaximal
concentration of amiloride (100 nM) shortens the transition times but
does not alter the single-channel conductance.
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To test the amiloride sensitivity of the single channels, we formed
outside-out patches, because amiloride interacts with the outside
surface of the channels. We experienced the "rundown" phenomena
typical of ENaC in the inside-out patch experiments. To inhibit lipid
peroxidation and delay channel rundown, we added vanadate (5 mM) and
fluoride (0.1 mM) to the Na-gluconate pipette and bath solutions.
Outside-out patches had channels with the same conductance and with the
long open and closed times observed in inside-out patches, typical of
ENaC. When the bath solution was supplemented with 100 nM amiloride,
the open-closed transition times were shortened (Fig. 13,
bottom) and channel open probability (NPo) was reduced by 60% from 1.97 to
0.77. This finding shows the incomplete inhibitory action of
amiloride on these channels at an amiloride concentration close to the
IC50 for amiloride and ENaC (75 nM). When the amiloride
concentration was increased to 2 µM, single-channel activity was
completely abolished.
When the cells were treated with 100 nM aldosterone, a somewhat
different channel behavior was observed. In the absence of aldosterone,
the frequency of encountering single channels was less than 1 in 20 patches. After treatment with aldosterone, the frequency increased to
~8 in 10 patches. In outside-out patches, the unitary conductance was
unchanged, but the channels opened and closed in groups (Fig.
14, top). Single-channel
activity was virtually abolished when amiloride (2 µM, final
concentration) was added to the bath solution (Fig. 14,
bottom). We have demonstrated that this single-channel
behavior produces the "ragged" whole cell currents that are
characteristic of the sodium conductance of principal cells and
lymphocytes by reconstructing the whole cell currents from
single-channel recordings (6). This behavior of opening
and closing in groups should not be too surprising because it has been
shown by freeze fracture that ENaC aggregates into groups when
expressed in oocytes (12). Finally, it should be mentioned
that the vast majority of patches (>80%) were devoid of
single-channel activity in cells that were not pretreated with aldosterone. These single-channel findings are completely consistent with the whole cell findings and establish by biophysical
characteristics that the channels that are activated by aldosterone are
ENaC.

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Fig. 14.
Single-channel records obtained from an outside-out patch made
from a lymphocyte treated with 100 nM aldosterone. Top:
records showing that the single-channel conductance is not altered by
aldosterone treatment; rather, the channel openings appear in groups.
Bottom: records showing that 2 µM amiloride virtually
eliminates channel opening. These records show that single-channel
kinetics behavior accounts for the "ragged" appearance of the whole
cell currents induced by aldosterone at hyperpolarized membrane
potentials.
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DISCUSSION |
The direct electrophysiological findings from these
studies address three major issues concerning the role of aldosterone in the regulation of sodium currents by principal cells. First, the
findings demonstrate directly an acute, nongenomic activation of an
amiloride-sensitive sodium conductance (ENaC) in renal principal cells
of the rabbit. In contrast, the findings also show that aldosterone
fails to activate any current in principal cells of the rat. Because
the kidneys of both species are used extensively to study renal salt
and water regulation, this difference has wide-ranging implications for
our understanding of the role of aldosterone in controlling sodium
reabsorption in the collecting duct. For example, on the basis of
numerous previous studies indicating that aldosterone increases
expression of sodium channels by interaction with the cytosolic
mineralocorticoid receptor (1, 17, 26), inhibition of this
receptor with spiranolactone has been a recognized treatment for
primary aldosteronism. However, since the experiments presented here
show that aldosterone has the ability to activate nascent channels in
the presence of spiranolactone, a more effective therapy may be the
combination of spiranolactone plus amiloride for cases of primary or
secondary aldosteronism where surgery is not warranted or cannot be performed.
We are aware of previous experiments utilizing rabbit cortical
collecting ducts that showed delayed effects of aldosterone on sodium
conductance but that failed to show the immediate effect demonstrated in our whole cell experiments (7, 27, 34). Because some of the experiments measured transepithelial potential (TEP) and could demonstrate an acute change in TEP induced by vasopressin but not by aldosterone, the findings suggested that aldosterone did not produce an acute ENaC activation in rabbit cortical
collecting ducts, contrary to the whole cell clamp findings presented
in this paper. We hypothesized that by resupplying cytosolic ATP with
the use of ATP-supplemented pipette solutions, we were able to replete
ATP to a concentration that was apparently higher than the ATP
concentration in intact cortical collecting ducts in vitro.
Also, these findings support the hypothesis that the depleted ATP was
essential for signal transduction between aldosterone and ENaC to
produce nongenomic ENaC activation. We found that in the absence of
pipette ATP, we obtained the same result as has been reported for
intact collecting ducts, i.e., no effect of aldosterone but an acute
increase in ENaC current by vasopressin. By titrating back the pipette
ATP, we were able to establish that for full aldosterone-mediated acute
ENaC activation, a minimum concentration of 2 mM ATP was required.
One question that remains is, how can vasopressin or 8-CPT-cAMP
activate ENaC current in the absence of pipette solution ATP? The only
established intracellular effect of cAMP that we are aware of is to
bind to the regulatory subunit of protein kinase A, thereby inducing
activity of the catalytic subunit. The activated catalytic subunit, in
turn, catalyzes phosphorylation of a variety of substrates. This
reaction requires ATP. We speculate that there must be separate pools
of ATP, one that becomes depleted and one that does not. Our findings
indicate that aldosterone-stimulated signal transduction for ENaC
regulation requires ATP from the depletable pool but that
vasopressin-stimulated signal transduction for ENaC activation utilizes
ATP from a pool that is not depleted. This line of reasoning reconciles
the difference between the whole cell clamp findings and the findings
obtained in intact collecting ducts. In intact tubules the in vitro
metabolism may not be robust (due to reduced oxygen delivery) enough to
continuously provide the cellular concentrations of ATP that are
required for aldosterone-mediated ENaC regulation.
Previous studies have shown that SAH inhibits substrate methylation
(24, 25, 30, 31). In the present study aldosterone failed
to activate ENaC currents when the pipette solution was supplemented
with SAH. Thus methylation appears to play a role in the signal
transduction pathway between aldosterone and ENaC for nongenomic
signaling. This finding is consistent with the findings of others who
also suggest that methylation reactions regulate ENaC. For example,
with the use of purified ENaC protein subunits, it has been
demonstrated directly that carboxymethylation of the
-subunit alters
the biophysical properties of ENaC (18, 22). It also has
been shown that isoprenlycysteine-O-carboxylmethyl transferase regulates aldosterone-sensitive sodium reabsorption (31). While the findings of the present study do not
identify the specific components of the signal transduction pathway,
they are consistent with these previous findings in suggesting that aldosterone-mediated nongenomic ENaC regulation utilizes methylation as
one component of the signal transduction pathway.
Another interesting finding in the present study is that in
lymphocytes, aldosterone-activated sodium currents were inhibited by
subsequent exposure to 8-CPT-cAMP. The same phenomenon was observed
when lymphocyte sodium currents were activated by using pertussis toxin
to activate the channels initially (5). Because pertussis
toxin ADP-ribosylates G proteins, the implication of the present study
is that, at least in lymphocytes, a G protein may be involved in the
aldosterone-mediated regulation of the sodium conductance. Others also
have implicated guanine nucleotides and GTP binding proteins in
aldosterone-mediated regulation of ENaC (24) and have
demonstrated directly that treatment with aldosterone plus GTP induces
methylation in purified ENaC polypeptides from A6 epithelia
(25).
The observation that the amiloride-sensitive sodium conductance of
lymphocytes can be activated by aldosterone is important for a number
of reasons. First, the finding implies that lymphocytes are affected
when circulating aldosterone levels rise, such as during periods of
salt deprivation. Lymphocyte sodium conductance can be activated by
norepinephrine via
1-adrenergic receptors (3). The findings of the experiments described here
suggest that another receptor (i.e., a plasma membrane steroid
receptor) can also regulate ENaC by a distinct and independent signal
transduction pathway. Why two independent pathways have evolved for the
acute regulation of ENaC is not clear. Rats do not appear to express a
signal transduction pathway for acute aldosterone-mediated ENaC activation, and yet they have no apparent deficiencies in renal function. Rats may not need acute ENaC activation to the extent that
rabbits, dogs, and humans do. Alternatively, the presence of both
pathways may provide more precise regulation of the sodium-retentive mechanisms in rabbits and humans. Whatever the ultimate explanation for
this difference, it remains important to recognize that aldosterone induces nongenomic activation of principal cells from rabbits but does
not do so in principal cells from rats.
In addition to the rat-rabbit differences and to the direct stimulation
of ENaC in the absence of mineralocorticoid receptor involvement and
its role in blood pressure regulation is the possibility that this
mechanism may be involved in other systemic pathophysiology associated
with aldosteronism, such as vascular damage, and in cardiac
pathophysiology. These experiments show that lymphocytes are sensitive
to aldosterone. Thus, if the plasma level of aldosterone is increased,
there is a possibility that the steroid can provoke an unwarranted
immunological response. It is well known that glucocorticoids are
immunosuppressive. We show here that a mineralocorticoid has agonistic
effects that mimic the actions of cytokines. Thus the basic findings
described here may have even broader physiological implications than we
currently understand.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Janos Peti-Peterdi and P. Darwin Bell for providing
renal tubules and Drs. Dale J. Benos and James A Schafer for critical
input into these studies.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant RO1-DK-52789 (J. K. Bubien). J. K. Bubien is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: J. K. Bubien, Dept. of Physiology and Biophysics, 876 McCallum Bldg., Univ. of Alabama at Birmingham, Birmingham, Alabama 35294 (E-mail: bubien{at}uab.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 22 March 2001; accepted in final form 25 May 2001.
 |
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