Time-dependent stimulation by aldosterone of
blocker-sensitive ENaCs in A6 epithelia
Sandy I.
Helman1,
Xuehong
Liu1,
Kieron
Baldwin3,
Bonnie L.
Blazer-Yost2, and
Willem J.
Els3
1 Department of Molecular and
Integrative Physiology, University of Illinois at Urbana-Champaign,
Urbana, Illinois 61801;
2 Department of Biology, Indiana
University-Purdue University at Indianapolis and Veterans Affairs
Medical Center, Indianapolis, Indiana 46202; and
3 Department of Anatomy and Cell
Biology, University of Cape Town Medical School, Cape Town, South
Africa
 |
ABSTRACT |
To study and define the early time-dependent response (
6 h) of
blocker-sensitive epithelial Na+
channels (ENaCs) to stimulation of
Na+ transport by aldosterone, we
used a new modified method of blocker-induced noise analysis to
determine the changes of single-channel current (iNa) channel open probability
(Po), and
channel density
(NT) under
transient conditions of transport as measured by macroscopic short-circuit currents
(Isc). In three
groups of experiments in which spontaneous baseline rates of transport
averaged 1.06, 5.40, and 15.14 µA/cm2, stimulation of transport
occurred due to increase of blocker-sensitive channels.
NT varied
linearly over a 70-fold range of transport (0.5-35
µA/cm2). Relatively small and
slow time-dependent but aldosterone-independent decreases of
Po occurred
during control (10-20% over 2 h) and aldosterone experimental
periods (10-30% over 6 h). When the
Po of control and
aldosterone-treated tissues was examined over the 70-fold extended
range of Na+ transport,
Po was observed
to vary inversely with
Isc, falling from
~0.5 to ~0.15 at the highest rates of
Na+ transport or ~25% per
3-fold increase of transport. Because decreases of
Po from any
source cannot explain stimulation of transport by aldosterone, it is
concluded that the early time-dependent stimulation of
Na+ transport in A6 epithelia is
due exclusively to increase of apical membrane
NT.
electrophysiology; epithelial sodium channels; tissue culture; cortical collecting ducts; kidney; noise analysis; amiloride
 |
INTRODUCTION |
APICAL MEMBRANES OF A variety of tight epithelia
express epithelial Na+ channels
(ENaCs) that function in regulating the rate of
Na+ entry into the cells and its
subsequent transport through basolateral membranes. Aldosterone is
known to play a key role in regulation of baseline rates of transport
at both surfaces of the cells (Refs. 3, 5, 18, 20, 24, 27, 28 and
references therein), acting to modulate apical membrane transport by
way of change of the permeability to
Na+. To understand the underlying
mechanisms involved, it is necessary to distinguish between changes of
permeability due to changes of channel density
(NT) and
changes of channel open probability (Po), as
changes of either will lead to changes of the open channel density
(No = PoNT).
Aldosterone stimulates Na+
transport through at least two populations of channels (amiloride
sensitive and insensitive) in native tissues like frog skin and toad
urinary bladder and in cell-cultured A6 epithelia grown on permeable
supports. Greater than 95% of transport occurs through
blocker-sensitive channels (16). Regardless of the origin of the pool
of blocker-sensitive channels (activation of channels preexisting at
the apical membranes and/or vesicle trafficking of channels to
the membrane), it has been observed after chronic exposure to
aldosterone (~24-48 h) that the density of channels is increased
with no measurable change of
Po (2, 23). In
oocytes expressing ENaCs, aldosterone causes the appearance of channels
with long open and closed times (7, 8) with
Po similar to
those that have been observed by patch clamp of rat renal cortical
collecting ducts (23) and A6 epithelia (9, 19, 21, 22) and by noise
analysis of A6 epithelia (2, 13).
We have in the present set of experiments examined the early or initial
response of A6 epithelia to aldosterone during the first 6 h following
stimulation of transport by this steroid. Previous methods of noise
analysis were limited to experiments done under conditions in which
transport rates were stable (unchanged) for at least 30 min. We
modified these methods so that channel densities and
Po could be
measured noninvasively during transient changes of transport. Three
groups of A6 epithelia were studied, with spontaneous baseline rates of
Na+ transport averaging 1.06, 5.40, and 15.14 µA/cm2. The
results of our experiments indicated that, despite large differences in
baseline values of density of blocker-sensitive channels, stimulation
of transport during the first 6 h could be attributed almost entirely
to an increase of
NT with
relatively minor compensatory decreases of single-channel current and
channel Po.
Theoretical Considerations
Equilibrium distribution of channels.
Spontaneous gating of blocker-sensitive ENaCs between open and closed
states is sluggish, with mean open and closed times of several seconds.
When a blocker inhibits the open state of the channels (2, 14),
channels must redistribute among open, closed, and blocked states,
giving rise to time-dependent changes of open channel density and hence
Na+ entry into the cells. For a
three-state scheme with open-to-closed, closed-to-open, blocker on, and
blocker off rate coefficients,
,
,
kob, and
kbo,
respectively, and blocker concentration
B
|
(1)
|
the
equilibrium density of open channels at any
B
(NBo) relative to
the density of open channels in the absence of blocker
(No) is
|
(2)
|
(14),
where Po =
/(
+
) and the blocker equilibrium constant
KB = kbo/kob.
Because channels can be recruited from closed to open states, the
fractional inhibition of open channel density and hence
blocker-inhibitable transport is dependent on
Po. Under conditions in which single-channel currents in the absence of blocker
(iNa) and in
the presence of blocker
(iBNa) remain essentially constant, Eq. 2
can be rewritten as Eq. 3, since
macroscopic current in the absence of blocker
INa = iNaNo and macroscopic current in the presence of blocker
IBNa = iBNaNBo
|
(3)
|
Hence
Po can be
determined from the fractional inhibition of the blocker-sensitive
short-circuit current when
KB is known. Because this relationship is not restricted to increases of blocker concentration from zero to B, it can
be rewritten more generally for changes of solutions containing blocker
concentrations B1 and B2 as
|
(4)
|
Solving
for Po and using
the shortened notation
IB2/B1Na
to indicate the ratio of currents at
B2 with respect
to those at B1
gives
|
(5)
|
Kinetics of channel redistribution.
With ideal instantaneous step increases of
B, channels will redistribute between
closed, open, and blocked states with time constants determined by the
rate coefficients. For the blocker 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), for which
kob and
kbo average near
7 s
1 · µM
1
and 210 s
1, respectively
(Ref. 15; see also CDPC rate
coefficients), the density of open
channels will decrease promptly by 50% with a time constant near 2.5 ms when B is increased from 0 to
B = KB = 30 µM.
With
and
reflecting mean open and closed times of, for example,
3 s, open channel density will increase thereafter with a
time constant of ~1.5 s, as illustrated in Fig.
1A. The secondary long time constant for redistribution of the channels toward
equilibrium will depend on the absolute values of
and
, with the
final equilibrium value of
NBo determined by
the Po. For
Po of 0.1, 0.3, or 0.5 illustrated in Fig. 1A, the
fractional
NBo/No
at equilibrium expressed as percentages are 90.1, 76.9, and 66.7%, respectively. It may be emphasized, as indicated in Fig.
1B, that the time constants for
equilibration vary with (
+
)
1 and the
Po is determined
by
/(
+
). Thus
Po may appear to be constant as assessed from fractional changes of
NBo/No but may be associated with a range of relaxation times dictated by the
actual mean open and closed times of the channels. For this to occur,
however,
and
must change identically.

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Fig. 1.
Theoretical time rates of change of open channel density (expressed as
fractional inhibition of open channel density) according to a 3-state
model of closed, open, and blocked states in which blocker
[6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC)]
interacts only with open state of channel (2). Blocker on
(kob) and off
(kbo) rate
coefficients were assumed to be equal to 7 s 1 · µM 1
and 210 s 1, respectively,
where the blocker equilibrium constant
KB = 30 µM.
Time constants for redistribution of channels among closed, open, and
blocked states and equilibrium value of fractional inhibition depend on
open-to-closed ( ) and closed-to-open ( ) rate coefficients.
NBo and
No, open channel
density in presence and absence of blocker, respectively.
A: was assumed to be constant
(0.333 s 1; mean closed time
of 3 s) and values of were 0.333, 0.777, and 2.997 s 1 (mean open times of 3, 1.287, and 0.334 s, respectively), with channel open probabilities
(Po) of 0.5, 0.3, and 0.1, respectively. B: values
of and were chosen to give same
Po but variable
time constants for redistribution of channels between closed, open, and
blocked states. For epithelial Na+
channels with mean open and closed times of several seconds,
redistribution of channels to equilibrium requires ~15-20 s
following step increases of blocker concentration
(B).
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Because unstirred layers at the apical surface of the cells and
exchange of solutions within the experimental chambers prevent instantaneous changes of B at the
channel sites, time-dependent changes of
NBo will reflect
not only redistribution of channels between closed, open, and blocked
states but also the time constant for exchange of the apical solution (
chamber). For infinitely
well mixed chambers with
chamber of between 1 and 4 s,
illustrated in Fig.
2A, where
B increases exponentially from 10 to
30 µM, the equilibrium value of
NBo is the same,
although the transient approach to equilibrium may be complex (Fig.
2B). In practice, with chamber
volumes of ~0.6 ml (1) that are perfused continuously at flow rates
of ~4-6 ml/min, the time required for complete exchange would
fall into a range of roughly 20-40 s. Accordingly, our experiments
were limited to measurements of fractional inhibitions of
blocker-sensitive open channel densities and short-circuit currents at
times consistent with equilibrium redistributions of channels among
closed, open, and blocked states.

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Fig. 2.
A: theoretical calculations of
influence of chamber mixing and exchange of solutions on time rate of
increase of B as
B is increased from 10 to 30 µM at a
constant flow rate. It was assumed that chambers are mixed infinitely
well, with time constants for exchange of apical solution
( chamber) of 0, 1, 2, and 4 s. B: time rates of change of
fractional inhibition of open channel density were calculated for
channels with Po
of 0.3 and chamber of 0, 1, 2, and 4 s. N10o and
N30o, open channel
density in presence of 10 and 30 µM CDPC, respectively. At slower
perfusion rates relative to chamber volume, fractional inhibition of
open channel density decreases monotonically as
chamber becomes rate-limiting
factor that determines time required for redistribution of channels to
equilibrium at 30 µM CDPC. Unstirred layers at apical surface of
cells have not been taken into account in these calculations because
their contribution to chamber
is negligible relative to mixing and exchange time constants and
because achieving infinitely well mixed compartments is unattainable in
working with living tissues.
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Graphic relationship between fractional inhibition of transport and
the channel Po.
Illustrated in Fig. 3 is the relationship
between the fractional inhibition of blocker-sensitive
Na+ entry into the cells caused by
increasing B from 10 to 30 µM CDPC
at KB ranging
between 20 and 60 µM. At a
KB of 30 µM,
blocker-sensitive Na+ entry would
be inhibited by 15.4% if
Po is 0.3 (I30/10Na = 0.846). Because
short-circuit currents can be measured precisely and with high
resolution (<0.01 µA/cm2),
small changes of
Po can be
resolved.

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Fig. 3.
Relationships between fractional inhibition of macroscopic
blocker-sensitive short-circuit currents
(I30/10Na) and channel
Po at various
KB in response
to increases of CDPC from 10 to 30 µM.
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Our theory is predicated on the assumption that blockers interact
solely with the open state of the channel, with the expectation that
the concentration of blocker required to inhibit 50% of the macroscopic rate of transport
(Ktransport) is
greater than the KB = kbo/kob
for interaction of the blocker with the open state of the channel.
Consequently,
Ktransport = KB/Po.
We know from our own studies that this is the case for ENaCs in both
frog skin and A6 epithelia under a wide variety of transport rates and
conditions of study. To our knowledge, this is the general case,
without exception, when comparisons are made of macroscopic and
microscopic blocker inhibition constants. Consequently, for
Po that range between 0.5 and 0.1, the
Ktransport would
be two to five times greater than the
KB for blocker
interaction between open and blocked states of the channel, provided
that single-channel current and NT remain
constant.
 |
MATERIALS AND METHODS |
Cell culture.
Three groups of experiments (groups
1, 2,
and 3) were done, with
differences among groups in passage number, growth medium, and the
permeabilized substrates on which the tissues were grown. Cells in
group
1 at
passages
84-88
originated in B. L. Blazer-Yost's laboratory, where confluent tissues
were grown on Transwell tissue culture treated inserts (Tr-tct, Costar,
Cambridge, MA). Confluent monolayers were brought to Urbana, IL, for
the experimental part of the studies. Cells in
group
2 were purchased from the American Type Culture Collection at passage
69, subcultured, and used at passage
73 with tissue growth on Millicell HA
substrates (Millipore, Bedford, MA) in Urbana. Cells in
group
3 were obtained as a gift to W. J. Els
from W. Van Driessche, used at
passages
108 with tissue growth on Millicell HA
substrates, and studied in Cape Town, South Africa.
Growth and perfusion media.
The growth medium for group
1 experiments was a Dulbecco's
modified Eagle's medium (91-5055EC; GIBCO, Grand Island, NY) with penicillin (25 U/ml) and streptomycin (25 µg/ml; GIBCO); 10% calf serum (CELLect, iron-supplemented calf serum, ICN Biomedicals, Aurora,
OH) was added to this medium. Cells and tissues were maintained in a
humidified incubator at 28°C with air containing 5.0%
CO2. The tissues were studied on
days
14-26
after an overnight incubation in serum-free medium.
The growth medium for group
2 and group
3 experiments was a Dulbecco's modified Eagle's
medium (84-5022EC, GIBCO) with 4 mM HEPES, 25 U/ml penicillin, 25 µg/ml streptomycin (BioWhittaker, Walkersville, MD), and 10% fetal
bovine serum (Hyclone, Logan, UT). Cells and tissues were grown in the
presence of humidified air containing 1%
CO2 in an incubator at 28°C.
Electrical measurements and experimental protocol.
The methods of study with blocker-induced noise analysis were identical
to those described in detail previously (10, 11, 14), except for the
pulse protocol of exposure of the tissues to CDPC. After transfer to
perfusion chambers designed for noise analysis (1), the tissues were
short-circuited continuously for at least 1 h to allow the macroscopic
short-circuit currents (Isc) to
stabilize. The tissues were perfused with growth medium minus the fetal
bovine serum and antibiotics.
Each tissue served as its own control with 2-h control periods and 6-h
experimental periods during which the tissues were exposed to 2.7 µM
aldosterone. About 30 min before the beginning of the control periods,
10 µM CDPC (Aldrich Chemical, Milwaukee, WI) was added to the apical
perfusion solution; 10 µM CDPC caused an immediate small inhibition
of the Isc
followed by an autoregulatory return of the short-circuit current at 10 µM CDPC (I10sc) to the
original value of
Isc. During
control and experimental periods, the apical perfusion solution was
switched at intervals of 20 min to the same solution containing 30 µM
CDPC for pulse intervals of 3 min and returned thereafter to the 10 µM CDPC-containing solution. Values of
I10sc and the currents in the
presence of 30 µM CDPC
(I30sc) were recorded
continuously on an analog strip-chart recorder and digitally at
intervals of 10 s from digital meters before and during pulse inhibition of the short-circuit currents to assess the fractional inhibition of Na+ transport
(I30/10Na) after subtraction
of the amiloride-insensitive currents.
Noise analysis and blocker rate coefficients.
Regardless of Na+ channel blocker,
including CDPC, corner frequencies
( fc) of
induced current noise Lorentzians vary linearly with
B (15). Accordingly, the
kob and
kbo can be
determined from a two-point analysis, where
2
fc = kobB + kbo. In the
design of the pulse protocol presented here, it was convenient to
expose the tissues chronically to 10 µM CDPC, since
1) this concentration of CDPC gave
Lorentzians that could be analyzed reliably even at the lowest rates of
transport (<1 µA/cm2),
2) switching between 10 µM and a
single higher concentration of CDPC was sufficient to obtain all data
required for determination of
Po, and
3) the differences of
fc at 30 and 10 µM CDPC ( f 30c and f 10c, respectively)
were sufficient to determine the blocker rate coefficients while
minimally inhibiting the short-circuit current to assure that
single-channel currents remained practically constant at 10 and 30 µM CDPC (2).
Current noise measurements were made in pairs, at 10 µM CDPC before
each pulse and after ~60 s of exposure to 30 µM CDPC. It became
evident during these experiments (see
Blocker-dependent short-circuit
currents) that the autoregulatory
increases of short-circuit currents in response to blocker inhibition
of Na+ entry were delayed by
~60-90 s, so that the fractional inhibitions of transport could
be assessed before onset of autoregulatory changes of transport.
Because fc are
independent of short-circuit current magnitudes, pairs of
f 30c and
f 10c at each pulse interval
could be used to assess the time rates of change of the
blocker rate coefficients and thus
KB.
Current noise was amplified after being filtered at the Nyquist
frequency, digitized, and Fourier transformed to yield power density
spectra. Low-frequency plateaus
(S0) and
fc of the
blocker-induced Lorentzians were determined by nonlinear curve fitting
of the spectra to the Lorentzian,
"1/f " noise at the lower
frequencies and amplifier noise at the higher frequencies. The average
of 60 2-s sweeps of current noise gave Lorentzians with uncertainty of
fc of ±1 to
±2 Hz. The sequential measurements of
f 10c and
f 30c were fit by nonlinear
regression (TableCurve, Jandel Scientific) to smooth curves, thereby
filtering small uncertainties in estimation of the blocker rate
coefficients (see CDPC rate
coefficients). The
kob and
kbo were
calculated from the slopes and intercepts of the rate-concentration
plots (2
fc = kobB + kbo) using
the filtered f 10c and
f 30c at 10 and 30 µM CDPC,
respectively, and yielding
KB = kbo/kob.
Single-channel current and channel densities.
Blocker-insensitive Na+ transport
(IAmilsc) was measured at the
end of each experiment by addition of 100 µM amiloride to the apical
solution. Defining blocker-sensitive macroscopic currents
(IBNa) as
IBsc
IAmilsc and where
S100 is
S0 at 10 µM
CDPC, the single-channel current through blocker-sensitive channels at
a B1 of 10 µM
CDPC is
|
(6)
|
since
the iNa in the
absence of blocker is not significantly different from the
i10Na (2). Blocker-sensitive open channel density at 10 µM CDPC is
N10o = I10Na/i10Na,
expressed in units of open channels per planar square centimeter or per 100 square micrometers, where the latter approximates the area per
cell. No was
calculated with Eq. 2. The total
density of functional channels
(NT) was
calculated from the quotient
No /Po.
It should be emphasized according to
Eq. 6
that all calculations of single-channel currents and channel
densities were done with data obtained before onset of the
blocker-related autoregulatory increases of transport, with the
exception of the f 30c that were used to calculate
KB and
where, as noted above,
fc are
independent of transport rate.
Summary data are reported as means ± SE.
 |
RESULTS |
Blocker-dependent short-circuit currents.
Illustrated in Fig. 4 are the short-circuit
current (I10Na) responses to
aldosterone in the three groups of experiments of markedly different
baseline rates of transport. After a delay of ~40 min, the currents
increased steadily over 6 h from baseline rates of 1.06 ± 0.11 µA/cm2
(group 1),
5.40 ± 0.37 µA/cm2
(group 2),
and 15.14 ± 3.33 µA/cm2
(group 3).
The factors responsible for governing baseline rates of transport are
unknown, although the substrate of tissue growth is of importance and
is at least in part responsible for the very low rates of transport in
group
1 tissues grown on Tr-tct membranes (16). Nevertheless, we were presented with the opportunity to test for
similarities and differences in the responses to aldosterone at the
earliest stages of stimulation of transport in tissues expressing
vastly different rates of transport at the apical membranes of
their cells.

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Fig. 4.
Aldosterone stimulation of blocker-sensitive
Na+ transport in tissue of
groups 1, 2,
and 3 (A,
B, and
C, respectively). Note differences in
baseline rates of Na+ transport
(INa).
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Representative strip-chart recordings presented in Fig.
5 document the typical delay in onset of
stimulation of
Isc caused by
aldosterone and indicate the times at which CDPC was increased from 10 to 30 µM and returned to 10 µM CDPC. Although not the focus of the
experiments, it was clearly apparent that the tissues responded with
autoregulatory increases of
Isc in response
to inhibition of transport with transient overshoots of the
Isc following return to 10 µM CDPC. The interesting point that emerged, as
indicated in Fig. 6, was the delay in onset
of the autoregulatory increase of
Isc. After the
initial decline of the
Isc toward
plateau values that persisted for ~20-100 s, the
Isc began to
increase. This autoregulatory phenomenon has been reported previously
and is associated with inhibition of transport resulting in long time constant transients of the
Isc in the range
of 10-20 min (2, 14). Because the autoregulatory transients were
clearly separable in time from the initial changes of
Isc caused by the
blocker, the fractional inhibitions of
Isc could be
calculated from the changes of
Isc at the
plateaus before and ~30-50 s after elevation of CDPC to 30 µM.
For the pulses illustrated in Fig. 6, the fractional inhibitions
(I30/10sc) were
0.827, 0.861, and 0.879 at the respective
Isc of 1.68, 5.02, and 13.16 µA/cm2.

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Fig. 5.
Strip-chart recordings of short-circuit currents
(Isc) during
control periods and after treatment of tissues with aldosterone in A6
epithelia grown on HA (A) and Tr-tct
(B) inserts.
Na+ transport was inhibited by 100 µM amiloride (Amil.) at ends of experiments. CDPC was increased from
10 to 30 µM for 3 min at intervals of 20 min, causing pulse
inhibitions of current.
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Fig. 6.
Measurements of fractional inhibition of
Na+ transport in tissue of
groups 1, 2,
and 3 (A,
B, and
C, respectively). Values of
Isc were recorded
at intervals of 10 s before and after increasing CDPC from 10 to 30 µM. Dashed lines, plateau values at 30 µM before onset of
autoregulatory increases of transport. Note differences of baseline
rates of Na+ transport and similar
fractional inhibitions of
Isc caused by
CDPC. See also completely reversible transient overshoots of
Isc in Fig. 5
following washout of 30 µM CDPC and return to 10 µM CDPC.
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CDPC rate coefficients.
Typical pairs of current noise-power density spectra are shown in Fig.
7 for tissues expressing very low (near 1 µA/cm2; Fig.
7A) and higher rates of transport.
Each pair of spectra yielded
f 10c and
f 30c together with their
respective S0
values, S100 and
S300. As indicated in Fig.
8A, the
f 10c and
f 30c of a typical experiment
were fit to smooth curves, thereby filtering the variance of
fc of individual
measurements. The
kob,
kbo, and
KB were
calculated at the respective times of measurement of the fractional
inhibitions of
Isc (Fig. 8,
B and
C) with the projected values of
fc of the smooth curves.

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Fig. 7.
Paired current noise-power density spectra
[S(f)]
at 10 µM (solid circles) and 30 µM CDPC (shaded circles) at low
(A; 0.99 µA/cm2) and intermediate
(B; 6.23 µA/cm2) rates of
Na+ transport.
f, Frequency. Corner frequencies
( fc) were
47.3 and 75.3 Hz (A) and 60.9 and
89.5 Hz (B) at 10 and 30 µM CDPC,
respectively. Data were fit by nonlinear regression to 3 components,
including a Lorentzian
{S0/[1+( f /fc)2]},
noise at low frequencies
(S1/f ),
and noise at higher frequencies
(S2 f ),
originating at input stage of voltage amplifier.
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Fig. 8.
Determination of CDPC blocker rate coefficients and
KB.
A: pairs of
fc from a single
experiment measured at 10 and 30 µM CDPC as a function of time during
control and experimental periods. Smooth curves were fit to data, and
projected values on fitted curves were used to calculate
kob and
kbo shown in
B. rad, Radians.
C: time-dependent changes of
KB = kbo/kob.
In the face of small random sampling errors in measurement of
fc, it is
appropriate to filter or smooth data to best determine
fc at each time
point. Given the simplicity of the data, we used TableCurve (Jandel
Scientific) to fit simple curves, linear or nonlinear, to data points.
More complex algorithms are available for data smoothing but were not
required for data of present experiments.
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In all groups of experiments, the mean
KB increased
slightly and continuously during the 8-h periods of observation. As
illustrated in Fig. 9, this was due to
small increases of
kbo (Fig.
9B) and small decreases of
kob in
group
1 but not
group
2 and
3 tissues (Fig.
9A). The changes of rate
coefficients were, however, unrelated to aldosterone. The trends
established during the control periods continued unchanged following
exposure of the tissues to aldosterone. The zero time
control values of
kob,
kbo, and
KB summarized in Table 1, determined just before addition of
aldosterone to the apical solution, are similar to those reported
previously, when kob and
kbo were
determined from the linear relationship between the
fc measured over
a larger range of CDPC concentrations (2). Accordingly, the channels
recruited by aldosterone possess the same CDPC blocker
kinetics as those present in the apical membrane before steroid
stimulation of transport.

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Fig. 9.
Summary of time-dependent changes of
kob
(A),
kbo
(B), and
KB
(C) in
group 1, 2,
and 3 tissues. Values are means ± SE. Error bars were omitted for group 3 for clarity of presentation.
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Single-channel current and open-channel density.
Stimulation of Na+ transport by
aldosterone could not be attributed to changes of single-channel
current. Zero time control iNa averaged
0.37, 0.30, and 0.27 pA in group
1, 2,
and 3 tissues, respectively (Fig.
10), with corresponding open-channel
densities of 3.1, 18.2, and 59.1 channels/100
µm2 (Table 1). The
iNa remained
practically constant during control and aldosterone treatment periods
in group
1 and
2 tissues but was decreased
significantly by aldosterone by ~10% in
group
3 tissues (Fig. 10). Accordingly, the
changes of open-channel density (not shown),
No = INa/iNa,
paralleled those of
INa from markedly different baseline values of
No.

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Fig. 10.
Summary of time-dependent changes of single-channel currents
(iNa) in
group 1, 2,
and 3 tissues. Decreases of
iNa in
group 3 tissues after aldosterone are
significant and are expected in tissues transporting
Na+ at higher rates, where changes
of apical membrane conductance lead to significant changes of
fractional transcellular resistance and hence apical membrane
voltage.
|
|
Channel open probability.
Stimulation of Na+ transport by
aldosterone could not be attributed to changes of channel
Po. As indicated
in Fig.
11A
(and in normalized form in Fig.
11B),
Po fell slowly
and progressively in group
1 and
3 tissues during control and
experimental periods and appeared to stabilize during the aldosterone
treatment period in group
2 tissues. When expressed as
experimental values divided by zero time control values (Fig.
11B),
Po continued to
fall ~20-25% after treatment of
group
1 and
3 tissues and to remain essentially unchanged in group
2 tissues. Although we do not know the
reason(s) for the chronic time-dependent decreases of
Po, it was
evident that stimulation of transport could not be due to changes of
channel Po in any
group of tissues, regardless of their baseline rates of transport or
Po. The zero time
values of Po
averaged 0.44, 0.33, and 0.18 in group
1, 2,
and 3 tissues, respectively
(Table 2), and appeared to be
inversely related to the macroscopic
INa (see
Dependence of
Po and
NT on macroscopic
INa).

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Fig. 11.
Summary of time-dependent decreases of
Po in
group 1, 2,
and 3 tissues.
A: values are means ± SE.
Po was also
measured in group 1 tissues 24 h after treatment with
aldosterone (bar). B:
Po were
normalized to interpolated zero time control values and expressed as
experimental values/zero time control values (means ± SE) for
baseline-independent comparison of time rates of change of
Po. Error bars
were omitted for group 3 in
B for clarity.
|
|
Functional channel densities.
Because stimulation of transport by aldosterone could not be attributed
to changes of single-channel current or channel
Po, changes of
transport must be due to increases of
NT
(NT
channels in open and closed states) from baseline values of 10.9, 60.5, and 350 channels/100 µm2 (Table 2). To
compare changes of
NT and
INa caused by
aldosterone, the experimental values of
NT and
INa were
normalized to zero time control values as illustrated in Fig.
12. In
group
1 tissues expressing very low rates of
transport, NT was
increased nearly fivefold within 6 h of treatment with aldosterone
(Fig. 12A). The fractional increases
of NT were less
in group
2 and
3 tissues, averaging near 2.8-fold
(Fig. 12, B and
C). The fractional increases of
NT in
group
1 and
3 tissues were greater than those of
INa, due
primarily to the chronic time-dependent decreases of
Po that occurred
during the 6-h intervals that tissues were treated with aldosterone
(Fig. 12, A and
C), but were similar in
group
2 tissues, where the
Po had stabilized
during this time (Fig. 12B).
Although the fractional increases of
NT were largest
in group
1 tissues with very low baseline
values of NT, the
largest absolute increases of
NT occurred in
group
3 tissues with the largest baseline
values of NT.
Thus on average
NT (in
channels/100 µm2) was
increased by aldosterone from 10.9 ± 3.0 to 43.5 ± 9.0 (group 1),
from 60.5 ± 5.7 to 166 ± 13.7 (group
2), and from 350 ± 92 to 1,040 ± 414 (group
3).

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Fig. 12.
Summary of time-dependent increases of channel density
(NT) and
blocker-sensitive Na+ transport
(INa) in
group 1, 2,
and 3 tissues
(A,
B, and
C, respectively). Values are means ± SE expressed as experimental values/zero time control values.
|
|
Three additional tissues from group
1 were treated overnight with
aldosterone, after which time further changes of transport do not
occur. INa
averaged 4.95 ± 0.23 µA/cm2,
iNa averaged 0.31 ± 0.02 pA, Po
averaged 0.31 ± 0.01, and
NT averaged 56.0 ± 4.5 channels/100 µm2. When
these values were compared with mean values obtained within 6 h of
aldosterone treatment (3.45 ± 0.51 µA/cm2, 0.34 ± 0.02 pA, 0.27 ± 0.01, and 43.5 ± 9.0 channels/100
µm2), it was apparent that
aldosterone exerted its major effects on
Na+ transport and
NT within 6 h in
these tissues.
Dependence of Po and
NT on macroscopic
INa.
We noted previously (see Channel open
probability) that Po
appeared to be inversely related to the macroscopic rates of
Na+ transport in the absence of
steroid treatment of the tissues. To examine this relationship in more
detail, we plotted against INa (Fig.
13A)
both the zero time control values of
Po and the values
measured after 6-h periods of aldosterone stimulation of transport
(Fig. 13A). Plotted also (Fig.
13B) are the zero time values of
NT and
INa and those
after stimulation by aldosterone. In both sets of data, a linear
log-log relationship existed between Po and
INa and between
NT and
INa. The
regressions shown indicate the slopes and 99% confidence intervals.
Over the range of
INa between
~0.5 and 35 µA/cm2,
Po appears to
decrease with increases of Na+
transport regardless of the presence or absence of aldosterone. Relative to the
INa-related
changes of NT
shown in Fig. 13B, changes of
Po are, however,
relatively small, so that transport is determined primarily by changes
of NT whether
caused by aldosterone and/or other factors that determine
baseline rates of transport.

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Fig. 13.
Relationship between
INa and
Po
(A) and between
INa and
NT
(B). Zero time control values and
paired values 6 h after treatment of
group 1, 2,
and 3 tissues with aldosterone are
plotted. Scales are log-log. Solid lines, linear regressions; dashed
lines, 99% confidence intervals.
|
|
 |
DISCUSSION |
From markedly different baseline rates of
Na+ transport observed in the
present studies and those reported in the literature, aldosterone
stimulates transport severalfold regardless of the presence or absence
of serum in the growth medium (4). For A6 epithelia treated chronically
overnight with aldosterone, stimulation of transport has been
attributed to increases of
NT with no
measurable difference in
Po of the
channels supporting baseline rates of transport from those recruited by
aldosterone into the pool of channels responsible for blocker-sensitive
Na+ entry into the cells (2). We
arrive at the same conclusion for the present series of experiments in
which increases of transport are mediated by aldosterone or by other
factors that govern baseline rates of transport. Regardless of
baseline, aldosterone caused 2.8- to 5-fold increases of
NT within the
first 6 h. The increases of
NT paralleled the
increases of transport. Because single-channel currents either remained
essentially constant at lower baselines of transport for which the
fractional transcellular resistance approaches unity
(groups 1 and
2) or decreased slightly, as is to
be expected in tissues transporting
Na+ at higher rates
(group 3),
there was no indication of either changes of single-channel conductance
of the newly recruited channels or a major effect of aldosterone at the
basolateral membranes that could have altered intracellular voltage and
hence the single-channel currents.
We observed time-dependent decreases of
Po during the
control periods that continued during the experimental periods after tissues were treated with aldosterone. Within the 6-h experimental periods, Po
stabilized only in group
2 tissues (Fig. 11). Although the
fractional decreases of
Po were
relatively small (~3-10%/h during control periods), it became
apparent that these
Po transients required many hours for complete stabilization. When
aldosterone-treated group
1 tissues were studied the following
day, their values of Po were similar
to those measured 6 h following treatment of tissues with aldosterone
(Fig. 11A). Regardless of the
reason for such long-term transients during control and experimental
periods, it was clearly apparent that aldosterone itself did not alter the Po of the
channels recruited to the pool of apical membrane channels subserving
Na+ entry into the cells. In the
face of decreasing or constant values of
Po, stimulation
of transport by aldosterone must occur by increase of blocker-sensitive
ENaC NT.
It was also apparent over considerably larger ranges (~70-fold) of
transport than could be elicited by aldosterone (3- to 5-fold) that
Po was inversely
related to Na+ transport, with the
highest values of
Po observed at
very low rates of transport (Fig.
11A). Transport-related decreases
of Po would be
expected to be ~50% per decade increase of
INa or ~25% per threefold increase of
INa. Over any
range of transport, it was nevertheless clear that
Po could vary
substantially, due perhaps to a variety of factors involved in
regulating Po and
unrelated to the direct action of aldosterone in stimulation of
transport. Accordingly, over three- to fivefold consistent increases of
transport caused by aldosterone, the aldosterone-related decreases of
Po would not be
readily apparent and could be masked by other factors involved in
regulation of Po.
Clearly, Po is
not constant, and it appears to vary with the rate of
Na+ entry into the cells
regardless of the presence or absence of aldosterone stimulation of
transport.
Although it is well appreciated that A6 epithelia express baseline
rates of transport that can vary enormously due to differences in
growth media, serum, and other unknown factors, it has recently been
shown that the substrate on which the cells are grown is a major
determinant in expression of Na+
transport (16). Regardless of substrate and baseline rate of transport,
all tissues respond to aldosterone, consistent with all reports in the
literature. Although we attempted to diversify our experiments by use
of different substrates and different growth media, it remains unknown
whether the transport-related dependence of
Po is due solely
to differences of apical membrane
Na+ entry and/or to other
factors associated with the substrate and the conditions of growth of
the tissues. Nevertheless, regardless of the reasons for differences of
baseline transport,
NT was directly related to transport rate both in the presence and absence of aldosterone stimulation of transport.
The modified pulse method used in the present studies has permitted for
the first time a mechanically noninvasive method of analysis of
time-dependent changes not only of the blocker rate coefficients,
single-channel currents, and open channel densities but also of the
Po and
NT of apical
membrane ENaCs. Previous pulse method studies had indicated that
Po was
independent of the fractional inhibition of open channel density (and
hence fractional inhibition of
INa) over
larger ranges of CDPC concentration than those used in the present
studies (14). Po
has been shown to be independent of B
and the time of exposure of the tissues to the blocker when studies
were carried out with staircase protocol exposures to CDPC at
concentrations exceeding 50 µM (2, 10, 11, 13, 14). Chronic exposure
of tissues to CDPC at their basolateral surface is without effect on
short-circuit currents, indicating the nonresponsiveness of the cells
to this agent if CDPC permeates the cells (unpublished observations).
It is clear under all conditions so far studied that the effects of
CDPC on transport are completely reversible regardless of the time of
exposure of the tissues to this blocker. It is also quite apparent from
the results of the present experiments compared with our own previous
experiments with aldosterone (2) and those of others that the
short-circuit current responses are the same regardless of the presence
or absence of CDPC in the apical solution at comparable baseline rates
of transport. Many blockers, including CDPC, have been used to
characterize ENaCs, and there are to our knowledge no exceptions to the
findings that blockers at any concentration do not alter the blocking
site, as judged from the rate coefficients, and do not alter the
single-channel conductance of the channel (17). Thus we know of no
circumstance in which chronic exposure to 10 µM CDPC would compromise
the response of the tissues to hormonal stimulation by aldosterone in
particular or to other drugs or hormones to which the responses are the
same as those measured in the complete absence of CDPC (Refs. 25, 26;
Blazer-Yost, Liu, and Helman, unpublished observations; Els, Liu, and
Helman, unpublished observations). Accordingly, it should
not be surprising that the results of the present studies are both
qualitatively and quantitatively similar to previous reports that have
used blocker-induced noise analysis as a way to study regulation of
Na+ transport at the apical
membranes of the cells, independent of the protocol used to analyze the
tissues.
Our analysis has indicated that the principal mechanism underlying the
early time-dependent increase of transport caused by aldosterone can be
attributed to increase of the population density of blocker-sensitive
ENaCs at the apical membranes of the cells with relatively little, if
any, decrease of channel
Po.
 |
ACKNOWLEDGEMENTS |
We thank A. L. Helman for excellence in maintaining our tissue
culture facility (Urbana, IL), the care and feeding of the cells, and
assistance in the preparation of this manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-30824 to S. I. Helman and also by a
Department of Veterans Affairs merit review grant and an American Heart
Association (Indiana Affiliate) grant-in-aid to B. L. Blazer-Yost.
X. Liu is a doctoral student in the Dept. of Molecular and Integrative
Physiology, University of Illinois at Urbana-Champaign, Urbana, IL.
Address for reprint requests: S. I. Helman, Dept. of Molecular and
Integrative Physiology, 524 Burrill Hall, 407 S. Goodwin Ave.,
University of Illinois at Urbana-Champaign, Urbana, IL 61801.
Received 5 August 1997; accepted in final form 3 December 1997.
 |
REFERENCES |
1.
Abramcheck, F. J.,
W. Van Driessche,
and
S. I. Helman.
Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270.
J. Gen. Physiol.
85:
555-582,
1985[Abstract].
2.
Baxendale-Cox, L. M.,
R. L. Duncan,
X. Liu,
K. Baldwin,
W. J. Els,
and
S. I. Helman.
Steroid hormone-dependent expression of blocker-sensitive ENaCs in apical membranes of A6 epithelia.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1650-C1656,
1997[Abstract/Free Full Text].
3.
Beron, J.,
and
F. Verrey.
Aldosterone induces early activation and late accumulation of Na-K-ATPase at surface of A6 cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1278-C1290,
1994[Abstract/Free Full Text].
4.
Bindels, R. J. M.,
J. M. Schafer,
and
M. C. Reif.
Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells.
Biochim. Biophys. Acta
972:
320-330,
1988[Medline].
5.
Broillet, M.-C.,
A. Berger,
and
J.-D. Horisberger.
Early effects of aldosterone on the basolateral potassium conductance of A6 cells.
Pflügers Arch.
424:
91-93,
1993[Medline].
7.
Canessa, C. M.,
J. D. Horisberger,
L. Schild,
and
B. C. Rossier.
Expression cloning of the epithelial sodium channel.
Kidney Int.
48:
950-955,
1995[Medline].
8.
Canessa, C. M.,
L. Schild,
G. Buell,
B. Thorens,
I. Gautschi,
J.-D. Horisberger,
and
B. C. Rossier.
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
Nature
367:
463-466,
1994[Medline].
9.
Eaton, D. C.,
and
Y. Marunaka.
Ion channel fluctuations: "noise" and single-channel measurements.
In: Channels and Noise in Epithelial Tissues, edited by S. I. Helman,
and W. Van Driessche. San Diego, CA: Academic, 1990, p. 61-113. (Curr. Top. Membr. Transp., vol. 37)
10.
Els, W. J.,
and
S. I. Helman.
Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action.
J. Gen. Physiol.
98:
1197-1220,
1991[Abstract].
11.
Els, W. J.,
and
S. I. Helman.
Dual role of prostaglandins (PGE2) in regulation of channel density and open probability of epithelial Na+ channels in frog skin (R. pipiens).
J. Membr. Biol.
155:
75-87,
1997[Medline].
13.
Granitzer, M.,
I. Mountian,
and
W. Van Driessche.
Effect of dexamethasone on sodium channel block and densities in A6 cells.
Pflügers Arch.
430:
493-500,
1995[Medline].
14.
Helman, S. I.,
and
L. M. Baxendale.
Blocker-related changes of channel density. Analysis of a three-state model for apical Na channels of frog skin.
J. Gen. Physiol.
95:
647-678,
1990[Abstract].
15.
Helman, S. I.,
and
N. L. Kizer.
Apical sodium ion channels of tight epithelia as viewed from the perspective of noise analysis.
In: Channels and Noise in Epithelial Tissues, edited by S. I. Helman,
and W. Van Driessche. San Diego, CA: Academic, 1990, p. 117-155. (Curr. Top. Membr. Transp., vol. 37)
16.
Helman, S. I.,
and
X. Liu.
Substrate-dependent expression of Na+ transport and shunt conductance in A6 epithelia.
Am. J. Physiol.
273 (Cell Physiol. 42):
C434-C441,
1997[Abstract/Free Full Text].
17.
Helman, S. I.,
and
W. Van Driessche
(Editors).
Channels and Noise in Epithelial Tissues. San Diego, CA: Academic, 1990. (Curr. Top. Membr. Transp., vol. 37)
18.
Horisberger, J.-D.,
and
C. Kaufmann.
Early effects of aldosterone on apical and basolateral membrane conductances of TBM cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C384-C388,
1992[Abstract/Free Full Text].
19.
Kemendy, A. E.,
T. R. Kleyman,
and
D. C. Eaton.
Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia.
Am. J. Physiol.
263 (Cell Physiol. 32):
C825-C837,
1992[Abstract/Free Full Text].
20.
Leal, T.,
and
J. Crabbé.
Effects of aldosterone on (Na++K+)-ATPase of amphibian sodium-transporting epithelial cells (A6) in culture.
J. Steroid Biochem.
34:
581-584,
1989[Medline].
21.
Ling, B. N.,
and
D. C. Eaton.
Effects of luminal Na+ on single Na+ channels in A6 cells, a regulatory role for protein kinase C.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F1094-F1103,
1989[Abstract/Free Full Text].
22.
Marunaka, Y.,
and
D. C. Eaton.
Effects of vasopressin and cAMP on single amiloride-blockable Na channels.
Am. J. Physiol.
260 (Cell Physiol. 29):
C1071-C1084,
1991[Abstract/Free Full Text].
23.
Pacha, J.,
G. Frindt,
L. Antonian,
R. B. Silver,
and
L. G. Palmer.
Regulation of Na channels of the rat cortical collecting tubule by aldosterone.
J. Gen. Physiol.
102:
25-42,
1993[Abstract].
24.
Palmer, L. G.,
L. Antonian,
and
G. Frindt.
Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone.
J. Gen. Physiol.
102:
43-57,
1993[Abstract].
25.
Paunescu, T. G.,
and
S. I. Helman.
Dual role of prostaglandin E2 in regulation of Na+ transport in A6 epithelia (Abstract).
Biophys. J.
72:
A230,
1997.
26.
Paunescu, T. G.,
X. Liu,
and
S. I. Helman.
Nonhormonal regulation of apical membrane sodium transport in A6 epithelia (Abstract).
FASEB J.
11:
A8,
1997.
27.
Shahedi, M.,
K. Laborde,
L. Bussières,
and
C. Sachs.
Acute and early effects of aldosterone on Na-K-ATPase activity in Madin-Darby canine kidney epithelial cells.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F1021-F1026,
1993[Abstract/Free Full Text].
28.
Verrey, F.,
and
J. Beron.
Activation and supply of channels and pumps by aldosterone.
News Physiol. Sci.
11:
126-133,
1996.[Abstract/Free Full Text]
AJP Cell Physiol 274(4):C947-C957