K+ transport and capacitance of the basolateral
membrane of the larval frog skin
Stanley D.
Hillyard1,
Horacio F.
Cantiello2, and
Willy
Van Driessche3
1 Department of Biological
Sciences, University of Nevada, Las Vegas, Nevada 89154;
2 Renal Unit, Massachusetts
General Hospital East and Harvard University Medical School, Boston,
Massachusetts 02129; and
3 Laboratorium voor Fysiologie,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
 |
ABSTRACT |
Skin from larval bullfrogs was mounted in an Ussing-type chamber
in which the apical surface was bathed with a Ringer solution containing 115 mM K+ and the
basolateral surface was bathed with a Ringer solution containing 115 mM
Na+. Ion transport was measured as
the short-circuit current
(Isc) with a
low-noise voltage clamp, and skin resistance
(Rm) was
measured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine waves
to the command stage of the voltage clamp. From the ratio of the
Fourier-transformed voltage and current signals, it was possible to
calculate the resistance and capacitance of the apical and basolateral
membranes of the epithelium
(Ra and
Rb,
Ca and Cb,
respectively). With
as the anion,
Rm decreased
rapidly within 5 min following the addition of 150 U/ml nystatin to the
apical solution, whereas
Isc increased
from 0.66 to 52.03 µA/cm2 over a
60-min period. These results indicate that nystatin becomes rapidly
incorporated into the apical membrane and that the increase in
basolateral K+ permeability
requires a more prolonged time course. Intermediate levels of
Isc were obtained
by adding 50, 100, and 150 U/ml nystatin to the apical solution. This
produced a progressive decrease in Ra and
Rb while
Ca and
Cb remained
constant. With Cl
as the
anion, Isc values
increased from 2.03 to 89.57 µA/cm2 following treatment with
150 U/ml nystatin, whereas with gluconate as the anion
Isc was only
increased from 0.63 to 11.64 µA/cm2. This suggests that the
increase in basolateral K+
permeability produced by nystatin treatment, in the presence of more
permeable anions, is due to swelling of the epithelial cells of the
tissue rather than the gradient for apical
K+ entry. Finally,
Cb was not
different among skins exposed to
Cl
,
, or gluconate, despite the large
differences in
Isc, nor did
inhibition of Isc
by treatment with hyperosmotic dextrose cause significant changes in
Cb. These results
support the hypothesis that increases in cell volume activate
K+ channels that are already
present in the basolateral membrane of epithelial cells.
cell volume regulation; potassium channels; nystatin; epithelial
transport
 |
INTRODUCTION |
POTASSIUM CHANNELS in the basolateral membrane of
epithelial cells allow K+ that is
transported into the cell by the
Na+-K+
pump to exit and establish a negative potential within the cell (14).
Basolateral K+ channels are also
involved in the regulation of cellular volume. For example, the
basolateral membrane of turtle colon has three types of
K+ channels, one of which is
opened under conditions that promote cell swelling (5, 8, 9). The
volume-sensitive K+ channel was
also found to be blocked by lidocaine. Dawson et al. (6) subsequently
showed that substitution of a less permeant ion, gluconate, for
Cl
inhibited the
lidocaine-sensitive component of the basolateral K+ conductance and used
fluctuation analysis to demonstrate that lidocaine inhibition under
swelling conditions (i.e.,
Cl
as the anion) obeyed
pseudo-first-order kinetics, as would be expected from a direct channel
blockage.
The above studies of basolateral
K+ permeability were conducted
with epithelial tissues that were mounted in an Ussing-type chamber
with K+ Ringer bathing the mucosal
surface of the tissue and Na+
Ringer bathing the serosal surface. The mucosal solution also contained
the ionophore amphotericin B, which formed
K+-conducting channels in the
apical membrane so that the basolateral membrane could be effectively
voltage clamped and the short-circuit current
(Isc) could be
related to the rate of basolateral
K+ transport. We have used a
similar approach to study basolateral K+ channels in the larval frog
skin (12, 25), using the ionophore nystatin to increase apical
K+ conductance. We have found that
a period of ~1 h following nystatin treatment is required for
Isc to reach a
maximum value. This suggests that the activation of basolateral
K+ channels is a relatively slow
process that might result from cellular swelling as
K+ and permeant anions enter the
cells across the apical membrane. Alternatively, the prolonged time
course for the stimulation of Isc could result
from a gradual insertion of nystatin into the apical membrane. The
present study was initiated to determine how quickly nystatin forms
K+-conducting channels in the
apical membrane relative to the time course for the development of
maximal K+ transport across the
basolateral membrane. Impedance analysis (24) was used to measure the
resistance and capacitance of the apical and basolateral membranes
(Ra and
Rb,
Ca and
Cb, respectively) as Isc increased
following nystatin treatment. An increase in Cb would suggest
that the increase in basolateral
K+ transport was due to the
addition of membrane vesicles, whereas lack of change in
Cb would suggest
that channels had become activated in situ. The larval bullfrog skin is
particularly useful for this study because the limiting barrier to ion
transport is a single layer of apical cells (20), the apical membrane
resistance can be easily reduced by nystatin (4), and the basolateral
membrane contains K+ channels that
are activated by conditions that result in cell swelling (12).
In our studies to date,
has been
used as the anion, since the basolateral membrane is highly permeable to Cl
(11) and the low
resistance of the tissue complicates fluctuation analysis measurements.
We have found that quinine, quinidine, lidocaine (26), and more
recently verapamil (12) inhibit that fraction of the
Isc that can also
be inhibited by treatment with hyperosmotic sucrose solutions. A second
goal of the study was to determine whether substitution of anions that
are progressively less permeant (i.e.,
Cl
,
, and gluconate) or the addition
of hypersomotic solute concentrations results in a lower stimulation of
basolateral K+ conductance and to
measure Rb and
Cb under these
conditions. If the stimulation of basolateral
K+ transport is the result of cell
swelling due to the entry of more permeant anions, there should be a
progressive decrease in Isc and increase
in Rb with the
less permeant anions or the addition of an impermeant solute. If these
changes in Isc
and Rb are the result of removal of membrane vesicles,
Cb would be
predicted to decrease in proportion to the decrease in
Isc.
 |
METHODS |
Larval bullfrogs, Rana catesbeiana,
were obtained from commercial suppliers and maintained in aquaria at
15°C. The ventral skin was dissected with the abdominal musculature
attached (4), using MS 222 anesthesia. The skin was mounted in an
Ussing-type chamber that minimized edge damage and permitted continuous
perfusion of the mucosal and serosal solutions (7). The mucosal
solution contained 115 mM K+ and
1.0 mM Ca2+, with either
Cl
,
, or gluconate as the anion, and
2.5 mM KHCO3. The serosal solution
contained 115 mM Na+ and 1.0 mM
Ca2+, with either
Cl
,
, or gluconate as the anion, and
2.5 mM KHCO3. The pH of both
solutions was 8.0. Once stable values for Isc had been
obtained, nystatin (Sigma Chemical) was added to the mucosal Ringer to
give the desired concentration. The appropriate amount of nystatin was
dissolved in 100 µl dimethyl sulfoxide per 100 ml Ringer.
The macroscopic
Isc was applied
with a low-noise voltage clamp and was recorded on a chart recorder.
The direct current (DC) resistance of the entire tissue
(Rm) was
measured by applying a 10-mV pulse to the command stage of the voltage
clamp and dividing by the change in
Isc. For
impedance measurements, the command stage of the voltage clamp was
connected to a computerized voltage signal composed of 53 sine waves,
and the voltage and current signals from the clamp were sampled
consecutively. The voltage and current signals were subjected to a fast
Fourier transform, and the impedance
(Z) was calculated as the ratio of
the transformed voltage and current signals (see Refs. 16 and 22 for
detailed description of the synchronization of the voltage and current signals). Two impedance curves were merged and plotted as impedance loci on Nyquist plots. Before nystatin treatment, the Nyquist plots
contained a single semicircle. This semicircle could be fitted with
Eq.
1, which assumes a single membrane
with parallel resistance (R) and
capacitance (C) elements (Ref. 24,
adapted from Ref. 3)
|
(1)
|
In
this equation, j =
and
is the angular
frequency = 2
f, where
f is frequency. The exponent
= 1
2
/
, where
is the angle, in degrees, between the line that connects the center of the semicircle with its intercept on the
real (resistance) axis. This equation provides an accurate measurement
of tissue capacitance
(Cm) that is
equivalent to Ca before nystatin treatment, since the apical membrane is the dominant resistance element (24). It should be noted, however, that the resistance elements calculated by Eq.
1 include that of
Ra and Rb in series and
the paracellular pathway
(Rp) that is in
parallel with Ra
and Rb. Thus the
resistance calculated by Eq.
1, before the addition of nystatin,
should be equal to
Rm and, since the Ra is very high,
should provide an approximate measure of
Rp.
After nystatin treatment, two semicircles frequently appeared in the
Nyquist plots. To fit the two semicircles,
Eq. 1 can be used separately for each (24), and the resistance values calculated by this equation approximate
Ra and
Rb.
Alternatively, Margineanu and Van Driessche (17) developed an
expression for transepithelial impedance
[Z(t)]
that fits two semicircles, with the assumption that they represent two
membranes in series, each with a parallel resistance and capacitance
(e.g., Ra,
Rb,
Ca, Cb). This model
also incorporates an expression for the paracellular shunt resistance
(Rp) that is in
parallel with the apical and basolateral membranes
|
(2)
|
In
Eq.
2, s
is the product j
, and A, B, C, and
D contain terms that relate to
Rp,
Ra,
Rb,
Ca, and
Cb
A,
B, C, and D were obtained by nonlinear curve fitting of
Eq. 2 to the impedance data as described by Margineanu and Van Driessche
(17). Values for
Rp were taken to
be the resistance values
(Rm) calculated
from the DC voltage pulses before nystatin treatment. As indicated
above, the apical membrane of the larval skin has a very high DC
resistance (4), and it was assumed that the resistance of the skin with
an intact apical membrane approximates
Rp.
To follow the time course of impedance changes as
Isc was
stimulated, Nyquist plots were generated at 5-min intervals for 60 min
following the addition of nystatin at a concentration of 150 U/ml,
which we have found to produce maximal
Isc values (25). In a second series of experiments,
Isc was more
gradually stimulated by sequentially adding 50, 100, and 150 U/ml
nystatin. Impedance was determined when
Isc had reached
stable values at each concentration. The above experiments were
conducted with
as the anion.
The effect of different anions was assessed by conducting separate sets
of experiments using Cl
,
, or gluconate as the anion. Skins
were treated with 150 U/ml nystatin, and impedance was determined after stable Isc values
had been achieved.
Finally, the effect of increasing the osmotic concentration was
evaluated by adding dextrose, in concentrations between 10 and 50 mM,
to the mucosal and serosal solutions of preparations having
as the anion. Dextrose was added after Isc had
equilibrated following mucosal treatment with 150 U/ml nystatin.
Statistical comparisons between current, resistance, and capacitance
values were made with Student's
t-test.
 |
RESULTS |
The time course for the stimulation of
Isc by the
addition of nystatin to the mucosal Ringer is shown in Fig.
1. Note the immediate rise in
Isc followed by a
slower but larger increase in
Isc over a 1-h
period. Note also that the nystatin effect could be reversed after
removal from the mucosal Ringer. Nyquist plots obtained before nystatin
treatment and after stimulation of
Isc with 150 U/ml
nystatin are shown in Fig. 2. The
magnitudes of the resistive and capacitive components of the plot
obtained after nystatin treatment were multiplied by a scale factor of
five to illustrate the presence of a second semicircle, and the points were fitted by Eq.
2. The plot obtained before nystatin
treatment was fitted with Eq.
1.

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Fig. 1.
Tracing of short-circuit current
(Isc) recorded
when
K2SO4
Ringer replaced
Na2SO4
Ringer as mucosal solution and when 150 U/ml nystatin was subsequently
added to mucosal solution. Note also that
Isc returns
toward control levels when nystatin is removed from mucosal solution.
|
|

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Fig. 2.
Nyquist plots obtained before nystatin treatment [control
(CTRL)] and after maximal
Isc values were
obtained following nystatin treatment. Control plot was fitted with
Eq.
1, and plot obtained after nystatin
treatment was fitted with Eq.
2. Plot obtained after nystatin
treatment was amplified by a scaling factor of 5 to illustrate presence
of 2nd semicircle that represents apical membrane.
R, resistance;
X, capacitance.
|
|
Rm decreased to
minimal values within 5 min following nystatin treatment. It was found
that resistance values calculated from the fit of the pretreatment
plots by Eq.
1 were almost identical with
Rm (Fig.
3A).
After nystatin treatment, the fit with
Eq. 2 showed that the smaller semicircle nearer the origin of the Nyquist
plot (the higher-frequency impedance loci) represented the apical
membrane, since
Ca values were
the same as those seen before nystatin treatment (see below) and
Ra values
declined in parallel with
Rm as would be
expected when nystatin formed channels in the apical membrane. The
larger semicircle represented the basolateral membrane, which
constitutes most of the resistance (Rb) following
150 U/ml nystatin treatment.

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Fig. 3.
A: changes in apical, basolateral, and
skin resistance
(Ra,
Rb, and
Rm, respectively)
produced by addition of 150 U/ml nystatin to mucosal Ringer (at time
indicated by arrow at top). See text
for description of methods used to determine these terms.
B: changes in apical, basolateral, and
tissue capacitance
(Ca,
Cb, and
Cm, respectively)
that were calculated from same Nyquist plots used to measure
Ra and
Rb in
A.
Cm was calculated
from fit of larger semicircle of Nyquist plot with
Eq.
1. Arrow, time of nystatin addition.
|
|
Apical capacitance was calculated from
Eq. 1 before nystatin treatment and by Eq.
2 following nystatin treatment (Fig.
3B). The two fits gave values for
apical capacitance that were not significantly different. The presence
of the second semicircle following nystatin treatment allowed the
calculation of Cb
by Eq.
2. It was also possible to use
Eq. 1 to fit the larger semicircle during the time course of nystatin
treatment and obtain a measure of
Cm when the
basolateral membrane was the primary resistance element of the tissue.
To demonstrate that the single and double fits gave similar values for
basolateral membrane capacitance, a series of 22 preparations was
evaluated after
Isc had
equilibrated with 150 U/ml nystatin in the mucosal Ringer. The values
for Cb were 12.73 ± 0.77 and 13.41 ± 0.74 µF/cm2 with
Eqs.
1 and
2, respectively, and were not
significantly different.
Both Cm and
Cb increased
during the first 15 min following nystatin treatment as the basolateral
membrane became the major component of the Nyquist plot.
Also, Isc was
rapidly increasing during this period as the cells responded to the
rapid increase in K+ entry across
the apical membrane. Given the sampling time required to obtain values
at the lower frequencies (~150 s), it was desirable to measure
resistance and capacitance at stable values of
Isc that were
intermediate between pretreatment and maximal values. This was
accomplished by exposing the apical membrane to intermediate concentrations of nystatin. It can be seen in Fig.
4 that sequential treatment with 50, 100, and 150 U/ml nystatin in the mucosal Ringer produced progressively
higher Isc values
that were stable throughout the sampling period required for impedance
measurements. Mean values for the stable
Isc and
Rm values for
five such preparations are given in Table
1. Impedance measurements at these
intermediate Isc
values showed the appearance of a second semicircle that became the
dominant component at the highest nystatin concentration (Fig. 5). Results from 13 preparations showed that
Ra decreased to a small fraction of the tissue resistance; however,
Ca remained relatively constant (Table 2). The decrease in
Ra with
increasing nystatin concentrations corresponds to the increase in
Isc and a
decrease in Rb.
The values for
Cb, however, were
not found to be significantly different.

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Fig. 4.
A tracing of Isc
obtained when nystatin concentration of mucosal solution was raised
incrementally from 50 to 100 to 150 U/ml. Nyquist plots were obtained
when Isc had
stabilized at each concentration.
|
|
When the Ringer solutions contained
Cl
as the anion, the
Isc values
obtained after treatment with 150 U/ml nystatin were greater and
Rb was lower than
those observed with
(Fig.
6). In contrast, lower values for
Isc and higher
values for Rb
were noted when gluconate was substituted for
. Values for
Cb obtained with
the three anions were not significantly different, despite
the large differences in
Isc and
Rb. We also observed, as seen with
in Fig.
3B, that the elevation of
Cb recorded
during the first 10-20 min following the addition of 150 U/ml
nystatin was seen regardless of the anion and the resulting degree to
which Isc was
stimulated (not shown).
The addition of dextrose produced a dose-dependent inhibition of
Isc in
conjunction with an increase in
Rm
(Fig. 7). The values for
Cm (fitted with
Eq.
1) that were obtained when stable
Isc values were
obtained at each dextrose concentration were not significantly different from that obtained before dextrose treatment.

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Fig. 5.
Nyquist plots obtained before nystatin treatment (control) of mucosal
Ringer (curve 1; ) and under
conditions of stable
Isc following
treatment with 50 (curve 2; ), 100 (curve 3; ), and 150 (curve 4; ) U/ml nystatin. Scales
of Nyquist plots obtained after treatment with 100 and 150 U/ml
nystatin were multiplied by a factor of 5 to better illustrate
emergence of semicircle that represents basolateral membrane.
|
|
 |
DISCUSSION |
The decrease in tissue resistance
(Rm or by
Eq. 1 or Eq.
2) within 5 min after the addition
of 150 U/ml nystatin to the mucosal Ringer shows that nystatin inserts
rapidly into the apical membrane. It is also evident from Fig. 1 that
nystatin washes out of the apical membrane when removed from the
mucosal Ringer. The development of maximal
Isc values during
the 1 h following treatment with the highest nystatin concentration is
thus the result of a more prolonged time course for the stimulation of
basolateral K+ permeability. After
the initial decline in
Ra,
Rm values were essentially identical with
Rb but did not
decrease further, despite the continued increase in
Isc. This could
be a result of other ions becoming permeable across the basolateral
membrane as Ra became maximally reduced or possibly is a result of the increasing Isc interfering
with the impedance measurements.

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Fig. 6.
Effect of substituting more permeant cations
[Cl > > gluconate (gluc)] on
Isc
(left),
Rb
(middle), and
Cb
(right) in preparations treated with
150 U/ml nystatin in mucosal Ringer.
Rb and
Cb were
calculated from fits of Nyquist plots with
Eq.
2.
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Fig. 7.
Adding dextrose to mucosal and serosal Ringer in preparations having
as anion and 150 U/ml nystatin in
mucosal solution causes a decrease in
Isc ( ) and an
increase in Rm
( ) but no change in
Cb ( ).
Cb was calculated
with Eq.
1.
|
|
In contrast, when the nystatin concentration was increased
incrementally, Rb
did decrease as
Isc values
increased when Ra was lowered by the ionophore, suggesting that
K+ permeability was a major
component of basolateral resistance under these conditions. In either
case, an increase in the entry of
K+ into the cells was compensated
for by an increase in K+
permeability across the basolateral membrane. It is unlikely, however,
that the K+ gradient per se is the
determining factor for the reduction of Rb, since the
increase in Isc
(Cl
>
> gluconate) and decrease in
Rb
(Cl
<
< gluconate) that were observed
with anion substitution occurred with the highest nystatin
concentration and with comparable gradients for
K+ transport across the tissue.
Gluconate substitution for
Cl
is known to reduce
cellular swelling in turtle colon cells after treatment with
amphotericin B (6). The increase in basolateral K+ permeability of the larval frog
skin bathed either with more permeant anions or with increasing
nystatin concentrations is probably regulated as part of a volume
regulatory decrease, as is the case for most cells faced with
conditions that promote swelling (15, 25). The reduction of
Isc and the
increase in Rm
that accompanied the addition of dextrose to the bathing solutions further support the hypothesis that the cells are responding to the
osmotic rather than an ionic effect of anion substitution.
There was an apparent increase in
Cb and
Cm during the 15 min following the addition of 150 U/ml nystatin. This occurred during the transition from the pretreatment conditions, in which the apical
membrane is the dominant component of the Nyquist plots, to the
equilibrium condition, in which the basolateral membrane is the major
component and Isc
values are increasing rapidly. The collection of impedance data
requires that two curves be generated with fundamental frequencies of
3.2 and 0.5 Hz (17). Multiple sweeps are taken for each record, so that
a total of ~150 s is needed for data acquisition. Changes in
Isc that result
from increasing K+ conductance
across the membrane during this sampling interval could result in
errors in measurement of capacitance. This transient increase in
Cb was comparably
seen in experiments with all three anions, even though the increase in
Isc was markedly
different among gluconate,
, and
Cl
. Thus, if the increase
in capacitance represents an addition of membrane vesicles to the
basolateral membrane, it does not appear to be associated with the
magnitude of the increase in K+
permeability. In contrast,
Cb values that
were calculated after stable
Isc values had
been obtained, either from preparations treated with 150 U/ml nystatin
or with stepwise addition of 50, 100, and 150 U/ml nystatin, were not
different, even though
Rb was lowered by
a factor of eight. Values for
Cb that were
calculated from either Eq.
1 or
Eq. 2 were not significantly different, and the value for
Rp chosen for
Eq. 2 was assumed not to change as Rb decreased. The
similar values for
Cb obtained by
both calculations suggest that changes in
Rp, if they
occur, are small.
That Cb was not
different despite large changes in
Rb and
Isc provides
evidence that the activation of basolateral
K+ channels does not require the
net addition of vesicles to the basolateral membrane and that channels
are activated in situ by cell swelling. These results are consistent
with observations by Cantiello et al. (2) that activation of
volume-sensitive K+ channels in
melanoma cells is dependent on actin-binding protein and their
suggestion that volume activation of channels involves an interaction
between the actin cytoskeleton and volume-sensitive channels in the
membrane.
As noted above, there was a transient increase in
Cb as
Isc values
increased in the first 15 min following the addition of 150 U/ml
nystatin in experiments with all three anions. If this does, in fact,
represent the addition of basolateral vesicles (as opposed to being the
result of increasing
Isc values during data acquisition), it could be that insertion of new
K+ channels is taking place but
the activation of these channels requires the volume change associated
with the more permeant anions. Experiments to date have
shown that blocker-sensitive channels (i.e., verapamil, quinidine,
quinine) are also inhibited by the addition of hyperosmotic sugar
solutions (12) or gluconate as the anion (unpublished observations). In
these experiments, a two-state model for blocker-induced noise analysis
has been used, and measurements of open probability are not available.
Furthermore, comparison of the kinetics of blocker action on current
fluctuations and on the inhibition of
Isc indicates
that the kinetics of basolateral K+ channels in larval frog skin
and in turtle colon (6, 12) are complex, so that the specific mechanism
of channel activation remains elusive.
The lack of change in
Cb does not
completely rule out the possibility that membrane retrieval and
insertion are occurring at comparable rates so that new channels are
added as membrane vesicles with a lower channel density are retrieved.
Increases in membrane capacitance have been shown to correlate with the addition of water-conducting aggrephores in the amphibian urinary bladder (13, 19, 22), and it has been suggested that the stimulation of
apical membrane Na+ conductance of
A6 cells by antidiuretic hormone is associated with the addition of
membrane vesicles containing amiloride-sensitive Na+ channels (18). Harris et al.
(10) found that conditions that favor cellular swelling result in
greater rates of exocytosis and endocytosis of apical membrane vesicles
by toad urinary bladder granular cells when osmotic water flux is
stimulated by antidiuretic hormone.
Using noise analysis, Van Driessche and Hillyard (25) estimated the
density of basolateral K+ channels
in the larval frog skin to be 7.7 channels/µm2 of cross-sectional
area. Because the capacitance measurements used to estimate the
basolateral membrane area suggest that the actual basolateral membrane
area is ~10- to 15-fold greater than the cross-sectional area, the
density of channels would be on the order of 0.5-1
µm
2. Thus a small
turnover of vesicles having a high density of channels could result in
substantial changes in K+
transport as the channels became activated.
Another caveat in the interpretation of the capacitance values is the
possibility that the dielectric behavior of cell membranes in this
tissue is frequency dependent, so that capacitance values obtained over
a range of frequencies would give rise to errors in capacitance
measurements (1). With Eqs.
1 and
2, membrane resistance and capacitance
values are determined over the same range of frequencies, and the
resistance values,
Ra and
Rb, are very
similar to the values for
Rm that were
obtained with a single DC voltage pulse. Even with errors in the
determination of absolute values for
Cb, the lack of
observed differences in this parameter over the wide range of
resistances and
Isc values
produced in the experimental treatments is most consistent with the
activation of in situ channels and/or a very precise mechanism
for the control of basolateral membrane area.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the technical assistance of J. De Beir
Simaels.
 |
FOOTNOTES |
S. D. Hillyard was supported in part by National Science Foundation
Grant IBN-9215023 and National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-38977-01. H. F. Cantiello was supported in
part by National Institute of Diabetes and Digestive and Kidney
Diseases Grant DK-48040, and W. Van Driessche was supported by Grant
G.023.95 from the Fonds Wetenschappelijk-Vlaanderen
(Belgium).
Address for reprint requests: S. D. Hillyard, Dept. of Biological
Sciences, University of Nevada, 4505 Maryland Pkwy., Las Vegas, NV
89154-4004.
Received 30 September 1996; accepted in final form 21 August 1997.
 |
REFERENCES |
1.
Aywada, M.,
and
S. I. Helman.
Na+ transport related changes of apical membrane capacitance in the tight epithelium of frog skin (Abstract).
FASEB J.
6:
A1239,
1992.
2.
Cantiello, H. F.,
A. G. Prat,
J. V. Bonventure,
C. C. Cunningham,
J. H. Hartwig,
and
D. A. Ausiello.
Actin-binding protein contributes to cell volume regulatory ion channel activation in melanoma cells.
J. Biol. Chem.
268:
4596-4599,
1993[Abstract/Free Full Text].
3.
Cole, K. S.,
and
R. H. Cole.
Dispersion and absorption in dielectrics. I. Alternating current characteristics.
J. Chem. Phys.
9:
341-351,
1940.
4.
Cox, T. C.,
and
R. H. Alvarado.
Electrical and transport characteristics of skin of larval Rana catesbeiana.
Am. J. Physiol.
237 (Regulatory Integrative Comp. Physiol. 6):
R74-R79,
1979[Medline].
5.
Dawson, D. C.,
and
N. W. Richards.
Basolateral K conductance: role in regulation of NaCl absorption and secretion.
Am. J. Physiol.
259 (Cell Physiol. 28):
C181-C195,
1990[Abstract/Free Full Text].
6.
Dawson, D. C.,
W. Van Driessche,
and
S. I. Helman.
Osmotically induced basolateral K+ conductance in turtle colon: lidocaine-induced K+ channel noise.
Am. J. Physiol.
254 (Cell Physiol. 23):
C165-C174,
1988[Abstract/Free Full Text].
7.
De Wolf, I.,
and
W. Van Driessche.
Voltage-dependent Ba2+ block of K+ channels in the apical membrane of frog skin.
Am. J. Physiol.
251 (Cell Physiol. 20):
C696-C706,
1986[Abstract/Free Full Text].
8.
Germann, W. J.,
S. A. Ernst,
and
D. C. Dawson.
Resting and osmotically-induced basolateral K conductances in turtle colon.
J. Gen. Physiol.
88:
253-274,
1986[Abstract].
9.
Germann, W. J.,
M. E. Lowy,
S. A. Ernst,
and
D. C. Dawson.
Differentiation of two distinct K conductances in the basolateral membrane of turtle colon.
J. Gen. Physiol.
88:
237-252,
1986[Abstract].
10.
Harris, H. W.,
J. B. Wade,
and
J. S. Handler.
Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated urinary bladder.
Am. J. Physiol.
251 (Cell Physiol. 20):
C274-C284,
1986[Abstract/Free Full Text].
11.
Hillyard, S. D.
Osmotically regulated Cl
conductance across the basolateral membrane of the tadpole skin epithelium (Abstract).
Physiologist
30:
156,
1987.
12.
Hillyard, S. D.,
and
W. Van Driessche.
Verapamil blocks basolateral K+ channels in the larval frog skin.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1161-C1166,
1992[Abstract/Free Full Text].
13.
Kachadorian, W. A.,
J. B. Wade,
and
V. A. DiScala.
Vasopressin-induced structural change in toad bladder luminal membrane.
Science
190:
67-69,
1975[Medline].
14.
Koefoed-Johnsen, V.,
and
H. H. Ussing.
The nature of the frog skin potential.
Acta Physiol. Scand.
42:
298-308,
1958.
15.
Lewis, S. A.,
and
P. Donaldson.
Ion channels and cell volume: chaos in an organized system.
News Physiol. Sci.
5:
112-119,
1990.[Abstract/Free Full Text]
16.
Lindemann, B.,
and
W. Van Driessche.
Sodium-specific membrane channels of frog skin are pores: current fluctuations reveal high turnover.
Science
195:
292-294,
1977[Medline].
17.
Margineanu, D.-G.,
and
W. Van Driessche.
Effects of millimolar concentrations of glutaraldehyde on the electrical properties of frog skin.
J. Physiol. (Lond.)
427:
567-581,
1990[Abstract].
18.
Marunaka, Y.,
and
D. C. Eaton.
Effects of vasopressin and cAMP on single amiloride-blockable channels.
Am. J. Physiol.
260 (Cell Physiol. 29):
C1071-C1084,
1991[Abstract/Free Full Text].
19.
Palmer, L. G.,
and
M. Lorenzen.
Antidiuretic hormone-dependent membrane capacitance and water permeability in the toad urinary bladder.
Am. J. Physiol.
244 (Renal Fluid Electrolyte Physiol. 13):
F195-F204,
1983[Medline].
20.
Robinson, D. A.,
and
M. B. Heintzelman.
Morphology of the ventral epidermis of Rana catesbeiana during metamorphosis.
Anat. Rec.
217:
305-317,
1987[Medline].
21.
Schultz, S. G.
Volume preservation then and now.
News Physiol. Sci.
4:
169-171,
1989.[Abstract/Free Full Text]
22.
Stetson, D. L.,
S. A. Lewis,
W. Alles,
and
J. B. Wade.
Evaluation by capacitance measurements of antidiuretic hormone-induced membrane area changes in toad bladder.
Biochim. Biophys. Acta
689:
267-274,
1982[Medline].
23.
Van Driessche, W.
Noise and impedance analysis.
In: Methods in Membrane and Transporter Research, edited by J. A. Schafer,
G. Giebisch,
P. Kristensen,
and H. H. Ussing. Austin, TX: Landes, 1994.
24.
Van Driessche, W.
Lidocaine blockade of basolateral potassium channels in the amphibian urinary bladder.
J. Physiol. (Lond.)
381:
575-593,
1986[Abstract].
25.
Van Driessche, W., and S. D. Hillyard. Quinidine
blockage of K+ channels in the
basolateral membrane of larval bullfrog skin.
Pflügers Archiv 405, Suppl. 1: S77-S82, 1985.
26.
Van Driessche, W.,
S. D. Hillyard,
and
H. Cantiello.
Investigations of basolateral K+ channels of the tadpole skin epithelium with noise analysis (Abstract).
Federation Proc.
44:
1567,
1985.
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