Hypotonicity activates a lanthanide-sensitive pathway for
K+ release in A6
epithelia
Patrick
De Smet,
Jinqing
Li, and
Willy
Van Driessche
Laboratory of Physiology, Katholieke Universiteit Leuven, Campus
Gasthuisberg, B-3000 Louvain, Belgium
 |
ABSTRACT |
The nature of the pathway for
K+ release activated during
regulatory volume decrease (RVD) in A6 epithelia was investigated by
measuring cell thickness (Tc) as
an index of cell volume and by probing
K+ efflux with
86Rb as tracer for
K+
(RRb). Cell swelling was induced
by sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH2O). Experiments were
performed in the absence of Na+
transport. Apical RRb was
negligible in iso- and hyposmotic conditions. On the other hand,
osmotic shock increased basolateral
RRb
(RblRb) rapidly, reaching a maximum 7 min
after the peak in Tc. Quinine (0.5 mM) completely inhibited RVD and RblRb.
Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine
(0.2 mM) did not exert a noticeable effect. The
K+ channel blocker
Ba2+ (30 mM) delayed RVD but could
not prevent complete volume recovery. Cs+ inhibited RVD noticeably at
concentrations <40 mM. With large Cs+ concentrations (>40 mM), the
initial osmometric swelling was followed by a gradual increase of
Tc, suggesting activation of Cs+ influx. Chronic exposure of
the basolateral surface to 0.5 mM La3+ or
Gd3+ completely abolished RVD and
RblRb. Acute administration of
lanthanides at the time of osmolality decrease did not affect the
initial phase of RVD and reduced RblRb
only slightly. Apical Gd3+ exerted
an inhibitory effect on RVD and RblRb.
The effect of Gd3+ should
therefore be localized at an intracellular site. The role of
Ca2+ entry could be excluded by
failure of extracellular Ca2+
removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administered
Mg2+ (0.5 mM) inhibited RVD and
RblRb by ~50%. These data suggest that
K+ excretion during RVD occurs
through a rather poorly selective pathway that does not seem to be
directly activated by membrane stretch.
barium; hypotonic shock; rubidium-86 efflux; gadolinium; cesium; lanthanum; magnesium; calcium; regulatory volume decrease
 |
INTRODUCTION |
THE CELLULAR INTEGRITY and epithelial structure of A6
epithelial cells are protected against extracellular osmotic
perturbations by activation of volume regulatory mechanisms. Similar to
many other tissues (22, 27, 30), this renal epithelial cell line exhibits a biphasic response to an abrupt reduction of the
extracellular osmolality. During the fast initial phase, cell volume
increases rapidly as a result of the equilibration of water across the
basolateral membrane, which has a large hydraulic conductivity. During
the following phase, termed regulatory volume decrease (RVD), efflux of
intracellular solutes and osmotically obliged water causes the cells to
shrink to their initial size.
Previous studies demonstrated that A6 epithelia regulate their volume
by excreting predominantly inorganic ions, mainly
K+ and
Cl
, and to a smaller extent
by the loss of amino acids (8, 9). In addition, these studies showed
that swelling-activated K+ and
Cl
excretion occurred
through conductive pathways that are highly sensitive to quinine but
rather resistant to Ba2+. In many
different cell types, mechanical stretch of the membrane as a result of
cell swelling was thought to trigger the activation of the
volume-activated pathways. These mechanosensitive channels not only
mediate inorganic ion efflux but also have a substantial permeability
for organic anions and uncharged osmolytes (20, 31). Micromolar
concentrations of La3+ or
Gd3+ block this pathway from the
intracellular side (6, 19, 41). Besides lanthanides,
Ca2+ and
Mg2+ were also found to inhibit
stretch-activated channels (SACs) (40, 41). On the other hand, volume
regulation in many cell types is mediated by
Ca2+ influx through
stretch-activated nonselective channels (7), whereas these
Ca2+-permeable SACs were proposed
to be the sensing elements of the hypotonic cell swelling-induced
Ca2+ increase (6).
The present study aims to further investigate the properties of the
volume-activated pathway for K+
efflux probed by the use of 86Rb
as a substitute for K+. Evidence
is given for the activation of a poorly selective cation channel
responsible for K+ excretion
during RVD, which, as far as its sensitivity to lanthanides is
concerned, has some resemblance to the SACs. Although the activation of
the channel depends on cell swelling, the maximum
86Rb efflux is noticeably delayed
compared with the peak in cell volume, suggesting that membrane stretch
is not directly involved in the activation of the volume regulatory
mechanism.
 |
MATERIALS AND METHODS |
Cell preparation.
As described previously (34), A6 cells obtained from Dr. J. P. Johnson
(University of Pittsburgh, Pittsburgh, PA) were cultured on permeable
filter supports (pore size = 0.2 µm; Nunc Intermed, Roskilde,
Denmark). For this study we utilized passages
103-113. The cells were seeded at a density of
105
cells/cm2 and used for our
experiments after 10-20 days of growth.
Cell thickness measurements.
Before the cells were seeded, the filters were coated with
1-µm-diameter fluorescent microspheres (catalog no. L-5081, Latex Fluospheres, sulfate, Molecular Probes, Eugene, OR). The apical surface
of the epithelium was labeled with fluorescent avidin-coated microbeads
(catalog no. F-8776, Molecular Probes). Cell thickness (Tc) was recorded as the
vertical distance between the lower and the upper beads, as described
previously (36). Taking into account the diameter of the microspheres,
we corrected the Tc values by subtracting 1 µm from the measured thickness. An improved version of
the software used in the present experiments enabled us to record
simultaneously and continuously Tc
of up to 20 cells. Only records where
Tc was followed over the entire
duration of the experiments were used for evaluation of cell volume.
Cell volume is expressed as percentage of the value recorded just
before the hyposmotic challenge is imposed. The different periods of
the experiment were precisely timed to enable us to average the
Tc records.
Tc was measured in the absence of
apical Na+. Transepithelial
conductance
(Gt) was
recorded at 14-s intervals with 5-mV pulses under voltage clamp. Under
these conditions, Gt mainly
reflects the conductance of the paracellular pathway. Continuous
monitoring of Gt
enabled us to verify the integrity of the structure of the epithelium.
86Rb efflux experiments.
Cells were loaded by exposure of the basolateral surface to a
K+-free isotonic solution that
contained 50 µCi of 86Rb. The
loading solution had the same composition as the control Cl
solution (Table
1), with
KHCO3 replaced by
NaHCO3. Depending on the specific
activity of the isotope, the Rb+
concentration varied between 0.6 and 1.3 mM. Tissues were removed from
the filter cups and mounted in a two-compartment chamber. The membrane
area exposed to the perfusates was 1.54 cm2. Both compartments were
continuously perfused. Rapid basolateral washout was achieved by
vigorous stirring with a motor-driven magnetic stirring bar.
The apical and basolateral perfusates were collected in
test tubes that were replaced at 1-min (Fig.
1) or 3-min intervals.
86Rb was measured by gamma
counting. The data are presented as release per unit time and expressed
as radioactivity released in each collected sample as percentage of the
total amount in the cells at that time. At the end of the experiment
the cells were lysed with a 5% (wt/vol) TCA solution, and five
additional samples of the chamber perfusate were collected to determine
the total amount accumulated in the cells, which was on average
105 counts.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Time course of 86Rb efflux and
volume changes during a hypotonic shock. Experiments were done with
HCO 3-buffered solutions.
86Rb release
(RRb) was sampled at 1-min time
intervals. A:
RRb increased from 0.17 ± 0.02 min 1 in isosmotic
conditions to a peak of 5.49 ± 0.23 min 1 [number of
experiments (N) = 4] 11 min
after initiation of hypotonic challenge. Apical RRb
(RapRb) was 0.12 ± 0.01 min 1 in isosmotic
conditions and was not noticeably affected by hypotonic shock. Cell
thickness (Tc) increased to
164% and reached its peak 4 min after osmolality was reduced. Mean
absolute value of Tc in isosmotic
conditions is 6.31 ± 0.19 µm
[N = 6, number of cells
(n) = 73].
Tc data recorded during final
isosmotic period are omitted for clarity.
RblRb, basolateral
RRb;
bl, basolateral osmolality.
B: relation between
Tc and
RRb values recorded during
hypotonic period. Even after volume recovery,
RblRb remained markedly above its
control.
|
|
Experimental protocols.
For cell volume and isotope efflux measurements, cells were subjected
to hypotonic conditions only after a stable baseline in isotonic
conditions (260 mosmol/kgH2O) was
recorded. The last 25- or 30-min period of this phase is shown. During
the subsequent 60-min period, we reduced the basolateral osmolality
(
bl) to 140 mosmol/kgH2O;
bl was restored to 260 mosmol/kgH2O during the final
30-min period.
Solutions.
The composition of the apical and basolateral solutions is depicted in
Table 1. Cl
was the
principal anion in all solutions. Apical solutions were hyposmotic and
Na+ free, with
N-methyl-D-glucamine
(NMDG+) as the major cation. In
experiments with large concentrations of inorganic blockers, we kept
the osmolality constant by reducing the
NMDG+ concentration. The
osmolality of the solutions was verified with a cryoscopic osmometer
(Osmomat 030, Gonotec, Berlin, Germany). Osmolality of the iso- and
hyposmotic basolateral solutions was 260 ± 2 and 140 ± 2 mosmol/kgH2O, respectively.
Basolateral Ca2+-free experiments
as well as experiments with Gd3+
and La3+ were executed with
HEPES-buffered solutions. Quinine (0.5 mM), lidocaine (0.2 mM), and
verapamil (0.2 mM) were added to both sides of the epithelium.
Ba2+ (30 mM) and
Mg2+ (0.5 or 5 mM) were added as
Cl
salts. All experiments
were performed at room temperature (22°C) in air-conditioned rooms.
Values are means ± SE; N is the
number of experiments, and n is the
number of cells (or Tc records)
used to calculate the averaged curves.
 |
RESULTS |
To avoid interference of Na+
uptake with the volume regulatory mechanisms (10), we used
Na+-free apical solutions in all
experiments. Because the water permeability of the apical barrier is
negligible (10), we could keep the osmolality of the apical bath at 140 mosmol/kgH2O during the entire experiment. With this procedure, no osmotic or ionic gradients are
created during the hyposmotic phase of the experiment. Transepithelial anion gradients during the hypotonic phase were also avoided by matching the anion composition of the apical and basolateral hyposmotic solutions; in this way, possible interference of transepithelial anion
currents with the volume regulatory processes was prevented. Cell
swelling was invoked by lowering the
bl.
Figure 1A compares the time course
of Tc and
RRb recorded with NMDG-Cl and NaCl
solutions applied to the apical and basolateral sides, respectively. On
reduction of
bl, cells swell
and regulate their volume completely back to their normal size. The
peak Tc was achieved 4 min after
the osmolality was reduced. The time constant of the
Tc decay is 4.6 min (Table
2). The
RRb data in Fig.
1A demonstrate that in isosmotic
conditions the basolateral 86Rb
release (RblRb) is relatively small (0.17 ± 0.02 min
1).
RblRb was drastically augmented by
hypotonicity and reached a maximum (5.49 ± 0.23 min
1) 11 min after
initiation of the osmotic perturbation. This relatively large delay in
RblRb suggests that cell volume expansion does not directly activate the pathway for cation release. This is
conspicuously illustrated in Fig. 1B,
which depicts the relation between
Tc and
RblRb during the hypotonic phase of the
experiment. Figure 1B shows that the
maximal isotope efflux was recorded only after
Tc declined to 15% above control.
Furthermore, during the last phase of the hypotonic period, when cell
volume had completely recovered, RblRb
remained clearly above its control. On the other hand,
RblRb only reached 0.92 min
1 (20% of its full
response) at the moment when we recorded the maximal
Tc increase. The data in Fig.
1A also show that the apical efflux
of 86Rb was extremely low (0.06 ± 0.01 min
1) during
the isosmotic period. Moreover, during the hypotonic treatment of the
tissue, apical efflux of 86Rb did
not increase (0.08 ± 0.01 min
1). Because of the
negligible amount of 86Rb release
at the apical barrier, we confine this report to
RblRb.
During the loading period of the
86Rb release experiments, the
tissues accumulated large amounts of cold
Rb+. We calculated that, depending
on the activity of 86Rb,
intracellular accumulated Rb+
concentration was as much as 50-85 mM. Therefore, we verified whether volume regulation was affected by this marked change in cell
K+ and loaded cells with cold
Rb+ to obtain concentrations
similar to those reached in the tracer experiments. Figure
2 compares volume recovery in control with that in Rb+-loaded tissues. It is
clear that the replacement of large amounts of cellular
K+ by
Rb+ did not affect the RVD. This
finding justifies the use of Rb+
as a tracer for studying K+
release during RVD.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
Volume regulation in cells loaded with
Rb+. Cells were loaded with cold
Rb+ as in
86Rb release experiments. Before
and during experiment, apical and basolateral sides of epithelium were
exposed to solutions in which Rb+
(2.5 mM) replaced K+. Incubation
time before hypotonic challenge was 60 min. From measurements of total
86Rb accumulated in cells and
concentration of cold Rb+ in
loading solution, we estimated a cell
Rb+ concentration of 65 mM.
Tc values in isosmotic conditions
are 6.31 ± 0.19 (N = 6, n = 73) and 6.81 ± 0.23 µm
(N = 6, n = 54) for control (Ctrl) and
Rb+-loaded cells, respectively.
|
|
Previously, we demonstrated that submillimolar concentrations of
quinine abolish the RVD completely (9, 26), whereas with high
concentrations of Ba2+, cells
remained swollen during the 25-min hyposmotic period (9). In this study
(Fig. 3) we show that
Ba2+ is not able to prevent RVD
and that volume recovery can take place over an extended period of 60 min. With Ba2+ as well as quinine,
the initial volume increase was markedly larger than in control (Table
2). The increase of Tc above the expected ideal osmometric swelling could be due to an increased osmotic
activity of the cell as a result of a change in cell composition and/or complex formation of the intracellular
solutes. Alternatively, it is conceivable that
Ba2+ and quinine block an osmolyte
release that might take place during the rising phase of
Tc.
Ba2+ markedly retarded the volume
recovery (time constant = 20.0 min
1), and at the end of
the hypotonic phase, Tc remained
clearly above control. RblRb was
noticeably inhibited by Ba2+ and
almost completely abolished by quinine. The peak value of RblRb of 4.69 ± 0.25 min
1 recorded in control
was reduced to 2.27 ± 0.08 min
1 in the presence of
Ba2+. This reduced
RblRb still enables an almost complete, but markedly delayed, RVD. On the other hand, quinine almost completely blocked RblRb and totally abolished the
RVD. This result supports the idea that quinine is an efficient blocker
of volume-activated K+ channels,
whereas Ba2+ is a poor inhibitor
of this channel type. Volume-activated
K+ channels studied in epithelia
by permeabilization of apical membranes are inhibited not only by
quinine (14) but also by verapamil (21) and even more effectively by
lidocaine (33). In these studies the apical membranes were treated with
nystatin in the presence of high
K+ concentrations, which were
assumed to increase cell K+ and
cell volume. Figure 4 illustrates the
effect of these agents on volume recovery. It is clear that lidocaine
only slightly affected the RVD, whereas verapamil exerted a marked
effect on volume recovery. These supposedly contradictory effects are
most likely due to the fact that volume increase induced by osmotic
cell swelling gives rise to the activation of pathways that differ from
those stimulated by permeabilization of the apical membranes.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of quinine and Ba2+ on
volume regulation and 86Rb efflux.
BaCl2 (30 mM,
Ba2+) and 0.5 mM quinine (QNE)
were administered to both compartments. Quinine was added to control
solution, and Ba2+ was used in 30 Ba2+ solution (Table 1).
A:
Tc records. In control and
Ba2+-treated tissues,
Tc reached a peak and returned
toward control. Peak values were 164 and 200% for control and
Ba2+-treated cells, respectively.
At end of hypotonic phase, Tc
reached plateau values of 98 and 118% in control and during
Ba2+ inhibition, respectively. In
presence of quinine, Tc initially
increased to 205% and gradually increased further to 220% at end of
hyposmotic shock. Initial absolute
Tc values are 6.31 ± 0.19 (N = 6, n = 73), 4.91 ± 0.20 (N = 6, n = 83), and 6.33 ± 0.74 µm
(N = 6, n = 78) for control and
Ba2+- and quinine-treated cells,
respectively. B:
RblRb sampled at 3-min intervals.
RapRb was negligible and not affected by
osmotic perturbation (see Fig. 1). For clarity,
RapRb is omitted. In isosmotic
conditions, quinine and Ba2+
reduced basal RblRb from 0.46 min 1 (control) to 0.36 and
0.16 min 1, respectively. In
control, RblRb reached a peak of 4.69 min 1.
Ba2+ reduced peak in
RblRb to 2.27 min 1, whereas quinine
almost completely abolished 86Rb
release.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of lidocaine and verapamil on volume recovery. Lidocaine (0.2 mM) and verapamil (0.2 mM) were added to both sides of epithelium. Mean
absolute Tc values in isosmotic
conditions are 6.31 ± 0.19 (N = 6, n = 73), 5.57 ± 0.22 (N = 6, n = 86), and 5.26 ± 0.25 µm
(N = 5, n = 36) for control and verapamil- and
lidocaine-treated cells, respectively.
|
|
Our data show that only quinine is able to block completely the RVD and
the related 86Rb efflux, whereas
Ba2+, a potent
K+ channel blocker, was unable to
prevent volume recovery. Therefore, we assayed the effect of
Cs+, which is also known to
inhibit certain types of K+
channels (11). Figure
5A
illustrates the effect of 10, 30, and 60 mM
Cs+ on volume regulation. It is
clear that high concentrations (30 mM) of
Cs+ are required to block volume
regulation. On the other hand, after replacement of 60 mM
Na+ in the basolateral solution by
Cs+, hypotonicity leads to a
massive increase of Tc. As early
as 25 min after the beginning of the osmotic challenge, this
augmentation of cell volume increased
Tc to 305 ± 14%. This extreme
increase in cell volume could be caused by an additional cellular
accumulation of osmolytes during hypotonicity, which under these
conditions is most likely caused by the entry of
Cs+ across the basolateral
membrane. The replacement of 60 mM basolateral Na+ by
Cs+ did not affect cell volume in
isosmotic conditions, indicating that the basolateral membranes are
impermeable for Cs+ in isosmotic
conditions. However, hyposmotic swelling seems to open a pathway with
poor selectivity that enables Cs+
to enter the cell. Comparison of
86Rb release data and
Tc measurements in Fig.
5B confirms this idea. Here 40 mM
Cs+ blocked the RVD almost
completely. On the other hand, RblRb increased to a level approaching the peak release in control and remained relatively high during the entire osmotic challenge. If
Cs+ acted as a pure blocker of
K+ efflux,
RblRb would have been reduced, as in the
experiment with quinine (Fig. 3). Thus
Cs+ seems to permeate the cell
membrane, thereby keeping intracellular cation content high and, in
this way, preventing net osmolyte loss. The lack of cellular osmolyte
decrease keeps cells swollen, as demonstrated in the
Tc record, which maintains the
86Rb release pathway activated.
Also, in the initial phase of the hyposmotic shock, the increase of
RblRb is noticeably delayed compared with
control. This observation suggests a direct inhibitory effect of
Cs+ on the
86Rb efflux.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Influence of Cs+ on regulatory
volume decrease (RVD) and 86Rb
efflux during a hypotonic shock. Solution is described in Table 1. To
avoid a transepithelial gradient, both sides of epithelium were exposed
to Cs+.
A: inhibitory effect of 10, 30, and 60 mM Cs+ on RVD. Initial absolute
Tc values are 6.31 ± 0.19 (N = 6, n = 73), 5.42 ± 0.30 (N = 3, n = 47), 7.76 ± 0.19 (N = 4, n = 73), and 7.45 ± 0.40 µm
(N = 4, n = 46) for 0 (control), 10, 30, and
60 mM Cs+, respectively.
B: RblRb
and Tc traces in control and in
presence of 40 mM Cs+. At end of
hyposmotic pulse, RblRb was 0.46 ± 0.02 (N = 7) and 1.65 ± 0.08 min 1
(N = 4) in control and
Cs+-treated cells, respectively.
Initial absolute Tc with 40 mM
Cs+ is 5.76 ± 0.22 µm
(N = 5, n = 57).
|
|
Inasmuch as classical K+ channel
blockers (Ba2+ and
Cs+) were not able to abolish
volume regulation and because the
Cs+ experiments indicated the
rather poorly selective nature of the regulatory pathway, we decided to
test the effect of lanthanides, which are known to block poorly
selective and stretch-activated ion channels in other tissues (7, 40,
41). Figure 6 demonstrates that chronic
exposure of the basolateral side to 0.5 mM
Gd3+ completely abolished volume
regulation and RblRb. On the other hand,
the peak in the RblRb response was only
slightly reduced if Gd3+ was
acutely added to the bath when the osmolality was reduced. However,
acute application also blocked the RblRb
fully toward the end of the osmotic pulse, indicating a marked delay in
the inhibitory action of the lanthanide. This delay also appears from
the Tc record (Fig.
6A), showing a clear onset of volume regulation, which, however, was interrupted after
Tc had declined from 171 to 122%.
This striking difference between the two modes of application suggests
an intracellular site of action. Whether Gd3+ exerts its action on the
signaling pathway or directly on the cation channels remains
unresolved. Effects similar to those shown with
Gd3+ were obtained with
La3+. Figure
6C compares the chronic and acute
effects on RVD of addition of the lanthanide at 0.5 mM to the
basolateral side. Here also chronic preincubation with 0.5 mM
lanthanide fully blocked RVD, whereas acute administration did not
affect the initial phase of volume recovery. This result confirms an
intracellular site of action of the lanthanides.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6.
Chronic and acute effect of 0.5 mM basolateral
Gd3+ and
La3+. In chronic experiment,
lanthanide was present in iso- and hyposmotic solution and added 60 min
before hypotonic challenge. Acute effects of
Gd3+ and
La3+ were tested by adding
lanthanide when osmolality was reduced. Experiments were performed in
HEPES-buffered solutions (Table 1). A:
effect of Gd3+ on volume
regulation. Initial absolute Tc
values are 7.46 ± 0.25 (N = 6, n = 59), 6.27 ± 0.35 (N = 6, n = 51), and 6.06 ± 0.22 µm
(N = 5, n = 59) for control and acutely and
chronically administered Gd3+,
respectively. B: effect of
Gd3+ on
RblRb. Peak
RblRb was 5.17 (N = 4), 3.65 (N = 6), and 0.47 min 1
(N = 4) for control and acutely and
chronically administered Gd3+,
respectively. C: effect of
La3+ on volume regulation. Initial
absolute Tc values are 7.46 ± 0.25 (N = 6, n = 59), 7.07 ± 0.22 (N = 7, n = 70), and 5.33 ± 0.25 µm
(N = 6, n = 68) for control and acutely and
chronically administered La3+,
respectively.
|
|
The fact that Gd3+ acts on an
intracellular site implies that the lanthanide could enter the cell and
that the cell membrane has a finite permeability for this cation. The
permeability of the basolateral membrane for
Gd3+ also appears in experiments
where the effect of Gd3+ on the
86Rb release is measured in
isotonic conditions (Fig. 7). The addition of 0.5 mM Gd3+ to the basolateral
side transiently increased RblRb, which
reached a maximum 15-18 min after addition of the lanthanide. The
apical release of 86Rb remained
unaffected by this treatment. We also tested the effect of 10 µM
nifedipine on the basal and
Gd3+-induced
RblRb. The data in Fig. 7 show that, in
the absence of Gd3+, nifedipine
only slightly decreased RblRb from 0.19 ± 0.01 to 0.15 ± 0.01 min
1. On the other hand,
the peak value reached during Gd3+
treatment was reduced from 0.81 ± 0.08 to 0.45 ± 0.07 min
1
(N = 4). This result is in agreement
with the idea that Gd3+ entry
proceeds at least partly through L-type
Ca2+ channels. Addition of
Gd3+ to the basolateral solution
in hypotonic conditions did not noticeably increase
RblRb (data not shown). This result
suggests that RblRb after acute
administration should not be overestimated by an effect of
Gd3+.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of basolaterally applied
Gd3+ on
Rb+ efflux in isosmotic
conditions. Gd3+ (0.5 mM) was
present during period indicated by open bar. Experiments were performed
in HEPES-buffered solutions. Effect of nifedipine was tested by
exposing basolateral membrane to 10 µM nifedipine during entire
experiment. Only effect of the
Ca2+ channel blocker on
RblRb is shown, because
RapRb was negligible.
|
|
Experiments with reduced amounts of
Gd3+ (Fig.
8) demonstrated that 0.5 mM was needed to
block the RVD completely. The chronic addition of 0.2 mM
Gd3+ to the basolateral surface
reduced the RVD markedly. During the hypotonic shock,
Tc increased to 177% and remained
markedly above control, reaching 136% at the end of the recovery
phase. Gd3+ (10 µM) had only a
small effect on RVD, which returned
Tc to 108%.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 8.
Dose dependence of basolaterally applied
Gd3+.
Tc was recorded with addition of
0.01, 0.2, and 0.5 mM Gd3+ to
basolateral side. Experiments were performed in HEPES-buffered
Cl solutions. Numerical
data are shown in Table 2. Mean absolute
Tc values in isosmotic conditions
are 7.46 ± 0.25 (N = 6, n = 59), 8.67 ± 0.25 (N = 5, n = 59), 7.34 ± 0.60 (N = 2, n = 18), and 6.27 ± 0.35 µm
(N = 6, n = 51) for control and 0.01, 0.2, and
0.5 mM Gd3+, respectively.
|
|
Inasmuch as the comparison of effects of an acute and chronic addition
of Gd3+ suggested an intracellular
effect, we also tested whether
Gd3+ could reach this site when
added to the apical solution. Figure 9
illustrates effects of chronic application of
Gd3+ to the apical surface of the
epithelium. The Tc records show that the lanthanide at 0.5 mM hardly affected volume recovery. Also
RblRb was not influenced by this amount
of Gd3+. A 10-fold higher dose of
this trivalent cation was needed to reduce
RblRb markedly and to exert a pronounced
effect on volume recovery. The fact that this rather high dose of
apical Gd3+ could exert this
inhibitory effect demonstrates that the apical border has a finite
permeability for the lanthanide, which enables the cation to reach its
intracellularly located inhibitory site.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 9.
Chronic application of Gd3+ to
apical surface. Experiments were performed in HEPES-buffered solutions.
A:
Tc in control and with 0.5 and 5 mM Gd3+ added to apical perfusate.
Initial absolute Tc values are
7.46 ± 0.25 (N = 6, n = 59), 7.20 ± 0.22 (N = 8, n = 114), and 7.17 ± 0.33 µm
(N = 5, n = 60) for control and 0.5 and 5 mM
Gd3+, respectively.
B: RblRb
with 0.5 and 5 mM Gd3+ added to
perfusate. Peak RblRb values were 5.17 ± 0.21 (N = 4) and 2.70 ± 0.09 min 1
(N = 4) for 0.5 and 5 mM apical
Gd3+, respectively.
|
|
Several reports (1, 6, 7, 40) demonstrate that lanthanides exert an
inhibitory effect on Ca2+ entry
through SACs. Therefore, it is conceivable that the effects of
Gd3+ and
La3+ added to the basolateral bath
are related to the blockage of Ca2+ entry. To verify this
hypothesis, we performed experiments with basolateral
Ca2+-free solutions prepared by
removing Ca2+ and chelating the
remaining Ca2+ with 0.5 mM EGTA.
In one type of experiment we added 0.5 mM
Gd3+ to the apical solution. This
treatment kept the transepithelial resistance above 300
· cm2,
indicating that the epithelial structure was maintained. Similarly, an
inhibition by La3+ of the increase
in Gt after
Ca2+ removal has recently been
reported for the epithelium of frog urinary bladder (25). However, the
nature of the protective effect of
Gd3+ remains unexplained. In the
absence of the lanthanide in the apical bath, we limited the duration
of the hyposmotic shock to 30 min because of an extremely large drop in
transepithelial resistance. The records in Fig.
10 demonstrate a considerable increase of
the peak value of Tc, indicating
that Ca2+ removal markedly
augments the osmotic activity of the cell. During the hypotonic period,
cell volume almost completely recovered, with a time constant of 8.2 min (Table 2). In the experiment with
Gd3+ added to the apical surface,
where we could extend the osmotic perturbation to 60 min, volume
recovery was noticeably delayed but still almost complete. These
results demonstrate that the development of the RVD does not require
basolateral Ca2+ entry. Moreover,
the inhibitory effect of the lanthanides does not seem to occur through
the blockage of Ca2+ entry.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of Ca2+ removal from
basolateral bathing solution. Control trace is from Fig. 6. Basolateral
Ca2+-free solutions were prepared
by removal of Ca2+ and addition of
0.5 mM EGTA. In one type of experiment, we added 0.5 mM
Gd3+ to apical solution. This
treatment kept transepithelial resistance high (>300
· cm2)
during entire hyposmotic shock of 60 min. In experiment performed
without Gd3+, we limited hypotonic
exposure time to 30 min because of a marked drop of transepithelial
resistance to 270 · cm2. Mean
absolute Tc values in isosmotic
conditions are 7.46 ± 0.25 (N = 6, n = 59), 5.76 ± 0.29 (N = 2, n = 27), and 4.78 ± 0.32 µm
(N = 3, n = 30) for control, 0 Ca2+, and 0 Ca2+ + apical
Gd3+, respectively.
|
|
Data reported by Basavappa and Strange (2) demonstrate that
volume-activated channels are sensitive to
Mg2+. Effects of the acutely and
chronically administered Mg2+ on
volume recovery and RblRb are illustrated
in Fig. 11. In contrast to the
lanthanides, the action of Mg2+
did not depend on the preincubation time of the divalent cation. Indeed, acute as well as chronic administration delayed volume recovery, which is reflected in the time constant of the
Tc decay (Table 2). Moreover, the
volume recovery was incomplete in both types of experiments, keeping
Tc 17% above control. The effects on volume recovery are reflected in the
86Rb release, which demonstrates
an almost identical reduction of RblRb
for both types of treatment (Table 2). Inasmuch as we did not find any
difference between the acute and chronic administration, we cannot
exclude an extracellular effect of the divalent cation. Therefore, we
tested a 10-fold higher dose. If Mg2+ exerted its effect directly
on an extracellular site of the pathway for osmolyte release, the
increased dose would exert a more pronounced effect on RVD as well as
RblRb. However, increasing the dose of
Mg2+ from 0.5 to 5 mM did not
improve the inhibitory effect of
Mg2+ (Table 2).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 11.
Chronic and acute effects of 0.5 mM
Mg2+. Divalent cation was added to
both sides of epithelium. In acute experiment,
Mg2+ was added when basolateral
osmolality was reduced. In chronic experiment, tissue was preincubated
with Mg2+ 30 min before hyposmotic
shock. A:
Tc. Mean absolute
Tc values in isosmotic conditions
are 6.31 ± 0.19 (N = 6, n = 73), 7.48 ± 0.43 (N = 3, n = 23), and 7.14 ± 0.32 µm
(N = 4, n = 29) for control and acutely and
chronically administered Mg2+,
respectively. B: effect of
Mg2+ on
RblRb. Peak
RblRb was 2.40 (N = 4) and 2.03 min 1
(N = 4) for acutely and chronically
administered Mg2+, respectively.
|
|
 |
DISCUSSION |
We have shown that cell swelling caused by a basolateral hyposmotic
challenge results in a substantial increase of the unidirectional efflux of K+
(86Rb), which takes place during
the volume regulatory phase. A striking agreement between the
86Rb efflux data and the
Tc records was observed: Except
for the Cs+ experiments, the
inhibition of volume recovery was reflected in all cases in a reduction
of the 86Rb release. Quinine
completely blocks the RVD and 86Rb
efflux, whereas lidocaine does not noticeably affect volume regulation.
Also Ba2+, even at high
concentrations, only displays a weak inhibitory effect. Furthermore,
chronic administration of lanthanides
(La3+ and
Gd3+) blocks
RblRb, resulting in a complete inhibition of the RVD. No difference was observed between chronic and acute administration of Mg2+, which
significantly affected RVD and RblRb. The
inhibition of RVD observed with high
Cs+ concentrations without
blocking the 86Rb release suggests
that hypotonicity opens a pathway for the entrance of this monovalent
cation into the cell. These results suggest that hypotonicity activates
a poorly selective cation channel that is highly sensitive to
lanthanides and quinine and markedly inhibited by
Mg2+.
In a variety of tissues, Ba2+
reduces the basolateral K+
conductance and affects volume regulation in a concentration-dependent manner (12, 39). We recorded a delayed and partially inhibited RVD with
30 mM Ba2+. The failure of
Ba2+ to exert a distinct effect on
RVD was shown in a previous study, where lower concentrations (1-5
mM) of the divalent cation were utilized (9). The low potency of
Ba2+ in blocking volume regulation
was also shown in HeLa cells (20), rabbit proximal straight tubules
(38, 39), cultured astrocytes (29), and nonpolarized A6 cells (12). The
failure of Ba2+ to inhibit volume
regulation was also found in experiments where the basolateral
osmolality was gradually reduced, which enabled the cell to adjust its
volume without noticeable cell swelling (35). The low sensitivity to
Ba2+ of the swelling-activated
pathway compared with the effect of the divalent cation on the native
K+ permeability of the basolateral
membrane suggests that cell swelling opens a pathway for
K+ excretion that has
characteristics completely different from the native
K+ channels. A similar conclusion
was derived from experiments in which the basolateral
K+ permeability was studied after
the apical border was permeabilized with nystatin (13, 14). These
studies mainly discriminated between the pathways on the basis of their
degree of sensitivity to quinine.
Within this context, we compared pharmacological effects on volume
regulation and on basolateral quinine-sensitive
K+ channels studied in
nystatin-treated tissues. Several reports demonstrated inhibitory
effects of quinine as well as verapamil and lidocaine (13, 21, 33, 37).
In these studies, K+ currents were
generated by establishment of an electrochemical gradient for
K+ and by permeabilization of the
apical membrane with ionophores. The latter treatment enabled
K+ to cross the apical barrier.
K+ entering the cell in this way
was assumed to cause cell swelling and activation of the
quinine-sensitive pathway. Therefore, the quinine-sensitive pathway and
volume regulation should display similar pharmacological properties.
Our results, however, showed that lidocaine, which was the most potent
inhibitor of the quinine-sensitive K+ channels in the studies
mentioned above, failed to have a significant effect on RVD. Therefore,
it appears that the basolateral K+
channels stimulated by permeabilization of the apical membrane and the
subsequent cell swelling are different from channels activated by the
hypotonic treatment. In this regard, besides its inhibitory effect
exerted on volume-activated K+
channels (12, 29), quinine has been found to inhibit nonselective cation channels in rat distal colon cells (15). The pharmacological resemblance between the nonselective pathway in rat colon and the
pathway for K+ excretion during
volume recovery is quite striking and agrees with the above-stated
hypothesis that volume recovery occurs through a poorly selective
cation pathway. On the other hand, it appears that lidocaine-sensitive
channels, activated by permeabilization of the apical membrane, are of
a different nature. However, besides the direct blockage of ion
channels, quinine might inhibit the volume-sensing mechanisms
and/or transduction pathways.
The fact that the swelling-activated
K+ release utilizes a pathway that
differs from the native K+
channels appears also from the Cs+
data. Several types of
K+-selective channels are capable
of binding Cs+ (5, 11), resulting
in a blockade of K+ current with
millimolar concentrations of the inhibitor. Our data show that high
concentrations of extracellular
Cs+ were needed to reduce RVD
significantly (Fig. 5). Although volume recovery was completely
abolished at high Cs+
concentrations, the RblRb was not
blocked. On the contrary, in the presence of 40 mM
Cs+,
86Rb efflux was activated to a
degree similar to that in control and remained high during the entire
hypotonic period. The apparently contradictory effects of
Cs+ on RVD and
RblRb seem to find their origin in the combination of two opposing phenomena.
1) In the initial phase of the
hyposmotic shock, Cs+ clearly
inhibits RblRb, most likely by occupying
the binding site of Rb+ in the
channel. 2) Then, when the pathway
is fully activated, Cs+ permeates
through the channel and substitutes for the intracellular K+ and
Rb+, thereby keeping cell volume
high and enhancing the isotope efflux. The entrance of
Cs+ is favored by the
electrochemical driving force for
Cs+, which is inwardly directed.
This results in a gradual increase of cellular volume in the presence
of 60 mM Cs+. It is conceivable
that the Cs+ uptake at least
partly occurs through the
Na+-K+-ATPase,
as was reported for frog skeletal muscle (3). However, experiments not
shown here demonstrated that 0.1 mM ouabain did not prevent the gradual
increase of cell volume when the basolateral surface was exposed to 60 mM Cs+. This result supports the
evidence for the uptake of Cs+ via
the volume-activated pathway.
We found that the volume recovery was completely inhibited by
lanthanides, especially Gd3+,
which has been described as a potent blocker of stretch-activated K+ channels (SACs) (41). SACs act
as Ca2+ entry pathways and thus
contribute to the regulation of intracellular Ca2+ (1, 6). Therefore, an
indirect effect of the lanthanides on the RVD via the inhibition of
Ca2+ influx cannot be excluded.
Indeed, in many cell types, cell volume regulation is mediated by
Ca2+-activated
K+ channels (6, 7). Within this
context, the effect of Gd3+ on RVD
via the inhibition of Ca2+ entry
would presume the existence of SACs in the A6 epithelia. The
experiments depicted in Fig. 10 demonstrate that an almost complete
volume recovery could take place in the absence of extracellular Ca2+, suggesting that
Ca2+ entry does not play a major
role in volume regulation of the A6 epithelia. The initial volume
increase was much larger than in control, indicating that
Ca2+ removal markedly increases
the intracellular osmotic activity. Volume recovery was noticeably
delayed by using apical Gd3+ to
maintain transepithelial resistance at a high level and, presumably, conserving in this way the integrity of the epithelial structure.
The marked differences between the effects of acute and chronic
administration of lanthanides on the RVD and
RblRb suggest an intracellular site of
action. The intracellular concentration of
Gd3+ reported to inhibit
mechanosensitive ion channels in the yeast plasma membrane was 10 µM
(18). It is conceivable that such an intracellular concentration is
reached by addition of 500 µM Gd3+ to the basolateral bath.
Evidence for a finite permeability of the cell membrane for
Gd3+ and, thus, for an increase of
the intracellular concentration was found in the marked transient
stimulation of RblRb in isotonic
conditions with an onset at ~12 min after administration of
Gd3+ (Fig. 7). The noticeable
delay in RblRb stimulation suggests that
the lanthanide enters the cell and possibly activates the
86Rb efflux by its action on
Ca2+-activated
K+ channels or by screening of
negative surface charges (41). The latter concept assumes that the
neutralization of negative surface charges reduces the local
concentration of the permeant monovalent cation, which results in a
transient increase of efflux of cations. The mechanism of action of
Gd3+ and
La3+ that causes the inhibition of
RVD and RblRb during hypotonic conditions
is unclear. At least two different mechanisms can be proposed. The
first is a direct inhibitory effect of the lanthanides on the
volume-activated channel. In this relation, Gd3+ inhibits SACs of very
different conductances such as Xenopus (41), yeast (19), and bacteria (4). However, it seems unlikely that the
inhibition of channels with such different sizes or structure occurs
via a direct blocking mechanism. Alternatively, as proposed by Berrier
et al. (4), the lanthanides could affect the stretch sensor that
controls the gating of the cation pathway. Our data do not enable us to
distinguish between these two possibilities.
Relatively small extracellular
Mg2+ concentrations (0.5 mM)
exhibit a significant inhibitory effect on volume recovery (Fig. 11).
Increasing the amount of the divalent cation did not augment the effect
on RVD and RblRb.
Mg2+ acts much faster than
Gd3+ and
La3+. Indeed, in contrast to the
lanthanides, with the divalent cation we found no difference in the
responses to acute and chronic administration.
Our data show that the response of RblRb
clearly lags behind the increase of
Tc invoked by the hypotonic
challenge (Fig. 1). This observation demonstrates that the
86Rb efflux is not directly
related to the size of the cell, which can increase by changing its
shape or by increasing the membrane area. If cell swelling causes
membrane stretch, this stimulus does not seem to directly activate the
osmolyte release in A6 epithelia. Similarly, membrane bending and thus
induced membrane stretch did not affect
K+-Cl
cotransport in rabbit red blood cells (23). Furthermore, Parker and
co-workers (28) demonstrated that volume regulatory mechanisms are
already activated in dog red blood cells at volumes 5-10% above
normal. In this cell type the accommodation of this additional volume
should be possible by relatively small changes in cell shape and thus
without a noticeable increase in membrane stretch. Alternatively, it
cannot be excluded that the initial phase of volume recovery is
mediated by the excretion of organic osmolytes (31), whereas inorganic
ion release accounts for the subsequent phase of the RVD. Within this
context, membrane stretch cannot be excluded as the trigger for
osmolyte excretion. Moreover, membrane stretch was suggested to trigger
the increase of protein tyrosine kinase, which was demonstrated to
regulate swelling-induced osmolyte excretion (32). Although the initial
activation step of protein tyrosine kinase could take place at the
moment cell volume reaches its maximum, different steps could delay the
activation of the pathway for osmolyte excretion in the transduction
system.
We found that, after an osmotic challenge of 120 mosmol/kgH2O, cell volume returns
to almost exactly its original size. The osmotic loss during such a
complete RVD cannot be attributed solely to KCl but implies a major
contribution of organic solutes (24, 31). The observation that
Gd3+,
La3+, and quinine are able to
completely block the RVD thus implies that these inhibitors not only
prevent the release of KCl but also abolish the organic osmolyte loss.
This observation favors the hypothesis of an action of the lanthanide
on the signal transduction pathway.
Two alternative mechanisms can be proposed for the mode of action of
the inhibitors. 1) As suggested
above, the lanthanides seem to exert their action on an intracellular
site, and it is well documented that quinine can reach intracellular
receptors. Therefore, it is conceivable that the inhibitors interfere
with an intracellularly located volume sensor or with the signal
transduction pathway, which activates the osmolyte release.
2) The inhibitors are known to be
blockers of nonselective pathways (15, 16). This action would agree
with the concept of a very unspecific large channel that enables the
excretion of all osmotic substances during RVD (20). Hall et al. (20)
suggested that this pore might be permeable for large anions and
cations. The activation of such a pathway could explain the partial but
pronounced volume recovery observed in
Cl
-depleted cells obtained
by using gluconate or sulfate as the main anion in the bathing
solutions (9).
 |
ACKNOWLEDGEMENTS |
We thank E. Larivière for executing the volume experiments,
R. Andries for manufacturing the specially designed Ussing chambers, and Ing G. Raskin for building the electronic equipment. The useful discussions with Dr. W. Zeiske are especially acknowledged.
 |
FOOTNOTES |
The project was financially supported by Fonds voor Geneeskundig
Wetenschappelijk Onderzoek Grant G.0235.95 and Levenslijn Program Grant
7.0030.94. P. De Smet is a postdoctoral fellow of the Fonds voor
Wetenschappelijk Onderzoek-Vlaanderen.
Address reprint requests to W. Van Driessche.
Received 6 November 1997; accepted in final form 30 March 1998.
 |
REFERENCES |
1.
Adorante, J. S.,
and
P. M. Cala.
Mechanisms of regulatory volume decrease in nonpigmented human ciliary epithelial cells.
Am. J. Physiol.
268 (Cell Physiol. 37):
C721-C731,
1995[Abstract/Free Full Text].
2.
Basavappa, S.,
and
K. Strange.
Regulation of VSOAC volume sensitivity by intracellular ATP.
J. Gen. Physiol.
110:
39A-40A,
1997.
3.
Beauge, L. A.,
A. Medici,
and
R. A. Sjodin.
The influence of external caesium ions on potassium efflux in frog skeletal muscle.
J. Physiol. (Lond.)
228:
1-11,
1973[Medline].
4.
Berrier, C.,
A. Coulombe,
I. Szabo,
M. Zoratti,
and
A. Ghazi.
Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch-activated channels in bacteria.
Eur. J. Biochem.
206:
559-565,
1992[Abstract].
5.
Cecchi, X.,
D. Wolff,
O. Alvarez,
and
R. Latorre.
Mechanisms of Cs+ blockade in a Ca2+-activated K+ channel from smooth muscle.
Biophys. J.
52:
707-716,
1987[Abstract].
6.
Chen, Y.,
S. M. Simasko,
J. Niggel,
J. W. Sigurdson,
and
F. Sachs.
Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1790-C1798,
1996[Abstract/Free Full Text].
7.
Christensen, O.
Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels.
Nature
330:
66-68,
1987[Medline].
8.
De Smet, P.,
J. Simaels,
P. E. Declercq,
and
W. Van Driessche.
Regulatory volume decrease in cultured kidney cells (A6): role of amino acids.
J. Gen. Physiol.
106:
525-542,
1995[Abstract].
9.
De Smet, P.,
J. Simaels,
and
W. Van Driessche.
Regulatory volume decrease in a renal distal tubular cell line (A6). I. Role of K+ and Cl
.
Pflügers Arch.
430:
936-944,
1995[Medline].
10.
De Smet, P.,
J. Simaels,
and
W. Van Driessche.
Regulatory volume decrease in a renal distal tubular cell line (A6). II. Effect of Na+ transport rate.
Pflügers Arch.
430:
945-953,
1995[Medline].
11.
De Wolf, I.,
and
W. Van Driessche.
Current-voltage relations of Cs+-inhibited K+ currents through the apical membrane of frog skin.
Pflügers Arch.
413:
111-117,
1988[Medline].
12.
Ehrenfeld, J.,
C. Raschi,
and
E. Brochiero.
Basolateral potassium membrane permeability of A6 cells and cell volume regulation.
J. Membr. Biol.
138:
181-195,
1994[Medline].
13.
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].
14.
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:
237-251,
1986[Abstract].
15.
Gögelein, H.,
and
K. Capek.
Quinine inhibits chloride and nonselective cation channels in isolated rat distal colon cells.
Biochim. Biophys. Acta
1027:
191-198,
1990[Medline].
16.
Goudeau, H.,
J. Wietzerbin,
and
C. M. Gary-Bobo.
Effects of mucosal lanthanum on electrical parameters of isolated frog skin. Mechanism of action.
Pflügers Arch.
379:
71-80,
1979[Medline].
17.
Granitzer, M.,
and
W. Nagel.
Dual effect of barium on basolateral membrane conductance of frog skin.
Pflügers Arch.
417:
207-212,
1990[Medline].
18.
Gustin, M. C.,
B. Martinac,
Y. Saimi,
M. R. Culbertson,
and
C. Kung.
Ion channels in yeast.
Science
233:
1195-1197,
1986[Medline].
19.
Gustin, M. C.,
X. L. Zhou,
B. Martinac,
and
C. Kung.
A mechanosensitive ion channel in the yeast plasma membrane.
Science
242:
762-765,
1988[Medline].
20.
Hall, J. A.,
J. Kirk,
J. R. Potts,
C. Rae,
and
K. Kirk.
Anion channel blockers inhibit swelling activated anion, cation, and nonelectrolyte transport in HeLa cells.
Am. J. Physiol.
271 (Cell Physiol. 40):
C579-C588,
1996[Abstract/Free Full Text].
21.
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].
22.
Hoffmann, E. K.,
and
P. B. Dunham.
Membrane mechanisms and intracellular signalling in cell volume regulation.
Int. Rev. Cytol.
161:
173-262,
1995[Medline].
23.
Jennings, M. L.,
and
R. K. Schulz.
Swelling-activated KCl cotransport in rabbit red cells: flux is determined mainly by cell volume rather than shape.
Am. J. Physiol.
259 (Cell Physiol. 28):
C960-C967,
1990[Abstract/Free Full Text].
24.
Kirk, K.
Swelling activated organic osmolyte channels.
J. Membr. Biol.
158:
1-16,
1997[Medline].
25.
Lacaz-Vieira, F.
Calcium site specificity. Early Ca2+-related tight junction events.
J. Gen. Physiol.
110:
727-740,
1997[Abstract/Free Full Text].
26.
Nilius, B.,
J. Sehrer,
P. De Smet,
W. Van Driessche,
and
G. Droogmans.
Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl
channels.
J. Physiol. (Lond.)
487:
367-378,
1995[Abstract].
27.
Parker, J. C.
In defense of cell volume?
Am. J. Physiol.
265 (Cell Physiol. 34):
C1191-C1200,
1993[Abstract/Free Full Text].
28.
Parker, J. C.,
T. J. McManus,
L. C. Starke,
and
H. J. Gitelman.
Coordinated regulation of Na/H exchange and K-Cl cotransport in dog red cells.
J. Gen. Physiol.
96:
1141-1152,
1990[Abstract].
29.
Sanchez-Olea, R.,
J. Moran,
A. Martinez,
and
H. Pasantes-Morales.
Volume-activated Rb+ transport in astrocytes in culture.
Am. J. Physiol.
264 (Cell Physiol. 33):
C836-C842,
1993[Abstract/Free Full Text].
30.
Sarkadi, B.,
and
J. C. Parker.
Activation of ion transport pathways by changes in cell volume.
Biochim. Biophys. Acta
1071:
407-427,
1991[Medline].
31.
Strange, K.,
and
P. S. Jackson.
Swelling-activated organic osmolyte efflux: a new role for anion channels.
Kidney Int.
48:
994-1003,
1995[Medline].
32.
Tilly, B. C.,
N. van den Berghe,
L. G. Tertoolen,
M. J. Edixhoven,
and
H. R. de Jonge.
Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances.
J. Biol. Chem.
268:
19919-19922,
1993[Abstract/Free Full Text].
33.
Van Driessche, W.
Lidocaine blockage of basolateral potassium channels in the amphibian urinary bladder.
J. Physiol. (Lond.)
381:
575-593,
1986[Abstract].
34.
Van Driessche, W.,
P. De Smet,
and
H. de Smedt.
Poorly selective cation channels in the apical membrane of A6 cells.
Pflügers Arch.
426:
387-395,
1994[Medline].
35.
Van Driessche, W.,
P. De Smet,
J. Q. Li,
S. Allen,
M. Zizi,
and
I. Mountian.
Isovolumetric regulation in a distal nephron cell line (A6).
Am. J. Physiol.
272 (Cell Physiol. 41):
C1890-C1898,
1997[Abstract/Free Full Text].
36.
Van Driessche, W.,
P. De Smet,
and
G. Raskin.
An automatic monitoring system for epithelial cell height.
Pflügers Arch.
425:
164-171,
1993[Medline].
37.
Van Driessche, W., and S. D. Hillyard. Quinidine
blockage of K+ channels in the
basolateral membrane of larval bullfrog skin.
Pflügers Arch. 405, Suppl. 1: S77-S82, 1985.
38.
Welling, P. A.,
and
M. A. Linshaw.
Importance of anion in hypotonic volume regulation of rabbit proximal straight tubule.
Am. J. Physiol.
255 (Renal Fluid Electrolyte Physiol. 24):
F853-F860,
1988[Abstract/Free Full Text].
39.
Welling, P. A.,
M. A. Linshaw,
and
L. P. Sullivan.
Effect of barium on cell volume regulation in rabbit proximal straight tubules.
Am. J. Physiol.
249 (Renal Fluid Electrolyte Physiol. 18):
F20-F27,
1985.
40.
Wellner, M. C.,
and
G. Isenberg.
Properties of stretch-activated channels in myocytes from the guinea-pig urinary bladder.
J. Physiol. (Lond.)
466:
213-227,
1993[Abstract].
41.
Yang, X. C.,
and
F. Sachs.
Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions.
Science
243:
1068-1071,
1989[Medline].
Am J Physiol Cell Physiol 275(1):C189-C199
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society