1 Facultad de Medicina, Organic osmolyte and halide permeability pathways activated in
epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl
cell volume regulation; taurine efflux; iodide efflux; organic
osmolyte
THE MECHANISMS OF CELL volume regulation involve the
release or uptake of intracellular organic osmolytes and ions by
activation of channels and transporters. In response to swelling
induced by extracellular hypotonicity, cells lose KCl, organic
osmolytes, and water to recover their normal volume in a process known
as regulatory volume decrease (RVD) (8, 28).
Taurine (2-aminoethanesulfonic acid) is the main organic osmolyte in
many mammalian cells and is actively accumulated in various tissues at
concentrations up to 65 mM (25, 34). Taurine has been shown to be
released from swollen cells together with
K+ and
Cl Data suggesting that the volume-activated release of taurine is
mediated by the same anion-selective channels responsible for
swelling-activated Cl Although the evidence for a common osmosensitive permeability pathway
for Cl In this report we extend our previous observations on the dependence of
swelling-activated taurine permeability on extracellular anions. In
addition, we study the time course of activation of taurine
permeability after cell swelling and compare it with that of the
osmosensitive Cl Cells.
HeLa cells were grown at 37°C in a 5%-95%
CO2-air atmosphere in DMEM (with
no taurine added) supplemented with 5% FCS, 80,000 IU/l penicillin,
and 50 µg/l streptomycin.
Efflux experiments.
Cells were grown to 80% confluence (usually 48-72 h after
plating) in 35-mm-diameter, six-well plates. They were loaded with [3H]taurine (1 µCi/ml) alone or with 125I (0.5 µCi/ml) by incubation for 2-2.5 h at 37°C in culture medium without FCS. Before each efflux experiment, the loading solution was
removed and the cells washed three times at room temperature with
isosmotic medium (see the figure legends for the compositions of the
solutions). The medium contained in each well (0.75 ml) was replaced at
timed intervals and saved for counting. Cells were allowed to
equilibrate for 3-5 min and then were challenged with hyposmotic
solutions. The radioactivity remaining in the cells at the end of the
experiment was determined after disruption of the cells with 0.75 ml of
0.25 M NaOH. Samples were counted in beta and gamma scintillation
counters, and appropriate corrections were applied to obtain pure
3H radioactivity. In isotonicity,
the amounts of
[3H]taurine and
125I remaining in the cells
decreased following single-exponential decay functions.
The data were expressed as efflux rate constants calculated from the
equation
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
by
anions that are more permeant through the volume-activated Cl
channel, as indicated by
electrophysiological measurements, significantly decreased
taurine efflux. In the presence of less-permeant anions, an increase in
taurine efflux was observed. Simultaneous measurement of the
125I, used as a tracer for
Cl
, and
[3H]taurine efflux
showed that the time courses for the two effluxes differed. In
Cl
-rich medium the increase
in I
efflux was transient,
whereas that for taurine was sustained. Osmosensitive
Cl
conductance, assessed by
measuring changes in cell volume, increased rapidly after hypotonic
shock. The influx of taurine was able to counteract
Cl
conductance-dependent
cell shrinkage but only ~4 min after triggering cell swelling. This
taurine-induced effect was blocked by DIDS. Differences in anion
sensitivity, the time course of activation, and sensitivity to DIDS
suggest that the main cell swelling-activated permeability pathways for
taurine and Cl
are separate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, suggesting that it may
play an important role in the RVD process (11). The molecular identity
of the membrane permeability pathway used by taurine to leave the cells
is not known. The pathway mediating swelling-dependent efflux of
taurine appears to be shared by a variety of structurally unrelated
organic molecules, catalyzes influx and efflux with similar
efficiencies, does not appear to saturate with increasing
concentrations of transported substance, does not present any
trans effects, and is not
stereoselective (11, 13). The characteristics of the transport
pathway(s) are unlike those of typical membrane transporters and are
more akin to those of porins.
efflux have been extensively reviewed (11, 24, 28). The strongest
evidence favoring a common pathway for organic osmolytes and
Cl
relies on the similar
pharmacological profiles of the two effluxes and on measurements of
single-channel and macroscopic currents mediated by taurine at an
alkaline intracellular pH in Madin-Darby canine kidney cells (3) and in
C6 glioma cells (10).
and organic osmolyte
efflux is strong, the question of the identity of the pathways involved
is not solved. Lambert and Hoffmann (15) reported the differential
activation and blockade by some unsaturated fatty acids of
swelling-activated taurine and
Cl
efflux in Ehrlich mouse
ascites tumor cells. In addition, DIDS is a strong inhibitor of the
taurine pathway in these cells but only inhibits the osmosensitive
Cl
permeability slightly.
These observations suggest the presence of two distinct types of
channels or permeability pathways of different natures in Ehrlich
cells. For rat mammary tissue, Shennan et al. (27) also demonstrated
that taurine efflux activated by cell swelling seems to occur via a
pathway different from the dominating volume-sensitive anion channel.
Sánchez-Olea et al. (26) noticed that in three different cell
lines there was a poor correlation between the abilities of a variety
of blockers to inhibit swelling-induced organic osmolyte and
125I efflux, an observation
inconsistent with the single common pathway hypothesis. Emma et al. (7)
have considered, to explain RVD dependence on intracellular
electrolytes, that Cl
channels separate from a taurine-permeable pathway could provide the
main route for swelling-activated
Cl
efflux. Recently, we
have shown that replacing extracellular Cl
with poorly permeant
anions differentially modulates the volume-activated Cl
current and taurine
efflux in HeLa cells (29). As for the Ehrlich cells, we have also
observed that DIDS is a stronger inhibitor of taurine efflux than of
Cl
conductance. These
observations are inconsistent with a single common pathway for
osmosensitive taurine and
Cl
efflux.
permeability. Our data suggest that the main taurine and
Cl
permeability pathways
activated during RVD are separate.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
where
ki is the efflux
rate constant at time i and
cpmi is the radioactivity counts
per minute at time i, in an experiment
where n samples are taken;
ti is the time
interval for sample
i in minutes. All the experiments were
carried out at room temperature (24°C).
Electrophysiology. For electrophysiological experiments, cells were seeded onto 35-mm-diameter cell culture petri dishes, which could be directly mounted on the stage of an inverted microscope. Standard whole cell patch-clamp recordings were performed as described before (5), except that the amplifier was used in the current-clamp mode.
Cell volume measurements. Changes in cell water volume in single cells were assessed by measuring changes in the concentration of an intracellularly trapped fluorescent dye (1). HeLa cells grown as for efflux experiments were plated on round coverslips, loaded with calcein-AM (5 µM; for 5 min), and then superfused with isosmotic solution for 30 min before the experiment was started. The experiments were performed with a confocal laser imaging system (Carl Zeiss). Excitation light was 488 nm, and emitted light was measured at wavelengths longer than 515 nm. Pictures were obtained at 10-s intervals, and the fluorescence of a ~10-µm2 area in the center of a cell was measured. The records were corrected for fluorescence decay independent of cell volume changes (primarily due to dye photobleaching). The data are presented as Vt/V0, where V0 = cell water volume in isosmotic solution at time 0 and Vt = cell water volume at time t. This was calculated from F0/Ft (F = fluorescence intensity) exactly as described previously (1).
Reagents. [3H]taurine was obtained from American Radiolabeled Chemicals (St. Louis, MO), and 125I was obtained from the Comisión Chilena de Energía Nuclear. All other reagents were of analytical grade and were purchased from Sigma (St. Louis, MO) and Merck (Darmstadt, Germany).
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RESULTS |
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Cell swelling-activated taurine efflux depends on extracellular
anions.
The cell swelling-activated
Cl conductances of HeLa and
other cells have been demonstrated to increase in the presence of anions more permeant than
Cl
in the extracellular
medium. The converse is observed for impermeant anions, such that the
predicted flux through the channel should be directly correlated with
the permeability of the main anion present in the medium (29, 30, 33).
To address the question of the influence of different extracellular
anions on the volume-activated taurine efflux, tracer experiments were
conducted. Cells loaded with
[3H]taurine were used
to monitor amino acid loss (29). After a period to measure basal efflux
rate, the medium bathing the cells was exchanged for a 30% hypotonic
solution to assess the increase in taurine permeability brought about
by cell swelling. Hypotonic solutions in which NaCl was replaced by KCl
(or the corresponding anion) and gramicidin were used to clamp the
membrane potential at ~0 mV and thus preclude the possible effects of
anion replacement on membrane potential. As shown in Fig.
1,
top, there was a marked increase in
the rate constant for taurine efflux in
Cl
-containing medium. The
permeability reached an apparently steady level in 5 min. When
Cl
was replaced by the
impermeant anions F
or
glutamate, there was an increase in the values of permeability reached.
On the other hand, when permeant anions were used, there was a decrease
in the maximal efflux rate achieved (Fig. 1,
bottom). Anion replacement could
directly affect the membrane transporter/channel mediating taurine
efflux or could affect the mechanisms signaling the permeability
change. In an attempt to distinguish these possibilities, the speeds of increase in the rate constant in the presence of different anions (I
and
glutamate, i.e., those producing maximal decrease and increase, respectively) were normalized to compare them with that in the presence
of Cl
. Figure 1,
top,
inset, shows that the anion
composition of the medium did not alter significantly the speed of
increase of the efflux rate constant, suggesting that the mechanism
transducing the change in volume signal into permeability activation
was not affected.1
Swelling-activated taurine permeability is therefore dependent on the
extracellular anion, following the sequence (derived from k10 min values)
glutamate > F
> Cl
> NO
3 > Br
> SCN
> I
. This sequence contrasts
with the permeability sequence
SCN
> I
> Br
> Cl
> F
> glutamate, described
for HeLa and a variety of other cell types for the volume-activated
Cl
current (5, 17, 18).
|
Volume-activated taurine and I
effluxes follow different time courses.
If taurine and Cl
share a
common pathway, it would be expected that volume-activated taurine and
iodide effluxes should follow similar time courses and be similarly
affected by anion replacement. Under control, isotonic conditions HeLa
cells lost previously loaded 125I
and [3H]taurine with
time courses well described by a single-exponential decay function
(29). In isotonicity, the respective rate constants were 0.05-0.08
min
1 for
125I and 0.01-0.02
min
1 for
[3H]taurine (Fig.
2; note the use of separate
y-axes). The rate constants remained
unaltered in the isosmotic medium for up to 14 min and were not
affected by the replacement of extracellular Cl
by glutamate. However,
challenging the cells with hypotonic solution markedly increased the
rates of efflux for both compounds. Figure 2A shows an experiment in which a high
(105 mM) Cl
concentration
was maintained throughout. The increase in efflux rate was transient
for iodide but appeared sustained for the amino acid within the time
frame of the experiments. When the hypotonic challenge experiment was
performed in a low-Cl
,
glutamate-rich hypotonic solution (Fig.
2B), an approximately similar
increase in the efflux rate constant for iodide was observed. The
increase in taurine efflux, however, was three-fold larger than that in
the Cl
-rich hypotonic
solution. For both compounds the increase was transient, the fall in
iodide efflux being faster in glutamate than in
Cl
medium. The decrease in
permeability for I
could be
adjusted to single-exponential functions with constants of 0.27 ± 0.04 min
1
(Cl
medium) and 0.74 ± 0.09 min
1 (glutamate
medium). The increase in taurine permeability was transient in
glutamate medium but not in
Cl
medium. This might be
due to RVD taking place in
low-Cl
glutamate medium
because of the larger outwardly directed
Cl
gradient. In both
hypotonic conditions, 125I efflux
reached its maximal value before
[3H]taurine efflux, a
result also shown by others (19). One possible explanation for the more
transient activation of 125I
efflux would be that the cells become depleted of this tracer more
rapidly than they do of
[3H]taurine. However,
this was not the case: for experiments in Cl
medium, radioactivity
levels remaining in the cell at the end of the experiments were 20 ± 1 and 16 ± 1% for
[3H]taurine and
125I, respectively. Corresponding
figures in glutamate medium were 6 ± 0.5 and 15 ± 1%.
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|
Cell swelling-activated taurine efflux is voltage dependent.
The voltage dependence of taurine efflux was studied by swelling the
cells with a NaCl-containing hypotonic solution. Once taurine efflux
was activated, the cells were exposed to hypotonic solutions containing
different K+ concentrations
[all Na+ replaced by
N-methyl-D-glucamine (NMDG)] and
gramicidin. Figure 4,
top, shows an experiment in which
taurine efflux was activated by hypotonicity in the usual manner. After
5 min in hypotonic medium, the cells were subjected to hypotonic
solutions containing different concentrations of
K+. Taurine efflux was dependent
on the external concentration of K+, decreasing as the cation
concentration increased, suggesting that the efflux rate was dependent
on membrane voltage
(Vm).
The effect of low concentrations of
K+ was transient, and this might
be due to dissipation of the gradient and/or the occurrence of RVD. In
order to quantify
Vm and correlate the taurine efflux rate constant to this parameter, current-clamp experiments were performed under the same experimental conditions. Figure 4, bottom, shows that cells
varied their Vm
between 100 and 5 mV (0 mM
K+ and 105 mM
K+, respectively). The maximal
efflux rate constant for taurine was significantly higher at
hyperpolarized potentials. The fact that taurine efflux is dependent on
membrane potential might indicate that a charged form of the amino acid
is transported. It has been argued, however, that taurine must be
leaving cells mainly in the neutral form, judging from the fact that a
small change or no change in intracellular pH occurs concomitantly
(15). Alternatively, an intrinsic voltage dependence of the transporter
or channel involved could account for our observations. In view of the
effect of Vm on
the rate constant of taurine efflux, the question of whether the
activation process of volume-induced taurine efflux was also affected
by hyperpolarization was addressed. Cells were swollen in a control
(NaCl) hyposmotic solution or in a solution of NMDG chloride plus
gramicidin. Figure 5 shows that, as
expected, the efflux rate constant reached was higher in
gramicidin-NMDG chloride solution than in NaCl-rich solution,
consistent with the expected depolarization and hyperpolarization,
respectively. However, by normalizing the NaCl to the experiment with
gramicidin-NMDG chloride, a similar time course for the development of
permeability was seen.
|
|
Cell swelling activates separate
Cl- and taurine-preferring
permeability pathways.
To compare the time courses of development of taurine permeability and
Cl
conductance, experiments
were conducted to monitor cellular volume by a single-cell,
fluorescence-based method (1). The approach uses the intracellularly
trapped fluorescent dye calcein. Figure 6,
top, shows a record of
F0/Ft
in normal (NaCl) isotonic medium and the effect of brief (<2 min)
exposures to anisosmotic solutions. Fast changes of fluorescence
intensity were observed for all anisosmotic challenges, reflecting
concentration or dilution of the dye within the element of volume under
consideration.
F0/Ft values are plotted against the osmotic pressure change
(
0/
t) in Fig. 6, bottom. The linear
relationship between these two variables gave a slope of 0.48 [correlation coefficient
(r) = 0.99], indicating that 52% of the dye was trapped
in an intracellular compartment insensitive to changes in osmolality.
This was used to correct data to yield changes in cell water volume as
follows (1): Vt/V0 = [(F0/Ft)
Fbkg]/(1
Fbkg) where
Fbkg = 0.52. This value, which
corresponds to dye trapped in an osmotically insensitive compartment,
compares with 0.66 for C6 glioma
cells and 0.7 for NG108-15 neuroblastoma × glioma hybrid
cells (1). Figure 6, bottom, also
shows that calculated
Vt/V0
data compared well with an ideal line predicting the behavior of a perfect osmometer.
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DISCUSSION |
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In the present work we have used HeLa epithelial carcinoma cells to
study the external anion dependence and time course of activation of
cell swelling-dependent permeability for taurine and contrasted them
with the same characteristics for osmosensitive Cl conductance. HeLa cells
have been demonstrated to undergo RVD when swollen by exposure to
hyposmotic solutions (32). Electrophysiological recordings have shown
that they possess Cl
and
K+ channels activated by cell
swelling and intracellular Ca2+,
respectively, which have been postulated to mediate KCl loss leading to
volume recovery (4, 5). HeLa cells swollen hypotonically increase their
taurine permeability and also their permeability to iodide. There is a
remarkable similarity in pharmacological sensitivity between these two
effluxes (12) and also an apparent good correlation with the
pharmacology of swelling-activated anion channels (5).
Swelling-activated anion channels of HeLa cells correspond to what have
been termed VSOAC (volume-sensitive organic osmolyte anion channels)
(28), which are postulated to be responsible also for swelling-induced
intracellular organic osmolyte release during RVD. Recent work,
however, points to differences in pharmacological sensitivity and other
characteristics between the two processes (7, 15, 26, 27) that might
suggest heterogeneity. Our previous work also suggested that HeLa cells
could have more than one volume-activated permeability pathway for
taurine and Cl
on the basis
of their differential modulation by external
Cl
(29, 30). Now we have
extended this to a range of anions and in addition present evidence
that the main Cl
conductance and taurine permeability pathways must be separate on the
basis of their different time courses of activation after hypotonic shock.
We have postulated that Cl
regulates the swelling-activated anion channel by interaction with a
site of unknown location within it so that its activity depends on the
presence of the anion (29, 30). This type of gating has been
demonstrated by elegant work on the ClC-0
Cl
channel expressed in
amphibian oocytes (22). The opening of the channel is strongly favored
by the presence of extracellular Cl
; other less permeant
anions are less effective in supporting channel activity, and the
reverse is true of more permeant anions. Pusch et al. (22) propose a
novel mechanism, in which gating is intrinsically linked to permeation,
to account for the data. Possibly a similar mechanism accounts for the
anion dependence of the activity of swelling-activated anion channels.
If the same conductance mediates organic osmolyte efflux, it would be
expected to affect similarly taurine permeability. The cell
swelling-activated taurine efflux, however, shows an almost inverse
effect when a series of anion replacements is used. This suggests that
the pathway mediating the efflux of amino acid is different from the
swelling-activated, outwardly rectifying
Cl
channel. In addition, it
suggests that the osmosensitive organic osmolyte permeability pathway
can be modulated by extracellular inorganic
anions.3
Much of the evidence put forward in support of the contention that the
osmosensitive taurine efflux pathway is identical to the
swelling-activated outwardly rectifying
Cl channels in HeLa and
other cells comes from the comparison of organic osmolyte and iodide
efflux. In the present work 125I
efflux was measured concomitantly with taurine movement.
I
efflux was little
affected by extracellular
Cl
removal, whereas in the
same cells taurine efflux was increased considerably. Other marked
differences in the behavior of the two effluxes suggest heterogeneity
of the pathways mediating the effluxes of these compounds. Iodide
efflux reaches a maximum at ~2 min after hypotonic challenge, whereas
taurine efflux is maximal only after 4-6 min. The effect of
hypotonicity on permeability was, however, much more transient for
I
efflux than for taurine
efflux. Taurine efflux was still elevated at 6-10 min after
hypotonicity, remaining elevated within the time frame of the
experiment. For I
, on the
other hand, the efflux rate had returned essentially to basal levels
during the same time. When
Cl
was replaced by
glutamate, the effect of hypotonicity on efflux was transient for both
compounds but the differences in rate of development of the full effect
remained: the I
peak efflux
rate was reached within 2-3 min, whereas it took 4-5 min for
taurine efflux to peak. The reestablishment of control values after the
maximum effect was also faster for
I
. These differences are
difficult to explain if the halide and the amino acid are leaving the
cell through a single pathway.
It might be reasonably argued that the conditions under which
patch-clamp whole cell recordings are carried out are so different from
those for intact cell efflux measurement as to render comparison unwarranted. Also, I
behaves differently from
Cl
, as observed in
electrophysiological studies (30, 31). For these reasons the time
course of activation of Cl
conductance was studied by measuring
Cl
conductance-limited
changes in cell volume (9). These experiments demonstrated that
swelling-induced Cl
conductance is fast to develop, reaching full activation within 1 min
of hyposmotic
stress.4
The counteraction of RVD by extracellular amino acids can be used to
assess their permeability (20). Using this approach, we
provide further evidence for the difference in time courses of
activation of Cl
conductance and taurine permeability after hypotonic shock. Whereas the
main Cl
conductance
activates rapidly (<1 min) after hypotonic cell swelling, the taurine
permeability pathway activates with a delay of 3-4 min. We have
previously shown that the stilbene DIDS is a reasonably good inhibitor
of the swelling-activated taurine permeability with
K1/2 values of
0.2 and 4 µM depending on the extracellular Cl
concentration (29). On
the other hand, DIDS is a voltage-dependent inhibitor of the
volume-activated Cl
channel, being particularly weak at hyperpolarizing potentials (2, 5).
In Ehrlich cells DIDS has been shown to block swelling-induced taurine
but not Cl
flux (15). The
results reported above demonstrate that DIDS abolished the taurine
permeability without affecting the main swelling-activated
Cl
conductance measured
simultaneously in the same cell under experimental conditions favoring
hyperpolarization. These data, taken together, are better interpreted
by assuming that separate permeability pathways mediate the main
Cl
and taurine
osmosensitive fluxes. DIDS was able to inhibit
I
efflux, albeit at higher
concentrations than those for inhibiting taurine
permeability. This is in contrast with the apparent
insensitivity of Cl
conductance to the stilbene and might indicate that
I
is a poor surrogate for
Cl
in cell
swelling-sensitive pathways.
The experimental evidence and the conclusion reached in the present
work appear at variance with the great deal of evidence suggesting that
cell swelling-activated Cl
channels (often referred to as VSOAC) are the organic osmolyte channels
mediating the osmosensitive efflux of taurine and other intracellular
substances. The excellent reviews that recently appeared
on the subject also point out the existence of other Cl
channels that might
participate in the RVD process and that do not correspond to the
so-called VSOAC (11, 13, 28). Recent work has identified the cloned
Cl
channel ClC-3 as the
putative molecular counterpart of VSOAC, but there is no information as
to its taurine permeability (6). It is therefore possible that, for
taurine and other organic osmolytes, osmosensitive efflux is via an
anion channel but that a separate channel accounts for the main pathway
for Cl
efflux under
hyposmotic conditions. The sensitivity of taurine permeability to membrane potential observed here and reported by others
previously (15) would be perhaps better explained by a channel being
the membrane agency mediating the efflux of the amino acid.
In conclusion we would like to propose, as a working hypothesis to explain our data, that cells possess at least two different permeability pathways with differing selectivities: a strictly anion-selective channel of early activation during RVD and a permeability allowing taurine permeation of later activation. The latter pathway might allow some halide permeation. Both pathways could be ion channels, but there is no reason to assume that taurine efflux is not carrier mediated.
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ACKNOWLEDGEMENTS |
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We are grateful to Luis Felipe Barros for critically reading the manuscript. We thank the Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, and the Sección de Medicina Nuclear, Departamento de Medicina, Hospital Clínico José Joaquín Aguirre, Universidad de Chile, for the use of their radioactivity counting facilities.
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FOOTNOTES |
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This work was supported by grants from Fondecyt (Grant 1970244; Chile) and the Volkswagen Stiftung (Germany). Institutional support to the Centro de Estudios Científicos de Santiago from a group of Chilean private companies (AFP Protección, CGE, Codelco, Copec, Empresas CMPC, Gener, Minera Collahuasi, Minera Escondida, Novagas, Business Design Associates, Xerox Chile), Fuerza Aérea de Chile, and Municipalidad de Las Condes is also acknowledged. The research of F. V. Sepúlveda was supported in part by an International Research Scholars grant from the Howard Hughes Medical Institute and a Cátedra Presidencial en Ciencias.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1 Exposure of cells to a high K+ concentration probably prevents RVD, but a secondary swelling might be expected in the presence of permeant anions. This would lead to an underestimation of the inhibitory effect of the permeant anions described here.
2
In addition, we have explored the effect of
DIDS on swelling-activated 125I
efflux and found a
K1/2 value of 20 µM (in high extracellular Cl solution; result not
shown), a fivefold difference in sensitivity compared with
swelling-activated taurine efflux. This is a higher sensitivity than
expected if I
were
permeating through the cell swelling-activated conductance and might
suggest that this halide is not an adequate surrogate for
Cl
. Clarification of this
point will have to await further experiments.
3
An alternative explanation for this result, as
already proposed previously (29), is that anions interact with taurine
within a single permeability pathway, which may be a multiple-occupancy anion channel, so that residency by low-permeability anions would favor
taurine flux and the converse would occur with permeable anions.
Interactions between acidic amino acids and
Cl within the pores of
volume-regulated Cl
channels have recently been demonstrated (16), but remain to be
explored for nonanionic amino acids or other organic osmolytes.
4
There is a discrepancy between the apparent
time courses for activation and inactivation of a swelling-activated
anion conductance estimated from cell volume experiments and those from
the 125I experiments. We ignore
the reason for this discrepancy, but I is more permeant than
Cl
through
swelling-activated Cl
channels and would be expected to exit more rapidly than the native
anion during hypotonicity. This will alter the ratio of the
concentration of I
to that
of Cl
during efflux, which
could affect apparent permeability.
Address for reprint requests and other correspondence: A. Stutzin, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 6530499, Chile (E-mail: astutzin{at}bitmed.med.uchile.cl).
Received 29 December 1998; accepted in final form 3 May 1999.
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