From the Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641
Necturus gallbladder epithelial cells bathed in 10 mM HCO3/1% CO2 display sizable basolateral
membrane conductances for Cl (GClb) and K + (GKb). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K +/Cl
selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca2+]i) increased transiently during hyposmotic swelling (peak at ~30 s, return to baseline within ~90 s), but chelation of
cell Ca2+ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium
by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10-12 min after osmotic swelling. Depolarization of
the basolateral membrane voltage (Vcs) produced a significant increase in the change in Vcs elicited by lowering
basolateral solution [Cl
], whereas hyperpolarization of Vcs had the opposite effect. These results suggest that: (a)
Hyposmotic swelling increases GKb and decreases G Clb. These two effects appear to be linked, i.e., the increase in G Kb
produces membrane hyperpolarization, which in turn reduces G Clb. ( b) Hyperosmotic shrinkage has the opposite
effects on GKb and G Clb. ( c) Cell swelling causes a transient increase in [Ca2+]i, but this response may not be necessary for the increase in GKb during cell swelling.
The Necturus gallbladder (NGB)1 epithelium is a useful
model system for functional studies of a family of epithelia that perform near-isosmotic fluid absorption.
The mechanisms accounting for ion and water transport in NGB epithelium under control conditions have
been characterized in detail and considerable information is also available on regulation of transport (for reviews, see Reuss, 1988; Reuss and Altenberg, 1995
). An
issue requiring further study is the regulation of basolateral membrane Cl
and K+ channels, especially its
relationship to the mechanism of cross talk between
apical and basolateral membranes, in which cell volume changes could play a major role (Schultz and
Hudson, 1991
). For instance, we have proposed that elevation of cAMP causes a reduction in Cl
efflux across
the basolateral membrane. This is based on the effects
of cAMP on fluid transport (Petersen and Reuss, 1983
), intracellular ion contents, and cell volume (reviewed in
Reuss and Altenberg, 1995
; Reuss et al., 1991
). Further,
exposure to cAMP reduces basolateral membrane conductance (Copello et al., 1993
). We suspected that this
may include a decrease in basolateral membrane GCl
(GClb) brought about by the cell shrinkage produced by
cAMP. In other words, the hypothesis is that the
changes in apical membrane conductances elicited by
cAMP result in a loss of cell KCl, which in turn causes cell shrinkage, and that the shrinkage results in a decrease in basolateral membrane G Cl. Accordingly, we assessed the effects of cell volume and membrane voltage
on the Cl
and K+ conductances of the basolateral
membrane. Specifically, we investigated the rapid effects of cell swelling and shrinkage on these conductances, in order to test the hypothesis that cell volume
changes are involved in cross talk between apical and
basolateral membranes. Inasmuch as GClb is small relative to the basolateral K + conductance (GKb), in addition to osmotic-shrinkage we carried out osmotic-swelling experiments. If cell swelling increases G Clb, then
swelling experiments would yield relative changes in
G Clb easier to measure than those elicited by cell shrinkage. In contrast with the apical membrane pathway
(Copello et al., 1993
; Heming et al., 1994
), the G Clb appears to be cAMP independent (Copello et al., 1993
)
and is activated by HCO 3
/CO2 (Stoddard and Reuss,
1988a
). The results of these studies demonstrate that
GClb is increased by cell shrinkage and decreased by cell
swelling, probably via changes in cell membrane voltage (depolarization during cell shrinkage, hyperpolarization during cell swelling). The basolateral K + conductance (GKb) is apparently voltage insensitive (Stoddard and Reuss, 1988
b), and possibly activated by
elevations in internal Ca2+ (Bello-Reuss et al., 1981
).
Here, we demonstrate that the K+ conductance is stimulated by cell swelling and inhibited by cell shrinkage.
General
Mudpuppies (Necturus maculosus) were purchased from Kon's
Scientific (Germantown, WI) or Nasco Biologicals (Ft. Atkinson, WI) and maintained at 5-10°C. Anesthesia was accomplished by
immersion in a 1 g/liter tricaine methanesulfonate solution. The
gallbladder was excised, opened, washed, and mounted in a modified Ussing chamber (Altenberg et al., 1990). When the gallbladders were mounted serosal side up, a patch of connective tissue
was removed by dissection, to allow for microelectrode impalements through the basolateral membrane (Altenberg et al.,
1990
). Some experiments were carried out with polarized epithelial cells isolated from the intact tissue ("figure-eight cells"), using a method previously described (Torres et al., 1996b
). The
control bathing solution (bPSS) contained, in mM: 90 NaCl, 10 NaHCO3, 2.5 KCl, 1.8 CaCl2, 1.0 MgCl2, and 0.5 NaH2PO4, gassed
with 1% CO2/99% air, pH 7.66, at room temperature, with an average osmolality of 207 mosmol/kg. A low-NaCl but isosmotic solution was prepared by replacing 35 mM NaCl with 70 mM sucrose. This solution was made hyposmotic (by ~17 or 34%) by removing sucrose, or hyperosmotic (also by ~17 or 34%) by adding
a further 35 or 70 mM sucrose. Changes in bathing solution osmolality were made only in the apical bathing medium, inasmuch as reductions in basolateral solution [NaCl], with or without changes in osmolality, elicited long-lasting oscillations in
membrane voltage and conductance. To assess cell membrane
ionic selectivity, Cl
was replaced with gluconate, and Na+ was replaced with K+. These replacements were isomolar.
Electrophysiological Techniques
Transepithelial voltage (Vms, referenced to the basolateral bathing solution; Vsm, referenced to the apical bathing solution) and
cell membrane voltages (apical = Vmc, basolateral = Vcs, referenced to the respective adjacent solutions) were measured as previously described (Altenberg et al., 1990). The ground electrode
was an Ag-AgCl pellet separated from the apical bathing solution
by a short Ringer-agar bridge. The basolateral bathing solution
electrode was a flowing, saturated-KCl bridge in series with a
calomel half-cell. Hence, liquid junction potentials upon changes
in the basolateral solution were minimized. The transepithelial
resistance, Rt (
Vms/It, where
Vms is the change in Vms elicited
by a constant current pulse of density It), and the apparent ratio
of cell membrane resistances (Ra/Rb, where the subscripts a and
b denote apical and basolateral membranes, respectively) were
determined from the steady-state voltage deflections elicited by
DC pulses of 50 µA/cm2 and 1-s duration, applied across the tissue through Ag-AgCl electrodes. The voltage deflections were
corrected for series resistances. The absolute values of Ra and Rb
were estimated from two-point cable analysis, as previously described (Petersen and Reuss, 1985
; Copello et al., 1993
). Intracellular-microelectrode studies were also carried out on isolated polarized cells attached with Cell-Tak® (Collaborative Medical Products, Bedford, MA) to a dialysis membrane mounted in the
microelectrode chamber (Torres et al., 1996b
).
Intracellular Cl activity (aCli) and tetramethylammonium
(TMA+) activity (aTMAi) were measured with double-barrel ion-sensitive microelectrodes constructed and calibrated as previously described (Altenberg et al., 1990
). Validation of impalements
was as described before (Altenberg et al., 1990
). To measure
changes in cell water volume, epithelia were loaded with TMA+
using transient exposure to nystatin, and the intracellular activity of TMA+ was determined with double-barrel, TMA+-sensitive microelectrodes (Reuss, 1985
; Altenberg et al., 1990
).
Fluorescence Techniques
Changes in cell water volume were assessed in isolated polarized
cells attached to a coverslip coated with Cell Tak®, loaded with
calcein,AM (3 µM, for 20 min) and then superfused with isosmotic solution for 15 min before starting the experiment. Calcein is a good choice among fluorescent indicators to assess changes in cell water volume because it undergoes less photobleaching than other probes and is retained better in the cells
(Altenberg et al., 1994; Alvarez-Leefmans et al., 1995
; Crowe et
al., 1995
). The experiments were performed using a digital video
confocal laser imaging system (Odyssey; Noran Instruments, Middleton, WI). Excitation light was 488 nm, and emitted light was
measured at wavelengths longer than 515 nm. Pictures were obtained at 30-s intervals, and fluorescence of a 15-20 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), which was fit by a single exponential. The data are presented as Fo/ Ft, where Fo = fluorescence in isosmotic solution, at t = 0, and Ft = corrected fluorescence at time = t. The ratio Fo/Ft is proportional to cell volume
and was ~75% of the ideal ("osmometric") responses after exposure to hypo or hyperosmotic solutions.
Intracellular free [Ca2+] ([Ca2+]i) was estimated from the
340/380 nm Fura-2 fluorescence ratio (F340/380) in isolated polarized cells. Isolated polarized cells (Torres et al., 1996b) were
loaded at room temperature with Fura-2,AM (5 µM for 1 h). The
cells were then attached to a coverslip coated with Cell-Tak®,
mounted in a chamber and superfused with isosmotic solution for ~15 min before starting the experiment. Measurements were carried out essentially as described (Altenberg et al., 1994
). Data
were acquired at 1 Hz. At the end of each experiment, cells were
exposed to 10-20 µM ionomycin to obtain saturating free
[Ca2+]i, and then to 1-2 mM MnCl2 in the continuous presence
of ionomycin, to quench the dye and thus correct for background fluorescence. Data are presented as F340/380 after background correction. In some experiments, cells were loaded with
acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N
,N
-tetraacetic acid (BAPTA,AM), Half-BAPTA,AM, or N,N,N
,N
-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), a heavy-metal
chelator (Kao, 1994
). All of these chemicals were obtained from
Molecular Probes (Eugene, OR).
Statistical Analysis
Results are given as means ± SEM. Statistical comparisons were done by t tests for paired or unpaired data, as appropriate. A value of P < 0.05 was considered significant.
Effects of Changes in Apical Bathing Solution Osmolality on Voltages and Resistances
The effects of lowering the apical solution osmolality
(from 200 to 135 mosmol/kg) on the basic electrophysiological properties of the epithelium are illustrated in
Fig. 1. In this experiment, a cell was impaled from the
apical side with a conventional microelectrode. Voltages and resistances were recorded before, during, and
after exposure to hyposmotic solution on the apical
side. Reducing bathing solution osmolality caused a hyperpolarization of Vcs and an increase in Ra/Rb. Both
effects were reversible. Table I summarizes the results
of experiments such as the one shown in Fig. 1. The hyperpolarization at 3 min was 8 ± 1 mV, and the repolarization was complete after 4 min of perfusion with isosmotic solution.
Table I. Effects of Apical Superfusion with Hyposmotic Solution on Electrical Properties of Necturus Gallbladder Epithelium |
The experiment shown in Fig. 2 illustrates the effects
of exposure to a hyperosmotic solution (the apical solution osmolality was increased from 200 to 270 mosmol/
kg), and Table II summarizes the results with this experimental protocol. The main effects of hyperosmotic
solution were cell membrane depolarization and a decrease in Ra/Rb, i.e., opposite changes to those elicited by hyposmotic solution. As in the experiments with hyp-osmotic solution, the effects were reversible. The effect
of anisosmotic solutions on Vms was small, but significant, apical side negative with hyposmotic solution and
opposite in polarity with hyperosmotic solution. These
changes can be explained by paracellular pseudo-streaming potentials caused by water flow from apical
to basolateral solution with hyposmotic solution and
from basolateral to apical solution with hyperosmotic
solution. These voltage changes, because of their small
magnitude, do not contribute significantly to the changes
in cell membrane voltages (Reuss et al., 1992a, b
). There were also significant changes in Rt: a decrease with hyp-osmotic solution and an increase with hyperosmotic solution. These changes are in the directions expected
for widening and narrowing of lateral-intercellular spaces, respectively (Reuss et al., 1992a
), but changes
in junctional resistance cannot be ruled out.
Table II. Effects of Apical Superfusion with Hyperosmotic Solution on Electrical Properties of Necturus Gallbladder Epithelium |
Effects of Changes in Apical Bathing Solution Osmolality on
Relative K+ and Cl Conductances of the
Basolateral Membrane
The changes in Vcs elicited by increasing basolateral
[K+] from 2.5 to 25 mM (VcsK), or by lowering basolateral [Cl
] from 98.1 to 8.1 mM (
VcsCl), were measured
in the same cells, during exposure to isosmotic apical
bathing solution and at 20 min of exposure to hyposmotic solution. Similar results were obtained between 5 and 20 min in hyposmotic bathing medium (not
shown). As illustrated in Fig. 3 and summarized in Table I, exposure to hyposmotic solution increased
VcsK
and decreased
VcsCl, relative to the respective values
measured in isosmotic solution. Further,
VcsCl in hyposmotic solution was not significantly different from zero.
Similar experiments were carried out to assess the
changes in basolateral membrane ionic selectivity after
a 5-10 min exposure to hyperosmotic solution. The results are illustrated in Fig. 4 and summarized in Table
II. In hyperosmotic medium, VcsK was reduced significantly, while
VcsCl was increased. Both changes are opposite to those elicited by exposure to hyposmotic solution.
Because of the low paracellular electrical resistance
of the NGB epithelium, the values of VcsK and
VcsCl are
less than the corresponding changes in zero-current
membrane voltages (the ionic substitutions produce
changes in intraepithelial current flow). There are also
concomitant paracellular diffusion potentials, but
these are small and in both instances (high-K+ and low-Cl
) tend to compensate in part for the shunting effect
of the paracellular pathway (see Reuss and Finn, 1975
).
These results demonstrate that the relative GKb increases and relative G Clb decreases during exposure to hyposmotic solution. Conversely, the relative G Kb decreases, and the relative G Clb increases during exposure to a hyperosmotic solution. To estimate the changes in absolute values of G Kb and G Clb in both sets of experiments, we performed the experiments described in the next section.
Effects of Exposure to Anisosmotic Solutions on Cell Membrane Resistances
Fig. 5, A and B, depicts representative two-point cable
analysis experiments under control conditions and during transient exposure to hyposmotic and hyperosmotic solutions. Two cells were simultaneously impaled, and both transepithelial and intracellular current pulses were applied at intervals. Downward
(hyperpolarizing) voltage spikes denote Vx, i.e., the
voltage changes elicited by current injection into another (electrically coupled) cell. Exposure to hyposmotic solution caused a decrease in
Vx, which reached
a stable value in ~4 min and returned to control levels within 4-5 min of restoring superfusion with isosmotic
solution (Fig. 5 A). In contrast, exposure to hyperosmotic solution increased
Vx. This effect was complete
within 4 min and returned to control levels after a 4-min
exposure to isosmotic solution (Fig. 5 B). Fig. 5 C depicts
fits of the Bessel function K0 to the two-point cable
analysis values obtained with hyposmotic and hyperosmotic solutions, respectively. From these data, we calculated Rz (resistance for current flow out of the cell, i.e.,
Ra and Rb in parallel); from Rz and the concomitant values of Ra/Rb and Rt, we calculated the resistances of the
cell membranes and the paracellular pathway. The
method used requires normalization of the data to the
values observed under control conditions. The bases,
assumptions, and limitations of this method have been
discussed in detail elsewhere (Petersen and Reuss, 1985
;
Copello et al., 1993
).
The results of these calculations are summarized in
Table III. Exposure to anisosmotic solutions elicits
mostly changes in basolateral membrane resistance: Rb
decreases during superfusion with hyposmotic solution
and increases during superfusion with hyperosmotic solution. Whereas hyposmotic solution causes exclusively a decrease in Rb, hyperosmotic solution both increases
Rb and decreases Ra. The latter effect could be due to
activation of apical membrane maxi-K+ channels by depolarization (Segal and Reuss, 1990).
Table III. Effects of Apical Superfusion with Anisosmotic Solutions on Cell Membrane Resistances |
The ionic-substitution experiments illustrated in Figs. 3 and 4 and summarized in Tables I and II demonstrated an increase in relative GKb with hyposmotic solution and a decrease in relative G Kb with hyperosmotic solution. These results, together with the resistance data, conclusively demonstrate that the changes in basolateral membrane conductance are principally due to changes in G Kb in the same direction: increase with hyposmotic solution and decrease with hyperosmotic solution. Rather unexpectedly, we found that the changes in GKb and G Clb were in opposite directions: when G Kb increased (hyposmotic solution), G Clb decreased; when G Kb decreased (hyperosmotic solution), G Clb increased. We next investigated the mechanism of the changes in G Kb and G Clb.
Cell Swelling Causes Elevation of [Ca 2+]i , But the Latter Is Not Necessary for the Increase in GKb
Entry of Ca 2+ has been shown to play a signaling role in
cell volume regulation from hyposmotic swelling by activation of a Ca2+-dependent GK (Christensen, 1987). Therefore, the effects of hyposmotic solution on [Ca2+]i in isolated polarized cells were estimated from the F340/F380 Fura-2 fluorescence ratio. The results are shown in Fig.
6. The cells were exposed to a 34%-hyposmotic solution under control conditions or after loading with the
Ca2+ chelator BAPTA,AM. After the onset of superfusion with hyposmotic solution, [Ca2+]i rose rapidly and
then (during continuous exposure to this solution) decreased monotonically to control levels in <2 min. In
cells preloaded with BAPTA, the elevation in [Ca2+]i by
exposure to hyposmotic solution was abolished. Experiments were also carried out with 50 µM Half-BAPTA,AM
and 50 µM TPEN, a heavy-metal chelator (Kao, 1994
).
Neither compound binds Ca2+ in the nanomolar range.
In both instances, the change in [Ca2+]i after exposure
to hyposmotic solution was similar to that observed under control conditions (not shown). Hence, the effect
of BAPTA cannot be attributed to nonspecific toxic effects or to heavy-metal chelation. Because of the effect
of pH on Fura-2 fluorescence (Reers et al., 1989
), we
also measured pHi with BCECF, using the same protocol as for the [Ca2+]i measurements. Hyposmotic solution did not cause measurable changes in pHi (data not
shown). These control experiments validate the conclusion that [Ca2+]i does in fact rise transiently during
exposure of NGB epithelial cells to hyposmotic solutions.
To determine the role of [Ca2+]i in the response of isolated polarized cells to hyposmotic swelling, we examined the effect of swelling (34% hyposmotic solution) on membrane voltage (Vm) with or without preloading the cells with 50 µM BAPTA (see METHODS). Without BAPTA, Vm hyperpolarized by 4 ± 1 mV; with BAPTA, the hyperpolarization was 3 ± 1 mV (not significantly different; n = 4 paired experiments).
GClb Is Modulated by Membrane Voltage
The changes in G Clb described above could result from:
( a) direct effect of changes in cell volume, (b) changes
in membrane voltage produced by the activation or inhibition of GKb, or ( c) other mechanisms. In other experiments, we have observed decreases in GClb associated with basolateral membrane hyperpolarization
(Stoddard and Reuss, 1989; Altenberg et al., 1992
; Altenberg et al., 1993
). Cell swelling activates G Kb; this in
turn would hyperpolarize the membrane (because E K \> Vcs), and the hyperpolarization would cause a decrease
in GClb. The opposite effects, i.e., decrease of G Kb, depolarization, and activation of G Clb, would be elicited by
cell shrinkage. To test the hypothesis that the effects of
cell volume changes on G Clb are mediated by changes in
membrane voltage, we studied the effects of baseline
V cs on
VcsCl. Depolarization of V cs was accomplished either by elevating apical solution [K+] from 2.5 to 25 mM (the change in Vcs results from a loop-current change, see Reuss and Finn, 1975
); hyperpolarization
of Vcs was accomplished by a transepithelial current
clamp. The results are shown in Fig. 7. With depolarization of Vcs,
VcsCl increased, and with hyperpolarization
of V cs, it decreased significantly. In the latter experiment, an extracellular electrode within 5 µm from the
impaled cell was used as reference to avoid the effect of
changes in voltage drops in the solutions (produced by
the different conductivities of high-Cl
and low-Cl
solutions). We conclude that GClb is voltage dependent:
decreased by hyperpolarization and increased by depolarization of V cs.
In conclusion, these experiments show that after
swelling of NGB epithelial cells GKb increases, causing
basolateral membrane hyperpolarization, which may
explain the parallel decrease in G Clb. In previous studies, we found no increase of apical membrane Cl conductance after cell swelling by exposure to hyposmotic
solutions (Heming et al., 1994
). This is consistent with
the lack of change in Ra (see above). Hence, our studies do not support the notion that NGB epithelial cells
undergo volume-regulatory decrease by loss of K+ and
Cl
via channels. However, cell volume measurements
using an optical-sectioning technique have been reported to show virtually complete cell volume regulation within a few minutes of the onset of the osmotic
perturbation (Persson and Spring, 1982
; Larson and
Spring, 1984
; Furlong and Spring, 1990
). Hence, it was
of interest to test whether cell volume regulation takes
place regardless of the decrease in GClb. This was done
in the next series of experiments.
Effects of Exposure to Hyposmotic Solution on Cell Water Volume and Intracellular aCl i
Cell water volume and aCli were measured before, during, and after exposure to hyposmotic solution (apical
side alone). In Fig. 8 A we illustrate a cell water volume
measurement in TMA+-loaded cells. Exposure to hyp-osmotic bathing solution caused an increase in cell volume, evidenced by the decrease in [TMA+]i. However,
there was no measurable cell volume regulation, although during this time GKb is clearly activated (see
Figs. 1 and 3 and Tables I and III). One could argue
that the experimental method, namely the TMA + loading by transient exposure to nystatin, caused a fall in
GKb or otherwise changed the properties of the cells,
abolishing the volume regulatory response. To test this
possibility, we measured aCl i before, during, and after
exposure to hyposmotic solution, without TMA+ loading. The expectations are that hyposmotic solution will
initially cause a dilution of intracellular Cl; if there is
cell volume regulation by KCl efflux, then aCli would
fall further during the regulatory response; if there is cell volume regulation by a different mechanism, without Cl
loss, then aCli would rise during the regulatory
response. Fig. 8 B illustrates the result of this experiment. Exposure to hyposmotic apical solution produced a monotonic change in aCli, i.e., there was no indication of more than one process (water influx)
changing aCli. Further, Fig. 8 C depicts the intracellular
TMA+ and Cl
activities during the first 5 min of exposure to the hyposmotic solution (records from Fig. 8, A
and B). The linear relationship indicates that the rates
of change in both ion activities are the same, i.e., that
the time courses of aCli and aTMAi mirror each other.
In addition, the maximal fractional changes in the two
activities did not differ significantly (see Fig. 8). Inasmuch as TMA+ is a cell water volume marker, i.e., effectively impermeant, this result indicates that during the
5-min period Cl
was also effectively impermeant, i.e.,
its cell content did not change. If cell volume regulation were very fast, one could envision a monotonic
"subosmometric" change in aTMAi after hyposmotic
swelling (cell water volume tends to increase because of
water influx and to decrease because of regulatory efflux of solute and water). However, the magnitude of
the change in aTMAi is that expected for a cell behaving as an ideal osmometer, and the parallel measurements of aCli do not show the higher rate of decrease
predicted if Cl
were involved in the regulatory response, or the lower rate of decrease expected if the
regulatory volume decrease did not involve Cl
.
The possibility of cell volume regulation after exposure to hyposmotic solution was further explored in a
preparation of isolated NGB epithelial cells that retain
structural and functional polarity (Torres et al., 1996a,
b
). Changes in cell water volume were estimated from
the changes in intracellular fluorescence in cells loaded with calcein, as illustrated in Fig. 9 A, B, and C. Exposure of isolated polarized cells to hyposmotic (Fig. 9 D)
or hyperosmotic solutions (Fig. 9 E) elicited rapid
changes in intracellular fluorescence, indicative of swelling and shrinkage, respectively, with no regulatory volume changes during the >10-min period of exposure
to anisosmotic solution. These experiments confirm the
results described in the preceding section, and argue
against the possibility that the lack of volume regulatory responses results from artefacts related to microelectrode impalements.
To eliminate the possibility of an artifactual inability
to measure a putative volume regulatory response, we
exposed cAMP-stimulated cells to an isosmotic, high-K+
external solution ([K+] was raised from 2.5 to 67.5 mM,
replacing Na+), at constant external [Cl] (63.1 mM).
The result, illustrated in Fig. 9 F, was rapid and reversible cell swelling, consistent with previous observations made with the TMA+ technique in the assembled epithelium (Cotton and Reuss, 1991
). These results further strengthen the conclusion that, in the experimental conditions of these studies, osmotically swollen NGB
epithelial cells do not undergo short-term regulatory
volume decrease.
Schultz (1981; see also Schultz and Hudson, 1991
) developed the important notion that changes in transport
rate at one of the membrane domains of an epithelium
must result in "matching" changes in transport rate at
the opposite membrane domain, so that in the steady
state cell volume and composition remain near constant. This adaptive mechanism requires a signaling system between the cell membranes, i.e., intermembrane
cross talk. The study of the mechanisms of cross talk between apical and basolateral membranes is of great importance to understand the regulation of salt and water
transport in gallbladder and other epithelia. Parameters possibly involved in cross talk are cell volume, intracellular ionic activities, and membrane voltage.
One instance in which parallel changes in transport
rates of apical and basolateral membranes occur in
NGB epithelium is that resulting from the effect of
cAMP. After an increase in cAMP levels, it has been
demonstrated that an apical Cl conductance, not
present under control conditions, develops and dominates the cell membrane conductances (Copello et al.,
1993
; Heming et al., 1994
; Reuss and Altenberg, 1995
).
This results in net Cl
loss across the apical membrane
(Cotton and Reuss, 1991
), because intracellular Cl
is
above the value predicted from electrochemical equilibrium as a consequence of the operation of the apical
membrane Cl
/HCO3
exchanger (Reuss, 1988
). Gar-vin and Spring (1992) have also suggested that cAMP
elevation results in a change in the dominant mechanism for Na + and Cl
transport across the apical membrane: from Na+/H+ and Cl
/HCO3
exchanges to
Na+-Cl
cotransport. Further, Dausch and Spring (1994)
have proposed that protein kinase C activation causes
the opposite effect. Regardless of these possibilities, the
increase in apical membrane GCl causes membrane depolarization and activation of voltage-sensitive apical membrane maxi-K+ channels (Cotton and Reuss, 1991
;
Reuss, 1991
). The parallel increases in apical membrane GCl and GK cause net KCl efflux, intracellular Cl
and K+ contents fall, the cells shrink (Cotton and
Reuss, 1991
), and transepithelial salt and water transport decrease. The latter result indicates that net transport across the basolateral membrane must also be reduced. Our starting hypothesis was that cAMP causes a
reduction in GClb. Hence, we assessed the effects of
changes in cell water volume on basolateral membrane
Cl
and K+ conductances.
Cell Swelling Stimulates and Cell Shrinkage Inhibits the Basolateral Membrane K+ Conductance
Our results show that cell swelling causes a selective increase in basolateral membrane conductance, attributable to an increase in K+ conductance. This effect is teleologically appropriate for cell volume regulation, inasmuch as it would facilitate K+ loss from the swollen cells.
The opposite experimental perturbation, i.e., cell shrinkage by exposure to a hyperosmotic solution, caused depolarization of both cell membranes, an increase in basolateral membrane resistance, and a decrease in the relative GKb. These effects are opposite to those elicited by exposure to hyposmotic solution and indicate that cell shrinkage decreases G Kb. Again, this effect is teleologically appropriate, i.e., net K + efflux across the basolateral membrane would decrease in a cell that has undergone shrinkage.
The elevation in [Ca2+]i after exposure to a hyposmotic solution could be solely or in part responsible for
the increase of the GKb, inasmuch as this conductance is
activated by maneuvers expected to elevate [Ca 2+]i
(Bello-Reuss et al., 1981). Activation of nonselective
cation channels, followed by Ca2+ entry and activation
of Ca2+-activated K+ channels, has been demonstrated
in other epithelia (Christensen, 1987
). Our measurements demonstrated that [Ca2+]i does rise during cell
swelling; chelation with BAPTA prevents the elevation
in [Ca2+]i but not the membrane hyperpolarization. Although this result could be interpreted to indicate that
[Ca2+]i plays no role in the activation of GKb, we cannot
rule out that, in the presence of BAPTA, [Ca 2+]i rose in
a small compartment, undetectable by our method. An alternative to Ca2+ activation is that the basolateral K+
channels are activated by stretch.
Cell Swelling Inhibits the Basolateral Membrane
Cl Conductance
Cell swelling reduced the relative GClb to a value not different from zero, indicating that the basolateral membrane Cl conductance decreases. This effect is contrary to expectations, inasmuch as it is opposite to the
effect of cell swelling on GKb and would, per se, tend to
prevent KCl efflux via conductive pathways.
The effect of cell swelling on G Clb could be indirect,
since it occurs during membrane hyperpolarization.
We found that membrane hyperpolarization per se reduces G Clb, as indicated by basolateral solution Cl -substitution experiments at three membrane voltages.
Atypical osmosensitivity of Cl
channels has been observed in other cell types (Chesnoy-Marchais and Fritsch,
1994
). It is possible that cell swelling and membrane hyperpolarization have separate effects on GClb, but the
membrane voltage changes appeared to dominate under the present experimental conditions.
Implications of the Results for Cell Volume Regulation
Osmotic swelling experiments yielded the expected increase in the total basolateral membrane conductance.
The unexpected feature was that changes in G Clb do not
parallel the changes in G Kb, because the basolateral
membrane Cl conductance is voltage sensitive. Hence,
it is possible that the effects of cell volume (e.g., via
membrane stretch) are directly exerted on GKb and that
the resulting change in membrane voltage is responsible for the change in G Clb.
Our cell volume measurements suggest that NGB epithelial cells do not regulate their volume after exposure to anisosmotic solution. Our measurements of intracellular [TMA +] and aCli are in apparent contradiction with the results of Spring and associates (Persson
and Spring, 1982; Larson and Spring, 1984
; Furlong
and Spring, 1990
), which suggested that the epithelial cells from NGB undergo regulatory volume decrease
from hyposmotic swelling by basolateral exit of K+ and
Cl
, probably via basolateral membrane channels (Furlong and Spring, 1990
). In contrast, our studies suggest
that there is no measurable short-term cell volume regulation in NGB epithelial cells undergoing osmotic swelling. The experiments on isolated, polarized cells strengthen our conclusions in that they rule out artifacts resulting from TMA+ loading and microelectrode
impalement as possible explanations for the lack of cell
volume regulation. Further, measurements of cell volume changes in isolated polarized cells in which apical membrane GCl was activated by cAMP showed that although these cells do not regulate rapidly their volume
after osmotic swelling (data not shown), they undergo
fast, sizable swelling after elevation of medium [K+],
i.e., under isosmotic conditions. This again indicates
that our results cannot be attributed to a methodological inability to detect cell swelling in isosmotic conditions. The explanation for the differences between our
results and those of Spring and associates (e.g., Furlong
and Spring, 1990
) is not clear.
Implications of the Results for Cross Talk
Our results indicate that cell swelling causes rapid and
reversible changes in basolateral membrane Cl- and
K+-channel activities. The demonstration that GKb is elevated by cell swelling supports the notion that cell volume is a signal responsible for cross talk between apical
and basolateral membranes. However, under these circumstances G Clb not only does not increase, but in fact
decreases. This would not be the expected response if
the cell had been swollen by increased solute entry
across the apical membrane. Under these conditions, the expected cross talk response would be to increase
solute exit across the basolateral membrane. It is in
principle possible that net solute exit is activated by cell
swelling, but not via conductive pathways. A pathway
for basolateral membrane Cl
efflux in NGB epithelium is KCl cotransport (Corcia and Armstrong, 1981
;
Reuss, 1981
). Activation or inactivation of this mechanism during cell swelling and cell shrinkage, respectively, would fulfill the requirements of intermembrane
cross talk, but would also result in cell volume regulation, and hence this possibility is not supported by our
results.
The present observations suggest that GClb is regulated mainly or exclusively by V m changes, although a
minor role of cell volume is possible. The dominant
role of membrane voltage suggests that this regulatory
mechanism is designed to preserve intracellular Cl
content instead of cell volume, i.e., there is an inverse
relationship between electrical driving force and electrodiffusive permeability. Because of the hyperpolarization elicited by activation of GKb after cell swelling, G Clb
falls, and this prevents Cl
loss and tends to maintain
aCli closer to control levels. The need for this mechanism from the point of view of cell homeostasis is not
obvious; cell Cl
content ends up being preserved at
the expense of cell volume. In other cell types, intracellular [Cl
] has been shown or claimed to modulate
membrane transporters (e.g., Robertson and Foskett,
1994
) and regulators, such as protein kinases (Treharne et al., 1994
). In NGB epithelium, in addition to
effects derived from these "messenger" roles of intracellular Cl
, a likely consequence of a decrease in intracellular [Cl
] is a fall in intracellular pH. The apical
membrane of this epithelium expresses a highly active
anion (Cl
/HCO3
) exchanger (Reuss, 1988
). A fall in
aCl i at constant [Cl
] in the apical bathing solution enhances Cl
influx and HCO3
efflux across the apical
membrane, which would cause cell acidification. In
conclusion, osmotic swelling of NGB epithelial cells increases G Kb, but reduces G Clb, and hence there is no appreciable short-term regulatory volume decrease. If cell
swelling is involved in cross talk between apical and basolateral membranes, then the effects of the increase in cell volume are not sufficient to provide an effective
negative-feedback mechanism to restore cell volume to
control levels, suggesting a more complex adaptive
mechanism.
Original version received 3 June 1996 and accepted version received 3 October 1996.
Address correspondence to Luis Reuss, M.D., Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641. Fax: 409-772-3381; E-mail: lreuss{at}mspo2.med.utmb.edu
Dr. Torres's present address is Department of Experimental Medicine, University of Chile, Independencia 1027, Santiago, Chile.We thank Drs. S.A. Lewis and S.A. Weinman for comments on a preliminary version of the manuscript, F. Bavarian and K. Spilker for technical support, and L. Durant for secretarial help.
This work was supported by National Institutes of Health Grant DK-38734.