Osmotic regulation of intestinal epithelial
Na+-K+-Cl
cotransport: role of Cl
and
F-actin
Jeffrey B.
Matthews,
Jeremy A.
Smith,
Edward C.
Mun, and
Jason K.
Sicklick
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, Massachusetts 02215
 |
ABSTRACT |
Previous data indicate that adenosine 3',5'-cyclic
monophosphate activates the epithelial basolateral
Na+-K+-Cl
cotransporter in microfilament-dependent fashion in part by direct action but also in response to apical
Cl
loss (due to cell
shrinkage or decreased intracellular
Cl
). To further address
the actin dependence of
Na+-K+-Cl
cotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed.
Na+-K+-Cl
cotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellular
Cl
concentration and also
to activation of K+ and
Cl
efflux pathways.
Stabilization of actin with phalloidin blunted cotransporter activation
by hypotonicity and abolished hypotonic activation of
K+ and
Cl
efflux. However,
phalloidin did not prevent activation of cotransport by hypertonicity
or isosmotic reduction of extracellular
Cl
. Conversely, hypertonic
but not hypotonic activation was attenuated by the microfilament
disassembler cytochalasin D. The results emphasize the complex
interrelationship among intracellular
Cl
activity, cell volume,
and the actin cytoskeleton in the regulation of epithelial
Cl
transport.
cytoskeleton; microfilament; bumetanide; NKCC1; BSC2
 |
INTRODUCTION |
THE BUMETANIDE-INHIBITABLE
Na+-K+-(2)Cl
cotransporter participates in the homeostatic control of transmembrane
ion gradients and cell volume in diverse cell types (10, 41). In
addition, Na+-K+-Cl
cotransport is an integral component of the salt-transporting apparatus
of many secretory and absorptive epithelia. Ion transfer by
Na+-K+-Cl
cotransporters generally obeys chemical potential. However, acute regulation of cotransport is influenced not only by thermodynamic driving force but also by a number of additional factors, including Na+-K+-Cl
cotransporter protein phosphorylation and, possibly, the function of
associated transmembrane regulatory proteins (6, 10, 41). In many
epithelia, Cl
secretion can
be elicited by agents acting via adenosine 3',5'-cyclic monophosphate (cAMP). Activation of apical
Cl
channels is generally
viewed as the primary regulatory event of cAMP-elicited
Cl
secretion. However,
basolateral
Na+-K+-Cl
cotransport must also increase to maintain cell electrolyte
composition, and therefore active salt secretion demands coordinated
control of apical Cl
exit
and basolateral Cl
entry.
The factors responsible for "cross talk" between apical and
basolateral transport events are incompletely defined but have been
proposed to include changes in cell volume, intracellular Cl
activity
([Cl
]i),
and the actin cytoskeleton (3, 10, 12, 26, 31, 40, 41).
Hypertonic cell shrinkage activates
Na+-K+-Cl
cotransport and mediates a compensatory regulatory volume increase
(RVI) in many epithelial and nonepithelial cells (36). Linkage of
apical and basolateral transport events during cAMP-elicited epithelial
salt secretion could involve signaling cascades similar to those evoked during hypertonic shrinkage (40). In other words, cAMP activation of
apical Cl
channels carries
an obligatory cell water loss, and the resultant cell shrinkage could
then secondarily trigger basolateral
Na+-K+-Cl
cotransport. However, regulation of
Na+-K+-Cl
cotransport during epithelial secretion appears to be more complex. For
example, in HT-29 intestinal (33) and canine tracheal epithelial cells
(11-13), cAMP has been shown to activate cotransport directly, that is, in the absence of cAMP-dependent salt efflux and cell shrinkage. Moreover, some cells have been shown to possess cAMP- and
shrinkage-independent pathways for cotransporter activation; in
tracheal epithelial cells, cotransporter protein phosphorylation can be
increased by manipulations that simply produce a fall in [Cl
]i
(12). Thus, in response to cAMP stimulation, epithelial
Na+-K+-Cl
cotransport may increase as a response to cell shrinkage and/or decreased
[Cl
]i
(both secondary to the activation of apical
Cl
channels) or as a direct
downstream effect of elevated intracellular cAMP.
We previously showed that stimulation of
Na+-K+-Cl
cotransport in T84 human intestinal epithelial cells under a number of
circumstances appears to involve dynamic reorganization of the
microfilamentous cytoskeleton and may be attenuated by the F-actin
stabilizer phalloidin. Thus activation of cotransport by cAMP,
5'-AMP, and guanosine 3',5'-cyclic monophosphate is
blunted in cells loaded with phalloidin (31, 32, 34, 46), and in each
of these cases, activation of transepithelial
Cl
secretion is
proportionately decreased. We also recently showed that F-actin
disassembly using cytochalasin D activates T84 cell Na+-K+-Cl
cotransport in the absence of secretagogue (29). The motivation of the
present study was to further address the actin dependence of regulation
of
Na+-K+-Cl
cotransport. Specifically, we wished to determine whether hypertonic cell shrinkage activates the cotransporter in T84 cells and whether such activation is attenuated by actin stabilization. In so doing, we
unexpectedly found that
Na+-K+-Cl
cotransport was activated not only by extracellular hypertonicity but
also by hypotonicity. Additionally, hypertonic and hypotonic activation
were found to display strikingly different sensitivity to chemical
manipulation of the actin cytoskeleton by phalloidin and cytochalasin
D.
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METHODS |
Cell culture and buffers.
T84 human intestinal epithelial cells obtained from American Type
Culture Collection and Dr. K. Barrett (University of California, San
Diego) were maintained in culture as previously described (7, 31). For
experiments, cells were seeded onto
1-cm2 collagen-coated permeable
supports (Costar, Cambridge, MA) and used after confluence and stable
transepithelial electrical resistances were achieved, ~7-14 days
after they were plated. A variety of buffered electrolyte solutions
were used for these experiments to examine the effects of changes in
extracellular tonicity and/or ionic composition. These buffers
are modifications of a
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-phosphate-buffered Ringer solution used in previous studies (29,
31-33) and are described in detail in Table
1. All solutions were prewarmed to
37°C, and experiments were carried out at 37°C, with
temperature control by thermal probe-coupled heat lamps.
Isotopic flux studies.
The functional activity of the basolateral
Na+-K+-Cl
cotransporter of confluent T84 monolayers was determined by
radioisotopic methods (bumetanide-sensitive
86Rb uptake), as previously
described (29, 31-33). Briefly, monolayers grown on permeable
supports were equilibrated in
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-phosphate-buffered Ringer solution for 20 min. At the beginning of
the experimental period, the apical buffer was removed, and monolayers
were then washed by three rapid dips into a beaker containing
100-200 ml of the appropriate experimental buffer. Inserts were
then placed in 12-well plates containing 1 ml of the appropriate
experimental solution with or without 20 µM bumetanide. The identical
buffer (1 ml, without bumetanide) was added to the apical side of the
insert. To initiate uptakes, inserts were transferred to new wells
containing the identical basolateral solution with the addition of
1-1.5 µCi/ml 86Rb. Uptake
has previously been shown to be linear for up to 5 min, and for the
experiments reported here a 3-min uptake period was routinely used.
Uptakes were terminated by rapidly washing the monolayers by 10 rapid
dips in ice-cold stop solution containing 100 mM
MgCl2 and 10 mM
tris(hydroxymethyl)aminomethane · HCl, pH 7.4. Filters were then cut out from the plastic support and placed in
scintillation vials containing 3 ml of Aquasol. Radioactivity was
counted using a Packard Liquid Scintillation counter. Representative monolayers were used for protein determination by bicinchoninic acid
assay (Pierce Chemical). Uptakes were expressed as nanomoles of
K+ per milligram of protein per
minute. Activation of Cl
and K+ efflux pathways in response
to osmotic challenge was measured by a modification of a method
previously described by Venglarik et al. (50), using
125I and
86Rb as tracers, respectively, and
previously reported techniques (31, 33). Efflux rate constants were
expressed per minute, as in previous studies.
Transepithelial transport.
Short-circuit current
(Isc), which
represents electrogenic Cl
secretion in the T84 model, was measured in monolayers grown on
0.33-cm2 permeable supports using
a dual voltage-current clamp and Ag-AgCl and calomel electrodes
interfaced via "chopstick" KCl-agar bridges, as previously
described (29, 31, 32).
Phalloidin loading.
Phalloidin (Sigma Chemical, St. Louis, MO) was dissolved in methanol as
a stock solution. For experiments, aliquots of the methanol stock
solution were dried under N2 and
dissolved in media to a final concentration of 3-10 µM.
Monolayers were loaded with phalloidin by overnight incubation, as
previously described (29, 31, 32). Adequacy of phalloidin loading was
assessed by measurement of inhibition of forskolin-stimulated
Isc responses
compared with unloaded control monolayers. The absence of monolayer
toxicity was confirmed by the preservation of transepithelial
resistance and the
Isc response to
carbachol, as previously described (31).
Fluorescence microscopy.
Fluorescent staining of the F-actin cytoskeleton was performed as
described previously (15, 29, 32). Briefly, monolayers grown on glass
coverslips were rinsed in phosphate-buffered saline, fixed in 3.7%
formaldehyde, and permeabilized in ice-cold acetone. Monolayers were
then dried and stained with rhodamine-phalloidin, and coverslips were
mounted upside down on glass slides in phosphate-buffered saline-glycerol-p-phenylendiamine.
Cells were examined using a Zeiss IM-35 inverted microscope equipped
for epifluorescence and photographed with Kodak Tri-X film (1,000 ASA).
Morphological analysis consisted of blinded review of slides by one
investigator without knowledge of the treatment groups.
Materials and statistical analysis.
Radionuclides were obtained from Dupont NEN (Boston, MA). All chemicals
were obtained from Sigma Chemical, with the exception of forskolin,
which was from Calbiochem (La Jolla, CA), and rhodamine-phalloidin, which was from Molecular Probes (Eugene, OR). Statistical analysis was
by Student's t-test for paired
variates and by analysis of variance, where appropriate, with
P < 0.05 considered statistically significant.
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RESULTS |
Response to hypertonicity.
The basal rate of
Na+-K+-Cl
cotransporter activity measured by bumetanide-sensitive
86Rb
(K+) uptake across the
basolateral membrane of confluent T84 monolayers was 4.63 ± 0.68 nmol K+ · mg
protein
1 · min
1
for 16 separate experiments each performed on duplicate or triplicate monolayers. We expected to demonstrate that hypertonic shock would activate
Na+-K+-Cl
cotransport in these cells. However, exposure of monolayers to hypertonic NaCl buffer for 10 min only marginally affected
Na+-K+-Cl
cotransport to a level that did not reach statistical significance (Fig. 1). We found that a more
substantial cotransporter response to hypertonicity could be uncovered
if the same degree of extracellular hypertonicity was imposed using
hypertonic mannitol buffer rather than hypertonic NaCl (Fig. 1). The
degree of activation of cotransport by hypertonic mannitol was
considerably lower than the response to a typical secretory agonist
such as forskolin (41.0 ± 9.6 nmol K+ · mg
protein
1 · min
1,
n = 3, 10 µM forskolin stimulation
for 10 min). Hypertonicity did not increase transepithelial
Isc (data not
shown).

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Fig. 1.
Response of T84 cell
Na+-K+-Cl
cotransporter to hypertonicity. T84 monolayers were equilibrated in
isotonic buffer and then transferred to identical buffer
(n = 15 experiments each performed on
triplicate monolayers), hypertonic NaCl buffer
(n = 5), or hypertonic mannitol buffer
(n = 11) for 10 min.
Bumetanide-sensitive uptake of K+
(86Rb) across basolateral membrane
was then measured. Buffer solutions are described in Table 1.
Significant increase over isotonic conditions was evident only in
hypertonic mannitol group [P < 0.005, by analysis of variance (ANOVA) with Fisher's preserved least
significant difference (PLSD) post hoc correction].
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Although the response to hypertonicity under high salt conditions
(hypertonic NaCl) was considerably less than under standard conditions,
we did not find evidence that the response to hypertonicity could be
meaningfully enhanced by a further reduction in extracellular Cl
concentration. For
example,
Na+-K+-Cl
cotransport after a 20-min equilibration in isotonic gluconate buffer
(76.5 mM Cl
) was 8.92 ± 1.6 nmol
K+ · mg
protein
1 · min
1,
and this increased to 16.9 ± 3.0 nmol
K+ · mg
protein
1 · min
1
after an increase in osmolarity to 445 mosM using 135 mM
added mannitol, a value comparable to that shown in Fig. 1.
Response to hypotonicity.
Hypotonic cell swelling evokes a regulatory volume decrease (RVD) in
most cells that usually involves inhibition of
Na+-K+-Cl
cotransport and activation of volume-sensitive
K+ and
Cl
channels (36).
Shrinkage-elicited activation of cotransport in some cell systems has
been shown to be enhanced by prior cell swelling (18). Because the T84
cotransporter response to hypertonicity seemed somewhat meager, we
examined the effect of isotonic cell shrinkage on T84 cell cotransport.
To do this, we initially exposed cells to hypotonic buffer, then
returned the cells to isotonic buffer, a so-called RVI-after-RVD
maneuver (18). To our surprise, we found that cotransport was
dramatically activated during the initial exposure to a nominally 175 mosM hypotonic buffer. As shown in Fig. 2,
the rate of bumetanide-sensitive
K+ uptake in response to this
hypotonic buffer was at least as great in magnitude as the response to
hypertonic mannitol buffer. Subsequent return to isosmotic conditions
after hypotonic shock in fact did not substantially increase
Na+-K+-Cl
cotransport beyond the already-stimulated activity (not shown). The
increase in cotransport evoked by hypotonicity was maximal ~10 min
after exposure (Fig. 2B).

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Fig. 2.
Response of T84 cell
Na+-K+-Cl
cotransporter to hypotonicity. A: T84
monolayers were equilibrated in isotonic buffer and then transferred to
identical buffer or hypotonic buffer (solutions described in Table 1)
for 10 min. Bumetanide-sensitive uptake of
K+
(86Rb) across basolateral membrane
was then determined. Values are means ± SE for 8 experiments
performed in triplicate, with P < 0.001 by paired t-test.
B: time course of activation of
Na+-K+-Cl
cotransport by hypotonic buffer. After monolayers were exposed to
hypotonic buffer for indicated time period, bumetanide-sensitive uptake
of K+
(86Rb) across basolateral membrane
was determined. Values are means ± SE for 3 monolayers.
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This unexpected activation of the
Na+-K+-Cl
cotransporter by hypotonicity could be due to the acute reduction in
extracellular tonicity and/or reduction in extracellular NaCl
concentration. To examine these possibilities, first, the effect of an
isosmotic reduction in extracellular NaCl concentration was studied.
Monolayers were bathed in isotonic buffer or in buffer in which 67.5 mM
NaCl was replaced with equimolar Na-gluconate (to decrease
Cl
concentration only),
N-methylglucamine (NMG)-Cl (to
decrease Na+ concentration only),
or mannitol (to decrease Cl
and Na+ concentrations). As shown
in Fig.
3A,
cotransporter activity markedly increased when
Cl
concentration was
reduced (isotonic gluconate and mannitol buffer) but not when
Na+ concentration alone was
decreased (isotonic NMG buffer). These data must be interpreted with
caution, inasmuch as it would be expected that isosmotic replacement of
a permeant ion (Cl
and/or Na+) with an
impermeant ion species induces a degree of cell shrinkage (28).
However, it seems unlikely that cell shrinkage could entirely account
for the activation response to these ion substitutions; if this were
the case, one would have expected to see substantial activation of
cotransport by isotonic NMG-Cl buffer. Nevertheless, a contribution
from cell shrinkage cannot be excluded; we did observe that isosmotic
substitution of Cl
with
failed to activate cotransport.
Because
permeation of anion
transport pathways more closely approximates
Cl
, little or no cell
shrinkage would be expected (11, 12). Unfortunately, and further
confounding the analysis,
has been
shown to display unusual inhibitory behavior with respect to the
outward-facing and, possibly, the inward-facing conformation of the
cotransporter compared with larger, more inert anion substituents (11,
16, 51). Hypotonic activation exceeded activation by isotonic gluconate
or isotonic mannitol buffer (P < 0.02). In fact, the degree of activation by hypotonicity exceeds the
degree of activation by isosmotic ion substitution of
Cl
(Na-gluconate)
throughout a range of extracellular
Cl
concentrations, as
indicated in Fig. 3B. This suggests
that the activation response to extracellular hypotonicity is in part
reflective of the reduction in extracellular
Cl
concentration but that
the hypotonic stimulus itself confers an additional component of
activation on the cotransporter.

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Fig. 3.
Response of
Na+-K+-Cl
cotransporter to isosmotic or hypotonic reduction in extracellular NaCl
concentration. A: activation of
Na+-K+-Cl
cotransport by isosmotic reduction in extracellular NaCl concentration
largely reflects decrease in
Cl rather than
Na+. T84 monolayers were
equilibrated in isotonic buffer and then transferred to identical
isotonic buffer, isotonic gluconate buffer, isotonic
N-methylglucamine (NMG) buffer,
isotonic mannitol buffer, isotonic
buffer, or hypotonic buffer for 10 min. Bumetanide-sensitive uptake of
K+
(86Rb) across basolateral membrane
was then determined. Buffers are described in Table 1; isotonic
buffer was similar to isotonic
gluconate buffer, except it contained
instead of gluconate. Each bar
represents mean and error bars represent SE of 3 separate experiments
performed in triplicate. Differences are statistically significant by
ANOVA with Fisher's PLSD post hoc correction for isotonic vs.
hypotonic (P < 0.0002), isotonic
gluconate (P < 0.015), and isotonic
mannitol (P < 0.003).
B: activation of cotransporter by
reduction of extracellular
Cl concentration
([Cl ]o)
is greater under hyposmotic than isotonic conditions. T84 monolayers
were equilibrated in isotonic buffer and then transferred to identical
isotonic buffer (140 mM
Cl ) or buffer in which
Cl was reduced to indicated
levels by removal of NaCl (hypotonic) or replacement with equimolar
Na-gluconate (isotonic). Bumetanide-sensitive uptake of
K+
(86Rb) across basolateral membrane
was then determined. Values are means ± SE for 5 hypotonic or 8 isotonic monolayers. Difference between hypotonic and isotonic
conditions is significant at P < 0.05 by ANOVA with Fisher's PLSD post hoc correction.
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We next wished to determine whether hypotonic shock in the absence of a
change in extracellular Cl
concentration affects
Na+-K+-Cl
cotransport. To this end, a series of experiments was performed to
explore the effect of hypotonicity at constant (but reduced) extracellular NaCl concentration. Monolayers were preequilibrated for
30 min in isotonic mannitol buffer in which 67.5 mM NaCl was replaced
by 135 mM mannitol. Monolayers were then transferred to hypotonic
buffer with the identical NaCl concentration (67.5 mM) but without
mannitol. As shown in Fig. 4, hypotonicity
persisted in activating
Na+-K+-Cl
cotransport, although the response was considerably less
than that elicited by hypotonicity with reduced extracellular
Cl
concentration (Fig. 3).
Others have reported that hypotonic stress activates transepithelial
Cl
secretion in T84
monolayers, possibly by a
Ca2+-dependent signaling pathway
(35). However, in our hands, no such stimulation could be elicited in
monolayers exposed to hypotonic buffer
(n = 6). Despite the absence of a
hypotonicity-elicited Isc, hypotonicity
did activate K+ and
Cl
efflux pathways.
Specifically, the rate constant of efflux of 86Rb from preloaded monolayers
substantially increased on exposure to hypotonic buffer (Fig.
5A).
This increase in 86Rb efflux was
confined to the basolateral aspect of the monolayers and also occurred
in monolayers pretreated with 20 µM bumetanide. This observation
suggests, first, that enhanced efflux of
86Rb likely reflects the
activation of volume-activated K+
channels that are basolaterally restricted and, second, that the
increase in cotransporter activity indeed represents enhanced net
inward flux of K+ (and presumably
the cotransported Na+ and
Cl
) through this pathway.
Activation of 125I efflux into the
basolateral but not apical buffer was also evident (Fig.
5B), suggesting that osmosensitive
Cl
channels are likely to
be present predominantly in the basolateral domain.

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Fig. 4.
Hypotonicity activates
Na+-K+-Cl
cotransport even at constant extracellular
Cl concentration. T84
monolayers were equilibrated in isotonic mannitol buffer, as described
in Table 1, and then transferred to identical buffer or hypotonic
buffer. Thus, under both conditions, extracellular
Cl concentration was 72.5 mM. Bumetanide-sensitive uptake of
K+
(86Rb) across basolateral membrane
was then determined. Each bar represents mean and error bars represent
SE of 3 experiments performed on triplicate monolayers. Difference is
small but statistically significant (P < 0.02, by paired t-test).
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Fig. 5.
Hypotonicity increases bumetanide-resistant
K+ efflux and
Cl efflux.
A: T84 monolayers were preloaded with
86Rb for 180 min. After 4 rapid
washes, monolayers were transferred to isotonic or hypotonic buffer
with or without 20 µM bumetanide, and efflux of
86Rb into basolateral buffer was
determined. Values are means ± SE of 4 monolayers in each group.
Difference between hypotonic and isotonic conditions is significant
(P < 0.0001, by ANOVA with Fisher's
PLSD post hoc correction). Bumetanide had no effect on efflux rate
constant in either buffer. Efflux of
86Rb into apical buffer for these
same monolayers was <5% of total efflux and did not differ between
isotonic and hypotonic buffers (not shown).
B: T84 monolayers were preloaded with
125I for 180 min, rapidly washed 4 times over 2 min, and then transferred to isotonic buffer. Two baseline
1-min efflux determinations were made; then at time indicated by arrow,
monolayers were transferred to hypotonic buffer, and 1-min
125I rate constants for apical and
basolateral efflux were measured. Values are means ± SE for 3 monolayers. Lines are statistically different
(P < 0.0005, by
ANOVA).
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Influence of the actin cytoskeleton.
We previously showed that activation of
Na+-K+-Cl
cotransport by cyclic nucleotide-dependent agonists in T84 monolayers
is markedly attenuated in monolayers loaded with the F-actin stabilizer
phalloidin (31, 32). Activation of
Cl
secretion by cyclic
nucleotide agonists has been shown to be paralleled by a rearrangement
of F-actin in the basal pole of T84 monolayers, an event that is also
blunted in phalloidin-loaded cells (31, 32). Hypotonicity, but not
hypertonicity, was associated with a rearrangement of basal F-actin
that resembled that seen in our earlier reports for
secretagogue-induced rearrangements (Fig.
6) (31, 32) and previously described in
other cell systems (20, 53). Isosmotic reduction of extracellular
Cl
concentration using
gluconate buffer, however, did not induce a significant rearrangement
of microfilament architecture (Fig. 6).

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Fig. 6.
Hypotonicity but not reduced extracellular
Cl concentration remodels
F-actin. En face fluorescently labeled images of F-actin in T84
monolayers are shown. Optical plane of focus is at basal-most pole of
cells. In control monolayer (A) and
in monolayer incubated in isotonic gluconate buffer for 30 min
(B), basal actin stress fibers
appear as randomly dispersed homogeneous filaments (open arrowheads).
No significant difference in morphological appearance could be
discerned by blinded review of 6 monolayers in each group. In contrast,
monolayers exposed to hypotonic buffer for 30 min
(C) showed evidence of dramatic
rearrangement of F-actin, with peripheral displacement and shortening
of actin filaments into clumps (filled arrowhead) around a relatively
cleared central zone (*). These changes were easily distinguished
from controls in 6 of 6 monolayers so treated. Images are magnified
×1,000.
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We then examined whether phalloidin would also affect osmotic
activation of cotransport. As shown in Fig.
7, phalloidin reduced the increase in
bumetanide-sensitive 86Rb uptake
induced by hypotonic buffer by ~40%. In contrast, hypertonic activation of cotransport was not inhibited by phalloidin; in fact, it
was enhanced. Phalloidin marginally reduced the activation of
Na+-K+-Cl
cotransport induced by isosmotic reduction in extracellular
Cl
concentration, but this
did not reach statistical significance (8.1 ± 1.0 vs. 6.4 ± 0.32 nmol
K+ · mg
protein
1 · min
1
for control vs. phalloidin treatment after 10 min of exposure to
isotonic gluconate buffer, each n = 6, P = NS). Hypotonic stimulation of
bumetanide-insensitive Cl
(125I) and
K+
(86Rb) efflux apparently required
F-actin remodeling, inasmuch as both responses were completely blocked
in phalloidin-loaded cells (Table 2). In
contrast to results with phalloidin, the F-actin disassembler
cytochalasin D, which was recently reported to enhance bumetanide-sensitive 86Rb uptake
in T84 cells (29), did not affect hypotonic activation of cotransport;
however, the ability of hypertonicity to activate cotransport was
diminished or abolished (Fig. 8).

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Fig. 7.
Stabilization of F-actin inhibits hypotonic but not hypertonic
activation of
Na+-K+-Cl
cotransport. Subsets of T84 monolayers were loaded with 15 µM
phalloidin in media by overnight coincubation; control monolayers
underwent simple media change. Monolayers were equilibrated in isotonic
buffer for 20 min and then transferred to identical buffer, hypotonic
mannitol buffer, or hypertonic mannitol buffer for 10 min with or
without 20 µM bumetanide. Bumetanide-sensitive uptake of
K+
(86Rb) across basolateral membrane
was then determined. Values are means ± SE for 6 monolayers in each
group performed in 1 day. Loading of phalloidin was confirmed by loss
of forskolin-stimulated short-circuit current and absence of toxicity
by preservation of carbachol-elicited short-circuit current. Similar
results were obtained in 3 additional experiments. Differences between
control and phalloidin conditions are significant for hypotonic
(decreased by phalloidin at P < 0.01) and hypertonic conditions (increased by phalloidin at
P < 0.002) by paired
t-test.
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Fig. 8.
Disassembly of F-actin inhibits hypertonic but not hypotonic activation
of cotransport. Subsets of T84 monolayers equilibrated in isotonic
buffer for 20-30 min were pretreated with or without 20 µM
cytochalasin D for 10 min. Monolayers were then transferred to isotonic
buffer or hypotonic buffer (A) or
isotonic or hypertonic mannitol buffer
(B). Bumetanide-sensitive uptake of
K+
(86Rb) across basolateral membrane
was then determined. Hypotonicity activates cotransport regardless of
presence of cytochalasin D, although cytochalasin D significantly
increases cotransport in isotonic buffer
(P < 0.001, by paired
t-test). In contrast, hypertonic
conditions fail to activate cotransport after cytochalasin D
pretreatment. Values are means ± SE for 3 experiments performed on
triplicate monolayers in each group.
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DISCUSSION |
Recent molecular studies have identified two major isoforms of the
Na+-K+-Cl
cotransporter (8, 27, 41, 42, 52). The more widely distributed NKCC1
(or BSC2) represents the major
Cl
loading pathway in
secretory epithelia and mediates cotransport in a wide range of
nonepithelial cell types. NKCC2 (or BSC1) is restricted to the kidney
and mediates apical salt uptake in absorptive renal epithelia. A number
of reports indicate that alterations in
Na+-K+-Cl
cotransporter function may substantially modulate transepithelial Cl
secretory capacity (14,
30, 31, 46); it is becoming apparent that NKCC1 is not simply a passive
element responding to apical membrane events but that it may serve as
an important independent regulatory site. In this study we establish
that osmotic stress, both hypertonic and hypotonic, activates inward
Na+-K+-Cl
cotransport across the basolateral membrane of polarized T84 human
intestinal epithelial cells without eliciting a corresponding increase
in transepithelial Cl
secretion. Clearly, cotransporter activation under these conditions is
dissociated from events at the apical membrane, confirming that
transport function of NKCC1 is not invariably linked to the secretory
state of the epithelium. Moreover, such data serve to illustrate that,
in epithelial cells, NKCC1 has the capacity to be regulated
independently of apical Cl
channels, although such regulation is not sufficient for activation of
transepithelial salt secretion. Similar conclusions have been drawn on
the basis of results obtained in airway epithelia (22) and shark rectal
gland (24).
Hypertonic cell shrinkage and cAMP lead to enhanced serine/threonine
phosphorylation of NKCC1 in models such as the shark rectal gland, an
event associated with an increase in the number of binding sites for
[3H]benzmetanide
in parallel with increased cotransport of ions (14, 24, 25, 30). The
specific kinases and/or phosphatases that directly affect the
state of NKCC1 phosphorylation in response to these stimuli remain
undefined. Although cAMP increases cotransport, NKCC1 phosphorylation,
and [3H]benzmetanide
binding, no region of the cloned shark NKCC1 isoform conforms to
consensus sequences for cAMP-dependent protein kinase (PKA) (41, 42,
52). Human NKCC1 contains one such sequence, although whether this site
plays a role in cAMP-dependent regulation is not established. Direct
evidence for a putative kinase downstream from PKA has been lacking.
Activation of Cl
secretion
by cAMP results in cell shrinkage due to apical
Cl
loss. Theoretically, a
shrinkage-activated kinase [such as the "V-kinase"
postulated to regulate transport events in erythrocytes (19, 23)]
could participate in shrinkage- and cAMP-dependent activation of
Na+-K+-Cl
cotransport. Indeed, Lytle (23) recently demonstrated that cAMP and
hypertonicity phosphorylate erythrocyte NKCC1 at common sites. Klein
and O'Neill (21) reported that myosin light chain kinase (MLCK) is a
shrinkage-activated kinase in endothelial cells and that the MLCK
inhibitor ML-7 inhibits shrinkage activation of cotransport. However,
MLCK is unlikely to be directly involved in shrinkage- and
cAMP-dependent activation of NKCC1, since inhibition by ML-7 did not
decrease hypertonic phosphorylation of NKCC1; moreover, PKA decreases
MLCK activity in a number of cells (38).
A decrease in
[Cl
]i
secondary to activation of cAMP-dependent apical channels may also
account for enhanced
Na+-K+-Cl
cotransport. Recent data from a number of cell types suggest the
possibility that, beyond its role in setting thermodynamic driving
force,
[Cl
]i
may modulate cotransport activity through direct kinetic inhibition of
NKCC1 itself or of a key regulatory protein (2, 3, 11, 12, 41). This
concept was first put forth by Breitwieser et al. (2) for hypertonic
regulation of cotransport in squid giant axon. Lytle and Forbush (24,
25) demonstrated an increase in cotransport activity, NKCC1
phosphorylation, and
[3H]bumetanide binding
in response to Cl
-free
media in shark rectal gland. Haas et al. (11, 13) demonstrated activation of cotransport and enhanced bumetanide binding in dog tracheal epithelial cells in response to apical
Cl
efflux elicited by the
cAMP-independent agonist UTP. More recently, Haas et al. (12) reported
[Cl
]i-dependent
phosphorylation of NKCC1 in nystatin-permeabilized airway epithelial
cells. How a change in
[Cl
]i
kinetically modifies cotransport and/or NKCC1 phosphorylation is a matter of speculation, but the presence of a
[Cl
]i-sensitive
regulatory element has now been postulated not only in the case of
NKCC1 but also for
Na+/H+
exchange and a nonselective cation channel in rat salivary acinar cells
(45) and for
Na+/H+
exchange in rat distal colonic crypts (44). Low
[Cl
]i
may create a "permissive" environment for
phosphorylation-dependent regulation of such pathways (2). Treharne et
al. (49) reported two novel protein kinases in airway epithelia that
were progressively inhibited as
Cl
concentration increased
above 50 mM and suggested that such anion-sensitive kinase activity may
explain the
[Cl
]i-dependent
regulation of various ion transporters. In our experiments, [Cl
]i
(to the extent that it was affected by changes in extracellular Cl
concentration) appeared
to exert some influence over hypertonic activation of cotransport. That
is, at the same level of extracellular hypertonicity (445 mosM), inward
bumetanide-sensitive fluxes were twice as large in buffer containing
added mannitol as in buffer containing added NaCl. However, hypertonic
stimulation of cotransport was not significantly enhanced under
conditions of reduced (76 mM) extracellular
Cl
concentration.
In this study,
Na+-K+-Cl
cotransport increased sharply in response to hypotonic stress. Although
in numerous cell systems, including endothelia (39), erythrocytes (1),
and Xenopus oocytes (48), cotransport
has been found to be inhibited by hypotonicity, this finding is not
unprecedented. Lytle and Forbush (24) reported that hypotonicity
increases
[3H]benzmetanide
binding and NKCC1 phosphorylation in shark rectal gland, an effect
attributed to a decrease in
[Cl
]i,
although the functional correlate of this finding in terms of ion
translocation was not addressed. Experimentally, it is difficult to
dissociate changes in
[Cl
]i
from changes in cell volume. However, we suspect that cotransport is
stimulated not by cell swelling per se, but rather by the events brought about by cell swelling, specifically KCl extrusion. Comparison of time course data shows that the increased rate of
86Rb and
125I efflux induced by
hypotonicity wanes after ~8 min (suggesting that RVD has largely been
accomplished), whereas the stimulated rate of cotransport does not
reach its peak until 10 min and appears to persist. Although we did not
directly measure
[Cl
]i
or cell volume in the present study, it seems reasonable to expect that
hypotonicity indeed decreases
[Cl
]i,
since extracellular Cl
concentration is reduced and 125I
efflux is enhanced. Isosmotic substitution of
Cl
but not
Na+ also activated cotransport,
although such manipulations also likely induced cell shrinkage. Cell
shrinkage occurs to a lesser extent or not at all when
rather than gluconate is used as
the replacement anion (11). However, the failure of isotonic
buffer to stimulate cotransport in
our experiments could also be attributable to an inhibitory interaction
of this species with the external- or internal-facing conformation of
the cotransporter (12, 16, 51) or to
inhibition of a putative
anion-sensitive kinase, such as was the case for one of the two kinases
identified by Treharne et al. (49).
The mechanism by which cotransporter function is influenced by the
actin cytoskeleton remains obscure and may be indirect. Inhibition of
MLCK by ML-7 suppresses hypertonic stimulation of cotransport but not
phosphorylation of NKCC1 (21). This is consistent with our previous
finding that the actin stabilizer phalloidin attenuates cAMP-dependent
activation of cotransport but not
[3H]bumetanide binding
(33). We previously demonstrated that cytochalasin D activates
Na+-K+-Cl
cotransport in T84 cells (29), and in the present study, cytochalasin D
was found to attenuate hypertonic but not hypotonic activation of
cotransport. Jessen and Hoffmann (20) noted similar behavior in Ehrlich
ascites cells, where cytochalasin B activated basal cotransport but attenuated hypertonic activation. In contrast, phalloidin attenuated only hypotonic but not hypertonic activation of
Na+-K+-Cl
cotransport. This implies that the signal transduction cascades required for hypertonic activation in T84 cells require the presence of
an intact or organized filament system but does not involve active
cytoskeletal remodeling per se. Indeed, hypotonicity but not
hypertonicity elicits F-actin rearrangements reminiscent of the
phalloidin-sensitive cytoskeletal reorganization previously demonstrated in cAMP-stimulated T84 cells (46); these changes resemble
hypotonic actin remodeling observed by others in shark rectal gland and
Ehrlich ascites cells (20, 53). Hypotonic stimulation of cotransport
appears to require this actin remodeling, since it is attenuated by
phalloidin. Hypotonic stimulation of cotransport may depend in part on
stimulation of bumetanide-insensitive K+ and
Cl
efflux pathways, both of
which were blocked in phalloidin-loaded cells. How a change in filament
structure affects the function of ion transport proteins is poorly
understood, but increasing numbers of examples of such regulation have
been identified (4, 5, 43).
Hypotonic and cAMP-dependent activation of the T84 cotransporter are
likely related at least in part to a decrease in
[Cl
]i.
This raises the speculative possibility that the
[Cl
]i-sensitive
regulatory site could also be F-actin dependent. If this is true, this
cytoskeletal dependence of the
Cl
sensor could account for
our previous finding that phalloidin inhibited cAMP-stimulated
cotransport more profoundly in the HT-29 subclone that expressed the
regulated Cl
efflux pathway
than in the undifferentiated parent line (33). Of interest
in this regard is the recent finding that stimulated Cl
efflux appears to be
required for the actin remodeling that accompanies cytokine-elicited
neutrophil activation (37). Moreover,
Cl
-depleted buffer in the
absence of cytokine has been shown to elicit neutrophil actin
polymerization, protein phosphorylation, lysozyme secretion, and
respiratory burst activity (9, 37). Similar to its aforementioned role
as a possible kinetic regulator of ion transport pathways,
[Cl
]i
could modulate the activity of the kinases, phosphatases, or other
regulatory proteins that subsequently affect actin nucleation cascades.
Halide binding to heterotrimeric G proteins has been shown by
Higashijima et al. (17) to affect intrinsic guanosine 5'-triphosphatase activity, and numerous GTP-binding proteins are
known to affect cytoskeletal organization. Against this hypothesis, however, stands our observation in the present study that an isosmotic reduction in extracellular
Cl
concentration does not
appear in and of itself to cause a marked actin rearrangement, and
stimulation of cotransport by this manipulation was only marginally if
at all blunted by phalloidin. In the absence of direct measurements of
[Cl
]i
and cell volume, it is difficult to fully interpret these data, however. As stated above, part of the activation of cotransport by the
isotonic gluconate buffer may involve cell shrinkage in addition to a
lowering of
[Cl
]i;
phalloidin appeared to exert an intermediate effect on isotonic gluconate stimulation of cotransport compared with phalloidin's augmentation of the hypertonic response and blunting of the hypotonic response.
In summary, we have shown in model T84 intestinal epithelial cells that
Na+-K+-Cl
cotransport is activated by hypertonic stress and reduced extracellular Cl
concentration in the
absence of a stimulus for transepithelial Cl
secretion. However, cAMP
(forskolin stimulation) in this model elicits a far greater activation
of cotransport than either hypertonic shrinkage or low extracellular
Cl
concentration, even when
hypertonic stress and low extracellular Cl
concentration stimuli
are presented in combination. This implies that cAMP may well act
through a pathway other than simply reduced cell
Cl
and shrinkage. Our
previous findings that cAMP-dependent activation of cotransport is
associated with F-actin remodeling raise the speculative consideration
that the additional pathway could involve the cytoskeleton. In this
context, we found that activation of cotransport by hypertonic stress
and by reduced extracellular Cl
concentration is not
associated with obvious actin rearrangements, and in neither case is
the response diminished by phalloidin-induced actin stabilization. In
contrast, we showed that a hypotonic stimulus activates
Na+-K+-Cl
cotransport in phalloidin-sensitive fashion and also induces F-actin
remodeling reminiscent of that previously observed with cAMP.
Activation of cotransport under these conditions may reflect an
associated fall in
[Cl
]i
due to reduced extracellular
Cl
concentration as well as
hypotonic-stimulated KCl efflux. Our findings emphasize the complex
interrelationship among
[Cl
]i,
cell volume, and the cytoskeleton in the regulation of
Na+-K+-Cl
transport and, by extension, epithelial
Cl
secretion.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-48010 and by the George H. A. Clowes, Jr., MD, FACS, Memorial Career Development Award from the American College of Surgeons (to J. B. Matthews).
 |
FOOTNOTES |
Some of these data have appeared in abstract form (47).
Address for reprint requests: J. B. Matthews, Dept. of Surgery, Beth
Israel Deaconess Medical Center, East Campus, 330 Brookline Ave.,
Boston, MA 02215.
Received 1 July 1997; accepted in final form 5 November 1997.
 |
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