Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through
a novel Cl
-dependent mechanism
Gengru
Jiang1,
Janet D.
Klein1, and
W. Charles
O'Neill1,2
1 Renal Division, Department of Medicine, and
2 Department of Physiology, Emory University School of
Medicine, Atlanta, Georgia 30322
 |
ABSTRACT |
The Na-K-2Cl
cotransporter NKCC1 is an important volume-regulatory transporter that
is regulated by cell volume and intracellular Cl
. This
regulation appears to be mediated by phosphorylation of NKCC1, although
there is evidence for additional, cytoskeletal regulation via myosin
light chain (MLC) kinase. NKCC1 is also activated by growth factors and
may contribute to cell hypertrophy, but the mechanism is unknown. In
aortic endothelial cells, NKCC1 (measured as bumetanide-sensitive
86Rb+ influx) was rapidly stimulated by serum,
lysophosphatidic acid, and fibroblast growth factor, with the greatest
stimulation by serum. Serum increased bumetanide-sensitive influx
significantly more than bumetanide-sensitive efflux (131% vs. 44%),
indicating asymmetric stimulation of NKCC1, and produced a 17%
increase in cell volume and a 25% increase in Cl
content
over 15 min. Stimulation by serum and hypertonic shrinkage were
additive, and serum did not increase phosphorylation of NKCC1 or MLC,
and did not decrease cellular Cl
content. When cellular
Cl
was replaced with methanesulfonate, influx via NKCC1
increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we propose
a novel mechanism whereby serum activates NKCC1 by reducing its
sensitivity to inhibition by intracellular Cl
. This
resetting of the Cl
set point of the transporter enables
the cotransporter to produce a hypertrophic volume increase.
Na-K-2Cl cotransport; serum; lysophosphatidic acid; fibroblast
growth factor; intracellular chloride; vascular endothelium
 |
INTRODUCTION |
THE UBIQUITOUS FORM
of the Na-K-2Cl cotransporter (NKCC1) has several important functions,
including regulation of cell volume and intracellular Cl
concentration ([Cl
]) and the vectorial transport of
salt across secretory epithelia. Consequently, the transporter is
regulated both by cell volume and intracellular Cl
. The
mechanisms responsible for this regulation are complex and poorly
understood. Activity of NKCC1 is increased by inhibitors of protein
phosphatases and reduced by kinase inhibitors (10, 26),
and phosphorylation of the transporter is increased by cell shrinkage
or by a reduction in intracellular [Cl
] (6, 9,
16, 17). Phosphorylation occurs at the same site or sites with
either stimulus (14), suggesting that NKCC1 is activated
by a specific protein kinase. We have recently identified c-jun
amino-terminal kinase as a volume-sensitive kinase capable of
phosphorylating NKCC1 in vitro (8).
Additional regulation of NKCC1 occurs through myosin light chain (MLC),
the phosphorylation of which is increased by cell shrinkage (9,
27). Inhibition of MLC kinase (MLCK) blocks NKCC1 activation in
endothelial cells (9), Ehrlich ascites cells
(11), colonic epithelial cells (7), and
vascular smooth muscle (1), and in endothelial cells this
is independent of cotransporter phosphorylation. Activation of the
cotransporter in colonic epithelial cells is also affected by agents
that alter F-actin (7, 18, 19). The mechanism by which the
cellular contractile apparatus or cytoskeleton regulates NKCC1 is not known.
Although intracellular Cl
affects phosphorylation of the
cotransporter (6, 17), regulation by this ion is probably
more complex and has important implications. Despite being
phosphorylated and activated by hypertonic shrinkage, NKCC1 does not
produce a net influx of ions, despite being thermodynamically favored (13, 21). Net influx and volume recovery after shrinkage
only occur when intracellular [Cl
] is reduced
(isosmotic shrinkage). This is consistent with the asymmetric
activation of NKCC1 observed in endothelial cells (21). Influx and efflux via NKCC1 are stimulated in parallel by hypertonic shrinkage (increased intracellular [Cl
]), but with
isosmotic shrinkage (reduced intracellular [Cl
]) influx
is stimulated more than efflux. This has led us to hypothesize that
intracellular Cl
, independent of NKCC1 phosphorylation,
blocks outward translocation of the unloaded cotransporter, thereby
preventing net influx when intracellular [Cl
] is high.
Unidirectional influx can still be stimulated but only represents
exchange with intracellular ions. Inhibition of NKCC1 by
Cl
therefore provides a simple feedback mechanism for
regulating cell volume and intracellular [Cl
].
The regulation of NKCC1 by intracellular Cl
becomes
important in considering the activation of NKCC1 by growth factors
(2, 23, 25). Cotransport-mediated increases in cell volume
may occur (22) and may be important for subsequent growth,
since inhibition of NKCC1 can block proliferation (5, 24)
and overexpression of cotransporters can cause transformation
(12). Activation of NKCC1 in the absence of cell shrinkage
should lead to an increase in cell volume, but this could be prevented
or limited by intracellular Cl
. For instance, direct
phosphorylation and activation of NKCC1 in endothelial cells by
calyculin does not increase cell volume (10). Thus an
increase in cell volume may require either a decrease in intracellular
[Cl
] or an increase in the Cl
set point
of the cotransporter. Despite the potential importance of NKCC1 in cell
growth, the mechanism by which it is activated by growth signals and
mediates a hypertrophic volume increase remains unknown.
 |
MATERIALS AND METHODS |
Cell culture.
Endothelial cells were cultured from bovine aortas as previously
described in DMEM medium with 10% fetal bovine serum
(21). Subconfluent cells were washed twice with serum-free
medium (50% DMEM, 50% Ham's F-12, 0.5 mg/ml transferrin, 0.2 mM
ascorbate) and then incubated in the same for 72 h with medium
changes at 24 and 48 h.
Na-K-2Cl cotransporter activity.
Cotransporter activity was measured as unidirectional K+
influx inhibited by 50 µM bumetanide, using
86Rb+ as a tracer, as previously described
(21). After a 5-min incubation in assay medium,
86Rb+ was added for 10 min, followed by washing
in ice-cold 110 mM MgCl2. Efflux of
86Rb+ was measured as previously described
after a 2-h loading period. Medium was removed and replaced at 2-min
intervals, and rate coefficients (the fraction of
86Rb+ leaving the cells/min) were calculated
before and 5 min after addition of serum. Standard isotonic solution
was Earle's salts with HEPES substituted for
HCO
, containing (in mM) 130 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 1 NaH2PO4, 5 glucose, and 26 HEPES, pH 7.4. The
measured osmolality was 290 mosmol/l. Hypertonic medium
contained an additional 150 mM sucrose so as not to alter the
concentration of any transported ion.
Cell volume and Cl
content.
Cell water was measured by equilibration of [14C]urea as
previously described (21). Cells were incubated with
[14C]urea with or without serum or added NaCl for 15 min,
followed by rapid washing in ice-cold medium to remove extracellular
urea. Medium was made hypertonic by the addition of 75 mM NaCl instead of sucrose, to avoid altering the thermodynamics of the cotransporter. For measurement of Cl
content, cells were preincubated
with 36Cl
in standard isotonic medium for 30 min and then washed with ice-cold, isosmotic sodium gluconate 15 min
after addition of serum and/or bumetanide.
Phosphorylation assays.
These assays were performed as previously described (9),
with some modifications. Cells were incubated in phosphate-free DMEM
containing 0.1 mCi/ml [32P]orthophosphate for 3 h at
37°C. To determine phosphorylation of NKCC1, cells were extracted
with 0.1% SDS in K+-free PBS for 20 min at room
temperature. A sixfold volume excess of 2%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in
buffer A [ 250 mM NaCl, 25 mM Tris · HCl, pH 7.5, 100 mM NaF, 10 mM EGTA, 5 mM EDTA, 100 mM sodium pyrophosphate, 0.2 mM
phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 10 µg/ml soybean trypsin inhibitor, 0.5 mM
benzamidine] was added, and the samples were sheared with a 26 G needle and incubated on ice for 60 min. After centrifugation to
remove insoluble material, supernatants were incubated overnight with
monoclonal anti-NKCC1 (T4) and then for 2 h at 4°C with protein
G-Sepharose (GIBCO, Grand Island, NY). The Sepharose was washed with
buffer A containing 1% Triton X-100 to remove all unbound
radioactivity and then suspended in sample buffer (2% SDS, 5%
2-mercaptoethanol, 10% glycerol, in Tris buffer) and boiled. To
examine phosphorylation of MLC, cells were extracted with a buffer
containing 0.2% Triton X-100, 10 mM PIPES, pH 6.8, 100 mM KCl, 300 mM
sucrose, 1 mM EDTA, 1 mM PMSF, 10 mM NaF, and 1 µg/ml leupeptin. The
remaining cytoskeletons were solubilized in sample buffer, sheared with
a 26 G needle, and boiled. Proteins were separated on 7.5% (NKCC1) or
15% (MLC) SDS polyacrylamide gels, and phosphorylated proteins were
identified by autoradiography.
Reagents.
86Rb+, [32P]orthophosphate,
[14C]urea, and 36Cl
were
purchased from New England Nuclear Life Sciences (Boston, MA). T4
antibody was obtained from the Developmental Studies Hybridoma
Bank (University of Iowa). Fetal bovine serum was obtained from Atlanta
Biologicals (Norcross, GA) and was heat-inactivated at 60°C for 30 min. All studies were performed with a single lot of serum.
Lysophosphatidic acid (LPA) was purchased from Avanti Polar Lipids
(Alabaster, AL) and resuspended in standard isotonic medium without
calcium and with 0.1% albumin (fatty acid-free) at a concentration of 1 mM. Fibroblast growth factor was a gift from Dr. Robert Swerlick.
 |
RESULTS |
The addition of serum to cells maintained for 3 days in a
serum-free medium devoid of growth factors produced a rapid stimulation of bumetanide-sensitive Rb+ influx that peaked at 5 min and
then declined but remained elevated for at least 20 min (Fig.
1). Maximal stimulation of NKCC1 by serum occurred at 3% (data not shown). LPA and basic
fibroblast growth factor also stimulated bumetanide-sensitive
Rb+ influx (Fig. 2), but to a
lesser extent than serum. Stimulation by LPA occurred at 3 µM and
increased up to 100 µM (Fig. 3).
Because of the greater stimulation by serum and the solubility problems with LPA, 3% serum was used for all subsequent assays. The stimulation of NKCC1 by serum resulted in an increase in cell volume (Fig. 4). In the absence of serum, inhibition
of NKCC1 with bumetanide produced a small, insignificant reduction in
cell volume, as previously described (21). Cell volume
increased 17% after exposure to serum for 15 min, and almost all of
this increase was inhibited by bumetanide. This indicated that
stimulation of NKCC1 by serum increases cell volume, ruling out the
possibility that serum stimulates NKCC1 through cell shrinkage. Serum
also stimulated NKCC1 in hypertonic medium (Fig.
5), indicating that the effects of
shrinkage and growth factors are additive. However, serum did not
produce a bumetanide-sensitive increase in cell volume in hypertonic
medium (data not shown).

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Fig. 1.
Time course of the stimulation of Na-K-Cl cotransporter
(NKCC1) by serum. Aortic endothelial cells were preincubated with 3%
serum for the times indicated, and then 86Rb+
influx was measured over 10 min in the absence or presence of 50 µM
bumetanide. Results are the means of 6 independent assays. Error bars,
SE.
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Fig. 2.
Stimulation of NKCC1 by different growth factors. Aortic
endothelial cells were preincubated with growth factors for 5 min, and
then 86Rb+ influx was measured over 10 min in
the absence or presence of 50 µM bumetanide. Results are the means of
at least 4 independent assays. Serum, 3% heat-inactivated fetal bovine
serum; LPA, 100 µM lysophosphatidic acid; FGF, 100 ng/ml basic
fibroblast growth factor. Error bars, SE.
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Fig. 3.
Concentration dependence of the stimulation of NKCC1 by
LPA. Aortic endothelial cells were preincubated with LPA for 5 min, and
then 86Rb+ influx was measured over 10 min in
the absence or presence of 50 µM bumetanide. Results are the means of
7 independent assays. Error bars, SE.
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Fig. 4.
Effect of serum on cell volume. Aortic endothelial cells
were incubated with [14C]urea and 3% serum for 15 min in
the absence and presence of 50 µM bumetanide as described in
MATERIALS AND METHODS. Results are the means of 6 independent assays. Error bars, SE. *P < 0.02 vs.
control. **P < 0.01 vs. serum.
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Fig. 5.
Additivity of the effects of serum and hypertonic
shrinkage on NKCC1. Aortic endothelial cells were preincubated with 3%
serum and/or 150 mM sucrose for 5 min, and then
86Rb+ influx was measured over 10 min in the
absence or presence of 50 µM bumetanide. Results are the means of 5 independent assays. Error bars, SE. *P = 0.03 vs. no
serum.
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|
Because NKCC1 is a bidirectional cotransporter, the effects of serum on
influx and efflux of Rb+ were compared in matched plates of
cells (Fig. 6). We have previously used
this approach to show that influx and efflux through NKCC1 are equal in
endothelial cells under basal conditions and after hypertonic shrinkage
(21). Thus, in the present study, no attempt was made to
quantitatively compare influx to efflux, which are expressed in
different units. However, the stimulation of each by serum can be
compared and was threefold greater for influx than for efflux (131 ± 35% vs. 44 ± 17%, P < 0.02), indicating that the activation of NKCC1 by serum is indeed asymmetric.

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Fig. 6.
Comparison of the effect of serum on influx and efflux
via NKCC1. Aortic endothelial cells were preincubated with serum for 5 min, after which influx and efflux of 86Rb+
were measured over 5 min in the absence or presence of 50 µM
bumetanide. Results are the means of 13 independent assays. Error bars,
SE.
|
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To determine how serum activates NKCC1, we examined three known
regulatory mechanisms: NKCC1 phosphorylation, MLC phosphorylation, and
intracellular [Cl
]. Phosphorylation of NKCC1 was
examined by immunoprecipitation from cells preincubated with
[32P]orthophosphate. In the autoradiogram shown in Fig.
7, phosphorylated NKCC1 appears as a
doublet at ~170 kDa, the intensity of which increased only slightly
after addition of serum to serum-starved cells. By comparison, a far
greater increase in phosphorylation occurred after hypertonic shrinkage
of serum-replete cells. Densitometry performed on 11 separate
experiments revealed a 25 ± 11% decrease in NKCC1
phosphorylation after addition of serum. Previous studies in
endothelial cells have shown that NKCC1 activity is also regulated by
phosphorylation of MLC independent of NKCC1 phosphorylation (9). Phosphorylation of MLC was also assessed in
32P-loaded cells and is apparent as a band at 20 kDa in the
autoradiogram shown in Fig. 8. There was
a clear increase in phosphorylation after hypertonic shrinkage, as
previously described, but no increase after addition of serum to
serum-starved cells. Because phosphorylation of NKCC1 and MLC appear to
be the principal pathways for activation of NKCC1 by cell shrinkage,
these results are consistent with the finding that serum does not
produce cell shrinkage. This indicates that serum does not act through
a volume-sensitive pathway, which is supported by the fact that
stimulation of NKCC1 by hypertonic shrinkage and serum are additive
(Fig. 5).

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Fig. 7.
Phosphorylation of NKCC1. NKCC1 was immunoprecipitated
from aortic endothelial cells preloaded with
[32P]orthophosphate as described in MATERIALS AND
METHODS after incubation with 150 mM sucrose or 3% serum for 15 min. NKCC1 appears as a doublet at ~170 kDa (arrow) on this
autoradiogram of a 7.5% acrylamide gel.
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Fig. 8.
Phosphorylation of myosin light chain (MLC). Cytoskeletal
proteins were extracted from aortic endothelial cells preloaded with
[32P]orthophosphate as described in MATERIALS AND
METHODS after incubation with 150 mM sucrose or 3% serum for 15 min. MLC appears as a band at 20 kDa (arrow) on this autoradiogram of a
15% acrylamide gel.
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To examine the potential role of intracellular Cl
,
changes in 36Cl
content were measured in
cells preequilibrated with 36Cl (Fig.
9). In the basal state, addition of
bumetanide produced a small but significant decrease in
Cl
content. The fact that there was not a corresponding
decrease in cell volume (Fig. 4) can be explained by the fact that
Cl
is a minority anion in cells and is thus altered to a
much greater extent by changes in Cl
flux than is cell
volume. Addition of serum produced a 25 ± 5% increase in
Cl
content, almost all of which was prevented by
bumetanide. The increase in Cl
content was greater than
the increase in cell water, indicating that intracellular
[Cl
] increased. This is consistent with a net influx of
Cl
through NKCC1 and indicates that a decrease in
intracellular [Cl
] cannot explain the stimulation of
NKCC1 by serum.

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Fig. 9.
Effect of serum on cell Cl content.
Cellular 36Cl was measured as described in
MATERIALS AND METHODS with or without 3% serum in the
absence and presence of 50 µM bumetanide (bumet). Results are the
means of 6 independent assays. Error bars, SE.
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|
Another possible mechanism for stimulation of NKCC1 is a decrease
in the sensitivity of the transporter to inhibition by intracellular Cl
. To test this, cells were preincubated in isotonic
medium containing methanesulfonate, a cell-permeant anion, in place of
Cl
for 30 min. Bumetanide-sensitive influx increased in
Cl
-depleted cells, consistent with inhibition of NKCC1 by
intracellular Cl
, and there was no additional stimulation
by serum (Fig. 10). This could not be
explained by the possibility that NKCC1 is already maximally stimulated
by depletion of Cl
, since further stimulation occurred
after hypertonic shrinkage. These results demonstrate that serum
stimulates NKCC1 by modulating the inhibitory effect of intracellular
Cl
.

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Fig. 10.
Effect of intracellular Cl depletion on
activation of NKCC1. Aortic endothelial cells were preincubated in
standard medium or in Cl -free medium containing
methanesulfonate for 30 min, after which 86Rb+
influx was measured as described above. A: influx in the
absence or presence of 3% serum. Results are the means of 5 independent assays. Error bars, SE. B: influx in the absence
or presence of 150 mM sucrose. Results are the means of 3 independent
assays. Error bars, SE. *P < 0.01, **P < 0.05 vs. isotonic.
|
|
 |
DISCUSSION |
Investigation of the regulation of NKCC1 has focused primarily on
cell volume and intracellular Cl
. Although these
parameters are linked under physiological conditions, they are clearly
independent regulators of NKCC1. A decrease in cell volume can increase
NKCC1, even in the face of an increase in intracellular
[Cl
] (i.e., hypertonic shrinkage), and a decrease in
intracellular [Cl
] can stimulate NKCC1 in swollen cells
(i.e., hypotonic swelling). Our results demonstrate that growth factors
are an additional, independent stimulus for NKCC1, since neither cell
volume nor intracellular [Cl
] was decreased.
Furthermore, this stimulation is independent of NKCC1 phosphorylation,
which has been thought to be the principal mechanism for regulation by
both cell volume and intracellular Cl
(6, 14, 16,
17), and independent of MLC phosphorylation, an additional
volume-sensitive regulatory pathway (1, 9). The fact that
the stimulation by serum was abolished by removing intracellular
Cl
points instead to modulation of the Cl
sensitivity of NKCC1 that is independent of phosphorylation.
This novel mechanism for regulation of NKCC1 enables cells to
respond appropriately to growth stimulation. While regulation of NKCC1
by cell volume and intracellular Cl
is homeostatic,
maintaining cell volume and intracellular [Cl
],
activation by growth factors is independent of either and is therefore
dynamic, with the potential to increase cell volume and intracellular
[Cl
]. This potential is only realized because the
normal feedback inhibition that regulates intracellular
[Cl
] (and therefore cell volume as well) is abrogated,
thus explaining the hypertrophic volume increase that is observed. A
rapid increase in cell volume is an early response to growth stimuli
and may be required for normal growth (20). Inhibition of
NKCC1 by either furosemide or bumetanide blocks the proliferative
response of endothelial cells to fibroblast growth factor
(24).
Although changes in intracellular [Cl
] can alter
phosphorylation of NKCC1, some previous data have supported, albeit
indirectly, an additional effect of Cl
on NKCC1. Of
particular interest are the differing effects on NKCC1 of hypertonic
and isosmotic shrinkage, between which the only major difference is the
intracellular [Cl
] (increasing in the former and
decreasing in the latter). Although NKCC1 phosphorylation and
unidirectional influx increase, and net influx is thermodynamically
favored in each case, net influx of ions only occurs after the latter
(21). This suggests an effect of Cl
on the
kinetics of NKCC1 (for review, see Ref. 20). Further evidence for this is the trans effect of intracellular
anions on the Na-K-Cl cotransporter in internally perfused squid axons (3, 4), although an effect on cotransporter
phosphorylation cannot be ruled out in these studies. One simple
explanation is that Cl
, either directly or indirectly,
blocks translocation of the empty cotransporter to the outer aspect of
the plasma membrane, preventing net influx and permitting only ion
exchange, thereby providing a simple, negative-feedback regulation of
intracellular [Cl
]. Removal of this block would produce
an asymmetric stimulation of influx and an increase in cell volume and
Cl
content, as was observed. However, this cannot be the
only mechanism, since efflux also increased, and the increase in
unidirectional influx by serum in hypertonic medium was not accompanied
by any net influx.
The mechanism by which serum alters the Cl
sensitivity of
NKCC1 is unclear, in part because the Cl
-sensing pathway
is poorly understood. Although many effects of growth factors are
mediated through phosphorylation, our data do not support changes in
the phosphorylation of NKCC1. However, we cannot rule out the
possibility that there is a change in phosphorylation sites without a
change in overall phosphorylation. If intracellular Cl
affects NKCC1 through direct binding, it is difficult to envision how
this could be altered by serum in the absence of NKCC1 phosphorylation. Alternatively, NKCC1 may be regulated by a Cl
-sensing
protein whose properties are regulated by growth factor-dependent phosphorylation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-47994.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: W. C. O'Neill, Emory Univ., Renal Division WMB 338, 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: woneill{at}emory.edu).
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. Section 1734 solely to indicate this fact.
Received 11 January 2001; accepted in final form 27 July 2001.
 |
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