Contractile regulation of the
Na+-K+-2Cl
cotransporter in
vascular smooth muscle
Fatma
Akar1,
Gengru
Jiang1,
Richard J.
Paul2, and
W. Charles
O'Neill1,3
1 Renal Division, Department of Medicine, and
3 Department of Physiology, Emory University School of
Medicine, Atlanta, Georgia 30322; and 2 Department of Biophysics
and Molecular Physiology, University of Cincinnati, Cincinnati, Ohio
45267
 |
ABSTRACT |
Vasoconstrictors activate the
Na+-K+-2Cl
cotransporter NKCC1 in
rat aortic smooth muscle, but the mechanism is unknown. Efflux of
86Rb+ from rat aorta in response to
phenylephrine (PE) was measured in the absence and presence of
bumetanide, a specific inhibitor of NKCC1. Removal of extracellular
Ca2+ completely abolished the activation of NKCC1 by PE.
This was not due to inhibition of Ca2+-dependent
K+ channels since blocking these channels with
Ba2+ in Ca2+-replete solution did not prevent
activation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited
70% by 75 µM ML-9, 97% by 2 µM wortmannin, and 70% by 2 mM
2,3-butanedione monoxime, each of which inhibited isometric force
generation in aortic rings. Bumetanide-insensitive Rb+
efflux, an indication of Ca2+-dependent K+
channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to
120% of normal almost completely blocked the stimulation of NKCC1 by
PE without inhibiting the stimulation by hypertonic shrinkage. We
conclude that activation of the
Na+-K+-2Cl
cotransporter by PE is
the direct result of smooth muscle contraction through
Ca2+-dependent activation of myosin light chain kinase.
This indicates that the
Na+-K+-2Cl
cotransporter is
regulated by the contractile state of vascular smooth muscle.
sodium-potassium-2-chloride cotransport; myosin light chain kinase; phenylephrine; contraction
 |
INTRODUCTION |
NKCC1, the
secretory or basolateral form of the
Na+-K+-2Cl
cotransporter,
participates in salt transport in secretory epithelia, but is also
present in nonepithelial cells where it functions to regulate cell
volume and intracellular [Cl
]. Consistent with this
role, NKCC1 is activated by cell shrinkage and inhibited intracellular
Cl
. The transporter is also activated by inhibitors of
protein phosphatases and is inhibited by kinase inhibitors (9,
22), indicating that regulation occurs through protein
phosphorylation. Stimuli that activate NKCC1 also phosphorylate the
transporter (3, 13, 14), probably at the same site or
sites (12), suggesting that NKCC1 is activated by a
specific protein kinase. We have recently identified c-Jun
NH2-terminal kinase as a volume-sensitive kinase capable of
phosphorylating NKCC1 in vitro (7).
There is also evidence for an additional regulatory pathway that may be
independent of NKCC1 phosphorylation. In several cell types, shrinkage
increases phosphorylation of myosin light chain (MLC) (8,
28), and inhibition of myosin light chain kinase (MLCK) blocks
NKCC1 activation in endothelial (8), Ehrlich ascites
(10), and colonic epithelial (5) cells.
Activation of the cotransporter in colonic epithelial cells is also
affected by alterations in F-actin (5, 15, 16). The
mechanism by which the cellular contractile apparatus or cytoskeleton
regulates NKCC1 is not known.
To help elucidate this putative contractile regulation of NKCC1,
we have turned to a contractile tissue, vascular smooth muscle. We have
previously shown that NKCC1 is present in smooth muscle from rat
aorta, where it is activated by vasoconstrictors and inhibited by
nitrovasodilators (1), consistent with contractile regulation of NKCC1. This regulation of NKCC1 is important in smooth
muscle function since inhibition of the cotransporter reduces force
generation. We speculate that NKCC1 may promote contraction by
maintaining or increasing intracellular [Cl
]. However,
it is possible that this regulation of NKCC1 could be mediated by other
actions of these compounds, including changes in cell volume. The
present studies were undertaken to determine the role of the
contractile apparatus in the activation of NKCC1 in rat aorta by the
vasoconstrictor phenylephrine.
 |
METHODS |
Tissue preparation.
The descending aorta was excised from male Sprague-Dawley rats
(200-350 g), dissected free of the adventitia, and opened
lengthwise. The endothelium was removed with a cotton swab, after which
the vessel was cut into 6-12 pieces that were used immediately for assays.
Na+-K+-2Cl
cotransporter activity.
Cotransporter activity was measured as unidirectional K+
efflux, using 86Rb as a tracer, as previously described
(1). In each assay, the basal rate of efflux was measured
over the 6 min before addition of test conditions, and the stimulated
rate of efflux was measured between 8 and 10 min after stimulation.
Data are presented as means ± SE. 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/kgH2O. 86RbCl was obtained from
DuPont-NEN. Bumetanide was a gift of Dr. Peter Sorter
(Hoffmann-LaRoche, Nutley, NJ) and stored as a stock solution of 100 mM
in dimethyl sulfoxide (DMSO). ML-9 was obtained from Biomol (Plymouth
Meeting, PA) and stored as a stock solution of 30 mM in DMSO.
2,3-Butanedione monoxime (BDM) was obtained from Sigma (St. Louis, MO)
and stored as a stock solution of 1 M in 15% ethanol. Wortmannin was
also purchased from Sigma and used as an aqueous solution.
Isometric force measurements.
Male Sprague-Dawley rats (178-430 g) were anesthetized in a
precharged CO2 chamber and killed by cervical
dislocation. The aorta was dissected and cleaned of loose fat and
connective tissue, and endothelium was removed. A ring of 5 mm was cut
from the thoracic aorta; blot weights were between 4 and 5 mg. Rings
were mounted between a fixed stainless steel post and one connected to
a Kistler-Morse force transducer whose output was monitored with a
computer-based data collection system (BioPac). The length between the
wires could be adjusted by a micrometer-driven device. Force
measurements were then performed as previously described
(17). The rings were mounted in a 15-ml organ bath and
allowed to equilibrate at 37°C for 1 h, during which the tension
was adjusted to 50 mN. This force was chosen because it places the ring
at a length within the range for optimal active force generation. The
aorta was then stimulated by addition of KCl (3 M) to bring the bath
concentration to 50 mM. The arteries were then relaxed by exchanging
the bathing solution; this contraction/relaxation cycle was repeated
until reproducible forces were generated.
The protocol for studies with ML-9, wortmannin, and BDM involved a
control contraction/relaxation cycle to 10 µM phenylephrine. The
aortas were then preincubated with these agents for 10 min, 2 h,
and 10 min, respectively, to parallel the design of the K+
efflux experiments. In the continued presence of inhibitor, a second
test contraction/relaxation to phenylephrine (10 mM) was conducted. The
effects of these agents are reported as a percentage of the initial
control force. After each experiment, ring dimensions and blot weight
were measured. Aortic wall thickness (t) was estimated using
the formula t = blot weight/(1.05 × length × circumference), and the cross-sectional area (CSA) for force
normalization was taken as CSA = 2 × t × length. Studies were performed at 37°C in the standard
isotonic solution (above), bubbled with 100% O2.
 |
RESULTS |
Phenylephrine produced a rapid increase in the efflux of
Rb+ from rat aortic smooth muscle (Fig.
1). Much of this was not
inhibited by bumetanide, but there was also a significant increase in
bumetanide-sensitive efflux as previously reported (1),
indicating activation of NKCC1. To test the role of Ca2+ in
this activation of NKCC1, Ca2+ was omitted from the medium
and EGTA was added to chelate any contaminating Ca2+. In
initial experiments, this resulted in a very high basal K+
efflux as previously reported in vascular smooth muscle
(27), which could be minimized by increasing the
concentration of Mg2+ to 11 mM isosmotically
(27). Ca2+-free conditions were initiated at
the beginning of the flux measurements, 10 min before adding
phenylephrine. As shown in Fig. 2, the
increase in both bumetanide-sensitive and bumetanide-insensitive efflux of Rb+ by phenylephrine was completely blocked by removal
of Ca2+. The inhibition of the bumetanide-insensitive
efflux is indicative of Ca2+-dependent K+
channels. We have previously shown that these channels are responsible for activation of NKCC1 by Ca2+-mobilizing agonists in
vascular endothelial cells, probably through loss of cell chloride and
cell shrinkage (19). To determine whether a similar
mechanism was operant in vascular smooth muscle, efflux was measured in
the presence of 3 mM Ba2+. As shown in Fig.
3, Ba2+ completely blocked
the increase in bumetanide-insensitive Rb+ efflux by
phenylephrine, providing further evidence that this flux is mediated by
K+ channels, but did not prevent activation of NKCC1. Our
previous demonstration that NKCC1 in rat aorta is also activated by KCl at concentrations that would actually increase cell volume and [Cl
] (up to 80 mM isosmotically substituted for
Na+) also indicates that shrinkage cannot explain the
activation of NKCC1 by agonists (1). These results
indicate that vasoconstrictors directly activate NKCC1 in aortic smooth
muscle via a Ca2+-dependent pathway.

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Fig. 1.
Effect of phenylephrine (PE) on the efflux of
Rb+ from rat aortic smooth muscle. Aortic segments were
loaded for 2 h with 86Rb+ after which
efflux was measured before and after addition of 10 µM PE, in the
absence (Total) and presence (+bumetanide) of 50 µM bumetanide
(A). B: bumetanide-sensitive efflux. Results are
means of 4 separate experiments, each performed in triplicate. Error
bars, SEs.
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Fig. 2.
Effect of Ca2+-free medium on the efflux of
Rb+ from rat aortic smooth muscle. Aortic segments
were loaded for 2 h with 86Rb+ after which
efflux was measured before and after addition of 10 µM PE, in the
absence (Total) and presence (+bumetanide) of 50 µM bumetanide in
Ca2+-free medium containing 1 mM EGTA. Results are means of
4 separate experiments, each performed in triplicate. Error bars,
SEs.
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Fig. 3.
Effect of Ba2+ on the activation of NKCC1 by
PE. A: efflux in the absence (Total) and presence
(+bumetanide) of 50 µM bumetanide. B:
bumetanide-sensitive efflux. Data are means of 3 independent
experiments. Error bars, SEs.
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We have previously shown in endothelial cells (8) that
activation of NKCC1 by shrinkage is associated with phosphorylation of
myosin light chain and blocked by inhibition of myosin light chain
kinase (MLCK). To test the role of MLCK in rat aorta, two unrelated
inhibitors of this kinase were employed: ML-9 and wortmannin. ML-9 is a
relatively specific inhibitor (6, 25) that is very closely
related to ML-7, the inhibitor used in previous studies in endothelial
cells (8). Wortmannin is also a potent inhibitor of MLCK
but has other actions including inhibition of phosphatidylinositol-3 kinase (2). ML-9 produced a dose-dependent inhibition of
NKCC1 activation by phenylephrine without altering basal efflux (Fig. 4). The inhibition by 75 µM ML-9 was
70%. Wortmannin at 2 µM almost completely blocked (97%) stimulation
of NKCC1 by phenylephrine (Fig. 5). There
was a small increase in basal NKCC1 activity that was not statistically
significant. Activation of NKCC1 was also substantially inhibited (70%
at 2 mM) by BDM (Fig. 6), a compound that
inhibits actin-myosin interaction in skeletal muscle and also inhibits
vascular smooth muscle contraction (21). ML-9, wortmannin,
and BDM each inhibited isometric force generation by phenylephrine in
aortic rings (Fig. 7). In the case of
ML-9 and wortmannin, inhibition of force generation correlated closely with inhibition of NKCC1 activation, while higher concentrations of BDM
were required to inhibit force generation.

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Fig. 4.
Effect of ML-9 on the activation of NKCC1 by PE. ML-9 was
added at the start of the efflux measurements. Each set of bars
represents the means of 5, 3, and 5 independent experiments for 0, 37.5, and 75 µM ML-9, respectively. Error bars, SEs.
* P < 0.02 vs. control (0 µM ML-9) for
stimulation by PE.
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Fig. 5.
Effect of wortmannin on the activation of NKCC1 by PE.
Wortmannin (2 µM) was added 2 h before the flux measurements.
Data are means of 5 independent experiments. Error bars, SEs.
* P < 0.01 vs. control for stimulation for
PE.
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Fig. 6.
Effect of 2,3-butanedione monoxime (BDM) on the
activation of NKCC1 by PE. BDM was added at the start of the efflux
measurements. Each set of bars represents the means of 5 independent
experiments except for 10 mM, which is a single experiment performed in
triplicate. Error bars, SEs. * P < 0.02, ** P < 0.001 vs. control (0 mM BDM) for stimulation
by PE.
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Fig. 7.
Inhibition of isometric force generation in rat aorta.
Aortic rings were treated with 10 µM PE to determine maximum force
generation. The PE was removed and the rings were then treated with
ML-9, wortmannin (WORT), and BDM as described for efflux measurements
before reexposure to PE. Each bar is the mean of triplicate
measurements. Error bars, SEs.
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BDM and ML-9 have also been reported to decrease Ca2+
influx (4, 29) and could inhibit contraction by reducing
intracellular [Ca2+]. The activity of
Ca2+-dependent K+ channels, which is measurable
as bumetanide-insensitive efflux, can serve as an indirect measure of
intracellular [Ca2+]. As shown in Fig.
8, both basal and stimulated
bumetanide-insensitive efflux was reduced by 75 µM ML-9, consistent
with a reduction in intracellular [Ca2+]. However,
stimulated efflux was unaffected by 2 mM BDM or 2 µM wortmannin,
indicating that sizable reductions in Ca2+ influx or
intracellular Ca2+ release were probably not occurring with
these compounds at these concentrations. Wortmannin actually increased
basal bumetanide-insensitive efflux (P < 0.01), the
mechanism of which is unknown.

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Fig. 8.
Bumetanide-insensitive K+ efflux. Efflux in
the presence of 50 µM bumetanide was measured before and after
addition of 10 µM PE. ML-9 (75 µM) and BDM (2 mM) were added at the
start of the assay while wortmannin (2 µM) was added 2 h before
assay. Results are the means of 5 individual experiments.
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The link between smooth muscle contraction and activation of NKCC1 was
investigated further by mounting aortic rings on tubing of different
sizes before measurement of efflux. Aortic rings (without endothelium)
were everted and placed over the tubing before loading with
86Rb+, with the luminal surface facing outward.
Tubing with the following compositions and outer diameters was used:
1.91 mm and 2.08 mm polyethylene (Becton Dickinson, Parsippany, NJ),
1.96 mm Silastic (Dow Corning, Midland, MI), and 2.00 mm Nalgene PVC
and 2.16 mm Nalgene silicone (Nalge, Rochester, NY). The measured lumen
diameter of the rat aortic segments used in this study was ~1.80 mm.
As shown in Fig. 9, stimulation of NKCC1
by phenylephrine was maintained up to a lumen diameter of 1.96 mm
(approximately a 10% increase), after which there was progressive
inhibition. By comparison, the stimulation of NKCC1 by hypertonic
shrinkage remained intact and actually increased. This indicates that
stretching of aorta is not producing a nonspecific inhibitory effect on
NKCC1. Bumetanide-insensitive efflux was not affected by rigid tubing
(data not shown).

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Fig. 9.
Effect of lumen diameter on the activation of NKCC1.
Aortic rings (without endothelium) were everted and placed over tubing,
as described in RESULTS, with the luminal surface facing
outward. Efflux of 86Rb+ was then measured as
described in METHODS, and the increase in
bumetanide-sensitive efflux with either PE (10 µM) or hypertonic
medium (150 mM sucrose) is plotted as a function of lumen diameter.
Results are the means of at least 3 experiments. Error bars, SEs.
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 |
DISCUSSION |
The present study demonstrates that inhibition of smooth muscle
contraction at multiple steps can block activation of the Na+-K+-2Cl
cotransporter by
phenylephrine. The Ca2+ dependence of the stimulation of
NKCC1 by phenylephrine is consistent with the Ca2+
dependence of
1-adrenergic contraction
(18). Although contraction by phenylephrine can be
mediated by release of Ca2+ from internal stores in the
absence of external Ca2+, force is reduced about 75% and
is not sustained (17). It is unlikely that
Ca2+ is having a direct effect on NKCC1 because we were not
able to demonstrate any stimulation of NKCC1 by Ca2+ in
endothelial cells provided that cell volume was held constant (19). Ca2+ could activate NKCC1 through
opening of Ca2+-dependent K+ channels with
resulting decreases in cell volume and intracellular [Cl
], as we have previously shown in endothelial cells
(19). However, this is not the case in vascular smooth
muscle since blockade of the channels by Ba2+ did not
prevent activation of NKCC1 by phenylephrine. Ba2+ would be
expected to increase aortic contraction by inhibiting Ca2+-dependent K+ channels and depolarizing the
membrane, and through direct activation of the contractile mechanism
(24). Furthermore, NKCC1 is also activated by isosmotic
KCl, which raises intracellular [Ca2+] and also increases
cell volume and intracellular [Cl
]. It is clear then
that vasoconstrictors can activate NKCC1 in vascular smooth muscle in a
Ca2+-dependent manner that does not involve activation of
K+ channels and decreases in cell volume or intracellular
[Cl
].
This Ca2+-dependent, volume-independent activation of NKCC1
by phenylephrine suggests involvement of MLCK, consistent with our previous results showing that activation of NKCC1 in cultured endothelial cells is dependent on myosin phosphorylation
(8). This was confirmed by showing that two different
inhibitors of MLCK reduced the stimulation of NKCC1 in aorta.
Furthermore, activation of NKCC1 was also blocked by BDM, an agent that
interferes with the interaction between actin and myosin in skeletal
muscle and inhibits contraction of smooth muscle (21).
However, inhibition of NKCC1 did not correlate precisely with
inhibition of contraction, with NKCC1 stimulation being more sensitive
to BDM, suggesting that contraction and inhibition of NKCC1 are not
tightly coupled or may occur through different mechanisms.
It is important to note that the mechanism by which BDM inhibits smooth
muscle contraction may be distinctly different from that in skeletal
muscle. There is no inhibition of contraction in permeabilized smooth
muscle, suggesting that the major effect of BDM is not on the
contractile proteins themselves, but rather on Ca2+
delivery during excitation (11, 26). BDM can inhibit
Ca2+ influx (4), but this may only occur at
high concentrations. Although we did not directly measure intracellular
[Ca2+], the activity of Ca2+-dependent
K+ channels provided an indirect measure. The fact that 2 mM BDM did not lower bumetanide-insensitive efflux indicates that
Ca2+-dependent K+ channel activity was not
reduced and that BDM probably did not substantially affect
intracellular [Ca2+] at a concentration that blocked
stimulation of NKCC1 and reduced force generation. Our results do not
rule out an effect of BDM on local Ca2+ concentrations or
Ca2+ "sparks" that might not affect
Ca2+-dependent K+ channels. A higher
concentration of BDM (10 mM) completely abrogated the stimulation of
bumetanide-insensitive K+ efflux by phenylephrine (data not
shown), indicating that effects on Ca2+ influx or
intracellular release can occur at higher concentrations.
The evidence that stimulation of NKCC1 is dependent on the contractile
apparatus was bolstered by studies showing a length dependence for this
stimulation. Increasing lumen diameter beyond 10% produced a
progressive decrease in the stimulation of NKCC1 by phenylephrine. The
basis for this length dependence is unknown but could be due to
prevention of contraction. Although there was no measurable contraction
of aorta on even the narrowest tubing, cell shortening could still be
occurring, counterbalanced by stretching of the extracellular elastic
component of the muscle. Larger tubing may stretch this elastic
component to the point where it cannot stretch further to accommodate
cell shortening, thus creating a state of true isometric contraction.
It is of interest that stimulation of NKCC1 by hypertonic shrinkage was
not only maintained in stretched aortas but was actually enhanced. Thus
the volume sensitivity of NKCC1 is also length dependent, but in the
opposite direction from agonist sensitivity.
On the basis of our data, we propose a novel pathway for regulation of
NKCC1 in vascular smooth muscle involving the contractile apparatus.
Agonists increase intracellular [Ca2+], activating MLCK
and resulting in MLC phosphorylation, stimulation of myosin ATPase,
cell contraction, and activation of NKCC1. The mechanism by which
isotonic contraction activates NKCC1 is unclear. One possibility is an
interaction between the cotransporter and the contractile apparatus or
cytoskeleton. This is consistent with the observation that agents that
perturb the actin cytoskeleton can alter NKCC1 activity in other cells
(5, 15, 16). Another possibility is that contraction of
vascular smooth muscle initiates a kinase signaling cascade that
results in phosphorylation of NKCC1. This is suggested by our previous
demonstration that phenylephrine and KCl increase phosphorylation of
NKCC1 in rat aorta (1) and is consistent with the ability
of mechanical strain to activate protein kinases in cultured vascular
smooth muscle cells (23). Further studies in vascular
smooth muscle should help elucidate the contractile regulation of the
Na+-K+-2Cl
cotransporter
 |
ACKNOWLEDGEMENTS |
This research was supported by National Institutes of Health Grants
HL-47449 and DK-07656 (to W. C. O'Neill) and HL-54829 and
HL-61974 (to R. J. Paul). F. Akar was supported by the North Atlantic Treaty Organization Scientific Fellowship Program of the
Scientific and Technical Research Council of Turkey (TUBITAK).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: W. C. O'Neill, Emory Univ. School of Medicine, 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 22 August 2000; accepted in final form 8 March 2001.
 |
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