Vasoconstrictors and nitrovasodilators reciprocally regulate
the
Na+-K+-2Cl
cotransporter in rat aorta
Fatma
Akar1,
Elizabeth
Skinner1,2,
Janet D.
Klein1,
Madhumita
Jena1,
Richard J.
Paul3, and
W. Charles
O'Neill1,4
1 Renal Division, Department of
Medicine, 4 Department of
Physiology, and 2 Department
of Pathology and Laboratory Medicine, Emory University School of
Medicine, Atlanta, Georgia 30322; and
3 Department of Molecular and
Cellular Physiology, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267
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ABSTRACT |
Little is known
about the function and regulation of the
Na+-K+-2Cl
cotransporter NKCC1 in vascular smooth muscle. The
activity of NKCC1 was measured as the bumetanide-sensitive efflux of
86Rb+
from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH2O) rapidly
doubled cotransporter activity, consistent with its volume-regulatory
function. NKCC1 was also acutely activated by the vasoconstrictors ANG
II (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl
(54%). Both nitric oxide and nitroprusside inhibited basal NKCC1
activity (39 and 34%, respectively), and nitroprusside
completely reversed the stimulation by phenylephrine. The
phosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced by
phenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. We
conclude that the
Na+-K+-2Cl
cotransporter in vascular smooth muscle is reciprocally regulated by
vasoconstrictors and nitrovasodilators and contributes to smooth muscle
contraction, indicating that alterations in NKCC1 could influence
vascular smooth muscle tone in vivo.
sodium-potassium-chloride cotransport; vascular smooth muscle; contraction; cell chloride; phenylephrine; angiotensin II; endothelin; nitric oxide
 |
INTRODUCTION |
IN ADDITION TO ION channels, vascular smooth muscle
cells (VSMC) possess a variety of ion transporters, the activities of which are influenced by vasoactive substances and growth factors, indicating potential roles in vasoconstriction and smooth muscle hypertrophy. Particular attention has been focused on monovalent cation
transporters including the
Na+-K+
pump,
Na+/H+
antiporter NHE1, and
Na+-K+-2Cl
cotransporter NKCC1, in part because they transport
Na+ (37). The importance of these
transporters may lie in their ability to regulate not only
intracellular Na+ concentration
([Na+]) but also
intracellular Cl
concentration
([Cl
]), cell
volume, and membrane potential. Although various abnormalities in the
function of these transporters in essential hypertension and in
hypertension models have been described, their significance remains
unclear because the findings have generally been limited to nonvascular
cells or to VSMC in culture. Because VSMC rapidly lose their
contractile phenotype in culture and convert to a proliferative and
synthetic phenotype (46), findings for cultured cells may not be
indicative of the function and regulation of ion transporters in vivo.
NKCC1 is a prominent transporter in VSMC in culture (29, 31, 32, 42).
It is activated by cell shrinkage (29, 30) and is responsible for
volume recovery (30), and it is also activated by ANG II both acutely
and chronically (2, 32, 48), suggesting a role in smooth muscle
contraction and hypertrophy. NKCC1 activity is reduced in cells
cultured from aortas of spontaneously hypertensive rats (37, 41).
Because of the proliferative phenotype in culture, and particularly
because NKCC1 is activated by growth factors and may participate in
cell growth, these findings are of questionable relevance to smooth
muscle in vivo (33, 34). However, few studies of NKCC1 in intact
vascular smooth muscle have been performed. Deth et al. (8) observed a
Na+-dependent,
Cl
-dependent
Rb+ influx in rat and rabbit
aortas that was inhibited by furosemide, thereby establishing the
presence of NKCC1 in intact smooth muscle. Furosemide reduced both the
Ca2+ and contractile responses to
phenylephrine in this study and has been shown to inhibit smooth muscle
contraction in other studies (9, 47), consistent with its vasodilatory
action in vivo. However, furosemide is not a specific inhibitor of
NKCC1 and can block Cl
transport through other pathways. Davis et al. (6) provided additional
evidence for the cotransporter in the rat femoral artery by showing
that bumetanide lowered intracellular
[Cl
]. This
decrease in intracellular
[Cl
] was
augmented in rats made hypertensive by the administration of
deoxycorticosterone and a high-salt diet, suggesting upregulation of
NKCC1. Norepinephrine produces an increase in intracellular [Cl
] that is
partly blocked by bumetanide (7), indicating activation of NKCC1, but
the response of the cotransporter in intact smooth muscle to other
vasoconstrictors or to vasodilators has not been examined.
Studies of ion transport in vascular tissue have been limited by
technical considerations. NKCC1 activity is usually measured as the
influx of
86Rb+,
a tracer for K+, but measurements
of influx are hampered by problems with trapped extracellular tracer,
with normalization (various proportions of cells vs. matrix), and with
the large amount of tissue required. NKCC1 activity has also been
assayed as changes in intracellular [Cl
]
measured with intracellular electrodes (6), but this is technically very demanding and time consuming. Because NKCC1 mediates bidirectional transport, NKCC1 activity can also be measured as the efflux of 86Rb+
(15). Although net transport is inward under physiological conditions,
there is a sizable efflux representing
Rb+/K+
exchange. The measurement of efflux avoids the problems associated with
influx measurements, particularly sample heterogeneity, because results
can be normalized to intracellular tracer content. The validity of
measuring NKCC1 activity as
86Rb+
efflux was established by previous studies of vascular endothelial cells (15, 28). The efflux of Rb+
in rat aorta has previously been measured and, although somewhat lower
than the efflux of K+, showed
qualitatively similar responses to norepinephrine and KCl
depolarization (43). We have now employed this technique to measure
NKCC1 and its regulation by vasoconstrictors and vasodilators in intact
smooth muscle from rat aorta. Coupled with the measurement of force
generation, this enabled us to demonstrate that the cotransporter has a
functional role in vascular smooth muscle.
 |
MATERIALS AND METHODS |
Tissue preparation.
Descending aortas were 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.
Cell culture.
VSMC were cultured from aortas of Sprague-Dawley rats and were grown in
DMEM (high glucose) containing 10% fetal bovine serum. Once the cells
reached ~70% confluency, they were serum starved for 72 h in
serum-free medium containing insulin, transferrin, and ascorbate (19),
with daily medium changes. All studies were performed with monolayers
in plastic multiwell plates.
NKCC1 activity.
Cotransporter activity was measured as unidirectional
K+ efflux as previously described
(35), with
86Rb+
as a tracer. The standard isotonic solution was Earle's salts, with
HEPES substituted for HCO
3, 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. The solution was
made hypertonic by adding 150 mM sucrose.
86RbCl was obtained from DuPont
NEN. Bumetanide was a gift from Dr. Peter Sorter (Hoffmann-LaRoche,
Nutley, NJ) and was stored as a 100 mM stock solution in DMSO.
Loading of cells with
86Rb+
was accomplished with a 2-h preincubation. Although this did not label
the intracellular pool to equilibrium, preliminary studies showed
similar responses after loading for 2 and 4 h. For studies requiring
preincubation with ANG II, losartan, or lisinopril, these compounds
were added to the loading solution. After the loading, the tissue was
washed four times over several minutes to remove extracellular
radioactivity. Thereafter, medium was removed at 2-min intervals and
the tissue was washed with an additional aliquot of medium. The
aliquots were combined for counting, and fresh medium was placed on the tissue. The radioactivity in each fraction plus that remaining in the
tissue at the end of the assay were measured as Cerenkov radiation in a
scintillation counter. These values were used to calculate the amount
of
86Rb+
present in the tissue at the start of the assay and at each subsequent time point. Rate coefficients at each time point were calculated by
dividing the amount of
86Rb+
in the medium by the amount in the tissue before that time point. Efflux measurements for cultured cells were performed similarly except
that medium was changed at 1-min intervals without intervening washes.
In each assay, basal efflux was derived by averaging the last three
rate constants before the establishment of test
conditions. The new level of efflux was derived by
averaging the rate constants at 8 and 10 min afterward (4 and 5 min in
cells). Data are presented as means ± SE, and significance was
determined by paired t-testing.
Cotransporter phosphorylation.
Aortas were prepared as described above and divided lengthwise, and
each half was incubated in 1-2 ml of phosphate-free Dulbecco's MEM containing 0.1 mCi/ml of
[32P]orthophosphate
for 3 h at 37°C in a CO2
incubator. Test substances were added at the end of this incubation.
Each half-aorta was homogenized in a ground-glass homogenizer in 250 µl of 250 mM sucrose, 10 mM triethanolamine, 1 µg/ml leupeptin, and
100 µg/ml phenylmethylsulfonyl fluoride (PMSF), pH 7.6. The
homogenizer was rinsed with an additional 250 µl, and the two
aliquots were combined. SDS was added to a final concentration of 1%,
and the mixture was then incubated for 10 min at room temperature. The mixture was passed through an insulin syringe several times and then
added to 5 ml of a solution of 2%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in extraction buffer containing (in mM) 25 Tris · HCl (pH 7.9), 100 sodium pyrophosphate, 100 NaF, 250 NaCl, 10 EGTA, 5 EDTA, 0.2 PMSF, and 0.5 benzamidine, with 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml soybean trypsin
inhibitor. After incubation on ice for 1 h, particulate
matter was removed by centrifugation and the supernatant
was incubated at 4°C for 12 h with a monoclonal antibody prepared
against the
Na+-K+-2Cl
cotransporter from human colonic carcinoma cells (T4 antibody; kindly
provided by Dr. C. Lytle). Protein G-Sepharose (GIBCO, Grand Island,
NY), washed and equilibrated in the 2% CHAPS extraction buffer, was
added (50 µl of a 40% suspension per sample) with gentle agitation
for 3 h at 4°C. The Sepharose was washed six times with 1% Triton
X-100 in extraction buffer and once with K+-free PBS. Bound material was
removed by heating the Sepharose in SDS electrophoresis
sample buffer and was separated on 7.5% polyacrylamide gels according
to the method of Laemmli (17).
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. A ring of 5 mm was cut
from the thoracic aorta; blot weights were 4-5 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 (24). 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. Then the aorta was
stimulated by the 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. Then a cumulative
concentration-force relation for phenylephrine was generated.
Bumetanide (10 µM) was added to the bath, and after 20 min the
concentration-force relation was again measured. This experimental
protocol for KCl stimulation was repeated in a separate study. After
each experiment, ring dimensions and blot weight were measured. Aortic
wall thickness (t) was estimated
from 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 with a physiological saline solution consisting of (in
mM) 118 NaCl, 4.73 KCl, 1.2 MgCl2, 0.026 EDTA, 1.2 KH2PO4,
2.5 CaCl2, 5.5 glucose, and 25 NaH2CO3,
which was bubbled with 95% O2-5%
CO2 at pH 7.4 and 37°C.
 |
RESULTS |
Although normal physiological conditions dictate a net influx of ions
through NKCC1, the transporter is bidirectional and exhibits a
considerable rate of
K+/K+
(Rb+) exchange. Thus
cotransporter activity can be measured as
Rb+ efflux, which has distinct
advantages for intact smooth muscle. Multiple time points for the same
segment of aorta can be obtained, substantially reducing the amount of
tissue required, and measurement of dry weight or protein or DNA
content is not required because the data are normalized to
Rb+ content (rate coefficients). A
typical assay is shown in Fig. 1. Data are
presented as fractions of tracer remaining in the cells
(top) and as rate coefficients
(bottom). A stable basal efflux was
obtained, after which the addition of 50 µM bumetanide, a specific
inhibitor of NKCC1, reduced efflux to a new, stable level. Subtraction
of the two rate coefficients yields the bumetanide-sensitive flux,
which accounts for approximately one-third of the total efflux. The
remaining bumetanide-insensitive efflux represents fluxes through
K+ channels and
K+ leak pathways. This procedure
permits the calculation of bumetanide-sensitive efflux for each aortic
segment. It has previously been shown that bumetanide-sensitive
Rb+ influx in aorta is dependent
on extracellular Na+ and
Cl
(8), consistent with
NKCC1 activity.

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Fig. 1.
Efflux of
86Rb+
from rat aortic smooth muscle. Efflux was measured at 2-min intervals,
before and after addition of 50 µM bumetanide (arrows), from rat
aorta prepared and loaded with
86Rb+
as described in MATERIALS AND METHODS.
Top: fraction of
86Rb+
remaining after each time point.
Bottom: fraction of
86Rb+
recovered in medium at each time point expressed as rate coefficient.
Results are means ± SE of a representative experiment performed in
triplicate.
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To measure acute changes in cotransport, parallel assays were performed
in the presence and absence of bumetanide (Fig.
2). In this experiment, the addition of 150 mM sucrose to produce hypertonic shrinkage increased total efflux and
reduced bumetanide-insensitive efflux, indicating a rapid doubling of
cotransporter activity. Cotransporter activity returned to baseline
when isotonicity was restored (data not shown). This is consistent with
the volume sensitivity of NKCC1 in virtually all cells, including
cultured VSMC (29), and indicates that
Rb+ efflux provides a reliable
assay of NKCC1 activation in intact vascular smooth muscle.

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Fig. 2.
Effect of hypertonic medium on Rb+
efflux in rat aorta.
86Rb+
efflux was measured in isotonic and hypertonic medium (arrows indicate
transition). Top: efflux in absence or
presence of bumetanide (50 µM).
Bottom: bumetanide-sensitive efflux.
Results are means ± SE from 3 aortas, each assayed in
triplicate.
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ANG II (100 nM) significantly increased the efflux of
Rb+ (Fig.
3). A portion of the increase was inhibited
by 50 µM bumetanide, revealing a transient increase in NKCC1 activity
that peaked at 152 ± 11% of control
(n = 7, P < 0.001). The increase in
bumetanide-insensitive efflux presumably represents the opening of
K+ channels. More prolonged
incubation with ANG II (performed during loading with
86Rb+)
revealed a second, delayed activation of NKCC1 (Fig.
4). There was no stimulation after 1 h of
incubation with 100 nM ANG II, but significant stimulation was apparent
after 2 h (82 ± 20%; n = 9, P < 0.02) and 4 h (22 ± 3%;
n = 3, P < 0.02). Losartan, an antagonist
of type 1 ANG II receptors, completely blocked both the acute and
delayed stimulation of NKCC1 at a concentration of 10 µM (Fig.
5). There was a small but statistically
significant reduction in basal NKCC1 activity (22%) with losartan.
However, neither 1 µM saralasin (a peptide antagonist of ANG II) nor
4 µM lisinopril, an inhibitor of ANG II-converting enzyme, reduced basal NKCC1 activity (data not shown). For comparison, efflux in VSMC
cultured from rat aorta was also measured. Bumetanide-insensitive efflux was much higher in cells (0.0189 ± 0.0012 min
1) than in aorta,
rendering bumetanide-sensitive efflux difficult to measure. However,
values for bumetanide-sensitive efflux under basal conditions
(0.00228 ± 0.00067
1),
after acute ANG II stimulation (0.00423 ± 0.00089
1), and after 2 h
of ANG II (0.00544 ± 0.00080
1) were similar to
those observed in intact vascular smooth muscle.

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Fig. 3.
Effect of ANG II on Rb+ efflux in
rat aorta. Top: efflux of
86Rb+
was measured in absence or presence of bumetanide (50 µM).
Bottom: bumetanide-sensitive efflux
derived from top data. ANG II (100 nM)
was added at time indicated (arrows). Results are means ± SE from 7 aortas, each assayed in triplicate.
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Fig. 4.
Delayed stimulation of
Na+-K+-2Cl
cotransporter NKCC1 by ANG II. Aortas were preincubated with (ANG II)
or without (control) ANG II (100 nM) for times indicated, after which
efflux of
86Rb+
was measured in absence or presence of bumetanide (50 µM). Results
are means ± SE from 3 aortas, each assayed in triplicate.
* P < 0.02 vs. control.
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Fig. 5.
Effect of losartan on bumetanide-sensitive
Rb+ efflux in rat aorta. Aortic
segments were preincubated for 2 h with (ANG II) or without (basal) ANG
II (100 nM) and with (losartan) or without (control) losartan (10 µM), and efflux of
86Rb+
was measured in presence and absence of bumetanide (50 µM). Results
are means ± SE of 7 aortas for basal flux and 4 aortas for flux in
presence of ANG II, each measurement performed in triplicate.
* P < 0.01 vs. basal control;
** P < 0.001 vs. basal
control; *** P < 0.001 vs. ANG II
control.
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NKCC1 activity in rat aorta was also stimulated acutely by 10 µM
phenylephrine (50 ± 13%; P < 0.01, n = 8), 50 nM
endothelin (53 ± 17%; P < 0.02, n = 6), and 30 mM KCl (54 ± 6%;
n = 5, P <0.01) as shown in Fig.
6. For the KCl experiments, KCl was
isosmotically substituted for NaCl to avoid any hypertonic stimulation
of NKCC1. However, some cell swelling would be expected in this medium, and this swelling would reduce NKCC1 activity. Thus the stimulation of
NKCC1 by membrane depolarization may be greater than that achieved in
these experiments. The stimulation of
Rb+ efflux by extracellular
K+ is unlikely to be due to direct
enhancement of
K+/Rb+
exchange (trans stimulation) by NKCC1
because stimulation was quantitatively similar at 80 mM (not shown) and
because raising extracellular K+
concentration ([K+])
does not increase bumetanide-sensitive
Rb+ efflux in endothelial cells
(28), which lack voltage-sensitive Ca2+ channels (1). As in the case
of ANG II, there was a significant increase in bumetanide-insensitive
efflux. Unlike the case of ANG II, there was no delayed stimulation by
phenylephrine or endothelin (data not shown).

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Fig. 6.
Effect of vasoconstrictors on bumetanide-sensitive
Rb+ efflux in rat aorta.
Phenylephrine (10 µM), endothelin (50 nM), and KCl were added at time
indicated (arrows). Results are means ± SE from 8 (phenylephrine), 6 (endothelin), and 3 aortas (KCl), each assayed in triplicate.
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Nitrovasodilators had an opposite effect on NKCC1. Nitric oxide was
tested by using 10 µM
2-(N,N-diethylamino)diazenolate-2-oxide (DEA-NO) as a donor. As shown in Fig. 7,
bumetanide-sensitive efflux was inhibited 34%. A similar degree of
inhibition was observed with 8 µM nitroprusside, with no additional
inhibition at higher concentrations of nitroprusside.
S-nitroso-N-acetyl-D,L-penicillamine also inhibited NKCC1, but this did not reach statistical significance. The inhibition of NKCC1 was more apparent when aortas were pretreated with phenylephrine, a process akin to relaxation assays of
preconstricted vessels. As shown in Fig.
8, nitroprusside (8 µM)
completely reversed the stimulation of NKCC1 by phenylephrine (10 µM).

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Fig. 7.
Effect of nitrovasodilators on bumetanide-sensitive
Rb+ efflux in rat aorta. Efflux
was measured before and after addition (arrow) of 10 µM
2-(N,N-diethylamino)diazenolate-2-oxide
(DEA-NO). Inset: mean
bumetanide-sensitive efflux before (control) and after (vasodilator)
addition of nitrovasodilators from 6 aortic segments. SNP, sodium
nitroprusside; SNAP,
S-nitroso-N-acetyl-D,L-penicillamine.
* P < 0.01 vs. control. Error
bars, SE.
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Fig. 8.
Effect of SNP on stimulation of
Rb+ efflux in rat aorta by
phenylephrine (PE). PE (10 µM) and SNP (8 µM) were added at times
indicated (arrows). Results are from 1 experiment performed in
triplicate and are representative of 3 additional experiments.
Inset: composite data from 4 experiments. Solid bar, mean basal efflux (2-6 min); open bar,
mean peak PE efflux (10 min); cross-hatched bar, mean efflux at 16 min.
Error bars, SE.
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In other cells, stimulation of NKCC1 is associated with phosphorylation
of the transporter (10, 14, 20). To confirm this relationship in smooth
muscle and to provide definitive evidence for NKCC1 in this tissue,
NKCC1 was immunoprecipitated from aortic smooth muscle preincubated
with
[32P]orthophosphate.
As shown in Fig. 9, changes in NKCC1
phosphorylation paralleled changes in bumetanide-sensitive
Rb+ efflux, with significant
stimulation by phenylephrine, KCl, and hypertonicity and inhibition by
nitroprusside. These data confirm that the efflux results accurately
represent changes in NKCC1 activity.

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Fig. 9.
Phosphorylation of NKCC1 in vascular smooth muscle. Rat aortas were
labeled with
[32P]orthophosphate
and treated for 5 min with 30 mM KCl, 10 µM PE, or 10 µM SNP or for
15 min in hypertonic medium (150 mM sucrose; HYPER). This was followed
by immunoprecipitation of NKCC1 as described in
MATERIALS AND METHODS.
A: representative autoradiogram. kD,
kilodaltons. B: change in
phosphorylation, expressed as percentages of concurrent control
(isotonic) samples and measured by densitometry. Bars are means ± SE
of 3-5 separate experiments.
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To determine the functional significance of cotransporter activation by
vasoconstrictors, force generation in aortic rings was measured before
and after a 20-min incubation with 10 µM bumetanide (Fig.
10). Bumetanide significantly reduced the
contractile response to submaximal concentrations of phenylephrine (63 and 26% at 10 and 30 nM, respectively) without altering maximal
contraction. This resulted in approximately a doubling of the
half-maximal phenylephrine concentration. In contrast, there was no
inhibition of contraction by KCl, under which condition bumetanide
would not be expected to lower cell volume or intracellular
[Cl
].

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Fig. 10.
Effect of bumetanide on aortic contraction. Isometric force in aortic
rings was measured as described in MATERIALS AND
METHODS before ( ) and after ( ) 20 min in 10 µM
bumetanide. * P < 0.05, ** P < 0.02 vs.
force at same concentration of PE in absence of bumetanide. Error bars,
SE.
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DISCUSSION |
Rb+ efflux from rat aortic smooth
muscle was inhibited 30-40% by bumetanide, a highly specific
inhibitor of
Na+-K+-2Cl
cotransport, indicating the presence of NKCC1 in vascular smooth muscle. Definitive evidence was provided by immunoprecipitation of the
phosphorylated cotransporter from the aorta. This extends previous
findings by Deth et al. (8) of furosemide-sensitive, Na+- and
Cl
-dependent
Rb+ influx in aortas from rats and
rabbits and the findings by Davis et al. (5) that bumetanide decreases
intracellular
[Cl
] in
vascular smooth muscle from the rat femoral artery. The significant stimulation of bumetanide-sensitive efflux in hypertonic medium is
further evidence for the
Na+-K+-2Cl
cotransporter, which is universally stimulated by cell shrinkage. The
present findings and previous studies contradict the recent contention
that NKCC1 is present only in cultured smooth muscle cells and not in
intact vascular smooth muscle (38). There are two forms of this
transporter, encoded by separate genes (11). Because one form (NKCC2)
is found only in the apical membrane of the thick ascending limb of
Henle in the kidney, it is assumed that NKCC1 is the form present in
vascular smooth muscle, and this is consistent with the presence of
NKCC1 mRNA in cultured rat aortic smooth muscle cells (38). The
inability of Raat et al. (38) to detect NKCC1 mRNA in
freshly isolated rat aortic smooth muscle cells is difficult to explain
because we observed similar rate coefficients for bumetanide-sensitive
efflux in rat aortas and in rat aortic smooth muscle cells in culture.
All vasoconstrictors tested produced a rapid increase in total
Rb+ efflux, consistent with
previous studies (3, 21-23, 44). Although this had been ascribed
to the opening of K+ channels, we
find that NKCC1 contributes substantially to this increase, a
contribution ranging from 30% (ANG II) to 36% (phenylephrine). That
portion of the KCl-induced K+
efflux previously described as
Ca2+ independent and swelling
independent (21) may in fact be mediated by NKCC1. The stimulation of
NKCC1 by KCl, which acts by depolarizing smooth muscle and activating
voltage-sensitive Ca2+ channels to
raise intracellular
[Ca2+], indicates that
the stimulation by other vasoconstrictors is a direct effect of
increased intracellular
[Ca2+] and not an
effect of other receptor-mediated actions. In addition to
immediate stimulation of NKCC1, ANG II also demonstrated a second,
delayed stimulation between 2 and 4 h that was not observed with
phenylephrine. This suggests at least two modes of activation of NKCC1
in vascular smooth muscle and is consistent with both immediate and
delayed signaling by ANG II in VSMC (40).
NKCC1 is known to be activated by cell shrinkage or a decrease in
intracellular
[Cl
]. We have
previously shown that activation of NKCC1 by
Ca2+-mobilizing agonists in
endothelial cells is the result of cell shrinkage or loss of
Cl
due to the opening of
Ca2+-dependent
K+ channels and possibly
Cl
channels as well (28).
This does not appear to be the case in vascular smooth muscle because
equivalent stimulation of NKCC1 occurred with KCl (up to 80 mM), which
would prevent cell shrinkage and loss of
Cl
through
K+ and
Cl
channels (28).
Furthermore, intracellular
[Cl
] in rat
arterial smooth muscle does not decrease and actually increases in the
presence of phenylephrine (7). Thus stimulation of NKCC1
by vasoconstrictors appears to be a direct effect of raising
intracellular [Ca2+]
rather than a secondary response to decreased intracellular [Cl
] or cell
volume. One possible mechanism may be the recently described link
between myosin light chain phosphorylation and NKCC1 activity in
endothelial cells (14).
Nitrovasodilators had the opposite effect on NKCC1, inhibiting its
basal activity and reversing its stimulation by phenylephrine. The
inhibition of NKCC1 by nitroprusside or nitric oxide has not previously
been described. cGMP, a mediator of nitrovasodilator effects,
stimulates NKCC1 in VSMC in culture (27), which is the opposite of the
effect of nitroprusside in rat aorta. However, cGMP is reported to
inhibit NKCC1 in cultured endothelial cells (26). Thus the effect of
cGMP may vary between cells and with culture conditions. Nitroprusside
is reported to lower intracellular [Ca2+] in vascular
smooth muscle (22), which would be consistent with the role of
intracellular Ca2+ in the
stimulation of NKCC1 by vasoconstrictors. Whether nitroprusside has
effects on cell volume or intracellular
[Cl
] that could
inhibit NKCC1 is unknown.
Activation of NKCC1 in other cells depends on phosphorylation (20, 36)
and is associated with the phosphorylation of the transporter (10, 14,
20), although it is not known whether the phosphorylation of NKCC1 is
required for activation. Consistent with this, we observed changes in
the phosphorylation of NKCC1 that paralleled changes in activity. This
suggests that vasoconstrictors and nitrovasodilators affect NKCC1
through a common pathway that alters its phosphorylation. The results
also indicate that the changes in NKCC1 activity observed in smooth
muscle are not due to kinetic effects related to changes in
intracellular ion activities.
The opposing effects of vasoconstrictors and vasodilators on NKCC1
suggest that the cotransporter has an important role in smooth muscle
contraction, which was confirmed in direct measurements of contraction
by phenylephrine. The inhibition by bumetanide was most apparent at low
agonist concentrations likely to be physiologically relevant.
Bumetanide was recently shown to inhibit the contraction of rat aorta
induced by norepinephrine (18), but the response to
different doses of agonist and the effect on maximal contraction were
not studied. The effect of bumetanide is consistent with the known
vasodilatory effect of furosemide in vitro and in vivo (8, 9). However,
furosemide is not specific for NKCC1 and can inhibit other
Cl
transport pathways.
Therefore the effect of bumetanide provides definitive proof that
inhibition of NKCC1 can produce vasodilation and indicates that this is
the likely mechanism for the vasodilatory effect of furosemide.
Vasodilation is not a prominent feature of "loop" diuretics
because extensive protein binding limits systemic inhibition of NKCC1.
Assuming that 90% of bumetanide is protein bound (4) and that the
volume of distribution is 0.068 l/kg (39), a standard dose of 0.015 mg/kg in humans would produce a free plasma concentration of ~60
nM. This is well below the half-inhibitory concentration of ~200 nM
(13), but a maximal dose could approach this concentration.
The lack of inhibition of vasoconstriction by KCl indicates possible
mechanisms by which NKCC1 promotes contraction. Because the
cotransporter would be expected to mediate a net influx under physiological conditions, bumetanide could have several effects in
smooth muscle, specifically, a decrease in intracellular
[Na+], intracellular
[Cl
], or cell
volume. The last two would be prevented or significantly reduced by
raising external [K+],
but the decrease in intracellular
[Na+] would not. The
fact that the vasodilatory effect of bumetanide is not observed after
KCl contraction therefore implicates intracellular [Cl
] or cell
volume. It also eliminates the possibility that bumetanide is acting
nonspecifically through an effect independent of NKCC1. Bumetanide has
been shown to decrease intracellular
[Cl
] in
vascular smooth muscle (6), indicating a role for NKCC1 in regulating
this ion. The activation of NKCC1 by vasoconstrictors may therefore
serve to raise intracellular
[Cl
], thereby
maintaining or increasing the
Cl
current that contributes
to membrane depolarization and subsequent Ca2+ influx (16, 18, 45). This
raises the possibility that alterations in NKCC1 could influence
vascular smooth muscle function and blood pressure in vivo.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institutes of Health Grants
HL-47449 and DK-07656.
 |
FOOTNOTES |
F. Akar was supported by the NATO Scientific Fellowship Program of the
Scientific and Technical Research Council of Turkey (TUBITAK).
Preliminary accounts of this work have previously been presented in
abstract form (12, 25).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. C. O'Neill,
Renal Division, Emory Univ. School of Medicine, WMB 338, 1639 Pierce
Dr., Atlanta, GA 30322 (E-mail: woneill{at}emory.edu).
Received 28 December 1998; accepted in final form 25 February
1999.
 |
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