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cotransporter
Departments of 1 Molecular Genetics, Biochemistry, and Microbiology and 2 Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The basolateral
Na+-K+-2Cl
cotransporter (NKCC1)
functions in the maintenance of cellular electrolyte and volume
homeostasis. NKCC1-deficient (Nkcc1/) mice
were used to examine its role in cardiac function and in the
maintenance of blood pressure and vascular tone. Tail-cuff measurements
demonstrated that awake Nkcc1/ mice had
significantly lower systolic blood pressure than wild-type (Nkcc1+/+) mice (114.5 ± 2.2 and
131.8 ± 2.5 mmHg, respectively). Serum aldosterone levels were
normal, indicating that extracellular fluid-volume homeostasis was not
impaired. Studies using pressure transducers in the femoral artery and
left ventricle showed that anesthetized
Nkcc1/ mice have decreased mean arterial
pressure and left ventricular pressure, whereas myocardial contraction
parameters were not significantly different from those of
Nkcc1+/+ mice. When stimulated with
phenylephrine, aortic smooth muscle from
Nkcc1+/+ and Nkcc1/
mice exhibited no significant differences in maximum contractility and
only moderate dose-response shifts. In phasic portal vein smooth muscle
from Nkcc1/ mice, however, a sharp reduction
in mechanical force was noted. These results indicate that NKCC1 can be
important for the maintenance of normal blood pressure and vascular tone.
vasculature; hypotension; bumetanide
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE BASOLATERAL
ISOFORM of the Na+-K+-2Cl
cotransporter (NKCC1) mediates the transport of 1 Na+, 1 K+, and 2 Cl into the cell
under normal conditions. NKCC1 is expressed in most tissues, including
heart (10, 42), vascular smooth muscle (39),
and endothelial cells (37). Although the roles of NKCC1 in
transepithelial ion transport and in the regulation of cell volume and
intracellular ion concentrations are well established (9, 14, 15,
17, 19-21, 24, 36, 37), its cardiovascular functions are
not well understood. Vasoactive agents alter the activity of NKCC1 in
vascular smooth muscle (2, 39, 47) and in endothelial
cells (6, 27, 35). There is evidence that NKCC1 is
responsible for the maintenance of intracellular Cl
concentrations and cell volume during agonist stimulation of endothelial cells (38) and that its activity is regulated
in response to changes in vascular smooth muscle contractility
(1). The loop diuretic bumetanide, an inhibitor of NKCC1,
causes a reduction in the sensitivity of rat aortic rings to
phenylephrine-induced contraction (2). Furosemide, another
loop diuretic, has been shown to relax canine venous smooth muscle
preparations while having little effect on arteries (18).
The results of these and other in vitro studies (reviewed in Ref.
13) suggest that NKCC1 could play a direct role in the
modulation of vascular tone.
In vivo studies using bumetanide and other loop diuretics provide support for this hypothesis. In patients with congestive heart failure, furosemide caused a reduction in left ventricular filling pressure and an increase in calf venous compliance, which preceded the natriuretic and diuretic effects (11). After acute myocardial infarction, furosemide caused a decrease in left ventricular filling pressure, which was attributed to venodilation, but also led to a rapid increase in blood pressure and systemic vascular resistance (34). These and other studies (reviewed in Ref. 13) indicate that loop diuretics affect systemic hemodynamics and cardiac function not only by their natriuretic and diuretic activities but also by effects on the vasculature. Although some of these hemodynamic effects appeared to be caused by the release of vasoactive compounds from the kidney (5), it has been suggested that direct inhibition of NKCC1 by loop diuretics may cause vasodilation of capacitance veins (13).
Gene-targeted mice lacking NKCC1 have been developed by several groups
(9, 15, 40). NKCC1-deficient
(Nkcc1/) mice exhibit reduced epithelial
chloride secretion (15, 17, 19, 20), male sterility
(40), and both profound deafness and a balance defect
(9, 15). In our own study (15), we also
reported a significant reduction in mean arterial pressure (MAP) of
anesthetized Nkcc1/ mice, measured using a
femoral artery catheter; however, another group of investigators
(40) observed no significant difference in systolic blood
pressure of awake Nkcc1/ and wild-type
(Nkcc1+/+) mice, measured using a
tail-cuff apparatus. With the exception of these limited analyses of
blood pressure carried out using different procedures, the
cardiovascular phenotype of Nkcc1/ mice has
not been examined. Thus the major objectives of the present study were
to perform a comprehensive analysis of blood pressure to resolve the
apparent discrepancies between the two studies and to determine whether
the loss of NKCC1 leads to alterations of cardiac and/or vascular
smooth muscle contractility. The results demonstrate that the
Nkcc1/ mouse has a hypotensive phenotype and
suggest that this may be due, at least in part, to a reduction in
vascular tone.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Mice and genotype analysis. Development of the NKCC1 null mutant mouse line by gene targeting was described previously (15). Null mutant and wild-type control mice were generated by breeding of heterozygous mutant mice that were on a mixed background of 129/SvJ and Black Swiss strains. Genotypes were determined by PCR analysis of DNA from tail biopsies as described previously (15). The use of mice in these experiments was approved by the University of Cincinnati Animal Care and Use Committee.
Tail-cuff blood pressure measurements.
Systolic blood pressure of adult Nkcc1+/+
(n = 17) and Nkcc1/ mice
(n = 16) was recorded for 21 days using a Visitech
Systems (Apex, NC) computerized tail-cuff apparatus (28).
Because of the defect in the vestibular system of the inner ear of
Nkcc1/ mice (9, 15, 40), the
mutants were easily agitated and would sometimes attempt to spin in the
apparatus. Thus extreme care in handling the mice was needed to obtain
accurate recordings of systolic pressure. Each day, 10 preliminary
blood pressure measurements were performed to acclimate the mice to the
apparatus, and these were followed by 10 recorded systolic blood
pressure and heart rate measurements. There were no time delays between each set of measurements. The blood pressure waveform was carefully observed during the preliminary measurements to identify mice that were
not retaining the proper position in the apparatus. During the second
set of measurements, data were accepted if a blood pressure was
identified by the computer in at least 5 of the 10 measurements and was
>30 or <200 mmHg. Recordings not meeting these criteria (fewer than
1%) were discarded.
Analysis of blood pH, gases, and electrolytes. Conscious mice of both sexes ranging in age from 8-16 wk were placed on a 37°C heating pad to enhance peripheral blood circulation. Blood (50 µl) was collected from the tail vein in a heparinized capillary tube (Ciba-Corning; Medfield, MA) and immediately analyzed for acid/base status, blood gases, and plasma electrolytes using a blood gas analyzer (model 348; Chiron Diagnostics, Oberlin, OH).
Serum aldosterone levels. Concentrations of aldosterone in serum from mice of both sexes ranging in age from 8 to 16 wk were determined using a 125I radioimmunoassay, performed in duplicate according to the manufacturer's suggested protocol (Diagnostics Products; Los Angeles, CA).
Measurement of mean arterial blood pressure and cardiac function
in the intact closed-chest mouse.
Adult male and female mice weighing 
20 g were anesthetized with an
intraperitoneal injection of 50 µg ketamine/g body wt and 100 µg
thiobutabarbital/g body wt (Inactin; Research Biochemicals International, Natick, MA). As described earlier (30),
polyethylene tubing (0.4 mm outer diameter) was inserted into the
abdominal aorta from the right femoral artery and connected to a
low-compliance pressure transducer (COBE Cardiovascular; Arvada, CO)
for measurement of MAP. A high-fidelity, 1.8-French Millar Mikro-Tip
transducer (model SPR-612; Millar Instruments, Houston, TX) was
inserted into the right carotid artery and advanced into the left
ventricle to monitor cardiac performance. The right femoral vein was
cannulated for infusion of dobutamine. MAP and intraventricular
pressure signals from the COBE transducer and the Millar transducer
were analyzed using a MacLab 4/s data acquisition system connected to a
Macintosh 7100/80 computer. Average values for heart rate, MAP, and
systolic left ventricular pressure (LVP) were measured directly from
the pressure waveforms and were determined for each animal from at
least 50 consecutive beats during the final 30 s of each 3-min
dosage period. Maximum dP/dt (+dP/dt) and
dP/dt at 40 mmHg (dP/dt40) were
calculated from the first derivative of the pressure waveforms.
Analysis of blood vessel contractile properties. Mice of 8-12 wk of age were euthanized by CO2 inhalation followed by cervical dislocation. Thoracic aortas, in segments of 5-7 mm, were dissected and mounted for isometric force recording as described previously (29). Studies were completed in both intact and endothelium-denuded aortas. Phenylephrine concentration-isometric force relationships were generated in the absence and presence of bumetanide (10 µM; 20-min incubation). The portal vein was dissected from each mouse by tying 4-0 sutures at the hepatic bifurcation and the anterior mesenteric vein. The portal vein was cut free, and each end was secured with the suture material to the myograph. From the time of the dissection, the vessel was maintained in physiological salt solutions (PSS). PSS contained the following (in mmol/l): 118 NaCl, 4.73 KCl, 1.2 MgCl2, 0.026 EDTA, 1.2 KH2PO4, 2.5 CaCl2, 5.5 glucose and was buffered with 25 NaH2CO3; pH was 7.4 at 37°C, when bubbled with 95% O2/5% CO2. Experiments were completed at optimal tension based on adjusting the vessels to a point where maximum peak-to-peak oscillations were observed. Force measurements were obtained using a Harvard Apparatus differential capacitor force transducer (South Natick, MA) connected to a Biopac MP100 data acquisition system that allowed measurements or calculations of contractile parameters including frequency of spontaneous contractions and tension-time integral (T-t integral). The effects of bumetanide on contractile parameters were examined after a 20-min preincubation with 10 µM bumetanide.
Statistics. Data are presented as means ± SE. Student's t-test was used to compare mutant mice to the corresponding control mice. Mixed two-factor ANOVA with repeated measures on the second factor was used to compare genotype and dosage in dobutamine-treated, closed-chest, anesthetized mice and tail-cuff blood pressure studies.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Tail-cuff measurements of systolic blood pressure.
In a recent study (40), it was reported that systolic
blood pressure in Nkcc1/ mice, measured
using a tail-cuff apparatus, was not significantly different from that
of wild-type controls. Because this result differed from our previous
results showing that MAP in anesthetized Nkcc1/ mice was significantly reduced
(15), we measured tail-cuff pressures in
Nkcc1/ and Nkcc1+/+
mice to determine whether the apparent discrepancy was due to the
different methods that were used.
/ mice
(n = 16) had a significantly lower systolic blood
pressure than Nkcc1+/+ mice (n = 17) throughout the course of the 21-day period (Fig. 1A), with average blood
pressures of 114.5 ± 2.2 mmHg in
Nkcc1/ mice and 131.8 ± 2.5 mmHg
in Nkcc1+/+ mice. There were no significant
differences in heart rates (data not shown). Because the original study
describing use of the tail-cuff apparatus included a 7-day period for
acclimation of the mice to the apparatus (28), we
performed separate analyses of the data for the first 7 days and the
following 14 days. As shown in Fig. 1B, blood pressures for
both genotypes were slightly lower during the initial 7-day period;
however, the differences between the two genotypes were essentially the
same during both periods. When the data from individual experiments
were analyzed, systolic blood pressure was significantly reduced
(P < 0.01) in the Nkcc1/
mice in all three experiments (Nkcc1+/+ and
Nkcc1/, respectively: experiment
1, 134.8 ± 2.6 and 116.8 ± 3.4 mmHg; experiment
2, 138.4 ± 4.3 and 119.3 ± 2.9 mmHg; experiment
3, 122.5 ± 2.5 and 106.3 ± 2.4 mmHg). On the basis of
these data, we conclude that the loss of NKCC1 causes a significant
reduction in systolic blood pressure.
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Blood acid-base, electrolyte status and serum aldosterone levels.
Although NKCC1 does not play a direct role in NaCl reabsorption in the
kidney, as in the case of NKCC2 (51), it is conceivable that its absence impairs Na+-fluid volume homeostasis by
some indirect means, thereby leading to the blood pressure defect. To
examine this possibility, we analyzed blood from
Nkcc1+/+ and Nkcc1/
mice (Table 1). There were no significant
differences in blood gases, pH, HCO
, or Ca2+; however,
K+ concentrations were significantly elevated in the
Nkcc1/ mice. Serum aldosterone levels were
essentially the same in both genotypes, indicating that extracellular
volume depletion was unlikely to be a significant factor in the
observed hypotension.
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Cardiac performance in the intact closed-chest anesthetized mouse.
To determine whether Nkcc1/ mice might
exhibit a cardiac disease phenotype that could be a contributing factor
in the blood pressure defect, we analyzed heart rate, MAP, LVP, left
ventricular end-diastolic pressure (LVEDP), and maximum and minimum
dP/dt, and dP/dt40 under basal
conditions and after the administration of dobutamine, a 
-adrenergic
agonist. There were no significant differences between the heart rates
of Nkcc1/ and
Nkcc1+/+ mice under either basal conditions or
after -adrenergic stimulation (Fig.
2A). MAP was significantly
decreased in Nkcc1/ mice under basal
conditions (Nkcc1/, 70.4 ± 3.0 mmHg;
Nkcc1+/+, 84.0 ± 3.9 mmHg) and at all but
the highest doses of dobutamine (Fig. 2B). Systolic LVP
(Fig. 2C) was significantly reduced in Nkcc1/ mice under basal conditions
(Nkcc1/, 95.6 ± 3.0 mmHg;
Nkcc1+/+, 107.3 ± 4.1 mmHg) and at all
dobutamine doses, consistent with a reduction in afterload (indicated
by the reduced MAP). There were no significant differences in LVEDP
(Fig. 2D), maximum dP/dt (Fig. 2E),
minimum dP/dt (data not shown), or
dP/dt40 (Fig. 2F). These data suggest
that the reduced blood pressure in anesthetized Nkcc1/ mice is not the result of an impaired
myocardium.
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Mechanical studies in Nkcc1/ aortas and portal
veins.
Regulation of vascular smooth muscle contractility plays a major role
in the maintenance of normal blood pressure. The observations that
NKCC1 activity is altered in response to vasoactive compounds (1) and that inhibitors of NKCC1 alter vascular
contractility (reviewed in Ref. 13) suggest that
eliminating NKCC1 could affect vascular tone. To test this hypothesis,
we examined the mechanical properties of aortas (tonic smooth muscle)
and portal veins (phasic smooth muscle) from
Nkcc1/ and Nkcc1+/+ mice.
/
and Nkcc1+/+ aortic rings after stimulation with
phenylephrine in either the presence or absence of 10 µM bumetanide,
an inhibitor of NKCC1. When aortic rings with intact endothelium (Fig.
3A) were examined, the Nkcc1/
aorta appeared to be more sensitive to phenylephrine than the Nkcc1+/+ aorta, regardless of whether the
experiment was performed in the absence or presence of bumetanide;
however, the concentration yielding 50% of maximum contraction
(EC50) was significantly different (P < 0.01) only in the presence of bumetanide. After treatment with
bumetanide, there was a slight leftward shift in the phenylephrine concentration-force curve for Nkcc1/ aortas
and a slight rightward shift in the curve for
Nkcc1+/+ aortas; however, neither of these
shifts were statistically significant.
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/
and Nkcc1+/+ aortic rings devoid of endothelium,
using the same pharmacological conditions as in Fig. 3A.
Relative to intact aortas, both genotypes exhibited greater
sensitivity to phenylephrine. Endothelium-denuded Nkcc1/ and
Nkcc1+/+ aortas treated with bumetanide
exhibited a rightward shift in the phenylephrine concentration-force
curve (toward reduced sensitivity) when compared with aortas of the
same genotype that were not treated with bumetanide. The
Nkcc1+/+ aorta exhibited a greater sensitivity
to bumetanide (approximately fourfold shift in EC50) than
the Nkcc1/ aorta (approximately twofold
shift in EC50); the differences were significant in the
Nkcc1+/+ (P < 0.001) but not in
the Nkcc1/ mice.
Figure 4 shows the maximum force/area (in
mN/mm2) generated by Nkcc1/ and
Nkcc1+/+ aortic rings, with or without
endothelium, after stimulation with 10 µM phenylephrine and in the
presence or absence of 10 µM bumetanide. Although
Nkcc1/ aortas exhibited a trend toward a
decreased maximum force of contraction relative to
Nkcc1+/+ controls, the differences were slight
and not significant. Similarly, bumetanide had no significant effect on
maximal force generated by aortas of either genotype.
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/
portal veins was examined before and after the administration of 10 µM bumetanide. Tracings of the spontaneous contraction profiles are
shown in Fig. 5. Within 10 min of
exposure to bumetanide, a marked decrease in mechanical force was
apparent for Nkcc1+/+ portal veins, whereas
little change was observed in Nkcc1/ portal
veins. In the absence of bumetanide, mechanical force (Fig.
6A), as estimated by the
T-t integral, was significantly lower in
Nkcc1/ portal veins (94.0 ± 18.4 mN · s/mm2) than in
Nkcc1+/+ controls (237.7 ± 37.7 mN · s/mm2). In the presence of 10 µM bumetanide, there was an 85% reduction in force in the
Nkcc1+/+ portal vein when compared with basal
force, whereas only a 30% reduction (from a much lower basal level)
was observed in Nkcc1/ portal veins (Fig.
6A). Measurements of on-time (Fig. 6B) and off-time (Fig. 6C) demonstrated that the two genotypes had
different contraction cycles. On-time (contraction phase) for
Nkcc1/ portal veins (10.5 ± 2.0 s) was significantly less than that of the
Nkcc1+/+ (16.8 ± 4.2 s), and off-time
(relaxation phase) for Nkcc1/ portal veins
(23.4 ± 7.7 s) was significantly greater than that of the
Nkcc1+/+ (10.4 ± 3.3 s). In the
presence of 10 µM bumetanide, there was a significant change in both
Nkcc1+/+ on time (9.41 ± 0.43 s) and
off time (41.8 ± 6.0 s) when compared with the basal state.
Nkcc1/ on time (9.03 ± 1.2 s) and
off time (31.4 ± 3.6 s) after bumetanide administration was
not significantly different from the baseline Nkcc1/ values.
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DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Our major objectives were to determine whether the loss of NKCC1
causes a hypotensive phenotype and whether NKCC1 null mice exhibit
impairments of cardiac performance and/or vascular contractility. This
is an important issue because loop diuretics, such as bumetanide and
furosemide, which inhibit NKCC1, are used in the treatment of a number
of cardiovascular diseases and lead to a reduction in blood pressure.
It is often assumed that the therapeutic effect of loop diuretics
results entirely from their well-established natriuretic and diuretic
activities, which are due to inhibition of NKCC2, the apical
Na+-K+-2Cl cotransporter of the
renal thick ascending limb. However, studies over the past three
decades suggest that these drugs may affect the vasculature
(13), either by stimulating the release of vasoactive compounds from the kidney or by direct action on vascular endothelium or smooth muscle, and there are indications that loop diuretics might
also affect cardiac function under certain conditions (3, 43).
There is controversy about whether the loss of NKCC1 in mice causes a
reduction in blood pressure. In our initial analysis of the
Nkcc1/ mice (15), in which mice
of a heterogeneous 129SvJ and Black Swiss background were used, we
found that MAP of a small group of anesthetized
Nkcc1/ mice was substantially lower than
that of Nkcc1+/+ mice; however, another group
reported that systolic blood pressure was not altered in awake
Nkcc1/ mice (of a heterogeneous 129SvJ,
C57BL/6, and DBA/2J background). In the latter study (40),
tail-cuff measurements yielded values of 100 ± 7 mmHg for
Nkcc1/ mice and 108 ± 8 mmHg for
Nkcc1+/+ mice. Although the mean value for the
Nkcc1/ mice was lower than that of
Nkcc1+/+ mice, the difference was not
statistically significant and it was concluded that blood pressure was
not affected by the loss of NKCC1. However, given the observed
variability, the small number of mice analyzed (n = 5 for each genotype), and the direction and magnitude of the measured
differences in blood pressure, those data were not inconsistent with a
hypotensive phenotype in the awake mouse. The results shown in Fig. 1,
using a larger number of mice and a 3-wk time period, demonstrate that
systolic blood pressure is, in fact, significantly reduced in
unanesthetized Nkcc1/ mice. Furthermore, the
magnitude of the reduction would be expected to be biologically
important. It is possible, as noted earlier (40), that
differences in mouse strains might affect the blood pressure phenotype.
However, our tail-cuff data negate the possibility that the reduction
in arterial pressure in the heterogeneous background used in our
studies occurs only when the mice are anesthetized, as might have
occurred if stimulation of the sympathetic nervous system was providing
compensation in the awake mouse.
Loss of ion transporters involved in Na+ reabsorption
across the apical membranes of renal epithelial cells leads to
extracellular volume depletion and consequent hypotension (7, 45,
46). NKCC1 is a basolateral transporter and does not contribute
directly to Na+ reabsorption; however, because it is
expressed in the kidney, we considered the possibility that it could
have an indirect effect on the maintenance of Na+-fluid
volume homeostasis. Also, NKCC1 has been implicated as part of the
hepatoportal system for sensing plasma Na+ and
K+ concentrations in the portal vein, which is involved in
the regulation of renal electrolyte excretion (32, 33).
Although plasma K+ concentrations were slightly elevated,
for reasons that remain unclear, analysis of blood electrolytes and
serum aldosterone gave no indication of a major impairment of
Na+-fluid volume homeostasis. Serum aldosterone levels
provide a sensitive indication of major alterations in
Na+-fluid volume homeostasis. For example, mice lacking the
Na+/H+ exchanger (NHE3), the primary mechanism
for NaCl absorption in the proximal tubule, have reduced blood pressure
and sixfold elevation in aldosterone levels as a result of impaired
Na+-fluid volume homeostasis (45). No
significant elevation of serum aldosterone was observed in
Nkcc1/ mice. On the basis of these data, it
seems unlikely that the blood pressure defect of NKCC1-deficient mice,
which is more severe than that of the NHE3 knockout mice, is due to
impaired Na+-fluid volume homeostasis.
An alternative hypothesis is that the loss of NKCC1 might cause a
direct impairment of the cardiovascular system. NKCC1 is expressed in
the heart (10), and there are data showing that inhibition
of Na+-K+-2Cl cotransport
activity with bumetanide can affect contractility of cultured cardiac
myocytes. The data are contradictory, however, with reversal of the
positive inotropic effect of low concentrations of ouabain in one study
(41) and a positive inotropic effect reported in another
study (26). Analysis of cardiac performance using the
closed-chest anesthetized mouse revealed similar values for maximum
dP/dt, but Nkcc1/ mice exhibited
a significant reduction in systolic LVP. Given the reduction in MAP,
however, this change is likely to be due to a reduction in afterload.
dP/dt40 was calculated to correct for the
reduction in afterload (30) and was found to be identical in Nkcc1/ and
Nkcc1+/+ mice. LVEDP (a measurement of
ventricular pressure just before contraction occurs, which is dependent
on the filling rate and compliance of the myocardium) was also similar
in the two groups of mice, with values well within the normal range
(30). Our in vivo data indicate that the genetic loss of
NKCC1 causes no major impairment of systolic or diastolic left
ventricular function. These results are consistent with an earlier
study (31) showing that high concentrations of furosemide
have no effect on cardiac performance in dogs.
To determine whether the loss of NKCC1 might affect vascular smooth
muscle tone, which could have a major impact on blood pressure, we
examined in vitro preparations of aorta and portal vein. In previous
studies of rat aorta, vasoactive compounds or alterations in aortic
smooth muscle contractility led to changes in the activity of NKCC1,
and bumetanide caused an approximately twofold reduction in sensitivity
to phenylephrine (1, 2), consistent with a role for NKCC1
in the regulation of vascular tone. In the intact mouse aorta, there
were only minor differences in phenylephrine dose-response curves
between the two genotypes, and bumetanide had little effect.
Furthermore, the sensitivity of Nkcc1/ mice
aortas to phenylephrine appeared to be slightly greater than that of
the Nkcc1+/+ mice aortas, rather than slightly
less as in the rat aorta treated with bumetanide (2). In
the endothelium-denuded Nkcc1+/+ mouse aorta,
bumetanide reduced the sensitivity to phenylephrine approximately
fourfold, suggesting that the activity of NKCC1 might be required for
normal sensitivity of smooth muscle to vasoconstrictors; however, a
reduction in sensitivity, albeit of lesser magnitude (approximately
twofold), was also observed in the Nkcc1/
mouse aorta. In response to phenylephrine, we observed no significant differences in maximum force of contraction of either intact or endothelium-denuded Nkcc1/ and
Nkcc1+/+ aortic rings, and bumetanide also had
no significant effect (Fig. 4). Overall, these data provide little
support for a major role for NKCC1 in regulating the contractility of
mouse aorta.
In contrast, treatment of Nkcc1+/+ mouse portal
veins with bumetanide caused a substantial reduction in mechanical
force and, relative to Nkcc1+/+ controls,
mechanical activity in untreated Nkcc1/
portal veins was significantly impaired (Figs. 5 and 6). Furthermore, bumetanide had little effect on contractility of
Nkcc1/ portal veins. These results are
consistent with previous studies using loop diuretics, which indicated
that NKCC1 plays a major role in the contractility of a subset of
vascular tissues. For example, furosemide inhibited the contractile
response to vasoconstrictors and reduced the contractility of
isolated rat portal vein (4). Furosemide caused the
selective relaxation of a number of isolated canine venous preparations
(pulmonary, splenic, mesenteric, saphenous) while having little effect
on the corresponding arteries (18), and it also inhibited
the contraction of human internal mammary artery and saphenous veins in
response to angiotensin II (48). Similarly, bumetanide
caused relaxation of canine carotid arteries (8). The
results of the present study, in which NKCC1 null mutant portal veins
were relatively insensitive to bumetanide, indicate that the sharp
reduction in contractility of wild-type portal veins after treatment
with bumetanide in vitro results from the inhibition of NKCC1.
It is unclear whether therapeutic concentrations of bumetanide observed in vivo are sufficient to have a major effect on vascular contractility, although this does appear to be the case for furosemide. NKCC1 has been reported to have a Ki for bumetanide of 0.044 µM (22). Peak plasma concentrations after a therapeutic dose of bumetanide to human subjects (12) ranged from 39 to 63 ng/ml (0.10-0.17 µM), which is ~2-4 times the levels required for 50% inhibition of the cotransporter. NKCC1 is completely inhibited at 10 µM bumetanide, which is commonly used for in vitro studies, whereas it would be only partially inhibited at therapeutic levels. Therapeutic concentrations of furosemide, which is more commonly used for treatment of cardiovascular diseases, can vary between 1 and 10 µM (52). Several studies have shown that the effects of furosemide on vascular contractility in vitro can be observed at doses within the upper range of therapeutic concentrations (10 µM), consistent with the possibility that part of their therapeutic activity might be due to direct effects on the vasculature (18, 48).
Results of numerous clinical studies support the hypothesis that the
therapeutic activity of loop diuretics is due in part, to alterations
in vascular tissues, although the mechanisms underlying the effects on
the vasculature in vivo are unclear. Treatment of congestive heart
failure patients with furosemide led to a rapid reduction in left
ventricular filling pressure, which correlated with an increase in
venous compliance (11). Other investigators have noted a
decrease in left ventricular filling pressure after the administration
of loop diuretics in humans (34, 52) or dogs
(5), which they also attributed, in part, to an increase in venous compliance. In addition to venodilation, several
investigators have reported increases in blood pressure and vascular
resistance (34, 52) after loop diuretic treatment. The
reduction in contractility of isolated
Nkcc1/ portal veins is consistent with the
hypothesis that the increased venous compliance in response to loop
diuretics is due to inhibition of NKCC1 in the vasculature; however,
the results of several studies (5, 16, 23) suggest that
these effects might also be due to the release of vasoactive compounds
from the kidney. If the kidney is involved, then inhibition of NKCC1,
which is expressed in the extraglomerular mesangium and the glomerular
afferent arteriole (25), could contribute to this effect.
Experiments presented here extend our understanding of the phenotypic consequences of the loss of NKCC1. These were previously shown to include impaired chloride secretion in the lung and intestine (15, 17, 19, 20, 53) with reduced fluid secretion in the lung (17) and a low incidence of intestinal blockage (15), severely impaired K+ secretion in the inner ear with accompanying deafness and imbalance (9, 15), male sterility resulting from defective spermatogenesis (40), a sharp reduction in the secretion of saliva (14), and impaired regulatory volume increase and release of excitatory amino acids from astrocytes (49). In the present study, we have shown that the loss of NKCC1 in the mouse causes both hypotension and a reduction in contractility of isolated portal veins but does not appear to impair cardiac performance. The simplest explanation for the hypotensive phenotype is that it is due to a reduction in vascular tone resulting from the loss of NKCC1 in vascular tissue, although the mechanism is likely to be more complex and could involve other organs, such as the kidney. If a vascular defect is the primary mechanism, then it is possible that impaired tone of capacitance veins contributes to the blood pressure deficit by reducing venous return (44) and/or that the tone of resistance vessels is impaired. In this regard, it has been reported that portal veins and resistance vessels share certain functional properties (50).
It should be noted that the observed reductions in blood pressure and
the contractility defect are only correlative; a cause and effect
relationship has not been established. Additional studies will be
needed to determine the extent of the deficit in vascular contractility, the ionic basis for this defect, and whether it is the
major mechanism of the observed hypotension. Given the expression of
NKCC1 in renin-secreting cells of the glomerular afferent arteriole, it
seems possible that alterations in the secretion of vasoactive
compounds from the kidney might, as suggested by others (5, 16,
23), account for some of observed vascular effects of loop
diuretics in vivo. The reduction in excitatory amino acid release
observed in Nkcc1/ astrocytes
(49) suggests that neuronal mechanisms could also be
involved. An understanding of the mechanisms by which the loss of NKCC1
activity affects blood pressure and vascular tone, which may be complex
and involve changes in multiple tissues, should be of clinical
importance in the treatment of chronic cardiovascular diseases.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Maureen Luehrmann and Angel Whitaker for expert animal husbandry.
| |
FOOTNOTES |
|---|
This research was supported by National Institutes of Health Grants HL-61974, DK-50594, and DK-57552.
Present address of R. L. Sutliff: Department of Pathology, Emory University, Atlanta, GA 30322.
Address for reprint requests and other correspondence: G. E. Shull, Dept. of Molecular Genetics, Biochemistry, and Microbiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 524, Cincinnati, OH 45267-0524 (E-mail: shullge{at}ucmail.uc.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.
July 11, 2002;10.1152/ajpheart.00083.2002
Received 25 February 2002; accepted in final form 3 July 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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