Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202
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ABSTRACT |
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First published August 9, 2001;
10.1152/ajprenal.0075.2001.We have reported that nitric oxide (NO)
inhibits thick ascending limb (THAL) chloride absorption
(JCl
). NaCl transport in the THAL
depends on apical Na+-K+-2Cl
cotransporters, apical K+ channels, and basolateral
Na+-K+-ATPase. However, the transporter
inhibited by NO is unknown. We hypothesized that NO decreases THAL
JCl
by inhibiting the
Na+-K+-2Cl
cotransporter. THALs
from Sprague-Dawley rats were isolated and perfused. Intracellular
sodium ([Na+]i) and chloride concentrations
([Cl
]i) were measured with sodium green and
SPQ, respectively. The NO donor spermine NONOate (SPM) decreased
[Na+]i from 13.5 ± 1.2 to 9.6 ± 1.6 mM (P < 0.05) and also decreased [Cl
]i (P < 0.01). We next
tested whether NO decreases
Na+-K+-2Cl
cotransporter activity
by measuring the initial rate of Na+ transport. In the
presence of SPM in the bath, initial rates of Na+ entry
were 49.6 ± 6.0% slower compared with control rates
(P < 0.05). To determine whether NO inhibits apical
K+ channel activity, we measured the change in membrane
potential caused by an increase in luminal K+ from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM,
increasing luminal K+ concentration depolarized THALs to
the same extent as it did in control tubules. We then tested whether a
change in apical K+ permeability could affect NO-induced
inhibition of THAL JCl
. In the
presence of luminal valinomycin, which increases K+
permeability, addition of SPM decreased THAL
JCl
by 41.2 ± 10.4%, not
significantly different from the inhibition observed in control
tubules. We finally tested whether NO alters the affinity or maximal
rate of Na+-K+-ATPase by measuring oxygen
consumption rate (QO2) in THAL suspensions in
the presence of nystatin in varying concentrations of Na+.
In the presence of 10.5 mM Na+, nystatin increased
QO2 to 119.1 ± 19.2 and 125.6 ± 23.4 nmol O2 · mg
protein
1 · min
1 in SPM- and
furosemide-treated tubules, respectively. In the presence of 145 mM
extracellular Na+, nystatin increased
QO2 by 104 ± 7 and 94 ± 20% in NO-
and furosemide-treated tubules, respectively. We concluded that NO
decreases THAL JCl
by inhibiting
Na+-K+-2Cl
cotransport rather
than inhibiting apical K+ channels or the sodium pump.
nitric oxide; chloride transport; sodium-potassium-2 chloride cotransporter; natriuresis; sodium-potassium-adenosinetriphosphatase
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INTRODUCTION |
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THE THICK ASCENDING LIMB (THAL)
plays an essential role in the maintenance of NaCl homeostasis. This
segment reabsorbs 25-30% of the filtered NaCl load while being
water impermeable (5). This capability allows the THAL to
dilute the urine and generate a high interstitial osmolality
(21). Net NaCl absorption in the THAL involves a secondary
active transport process in which luminal Na+ and
Cl enter the cell via the electroneutral
Na+-K+-2Cl
cotransporter
(8, 22). This cotransporter is abundantly expressed in the
apical membrane of medullary and cortical THALs (28). The
driving force for NaCl entry via the cotransporter is generated by
Na+ extrusion across the basolateral membrane through
Na+-K+-ATPase (20).
Cl
then exits the cell through basolateral channels and
K+-Cl
cotransport (16). The
apical membrane of the THAL has a large K+ conductance
given by the presence of two types of K+ channels
(35). K+ recycling across the apical membrane
is important for Na+-K+-2Cl
cotransport and generates a positive luminal potential that provides the force for paracellular transport of cations such as
Ca2+ and Mg2+ (15).
Nitric oxide (NO) plays an important role in modulating transport in
the THAL (11, 14, 25, 29, 31). We have previously reported
that endogenously produced NO inhibits net chloride absorption (JCl) in isolated, perfused THALs
(31). However, the specific transporter inhibited by NO in
the THAL is still unknown. We hypothesized that NO inhibits
JCl
by decreasing apical
Na+-K+-2Cl
cotransport activity.
Our findings indicate that NO inhibits THAL
JCl
by decreasing
Na+-K+-2Cl
cotransport activity
rather than inhibiting apical K+ channels or
Na+-K+-ATPase.
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METHODS |
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Animals. Male Sprague-Dawley rats, weighing 120 to 150 g (Charles River Breeding Laboratories, Wilmington, MA) and which had been fed a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) for at least 5 days, were used for THAL perfusion and preparation of THAL suspensions. On the day of the experiment, rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip).
Isolation and perfusion of rat THALs. After anesthesia, the abdominal cavity was opened, and the left kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline. Cortical THALs were dissected from the medullary rays under a stereomicroscope at 4-10°C. THALs (ranging from 0.5 to 1.0 mm in length) were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 ± 1°C as described previously (13, 29). The flow rate of the basolateral bath was 0.5 ml/min.
Measurement of intracellular Na and Cl.
Once perfused, THAL cells were loaded by bathing the tubules at 37 ± 1°C for 15 min in 1 µM sodium green tetraacetate, 0.01% Pluronic or 5 µM 6-methoxy-N-[3-sulfopropyl]quinolinium
(SPQ; Molecular Probes, Eugene, OR) for measurements of intracellular Na+ and Cl concentration ([Na]i
and [Cl]I), respectively. Loading was followed by a
15-min wash period. Solution A (Table
1) was used for the basolateral bath and
perfusate. The sodium-sensitive fluorescent dye sodium green was
prepared daily. Sodium green- or SPQ-loaded tubules were excited at 500 or 340 nm, respectively, and 510- or 400-nm dichroic mirrors were used
as emission filters as appropriate. Fluorescence was digitally imaged
with an image intensifier (Video Scope International, Herndon, VA) and
a charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan).
Measurements of the images were recorded utilizing an Image One
Metafluor system (Universal Imaging, West Chester, PA). Control
measurements were taken every minute for 5 min. Then,
1,3-propanediamine,
N-{4- [1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl} C10H26N6O2 (SPM;
Cayman Chemical, Ann Arbor, MI) was added to the bath, and measurements
were taken once every minute for 15 min. The final five measurements
were averaged and taken as the value for
[Na+]i or [Cl
]i.
In situ calibration was performed for [Na+]i
measurements using different Na+ calibration solutions and
the Na+ ionophore nystatin. The Na+ calibration
solutions contained (in mM) 70 KCl+NaCl, 80 N-methyl-D-glucamine, 1.2 MgSO4, 1 CaCl2, and 10 HEPES (pH 7.4). For each calibration solution, the concentrations of KCl and NaCl were changed to obtain the
appropriate concentration of Na+ such that the sum of the
two concentrations was 70 mM (e.g, 10 mM NaCl + 60 mM KCl).
Nystatin (Sigma, St. Louis, MO) was prepared daily as a 100× stock and
diluted in calibration solution before use. No calibration was
performed for [Cl
]i measurements.
Osmolality of all solutions was adjusted to 290 ± 3 mosmol/kgH2O and equilibrated with 95% O2-5%
CO2.
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Measurement of Na+-K+-2Cl
cotransporter activity.
Once perfused, THAL cells were loaded by bathing the tubules at 37 ± 1°C for 60 min in a solution containing 4.4 µM of the Na+-sensitive fluorescent dye benzofuran
isophthalate-acetoxymethyl ester (SBFI-AM; Molecular Probes, Eugene,
OR), 0.015% Pluronic, for ratiometric measurements of
[Na+]i. During the loading period, THALs were
perfused with solution E (Table 1). This solution prevents
Na+-K+-2Cl
cotransporter activity
and inhibits the apical Na+/H+ exchanger.
Loading was followed by a 20-min wash period. Solution B
(Table 1) was used for the basolateral bath. The sodium-sensitive fluorescent dye SBFI-AM was prepared daily. SBFI-AM-loaded tubules were
excited alternately at 340 or 380 nm, and a 400-nm dichroic mirror was
used as an emission filter. An Image One Metafluor system was used to
record 340/380 ratio measurements of the images. Experiments consisted
of two periods in which we measured the initial rate of
[Na+]i increase caused by a switch in luminal
Na+ and Cl
from 4.5 and 0 mM, respectively
(solution E, Table 1), to 130 mM NaCl (solution
F, Table 1). Because solutions E and F
contain 100 µM dimethylamiloride, which inhibits
Na+/H+ exchangers, the increase in
[Na+]i is caused by Na+ entry
through the Na+-K+-2Cl
cotransporter. Ratio measurements of multiple regions within a tubule
were recorded every 3 s for a 5-min control period, during which
luminal NaCl was increased. The luminal solution was then switched back
to solution E, and SPM was added to the bath. After 30 min,
the luminal solution was switched again, and measurements were recorded
for a second 5-min period. The initial rates of [Na+]i increase were calculated for each
individual region of a tubule and averaged. In the presence of
furosemide (1 mM) in the lumen, increasing luminal NaCl did not change
[Na+]i. Control experiments were performed
according to the same protocol, except that no SPM was added before the
second period.
Measurement of membrane potential. Once THALs were perfused, cells were loaded by perfusing them with 2 µM of the membrane potential-sensitive dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes) in 0.1% Pluronic at 37 ± 1°C for 30 min. Tubules were then washed for 5 min. Solution B (Table 1) was used for the basolateral bath. Frozen stock solutions of JC-1 (10 mg/ml) were thawed daily and diluted just before addition to the lumen. JC-1 was excited at 488 nm, and emission fluorescence was observed via the Oz with Intervision confocal scanning imaging system (Noran Instruments, Middleteon, WI) utilizing a 560-nm dichroic mirror and a 595-nm band-pass filter. Experiments consisted of two periods of depolarization, which is measured as a decrease in fluorescence induced by increasing luminal K+ from 1 mM (solution C, Table 1) to 25 mM (solution D, Table 1). Measurements were recorded for a 30-s period, during which luminal K+ was increased. The luminal solution was then switched back to 1 mM K+, and SPM was added to the bath. After 20 min, luminal K+ was increased again, and measurements were recorded for a second 30-s period. The percent change in fluorescence was calculated for both the control and experimental periods. A difference in the fluorescence change caused by a switch in luminal K+ is equivalent to a change in K+ permeability. Control experiments were performed according to the same protocol, except that no SPM was added before the second period.
JCl measurement.
THALs were mounted on concentric glass pipettes and perfused at 37°C
as described previously (13). The perfusion rate was set
at 5-10
nl · mm
1 · min
1. After
perfusion, THALs were equilibrated for 20 min alone or in the presence
of valinomycin (Sigma), and four measurements corresponding to the
basal absorption rates were made. SPM (10 µM) was then added to the
bath, and after a 20-min reequilibration period, four additional
collections were made. The concentration of valinomycin used was
determined as the maximal concentration that did not induce cell
swelling and allowed THALs to absorb chloride at normal rates (~100
pmol · mm
1 · min
1). Because
we wanted to reverse NO-induced inhibition of Cl
transport with valinomycin, the lowest concentration of SPM (10 µM)
that produced a maximal response was used. Cl
concentration in the perfusate and collected fluid was measured by
microfluorometry (11). All data were recorded and stored by using data-acquisition software (DATAQ instruments, Akron, OH). Data
analysis was performed with newly developed software specifically
designed for voltage-spike analysis. Because there is no water
reabsorption in the THAL, JCl
was calculated as follows
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Oxygen consumption measurements.
Suspensions of medullary THALs (mTHAL) were prepared according to a
modified protocol as described by Chamberlin et al. (7). Briefly, kidneys were perfused via retrograde perfusion of the aorta
with solution A (Table 1) containing 0.1% collagenase
(Sigma) and 100 U heparin. The inner stripe of the outer
medulla was cut from coronal slices of the kidney, minced, and
incubated at 37°C for 30 min in 0.1% collagenase. The tissue was
pelleted via 114-g centrifugation, resuspended in cold
solution, and stirred on ice for 30 min to release the tubules. The
suspension was filtered through 250-µm nylon mesh and centrifuged at
114 g. The pellet was washed, centrifuged again, and finally
resuspended in 0.1 ml of cold solution A (Table 1). The
mTHAL suspension was warmed to 37°C and equilibrated with 95%
O2-5% CO2. It was then added to a closed
chamber maintained at 37°C and oxygen consumption rate
(QO2) was recorded continuously. An initial
constant slope was established at the beginning of each experiment, and
SPM or furosemide (104 M) was then added. SPM was
prepared daily before use. A constant QO2 was
established before nystatin (135 U/ml) was added. All experiments were
completed within 15 min. Similar results were obtained with 10 and 100 µM SPM, indicating that 10 µM yields a maximal response. An aliquot
of the suspension was used to determine protein concentration using
Coomassie protein assay reagent (Pierce, Rockford, IL).
Statistics. Results are expressed as means ± SE. Data were evaluated with Student's paired t-test. P < 0.05 was considered significant.
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RESULTS |
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We have previously reported that NO inhibits THAL
JCl (31). A decrease
in JCl
could be due to a decrease
in apical ion entry or inhibition of basolateral ion exit. We first
tested whether NO decreases ion entry by measuring [Na+]i in the THAL. In isolated, perfused
THALs, addition of the NO donor SPM (100 µM) to the bath decreased
THAL [Na+]i from 13.5 ± 1.2 to 9.6 ± 1.6 mM, a reduction of 28.9 ± 10.0% (P < 0.05; n = 6) (Fig. 1). These
data indicate that NO decreases Na+ entry in the THAL.
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In this nephron segment, Na+ and Cl
entry are coupled through the
Na+-K+-2Cl
cotransporter;
therefore, we tested whether NO could also decrease [Cl
]i. In isolated, perfused THALs,
addition of SPM (100 µM) to the bath increased SPQ fluorescence by
19.3 ± 4.1% (P < 0.01, n = 6) (Fig.
2), indicative of a decrease in
[Cl
]i. These data indicate that NO
decreases Cl
entry in the THAL.
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Because NO addition decreased both THAL
[Na+]i and [Cl]i,
the effect of NO on THAL JCl
is
most likely due to inhibition of ion entry through
Na+-K+-2Cl
cotransporters. Thus
we tested whether NO could decrease
Na+-K+-2Cl
cotransporter activity
by measuring the initial rate of Na+ entry through the
cotransporter in the absence and presence of SPM. Initial rates were
measured by monitoring the increase in THAL
[Na+]i caused by a switch in luminal NaCl
concentration from 0 to 130 mM (Fig.
3A). Under control conditions,
increasing luminal NaCl from 0 to 130 mM increased
[Na+]i at an initial rate of 6.99 ± 1.84 × 10
3 arbitrary units (au)/s. In the same
tubules and in the presence of 100 µM SPM, increasing luminal NaCl
from 0 to 130 mM increased [Na+]i at an
initial rate of 3.19 ± 0.89 × 10
3 au/s,
49.5 ± 6.0% slower than the control period initial rates (Fig.
3B) (n = 5; P < 0.05).
Control experiments show no significant change in initial rates between
the two periods of measurement (7.00 ± 1.73 vs. 6.73 ± 0.73 × 10
3 au/s; n = 4; not
significant). The increase in [Na+]i caused
by a switch in luminal NaCl was completely inhibited by the presence of
furosemide in the luminal perfusion solution. These data indicate that
NO decreases Na+-K+-2Cl
cotransporter activity.
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Na+-K+-2Cl cotransporter activity
could be inhibited either directly or by a decrease in apical
K+ permeability (15, 32). Therefore, we tested
whether NO inhibits K+ channel activity by measuring the
change in apical membrane potential caused by an increase in luminal
K+ from 1 to 25 mM in the absence and presence of NO. Under
control conditions, increasing luminal K+ from 1 to 25 mM
decreased the fluorescence of JC-1 by 20 ± 1%, indicative of
membrane depolarization. In the same tubules, in the presence of 100 µM SPM, increasing luminal K+ from 1 to 25 mM decreased
the fluorescence of JC-1 by 18 ± 1% (data not shown). The same
results were obtained in control experiments in the absence of SPM.
These data indicate that NO does not decrease THAL apical
K+ permeability.
To ensure that a change in apical K+ conductance does
not alter NO effects in the THAL, we tested whether NO inhibits THAL JCl in the presence of an
artificially induced high apical K+ permeability. An
increase in apical K+ permeability was achieved by
perfusing THALs with a solution containing the K+ ionophore
valinomycin. In the absence of valinomycin, addition of 10 µM SPM to
the bath decreased THAL JCl
by
46.0 ± 6.7% (from 99.6 ± 14.7 to 52.3 ± 16.9 pmol · mm
1 · min
1;
P < 0.05; n = 4). In the presence of
5 × 10
5 M valinomycin in the lumen, addition of 10 µM SPM to the bath decreased THAL
JCl
by 41.2 ± 10.4% (from
113.0 ± 12.3 to 68.2 ± 15.2 pmol · mm
1 · min
1;
P < 0.05; n = 5) (Fig.
4). Taken together, these results
indicate that NO inhibits chloride entry even when apical
K+ permeability is greatly increased.
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To ensure that NO does not alter THAL sodium pump activity, we
investigated the effects of NO on Na+-K+-ATPase
activity. We first tested whether NO affects the affinity constant
(K1/2) of
Na+-K+-ATPase for Na+ in the THAL
by measuring the effects of nystatin on QO2 by
THALs in the presence of 10.5 mM extracellular Na+ (a
concentration similar to basal intracellular Na+, and the
reported K1/2 for Na+). As shown in
Fig. 5, 100 µM SPM decreased
QO2 from 87.2 ± 10.3 to 68.8 ± 8.9 nmol O2 · mg
protein1 · min
1, a 22 ± 2%
decrease (P < 0.01, n = 5). Similarly,
inhibiting Na+-K+-2Cl
cotransport
with 10
4 M furosemide reduced QO2
by 25 ± 4% (P < 0.01, n = 5).
In the presence of SPM, addition of nystatin (135 U/ml), which
equilibrates intra- and extracellular Na+, restored
QO2 to control values (81.3 ± 7 nmol
O2 · mg
protein
1 · min
1). Similarly,
nystatin returned QO2 to control levels in
furosemide-treated tubules (n = 5). Similar results were
obtained when 10 µM SPM was used (n = 5). The finding
that nystatin restores QO2 to control levels in
both SPM- and furosemide-treated tubules indicates that NO does not
affect the K1/2 of
Na+-K+-ATPase for intracellular
Na+.
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NO could also affect Na+-K+-ATPase by
decreasing its maximum turnover or by decreasing ATP concentration. To
test this, we measured the effect of nystatin on
QO2 by THALs in the presence of 145 mM
extracellular Na+ (normal extracellular Na+).
Under these conditions, addition of SPM reduced THAL
QO2 by 22 ± 2% (P < 0.01, n = 5) (Fig. 6).
Similarly, inhibiting the Na+-K+-2Cl cotransporter with
10
4 M furosemide reduced THAL QO2
by 25 ± 2%. In the presence of SPM, the addition of nystatin
(135 U/ml) increased THAL QO2 by 104 ± 7% (P < 0.05, n = 5). Similarly, nystatin
increased QO2 by 94 ± 20% in
furosemide-treated THALs (P < 0.05, n = 5).
Taken together, these results indicate that NO does not affect
Na+-K+- ATPase maximal rate nor its
K1/2 for [Na+]i.
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DISCUSSION |
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We found that the NO donor SPM decreased
[Na+]i and [Cl]i
in the THAL and inhibited
Na+-K+-2Cl
cotransporter
activity. Changing luminal K+ concentration from 1 to 25 mM
depolarized THALs to the same extent in the absence or presence of SPM,
indicating that NO does not decrease apical K+
permeability. In the presence of valinomycin in the lumen, addition of
SPM to the bath decreased THAL net
JCl
to the same extent as it did
in control tubules, indicating that NO decreases THAL
JCl
even when apical
K+ permeability is increased. Finally, we found no effect
of NO on the affinity of Na+-K+-ATPase for
Na+ nor the maximal rate. Taken together, our data show
that NO decreases JCl
by
inhibiting apical Na+-K+-2Cl
cotransporter activity rather than by inhibiting apical K+
channels or basolateral Na+-K+-ATPase.
We have previously reported that NO inhibits THAL net
JCl (31). A decrease
in JCl
may be caused by a decrease
in ion entry through the apical membrane or by inhibition of ion exit
across the basolateral membrane. To test whether NO inhibits ion entry
rather than exit, we measured the effect of NO on
[Na+]i and [Cl
]i.
We found that NO decreases THAL [Na+]i and
[Cl
]i. A decrease in
[Na+]i could be caused by inhibition of
Na+ entry or by increasing Na+ exit through
Na+-K+-ATPase. Because we found that NO does
not affect sodium pump activity, a decrease in
[Na+]i can only be caused by inhibiting
apical Na+ entry. Because there are two modes of
Na+ entry in the THAL, the
Na+-K+-2Cl
cotransporter and the
Na+/H+ exchanger, we measured
[Cl
]i. [Cl
]i in
the THAL is determined by the balance between apical entry through the
Na+-K+-2Cl
cotransporter and
basolateral exit through Cl
channels (16). A
decrease in [Cl
]i could be caused by
decreasing Cl
entry through the
Na+-K+-2Cl
cotransporter or by
increasing basolateral Cl
exit. Because we have
previously shown that NO inhibits net
JCl
in the THAL, a decrease in
[Cl
]i is most likely mediated by a decrease
in apical Cl
entry through the
Na+-K+-2Cl
cotransporter, and as
NO decreased [Cl
]i, our data also suggest
that NO does not affect basolateral Cl
channels.
Because we found that NO decreased apical ion entry, we tested whether
NO inhibits Na+-K+-2Cl
cotransporter activity. By measuring [Na+]i,
we determined the initial rates of Na+ transport through
the Na+-K+-2Cl
cotransporter in isolated, perfused THALs. We have found that in
the presence of SPM, initial rates were 49% slower than under control
conditions. These data indicate that NO decreases
Na+-K+-2Cl
cotransporter activity.
It has been reported that inhibition of apical K+ channels
with Ba2+ inhibits THAL
JCl (17). Thus a
decrease in cotransporter activity may be secondary to a decrease in
apical K+ channel activity. The apical membrane of the THAL
possesses a high K+ conductance, provided by the presence
of at least two types of K+ channels (35).
Therefore, we tested whether NO decreases apical K+
permeability by measuring the change in membrane potential caused by an
increase in luminal K+ from 1 to 25 mM. Due to the high
K+ permeability compared with that of other ions
(9), a change in voltage caused by a switch in luminal
K+ can be used to estimate apical K+
permeability. Our results show that the depolarization caused by
increasing luminal K+ was the same in the absence and
presence of NO, indicating that NO does not inhibit apical
K+ permeability.
To ensure that a change in apical K+ permeability does not
alter the effects of NO, we tested whether NO could inhibit THAL JCl when apical K+
permeability was artificially increased. We found that, in the presence
of valinomycin in the tubular lumen, NO decreases THAL JCl
to the same extent as it did
in control cells. These data indicate that NO inhibits
JCl
even when apical
K+ permeability is increased and supports the hypothesis of
a direct inhibitory effect of NO on the
Na+-K+-2Cl
cotransporter.
Contrary to our results, a stimulatory effect of NO on the THAL apical 70-pS K+ channel has been observed in cell-attached patch-clamp experiments (25). Although we have no explanation for these disparate results, this discrepancy is not due to a lack of sensitivity in our measurements because we are able to detect changes in K+ permeability as small as 10%. It is possible that differences in rat age or diet, which are known to influence the hormonal state of the animals, may account for differences in the response to NO.
Na+-K+-2Cl cotransporter activity
has been shown to be regulated by different mechanisms (19,
32). However, the molecular mechanism through which NO can
inhibit the Na+-K+-2Cl
cotransporter is still unknown. We have recently reported that the
mechanism through which NO inhibits THAL
JCl
is mediated by activation of
cGMP-stimulated phosphodiesterase, which, in turn, decreases basal cAMP
levels (30). Several studies indicate that stimulation of
the Na+-K+-2Cl
cotransporter by
hormones that increase cAMP is mediated by protein kinase A activation
(26, 27). Therefore, it is possible that NO-induced
inhibition of the THAL Na+-K+-2Cl
cotransporter may be mediated by a decrease in protein kinase A
activity. However, further studies are needed to define the exact
molecular effector that mediates NO-induced inhibition of Na+-K+-2Cl
cotransport.
Finally, to ensure that NO does not affect the sodium pump, we investigated whether NO inhibits Na+-K+-ATPase. We first tested whether NO could alter the affinity of Na+-K+-ATPase for Na+. The K1/2 of Na+-K+-ATPase for Na+ has been reported to be 10-15 mM (10). Therefore, we investigated the effect of nystatin on THAL QO2 in the presence of 10.5 mM extracellular Na+ after treatment with SPM. We found that, in the presence of either SPM or furosemide, nystatin restored QO2 to control values, indicating that NO does not affect the K1/2 of Na+-K+-ATPase for Na+. We then investigated whether NO could affect the maximal turnover of Na+-K+-ATPase or ATP production by measuring the effect of nystatin on THAL QO2 in the presence of 145 mM extracellular Na+ (normal extracellular Na+). Under these conditions, nystatin increases intracellular Na+ and generates maximal activation of Na+-K+-ATPase. We found that, in the presence of either SPM or furosemide, nystatin increased QO2 by 100%. Taken together, these data indicate that NO does not affect the K1/2 of Na+-K+- ATPase for intracellular Na+ nor maximal Na+-K+-ATPase turnover.
In agreement with the present results, we have previously reported that neither SPM nor nitroglycerin affects Na+-K+-ATPase activity in isolated cortical collecting ducts (34). However, other investigators have found different effects of NO on Na+-K+-ATPase activity. Liang and Knox (23, 24) reported that NO caused a decrease in the activity of Na+-K+-ATPase in a proximal tubule-like cell type. Guzman et al. (18) showed that the NO donor sodium nitroprusside and the peroxynitrite donor 3-morpholinosydnonimine inhibited Na+-K+-ATPase activity in cultured proximal tubule cells. A possible explanation for these disparate results could be given by the fact that a significant decrease in Na+-K+-ATPase activity is only observed after prolonged exposure to NO (1-2 h) in those studies. We found a decrease in ion entry 15 min after treatment with SPM. In addition, due to the presence of different protein kinases and protein phosphatases, the effect of NO on Na+-K+-ATPase activity could be different in the proximal compared with the distal nephron.
We have found that, in the presence of SPM, nystatin increased QO2 in tubules bathed with 145 mM Na+. These data indicate that NO by itself does not affect mitochondrial respiration, nor does it cause a decrease in ATP that is sufficient to prevent maximal Na+-K+-ATPase turnover. In agreement with these results, we have previously found that NO does not affect ATP production in cultured cortical collecting duct cells (33). However, it has been reported that NO can reversibly inhibit mitochondrial respiration in different cell types (3) and, in some cases, decrease cellular ATP concentration (1). Although we have no explanation for these differences, several factors, such as oxygen tension (4), light (2), and metabolic substrates, are known to influence the effect of NO on mitochondrial respiration. Therefore, it is possible that at the concentration of SPM we used, and in the presence of a high oxygen concentration, NO did not produce a direct effect on mitochondrial respiration.
We conclude that NO inhibits THAL
JCl by reducing apical
Na+-K+-2Cl
cotransporter activity
rather than by inhibiting apical K+ channels or basolateral
Na+-K+-ATPase.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-28982.
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FOOTNOTES |
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First published August 9, 2001;10.1152/ajprenal.0075.2001
Address for reprint requests and other correspondence: J. L. Garvin, Div. of Hypertension and Vascular Research, Dept. of Internal Medicine, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202 (E-mail: jgarvin1{at}hfhs.org).
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 8 March 2001; accepted in final form 6 July 2001.
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