Ca2+ mediates the effect of inhibition
of Na+-K+-ATPase on the
basolateral K+ channels in the rat
CCD
Yuan
Wei,
Ming
Lu, and
WenHui
Wang
Department of Pharmacology, New York Medical College, Valhalla, New
York 10595
 |
ABSTRACT |
We investigated the effect of
inhibiting Na+-K+-ATPase on the basolateral
18-pS K+ channel in the cortical collecting duct (CCD) of
the rat kidney. Inhibiting Na+-K+-ATPase with
strophanthidin decreased the activity of the 18-pS K+
channel and increased the intracellular Ca2+ to 420 nM.
Removal of extracellular Ca2+ abolished the effect of
strophanthidin. When intracellular Ca2+ was raised with 5 µM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the
18-pS K+ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism of
Ca2+-induced inhibition, the effect of 400 nM
Ca2+ on channel activity was studied in the presence of
calphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62,
inhibitors of calmodulin-dependent kinase II. Addition of calphostin C
or KN-93 or KN-62 failed to block the inhibitory effect of high
concentrations of Ca2+. This suggested that the inhibitory
effect of high concentrations of Ca2+ was not mediated by
protein kinase C or calmodulin-dependent kinase II pathways. To examine
the possibility that the inhibitory effect of high concentrations of
Ca2+ was mediated by the interaction of nitric oxide with
superoxide, we investigated the effect of 400 nM Ca2+ on
channel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or
N
-nitro-L-arginine methyl ester.
Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic
acid or N
-nitro-L-arginine methyl
ester completely abolished the inhibitory effect of 400 nM
Ca2+ on channel activity. Moreover, application of
4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effect
of strophanthidin. We conclude that the effect of inhibiting
Na+-K+-ATPase is mediated by intracellular
Ca2+ and the inhibitory effect of high concentrations of
Ca2+ is the result of interaction of nitric oxide with superoxide.
superoxide; peroxynitrite; protein kinase C; calmodulin-dependent
kinase; cortical collecting duct
 |
INTRODUCTION |
IT IS WELL
ESTABLISHED that the basolateral K+ conductance is
closely related to the activity of
Na+-K+- ATPase: an increase in the turnover
rate of the Na+-K+-ATPase augments, whereas a
decrease reduces, the basolateral K+ conductance (9,
12, 13, 25, 26). The mechanism by which the basolateral
K+ conductance is linked to the activity of the
Na+-K+-ATPase is not completely understood. In
the proximal tubule, ATP has been suggested to link the basolateral
K+ conductance to the activity of
Na+-K+-ATPase (1, 25). Stimulation
of the Na+-K+-ATPase tends to decrease the
intracellular ATP concentration, which in turn activates the
basolateral ATP-sensitive K+ channel (1). On
the other hand, inhibition of Na+-K+-ATPase
should increase ATP concentration, which could inhibit the channel
activity (13). However, this mechanism is unlikely to be a
mediator in the cortical collecting duct (CCD), since the basolateral
K+ channels are not sensitive to ATP (30).
Inhibition of Na+-K+-ATPase has been
demonstrated to increase intracellular Ca2+
(28). Moreover, an increase in intracellular
Ca2+ is responsible for coupling the activity of the apical
K+ channels with that of
Na+-K+-ATPase (28). However, it is
not clear whether Ca2+ is also responsible for linking the
activity of Na+-K+-ATPase to that of the
basolateral K+ channels.
Three types of basolateral K+ channels have been found in
the basolateral membrane of the CCD (10, 30). When the
channel conductance was measured in cell-attached patches with 140 mM KCl in the pipette and NaCl Ringer solution in the bath, conductance of
the three types of K+ channels was 145, 85, and 28 pS,
respectively. When the channel conductance was measured in inside-out
patches with symmetrical 140 mM KCl in the bath as well as in the
pipette, conductance of the three K+ channels was 85, 28, and 18 pS, respectively. Because the 18-pS K+ channel was
predominant in the CCD from rats fed a high-K+ diet, we
focused our study on the 18-pS K+ channel. The main purpose
of the present study is to explore whether Ca2+ is
responsible for linking the turnover rate of
Na+-K+-ATPase to the activity of the
basolateral 18-pS K+ channels as well as to identify the
Ca2+-dependent signal transduction pathways responsible for
the coupling.
 |
METHODS |
Preparation of CCDs.
Pathogen-free Sprague-Dawley rats of both sexes (6 wk old) were
purchased from Taconic Farms (Germantown, NY). They were placed on a
high-K+ (10%, wt/wt) diet (Harlan Teklad, Madison, WI) for
7 days before use. The reason for maintaining animals on a
high-K+ diet is that the basolateral membrane of principal
cells from animals fed a high-K+ diet is easier to patch
than that from animals fed a normal chow diet, because the area of the
lateral membrane increases. The animals used for experiments weighed
between 100 and 120 g. Rats were killed by cervical dislocation,
and kidneys were removed immediately. Several thin slices of the kidney
(<1 mm) were cut and placed in ice-cold Ringer solution until
dissection. The dissection was carried out at room temperature, and two
watchmakers' forceps were used to isolate the single CCD. To
immobilize the tubules, they were placed onto a 5 × 5 mm cover
glass coated with Cell-Tak (Becton-Dickinson, Bedford, MA). The cover
glass was transferred to a chamber (1,000 µl) mounted on an inverted
Nikon microscope. The CCDs were superfused with HEPES-buffered NaCl
solution, and the temperature of the chamber was maintained at 37 ± 1°C by circulating warm water surrounding the chamber. We followed
the methods described previously to prepare the basolateral membrane
for patch-clamp experiments (30).
Patch-clamp technique.
An Axon 200A patch-clamp amplifier was used to record channel current.
The current was low-pass filtered at 1 kHz using an eight-pole Bessel
filter (model 902LPF, Frequency Devices, Haverhill, MA) and digitized
at a sampling rate of 44 kHz using a VR-10B digital data recorder and
stored on videotape (model FX600, Hitachi). For analysis, data stored
on the tape were collected to an IBM-compatible Pentium computer
(Gateway 2000) at a rate of 4 kHz and analyzed using the pClamp
software system 6.04 (Axon Instruments, Burlingame, CA). Channel
activity was defined as open channel probability (NPo), which was calculated from data samples of
60-s duration in the steady state as follows
|
(1)
|
where ti is the fractional open time
spent at each of the current levels.
Measurement of intracellular
Ca2+.
The intracellular Ca2+ was measured with fura 2-AM
(Molecular Probes, Eugene, OR). Fluorescence was imaged digitally with
an intensified video imaging system including an SIT 68 camera,
controller, and HR 1000 video monitor. The exciting and emitted light
passed through a ×40 fluorite objective (NA 1.30; Nikon, Melville,
NY). We followed the method published previously to measure and
calculate the intracellular Ca2+ (19). We used
ionomycin or A-23187 (5 µM) to clamp the intracellular Ca2+ concentrations by changing the bath solution to a
medium containing 100, 200, 300, 400, and 500 nM free Ca2+.
Figure 1 is a representative trace to
demonstrate changes in intracellular Ca2+ induced by
raising extracellular Ca2+ from 100 to 400 nM in the
presence of 5 µM ionomycin. The intracellular Ca2+ was
measured every week or when new bath solutions were made. The variation
among measurements was <10%.

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Fig. 1.
Changes in intracellular Ca2+ when
extracellular Ca2+ was raised from 100 to 500 nM in the
presence of 5 µM ionomycin.
|
|
Experimental solution and statistics.
The pipette solution contained (in mM) 140 KCl, 1.8 MgCl2,
and 10 HEPES (pH 7.4). The bath solution for cell-attached patches was
composed of (in mM) 140 NaCl, 5 KCl, 1.8 CaCl2, 1.8 MgCl2, and 10 HEPES (pH 7.4). When we clamped the
intracellular Ca2+ concentrations, we switched the bath
solution to a medium containing corresponding low Ca2+
concentrations. The composition of the bath solution for inside-out patches was the same as that for cell-attached patches, except free
Ca2+ was reduced to 100 nM. Ionomycin, strophanthidin,
A-23187, 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), and
N
-nitro-L-arginine methyl ester
(L-NAME) were purchased from Sigma Chemical (St. Louis,
MO). Values are means ± SE, and the paired Student's
t-test was used to calculate the significance between the
control and experimental groups. Statistical significance was taken as
P < 0.05.
 |
RESULTS |
Figure 2 is a representative
recording showing the effect of strophanthidin, an inhibitor of
Na+-K+- ATPase, on the activity of the
basolateral 18-pS K+ channel. It is apparent that addition
of 100 µM strophanthidin decreased channel activity by 90 ± 10% (n = 4). We previously demonstrated that
inhibition of Na+-K+-ATPase raised
intracellular Ca2+ (28). This is further
confirmed by the present study, in which application of strophanthidin
increased the intracellular Ca2+ from the control value
(85 ± 8 nM) to 420 ± 50 nM (n = 4). To explore the role of Ca2+ in mediating the effect of
inhibiting Na+-K+-ATPase, we examined the
effect of raising Ca2+ on channel activity. Ionomycin or
A-23187 was used to clamp the intracellular Ca2+ to 100, 200, 300, 400, and 500 nM.

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Fig. 2.
Effect of 100 µM strophanthidin on the activity of the
basolateral 18-pS K+ channel. The experiment was carried
out in a cell-attached patch, and the holding potential was 0 mV.
Top trace, time course of the experiment; traces 1 and 2, data from the top trace extended at a
fast time resolution. C, channel closed level.
|
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Figure 3 shows the effect on the 18-pS
K+ channel of raising extracellular Ca2+ from
100 to 400 nM in the presence of 5 µM A-23187 or ionomycin in a
cell-attached patch. It is clear that 400 nM Ca2+ inhibited
the activity of the 18-pS K+ channel within 5 min. The
effect of high concentrations of Ca2+ was reversible,
because the channel activity was completely restored when
Ca2+ concentration returned to 100 nM. Figure
4 is a dose-response curve showing the
relationship between Ca2+ concentration and channel
activity. Raising Ca2+ concentration from 100 nM to 300, 400, and 500 nM reduced the channel activity in cell-attached patches
by 40 ± 4, 85 ± 10, and 96 ± 3% (n = 8), respectively. The notion that the effect of strophanthidin was
mediated by an increase in Ca2+ influx was further
supported by experiments in which removal of extracellular
Ca2+ abolished the effect of strophanthidin on channel
activity. Figure 5 is a representative
recording showing that inhibiting Na+-K+-ATPase
did not significantly decrease NPo (94 ± 5% of the control value, n = 4) when extracellular
Ca2+ decreased to 100 nM.

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Fig. 3.
Effect of raising Ca2+ on channel activity. The
experiment was performed in a cell-attached patch. At diagonal arrow,
cell Ca2+ was raised from 100 to 400 nM. Traces
1-4, data from the top trace extended at a fast
time resolution.
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Fig. 4.
Dose-response curve of the Ca2+-induced
inhibition of channel activity. Experiments are performed in
cell-attached patches (n = 9).
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Fig. 5.
Effect of strophanthidin on channel activity when the extracellular
Ca2+ was reduced to 100 nM. Top trace, time
course of the experiment; traces 1 and 2, data
from the top trace extended at a fast time resolution.
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After establishing that high concentrations of Ca2+ inhibit
the activity of the basolateral 18-pS K+ channel, we
explored the mechanisms by which Ca2+ inhibits the channel
activity. We previously demonstrated that high concentrations of
Ca2+ inhibited the apical K+ channels by a
protein kinase C (PKC)-dependent mechanism in the CCD
(28). Thus we first examined the role of PKC in mediating the effect of high concentrations of Ca2+. Figure
6 is a representative recording
demonstrating the effect of high concentrations of Ca2+ on
the channel activity in the presence of 100 nM calphostin C. We
confirmed the previous finding that addition of calphostin C decreased
the channel activity (17), and NPo
dropped by 40 ± 4% (n = 6). Moreover, increasing
Ca2+ to 400 nM further reduced the channel activity to
6 ± 1% (n = 6) of the control value. Figure
7 summarizes results from six experiments. It is apparent that the inhibition of PKC failed to
abolish the effect of high concentrations of Ca2+.

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Fig. 6.
Effect of 400 nM Ca2+ on channel activity in the
presence of calphostin C (100 nM). The experiment was carried out in a
cell-attached patch. Top trace, time course of the
experiment. Traces 1-4, data from the top
trace extended at a fast time resolution.
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Fig. 7.
Effect of 400 nM Ca2+ on channel activity in
the presence of calphostin C and KN-62 or KN-93. Experiments were
performed in cell-attached patches.
|
|
Also, we previously showed that calmodulin-dependent kinase II (CaMK
II) was involved in mediating the effect of high concentrations of
Ca2+ on the apical K+ channels in the CCD
(14). Thus we examined the role of CaMK II in mediating
the inhibitory effect of Ca2+ on the basolateral
K+ channels. Figure 7 summarizes the results of seven
experiments in which the effect of 400 nM Ca2+ was tested
in the presence of KN-62 or KN-93, agents that inhibit the CaMK II.
Application of KN-93 or KN-62 had no significant effect on channel
activity. Moreover, inhibition of CaMK II failed to abolish the effect
of 400 nM Ca2+, because the NPo
decreased by 90 ± 10%, a value that is not significantly different from that in the absence of KN-93 or KN-62.
Finally, high concentrations (>100 µM) of nitric oxide (NO) donors
have been shown to inhibit the basolateral 18-pS K+ channel
(18). The inhibitory effect of NO is mediated by forming peroxynitrite (OONO
) through the interaction with
superoxide. This possibility was tested by examining the effect of 400 nM Ca2+ in the presence of L-NAME, an agent
that inhibits NO synthase (NOS). Figure 8
is a typical recording showing the effect of 400 nM Ca2+
after the tubules were treated with 0.2 mM L-NAME for 20 min. From inspection of Fig. 8, it is clear that the inhibitory effect of 400 nM Ca2+ was significantly attenuated, since
NPo decreased modestly from the control value
(1.62 ± 0.2) to 1.45 ± 0.2. In the presence of
L-NAME, 400 nM Ca2+ had no significant effect
on channel activity (94 ± 8% of the control, n = 9). The notion that the inhibitory effect of high concentrations of
Ca2+ results from the interaction of NO with superoxide was
further confirmed by experiments in which the effect of
Ca2+ was examined in the presence of Tiron, a scavenger of
superoxide (8). Figure 9 is
a representative recording showing the effect of raising intracellular
Ca2+ on channel activity [from control (0.88 ± 0.1)
to 1.02 ± 0.1] in the CCDs that were pretreated with 10 mM
Tiron. In the presence of Tiron, application of high concentrations of
Ca2+ resulted in a slight increase in
NPo (115 ± 10% of the control value,
n = 7).

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Fig. 8.
Effect of 400 nM Ca2+ on channel activity in the
cortical collecting ducts (CCDs) pretreated with 0.2 mM
N -nitro-L-arginine methyl ester
(L-NAME). The experiment was performed in a cell-attached
patch, and the holding potential was 0 mV. Traces 1 and
2, data from the top trace extended at a fast
time resolution.
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Fig. 9.
Effect of 400 nM Ca2+ on channel activity in the CCDs
pretreated with 10 mM 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron).
Top trace, time course of the experiment; traces
1 and 2, data from the top trace extended at
a fast time resolution.
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|
That interaction between NO and superoxide is responsible for the
effect of Na+-K+-ATPase on the basolateral
K+ channels is also supported by observations that Tiron
could reverse the inhibitory effect of strophanthidin. Figure
10 is a continuous recording as shown
in Fig. 2 and demonstrates the effect of Tiron on channel activity in
the presence of strophanthidin. In the absence of Tiron, strophanthidin
reduced NPo by 90 ± 10%
(n = 4). Addition of 10 mM Tiron not only reversed the
strophanthidin-induced inhibition but also increased the channel
activity slightly (120 ± 10% of the control value,
n = 3).

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Fig. 10.
Effect of 10 mM Tiron on strophanthidin-induced inhibition of
channel activity. Continuous recording from Fig. 2 is shown. The
experiment was performed in a cell-attached patch, and the holding
potential was 0 mV. Top trace, time course of the
experiment; traces 1 and 2, data from the
top trace extended at a fast time resolution.
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 |
DISCUSSION |
The basolateral K+ channels serve several important
cell functions in the CCD (7). First, they participate in
generating the cell membrane potential. Because Na+
reabsorption and K+ secretion are electrogenic processes,
alteration in cell membrane potentials can affect Na+
reabsorption and K+ secretion. Second, the basolateral
K+ channels play a key role in K+ recycling
across the basolateral membrane. Third, the basolateral K+
channels could provide the second route for K+ entering the
cell across the basolateral membrane when the cell membrane potential
exceeds the K+ equilibrium potential. The regulation of the
basolateral K+ channels has been extensively studied
(29), and several studies have demonstrated that
cGMP-dependent kinase stimulates the three types of basolateral
K+ channels (11, 27). We further observed that
the activity of the basolateral 18-pS K+ channel increased
significantly by raising intracellular Ca2+ from 10 to 100 nM (19). The stimulatory effect of Ca2+ is
mediated by a cGMP-dependent protein kinase, because addition of cGMP
mimics the stimulatory effect of Ca2+ (27).
In the present study, we have shown that an increase in intracellular
Ca2+ can also inhibit the activity of the 18-pS
K+ channels in the CCD. Thus the effect of intracellular
Ca2+ on the basolateral 18-pS K+ channels is
biphasic: low concentrations (<100 nM) stimulate, while high
concentrations (>200 nM) inhibit, the channel activity. Because the
inhibitory effect of Ca2+ was absent in the presence of
L-NAME or Tiron, this excluded the possibility that
Ca2+ directly blocked the 18-pS K+ channel.
Thus it is most likely that the inhibitory effect of Ca2+
on channel activity is mediated by a Ca2+-dependent signal
transduction pathway. Moreover, we previously demonstrated that NO has
dual effects on the 18-pS K+ channel: low concentrations of
NO stimulated the 18-pS K+ channel via a protein kinase
G-dependent pathway (16), while high concentrations of NO
inhibited the 18-pS K+ channel by the interaction with
superoxide (18). Because NO production is stimulated by
high concentrations of Ca2+ (2), it is
conceivable that the effect of inhibiting
Na+-K+-ATPase on the basolateral K+
channels is mediated by Ca2+ NO signaling.
The presence of three Ca2+-dependent signal transduction
pathways, PKC, CaMK II, and NOS, has been reported in the CCD
(14, 19, 28). Moreover, PKC and CaMK II have been shown to
inhibit the apical secretory K+ channels (14,
28). However, it is unlikely that the inhibitory effect of
Ca2+ on the basolateral K+ channel is mediated
by PKC, because inhibition of PKC failed to abolish the effect of
Ca2+. Moreover, we confirmed the previous finding that
inhibiting PKC decreased the activity of the basolateral 18-pS
K+ channels (17). The effect of inhibiting PKC
is most likely the result of decreasing NO production, since addition
of exogenous NO donors reversed the effect of the PKC inhibitor
(17). Stimulation of PKC has been shown to increase the
activity of neuronal NOS (nNOS) (20) and to raise the
intracellular cGMP concentration (21). Also, observations
that addition of specific inhibitors of CaMK II did not block the
effect of 400 nM Ca2+ on channel activity excluded the
possibility that CaMK II was responsible for mediating the effect of
high concentrations of Ca2+ on the 18-pS K+
channels. Thus it is apparent that the regulation of basolateral K+ channels is different from that of the apical
K+ channels.
Two lines of evidence indicate that the inhibitory effect of high
concentrations of Ca2+ is mediated by an NO-dependent
pathway. First, we previously demonstrated that high concentrations of
NO blocked the basolateral K+ channels (18).
Second, pretreatment of the tubules with L-NAME abolished
the effect of 400 nM Ca2+ on channel activity. This
indicates that an increase in NO release is involved in mediating the
Ca2+-induced inhibition. It is well established that high
concentrations of NO could interact with superoxide to form
OONO
(22, 32). We showed previously that
OONO
is responsible for mediating the inhibitory effect
of NO (18), because addition of exogenous superoxide
donors reversed a stimulatory effect of NO to an inhibitory effect
(18). It is possible that an increase in intracellular
Ca2+ stimulates the activity of NOS and augments NO
formation, which in turn interacts with superoxide to form
OONO
and inhibits the channel activity. This notion is
supported by the observation that removal of superoxide with Tiron not
only completely abolished the inhibitory effect of high concentrations of Ca2+ but also slightly increased the channel activity.
Thus the interaction between NO and superoxide is responsible for
mediating the inhibitory effect of high concentrations of
Ca2+.
We previously reported that nNOS is expressed in principal cells of the
CCD (31). Because nNOS activity is stimulated by Ca2+, an increase in the intracellular Ca2+ is
expected to enhance the NO release. Thus it is conceivable that the
biphasic effect of Ca2+ is the result of a biphasic effect
of NO. OONO
is a highly active oxidant that reacts with a
variety of molecules (2). It is believed that
OONO
is responsible for mediating NO-induced cell injury
(33). In addition, OONO
has also been
suggested to play an important role in signal transduction mechanisms
to modulate a variety of cell functions (15, 24). Several
studies have suggested that OONO
modulates the
Ca2+-activated K+ channel in smooth muscle
cells (3, 4), L-type Ca2+ channels in myocytes
(6), and minK channels through thiol nitrosylation
(5).
Ca2+ has been shown to play a key role in linking the
activity of the apical K+ channels to the turnover rate of
the basolateral Na+-K+-ATPase. In the present
study, we have demonstrated that the intracellular Ca2+ is
also responsible for coupling the activity of the basolateral K+ channels to the Na+-K+-ATPase.
However, the mechanism by which raising Ca2+ inhibits the
basolateral K+ channels is different from the mechanism
that blocks the apical K+ channels. The different
regulatory mechanisms by Ca2+ between the apical and
basolateral K+ channels may be essential for achieving the
cell function in the CCD.
The Ca2+-induced inhibition of the basolateral
K+ channels may play a key role in maintaining a constant
intracellular K+ concentration. It has been demonstrated
that losing intracellular K+ is closely related to the cell
death induced by ischemia, because the inhibition of
K+ channels diminished the cell injury (23).
Ischemia is expected to inhibit the
Na+-K+-ATPase and raise the intracellular
Ca2+. An increase in intracellular Ca2+
stimulates Ca2+-dependent PKC and CaMK II, which block the
apical K+ channels (14, 28) and increase NO
release. On the other hand, ischemia also increases the
superoxide production (32). Accordingly, the interaction
between NO and superoxide is enhanced and results in an inhibition of
the basolateral K+ channels. Our studies have suggested
that NO plays a key role in linking the basolateral K+
conductance to the apical Na+ transport (19)
and turnover rate of Na+-K+-ATPase. An increase
in apical Na+ transport should raise intracellular
Ca2+, which enhances NO release and stimulates the
basolateral K+ conductance by a cGMP-dependent pathway. On
the other hand, a large increase in intracellular Ca2+
should decrease the basolateral K+ conductance by
interaction between NO and superoxide. Figure 11 depicts the mechanism by which
strophanthidin inhibits the basolateral 18-pS K+ channel.
Inhibition of Na+-K+-ATPase increases
intracellular Na+ concentration and diminishes the driving
force for Ca2+/Na+ exchanger. Accordingly, an
increase in intracellular Ca2+ stimulates NO formation,
which interacts with superoxide to form OONO
and blocks
the channel activity.

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Fig. 11.
Cell model illustrating the mechanism by which
inhibiting Na+-K+-ATPase decreases the
basolateral K+ conductance. NOS, nitric oxide synthase;
OONO , peroxynitrite.
|
|
We conclude that inhibiting Na+-K+-ATPase
decreased the basolateral 18-pS K+ channel in the CCD by a
Ca2+-dependent mechanism. An increase in intracellular
Ca2+ has a biphasic effect on channel activity: low
concentrations of Ca2+ stimulate, while high concentrations
of Ca2+ inhibit, the channel activity. The inhibitory
effect of high concentrations of Ca2+ is the result of
interaction between NO and superoxide.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health
Grants DK-47402 and P01 HL-34300.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: W.-H.
Wang, Dept. of Pharmacology, New York Medical College, Valhalla, NY
10595 (E-mail: wenhui_wang{at}nymc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 May 2000; accepted in final form 30 October 2000.
 |
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