From the * Department of Pharmacology, New York Medical College, Valhalla, New York 10595; and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport
on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD)
of the rat kidney. Application of 50 µM amiloride decreased the activity of SK, defined as nPo (a product of channel open probability and channel number), to 61% of the control value. Application of 1 µM benzamil, a specific
Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control
value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride
was not the result of a decrease in intracellular pH, since addition 50 µM 5-(n-ethyl-n-isopropyl) amiloride (EIPA),
an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of
amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM
to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular
Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+
from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and
the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP-dependent pathways. Addition of 10 µM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 µM 8-bromoguanosine 3:5
-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had
been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of
inhibiting the apical Na+ transport on the basolateral SK in the rat CCD.
The cortical collecting duct (CCD)1 plays an important
role in Na+ reabsorption and K+ excretion as evidenced by the fact that Na+ reabsorption and K+ secretion are finely regulated and controlled by several hormones, such as aldosterone and vasopressin (Schafer
and Hawk, 1992; Palmer et al., 1993
; Breyer and Ando,
1994
). Na+ reabsorption and K+ secretion are two-step
processes that involve several transport proteins such as
ion channels and Na-K-ATPase (Smith and Benos, 1991
; Palmer et al., 1993
; Giebisch, 1995
). Changes in
channel activity or turnover rate of Na-K-ATPase may
have a profound effect on K+ secretion and Na+ reabsorption in the CCD (Strieter et al., 1992a
, 1992b
).
Three functions are served by basolateral K+ channels.
First, they participate in generating the cell membrane potential. Since K+ secretion and Na+ reabsorption are
electrogenic processes, alteration of cell membrane potential has a significant effect on K+ secretion and Na+
reabsorption. It has been found that inhibition of the
basolateral K+ conductance with Ba2+ reduced the Na+
reabsorption rate (Schafer and Troutman, 1987
). Second, the K+ channels in the basolateral membrane are
involved in K+ recycling across the basolateral membrane (Dawson and Richards, 1990
), this recycling being important for maintaining the function of the Na-K-ATPase. Inhibition of K+ recycling diminished the
short circuit current, an index for active Na+ transport,
in frog skin (Urbach et al., 1996b
). Finally, the basolateral K+ channels provide a route for K+ entering the
cell under conditions, such as hyperaldonism, in which
the cell membrane potential exceeds the K+ equilibrium potential.
Three types of K+ channels, large conductance
(>198 pS), intermediate conductance (85 pS), and
small conductance (28 pS), have been found in the basolateral membrane of the CCD (Hirsch and Schlatter,
1993; Wang et al., 1994
; Wang, 1995
). The 28-pS K+
channel is predominant in the lateral membrane of the
CCD in rats on either a normal or high potassium diet
(Lu and Wang, 1996
). In contrast, the 198-pS K+ channel is predominant in the basolateral membrane of the
CCD in the rats on a low sodium diet (Hirsch and
Schlatter, 1993
). The small-conductance K+ channel
(SK) is activated by nitric oxide via a cGMP-dependent pathway (Lu and Wang, 1996
), but is insensitive to ATP
(Wang et al., 1994
).
It is well established that the basolateral K+ conductance is closely correlated with the activity of the basolateral Na-K-ATPase and Na+ transport across the apical membrane (Horisberger and Giebisch, 1988a, 1988b
;
Harvey, 1995
). Inhibition of the apical Na+ channels with
amiloride reduced the basolateral K+ permeability in
the toad urinary bladder (Davis and Finn, 1982
). Horisberg and Giebisch (1988a) have further shown that inhibition of Na-K-ATPase reduced basolateral K+ conductance. On the other hand, stimulation of Na+ transport has been shown to increase the basolateral K+
conductance (Tsuchiya et al., 1992
; Beck et al., 1993
).
Such "cross talk" between the apical Na+ transport and
the basolateral K+ conductance is important in maintaining salt and water transport and ion concentration
in the intracellular milieu (Schultz, 1981
). The mechanisms of cross talk have been extensively explored and several candidates, including changes in pH, Ca2+, and
ATP, for mediating the feed-back between apical Na+
transport and basolateral K+ channels, have been identified (Harvey, 1995
). In the present study, we investigate the role of NO in linking activity of the basolateral
K+ channels to apical Na+ transport.
Preparation of Rat CCD
The CCDs were isolated from kidneys of pathogen-free Sprague-Dawley rats purchased from Taconic Farms Inc. (Germantown, NY) and the animals kept on either a normal rat chow diet or a high K+ diet. The kidneys were removed immediately after killing and thin coronal sections were cut with a razor blade. The CCD was dissected in HEPES-buffered NaCl Ringer solution containing (mM) 140 NaCl, 5 KCl, 1.5 MgCl2, 1.8 CaCl2, 5 glucose, and 10 HEPES (pH 7.4 with NaOH) at 22°C and transferred onto a 5 × 5 mm cover glass coated with "Cell-Tak" (Collaborative Research Inc., Bedford, MA) to immobilize the tubules. The cover glass was placed in a chamber mounted on an inverted microscope (Nikon Inc., Melville, NY) and the tubules were superfused with HEPES-buffered NaCl solution. The CCD was cut open with a sharpened micropipette and intercalated cells were then removed to expose the lateral membrane of principal cells. The temperature of the chamber (1,000 µl) was maintained at 37 ± 1°C by circulating warm water around the chamber.
Patch-clamp Technique
We used a patch-clamp amplifier (200A; Axon Instruments, Foster City, CA) to record channel current. The current was low pass filtered at 1 kHz using an 8-pole Bessel filter (902LPF; Frequency Devices Inc., Haverhill, MA) and was digitized at a sampling rate of 44 kHz using a modified PCM-501ES pulse code modulator
(Sony Corp., Park Ridge, NJ) and stored on videotape (SL-2700;
Sony Corp.). For analysis, data stored on the tape were transferred to an IBM-compatible 486 computer (Gateway 2000, Sioux
Falls, South Dakota) at a rate of 4 kHz and analyzed using the
pClamp software system 6.03 (Axon Instruments). Channel activity is defined as nPo and no efforts were made to determine
whether alterations of channel activity were due to changes in
channel number (n) or channel open probability (Po). The nPo
was calculated from data samples of 30-60-s duration in the
steady state as follows: nPo = (t1 + t2 +......tn), where tn is the
fractional open time spent at each of the observed current levels.
Measurement of Intracellular Ca2+
Fluorescence was imaged digitally with an intensified video imaging system including a SIT 68 camera, controller, and HR 1000 video monitor (Long Island Industries, North Bellmore, NY). The exciting and emitted light passed through a 40× fluorite objective (NA = 1.30; Nikon Inc.). The microscope was coupled to an alternating wavelength illumination system (Ionoptix, Milton, MA). Digital images were collected at the rate of 10 ratio pairs/ min and analyzed with Ionoptix software (Ionoptix, Milton, MA).
The CCD was loaded with the fluorescent dye Fura-2 AM (5 µM) (Molecular Probes, Inc., Eugene, OR) at room temperature
(22°C) for 30 min. At the end of the incubation period, the tubules were washed with the Ringer solution and transferred to a
new cover glass coated with Cell-Tak. The cover glass was transferred to a chamber and the tubules were incubated for an additional 15 min before experiments. Three to seven cells were selected for each experiment. Dye in the tubule was excited with
light of 340/380-nM wavelengths using a 75-W xenon source, and
emission was recorded at 510 nM. The Ca2+i was measured from
the ratio of fluorescence at excitations of 340/380 nM and calculated using the equation described by Grynkiewicz et al. (1985):
Ca2+i = [(R
Rmin)/(Rmax
R)] × (Fmax/Fmin) × Kd, where R is
the measured ratio of emitted light, Fmax is the fluorescence at
380 nM with 0 mM Ca2+ bath solution, Fmin is the fluorescence at
380 nM with 2 mM Ca2+ bath solution, and Kd = 225 nM for the
Fura-2-calcium binding.
Measurement of Intracellular Na+ Concentrations
The same set-up used for measuring Ca2+ was employed to measure intracellular Na+. The split-open CCD was loaded with the fluorescent dye SBFI-AM (7 µM) and 0.001% pluronic acid (Molecular Probes, Inc.) at room temperature (22°C) for 60 min. At the end of the incubation period, the tubules were washed with the Ringer solution and transferred to a new cover glass coated with Cell-Tak. The cover glass was transferred to a chamber and the tubules were incubated for an additional 15 min before experiments. Three to seven cells were selected for each experiment. Dye in the tubule was excited with light of 340/380 nM wavelengths using a 75-W xenon source, and emission was recorded at 510 nM. Intracellular Na+ was measured from the ratio of fluorescence at excitations of 340/380-nM wavelengths. Fluorescence ratio was calibrated in situ by permeabilizing cells with 10 µM ionophore, lasalocid (Sigma Chemical Co., St. Louis, MO), and altering the Na+ concentration of the bath at the end of each experiment.
Experimental Solution and Statistics
The pipette solution contained (mM): 140 KCl, 1.8 MgCl2, 1 EGTA, and 10 HEPES (pH 7.40 with KOH). The bath solution
for cell-attached patches and for fluorescence measurements was
composed of (mM) 140 NaCl, 5 KCl, 1.8 CaCl2, 1.8 MgCl2, 5 glucose, and 10 HEPES (pH 7.40 with NaOH) under control conditions. The Ca2+-free bath was achieved by removal of Ca2+ and
addition of 1 mM EGTA. To study the effect of Ca2+ on channel
activity, the intracellular Ca2+ concentrations were clamped with
1 µM ionomycin when extracellular free Ca2+ was titrated to 10, 50, and 100 nM, respectively. Ionomycin, 8-bromoguanosine 3:
5
-cyclic monophosphate (8Br-cGMP), L-arginine, and N-acetyl-penicillamine were purchased from Sigma Chemical Co. S-nitroso-
N-acetyl-penicillamine (SNAP) was obtained from Calbiochem
Corp. (La Jolla, CA), and 5-(n-ethyl-n-isopropyl)amiloride (EIPA)
was obtained from LC laboratory (Woburn, MA). Ionomycin, SNAP, and EIPA were dissolved in pure ethanol (Ionomycin and
SNAP) or DMSO (EIPA). The final concentration of ethanol or
DMSO in the bath was 0.1% and had no effect on channel activity. The chemicals were added directly to the bath to reach the final concentration.
Data are shown as mean ± SEM and paired Student's t test was used to determine the significance between the control and experimental periods. Statistical significance was taken as P < 0.05.
Fig. 1 is a representative recording made in a cell-
attached patch showing the effect of 50 µM amiloride
on the activity of the SK. It is apparent that addition of
amiloride decreased the activity of the SK. In 10 experiments, we observed that 50 µM amiloride decreased
nPo from 2.1 ± 0.2 to 1.3 ± 0.1 within 3-5 min (Fig. 2).
The effect of amiloride was fully reversible and wash-out restored the channel activity. In addition to inhibiting Na+ channels, amiloride blocks Na/H exchange.
To exclude the possibility that the effect of amiloride
was the result of inhibiting the Na/H exchanger, we examined the effect of EIPA, an agent that specifically inhibits the Na/H exchanger without blocking Na+ channels (Gupta et al., 1989). Fig. 2 shows that addition of 50 µM EIPA had no significant effect on the SK in cell-attached patches (n = 5). To confirm further that the
effect of amiloride on the SK was the result of inhibition of the Na+ channels, we investigated the effect of
benzamil, a specific Na+ channel inhibitor (Kleyman
and Cragoe, 1988
). Fig. 3 shows that application of 1 µM benzamil mimicked the effect of amiloride and reduced channel activity in cell-attached patches by 38 ± 5% (n = 8). The notion that the effect of benzamil is
the result of blocking Na+ transport is further indicated by experiments in which addition of 1 µM benzamil significantly reduced intracellular Na+ concentration from 15 ± 2 to 11 ± 2 mM (n = 5) (Fig. 4).
Since EIPA failed to mimic the effect of amiloride, the role of intracellular pH in mediating cross talk in the CCD is largely excluded. It is also unlikely that ATP is involved in mediating the effect since the basolateral K+ channels are not sensitive to ATP. To examine the role of Ca2+, we studied the effects of amiloride on the SK in a Ca2+-free bath solution and the results are summarized in Table I. Removal of extracellular Ca2+ abolished the effect of amiloride since channel activity was not significantly different from the control value (110 ± 10%), whereas amiloride reduced channel activity by 39 ± 5% in the presence of Ca2+ (Fig. 2 and Table I).
Table I. The Effects of Amiloride (50 µM) on the Activity of the SK in the Presence or Absence of Extracellular Ca2+ |
Schlatter et al. (1996) found that amiloride decreased intracellular Ca2+. We have also examined the
effect of amiloride on the intracellular Ca2+ in the absence and presence of extracellular Ca2+. Fig. 5 a is one
representative trace out of four experiments showing
that addition of 0.5-1 µM amiloride significantly reduced intracellular Ca2+ from 75 ± 8 to 64 ± 5 nM.
Also, Fig. 5 a shows that removal of extracellular Ca2+
significantly reduced intracellular Ca2+ to 45 ± 5 nM,
and, moreover, amiloride had no significant effect on
intracellular Ca2+ in the absence of extracellular Ca2+.
Although we observed only a modest (15%) decrease
in the intracellular Ca2+ with 0.5-1 µM amiloride, a
higher concentration of amiloride might result in a
larger decrease. However, we were unable to use
amiloride at higher concentrations since fluorescence
emitted by amiloride at high concentrations interfered
with the measurement. That the amiloride-induced
small decrease in intracellular Ca2+ is due to incompletely inhibiting Na+ channels is supported by experiments in which adding 1 µM benzamil reduced intracellular Ca2+ by 30% from 82 ± 7 to 57 ± 6 nM (Fig. 5
b). The observation is consistent with the results reported by Frindt et al. (1993)
.
Data supporting the notion that the decrease in intracellular Ca2+ is responsible for the effects of inhibiting Na+ transport on the SK were obtained from experiments in which the effects of Ca2+ on the SK were investigated. We used 1 µM ionomycin to clamp the
intracellular Ca2+. Fig. 6 a is a representative recording
showing the changes in intracellular Ca2+ in the presence of 1 µM ionomycin when the extracellular Ca2+
increased from 10 to 50 nM. Fig. 6 b is a representative
recording showing the effect of raising Ca2+ on channel activity in a cell-attached patch. It is apparent that
the increase in intracellular Ca2+ to 50 nM significantly
stimulates the SK. Fig. 7 shows a relationship between
the channel activity and intracellular Ca2+ obtained
from eight experiments. Raising intracellular Ca2+ to
50 and 100 nM increases the nPo (0.9 ± 0.3, control
value) by 105 ± 11 and 190 ± 15%, respectively. Thus,
data strongly indicate that a decrease in intracellular
Ca2+ is responsible for the effect of inhibiting Na+
transport.
Having proposed that Ca2+ is involved in mediating
the effect of inhibiting Na+ channels on the SK, we explored the mechanism by which intracellular Ca2+
modulates channel activity. The effect of Ca2+ is not direct since in excised patches we did not find significant changes in channel activity when the Ca2+ concentration was increased from 0 to 100 nM (data not shown). Moreover, an increase in Ca2+ to 1 µM inhibited the SK
and led to channel run-down in excised patches (data
not shown). Thus, our data strongly suggest that the effect of Ca2+ is indirect and mediated by a Ca2+-dependent pathway. Our previous study had demonstrated
that NO stimulated the SK via a cGMP-dependent pathway (Lu and Wang, 1996), and we recently found that
neuronal NOS (nNOS) is expressed in the CCD (Wang
et al., 1997
). Since nNOS activity has been shown to depend critically on intracellular Ca2+ in the physiological range of Ca2+ concentration (50-250 nM) (Knowles
et al., 1989
), we examined the possible role of NO in
mediating the effect of inhibiting apical Na+ transport.
Fig. 8 shows the effect of SNAP, a NO donor, on channel activity that had been decreased by benzamil. It is
apparent that addition of 10 µM SNAP reversed the
benzamil-induced decrease of the channel activity.
Fig. 9 summarizes the effect of SNAP on SK in the
presence of either 50 µM amiloride or 1 µM benzamil.
Both decreased channel activity (nPo) from 2.1 ± 0.2 to
1.3 ± 0.1 (n = 12); however, application of SNAP restored channel activity (nPo) to 2.25 ± 0.25 (n = 12),
suggesting that the decrease in NO production may be
responsible for the effect of inhibiting Na+ channels.
We have previously shown that addition of 100 µM
L-NAME (L-N G-nitroarginine methyl ester) blocked the
SK channel (Lu and Wang, 1996). We have further extended our study to examine the effect of L-NAME in
the presence of L-arginine. Fig. 10 summarizes the results from such experiments, showing that application
of 400 µM L-arginine abolished the effect of L-NAME.
In addition, the effect of L-NAME can be reversed by 10 µM SNAP but not by N-acetyl-penicillamine (10 µM),
the byproduct of SNAP, suggesting that the effect of SNAP results from NO release.
Since cGMP has been shown to mimic the effect of
NO donors such as SNAP, we next investigated whether
cGMP can reverse the effect of inhibiting apical Na+
transport. Fig. 11 shows that addition of 100 µM 8Br-cGMP mimicked the effect of SNAP and reactivated the
SK in cell-attached patches in the presence of benzamil. Fig. 12 summarizes these results, showing inhibition of the Na+ channel-reduced nPo of the SK from
1.9 ± 0.4 to 1.2 ± 0.3 (n = 5), whereas addition of 100 µM cGMP restored channel activity (nPo = 2.0 ± 0.4).
Moreover, the effect of cGMP and SNAP is not additive
(data not shown), further supporting the notion that cGMP mediates the effect of NO.
The notion that NO may be involved in mediating
the effects of inhibition of the Na+ channel is further
supported by experiments in which addition of SNAP
stimulated channel activity in the Ca2+-free bath solution (Fig. 13). Application of 10 µM SNAP caused a significant increase in channel activity (nPo) from 0.5 ± 0.1 to 1.1 ± 0.2, suggesting that diminished NO formation associated with the decrease in intracellular Ca2+ is
responsible for the effect of inhibiting Na+ channels.
Three types of K+ channels have been found in the basolateral membrane of the rat CCD (Wang et al., 1994;
Hirsch and Schlatter, 1993
) and we confirmed previous
observations that in rats on either normal or high potassium diet the SK is predominant in the lateral membrane of the CCD. Accordingly, the SK plays an important role in determination of cell membrane potential.
We have previously shown that NO stimulates the SK
channel via a cGMP-dependent pathway (Lu and
Wang, 1996
). This finding is further confirmed by results in experiments in which the effect of L-NAME was
abolished in the presence of L-arginine, suggesting that
the effect of L-NAME is the result of competing for
NOS with the endogenous L-arginine in the CCD.
In the present study, we examined the effect of inhibiting Na+ transport on the SK to gain an insight into
the mechanism by which apical Na+ transport is linked
to the basolateral K+ conductance. Since it has been
observed that cGMP stimulates basolateral K+ channels
other than the SK (Hirsch and Schlatter, 1995), it is conceivable that the SK is not the only K+ channel that
is involved in the cross-talk mechanism.
The present study confirms other investigators' findings that the transepithelial Na+ transport is coupled to
the basolateral K+ conductance (Horisberger and Giebisch, 1988a, 1988b
; Harvey, 1995
; Beck et al., 1993
;
Tsuchiya et al., 1992
). The cross-talk mechanism by
which the apical Na+ transport links to the basolateral
K+ channel has been extensively explored and changes
in intracellular ATP, pH, and Ca2+ have been suggested to be involved (Beck et al., 1993
; Harvey, 1995
;
Schlatter et al., 1996
; Tsuchiya et al., 1992
). ATP has been shown to play a key role in linking apical Na+
transport to the basolateral ATP-sensitive K+ channels
in the proximal tubule cells of rabbit and rat kidneys (Tsuchiya et al., 1992
; Hurst et al., 1991
; Beck et al.,
1993
), and in the principal cells of amphibian tight epithelial cells (Urbach et al., 1996b
). However, since the
basolateral K+ channel in the CCD is not sensitive to
ATP, a role of ATP in linking apical Na+ transport to
basolateral K+ conductance is largely excluded.
Intracellular pH has been demonstrated to play an
important role in mediating the aldosterone-induced
stimulation of basolateral K+ conductance in amphibian distal nephron cells (Urbach et al., 1996a; Wang et
al., 1989
). Application of aldosterone to stimulate the
Na+ transport induces a significant alkalinization of intracellular pH and, accordingly, increases the basolateral pH-sensitive K+ conductance in the frog distal
nephron. Although the basolateral K+ channels are pH
sensitive, several lines of evidence indicate that the effect of amiloride is not the result of decreasing intracellular pH. First, EIPA, which selectively inhibits the
Na/H exchanger but not Na+ channels, has no effect
on the basolateral K+ channels. Second, benzamil, which
is a specific Na+ channel blocker, reduces the activity of
the SK. We also confirmed observations of Schlatter et
al. (1996)
that inhibition of Na+ transport significantly
reduced intracellular Na+ concentration. Finally, the
effect of amiloride on the SK is abolished in a Ca2+-free
bath solution, further suggesting that intracellular pH is not involved in mediating the effect of amiloride. In
addition, Frindt et al. (1993)
have shown that application of 10 µM amiloride has no effect on intracellular
pH. Thus, it is unlikely that intracellular pH plays a significant role in mediating the effect of inhibiting the
Na+ channels.
Three lines of evidence strongly suggest that Ca2+ is critically involved in mediating the effect on the SK of inhibition of the Na+ channels: first, the effect is correlated with a decrease in intracellular Ca2+; second, removal of Ca2+ abolishes the effect; and third, raising intracellular Ca2+ from 10 to 50 and 100 nM stimulates the SK. The amiloride-induced reduction of intracellular Ca2+ is presumably the result of an increase in the electrochemical gradient of Na+ that drives the Na/Ca exchanger. Removal of extracellular Ca2+ not only abolishes the Ca2+ influx, but also facilitates the extrusion of Ca2+ along its electrochemical gradient.
Although the present data indicate that the effect of
inhibiting the Na+ channels on the activity of the SK is
related to the decline of intracellular Ca2+, the effect of
Ca2+ on the SK is not a direct action since Ca2+-induced
increases in channel activity were absent in excised patches (data not shown). Moreover, the effect of raising Ca2+ from 10 to 100 nM is absent in the presence of
100 µM L-NAME (our unpublished observations), suggesting that the effect of Ca2+ is related to NO formation. Several lines of evidence suggest that NO could be
responsible for mediating the effect of inhibiting Na+
transport. The constitutive form of NOS has been
shown to be present in the kidney, including the CCD
(Terada et al., 1992), and we have confirmed this using
the reverse transcription-PCR and immunocytochemical methods (Wang et al., 1997
). It is well established
that the activity of nNOS dependents on Ca2+ in the
physiological ranges (50-250 nM) and a decrease in
Ca2+ significantly reduces the activity of nNOS (Knowles
et al., 1989
). NO has been found to stimulate the activity of the SK by a cGMP-dependent pathway (Lu and
Wang, 1996
), and addition of NO donors or cGMP reversed the effect of inhibiting Na+ channels. Finally,
NO donors mimic the effect of raising extracellular Ca2+ and increase the activity of the SK in a Ca2+-free
bath. Taken together, these data suggest that inhibition of Na+ channels leads to reduction of intracellular
Ca2+, which in turn decreases NO formation and inhibits basolateral K+ channels.
Ca2+ has also been found to play a key role in linking
the apical K+ conductance (Wang et al., 1993) and Na+
transport to the activity of Na-K-ATPase (Frindt et al.,
1996
; Silver et al., 1993
; Ling and Eaton, 1989
). Inhibition of Na-K-ATPase decreased the open probability of
the apical K+ channel in the CCD, and the effect of inhibiting the Na-K-ATPase was mediated by Ca2+-dependent PKC (Wang et al., 1993
). Inhibition of the Na-K-ATPase has also been shown to decrease the basolateral K+ transference number, an index of the basolateral
K+ permeability (Schlatter and Schafer, 1987
). This effect is believed to be mediated by raising intracellular
Ca2+ (Schlatter et al., 1996
). In the present study, we
show that a decrease in intracellular Ca2+ leads to a decline in the activity of the basolateral K+ channels.
Therefore, it is conceivable that the intracellular Ca2+
may have biphasic effects on basolateral K+ conductance. At a low concentration, an increase in intracellular Ca2+ activates the basolateral K+ conductance by
stimulating the cGMP pathway. On the other hand, at a
high concentration, intracellular Ca2+ may inhibit the
basolateral K+ channels. Further experiments are needed
to determine the precise relationship between intracellular Ca2+ and basolateral K+ channel activity.
Fig. 14 is a cell model to illustrate the mechanism by
which inhibition of apical Na+ channels reduces the activity of the SK. The blockade of the Na+ channels by
amiloride/benzamil decreases intracellular Na+ concentration and reduces the turnover rate of the Na-K-ATPase since the activity of the Na-K-ATPase has been shown to
be coupled to apical Na+ transport (Flemmer et al.,
1993). Such a decrease in intracellular Na+ increases
the electrochemical driving force for Ca2+/Na+ exchange and enhances the extrusion of intracellular
Ca2+ from the cell. Since the activity of nNOS is Ca2+
dependent, a decrease in intracellular Ca2+ is expected
to inhibit nNOS and reduce the formation of NO and cGMP. As a consequence, the activity of the basolateral
small conductance K+ channels decreases.
Address correspondence to Dr.WenHui Wang, Department of Pharmacology, New York Medical College, Valhalla, NY 10595. Fax: 914-347-4958; E-mail: wenhui_wang{at}nymc.edu
Received for publication 5 May 1997 and accepted in revised form 15 October 1997.
1 Abbreviations used in this paper: CCD, cortical collecting duct; SK, small-conductance K+ channel.We thank Dr. R.W. Berliner for help in preparation of the manuscript.
This work was supported by National Institutes of Health grants DK-17433 (G. Giebisch), DK-47402, and HL-34300 (W.H. Wang).
1. |
Beck, J.S.,
A.M. Hurst,
J.Y. Lapointe, and
R. Laprade.
1993.
Regulation of basolateral K channels in proximal tubule studied during
continuous microperfusion.
Am. J. Physiol.
264:
F496-F501
|
2. | Breyer, M.D., and Y. Ando. 1994. Hormonal signaling and regulation of salt and water transport in the collecting duct. Annu. Rev. Physiol. 56: 711-739 [Medline]. |
3. | Davis, W., and A.L. Finn. 1982. Sodium transport inhibition by amiloride reduces basolateral membrane K conductance in tight epithelia. Science. 26: 525-527 . |
4. |
Dawson, D.C., and
N.W. Richards.
1990.
Basolateral K conductance: role in regulation of NaCl absorption and secretion.
Am. J. Physiol.
259:
C181-C195
|
5. | Flemmer, A., A. Doerge, K. Thurau, and F.X. Beck. 1993. Transcellular sodium transport and basolateral rubidium uptake in the isolated perfused cortical collecting duct. Pflügers Arch. 424: 250-254 [Medline]. |
6. |
Frindt, G.,
R.B. Silver,
E.E. Windhager, and
L.G. Palmer.
1993.
Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry.
Am. J. Physiol.
264:
F565-F574
|
7. |
Frindt, G.,
L.G. Palmer, and
E.E. Windhager.
1996.
Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of
protein kinase C.
Am. J. Physiol.
270:
F371-F376
|
8. | Giebisch, G.. 1995. Renal potassium channels: an overview. Kidney Int. 48: 1004-1009 [Medline]. |
9. | Grynkiewicz, G., M. Ponie, and R.Y. Tsien. 1985. A new generation of Ca indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450 [Abstract]. |
10. | Gupta, S., E.J. Cragoe, and R.C. Deth. 1989. Influence of atrial natriuretic factor on 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na uptake in rabbit aorta. J. Pharmacol. Exp. Ther. 248: 991-996 [Abstract]. |
11. | Harvey, B.J.. 1995. Cross-talk between sodium and potassium channels in tight epithelia. Kidney Int. 48: 1191-1199 [Medline]. |
12. | Hirsch, J., and E. Schlatter. 1993. K+ channels in the basolateral membrane of rat cortical colleting duct. Pflügers Arch. 424: 470-477 [Medline]. |
13. | Hirsch, J., and E. Schlatter. 1995. K+ channels in the basolateral membrane of rat cortical collecting duct are regulated by a cGMP-dependent protein kinase. Pflügers Arch. 429: 338-344 [Medline]. |
14. | Horisberger, J.D., and G. Giebisch. 1988a. Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma. J. Gen. Physiol. 92: 643-665 [Abstract]. |
15. | Horisberger, J.D., and G. Giebisch. 1988b. Voltage dependence of the basolateral membrane conductance in the amphiuma collecting tubule. J. Membr. Biol. 105: 257-263 [Medline]. |
16. | Hurst, A.M., J.S. Beck, R. Laprade, and J.Y. Lapointe. 1991. Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule. Am. J. Physiol. 264: F760-F764 . |
17. | Kleyman, T.R., and E.J. Cragoe Jr.. 1988. Amiloride and its analogs as tools in the study of ion transport. J. Membr. Biol. 105: 1-21 [Medline]. |
18. | Knowles, R.G., M. Palacios, R.M. Palmer, and S. Moncada. 1989. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Natl. Acad. Sci. USA. 86: 5159-5162 [Abstract]. |
19. |
Ling, B.N., and
D.C. Eaton.
1989.
Effects of luminal Na+ on single
Na channels in A6 cells, a regulatory role for protein kinase C.
Am. J. Physiol.
256:
F1094-F1103
|
20. | Lu, M., and W.H. Wang. 1996. Nitric oxide regulates the low-conductance K channel in the basolateral membrane of the cortical collecting duct. Am. J. Physiol. 270: C1338-C1342 . |
21. | Palmer, L.G., L. Antonian, and G. Frindt. 1993. Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone. J. Gen. Physiol. 102: 43-57 [Abstract]. |
22. | Schafer, J.A., and C.T. Hawk. 1992. Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids. Kidney Int. 41: 255-268 [Medline]. |
23. |
Schafer, J.A., and
S.L. Troutman.
1987.
Potassium transport in cortical collecting tubules from mineralcorticoid-treated rat.
Am. J. Physiol.
253:
F76-F88
|
24. |
Schlatter, E.,
S. Haxelmans, and
I. Ankorina.
1996.
Correlation between intracellular activities of Ca2+ and Na+ in rat cortical
collecting duct![]() |
25. | Schlatter, E., and J.A. Schafer. 1987. Electrophysiological studies in principal cells of rat cortical collecting tubules. ADH increases the apical membrane Na+-conductance. Pflügers Arch. 409: 81-92 [Medline]. |
26. |
Schultz, S.G..
1981.
Homocellular regulatory mechanisms in sodium-transporting epithelia: avoidance of extinction by "flush-through."
Am. J. Physiol.
241:
F579-F590
|
27. |
Silver, R.B.,
G. Frindt,
E.E. Windhager, and
L.G. Palmer.
1993.
Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump.
Am. J. Physiol.
264:
F557-F564
|
28. | Smith, P.R., and D.J. Benos. 1991. Epithelial Na channels. Annu. Rev. Physiol. 53: 509-530 [Medline]. |
29. |
Strieter, J.,
J.L. Stephenson,
G. Giebisch, and
A.M. Weinstein.
1992a.
A mathematical model of the rabbit cortical collecting tubule.
Am. J. Physiol.
263:
F1063-F1075
|
30. |
Strieter, J.,
A.M. Weinstein,
G. Giebisch, and
J. Stephenson.
1992b.
Regulation of K transport in a mathematical model of the cortical collecting tubule.
Am. J. Physiol.
263:
F1076-F1086
|
31. | Terada, Y., K. Tomta, H. Nonoguchi, and F. Marumo. 1992. Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segments. J. Clin. Invest. 90: 659-665 [Medline]. |
32. | Tsuchiya, K., W.H. Wang, G. Giebisch, and P.A. Welling. 1992. ATP is a coupling-modulator of parallel Na/K ATPase-K channel activity in the renal proximal tubule. Proc. Nat. Acad. Sci. USA. 89: 6418-6422 [Abstract]. |
33. | Urbach, V., E.V. Kerkhove, D. Maguire, and B.J. Harvey. 1996a. Rapid activation of KATP channels by aldosterone in principal cells of frog skin. J. Physiol. (Camb.). 491: 111-120 [Abstract]. |
34. | Urbach, V., E.V. Kerkhove, D. Maguire, and B.J. Harvey. 1996b. Cross-talk between ATP-regulated K+ channels and Na+ transport via cellular metabolism in frog skin principal cells. J. Physiol. (Camb.). 491: 99-109 [Abstract]. |
35. | Wang, W.H., R.M. Henderson, J. Geibel, S. White, and G. Giebisch. 1989. Mechanism of aldosterone-induced increase of K conductance in early distal renal tubule cells of the frog. J. Membr. Biol. 111: 277-289 [Medline]. |
36. | Wang, W.H., J. Geibel, and G. Giebisch. 1993. Mechanism of apical K channel modulation in principal renal tubule cells: effect of inhibition of basolateral Na-K-ATPase. J. Gen. Physiol. 101: 673-694 [Abstract]. |
37. |
Wang, W.H.,
C.M. McNicholas,
A.S. Segal, and
G. Giebisch.
1994.
A novel approach allows identification of K+ channels in the lateral membrane of rat CCD.
Am. J. Physiol.
266:
F813-F822
|
38. | Wang, W.H.. 1995. Regulation of the hyperpolarization-activated K channel in the lateral membrane of the CCD. J. Gen. Physiol. 106: 25-43 [Abstract]. |
39. | Wang, X.H., M. Lu, A. Papapetropoulos, W. Sessa, and W.H. Wang. 1997. Neuronal nitric oxide synthase is expressed in the cortical collecting duct (CCD) of the rat kidney. FASEB (Fed. Am. Soc. Exp. Biol.) J. 11: A551 . (Abstr.) . |