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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Nomega -nitro-L-arginine methyl ester. Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic acid or Nomega -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
NP<SUB>o</SUB><IT>=</IT><LIM><OP>∑</OP></LIM>(<IT>t<SUB>1</SUB>+t<SUB>2</SUB>+... t<SUB>i</SUB></IT>) (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 Nomega -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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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 Nomega -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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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