Activity of the basolateral K channels is coupled to the Na -K -ATPase in the c - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:47 |只看该作者 |倒序浏览 |打印

作者：Shigeaki Muto, Yasushi Asano, WenHui Wang, Donald Seldin,  Gerhard Giebisch作者单位：1 Department of Nephrology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi, 329-0498 Japan; Department of Pharmacology, New York Medical College, Valhalla, New York 10595; Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Texas 75235; and Department o " C1 m4 c( [8 T% m" ]0 U
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      【摘要】; B- P7 Q' ~: Z! w* l. d9 K
   Microelectrode and patch-clamp techniques were used in the isolated cortical collecting duct to study the effects of stimulating Na -K -ATPase by raising bath K (Fujii Y and Katz AI. Am J Physiol Renal Fluid Electrolyte Physiol 257: F595-F601, 1989 and Muto S, Asano Y, Seldin D, and Giebisch. Am J Physiol Renal Physiol 276: F143-F158, 1999) on the transepithelial ( V T ) and basolateral membrane ( V B ) voltages and basolateral K channel activity. Increasing bath K from 2.5 to 8.5 mM resulted in an initial hyperpolarization of both V T and V B followed by a delayed depolarization. The effects of raising bath K on V T and V B were attenuated by decreasing luminal Na from 146.8 to 14.0 mM and were abolished by removal of luminal Na  , whereas those were magnified in desoxycorticosterone acetate (DOCA)-treated rabbits. Increasing bath K also led to a significant reduction of the intracellular Na and Ca 2  concentrations. The transepithelial conductance ( G T ) or fractional apical membrane resistance (f R A ) were unaltered during the initial hyperpolarization phase, whereas, in the late depolarization phase, there were an increase in G T and a decrease in f R A, both of which were attenuated in the presence of low luminal Na (14.0 mM). In tubules from DOCA-treated animals, bath Ba 2  not only caused a significantly larger initial hyperpolarization of V T and V B but also blunted the late depolarization by high bath K  . N -nitro- L -arginine methyl ester ( L -NAME) partially mimicked the effect of Ba 2  and decreased the amplitude of the late depolarization. Patch-clamp experiments showed that raising bath K from 2.5 to 8.5 mM resulted in an increased activity of the basolateral K channel, which was absent in the presence of L -NAME. We conclude that stimulation of Na -K -ATPase increases the basolateral K conductance and that this effect involves suppression of nitric oxide-dependent inhibition of K channels.
      【关键词】 basolateral potassium channels sodium transport nitric oxide potassium secretion1 Y, @4 R2 w' L) m/ l( L# t
   THE CORTICAL COLLECTING DUCT (CCD) plays a key role in the regulation of hormone-regulated Na transport and K secretion ( 6, 28 ). Transcellular Na transport involves passive Na entry through apical Na channels along a favorable electrochemical gradient and active extrusion across the basolateral membrane by Na -K -ATPase ( 21, 22 ). To safeguard intracellular Na concentrations, apical Na entry must match the rate of extrusion of Na across the basolateral membrane. Moreover, changes in intracellular Na modulate Na -K -ATPase activity and thus affect uptake of K across the basolateral membrane. To maintain intracellular concentrations of K constant, apical and basolateral K conductance must also work in concert with the Na -K -ATPase.
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In a previous study, we demonstrated that stimulation of Na -K -ATPase, by raising bath K content from 2.5 to 8.5 mM, increased the apical and basolateral K conductance in the CCD from desoxycorticosterone acetate (DOCA)-treated rabbits ( 16 ). This suggests that stimulation of Na transport effects coupling between the Na -K -ATPase activity and K conductance. However, the mechanism by which stimulation of the Na -K -ATPase augments the basolateral K conductance has not been explored. In the present study, we explored the effects of changes in the luminal Na on the coupling mechanism between Na -K -ATPase and basolateral K conductance and elucidated a significant role of Ca 2  -mediated changes in nitric oxide (NO).0 d) M' K( X8 f' R/ Y
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METHODS
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Isolation and perfusion of tubules. Female white rabbits (1.5-2.5 kg; Clea Japan, Tokyo, Japan) were maintained on a standard rabbit chow and tap water ad libitum, and, after a period of acclimation, they were divided into control and DOCA-treated groups. These latter animals received DOCA (Sigma, St. Louis, MO) dissolved in sesame oil at a dosage of 2 mg · kg -1 · day -1 im for 7-10 days before the experiment. Control animals were given vehicle in the same manner.

The animals of both groups were anesthetized with intravenous pentobarbital sodium (35 mg/kg), and both kidneys were removed. Thin slices (1-2 mm) were cut from the coronal section of each kidney and transferred to a dish containing dissecting solution composed of (in mM) 14 KCl, 44 K 2 HPO 4, 14 KH 2 PO 4, 9 NaHCO 3, and 160 sucrose, a medium that had been shown to improve the quality of the kidney tissue ( 16, 20 ). The CCD was dissected and transferred to a bath chamber mounted on an inverted microscope (Diaphot; Nikon, Tokyo, Japan). Methods used to perfuse the CCD have been described previously ( 16 - 19 ). Briefly, after suspending the tubule between two pipettes, the lumen was perfused at a rate exceeding 20 nl/min. The distal end of the CCD was held in a collecting pipette coated with unpolymerized Sylgard 184 (Dow Corning, Midland, MI). The volume of the bath chamber was 100 µl to permit rapid exchange of different bath solutions within 5 s. The composition of the perfusion solutions is listed in Table 1.# l0 Z+ C3 @/ {& S: y

Table 1. Solution composition8 y5 A+ e1 ?- ~" ?7 K
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Electrical measurements. The transepithelial and cellular electrical potentials were measured using methods described previously ( 16, 18 - 20 ). The transepithelial voltage ( V T ) was measured via a perfusion pipette connected to a dual channel electrometer (Duo 773; World Precision Instruments, Sarasota, FL) with a 3 M KCl-3% agar bridge and a calomel half-cell electrode. The basolateral membrane voltage ( V B ) was measured with 0.5 M KCl-filled microelectrodes that were fabricated from borosilicate glass capillaries (GD-1.5; 1.5-mm OD, 1.0-mm ID; Narishige Scientific Laboratory, Tokyo, Japan) by using a vertical puller (PE-2; Narishige Scientific Laboratory). Both V T and V B were referenced to the bath (0 mV) and were recorded on a four-pen chart recorder (R64; Rikadenki, Tokyo, Japan). The electrical potential difference across the apical membrane ( V A ) was calculated according to the following formula

The liquid junction potential induced by changing luminal Na was corrected with free-flowing 3 M KCl electrodes ( 22, 23 ).
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Cable analysis was used to calculate the transepithelial conductance ( G T ) and the fractional apical membrane resistance (f R A ), as described in detail previously ( 16, 18 - 21, 24 - 26 ). Constant-current pulses, 50 nA (300 ms in duration, 10-s intervals), were injected in the tubule lumen via the perfusion pipette. The f R A was estimated from the ratio of the voltage deflection across the apical membrane and the entire epithelium at the point of impalement., T' N1 X: L$ D
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The conductances of the apical and basolateral membranes ( G A and G B, respectively) and the tight junction conductance ( G Tj ) were estimated by the following equation described previously ( 16, 19, 20, 24, 25 )) b* W; h" V* z; P1 V( o6 h
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which is the equation of a straight line with a slope of G B and intercept of G Tj. Ba 2  (2 mM) was applied to the luminal perfusate to estimate G B and G Tj. The G B was calculated according to the following equation/ v: m( i2 L; S( ?$ [& A! I$ b! p

where G T (-Ba 2  ), G T ( Ba 2  ), f R A (-Ba 2  ), and f R A ( Ba 2  ) represent G T and f R A values in the absence and presence of luminal Ba 2 , respectively. In the present experiment, only principal collecting duct (CD) cells in the CCD were impaled, using methods of identification previously described ( 16, 18, 20 ).

Measurement of cell Ca 2 and Na  . We have followed the method described previously to measure the concentrations of intracellular Ca 2  and Na ( 14 ). Fluorescence was imaged digitally with an intensified video imaging system, including a SIT 68 camera, controller, and HR 1000 video monitor (Long Island, North Bellmore, NY). The excited and emitted light passed through a x 40 Nikon fluorite objective (numeric aperture = 1.30). The microscope (Nikon) was coupled to an alternating wavelength illumination system (Ionoptix, Milton, MA). We used the split-open CCD to measure the intracellular Ca 2  and Na  . The split-open CCDs were incubated with fura 2-AM (5 µM) or sodium-binding benzofuran isophthalate-AM (7 µM) at room temperature for 20-30 min. After being loaded, tubules were washed at least three times with the control solution. The dye in the specimen was excited with light of the desired wavelengths (340-380 nM) using a 75-watt Xenon source coupled to two monochromators with variable bandwidth. The light exciting the monochromators enters a computer-controlled high-speed electronic shutter that allows incident light to alternate between two excitation wavelengths. The intracellular Ca 2  was measured from the ratio of fluorescence at excitation of 340/380 nM and calibrated using the equation described previously ( 14 ). The cell Na was calibrated by equilibrating cell Na with the extracellular Na concentrations using 10 µM ionophore, lasalocid (Sigma), at the end of experiments. The measurement was performed by selection of three to five principal CD cells, and the results were averaged as one point.
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Patch-clamp experiment in rat CCDs. Patch-clamp experiments were performed in CCDs from DOCA-treated pathogen-free Sprague-Dawley rats of both sexes (age, 6 wk; Taconic Farms, Germantown, NY). After 7 days of recovery from travel-related stress, the rats were injected with DOCA (200 mg/100 g body wt) for an additional 7 days before use. The weight of animals used for experiments varied between 100 and 120 g. The methods for isolating the CCD and the patch-clamp experiments have been described previously ( 14 ). The reason for using rat CCD rather than rabbit tubule is that it is difficult to obtain a high-resistance seal for patching the rabbit CCD. In addition, rabbit has a low-K channel density in general. However, available results from patch-clamp studies have indicated that the K channel population is similar in the thick ascending limb of Henle's loop and the apical membrane of the CCD between rat and rabbit kidneys; it is reasonable to speculate that the same K channels are expressed in the basolateral membrane of rabbit CCD as that of rat CCD.2 M# J+ w) v0 n
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Statistics. Data are shown as means ± SE, and paired or nonpaired Student's t -test was used to determine the significance between the two groups. Statistical significance was taken as P6 F! u7 h8 a6 }7 d. ^
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RESULTS* x9 h& I4 ]. x+ e5 r

Effects of changes in basolateral K and luminal Na on electrical parameters ( V T and V B ) were studied in control and DOCA-treated animals. The data shown in Fig. 1 A and Table 2 confirmed previous observations that, at high luminal Na concentrations (146.8 mM), raising bath K from 2.5 to 8.5 mM resulted in a biphasic alteration in V T and V B : an initial hyperpolarization of V T and V B from -12.9 ± 1.2 to -16.1 ± 1.4 mV ( = -3.2 ± 0.3 mV, n = 34) and from -84.8 ± 1.2 to -92.8 ± 1.4 mV ( = -8.0 ± 0.8 mV, n = 34) was followed within 10 s by a delayed depolarization of V T and V B from -16.1 ± 1.4 to -9.1 ± 0.9 mV ( n = 34) and from -92.8 ± 1.5 to -82.3 ± 1.4 mV ( n = 34), respectively ( Fig. 1 ). During the initial hyperpolarization of V T and V B, neither G T (2.5 mM K : G T = 8.9 ± 0.2 mS/cm 2; 8.5 mM K : G T = 9.0 ± 0.2 mS/cm 2 ) nor f R A (2.5 mM K : f R A = 0.48 ± 0.03; 8.5 mM K : f R A = 0.47 ± 0.03) changed. The initial hyperpolarization of the basolateral membrane potential by high bath K had previously been shown to result from stimulation of Na -K -ATPase ( 16 ). This notion was fully supported by further experiments in our present study by examining the effect of high bath K in the presence of 14.0 mM luminal Na  , since it is known that reducing the apical luminal Na entry modulates Na -K -ATPase activity ( 8, 9 ). Figure 1 A is a typical tracing demonstrating that lowering luminal Na strongly attenuates the initial hyperpolarization of V T and V B from -9.5 ± 0.8 to -11.3 ± 0.9 mV ( = -1.8 ± 0.3 mV, n = 34, P 1.2 mV ( = -2.7 ± 0.3 mV, n = 34, P Removal of luminal Na completely abolished the hyperpolarization (data not shown).
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Fig. 1. Typical tracings showing effects of raising bath K on transepithelial ( V T ) and basolateral ( V B ) voltage of the cortical collecting duct (CCD) in the presence of low (14.0 mM) and high (146.8 mM) luminal Na from control rabbits ( A ) and desoxycorticosterone acetate (DOCA)-treated rabbits ( B ).

Table 2. Effects of raising bath K concentrations in the presence of low and high luminal Na concentrations on barrier voltages and conductances in the CCDs from DOCA-treated rabbits

In contrast to the response in principal CD cells in the control animal, raising bath K from 2.5 to 8.5 mM in the DOCA-treated rabbit initiates a significantly larger hyperpolarization of V T and V B from -53.0 ± 1.8 to -61.3 ± 2.6 mV ( = -8.3 ± 0.9 mV, n = 27, P ± 2.0 to -130.5 ± 2.6 mV ( = -12.6 ± 1.1 mV, n = 27, P Fig. 1 B and Table 2 ). This strongly suggests that initial hyperpolarization by high bath K is the result of stimulation of Na -K -ATPase, and its response is shown to depend on luminal Na delivery.
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In a previous study ( 16 ), DOCA did not enhance the magnitude of the initial hyperpolarization after high K in the bath. In these experiments, lumen Na was kept constant at 146.8 mM. It was argued that the observed increase in basolateral K conductance and the membrane potential exceeding the K equilibrium potential generated enough positive current from bath to cell to reduce the magnitude of the pump-generated hyperpolarization ( 16 ). According to this interpretation, the resulting changes in membrane potential are the balance between electrogenic pump activity and the opposing K current. In the present experimental setting, the initial lumen Na concentration was low (14.0 mM) and followed by sequential increases in both lumen Na and basolateral K  . Under these conditions, it is safe to assume that cell Na started out at reduced levels so that the combined effects of increments in lumen Na and bath K may have led to a larger change in pump stimulation that could have effectively opposed the depolarizing effect of K currents.

Inspection of Fig. 2 indicates that, in the control rabbit, G T significantly ( P 0.2 to 10.1 ± 0.3 mS/cm 2 ( = 1.1 ± 0.1 mS/cm 2, n = 34), and f R A significantly ( P 0.47 ± 0.03 to 0.41 ± 0.03 ( = -0.06 ± 0.006, n = 34) during the delayed depolarization phase. This suggests that stimulation of the basolateral Na -K -ATPase alters apical or basolateral ionic conductances. It is also apparent that the effect of high bath K on G T and f R A in the late depolarization phase is enhanced in the DOCA-treated rabbit CCD. These data summarized in Fig. 2 showed that, in the DOCA-treated animal, raising bath K to 8.5 mM significantly increased G T from 15.0 ± 0.3 to 19.0 ± 0.4 mS/cm 2 ( = 4.0 ± 0.2 mS/cm 2, n = 27, P R A significantly decreased from 0.35 ± 0.02 to 0.26 ± 0.02 ( = -0.09 ± 0.006, n = 27, P Fig. 2 and Table 2 ). The effects of high bath K on G T and f R A were significantly ( P diminished in the presence of low luminal Na (14.0 mM; Fig. 2 and Table 2 ). Under the conditions, raising bath K significantly ( P G T from 12.0 ± 0.3 to 14.3 ± 0.3 mS/cm 2 ( = 2.3 ± 0.1 mS/cm 2, n = 27) and significantly ( P R A from 0.47 ± 0.02 to 0.43 ± 0.03 ( = -0.04 ± 0.005, n = 27; Table 2 ). These results suggest that changes in G T and f R A depend on the luminal Na delivery. This interpretation is also supported by findings that further decreasing the luminal Na concentrations to 1.4 mM abolished the effect of high bath K on both G T and f R A (data not shown). These data strongly suggest that luminal Na supply, by modulating the activity of basolateral Na -K -ATPase activity, is tightly linked to the G B. We have taken advantage of the high Na transport rate in DOCA-treated animals to further study the mechanisms by which stimulation of Na -K -ATPase increases the basolateral K conductance.) M" l- g# N3 v5 K4 R9 H: B% s
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Fig. 2. Effect of raising bath K (2.5-8.5 mM) on changes in V T, V B, transepithelial conductance ( G T ), and fractional apical membrane resistance (f R A ) in the presence of low (14.0 mM) and high (146.8 mM) luminal Na in the CCDs from control and DOCA-treated rabbits. HYPER, initial hyperpolarization phase; DEPO, late depolarization phase. Difference between high and low luminal Na is significant ( * P ** P. ?4 I5 J4 `* L6 X7 b/ ]
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First, we examined the effect of rapidly stimulating the activity of Na -K -ATPase on the intracellular Na concentration. Data summarized in Fig. 3 showed that raising bath K significantly ( P concentration from 17.2 ± 1.7 to 10.4 ± 1.1 mM ( n = 4). This decline indicates that basolateral active Na extrusion stimulated by external K exceeds the rate of increased apical Na entry. When the basolateral concentration of K was abruptly raised in the presence of only 14.0 mM Na in the lumen, the decrease in intracellular Na concentration was significantly ( P 0.01) smaller, declining from an initial mean value of 14.9 ± 0.9 to 13.4 ± 0.7 mM ( n = 4, P smaller effect on cell Na concentration is best explained by the lesser stimulation of basolateral Na -K -ATPase activity after diminished Na entry along a small electrochemical gradient of Na across the apical membrane. It is safe to conclude that the stimulatory effect of high bath K on Na -K -ATPase turnover is modulated by the luminal Na concentration and the amount of Na available for basolateral pump activity. Because the f R A of the tubule remained unchanged during the initial phase of hyperpolarization, it is unlikely that the apical Na conductance was significantly altered and could affect the rate of apical Na entry.: }  I* C0 f& R6 v0 z3 E# v

Fig. 3. Effects of raising bath K on intracellular Na concentrations in the CCDs from DOCA-treated rabbits. "Low Na" and "High Na" represent 14.0 and 146.8 mM Na in the lumen, respectively. 2.5K and 8.5K represent 2.5 and 8.5 mM K in the bath, respectively. No. of tubules examined is 4.& V9 F8 V: `8 u6 ^( l. ?0 g5 C# n

As summarized in Fig. 2, raising bath K to 8.5 mM increased G T and decreased f R A in the late depolarization phase. From the changes in G T and f R A, we have calculated the G B and G Tj : at high luminal Na concentrations, the G B after stimulation of Na -K -ATPase rose significantly ( P ± 0.9 mS/cm 2 to 20.8 ± 1.6 mS/cm 2, but G Tj (2.5 mM K : 5.0 ± 0.2 mS/cm 2; 8.5 mM K : 4.7 ± 0.2 mS/cm 2 ) remained unchanged ( Fig. 4 ). The stimulatory effect of high bath K on G B could be shown to depend on the luminal Na concentrations because high bath K significantly ( P 0.001) increased G B in the presence of 146.8 and 14.0 mM luminal Na  , but the increase in G B in the presence of 146.8 mM Na (5.7 ± 0.7 mS/cm 2 ) was significantly ( P than in the presence of 14.0 mM Na (3.0 ± 0.4 mS/cm 2 ).% Q* d' f, I( n4 p6 @

Fig. 4. Effects of raising bath K on basolateral membrane conductance ( G B ) and tight junction conductance ( G Tj ) in the late depolarization phase in the presence of low (14.0 mM) and high (146.8 mM) luminal Na  . No. of tubules examined is 15. Difference between the low and high bath K is significant ( P

Because the K conductance is the main cation conductance in the basolateral membrane, it is likely that raising bath K from 2.5 to 8.5 mM increases the basolateral K conductance. This was tested in CCDs from DOCA-treated rabbits by examining the effect of increasing bath K on V T, V B, G T, and f R A in the presence of basolateral Ba 2 , an inhibitor of the basolateral K conductance. Application of 2 mM Ba 2  led to sustained hyperpolarization of V T and V B from -34.6 ± 2.5 to -40.4 ± 3.5 mV and from -107.4 ± 4.8 to -113.6 ± 6.0 mV, respectively ( Fig. 5 ). This hyperpolarization is different from the behavior of non-DOCA-treated animals in which blocking the K conductance had no effect on V B ( 16 ). Hyperpolarization observed in the DOCA-treated CCD after Ba 2  is best explained by a reduction of inward K current driven by the negative membrane potentials, which exceeds the K equilibrium potential. This notion is further supported by the finding that inhibition of the basolateral K conductance by Ba 2  not only potentiates the initial hyperpolarization but also attenuates the magnitude of the late depolarization when the bath K was raised from 2.5 to 8.5 mM ( Fig. 5 and Table 3 ). In the absence of Ba 2 , raising bath K to 8.5 mM caused -7.4 ± 1.4 mV ( V T ) and -11.0 ± 1.8 mV ( V B; n = 7) hyperpolarization. In contrast, in the presence of Ba 2 , the changes of V T and V B were -15.6 ± 1.8 mV and -25.0 ± 3.2 mV, respectively. In addition, Ba 2  significantly ( P (-Ba 2  : V T =  22.2 ± 2.3 mV, V B =  26.9 ± 2.8 mV;  Ba 2  : V T =  14.5 ± 2.4 mV, V B =  20.6 ± 2.8 mV). In addition, inspection of Fig. 5 and Table 3 demonstrates that Ba 2  has a smaller effect on high bath K -induced changes in initial hyperpolarization ( V T = -10.0 ± 1.5 mV, P V B = -12.8 ± 1.5 mV, P late depolarization ( V T =  6.8 ± 2.2 mV, P V B =  9.1 ± 1.4 mV, P luminal Na concentrations. This indicates that the coupling between the Na -K -ATPase activity and the basolateral K conductance depends on luminal Na entry.- x& N3 V, _: w- f1 ^: ~* D

Fig. 5. Typical tracings showing effects of raising bath K on V T and V B in the absence and presence of bath 2 mM Ba 2  in the CCD from the DOCA-treated rabbit.2 v$ S9 A" v% @7 x/ R/ o6 n- z- X0 p3 W
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Table 3. Effects of raising bath K concentrations in the absence and presence of bath Ba 2  under low and high luminal Na concentrations on barrier voltages and conductances in CCDs from DOCA-treated rabbits
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Stimulation of Na -K -ATPase decreased the intracellular Na concentration ( Fig. 3 ). As a consequence of decreasing intracellular Na  , the intracellular Ca 2  should also fall because the driving force for Na /Ca 2  exchanger increases. Experiments with respect to the effect of raising bath K on intracellular Ca 2  were studied with fura 2. Figure 6 summarizes the results demonstrating that raising bath K significantly ( P intracellular Ca 2  from 272.8 ± 64.9 to 109.6 ± 37.9 nM ( n = 6). We have previously demonstrated that high concentrations of intracellular Ca 2  200 nM) inhibit basolateral 18-pS K channels by an NO-dependent mechanism ( 33 ). Therefore, we tested possibility that raising bath K from 2.5 to 8.5 mM reduces the intracellular Ca 2  concentration and affects the NO-dependent inhibition on basolateral K channel activity. We examined the effect of raising bath K on basolateral K conductance in the presence of N -nitro- L -arginine methyl ester ( L -NAME; an inhibitor of endogenous NO synthase). Figure 7 is a typical tracing showing the effect of raising bath K on V T and V B in the absence and presence of 2 mM L -NAME. In the absence of L -NAME, raising bath K to 8.5 mM led to an initial hyperpolarization of V B from -113.2 ± 3.4 to -117.9 ± 3.3 mV ( V B = -4.7 ± 0.9 mV, n = 10) and delayed depolarization of V B from -117.9 ± 3.3 to -104.5 ± 3.5 mV ( V B = 13.4 ± 1.1 mV, n = 10). However, in the presence of L -NAME, raising bath K caused a significantly larger hyperpolarization of V B from -115.4 ± 1.2 to -120.9 ± 2.9 mV ( V B = -5.5 ± 0.9 mV, n = 10, P of V B from -120.9 ± 2.9 to -109.6 ± 3.3 mV ( V B = 11.3 ± 1.2 mV, n = 10, P in the absence of L -NAME. Therefore, L -NAME can partially mimic the effect of Ba 2  to increase the initial hyperpolarization and decrease the late depolarization. Moreover, the view that the high K -induced increase in basolateral K conductance is the result of removal of NO-dependent suppression is further supported by examining the effect of L -NAME on G T and f R A. Figure 8 summarizes the effect of L -NAME in the late depolarization phase: inhibition of NO synthase with L -NAME attenuated the effect of raising bath K on G T ( G T = 2.0 ± 0.2 mS/cm 2 ) and f R A ( f R A = 0.04 ± 0.01) in the late depolarization phase, values that were significantly ( P than those observed in control conditions ( G T = 2.5 ± 0.3 mS/cm 2; f R A = 0.06 ± 0.01), and were similar to those in the presence of bath Ba 2  ( G T = 2.2 ± 0.2 mS/cm 2; f R A = 0.04 ± 0.01).) ]! _) k8 B" F0 c

Fig. 6. Effects of raising bath K on intracellular Ca 2  concentrations in the presence of low Na (14.0 mM) and high Na (146.8 mM) in CCDs from the DOCA-treated rabbits. The difference is significant ( * P5 o/ Z! c& [: g( O: M+ c
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Fig. 7. Typical tracings showing effects of raising bath K on V T and V B in the absence and presence of bath 2 mM N -nitro- L -arginine methyl ester ( L -NAME) in the CCD from the DOCA-treated rabbit. The luminal Na was 146.8 mM.4 q6 G& p4 l5 W: [

Fig. 8. Effect of raising bath K on G T ( A ) and f R A ( B ) in control conditions and in the presence of bath Ba 2  or L -NAME in the presence of 146.8 mM luminal Na in the CCDs from the DOCA-treated rabbits. No. of tubules examined under control conditions and in the presence of bath Ba 2  and L -NAME is 27, 7, and 10, respectively. * P) {  V1 @/ R  T' Z

To further examine the hypothesis that stimulation of Na -K -ATPase increases the basolateral K conductance, we used the patch-clamp technique to study the effect of raising bath K from 2.5 to 8.5 mM on basolateral K channels in the CCD obtained from DOCA-treated rats. Figure 9 is a representative channel recording showing that raising bath K from 2.5 to 8.5 mM increases the activity [open probability number ( NP o )] of the basolateral 18-pS K channel from 0.26 ± 0.1 to 1.51 ± 0.2 ( n = 8). These patch-clamp experiments support the notion that stimulation of Na -K -ATPase increases the basolateral K conductance. Also, high bath K did not increase the channel activity in the presence of L -NAME from all nine experiments ( NP o = 0.3 ± 0.1).
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Fig. 9. Recording showing the effect of raising bath K to 8.5 mM on the activity of the basolateral 18-pS K channel in the rat CCD. Experiments were performed in a cell-attached patch, and the holding potential was -30 mV. C, channel closed level.

DISCUSSION
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The main finding of the present study is that acute stimulation of Na -K -ATPase by raising the concentration of K in the bath solution increases the basolateral K conductance and that the NO-dependent signaling pathway plays an important role in linking the activity of Na -K -ATPase to the basolateral K conductance. Stimulation of the basolateral Na -K -ATPase was shown to result in an initial hyperpolarization of the basolateral membrane followed by delayed depolarization ( 16 ). The following three lines of evidence suggest that the initial hyperpolarization is caused by stimulation of Na -K -ATPase activity: 1 ) raising K in the bath solution decreases intracellular Na  ; 2 ) the initial pump activation has also been shown to depend on the luminal Na concentration and can be amplified by pretreatment with DOCA; and 3 ) the observation that either G T or f R A remained unchanged during the initial hyperpolarization excludes the possibility that hyperpolarization was mediated by changes in apical or basolateral ion conductances." f: Z0 q0 j6 z" l+ R, A3 s
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After the initial hyperpolarization, raising the concentration of K in the bath also results in a delayed depolarization of the basolateral membrane potential. This effect is not only the result of the diminished gradient of K but also the result of a significant increase in K conductance. This is strongly suggested by the observation that bath Ba 2  attenuated the depolarization and by the results of patch-clamp studies demonstrating a sharp increase of the activity of the 18-pS K channel.7 z  @$ h7 T5 u; ?9 |) |* e5 X
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Linkage of the activity of Na -K -ATPase to the basolateral K conductance has an important physiological significance: an increase in luminal Na entry is expected to enhance the Na -K -ATPase ( 9, 10 ) and the uptake of K across the basolateral membrane. To maintain a constant intracellular K  , the basolateral K conductance must adjust to the turnover rate of the Na -K -ATPase by a mechanism best explained as pump-leak coupling ( 27 ). Although such pump-leak mechanism has been demonstrated in a variety of tissues, including proximal tubule cells ( 1, 11, 13, 30, 34 ) and the frog skin ( 7, 31 ), the mechanism underlying this phenomenon is not completely understood. There are at least three possible mediators, such as ATP, pH, and Ca 2 , that could be responsible for such couplings between Na -K -ATPase and basolateral K channels.

In the proximal tubule, it has been shown that intracellular ATP is a possible mediator for coupling between the basolateral K conductance and the Na -K -ATPase: this stimulation of Na -K -ATPase decreases the intracellular ATP concentration and, accordingly, activates the basolateral ATP-sensitive K channels ( 1, 13, 30, 31 ). The hypothesis has been supported by the finding that luminal addition of glucose, known to enhance apical Na entry, decreases the ATP concentration and simultaneously stimulates basolateral ATP-sensitive K channels in the proximal tubules. However, ATP is unlikely to mediate the coupling between Na -K -ATPase and basolateral K conductance in the CCD because the basolateral K channels are not sensitive to ATP ( 8, 32 ).

The second possible mediator is intracellular pH ( 7 ): stimulation of Na -K -ATPase could decrease intracellular Na and sequentially increase the driving force for Na /H exchanger in the basolateral membrane of the CCD ( 3 ). As a consequence, intracellular alkalization could stimulate basolateral pH-sensitive K channels ( 8 ). Although we cannot completely exclude the role of pH, two lines of evidence do not support this thesis. 1 ) High bath K did not increase the activity of the basolateral K channels in the presence of L -NAME. 2 ) The effect of high K on G T and f R A has been shown to be significantly blocked by L -NAME.# u( R5 v) y) L  O- v8 }# p
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Three lines of evidence suggest that a Ca 2  -dependent NO signaling pathway may be a possible mediator that links the activity of basolateral Na -K -ATPase activity to the basolateral K channels and K conductance. First, inhibition of NO synthase with L -NAME abolished the effect of high K in the bath on basolateral K channel activity. Second, L -NAME attenuated the amplitude of the delayed depolarization. This is consistent with a reduction of K conductance. Further evidence for a role of NO was the observation that L -NAME mimics the effects of Ba 2  in the bath on G T and f R A.- }  l' i* w& S* P, ~# q+ C

It is of interest that stimulation of Na -K -ATPase by raising both bath K and lumen Na leads to an increase in basolateral K conductance. Although cell Ca 2  levels increase with elevation of lumen Na  , Ca 2  decreases when raising bath K augments Na -K -ATPase activity. In the following, we propose a possible mechanism. When lumen Na increases, Na entry raises intracellular Na and thereby decreases the driving force for Ca 2  extrusion via the Na /Ca 2  exchanger ( 29 ). In this experimental setting, the rise in Ca 2  would be expected to enhance NO-cGMP production, as shown previously ( 14 ), and to stimulate basolateral K channel activity, as observed. The situation is different when basolateral Na -K -ATPase activity is stimulated by elevating the concentration of K in the bath. In the present experimental setting, intracellular Ca 2  is initially elevated because of low Na -K -ATPase activity as a consequence of low bath K (see Fig. 10 A ). We have used the cell Na and Ca 2  concentrations measured in the present study to estimate the driving force for 3Na exchanger with the equation published by Frindt et al. ( 4 ). It is estimated that, in the presence of high luminal Na  , the electrochemical gradient for Ca 2  was 456 mV at 2.5 mM bath K and increased to 512 mV at 8.5 mM bath K  . On the other hand, the electrochemical gradient for 3Na was 507 mV at 2.5 mM bath K and increased to 600 mV at 8.5 mM bath K  . Therefore, an increase in bath K significantly augments the driving force for Na /Ca exchanger and decreases the intracellular Ca 2 .9 U9 K" i3 H) P, J
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Fig. 10. Cell models linking the activity of Na -K -ATPase to basolateral K channels. Shown are conditions in which the basolateral K concentration is initially low ( A ) and after an increase in bath K ( B ). High lumen Na was maintained during the experiments. As shown in A, the intracellular Ca 2  concentration is initially high because of low Na -K -ATPase activity (see Fig. 6 ). As previously shown, cGMP levels are saturated under these conditions ( 12 ). It is likely that the high NO concentration induced by elevated cell Ca 2  200 nM) led to formation of inhibiting levels of peroxynitrite ( 2 ). It is proposed that raising K in the bath decreases intracellular Ca 2  to a level that diminishes the inhibitory effect of peroxynitrite; as a consequence, basolateral K channel activity increases. The solid and dotted arrows indicate an enhanced or diminished stimulation, respectively. For details, see text. NO, nitric oxide; OONO -, peroxynitrite.# ~* G; S: b( o" r

It has been demonstrated previously that the resulting high cell Ca 2  inhibits basolateral K channels ( 15, 33 ). This effect is best explained by the dual effect of Ca 2  : at low concentrations (Ca 2  2  is the stimulation of cGMP generation and enhancement of basolateral K channel activity ( 8, 14 ). In contrast, at high concentrations (Ca 2  200 nM), Ca 2  can also increase the generation of peroxynitrite ( 2 ), which has been shown to be a potent inhibitor of basolateral K channels. It is likely that, under these conditions, stimulation of K channels by the NO-cGMP pathway is opposed and even abolished by the potent inhibition by peroxynitrite ( Fig. 10 B ). A decrease in cell Ca 2  by stimulating Na -K -ATPase would now remove the inhibitory effect of peroxynitrite and permit augmentation of cGMP by NO to activate basolateral K channels.

In conclusion, increasing bath K stimulates basolateral Na -K -ATPase activity, and this effect depends on the concentration of Na in the lumen of the CCD. It is suggested that changes in cell Ca 2  play a role in mediating an increase in basolateral K channel activity (K conductance) that is observed during pump stimulation.! D* K" n) Z7 B! i
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DISCLOSURES+ r7 x+ \2 P5 [" \; o/ U
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This work was supported by a grant from the Takeda Science Foundation (S. Muto), the Salt Science Foundation (S. Muto), Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (S. Muto), and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47402 (W. H. Wang) and DK-17433 (G. Giebisch).

ACKNOWLEDGMENTS( B, l# T; |& g7 M. _- u9 R2 x

A part of this work has been presented at the Annual Meeting of the American Society of Nephrology in Philadelphia, PA, in 1998.
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