NO decreases thick ascending limb chloride absorption by reducing Na+-K+-2Clminus cotransporter activity

Pablo A. Ortiz, Nancy J. Hong, and Jeffrey L. Garvin

Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

First published August 9, 2001; 10.1152/ajprenal.0075.2001.---We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption (JCl-). NaCl transport in the THAL depends on apical Na+-K+-2Cl- cotransporters, apical K+ channels, and basolateral Na+-K+-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL JCl- by inhibiting the Na+-K+-2Cl- cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na+]i) and chloride concentrations ([Cl-]i) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na+]i from 13.5 ± 1.2 to 9.6 ± 1.6 mM (P < 0.05) and also decreased [Cl-]i (P < 0.01). We next tested whether NO decreases Na+-K+-2Cl- cotransporter activity by measuring the initial rate of Na+ transport. In the presence of SPM in the bath, initial rates of Na+ entry were 49.6 ± 6.0% slower compared with control rates (P < 0.05). To determine whether NO inhibits apical K+ channel activity, we measured the change in membrane potential caused by an increase in luminal K+ from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K+ concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K+ permeability could affect NO-induced inhibition of THAL JCl-. In the presence of luminal valinomycin, which increases K+ permeability, addition of SPM decreased THAL JCl- by 41.2 ± 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na+-K+-ATPase by measuring oxygen consumption rate (QO2) in THAL suspensions in the presence of nystatin in varying concentrations of Na+. In the presence of 10.5 mM Na+, nystatin increased QO2 to 119.1 ± 19.2 and 125.6 ± 23.4 nmol O2 · mg protein-1 · min-1 in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na+, nystatin increased QO2 by 104 ± 7 and 94 ± 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL JCl- by inhibiting Na+-K+-2Cl- cotransport rather than inhibiting apical K+ channels or the sodium pump.

nitric oxide; chloride transport; sodium-potassium-2 chloride cotransporter; natriuresis; sodium-potassium-adenosinetriphosphatase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE THICK ASCENDING LIMB (THAL) plays an essential role in the maintenance of NaCl homeostasis. This segment reabsorbs 25-30% of the filtered NaCl load while being water impermeable (5). This capability allows the THAL to dilute the urine and generate a high interstitial osmolality (21). Net NaCl absorption in the THAL involves a secondary active transport process in which luminal Na+ and Cl- enter the cell via the electroneutral Na+-K+-2Cl- cotransporter (8, 22). This cotransporter is abundantly expressed in the apical membrane of medullary and cortical THALs (28). The driving force for NaCl entry via the cotransporter is generated by Na+ extrusion across the basolateral membrane through Na+-K+-ATPase (20). Cl- then exits the cell through basolateral channels and K+-Cl- cotransport (16). The apical membrane of the THAL has a large K+ conductance given by the presence of two types of K+ channels (35). K+ recycling across the apical membrane is important for Na+-K+-2Cl- cotransport and generates a positive luminal potential that provides the force for paracellular transport of cations such as Ca2+ and Mg2+ (15).

Nitric oxide (NO) plays an important role in modulating transport in the THAL (11, 14, 25, 29, 31). We have previously reported that endogenously produced NO inhibits net chloride absorption (JCl-) in isolated, perfused THALs (31). However, the specific transporter inhibited by NO in the THAL is still unknown. We hypothesized that NO inhibits JCl- by decreasing apical Na+-K+-2Cl- cotransport activity. Our findings indicate that NO inhibits THAL JCl- by decreasing Na+-K+-2Cl- cotransport activity rather than inhibiting apical K+ channels or Na+-K+-ATPase.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animals. Male Sprague-Dawley rats, weighing 120 to 150 g (Charles River Breeding Laboratories, Wilmington, MA) and which had been fed a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) for at least 5 days, were used for THAL perfusion and preparation of THAL suspensions. On the day of the experiment, rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip).

Isolation and perfusion of rat THALs. After anesthesia, the abdominal cavity was opened, and the left kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline. Cortical THALs were dissected from the medullary rays under a stereomicroscope at 4-10°C. THALs (ranging from 0.5 to 1.0 mm in length) were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 ± 1°C as described previously (13, 29). The flow rate of the basolateral bath was 0.5 ml/min.

Measurement of intracellular Na and Cl. Once perfused, THAL cells were loaded by bathing the tubules at 37 ± 1°C for 15 min in 1 µM sodium green tetraacetate, 0.01% Pluronic or 5 µM 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ; Molecular Probes, Eugene, OR) for measurements of intracellular Na+ and Cl- concentration ([Na]i and [Cl]I), respectively. Loading was followed by a 15-min wash period. Solution A (Table 1) was used for the basolateral bath and perfusate. The sodium-sensitive fluorescent dye sodium green was prepared daily. Sodium green- or SPQ-loaded tubules were excited at 500 or 340 nm, respectively, and 510- or 400-nm dichroic mirrors were used as emission filters as appropriate. Fluorescence was digitally imaged with an image intensifier (Video Scope International, Herndon, VA) and a charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan). Measurements of the images were recorded utilizing an Image One Metafluor system (Universal Imaging, West Chester, PA). Control measurements were taken every minute for 5 min. Then, 1,3-propanediamine, N-{4- [1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl} C10H26N6O2 (SPM; Cayman Chemical, Ann Arbor, MI) was added to the bath, and measurements were taken once every minute for 15 min. The final five measurements were averaged and taken as the value for [Na+]i or [Cl-]i. In situ calibration was performed for [Na+]i measurements using different Na+ calibration solutions and the Na+ ionophore nystatin. The Na+ calibration solutions contained (in mM) 70 KCl+NaCl, 80 N-methyl-D-glucamine, 1.2 MgSO4, 1 CaCl2, and 10 HEPES (pH 7.4). For each calibration solution, the concentrations of KCl and NaCl were changed to obtain the appropriate concentration of Na+ such that the sum of the two concentrations was 70 mM (e.g, 10 mM NaCl + 60 mM KCl). Nystatin (Sigma, St. Louis, MO) was prepared daily as a 100× stock and diluted in calibration solution before use. No calibration was performed for [Cl-]i measurements. Osmolality of all solutions was adjusted to 290 ± 3 mosmol/kgH2O and equilibrated with 95% O2-5% CO2.

                              
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Table 1.   Solutions

Measurement of Na+-K+-2Cl- cotransporter activity. Once perfused, THAL cells were loaded by bathing the tubules at 37 ± 1°C for 60 min in a solution containing 4.4 µM of the Na+-sensitive fluorescent dye benzofuran isophthalate-acetoxymethyl ester (SBFI-AM; Molecular Probes, Eugene, OR), 0.015% Pluronic, for ratiometric measurements of [Na+]i. During the loading period, THALs were perfused with solution E (Table 1). This solution prevents Na+-K+-2Cl- cotransporter activity and inhibits the apical Na+/H+ exchanger. Loading was followed by a 20-min wash period. Solution B (Table 1) was used for the basolateral bath. The sodium-sensitive fluorescent dye SBFI-AM was prepared daily. SBFI-AM-loaded tubules were excited alternately at 340 or 380 nm, and a 400-nm dichroic mirror was used as an emission filter. An Image One Metafluor system was used to record 340/380 ratio measurements of the images. Experiments consisted of two periods in which we measured the initial rate of [Na+]i increase caused by a switch in luminal Na+ and Cl- from 4.5 and 0 mM, respectively (solution E, Table 1), to 130 mM NaCl (solution F, Table 1). Because solutions E and F contain 100 µM dimethylamiloride, which inhibits Na+/H+ exchangers, the increase in [Na+]i is caused by Na+ entry through the Na+-K+-2Cl- cotransporter. Ratio measurements of multiple regions within a tubule were recorded every 3 s for a 5-min control period, during which luminal NaCl was increased. The luminal solution was then switched back to solution E, and SPM was added to the bath. After 30 min, the luminal solution was switched again, and measurements were recorded for a second 5-min period. The initial rates of [Na+]i increase were calculated for each individual region of a tubule and averaged. In the presence of furosemide (1 mM) in the lumen, increasing luminal NaCl did not change [Na+]i. Control experiments were performed according to the same protocol, except that no SPM was added before the second period.

Measurement of membrane potential. Once THALs were perfused, cells were loaded by perfusing them with 2 µM of the membrane potential-sensitive dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes) in 0.1% Pluronic at 37 ± 1°C for 30 min. Tubules were then washed for 5 min. Solution B (Table 1) was used for the basolateral bath. Frozen stock solutions of JC-1 (10 mg/ml) were thawed daily and diluted just before addition to the lumen. JC-1 was excited at 488 nm, and emission fluorescence was observed via the Oz with Intervision confocal scanning imaging system (Noran Instruments, Middleteon, WI) utilizing a 560-nm dichroic mirror and a 595-nm band-pass filter. Experiments consisted of two periods of depolarization, which is measured as a decrease in fluorescence induced by increasing luminal K+ from 1 mM (solution C, Table 1) to 25 mM (solution D, Table 1). Measurements were recorded for a 30-s period, during which luminal K+ was increased. The luminal solution was then switched back to 1 mM K+, and SPM was added to the bath. After 20 min, luminal K+ was increased again, and measurements were recorded for a second 30-s period. The percent change in fluorescence was calculated for both the control and experimental periods. A difference in the fluorescence change caused by a switch in luminal K+ is equivalent to a change in K+ permeability. Control experiments were performed according to the same protocol, except that no SPM was added before the second period.

JCl- measurement. THALs were mounted on concentric glass pipettes and perfused at 37°C as described previously (13). The perfusion rate was set at 5-10 nl · mm-1 · min-1. After perfusion, THALs were equilibrated for 20 min alone or in the presence of valinomycin (Sigma), and four measurements corresponding to the basal absorption rates were made. SPM (10 µM) was then added to the bath, and after a 20-min reequilibration period, four additional collections were made. The concentration of valinomycin used was determined as the maximal concentration that did not induce cell swelling and allowed THALs to absorb chloride at normal rates (~100 pmol · mm-1 · min-1). Because we wanted to reverse NO-induced inhibition of Cl- transport with valinomycin, the lowest concentration of SPM (10 µM) that produced a maximal response was used. Cl- concentration in the perfusate and collected fluid was measured by microfluorometry (11). All data were recorded and stored by using data-acquisition software (DATAQ instruments, Akron, OH). Data analysis was performed with newly developed software specifically designed for voltage-spike analysis. Because there is no water reabsorption in the THAL, JCl- was calculated as follows
J<SUB>Cl<SUP><IT>−</IT></SUP></SUB><IT>=</IT>CR(C<SUB>oCl<SUP><IT>−</IT></SUP></SUB><IT>−</IT>C<SUB>lCl<SUP><IT>−</IT></SUP></SUB>)
where CR is the collection rate normalized per tubule length, Co Cl- is the Cl- concentration in the perfusion solution, and Cl Cl- is the Cl- concentration in the collected fluid.

Oxygen consumption measurements. Suspensions of medullary THALs (mTHAL) were prepared according to a modified protocol as described by Chamberlin et al. (7). Briefly, kidneys were perfused via retrograde perfusion of the aorta with solution A (Table 1) containing 0.1% collagenase (Sigma) and 100 U heparin. The inner stripe of the outer medulla was cut from coronal slices of the kidney, minced, and incubated at 37°C for 30 min in 0.1% collagenase. The tissue was pelleted via 114-g centrifugation, resuspended in cold solution, and stirred on ice for 30 min to release the tubules. The suspension was filtered through 250-µm nylon mesh and centrifuged at 114 g. The pellet was washed, centrifuged again, and finally resuspended in 0.1 ml of cold solution A (Table 1). The mTHAL suspension was warmed to 37°C and equilibrated with 95% O2-5% CO2. It was then added to a closed chamber maintained at 37°C and oxygen consumption rate (QO2) was recorded continuously. An initial constant slope was established at the beginning of each experiment, and SPM or furosemide (10-4 M) was then added. SPM was prepared daily before use. A constant QO2 was established before nystatin (135 U/ml) was added. All experiments were completed within 15 min. Similar results were obtained with 10 and 100 µM SPM, indicating that 10 µM yields a maximal response. An aliquot of the suspension was used to determine protein concentration using Coomassie protein assay reagent (Pierce, Rockford, IL).

Statistics. Results are expressed as means ± SE. Data were evaluated with Student's paired t-test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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We have previously reported that NO inhibits THAL JCl- (31). A decrease in JCl- could be due to a decrease in apical ion entry or inhibition of basolateral ion exit. We first tested whether NO decreases ion entry by measuring [Na+]i in the THAL. In isolated, perfused THALs, addition of the NO donor SPM (100 µM) to the bath decreased THAL [Na+]i from 13.5 ± 1.2 to 9.6 ± 1.6 mM, a reduction of 28.9 ± 10.0% (P < 0.05; n = 6) (Fig. 1). These data indicate that NO decreases Na+ entry in the THAL.


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Fig. 1.   Effect of the nitric oxide (NO) donor 1,3-propanediamine, N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl} C10H26N6O2 (SPM) on thick ascending limb of Henle (THAL) intracellular sodium concentration. Addition of SPM (100 µM) to the bath decreased intracellular sodium by 28.9 ± 10.0% in isolated perfused THALs (n = 5). *P < 0.05.

In this nephron segment, Na+ and Cl- entry are coupled through the Na+-K+-2Cl- cotransporter; therefore, we tested whether NO could also decrease [Cl-]i. In isolated, perfused THALs, addition of SPM (100 µM) to the bath increased SPQ fluorescence by 19.3 ± 4.1% (P < 0.01, n = 6) (Fig. 2), indicative of a decrease in [Cl-]i. These data indicate that NO decreases Cl- entry in the THAL.


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Fig. 2.   Effect of the NO donor SPM on THAL intracellular chloride concentration. Addition of 100 µM SPM to the bath decreased THAL intracellular chloride concentration, indicated by a 19.3 ± 4.1% increase in 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ) fluorescence (n = 6). au, Arbitrary units.*P < 0.05.

Because NO addition decreased both THAL [Na+]i and [Cl-]i, the effect of NO on THAL JCl- is most likely due to inhibition of ion entry through Na+-K+-2Cl- cotransporters. Thus we tested whether NO could decrease Na+-K+-2Cl- cotransporter activity by measuring the initial rate of Na+ entry through the cotransporter in the absence and presence of SPM. Initial rates were measured by monitoring the increase in THAL [Na+]i caused by a switch in luminal NaCl concentration from 0 to 130 mM (Fig. 3A). Under control conditions, increasing luminal NaCl from 0 to 130 mM increased [Na+]i at an initial rate of 6.99 ± 1.84 × 10-3 arbitrary units (au)/s. In the same tubules and in the presence of 100 µM SPM, increasing luminal NaCl from 0 to 130 mM increased [Na+]i at an initial rate of 3.19 ± 0.89 × 10-3 au/s, 49.5 ± 6.0% slower than the control period initial rates (Fig. 3B) (n = 5; P < 0.05). Control experiments show no significant change in initial rates between the two periods of measurement (7.00 ± 1.73 vs. 6.73 ± 0.73 × 10-3 au/s; n = 4; not significant). The increase in [Na+]i caused by a switch in luminal NaCl was completely inhibited by the presence of furosemide in the luminal perfusion solution. These data indicate that NO decreases Na+-K+-2Cl- cotransporter activity.


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Fig. 3.   Effect of the NO donor SPM on THAL Na+-K+-2Cl- cotransporter activity. A: representative trace showing the initial rates of Na+ entry in the same tubule, in the absence and presence of SPM. B: addition of 100 µM SPM to the bath decreased initial rates of Na+ entry to the THAL by 49.5 ± 6.0% (n = 5).*P < 0.05.

Na+-K+-2Cl- cotransporter activity could be inhibited either directly or by a decrease in apical K+ permeability (15, 32). Therefore, we tested whether NO inhibits K+ channel activity by measuring the change in apical membrane potential caused by an increase in luminal K+ from 1 to 25 mM in the absence and presence of NO. Under control conditions, increasing luminal K+ from 1 to 25 mM decreased the fluorescence of JC-1 by 20 ± 1%, indicative of membrane depolarization. In the same tubules, in the presence of 100 µM SPM, increasing luminal K+ from 1 to 25 mM decreased the fluorescence of JC-1 by 18 ± 1% (data not shown). The same results were obtained in control experiments in the absence of SPM. These data indicate that NO does not decrease THAL apical K+ permeability.

To ensure that a change in apical K+ conductance does not alter NO effects in the THAL, we tested whether NO inhibits THAL JCl- in the presence of an artificially induced high apical K+ permeability. An increase in apical K+ permeability was achieved by perfusing THALs with a solution containing the K+ ionophore valinomycin. In the absence of valinomycin, addition of 10 µM SPM to the bath decreased THAL JCl- by 46.0 ± 6.7% (from 99.6 ± 14.7 to 52.3 ± 16.9 pmol · mm-1 · min-1; P < 0.05; n = 4). In the presence of 5 × 10-5 M valinomycin in the lumen, addition of 10 µM SPM to the bath decreased THAL JCl- by 41.2 ± 10.4% (from 113.0 ± 12.3 to 68.2 ± 15.2 pmol · mm-1 · min-1; P < 0.05; n = 5) (Fig. 4). Taken together, these results indicate that NO inhibits chloride entry even when apical K+ permeability is greatly increased.


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Fig. 4.   Effect of SPM on THAL chloride absorption (JCl-) in the absence and presence of valinomycin (Val) in the lumen. In the absence of valinomycin, addition of 10 µM SPM decreased THAL JCl- by 46.0 ± 6.7% (n = 4). *P < 0.05. In the presence of valinomycin (5 × 10-5 M) in the lumen, SPM decreased JCl- by 41.2 ± 10.4% (n = 5). #P < 0.05.

To ensure that NO does not alter THAL sodium pump activity, we investigated the effects of NO on Na+-K+-ATPase activity. We first tested whether NO affects the affinity constant (K1/2) of Na+-K+-ATPase for Na+ in the THAL by measuring the effects of nystatin on QO2 by THALs in the presence of 10.5 mM extracellular Na+ (a concentration similar to basal intracellular Na+, and the reported K1/2 for Na+). As shown in Fig. 5, 100 µM SPM decreased QO2 from 87.2 ± 10.3 to 68.8 ± 8.9 nmol O2 · mg protein-1 · min-1, a 22 ± 2% decrease (P < 0.01, n = 5). Similarly, inhibiting Na+-K+-2Cl- cotransport with 10-4 M furosemide reduced QO2 by 25 ± 4% (P < 0.01, n = 5). In the presence of SPM, addition of nystatin (135 U/ml), which equilibrates intra- and extracellular Na+, restored QO2 to control values (81.3 ± 7 nmol O2 · mg protein-1 · min-1). Similarly, nystatin returned QO2 to control levels in furosemide-treated tubules (n = 5). Similar results were obtained when 10 µM SPM was used (n = 5). The finding that nystatin restores QO2 to control levels in both SPM- and furosemide-treated tubules indicates that NO does not affect the K1/2 of Na+-K+-ATPase for intracellular Na+.


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Fig. 5.   Effect of nystatin on SPM- and furosemide (Furos.)-induced inhibition of THAL oxygen consumption (QO2) in the presence of 10.5 mM extracellular Na+. Addition of nystatin (135 U/ml) to the bath restored QO2 to control values in SPM- and furosemide-treated tubules (n = 5). *P < 0.05 compared with control.

NO could also affect Na+-K+-ATPase by decreasing its maximum turnover or by decreasing ATP concentration. To test this, we measured the effect of nystatin on QO2 by THALs in the presence of 145 mM extracellular Na+ (normal extracellular Na+). Under these conditions, addition of SPM reduced THAL QO2 by 22 ± 2% (P < 0.01, n = 5) (Fig. 6). Similarly, inhibiting the Na+-K+-2Cl- cotransporter with 10-4 M furosemide reduced THAL QO2 by 25 ± 2%. In the presence of SPM, the addition of nystatin (135 U/ml) increased THAL QO2 by 104 ± 7% (P < 0.05, n = 5). Similarly, nystatin increased QO2 by 94 ± 20% in furosemide-treated THALs (P < 0.05, n = 5). Taken together, these results indicate that NO does not affect Na+-K+- ATPase maximal rate nor its K1/2 for [Na+]i.


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Fig. 6.   Effect of nystatin on SPM- and furosemide-induced inhibition of THAL QO2 in the presence of normal extracellular sodium (145.5 mM). Addition of nystatin (135 U/ml) to the bath increased QO2 in SPM- and furosemide-treated tubules (n = 5). *P < 0.05 compared with control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We found that the NO donor SPM decreased [Na+]i and [Cl-]i in the THAL and inhibited Na+-K+-2Cl- cotransporter activity. Changing luminal K+ concentration from 1 to 25 mM depolarized THALs to the same extent in the absence or presence of SPM, indicating that NO does not decrease apical K+ permeability. In the presence of valinomycin in the lumen, addition of SPM to the bath decreased THAL net JCl- to the same extent as it did in control tubules, indicating that NO decreases THAL JCl- even when apical K+ permeability is increased. Finally, we found no effect of NO on the affinity of Na+-K+-ATPase for Na+ nor the maximal rate. Taken together, our data show that NO decreases JCl- by inhibiting apical Na+-K+-2Cl- cotransporter activity rather than by inhibiting apical K+ channels or basolateral Na+-K+-ATPase.

We have previously reported that NO inhibits THAL net JCl- (31). A decrease in JCl- may be caused by a decrease in ion entry through the apical membrane or by inhibition of ion exit across the basolateral membrane. To test whether NO inhibits ion entry rather than exit, we measured the effect of NO on [Na+]i and [Cl-]i. We found that NO decreases THAL [Na+]i and [Cl-]i. A decrease in [Na+]i could be caused by inhibition of Na+ entry or by increasing Na+ exit through Na+-K+-ATPase. Because we found that NO does not affect sodium pump activity, a decrease in [Na+]i can only be caused by inhibiting apical Na+ entry. Because there are two modes of Na+ entry in the THAL, the Na+-K+-2Cl- cotransporter and the Na+/H+ exchanger, we measured [Cl-]i. [Cl-]i in the THAL is determined by the balance between apical entry through the Na+-K+-2Cl- cotransporter and basolateral exit through Cl- channels (16). A decrease in [Cl-]i could be caused by decreasing Cl- entry through the Na+-K+-2Cl- cotransporter or by increasing basolateral Cl- exit. Because we have previously shown that NO inhibits net JCl- in the THAL, a decrease in [Cl-]i is most likely mediated by a decrease in apical Cl- entry through the Na+-K+-2Cl- cotransporter, and as NO decreased [Cl-]i, our data also suggest that NO does not affect basolateral Cl- channels.

Because we found that NO decreased apical ion entry, we tested whether NO inhibits Na+-K+-2Cl- cotransporter activity. By measuring [Na+]i, we determined the initial rates of Na+ transport through the Na+-K+-2Cl- cotransporter in isolated, perfused THALs. We have found that in the presence of SPM, initial rates were 49% slower than under control conditions. These data indicate that NO decreases Na+-K+-2Cl- cotransporter activity.

It has been reported that inhibition of apical K+ channels with Ba2+ inhibits THAL JCl- (17). Thus a decrease in cotransporter activity may be secondary to a decrease in apical K+ channel activity. The apical membrane of the THAL possesses a high K+ conductance, provided by the presence of at least two types of K+ channels (35). Therefore, we tested whether NO decreases apical K+ permeability by measuring the change in membrane potential caused by an increase in luminal K+ from 1 to 25 mM. Due to the high K+ permeability compared with that of other ions (9), a change in voltage caused by a switch in luminal K+ can be used to estimate apical K+ permeability. Our results show that the depolarization caused by increasing luminal K+ was the same in the absence and presence of NO, indicating that NO does not inhibit apical K+ permeability.

To ensure that a change in apical K+ permeability does not alter the effects of NO, we tested whether NO could inhibit THAL JCl- when apical K+ permeability was artificially increased. We found that, in the presence of valinomycin in the tubular lumen, NO decreases THAL JCl- to the same extent as it did in control cells. These data indicate that NO inhibits JCl- even when apical K+ permeability is increased and supports the hypothesis of a direct inhibitory effect of NO on the Na+-K+-2Cl- cotransporter.

Contrary to our results, a stimulatory effect of NO on the THAL apical 70-pS K+ channel has been observed in cell-attached patch-clamp experiments (25). Although we have no explanation for these disparate results, this discrepancy is not due to a lack of sensitivity in our measurements because we are able to detect changes in K+ permeability as small as 10%. It is possible that differences in rat age or diet, which are known to influence the hormonal state of the animals, may account for differences in the response to NO.

Na+-K+-2Cl- cotransporter activity has been shown to be regulated by different mechanisms (19, 32). However, the molecular mechanism through which NO can inhibit the Na+-K+-2Cl- cotransporter is still unknown. We have recently reported that the mechanism through which NO inhibits THAL JCl- is mediated by activation of cGMP-stimulated phosphodiesterase, which, in turn, decreases basal cAMP levels (30). Several studies indicate that stimulation of the Na+-K+-2Cl- cotransporter by hormones that increase cAMP is mediated by protein kinase A activation (26, 27). Therefore, it is possible that NO-induced inhibition of the THAL Na+-K+-2Cl- cotransporter may be mediated by a decrease in protein kinase A activity. However, further studies are needed to define the exact molecular effector that mediates NO-induced inhibition of Na+-K+-2Cl- cotransport.

Finally, to ensure that NO does not affect the sodium pump, we investigated whether NO inhibits Na+-K+-ATPase. We first tested whether NO could alter the affinity of Na+-K+-ATPase for Na+. The K1/2 of Na+-K+-ATPase for Na+ has been reported to be 10-15 mM (10). Therefore, we investigated the effect of nystatin on THAL QO2 in the presence of 10.5 mM extracellular Na+ after treatment with SPM. We found that, in the presence of either SPM or furosemide, nystatin restored QO2 to control values, indicating that NO does not affect the K1/2 of Na+-K+-ATPase for Na+. We then investigated whether NO could affect the maximal turnover of Na+-K+-ATPase or ATP production by measuring the effect of nystatin on THAL QO2 in the presence of 145 mM extracellular Na+ (normal extracellular Na+). Under these conditions, nystatin increases intracellular Na+ and generates maximal activation of Na+-K+-ATPase. We found that, in the presence of either SPM or furosemide, nystatin increased QO2 by 100%. Taken together, these data indicate that NO does not affect the K1/2 of Na+-K+- ATPase for intracellular Na+ nor maximal Na+-K+-ATPase turnover.

In agreement with the present results, we have previously reported that neither SPM nor nitroglycerin affects Na+-K+-ATPase activity in isolated cortical collecting ducts (34). However, other investigators have found different effects of NO on Na+-K+-ATPase activity. Liang and Knox (23, 24) reported that NO caused a decrease in the activity of Na+-K+-ATPase in a proximal tubule-like cell type. Guzman et al. (18) showed that the NO donor sodium nitroprusside and the peroxynitrite donor 3-morpholinosydnonimine inhibited Na+-K+-ATPase activity in cultured proximal tubule cells. A possible explanation for these disparate results could be given by the fact that a significant decrease in Na+-K+-ATPase activity is only observed after prolonged exposure to NO (1-2 h) in those studies. We found a decrease in ion entry 15 min after treatment with SPM. In addition, due to the presence of different protein kinases and protein phosphatases, the effect of NO on Na+-K+-ATPase activity could be different in the proximal compared with the distal nephron.

We have found that, in the presence of SPM, nystatin increased QO2 in tubules bathed with 145 mM Na+. These data indicate that NO by itself does not affect mitochondrial respiration, nor does it cause a decrease in ATP that is sufficient to prevent maximal Na+-K+-ATPase turnover. In agreement with these results, we have previously found that NO does not affect ATP production in cultured cortical collecting duct cells (33). However, it has been reported that NO can reversibly inhibit mitochondrial respiration in different cell types (3) and, in some cases, decrease cellular ATP concentration (1). Although we have no explanation for these differences, several factors, such as oxygen tension (4), light (2), and metabolic substrates, are known to influence the effect of NO on mitochondrial respiration. Therefore, it is possible that at the concentration of SPM we used, and in the presence of a high oxygen concentration, NO did not produce a direct effect on mitochondrial respiration.

We conclude that NO inhibits THAL JCl- by reducing apical Na+-K+-2Cl- cotransporter activity rather than by inhibiting apical K+ channels or basolateral Na+-K+-ATPase.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-28982.


    FOOTNOTES

First published August 9, 2001;10.1152/ajprenal.0075.2001

Address for reprint requests and other correspondence: J. L. Garvin, Div. of Hypertension and Vascular Research, Dept. of Internal Medicine, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202 (E-mail: jgarvin1{at}hfhs.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 March 2001; accepted in final form 6 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 281(5):F819-F825
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