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(Hypertension. 1999;34:508-513.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Nitric Oxide–Induced Inhibition of Transport by Thick Ascending Limbs From Dahl Salt-Sensitive Rats

Néstor H. García; Craig F. Plato; Barbara A. Stoos; Jeffrey L. Garvin

From the Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Mich.

Correspondence to Jeffrey L. Garvin, Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202.

	Abstract

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Abstract—The factor responsible for salt sensitivity of blood pressure in Dahl rats is unclear but presumably resides in the kidney. We tested the hypotheses that (1) thick ascending limbs of Dahl salt-sensitive rats (DS) absorb more NaCl than those of Dahl salt-resistant rats (DR) and (2) NO inhibits transport to a lesser extent in thick ascending limbs from DS. We found that basal chloride absorption (J_Cl) by thick ascending limbs from DR was 105.8±10.0 pmol · mm^-1 · min^-1 (n=6). Ten and 100 µmol/L spermine NONOate, an NO donor, decreased J_Cl in DR to 65.8±8.5 and 46.8±7.0 pmol · mm^-1 · min^-1, respectively. Basal J_Cl in DS was 131.6±13.4 pmol · mm^-1 · min^-1 (n=7). In DS, 10 and 100 µmol/L spermine NONOate decreased J_Cl to 111.5±12.8 and 46.8±6.2 pmol · mm^-1 · min^-1, respectively. No difference was observed in basal or NO-inhibited Na absorption by cortical collecting ducts or in basal or NO-inhibited oxygen consumption by inner medullary collecting ducts. Because NO acts via generation of cGMP, we measured cGMP production by thick ascending limbs from DS and DR to see whether a difference in cGMP production could account for the difference in basal or NO-inhibited transport. Basal rates of cGMP production were similar between the 2 strains. Although NO increased cGMP production by thick ascending limbs from both strains, no difference existed between DS and DR. We concluded that the reduced ability of NO to block transport in thick ascending limbs in DS may account for at least part of the salt sensitivity of blood pressure in this strain.

Key Words: absorption, chloride • absorption, sodium • kidney tubules, collecting • cyclic GMP • nephron

	Introduction

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During normal circumstances, dietary salt intake does not dramatically affect blood pressure. However, in a certain percentage of the population, referred to as salt-sensitive, ingestion of large amounts of salt leads to an increase in blood pressure. Dahl salt-sensitive rats (DS) develop hypertension when they are fed a high-sodium diet, whereas the complementary inbred strain, Dahl salt-resistant rats (DR), does not. The underlying pathology in the Dahl strain is thought to reside in the kidney, because Dahl and Heine¹ demonstrated that transplanting a kidney from a DS to a DR transferred salt sensitivity as well, whereas transplanting a kidney from a DR to a DS normalized blood pressure. Although the exact origin of the salt sensitivity in this strain has not been fully explained, recent work has given some insight into possible causes. DS have been shown to have impaired pressure natriuresis compared with DR.^{2 3} This may in part be due to a deficiency in salt absorption by the nephron,^{4 5 6} including the loop of Henle.^{4 5}

Although the cause of the disparate salt absorption by the loops of Henle of DS and DR is unclear, both P450 metabolites⁷ and NO have been implicated.^{2 8 9} Patel et al² showed that L-arginine, the substrate for NO synthase (NOS), restores pressure natriuresis to normal levels in DS. Salazar et al⁸ showed that intravenous infusion of N-nitro-L-arginine methyl ester (an NOS inhibitor) confers salt sensitivity in dogs. Chen and Sanders⁹ showed that L-arginine, but not D-arginine, lowered blood pressure in DS fed a high-salt diet and that a competitive inhibitor of NOS raised blood pressure more in DR than in DS. These reports indicate that either production of or response to NO is diminished in the DS.

Because both the collecting duct and the thick ascending limb have been implicated as expressing a defect in the DS,^{5 6} we investigated basal rates of transport by cortical and medullary thick ascending limbs and collecting ducts. Also, because a defect in either the synthesis of or the response to NO is implicated in Dahl salt sensitivity⁹ and NO inhibits nephron transport,^{10 11} we examined the effect of NO on transport in each segment. We hypothesized that basal transport is greater in thick ascending limbs, whereas inhibition of transport by NO is diminished in DS.

	Methods

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Determination of Blood Pressure
To ensure that DS developed hypertension after NaCl loading, we measured blood pressure by tail cuff. Three days after the rats arrived, their blood pressure was measured at 103±5 mm Hg in DS and 80±5 mm Hg for DR. After the rats had drunk water with 1% NaCl added for 3 weeks, the blood pressure in DS increased to 176±11 mm Hg, whereas in DR, it increased to 104±5 mm Hg.

Tubule Perfusion
Male DS and DR that weighed 130 to 150 g (Harlan) were maintained on a diet that contained 0.2% sodium and 1.1% potassium (Ralston Purina) for 5 days before use. Rats used for cortical collecting duct experiments were injected with DOC acetate (5 mg/rat IM) 5 to 9 days before the experiment to enhance sodium transport.¹¹ Rats were anesthetized with ketamine (0.1 mg/kg; Parke Davis). The peritoneal cavity was opened and flushed with cold NaCl (150 mmol/L). The left kidney was removed and placed in cold dissection medium equilibrated with 95% O₂–5% CO₂. Coronal slices were transferred to a dissection dish that contained physiological saline at 6°C to 10°C. Medullary rays were dissected from the slices, and tubules were dissected from the rays. Tubules were transferred to a perfusion chamber, mounted on concentric pipettes, and perfused at 37°C.¹⁰ All protocols were performed in accordance with the guidelines of the Henry Ford Hospital Animal Care and Use Committee. The composition of the perfusion solution (in mmol/L) was: NaCl 114.0, KCl 4.0, NaH₂PO₄ 2.5, MgSO₄ 1.2, Na₃ citrate 1.0, NaHCO₃ 25.0, alanine 6.0, Ca lactate₂ 2.0, glucose 5.5, and raffinose 5.0. The perfusion and bath solutions were generally the same, except that spermine NONOate (SPM) or nitroglycerin (NTG) was added to the bath. The osmolality of all solutions was 295±3 mOsmol/L. The pH of the bath was 7.4. Solutions were gassed with 95% O₂–5% CO₂.

Chloride Absorption
Chloride concentration was measured in samples of the bath, perfusate, and collected fluid with a newly developed ultramicrofluorometric assay.¹² Net chloride absorption (J_Cl) was calculated according to the equation

where Co_Cl is the chloride concentration in the perfusate (mmol/L), Cl_Cl is the chloride concentration in the collected fluid (mmol/L), Vo is the perfusion rate (nL/min per mm), and VL is the collection rate (nL/min per mm).

Sodium Absorption
Sodium concentration was measured in samples of the bath, perfusate, and collected fluid as described previously.¹¹ Net sodium flux was calculated with the equation above.

Suspensions of Inner Medullary Collecting Duct
Rats were anesthetized with ketamine and treated with heparin (100 U). A midline incision was made, and the aorta was cannulated below the left kidney. The submesenteric artery and suprarenal aorta were clamped, and the left kidney was flushed with 10 mL of perfusion solution that contained 0.2% type I collagenase (Sigma). The kidney was removed, and the inner medulla was minced and then incubated in 0.2% collagenase at 37°C for 60 minutes, with mixing every 5 to 10 minutes. At the end of the incubation period, 1.5 vol of distilled water was added and the suspension was filtered through a 250-µm nylon mesh. The resulting suspension was centrifuged, and the pellet was washed twice with perfusion solution.¹³

Suspensions of Thick Ascending Limbs
Rats were anesthetized as described above. Kidneys were removed and placed in perfusion solution at 4°C. The outer medulla was minced into 1-mm pieces. Tissue chunks were incubated for six 10-minute periods in 0.04% type I collagenase with gentle agitation. At the end of each incubation period, tissue was allowed to settle for 30 seconds. The supernatant was then removed and stored at 4°C. When incubation was completed, tubules were filtered through a 180-µm mesh and pelleted via centrifugation. The pellets were washed twice with perfusion solution.¹⁴

Cyclic GMP
Tubules were incubated in 95 µL of perfusion solution that contained 1 mmol/L 3-isobutyl-1-methylxanthine at 37°C for 10 minutes before 5 µL of SPM or vehicle was added to yield 10^-7 mol/L, 10^-6 mol/L, or 10^-5 mol/L. After 30 minutes, the reaction was stopped with 100 µL of methanol and the solution was stored at -80°C. cGMP was determined with a radioimmunoassay (Biomedical Technologies). On the day of the assay, samples were centrifuged, the supernatant was transferred to another tube that was dried in a Savant dryer (Forma Scientific), and the pellet was reconstituted in 110 µL of sodium acetate buffer. cGMP standards were treated similarly to the samples. Protein was measured with Coomassie blue G-250 reagent (Pierce).

Oxygen Consumption
Tubules were incubated at 37°C to eliminate the bubbles caused when cold solution is added to the oxygen consumption (QO₂) chamber and then warmed. QO₂ was measured in a sealed chamber with a small injection port by a Clark-type electrode.¹⁵ Compounds were added to the suspension as indicated in the text. QO₂ was calculated from the decrease in oxygen tension with time. Protein was measured with Coomassie blue G-250 reagent (Pierce).

Statistics
Values are reported as mean±SEM. Two-way ANOVA for repeated measures, paired t test, and Student's t test were used to test for significant differences as appropriate.

	Results

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Because it has been reported that loops of Henle from DS absorb more NaCl than those from DR ^{4 5} and NO has been implicated in salt sensitivity,^{2 8 9} we first measured basal and NO-inhibited J_Cl by cortical thick ascending limbs. Initially, we measured basal rates of transport, and then we challenged tubules with 2 concentrations of SPM (10 and 100 µmol/L), the NO donor, to investigate whether the inhibition of J_Cl induced by NO is similar in the 2 strains. Basal J_Cl in DR was 105.8±10.0 pmol · mm^-1 · min^-1 (n=6). Ten and 100 µmol/L SPM decreased J_Cl in DR to 65.8±8.5 (P<0.004) and 46.8±7.0 pmol · mm^-1 · min^-1 (P<0.002), respectively. Basal J_Cl in DS rats was 131.6±13.4 pmol · mm^-1 · min^-1 (n=7). In DS rats, 10 and 100 µmol/L SPM decreased J_Cl to 111.5±12.8 pmol · mm^-1 · min^-1 (P<0.051) and 46.6±6.2 pmol · mm^-1 · min^-1 (P<0.001), respectively. Thus, the basal rate of J_Cl was not different in thick ascending limbs from DS versus DR (131.6±13.4 versus 105.8±10.0 pmol · mm^-1 · min^-1). These data indicate that DR are more susceptible to NO inhibition than DS, because 10 µmol/L SPM inhibited J_Cl by 38.2±5.8% in DR but only 15.4±7.4% in DS (P<0.04). The difference between the effects of 100 µmol/L SPM on transport by thick ascending limbs from the 2 strains was not significant (DR 35.1±4.6% versus DS44.3±5.8%; Figure 1). Controls did not change significantly with time.

View larger version (34K): [in this window] [in a new window]   Figure 1. Differential effect of NO donated by SPM on chloride transport by thick ascending limbs from DS and DR. *P<0.04 vs DS value. DS, n=7; DR, n=6.

We also tested for a difference in the ability of NTG, a chemically distinct NO donor, to inhibit transport. J_Cl by thick ascending limbs from DS rats increased 12.1±16.4% when 1 µmol/L NTG was added to the bath ( +13.0±16.8 pmol · mm^-1 · min^-1, n=8), whereas the same concentration of NTG inhibited J_Cl by thick ascending limbs from DR rats by 26.6±5.1% ( -32.5±6.2 pmol · mm^-1 · min^-1, n=5, P<0.03 versus DS; Figure 2). Similar to SPM, a larger concentration of NTG inhibited J_Cl by thick ascending limbs to the same extent in both strains. Basal rates of transport did not differ between strains.

View larger version (13K): [in this window] [in a new window]   Figure 2. Differential effect of NO donated by NTG on chloride transport by thick ascending limbs from DS and DR. *P<0.03 vs DS value. DR, n=5; DS, n=8.

It has been reported that in primary cultures of inner medullary collecting duct cells from prehypertensive DS, Na⁺ transport is greater than in monolayers from DR⁶ ; thus, we investigated the effect of SPM on basal QO₂ as an index of active transport by suspensions of inner medullary collecting duct cells (Figure 3). Basal QO₂ was 144.5±27.5 nmol/mg protein per min in DR (n=7) and 153.8±18.0 nmol/mg protein per min in DS (n=9) (P>0.80). When suspensions of DR inner medullary collecting ducts were treated with 10^-5mol/L SPM, QO₂ decreased to 118.4±28.9 nmol · mg^-1 · min^-1 · (P<0.02). When the concentration of SPM was increased to 10^-4 mol/L, QO₂ fell to 88.4±22.5 nmol · mg^-1 · min^-1 (P<0.001). After 10^-3 mol/L SPM was added, QO₂ decreased to 74.4±20.7 nmol · mg^-1 · min^-1 (P<0.001). When suspensions of inner medullary collecting duct cells from DS were treated with 10^-5, 10^-4, and 10^-3 mol/L SPM, QO₂ decreased from 153.8±18.0 to 118.7±12.5 (P>0.01), 90.5±12.5 (P<0.005), and 77.8±14.3 nmol/mg protein per min, respectively (P<0.001).Thus, no statistically significant difference existed between the effects of SPM in the 2 strains of rats.

View larger version (38K): [in this window] [in a new window]   Figure 3. Effect of NO on QO2 by inner medullary collecting ducts. ++P<0.025; #P<0.01; ###P<0.001 vs 0 value. DR, n=7; DS, n=9.

In separate experiments, we examined ouabain-sensitive QO₂ and whether SPM inhibited only transport-related QO₂. Tubules from DR consumed oxygen at a rate of 107.9±14.7 nmol/mg protein per min. When DR were treated with 1 mmol/L ouabain, QO₂ decreased by 40.8±8.7 to 67.9±7.0 nmol · mg^-1 · min^-1 (n=9). When SPM (1 mmol/L) was added in the presence of ouabain, there was no further decrease in QO₂ (60.6±5.0 nmol · mg^-1 · min^-1). Tubules from DS consumed oxygen at a rate of 99.9±20.8 nmol/mg protein per min (not significantly different from DR). When DS was treated with 1 mmol/L ouabain, QO₂ decreased by 48.3±12.5 to 51.6±10.2 nmol · mg^-1 · min^-1 (n=8; not significantly different from DR). When SPM (1 mmol/L) was added in the presence of ouabain, there was no further decrease in QO₂ (50.6±9.8 nmol · mg^-1 · min^-1). These data indicate that (1) the rate of total QO₂ does not differ between the 2 strains; (2) ouabain-sensitive QO₂, and thus active Na transport, does not differ between the 2 strains; and (3) the effects of SPM appear to be due to only inhibition of Na transport.

Finally, we investigated basal sodium reabsorption (J_Na) and the inhibition induced by SPM in isolated perfused cortical collecting ducts from DS and DR (Figure 4). We used 10 µmol/L SPM because this concentration resulted in greater inhibition of J_Cl by thick ascending limbs in DR than in DS. Basal J_Na by cortical collecting ducts from DR was 15.0±3.0 pmol · mm^-1 · min^-1 (n=6). Basal J_Na by cortical collecting ducts from DS was 13.9±1.8 pmol · mm^-1 · min^-1 (n=5) (not significantly different from DR). When 10 µmol/L SPM was added to the bath, basal J_Na decreased equally in both strains. In cortical collecting ducts from DR, J_Na fell to 5.7±1.4 pmol · mm^-1 · min^-1 (P<0.01 versus basal). In cortical collecting ducts from DS, it decreased to 6.1±1.1 pmol · mm^-1 · min^-1 (P<0.01 versus basal; not significantly different from DR). Controls showed no significant changes with time. Thus, neither basal nor SPM-inhibited J_Na differed significantly between the 2 strains.

View larger version (33K): [in this window] [in a new window]   Figure 4. Effect of NO on JNa by the cortical collecting duct. #P<0.01 vs control. DR, n=6; DS, n=5.

Because NO regulates transport in the kidney via cGMP,^{16 17} we investigated whether the disparate effect of NO on J_Cl by thick ascending limbs from DS and DR was the result of differential intracellular cGMP production (Figure 5). Basal cGMP content was 25.6±5.2 (n=14) and 22.0±2.6 (n=11) fmol/µg protein in thick ascending limbs from DR and DS (P>0.90), respectively. cGMP content increased to 42.2±8.3 and 39.6±5.7 (P<0.02 versus basal) fmol/µg protein in thick ascending limbs from DR and DS after treatment with 0.1 µmol/L SPM. cGMP content increased to 89.9±18.8 (P<0.005 versus basal) and to 98.7±12.1 (P<0.001 versus basal) fmol/µg protein in thick ascending limbs from DR and DS after treatment with 1 µmol/L SPM. After treatment with 10 µmol/L SPM, cGMP content of thick ascending limbs from DR and DS was 88.0±16.1 (P<0.005 versus basal) and 104.7±11.4 (P<0.001 versus basal) fmol/µg protein. Thus, SPM increased cGMP content in thick ascending limbs of DS and DR to a similar extent at all concentrations tested, including concentrations that induced different effects on J_Cl.

View larger version (39K): [in this window] [in a new window]   Figure 5. Effect of NO on cGMP production by thick ascending limbs. ++P<0.025; ##P<0.005; ###P<0.001 vs 0 value. DR, n=14; DS, n=11.

	Discussion

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To the best of our knowledge, this is the first report that shows that in isolated microperfused thick ascending limbs, basal J_Cl in DS is similar to DR when both are on a low-salt diet, but NO is less effective at inhibiting J_Cl by thick ascending limbs from DS versus DR. Basal J_Na by cortical and inner medullary collecting ducts was the same in both strains of rats and was inhibited by the same magnitude by NO. In addition, NO-induced increases in cGMP content in the thick ascending limb of DR and DS were not significantly different.

The cause of salt-sensitive hypertension is not completely understood. However, a common abnormality appears to be a defect in renal salt excretion that can lead to initial volume expansion and hypertension. Several hormones and factors have been proposed to explain the differing ability of DS and DR to eliminate a salt load,^{18 19 20 21 22 23} including NO.^{2 4 9} In vivo data indicate that NO can induce natriuresis and diuresis without altering glomerular filtration rate or renal blood flow, which shows that NO directly alters tubular transport.²⁴ This study and others have directly demonstrated that NO inhibits transport by various nephron segments, including the proximal tubule,²⁵ thick ascending limb,^{26 27} cortical collecting duct,¹⁰ and inner medullary collecting duct.¹⁷ In addition, inhibition of NO synthesis results in salt-sensitive hypertension.^{8 28} As a result, we tested whether the response to NO is different in the thick ascending limb and collecting duct. We found that NO decreased transport in the thick ascending limb, cortical collecting duct, and inner medullary collecting duct. However, we only observed a difference in the ability of NO to inhibit J_Cl by thick ascending limbs from DS and DR. SPM at 10 µmol/L reduced chloride reabsorption by only 15% in DS versus 38% for DR. This concentration of SPM will provide 50 nmol/L NO.¹⁰ A higher concentration of NO inhibited J_Cl to the same extent in thick ascending limbs from both strains. To ensure that the difference in the sensitivity of J_Cl to NO donated by SPM was due to NO and not artifactual, we also tested for a difference in the ability of NTG, a chemically distinct NO donor, to inhibit transport. J_Cl by thick ascending limbs from DS increased slightly when 1 µmol/L NTG was added to the bath, whereas J_Cl by thick ascending limbs from DR was inhibited by this concentration of NTG by 35%. Like SPM, a greater concentration of NTG inhibited chloride absorption by thick ascending limbs to the same extent in both strains. Thus, the defect that renders Dahl rats salt-sensitive may be due in part to the diminished response of the thick ascending limb to NO.

Our results may provide an explanation for the in vivo data that shows that salt sensitivity in DS can be corrected with L-arginine.^{2 9} These reports imply that the defect is due to reduced production of NO. However, they can also be explained by a decreased response to NO, as indicated by our data that shows that if the concentration of NO is high enough, there is no difference in its ability to inhibit chloride absorption by thick ascending limbs. Our findings appear to be at odds with reports implicating the kallikrein/kinin system²⁰ and 20-HETE.²¹ However, both kinins and arachidonic acid metabolites may affect the endogenous NO-generating system of the thick ascending limb.²⁷ Kinins are known to stimulate the release of NO in the kidney,²⁴ whereas 20-HETE purportedly counters the effects of NO. Thus, the diminished ability of NO to reduce chloride transport in the thick ascending limb may be due to increased production of a factor that counters its action, such as 20-HETE.

It is difficult to develop a rational explanation for a link between NO and dopamine^{19 22} or atrial natriuretic factor (ANF),²³ which have also been implicated in salt-sensitive hypertension in the Dahl rat by other investigators. However, note that Appel and Dunn,²³ who reported that collecting ducts from DS produce less cGMP in response to ANF than tubules from DR, found a difference only at a concentration of 1 µmol/L ANF, which is not in the physiological range.

Our data indicate that at least part of the defect responsible for salt-sensitive hypertension in the DS may be due to an increase in NaCl absorption by the thick ascending limb. These data are consistent with reports that show that J_Cl by the loop of Henle is greater in DS than in DR ^{4 5 29} and also with in vitro microperfusion studies that showed no difference in cortical collecting duct Na absorption between the 2 strains.³⁰ Interestingly, the thick ascending limb has been implicated in development of hypertension in the Milan hypertensive rat, which shows an increase in Na/K/2 Cl cotransport³¹ and Na,K-ATPase ³² compared with normotensive strains. We did not study the concentration-dependent response of the cortical collecting duct, because rats used in these experiments must be treated with DOC acetate. Consequently, a differential effect or the lack of a differential effect of NO on this segment cannot be unambiguously interpreted.

In contrast to the data that show that the thick ascending limb is involved in salt-sensitive hypertension in the Dahl rat, Husted et al⁶ reported that cultured inner medullary collecting duct cells from DS reabsorb more Na than those from DR. We found no difference in ouabain-sensitive QO₂ (a measure of Na transport) by inner medullary collecting ducts from DS and DR. Similarly, we found no difference in the ability of NO to inhibit QO₂. The explanation for the difference between our results and the results from Husted and colleagues may be that we studied freshly isolated tubules, whereas Husted et al⁶ studied cultured cells. Culture conditions may affect DS inner medullary collecting duct cells differently from DR. Culture conditions can alter the phenotype, and various types of cells are often affected differently when culture conditions are changed.³³ Finally, because the Dahl rat is an inbred strain, substrains may have developed because of inbreeding at different locations by different vendors. Dahl rat substrains might have different causes of hypertension. Recently, Dahl rats from Harlan were outbred, and at least 2 substrains have been reported³⁴ ; thus, the exact nature of the defect may depend on the vendor and generation number.

Along the nephron, NO acts via generation of cGMP.^{16 17} Consequently, we tested whether cGMP production by thick ascending limbs differed between the 2 strains. We found that cGMP production became saturated at a concentration of SPM that did not maximally inhibit transport. These data indicate that either (1) NO is not acting via cGMP to inhibit transport or (2) cGMP degradation is different between the 2 strains. In our experiments, phosphodiesterase activity was inhibited by 3-isobutyl-1-methylxanthine; therefore, we cannot eliminate the latter possibility. In addition, we found no difference between basal and NO-stimulated cGMP production. These data indicate that either the defect may reside at some step in the second messenger cascade beyond cGMP, such as at the level of cGMP-dependent protein kinase; NO may activate a different second messenger cascade; or an intrinsic defect exists in either the Na/K/2 Cl cotransporter or Na,K-ATPase. Additional investigation is required to resolve this issue.

In conclusion, we found that basal chloride reabsorption is not significantly different in the thick ascending limb of salt-sensitive and salt-resistant Dahl rats and that NO-induced inhibition of chloride reabsorption is deficient in this model. Because cGMP production was the same in both strains, the abnormality may reside at the level of cGMP-dependent protein kinase or at the site of its phosphorylation. Basal and NO-inhibited transport in the cortical and inner medullary collecting ducts were the same in both strains.

	Acknowledgments

 
This work was supported in part by a grant from the National Institutes of Health (HL-28982). J.L.G. was supported in part by a Research Career Development Award from the National Institutes of Health (HL-02891) during this study.

Received March 10, 1999; first decision March 29, 1999; accepted May 15, 1999.

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