Nitric oxide inhibits transcription of the Na+-K+-ATPase alpha 1-subunit gene in an MTAL cell line

Bruce C. Kone and Sandra Higham

Departments of Internal Medicine and Integrative Biology, Pharmacology, and Physiology, University of Texas Medical School at Houston, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) has been implicated as an autocrine modulator of active sodium transport. To determine whether tonic exposure to NO influences active sodium transport in epithelial cells, we established transfected medullary thick ascending limb of Henle (MTAL) cell lines that overexpressed NO synthase-2 (NOS2) and analyzed the effects of deficient or continuous NO production [with or without NG-nitro-L-arginine methyl ester (L-NAME) in the culture medium, respectively] on Na+-K+-ATPase function and expression. The NOS2-transfected cells exhibited high-level NOS2 expression and NO generation, which did not affect cell viability or cloning efficiency. NOS2-transfected cells were grown in the presence of vehicle, NG-nitro-D-arginine methyl ester (D-NAME), or L-NAME for 16 h, after which 86Rb+ uptake assays, Northern analysis, or nuclear run-on transcription assays were performed. The NOS2-transfected cells allowed to produce NO continuously (vehicle or D-NAME) exhibited lower rates of ouabain-sensitive 86Rb+ uptake (~65%), lower levels of Na+-K+-ATPase alpha 1-subunit mRNA (~60%), and reduced rates of de novo Na+-K+-ATPase alpha 1-subunit transcription compared with L-NAME-treated cells. These results have uncovered a novel effect of NO to inhibit transcription of the Na+-K+-ATPase alpha 1-subunit gene.

sodium pump; nitric oxide synthase; gene expression; kidney; sodium transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is a free radical gas that exerts potent effects on essential biological functions (36), including the regulation of ion transport in epithelia (22, 46). Recent data in animals and humans have documented that NO plays a critical role in the homeostatic control of renal sodium excretion and the extracellular fluid volume (23). Intrarenal NO synthesis is increased during periods of increased salt intake to facilitate natriuresis and maintain normal blood pressure (31, 41, 44, 49). Conversely, impaired NO synthesis or action appears to contribute to the maladaptive renal sodium handling that results in salt-sensitive hypertension (16, 53). Although many of these responses appear to be mediated by direct or indirect actions on the renal microcirculation, NO also exerts direct effects on solute and water transport by the renal tubules. For example, in vivo animal studies have shown that acute or chronic administration of NO synthase (NOS) inhibitors to animals, at concentrations that do not measurably affect glomerular or systemic hemodynamics, impairs urinary sodium excretion (41, 44, 49), whereas NO donors promote natriuresis (30). In humans, chronic administration of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) resulted in a 40% reduction in fractional excretion of sodium (3).

In vitro studies with NOS inhibitors and NO donors have shown that NO can inhibit the functional activity of transport proteins involved in transepithelial Na+ reabsorption (46). For example, NO inhibited Na+ entry in the cortical collecting duct (45) and Na+/H+ exchange in the proximal tubule (40, 50). NO was also shown to inhibit Na+-K+-ATPase activity in renal medullary slices (32), kidney enzyme preparations (6), and mouse proximal tubule cells that had been stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma (14). The mechanisms by which NO donors inhibit Na+-K+-ATPase activity in renal tissues include cGMP-dependent and -independent mechanisms (14, 32). The cGMP-independent effects on enzymatic activity might be mediated by the blockade of sulfhydryl groups within the enzyme, because the sulfhydryl reagents dithiothreitol and cysteine restored Na+-K+-ATPase activity and thiol content after exposure to thiol-containing NO derivatives (6). The possibility that NO might act at more proximal steps of Na+-K+-ATPase gene expression, such as gene transcription, however, has not been explored despite the fact that NO is known to influence the activities of transcription factors (7, 27, 38, 43, 47) that commonly regulate other genes.

To investigate the effects of constitutive NO production on the Na+-K+-ATPase, and by inference, transepithelial sodium transport, we used gene transfer to overexpress the gene encoding murine NO synthase-2 (NOS2; also known as "inducible" NOS) in a medullary thick ascending limb of Henle (MTAL) cell line that lacks basal NOS expression (25). This strategy eliminated the myriad of confounding cellular effects of immunoactive agents needed to induce NOS2 gene expression, allowed endogenously produced NO to interact with cellular constituents, and permitted the analysis of Na+-K+-ATPase biosynthesis and function at multiple levels. The results indicate that endogenously produced NO inhibits the expression and activity of the Na+-K+-ATPase, in large part, by constraining Na+-K+-ATPase alpha 1-subunit gene transcription. This previously unrecognized effect of NO may contribute to the known natriuretic and diuretic effects of NO in vivo.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. L-Glutamine, heat-inactivated fetal bovine serum (FBS), penicillin, streptomycin, G418, and DMEM lacking phenol red were from GIBCO (Grand Island, NY). LPS from Escherichia coli O111:B4, NG-nitro-D-arginine methyl ester (D-NAME), and L-NAME were from Sigma Chemical (St. Louis, MO). Radiochemicals were purchased from Amersham (Arlington Heights, IL). The Tfx-50 reagent was purchased from Boehringer Mannheim (Germany). RNAzol II was acquired from Tel-Test (Friendswood, TX). Mouse monoclonal antibody (MAb) against murine NOS2 was from Transduction Laboratories (Lexington, KY). Mouse MAb against Tamm-Horsfall glycoprotein was provided by Dr. John Hoyer (Children's Hospital, Philadelphia, PA). Affinity-purified antipeptide IgG directed against the 5-hydroxytryptamine 5-HT1A receptor was obtained from Dr. John Raymond (Duke University, Durham, NC). Mouse recombinant IFN-gamma was from Genzyme (Cambridge, MA).

Cell culture. The MTAL-derived cell line ST-1 (15, 25), a gift from Dr. Adam Sun, was maintained in DMEM supplemented with 10% FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin (complete medium). The mouse macrophage cell line RAW 264.7 (American Type Culture Collection) was grown in complete medium. For induction of NOS2, RAW 264.7 cells were treated with LPS (100 ng/ml) and IFN-gamma (0.5 U/ml) for 16 h before study.

Transfection procedures and selection of clones. The encoding DNA for murine NOS2 (25) was cloned into the Hind III and Sal I sites of the mammalian expression vector pcDNA3.1 (Invitrogen) downstream of the cytomegalovirus (CMV) promoter to yield the recombinant molecule pcDNA3.1/NOS2. Subconfluent ST-1 cells were grown on 10-mm culture dishes and transfected with pcDNA3.1 (as a vector control) or pcDNA3.1/NOS2 with the Tfx-50 reagent. Briefly, 10 µg of plasmid DNA and 22 µl of Tfx-50 reagent were mixed with 6 ml DMEM. The mixture was added to the monolayers and incubated for 2 h at 37°C in a 5% CO2 incubator. Twelve milliliters of prewarmed complete medium were then added to the cultures. After 48 h, the medium was replaced with complete medium containing 5 mM L-NAME and 600 µg/ml G418. L-NAME (5 mM) was included in the culture medium during the selection process to inhibit the activity of the overexpressed NOS2 enzyme, thus eliminating potential NO cytotoxicity during the selection of individual clones.1 The L-NAME-G418 medium was replaced every 3 days until individual resistant colonies were isolated and established in culture as individual lines. All lines were maintained in L-NAME-G418 medium and frozen after one to three in vitro passages. For the identification of NOS2 gene-positive cells, cells of each clonal cell line were plated into individual wells of 24-well plates. Once confluent, the cells were washed and incubated with complete medium lacking L-NAME. After specified times, culture supernatants were collected for nitrite analysis. We selected pcDNA3.1/NOS2-transfected cell lines that expressed functional NOS2, as determined by nitrite assays and immunoblot analysis, for further study.

Nitrite measurements. Nitrite, the stable metabolite of NO, was measured in culture supernatants by a modification of the Griess reaction (25).

Preparation of cell lysates and immunoblot analysis. Cell monolayers were washed twice with 10 ml ice-cold homogenization buffer (250 mM sucrose, 50 mM Tris · HCl, pH 7.4, 1 mM EGTA) and then scraped into 3 ml homogenization buffer. The cells were pelleted by centrifugation, and the pellet was resuspended in 1 ml lysis buffer (140 mM NaCl, 10 mM Tris · HCl, 1.5 mM MgCl2, 0.5% NP40, pH 8.6) and incubated on ice for 5 min. Homogenates were centrifuged at 10,000 g for 20 s at 4°C, and the resulting supernatants were collected, stored, and frozen at -80°C until used. The protein concentrations of the lysates were measured by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Twenty-milligram samples were diluted in electrophoresis sample buffer, boiled for 5 min, and electrophoresed through 0.1% SDS-7.5% polyacrylamide gels. The proteins were electrophoretically transferred to nitrocellulose membranes (Hybond ECL; Amersham). Equality of sample loading and blotting was verified by Ponceau S staining of the membranes. The blots were quenched in blocking solution (1% BSA in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, incubated for 1 h at room temperature with antibodies (5 µg/ml) against NOS2, Tamm-Horsfall glycoprotein, or the 5-HT1A receptor in blocking solution, and then washed extensively with a solution containing 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20. The antigen-antibody complexes were detected by the enhanced chemiluminescence protocol (ECL; Amersham) using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG, as appropriate.

Indirect immunofluorescence microscopy. ST-1 cells were plated on glass coverslips in 35-mm culture dishes and grown to confluence. Cells were fixed and permeabilized with methanol:acetone (1:1) at -20°C for 10 min, blocked in PBS containing 1% BSA (PBS-BSA) for 1 h at room temperature, and incubated for 1 h at room temperature with 0.5 µg/ml anti-NOS2 antibody in PBS-BSA. The primary antibody was removed, and, after three washes in PBS-BSA, the cells were incubated with 5 µg/ml FITC-conjugated goat anti-mouse IgG in PBS-BSA at room temperature for 1 h. The cells were washed for 30 min in several changes of PBS-BSA and mounted on glass slides in mounting medium. The sections were examined and photographed with a Nikon Labophot inverted fluorescence microscope.

86Rb+ uptake. Uptake of 86Rb+, a K+ congener, was measured in cells grown in 24-well plates according to a published protocol (24). Cells grown on 24-well plates in the presence of vehicle, D-NAME, or L-NAME for 16 h were preincubated at 37°C for 30 min with assay buffer containing (in mM) 140 NaCl, 1 KCl, 5.5 glucose, 1 MgSO4, 2 CaCl2, 2.5 NaH2PO4, and 10 HEPES, pH 7.4, containing vehicle, 1 mM ouabain, 0.1 mM Sch-28080, 0.2 mM bumetanide, or combinations of the inhibitors as indicated in RESULTS. Uptake was initiated by the addition of 0.2 ml uptake buffer containing ~1 µCi/ml 86Rb+ and vehicle or transport inhibitor (ouabain, Sch-28080, bumetanide) to each well. After 15 min at 37°C, the uptake was stopped by six rapid washes with ice-cold stop buffer (100 mM MgCl2, 10 mM Tris-HEPES, pH 7.4). Parametric studies indicated that this time point was in the linear range of uptake. The cells were solubilized in 2% SDS-0.1 N NaOH, and the resulting extracts were measured for 86Rb+ by Cherenkov radiation. To determine the absolute rates of bumetanide-sensitive and ouabain-sensitive 86Rb+ uptake, we assayed the extracts for protein content by the BCA protein assay reagent (Pierce), and normalized the uptake rates to these values. For the experiments to test the potential inhibitory effects of Sch-28080, the data were normalized to the wells. Triplicate or quadruplicate measurements were obtained in each uptake condition and represent a single observation.

Northern analysis. ST-1 cells were grown on 150-mm plates in the presence of vehicle, D-NAME, or L-NAME for 16 h at 37°C. Thereafter, RNA isolation and Northern blotting were performed according to our previously published methods (24), except that the blots were sequentially hybridized with 32P-labeled cDNA probes specific for the rat Na+-K+-ATPase alpha 1-subunit (nucleotides 3207-3529) (8) and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (nucleotides 469-984) (24) were used. The blots were stripped for analysis of Na+-K+-ATPase alpha 1-subunit mRNA before proceeding to the GAPDH analysis. The blots were washed to a final stringency of 0.1× SSC, 0.1% SDS (where 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) at 60°C. Autoradiographs of the blots were prepared at 70°C.

Nuclear run-on transcription. ST-1 cells were grown on 150-mm plates in the presence of vehicle, D-NAME, or L-NAME for 16 h at 37°C. Isolation of nuclei and the nuclear run-on transcription assay in the presence of [alpha -32P]UTP were performed as previously described (34). cDNA inserts specific for rat Na+-K+-ATPase alpha 1 (nucleotides 3207-3529) and rat beta -actin (nucleotides 2498-2765) were denatured and immobilized to nitrocellulose membranes with a dot-blot apparatus. Equal incorporated counts of 32P-labeled nuclear RNA were hybridized to the membranes at 60°C for 24 h, after which the filters were washed and exposed to X-ray film at -80°C for 72 h.

Data analysis. The intensities of bands on the autoradiograms were measured by whole-band densitometry software running on a SPARC Station IPC (Sun Microsystems, Mountain View, CA) equipped with an image analysis system (Bio Image, Ann Arbor, MI). Quantitative data are presented as means ± SE and were tested for significance by ANOVA. For P < 0.05, we used the post hoc Bonferroni-Dunn test to establish significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Additional phenotypic characterization of ST-1 cells. We previously reported that, like the MTAL in vivo, ST-1 cells express immunoreactivity for the 5-HT1A receptor and Tamm-Horsfall glycoprotein (25). To determine whether ST-1 cells also express a bumetanide-sensitive K+ uptake mechanism typical of the MTAL in vivo, we measured 86Rb+ uptake of confluent monolayers of ST-1 cells in the presence and absence of 0.2 mM bumetanide. Bumetanide inhibited 86Rb+ uptake by ~40% compared with vehicle-treated controls (16.6 ± 2.0 vs. 27.6 ± 1.8 nmol · mg protein-1 · min-1, respectively; n = 4, P < 0.05). This result is compatible with and extends the scope of earlier work showing that ST-1 cells express [3H]bumetanide binding sites at their apical membranes (15).

A recent study demonstrated the presence of Sch-28080-sensitive, ouabain-sensitive K+-ATPase activity in permeabilized MTAL segments microdissected from rats (54). In that study, Sch-28080, at a concentration of 0.1 mM, virtually abolished this K+-ATPase activity. To determine if ST-1 cells express a similar K+ transport mechanism, we tested the effects of 0.1 mM Sch-28080 on 86Rb+ uptake in confluent monolayers of ST-1 cells. Sch-28080 had no significant effect (NS) on total 86Rb+ uptake (4,015 ± 75 vs. 4,187 ± 99 cpm · well-1 · min-1 for controls; n = 4, P = NS) and showed no significant additive inhibition with ouabain (2,479 ± 65 vs. 4,280 ± 109 cpm · well-1 · min-1 for controls; n = 4, P = NS). These results indicated that the Sch-28080-sensitive component of 86Rb+ uptake is not expressed in ST-1 cells under these experimental conditions.

Transfection and selection of NOS2-expressing ST-1 cells. To establish stable transfectants, we transfected ST-1 cells with either pcDNA3.1 (as a vector control) or pcDNA3.1/NOS2 plasmids and selected them in medium containing G418. We switched G418-resistant clones to L-NAME-free medium and screened them for nitrite production and for NOS2 protein expression by Western blotting and indirect immunofluorescence microscopy using an anti-NOS2 MAb.

As shown in Fig. 1A, ST-1 cells transfected with pcDNA3.1 produced negligible amounts of nitrite, whereas the pcDNA3.1/NOS2-transfected cells tonically produced micromolar amounts of nitrite. The nitrite production by the pcDNA3.1/NOS2-transfected cells was virtually abolished by 5 mM L-NAME, indicating that the nitrite was produced from NOS.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Constitutive nitric oxide (NO) production and NO synthase-2 (NOS2) expression in NOS2-transfected ST-1 cells. A: basal nitrite production (as measured by modified Griess reaction of culture supernatants) of ST-1 cells stably transfected with pcDNA3.1 (vector control) or pcDNA3.1/NOS2 (n = 4). ST-1 cells transfected with pcDNA3.1 produced negligible amounts of nitrite, whereas pcDNA3.1/NOS2-transfected cells tonically produced micromolar amounts of nitrite. Nitrite production by pcDNA3.1/NOS2-transfected cells was virtually abolished by 5 mM L-NAME. * P < 0.05 vs. pcDNA3.1 and # P < 0.05 vs. pcDNA3.1/NOS2 + L-NAME. B: immunoblot of cell lysates with anti-NOS2 antibody (n = 3). Cell lysates were resolved by SDS-PAGE, blotted to nitrocellulose, and probed with anti-NOS2 monoclonal antibody (MAb) and rabbit anti-mouse IgG-horseradish peroxidase. Bound antibodies were detected by enhanced chemiluminescence (ECL) method. NOS2 is ~130-kDa protein. Lane 1, control RAW 264.7 cells; lane 2, RAW 264.7 cells treated for 16 h with lipopolysaccharide (LPS) + interferon (IFN)-gamma ; lane 3, ST-1 cells stably transfected with pcDNA3.1; lane 4, ST-1 cells stably transfected with pcDNA3.1/NOS2. Nos. at left in kDa. C: indirect immunofluorescence microscopy of ST-1 cells stably transfected with pcDNA3.1/NOS2 using anti-NOS2 MAb and rabbit anti-mouse IgG-FITC (n = 3).

Immunoblots of cell lysates prepared from the control and NOS2-expressing cell lines revealed that the pcDNA3.1/NOS2-transfected cells produced an abundant 130-kDa protein immunoreactive with an anti-NOS2 MAb, whereas no such protein was detected with the pcDNA3.1-transfected cells (Fig. 1B). By indirect immunofluorescence microscopy, the NOS2 immunoreactivity was distributed throughout the cytoplasm of the pcDNA3.1/NOS2-transfected cells (Fig. 1C). Like the naive ST-1 cells, the pcDNA3.1/NOS2-transfected cells retained expression of immunoreactivity for Tamm-Horsfall glycoprotein and the 5-HT1A receptor and did not exhibit a component of 86Rb+ uptake that was sensitive to 0.1 mM Sch-28080 (data not shown).

Effect of endogenous NO production on Na+ pump activity. To test the effects of tonic NO production on Na+-K+-ATPase function, we grew pcDNA3.1/NOS2-transfected ST-1 cells for 16 h in the presence of vehicle, L-NAME, or the inactive stereoisomer D-NAME. This maneuver created "NO-deficient" (L-NAME) and "NO-replete" (vehicle or D-NAME) conditions. Cell viability, as indexed by trypan blue exclusion and cloning efficiency, was not different between the NO-replete and NO-deficient cells (data not shown), indicating that prolonged exposure to NO in this system was not grossly cytotoxic. After the incubation period, the cells were first assayed for ouabain-sensitive 86Rb+ uptake to assess Na+-K+-ATPase activity in the intact cell. As shown in Fig. 2, pcDNA3.1/NOS2 ST-1 cells grown in the presence of vehicle or D-NAME exhibited rates of ouabain-sensitive 86Rb+ uptake that were ~35% lower than the cells grown in the presence of L-NAME. Moreover, no Sch-28080-sensitive component of 86Rb+ uptake was observed in the vehicle-treated (3,610 ± 123 vs. 3,420 ± 84 cpm · well-1 · min-1, control and Sch-28080-treated, respectively; n = 4, P = NS) or L-NAME-treated cells (4,797 ± 203 vs. 4,637 ± 114 cpm · well-1 · min-1, control and Sch-28080-treated, respectively; n = 4, P = NS). Therefore, the greater rate of ouabain-sensitive 86Rb+ uptake in the L-NAME-treated cells reflects enhanced Na+-K+-ATPase activity. In the aggregate, these data indicate that constitutive NO production inhibited sodium pump activity or expression.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   Ouabain-sensitive 86Rb+ uptake by NOS2-transfected ST-1 cells. ST-1 cells stably transfected with pcDNA3.1/NOS2 were incubated with vehicle, 5 mM NG-nitro-D-arginine methyl ester (D-NAME), or 5 mM NG-nitro-L-arginine methyl ester (L-NAME) for 16 h. 86Rb+ uptake was then measured in presence or absence of 1 mM ouabain as described in MATERIALS AND METHODS. 86Rb+ uptake inhibited by 1 mM ouabain was recorded as ouabain-sensitive (O.S.) component and taken to represent transport mediated by Na+-K+-ATPase. Value for L-NAME-treated cells (17.2 ± 1.2 nmol · min-1 · mg protein-1) was designated as 100%. Values are means of 5 observations. * P < 0.05 vs. L-NAME-treated cells.

Endogenous NO production inhibits expression of the Na+-K+-ATPase alpha 1-subunit gene. Diminished rates of ouabain-sensitive 86Rb+ uptake could represent an inhibitory effect of NO on mitochondrial ATP production, Na+ entry, Na+-K+-ATPase itself, or Na+-K+-ATPase gene expression. To determine whether the inhibitory effect of endogenous NO on sodium pump function occurred at the level of Na+-K+-ATPase gene expression, we perfomed Northern analysis to compare steady-state levels of Na+-K+-ATPase alpha 1-subunit mRNA in NOS2-transfected cells grown in the presence of vehicle, D-NAME, or L-NAME. As shown in Fig. 3, A and B, the abundance of Na+-K+-ATPase alpha 1-subunit mRNA in the cells exposed to vehicle or D-NAME was ~40% less than in the cells studied in the presence of L-NAME. GAPDH mRNA levels were not significantly different among the three groups, indicating that the effect of NO to suppress Na+-K+-ATPase alpha 1-subunit gene expression was not generalized. Because the effect of NO to limit Na+ entry could conceivably downregulate the Na+-K+-ATPase alpha 1-subunit gene, we used amiloride and bumetanide to inhibit Na+ entry mediated by epithelial Na+ channels, Na+/H+ exchangers, and the Na+-K+-2Cl+ cotransporter, and then measured the abundance of Na+-K+-ATPase alpha 1-subunit mRNA. As seen in Fig. 3C, incubation of NOS2-transfected cells studied in the presence of L-NAME together with 500 µM amiloride or 100 µM bumetanide for 16 h did not significantly alter steady-state levels of Na+-K+-ATPase alpha 1-subunit mRNA. A similar lack of effect of amiloride and bumetanide was observed in pcDNA3.1-transfected cells (n = 3, data not shown). Therefore, the inhibitory effect of NO on the expression of Na+-K+-ATPase alpha 1-subunit mRNA appears to be direct and unrelated to changes in amiloride- or bumetanide-inhibitable Na+ entry mechanisms.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of constitutive NO production on mRNA abundance of the Na+-K+-ATPase alpha 1-subunit. A: representative Northern blot (n = 5) of total RNA harvested from NOS2-transfected ST-1 cells preincubated with vehicle, 5 mM D-NAME, or 5 mM L-NAME for 16 h. Blots were hybridized sequentially with 32P-labeled cDNA probes for Na+-K+-ATPase alpha 1 (NKalpha 1) subunit and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: histogram showing results of densitometric analysis of Northern blots. Ratio of relative optical density (OD) of Na+-K+-ATPase alpha 1-subunit and GAPDH transcript bands are plotted, with value for L-NAME-treated cells designated as 100%. * P < 0.05 vs. L-NAME-treated cells. C: representative Northern blot (n = 3) of total RNA harvested from NOS2-transfected ST-1 cells preincubated with 500 µM amiloride or 100 µM bumetanide for 16 h. Blots were hybridized sequentially with 32P-labeled cDNA probes for NKalpha 1-subunit and GAPDH.

Endogenous NO production inhibits transcription of the Na+-K+-ATPase alpha 1-subunit gene. To determine whether the diminished expression of Na+-K+-ATPase alpha 1-subunit mRNA in the untreated NOS2-transfected cells resulted from lower rates of de novo gene transcription, we performed nuclear run-on transcription assays using nuclei isolated from NOS2-transfected cells treated with vehicle, D-NAME, or L-NAME for 16 h. The NOS2-transfected cells treated with vehicle or D-NAME exhibited significantly lower rates of Na+-K+-ATPase alpha 1-subunit gene transcription compared with those treated with L-NAME (Fig. 4). In contrast, all three groups exhibited comparable rates of beta -actin gene transcription (Fig. 4).


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of constitutive NO production on transcription of NKalpha 1-subunit gene. Nuclei were harvested from NOS2-transfected ST-1 cells preincubated in presence of vehicle, 5 mM D-NAME, or 5 mM L-NAME for 16 h. Run-off transcription rates were then measured directly as described in MATERIALS AND METHODS. 32P-labeled nuclear RNAs were hybridized to filter immobilized DNAs of rat NKalpha 1-subunit and beta -actin. Results shown are representative of 3 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The introduction of a functionally active NOS2 gene into cultured ST-1 cells allowed us to examine the effects of constitutive, high-output NO production on epithelial cell function without the confounding effects of LPS and cytokines needed to induce NOS2 expression, the limitations of NO donors (4, 19), or vascular effects that might indirectly modulate epithelial function. By using L-NAME to control the operation of NOS2 in these cells, we were able to explore, during a defined time period, the effects of continuously produced NO on Na+-K+-ATPase expression and function. The concordance of our ouabain-sensitive 86Rb+ uptake, Northern blot, and nuclear run-off transcription data indicates that the effect of NO in this model system is exerted principally on the regulation of Na+-K+-ATPase alpha 1-subunit gene expression rather than directly on the enzyme itself. Our results indicate that NO tonically produced by these cells selectively suppresses Na+-K+-ATPase alpha 1-subunit gene transcription and, consequently, sodium pump activity. This novel effect of NO may contribute to the known natriuretic and diuretic actions of NO in vivo, particularly under conditions in which the kidney is exposed to endogenous or exogenous inflammatory mediators known to provoke NOS2 expression.

The fact that expression of GAPDH (Fig. 3) and beta -actin (Fig. 4) transcripts was not affected by NO under the experimental conditions employed suggests that the inhibitory effect of NO on the Na+-K+-ATPase alpha 1-subunit gene does not result from a generalized suppression of the transcriptional machinery of the cell, but rather involves mechanisms that are necessary, and possibly specific, for transcription of the Na+-K+-ATPase alpha 1-subunit gene. In an in vivo model of NO excess, S. H. Liu and T. J. Sheu (26) reported reduced protein expression of Na+-K+-ATPase alpha 1-subunit, but not of alpha 2- or alpha 3-subunits, in the sciatic nerves of rats treated with LPS. These data suggest that the inhibitory effect of NO may be selective for the Na+-K+-ATPase alpha 1-subunit and may occur in other tissues. However, because LPS regulates the expression of numerous genes, further studies are needed to determine whether continuous NO exposure itself alters the expression of the Na+-K+-ATPase alpha 2-, alpha 3-, and alpha 4-subunits. Additionally, the present study did not specifically examine the effects of NO on other aspects of Na+-K+-ATPase bioregulation, which include pretranslational, translational, and posttranslational controls of alpha - and beta -subunit expression, as well as controls of alpha -beta -subunit assembly, subcellular distribution, and phosphorylation state of the holoenzyme (5, 10).

Because blockade of Na+ entry pathways with amiloride and bumetanide did not mimic the decrement in the steady-state abundance of Na+-K+-ATPase alpha 1-subunit mRNA observed in the NOS2-transfected cells treated with L-NAME, the NO effect on Na+-K+-ATPase alpha 1-subunit transcription in the NOS2-transfected ST-1 cells is largely independent of, but perhaps additive to, physiological changes in Na+ entry. It is noteworthy that NO has been shown to inhibit Na+/H+ exchange (40) and Na+-selective cation channels (9, 13, 17) in other epithelial cell types. Because the production of NO in our system was unregulated and, at micromolar concentrations, exceeded the concentrations of NO typically used for the signaling actions of the molecule, it is not known whether NOS2-generated NO production might coordinately regulate Na+ entry and Na+-K+-ATPase activity in response to changes in body sodium balance. The finding that NOS2 protein levels in the outer and inner medullae of rats maintained on a high-NaCl diet were 50% greater than in these renal zones of rats fed a low-NaCl diet (31) suggests such a possibility.

Our results parallel and extend those of Guzman and co-workers (14), who studied Na+-K+-ATPase activity in mouse proximal tubule cells pretreated with LPS and IFN-gamma . These authors observed that Na+-K+-ATPase activity decreased beginning 4 h after LPS and IFN-gamma treatment, presumably reflecting the time needed for induction of NOS2 protein expression, but that maximal inhibition was achieved after 24 h. The latter result suggests that the cumulative dose or duration of exposure to NO was important. The magnitude of inhibition (34%) of Na+-K+-ATPase activity observed in their study is comparable to the ~35% inhibition reported here. Because it was not determined whether Na+-K+-ATPase alpha 1-protein or mRNA abundance were also reduced, it is not known whether the effect observed in their studies was related to effects on Na+-K+-ATPase alpha -subunit gene expression.

We did not identify any Sch-28080-sensitive component of 86Rb+ uptake in either naive or NOS2-transfected ST-1 cells. These results contrast with those of Younes-Ibrahim and co-workers (54), who detected Sch-28080-sensitive K+-ATPase activity in permeabilized MTALs microdissected from the rat. The reasons for this apparent discrepancy are unclear, but may reflect species differences, dedifferentiation of the ST-1 cells, or the fact that we measured 86Rb+ uptake in intact cells, whereas they measured enzymatic activity in permeabilized tubules (i.e., it has not been established that the rat MTAL expresses a Sch-28080-sensitive, active K+ transport system in its plasma membrane). Because immunoreactivity for the known H+-K+-ATPase alpha -subunit isoforms has not been demonstrated in this nephron segment (42, 52), the molecular identity of the transporter mediating the Sch-28080-sensitive K+-ATPase activity in permeabilized rat MTALs remains unknown. These contrasting results highlight the need for caution in extrapolating the results obtained in the immortalized ST-1 cell line to the MTAL in vivo. Although ST-1 cells exhibit several of the phenotypic properties of the MTAL in vivo, they do not perfectly model the MTAL of normal kidney. For example, we previously detected in ST-1 cells expression of mRNA encoding mBSC2 (also termed NKCC1) (25), the "secretory" isoform of the bumetanide-sensitive Na-K-2Cl cotransporter that is not normally expressed in the MTAL in vivo (12, 18). Because cell culture itself has been shown to induce expression of BSC2 in proximal tubule and other cells (39), the significance of this finding is unknown. In addition, further studies will be needed to determine the specific Na-K-2Cl cotransporter(s) that mediates the bumetanide-sensitive 86Rb+ uptake of ST-1 that we observed in the present study.

In normal rats, the MTAL basally expresses high NOS2 mRNA levels (1, 33, 35), NOS2 immunoreactivity (48), and strong NADPH diaphorase activity (32), a histochemical marker of NOS activity, suggesting that the NOS2 expressed in this segment may be constitutively active. In contrast, the MTAL of normal rat does not appear to express NOS1 or NOS3 (2). Why the MTAL, and not other nephron segments, might tonically express NOS2 has not been established. The MTAL is the major nephron site responsible for the concentration and dilution of the urine, and it accomplishes these tasks, in large part, using extremely high rates of active sodium reabsorption (11). The electrochemical gradients established and maintained by the Na+-K+-ATPase in the basolateral membrane of the MTAL serve as the principal driving force for this work. An inhibitory effect of NO on Na+-K+-ATPase gene expression in the MTAL would be expected to inhibit NaCl absorption by this segment and facilitate natriuresis if NaCl absorption were also inhibited in the cortical TAL (20); if NaCl absorption in the cortical TAL were not concomitantly reduced, the inhibitory effect of NO on MTAL NaCl absorption may only reduce the medullary osmolality and impair urinary concentration. Such direct tubular effects, however, may be difficult to discriminate from the effects of NO on the renal microcirculation (37).

Whereas the present study was designed to study the effects of sustained NO production and exposure on the Na+-K+-ATPase of an MTAL-derived cell line, others have demonstrated rapid effects of NO on K+ channels of the MTAL. Lu and co-workers (28, 29) measured the acute effects of NO donors and NOS inhibitors on the activity of the apical 70-pS K+ channel in cell-attached patches of the rat MTAL. They concluded that NO, via a cGMP-dependent mechanism, rapidly activates this K+ channel (28). Moreover, they found that high angiotensin II concentrations, through stimulation of endogenous NO production, activate the channel (29). That NO could both activate an apical K+ channel and inhibit the Na+-K+-ATPase in the MTAL is perplexing, if indeed these actions occur simultaneously. Further studies to determine whether ST-1 cells express an apical K+ channel comparable to that of rat MTAL and to examine activity of the apical channel after more prolonged exposure to NO would help to clarify this issue.

We speculate that the inhibitory effect of NO on Na+-K+-ATPase alpha 1-subunit transcription is related to interference with DNA-protein interactions important for transcription of the Na+-K+-ATPase alpha 1-subunit gene. Several cis elements and trans factors have been implicated in transcriptional control of the Na+-K+-ATPase alpha 1-subunit gene (21, 51), and NO has been shown to modulate the binding or activity of several transcription factors in other cell types (7, 27, 38, 43, 47). Given the ubiquity of the Na+-K+-ATPase and its importance to the function of all eukaryotic cells, and the ubiquity of NOS2 in immune-stimulated mammalian cell types, this novel action of NO may have broad physiological and pathophysiological implications.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47981 and DK-50745 (to B. C. Kone), and was completed during the tenure of B. C. Kone as an Established Investigator of the American Heart Association.


    FOOTNOTES

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. §1734 solely to indicate this fact.

1 We were concerned that release of reactive oxygen species, such as superoxide anion and hydroxyl radical, from the large number of dying cells (those not successfully incorporating the transfected neomycin resistance gene) might react with NO from the successfully transfected cells to produce toxic metabolites that would limit clonal proliferation. However, we did not formally test whether inclusion of L-NAME in the culture medium during G418 selection was required for generation of the NOS2-expressing clones.

Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and Integrative Biology, Pharmacology, and Physiology, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030 (E-mail: bkone{at}heart.med.uth.tmc.edu).

Received 15 July 1998; accepted in final form 31 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahn, K. Y., M. G. Mohaupt, K. M. Madsen, and B. C. Kone. In situ hybridization localization of mRNA encoding inducible nitric oxide synthase in rat kidney. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F748-F757, 1994[Abstract/Free Full Text].

2.   Bachmann, S., H. M. Bosse, and P. Mundel. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F885-F898, 1995[Abstract/Free Full Text].

3.   Bech, J. N., C. B. Nielsen, and E. B. Pedersen. Effects of systemic NO synthesis inhibition on RPF, GFR, UNa, and vasoactive hormones in healthy humans. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F845-F851, 1996[Abstract/Free Full Text].

4.   Beckman, J. S., and W. H. Koppenol. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am. J. Physiol. 271 (Cell Physiol. 40): C1424-C1437, 1996[Abstract/Free Full Text].

5.   Bertorello, A. M., and A. I. Katz. Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F743-F755, 1993[Abstract/Free Full Text].

6.   Boldyrev, A. A., E. R. Bulygina, G. G. Kramarenko, and A. F. Vanin. Effect of nitroso compounds on Na/K-ATPase. Biochim. Biophys. Acta 1321: 243-251, 1997[Medline].

7.   Brendeford, E. M., K. B. Andersson, and O. S. Gabrielsen. Nitric oxide (NO) disrupts specific DNA binding of the transcription factor c-Myb in vitro. FEBS Lett. 425: 52-56, 1998[Medline].

8.   Clapp, W. L., P. Bowman, G. S. Shaw, P. Patel, and B. C. Kone. Segmental localization of mRNAs encoding Na+-K+-ATPase alpha  and beta  subunit isoforms in rat kidney using RT-PCR. Kidney Int. 46: 627-638, 1994[Medline].

9.   Ding, J. W., J. Dickie, H. O'Brodovich, Y. Shintani, B. Rafii, D. Hackam, Y. Marunaka, and O. D. Rotstein. Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L378-L387, 1998[Abstract/Free Full Text].

10.   Ewart, H. S., and A. Klip. Hormonal regulation of the Na+-K+-ATPase: mechanisms underlying rapid and sustained changes in pump activity. Am. J. Physiol. 269 (Cell Physiol. 38): C295-C311, 1995[Abstract/Free Full Text].

11.   Garg, L. C., M. A. Knepper, and M. B. Burg. Mineralocorticoid effects on Na-K-ATPase in individual nephron segments. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F40-F45, 1990[Abstract/Free Full Text].

12.   Ginns, S. M., M. A. Knepper, C. A. Ecelbarger, J. Terris, X. He, R. A. Coleman, and J. B. Wade. Immunolocalization of the secretory isoform of Na-K-Cl cotransporter in rat renal intercalated cells. J. Am. Soc. Nephrol. 7: 2533-2542, 1996[Abstract].

13.   Guo, Y., M. D. DuVall, J. P. Crow, and S. Matalon. Nitric oxide inhibits Na+ absorption across cultured alveolar type II monolayers. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L369-L377, 1998[Abstract/Free Full Text].

14.   Guzman, N. J., M. Z. Fang, S. S. Tang, J. R. Ingelfinger, and L. C. Garg. Autocrine inhibition of Na+/K+-ATPase by nitric oxide in mouse proximal tubule epithelial cells. J. Clin. Invest. 95: 2083-2088, 1995[Medline].

15.   Haas, M., and S. C. Hebert. [3H]bumetanide binding to a mouse medullary thick limb (MTAL) cell line (Abstract). J. Am. Soc. Nephrol. 3: 808, 1992.

16.   Higashi, Y., T. Oshima, M. Watanabe, H. Matsuura, and G. Kajiyama. Renal response to L-arginine in salt-sensitive patients with essential hypertension. Hypertension 27: 643-648, 1996[Abstract/Free Full Text].

17.   Jain, L., X. J. Chen, L. A. Brown, and D. C. Eaton. Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L475-L484, 1998[Abstract/Free Full Text].

18.   Kaplan, M. R., M. D. Plotkin, D. Brown, S. C. Hebert, and E. Delpire. Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium, and the glomerular afferent arteriole. J. Clin. Invest. 98: 723-730, 1996[Abstract/Free Full Text].

19.   Kishnani, N. S., and H. L. Fung. Nitric oxide generation from pharmacological nitric oxide donors. Methods Enzymol. 268: 259-265, 1996[Medline].

20.   Knepper, M., and M. Burg. Organization of nephron function. Am. J. Physiol. 244 (Renal Fluid Electrolyte Physiol. 13): F579-F589, 1983[Abstract/Free Full Text].

21.   Kobayashi, M., and K. Kawakami. Synergism of the ATF/CRE site and GC box in the housekeeping Na,K-ATPase alpha 1 subunit gene is essential for constitutive expression. Biochem. Biophys. Res. Commun. 241: 169-174, 1997[Medline].

22.   Kone, B. C. Nitric oxide in renal health and disease. Am. J. Kidney Dis. 30: 311-333, 1997[Medline].

23.   Kone, B. C., and C. Baylis. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am. J. Physiol. 272 (Renal Physiol. 41): F561-F578, 1997[Abstract/Free Full Text].

24.   Kone, B. C., and S. C. Higham. A novel N-terminal splice variant of the rat H+-K+-ATPase alpha 2 subunit. Cloning, functional expression, and renal adaptive response to chronic hypokalemia. J. Biol. Chem. 273: 2543-2552, 1998[Abstract/Free Full Text].

25.   Kone, B. C., J. Schwobel, P. Turner, M. G. Mohaupt, and C. B. Cangro. Role of NF-kappa B in the regulation of inducible nitric oxide synthase in an MTAL cell line. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F718-F729, 1995[Abstract/Free Full Text].

26.   Liu, S. H., and T. J. Sheu. The in vivo effect of lipopolysaccharide on Na+,K+-ATPase catalytic alpha  subunit isoforms in rat sciatic nerve. Neurosci. Lett. 234: 166-168, 1997[Medline].

27.   Liu, X. K., D. R. Abernethy, and N. S. Andrawis. Nitric oxide inhibits Oct-1 DNA binding activity in cultured vascular smooth muscle cells. Life Sci. 62: 739-749, 1998[Medline].

28.   Lu, M., X. Wang, and W. Wang. Nitric oxide increases the activity of the apical 70-pS K+ channel in TAL of rat kidney. Am. J. Physiol. 274 (Renal Physiol. 43): F946-F950, 1998[Abstract/Free Full Text].

29.   Lu, M., Y. Zhu, M. Balazy, K. M. Reddy, J. R. Falck, and W. Wang. Effect of angiotensin II on the apical K+ channel in the thick ascending limb of the rat kidney. J. Gen. Physiol. 108: 537-547, 1996[Abstract].

30.   Majid, D. S. A., A. Williams, P. J. Kadowitz, and L. G. Navar. Renal responses to intra-arterial administration of nitric oxide donors in dogs. Hypertension 22: 535-541, 1993[Abstract].

31.   Mattson, D. L., and D. J. Higgins. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 27: 688-692, 1996[Abstract/Free Full Text].

32.   McKee, M., C. Scavone, and J. A. Nathanson. Nitric oxide, cGMP, and hormone regulation of active sodium transport. Proc. Natl. Acad. Sci. USA 91: 12056-12060, 1994[Abstract/Free Full Text].

33.   Mohaupt, M. G., J. L. Elzie, K. Y. Ahn, W. L. Clapp, C. S. Wilcox, and B. C. Kone. Differential expression and induction of mRNAs encoding two inducible nitric oxide synthases in rat kidney. Kidney Int. 46: 653-665, 1994[Medline].

34.   Mohaupt, M. G., J. Schwobel, J. L. Elzie, G. S. Kannan, and B. C. Kone. Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F770-F777, 1995[Abstract/Free Full Text].

35.   Morrissey, J. J., R. McCracken, H. Kaneto, M. Vehaskari, D. Montani, and S. Klahr. Location of an inducible nitric oxide synthase mRNA in the normal kidney. Kidney Int. 45: 998-1005, 1994[Medline].

36.   Nathan, C. Inducible nitric oxide synthase: regulation subserves function. Curr. Top. Microbiol. Immunol. 196: 1-4, 1995[Medline].

37.   Navar, L. G., E. W. Inscho, S. A. Majid, J. D. Imig, L. M. Harrison Bernard, and K. D. Mitchell. Paracrine regulation of the renal microcirculation. Physiol. Rev. 76: 425-536, 1996[Abstract/Free Full Text].

38.   Ohki, K., K. Yoshida, M. Hagiwara, T. Harada, M. Takamura, T. Ohashi, H. Matsuda, and J. Imaki. Nitric oxide induces c-fos gene expression via cyclic AMP response element binding protein (CREB) phosphorylation in rat retinal pigment epithelium. Brain Res. 696: 140-144, 1995[Medline].

39.   Raat, N. J., E. Delpire, C. H. van Os, and R. J. Bindels. Culturing induced expression of basolateral Na+-K+-2Cl- cotransporter BSC2 in proximal tubule, aortic endothelium, and vascular smooth muscle. Pflügers Arch. 431: 458-460, 1996[Medline].

40.   Roczniak, A., and K. D. Burns. Nitric oxide stimulates guanylate cyclase and regulates sodium transport in rabbit proximal tubule. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F106-F115, 1996[Abstract/Free Full Text].

41.   Salazar, F. J., A. Alberola, J. M. Pinilla, J. C. Romero, and T. Quesada. Salt-induced increase in arterial pressure during nitric oxide synthesis inhibition. Hypertension 22: 49-55, 1993[Abstract].

42.   Sangan, P., V. M. Rajendran, A. S. Mann, M. Kashgarian, and H. J. Binder. Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion. Am. J. Physiol. 272 (Cell Physiol. 41): C685-C696, 1997[Abstract/Free Full Text].

43.   Sciorati, C., G. Nistico, J. Meldolesi, and E. Clementi. Nitric oxide effects on cell growth: cyclic GMP-dependent stimulation of the AP-1 transcription complex and cyclic GMP-independent slowing of cell cycling. Br. J. Pharmacol. 122: 687-697, 1997[Abstract].

44.   Shultz, P. J., and J. P. Tolins. Adaptation to increased dietary salt intake in the rat. Role of endogenous nitric oxide. J. Clin. Invest. 91: 642-650, 1993[Medline].

45.   Stoos, B. A., N. H. Garcia, and J. L. Garvin. Nitric oxide inhibits sodium reabsorption in the isolated perfused cortical collecting duct. J. Am. Soc. Nephrol. 6: 89-94, 1995[Abstract].

46.   Stoos, B. A., and J. L. Garvin. Actions of nitric oxide on renal epithelial transport. Clin. Exp. Pharmacol. Physiol. 24: 591-594, 1997[Medline].

47.   Tabuchi, A., E. Oh, A. Taoka, H. Sakurai, T. Tsuchiya, and M. Tsuda. Rapid attenuation of AP-1 transcriptional factors associated with nitric oxide (NO)-mediated neuronal cell death. J. Biol. Chem. 271: 31061-31067, 1996[Abstract/Free Full Text].

48.   Tojo, A., W. J. Welch, V. Bremer, M. Kimoto, K. Kimura, M. Omata, T. Ogawa, P. Vallance, and C. S. Wilcox. Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. Kidney Int. 52: 1593-1601, 1997[Medline].

49.   Tolins, J. P., and P. J. Shultz. Endogenous nitric oxide synthesis determines sensitivity to the pressor effect of salt. Kidney Int. 46: 230-236, 1994[Medline].

50.   Wang, T. Nitric oxide regulates HCO-3 and Na+ transport by a cGMP-mediated mechanism in the kidney proximal tubule. Am. J. Physiol. 272 (Renal Physiol. 41): F242-F248, 1997[Abstract/Free Full Text].

51.   Watanabe, Y., K. Kawakami, Y. Hirayama, and K. Nagano. Transcription factors positively and negatively regulating the Na,K-ATPase alpha 1 subunit gene. J. Biochem. (Tokyo) 114: 849-855, 1993[Abstract].

52.   Wingo, C. S., K. M. Madsen, A. Smolka, and C. C. Tisher. H,K-ATPase immunoreactivity in cortical and outer medullary collecting duct. Kidney Int. 38: 985-990, 1990[Medline].

53.   Yamada, S. S., A. L. Sassaki, C. K. Fujihara, D. M. Malheiros, G. De Nucci, and R. Zatz. Effect of salt intake and inhibitor dose on arterial hypertension and renal injury induced by chronic nitric oxide blockade. Hypertension 27: 1165-1172, 1996[Abstract/Free Full Text].

54.   Younes-Ibrahim, M., C. Barlet Bas, B. Buffin Meyer, L. Cheval, R. Rajerison, and A. Doucet. Ouabain-sensitive and -insensitive K-ATPases in rat nephron: effect of K depletion. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1141-F1147, 1995[Abstract/Free Full Text].


Am J Physiol Renal Physiol 276(4):F614-F621
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society