Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

My N. Helms,1 Ling Yu,1 Bela Malik,1 Dean J. Kleinhenz,2 C. Michael Hart,2 and Douglas C. Eaton1

1Department of Physiology, Emory University School of Medicine; and 2Department of Medicine, Atlanta Veterans Affairs Medical Center and Emory University Medical Center, Atlanta, Georgia

Submitted 6 January 2005 ; accepted in final form 12 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several studies have shown that nitric oxide (NO) inhibits Na+ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na+ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug L-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 µM dexamethasone and 10% bovine serum. After 1.5 µM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound (P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability (Po) from 0.186 ± 0.043 to 0.045 ± 0.009 (n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na+ transport is by altering iNOS production of NO.

aldosterone; epithelial sodium channel; serum- and glycocorticoid-inducible kinase


EPITHELIAL SODIUM CHANNELS (ENaC) are composed of three homologous subunits, designated {alpha}-, {beta}-, and {gamma}-ENaC (4), and are highly selective in transporting Na+ ions. The subunits are located in tight epithelial cells, and normal function of each subunit is important for maintaining electrolyte and fluid homeostasis. For example, inappropriate activation of ENaC in the distal renal tubule can cause hypertensive diseases such as Liddle's syndrome. On the other hand, decreased ENaC activity in renal epithelia leads to hypotensive disorders, and transgenic {alpha}-ENaC-knockout mice die within 40 h of birth as a result of an inability to clear lung fluid (21). We and others have recently shown, using single-channel patch-clamp techniques, that nitric oxide (NO) inhibits alveolar Na+ transport through a cGMP-dependent pathway (24, 31). Similarly, investigators in several studies have reported that NO decreases net active Na+ transport in renal epithelia (13, 35, 4043) and blocks collecting duct water permeability (14). However, single-channel analysis of NO's inhibitory effect on ENaC has not been reported in A6 cells, and the mechanisms by which NO alters ENaC activity are unclear. In our current study, we first examined the effect of NO donor on net Na+ transport in renal epithelial cells and observed single-channel activity in A6 cells using patch-clamp analysis.

NO is a highly diffusible, short-lived free radical that is synthesized from the amino acid L-arginine in a reaction catalyzed by NO synthase (NOS). In vivo NOS catalyzes the conversion of L-arginine, NADPH, and O2 to L-citrulline, NADP+, and NO. Two types of NOS have been identified: the constitutively active forms neuronal and endothelial NOS (nNOS and eNOS, respectively) are dependent on Ca2+ and calmodulin for activity; in contrast, inducible NOS (iNOS) is Ca2+ and calmodulin independent. Both aldosterone and glucocorticoids have been shown to inhibit iNOS activity and hence to decrease levels of NO without effecting iNOS mRNA expression (5, 8, 23, 30). Because corticosteroids are the principal physiological regulators of transepithelial Na+ transport, we reasoned that one effect of aldosterone and glucocorticoids is to decrease iNOS activity and thereby reduce NO inhibition of ENaC activity in Na+-reabsorbing epithelia. This model of NOS regulation has been reported in the neuronal isoform. Phosphorylation of nNOS by calmodulin kinase at S847 reportedly inhibits nNOS activity (17, 29). Because iNOS and nNOS share 57% homology, we hypothesized that corticosteroids might also mediate the phosphorylation of iNOS, possibly through serum- and glucocorticoid-inducible kinase (SGK1), to decrease NO production in Na+-transporting epithelia, which would otherwise inhibit ENaC function.

SGK1 was first described as an immediate, early induced transcript in mammary epithelial cells by serum and glucocorticoids (44). It also has been shown that aldosterone increases the level of SGK1 expression within minutes in A6 distal nephron cell lines (6) and mammalian cortical collecting duct (CCD) cells (33). Moreover, knockdown of kinase activity by dominant negative SGK1 (18) or antisense SGK1 (34) expression substantially decreases ENaC activity in renal epithelia, and SGK1-null mice experience impaired Na+ retention when fed a low-salt diet (45). It has been proposed that SGK1 positively regulates ENaC through direct interaction of its PY motif with the ubiquitin ligase Nedd4-2, which would lead to the eventual degradation of surface ENaC proteins (9, 39). However, because SGK1 is expressed in a wide range of tissues and several pathways may lead to the regulation of amiloride-sensitive Na+ transport, additional SGK1 effectors may be involved in SGK1's signal transduction cascade, leading to the upregulation of ENaC activity. Therefore, our current study examined the physiological interaction between SGK1 and iNOS to determine whether the NO inhibition of amiloride-sensitive Na+ channel activity and corticosteroid inhibition of NOS were mediated by SGK1.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and type II pneumocyte isolation. Low-passage Xenopus kidney distal nephron A6 and M1 CCD cells were purchased from the American Type Culture Collection (Manassas, VA). M1 cells were maintained in plastic flasks (Corning, Corning, NY) at 37°C in a humidified incubator with 5% CO2 in air. The M1 culture medium consisted of DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, glutamine, 1.5 µM aldosterone, and 1% streptomycin, and 0.6% penicillin. A6 cells were cultured in a mixture of Coon's F-12 medium (3 parts) and Leibovitz's L-15 medium (7 parts) (Irving Scientific, Santa Ana, CA) in a final concentration of 104 mM NaCl, 25 mM NaHCO3, 0.6% penicillin, 1.0% streptomycin, 10% (vol/vol) fetal bovine serum, and 1.5 µM aldosterone at 4% CO2 and 26°C.

Primary mouse alveolar type II (ATII) cells were isolated and maintained as described previously (24). All procedures involving animals were reviewed and approved by our Institutional Animal Care and Use Committee. Briefly, 3-mo-old BALB/c mice were anesthetized with pentobarbital sodium and then killed after the lungs were perfused with PBS. Lungs were removed from the animal, and subsequently enzyme digested with dispase and 0.1 mg/ml DNAse in DMEM. Dispersed cells were then passed through a 20-µm nylon mesh and purified using the differential adherence technique (10).

Electrophysiological measurements. With the use of patch-clamp techniques, cell-attached recordings were established on the apical membrane of A6 cells and grown on permeable supports. Polished micropipettes were pulled from filamented borosilicate glass capillaries (TW-150; World Precision Instruments) with a two-stage vertical puller (Narishige, Tokyo, Japan). The resistance of fire-polished pipettes were between 5 and 10 M{Omega} when filled with pipette solution containing (in mM) 96 NaCl, 3.4 KCl, 0.8 CaCl2, 0.8 MgCl2, and 10 HEPES, pH 7.4. Under the above culture conditions, a high-resistance seal (>20 G{Omega}) was usually formed after slight negative pressure was applied to the patch membrane. Channel currents were sampled at 5 kHz with a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Union City, CA) and filtered at 1 kHz with a low-pass Bessel filter. Data were recorded using a computer equipped with AxoScope8 software (Molecular Devices). The open probability (Po) of the channels was calculated using FETCHAN in pCLAMP6 software. Experiments were conducted at 22–23°C.

A6 and M1 cells were grown to confluence on Transwell-permeable supports (Corning, Acton, MA). After ~20 days in culture, the potential difference (PD) and transepithelial resistance (RTE) across cell monolayers were measured using an epithelial voltohmeter equipped with stick electrodes (World Precision Instruments, Sarasota, FL). The equivalent short-circuit current (Isc) was calculated according to Ohm's law (Isc = PD/RTE) and then corrected for the surface area of the Transwell insert.

Immunoprecipitation of iNOS and Western blot analysis. A6, M1, and ATII cells were rinsed three times with PBS before being lysed with 600 µl of lysis buffer (150 mM NaCl, 10 mM NaPO4, pH 7.4, 0.1% SDS, 1% Nonidet P-40, 0.25% Na+-deoxycholate, and freshly prepared 1x protease inhibitor cocktail), and protein concentration was determined using bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL). iNOS immunoprecipitations were performed with rabbit polyclonal iNOS antibody (Upstate, Lake Placid, NY). To coimmunoprecipitate iNOS with SGK1, 3 µl of rabbit polyclonal anti-SGK1 antibody were incubated as described previously (46) with the cell lysate overnight at 4°C. The next day, immunoprecipitated protein complexes were immobilized with ImmunoPure protein A beads (Pierce Chemical), electrophoresed on a 7.5% acrylamide gel under denaturing conditions, and then transferred to Protran nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was blocked in TBST buffer (10 mM Tris, pH 7.5, 70 mM NaCl, and 0.1% Tween) with 5% dry milk and then incubated with 1 µg/ml mouse monoclonal anti-iNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. IgG-horseradish peroxidase (HRP)-labeled secondary antibody (KPL, Gaithersburg, MD) was added at a concentration of 1 µg/10 ml TBST and incubated for another 1 h at room temperature. After being washed thoroughly, HRP signal was detected using the enhanced chemiluminescence substrate and Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunocytochemistry. Primary mouse ATII cells were allowed to adhere overnight on 1-mm round poly-D-lysine-coated coverslips (Becton Dickinson Labware, Bedford, MA) in growth medium and then were fixed with 4% paraformaldehyde for 10 min at room temperature. A 1:1,000 dilution of polyclonal rabbit anti-SGK1 and mouse monoclonal iNOS antibody (see above) was diluted in Ca2+- and MgCl-free PBS with 3% horse serum and 1% BSA and then was applied to the cells for 1 h at room temperature. The cells were then washed three times with blocking buffer, followed by application to the cells of a 1:10,000 mix of Alexa Fluor 488-labeled goat anti-rabbit and Alexa Fluor 568-labeled goat anti-mouse antibodies (Molecular Probes, Eugene, OR) for an additional 1 h at room temperature. Cells were washed and fixed again and then mounted onto a slide with antifade reagent (Molecular Probes). Subcellular localization of SGK1 and iNOS was analyzed using standard confocal microscopy, and 1-µm-thick sections throughout the cell were sequentially imaged. The emission data for SGK1 and iNOS were collected separately and subsequently superimposed using LSM 5 Image Browser software (Carl Zeiss, Thornwood, NY)

In vitro SGK1 kinase assay. Active SGK1 enzyme ({Delta}1–60, S422D) was purchased from Upstate, and the kinase assay procedures were performed as recommended by the manufacturer. Briefly, in a 50-µl reaction volume, 25 ng of SGK1, 10 µCi [{gamma}-32P]ATP, 10 µM cold ATP, and PKA/PKC inhibitors were incubated at 30°C for 10 min with either hiNOS enzyme (Alexis Biochemicals, San Diego, CA) or iNOS oligopeptides custom produced by Sigma Genosys (The Woodlands, TX) as described in the text. Subsequent to incubation, a 35-µl aliquot of the reaction was transferred to phosphocellulose squares, washed, and read in scintillation liquid. Data were recorded as counts per minute (cpm), which indicate {gamma}-32P incorporation into iNOS substrate. Assay buffer was substituted for hiNOS or oligopeptides in negative background control groups.

32P labeling. Confluent M1 cells were serum and hormone deprived for 72 h and then rinsed twice with sodium phosphate-free DMEM (Invitrogen). The cells were then labeled with 0.5 mCi 32P Pi (Amersham Biosciences) for 6 h and then treated with 1.5 µg of aldosterone (or remained in serum- and hormone-free medium as control) for an additional 4 h. Radioactively labeled lysates were then immunoprecipitated using anti-iNOS or SGK1 antibody, separated on 7.5% denaturing gel, and transferred to nitrocellulose using the procedures described above. Quantification of 32P-labeled iNOS protein was enhanced with the use of Molecular Dynamics PhosphorScreen (Sunnyvale, CA) and quantified using ImageQuant software obtained from Amersham Biosciences.

Measurement of NO release. Measurement of NO release was performed on freshly isolated ATII cells. Immediately after isolation, ATII cells were seeded onto Costar Transwell 12-mm inserts (Corning) at confluent densities. NO release was determined by measuring NO and its oxidation products, NO2 and NO3, from the culture medium as described in (27). Briefly, after incubation with or without aldosterone overnight, culture medium was collected and injected into a vessel containing 0.8% NaCl3 in 1 N HCl at 95°C. NO was detected using a chemiluminescence NO analyzer (model 280; Sievers, Boulder, CO), and standard curves were generated using 0.1–10 µM NaNO3 in serum- and hormone-free DMEM.

Statistical evaluation. Statistical analyses were performed using Student's t-test, with statistical significance defined as P < 0.05. Data are expressed as means ± SE. ANOVA among multiple parameters was performed using the Holm t-test, which allows sequential comparison of unadjusted P values.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PAPA-NONOate release of NO decreases transepithelial Na+ transport in A6 and M1 cells. Because of conflicting reports in the literature, we decided to examine the effect of NO and the role of SGK1 in regulating iNOS in three different cell systems. One type of cell studied was primary cultures of ATII cells because of the potential importance of NO in lung inflammation and because inhaled NO is often used to improve blood flow in neonates. A second type of cell studied was mouse M1 cells, a renal cell line derived from CCD, because of reports that NO can alter Na+ transport in the distal nephron. The third type of cells studied was A6 cells, a cell line originally derived from Xenopus kidney that shares some properties with both mammalian lung cells and mammalian distal nephron cells.

Several previous studies have shown opposing data regarding the effect of NO on Na+ transport. For example, infusion of substances such as acetylcholine (which causes NO release into the renal artery) increases urinary volume and decreases Na+ absorption in in vivo animal models (35). Application of acetylcholine and NO donors such as spermine NONOate and nitroglycerin to M1 cells also has shown that NO directly decreases net Na+ flux in mouse CCD cell lines (40). However, other studies have reported that sodium nitroprusside release of NO fails to change net Na+ flux (19) or that NO can even stimulate amiloride-sensitive Na+ channels in rat CCD cells (32). Because such differences could be attributed to differences in the properties of the cell types tested or even the specific NO donor used, we tested the effect of PAPA-NONOate on both the A6 Xenopus distal renal cells and in the mouse M1 CCD cell lines to better understand NO's role in regulating ENaC. Both cell lines are model systems for studying amiloride-sensitive Na+ transport. The NO donor PAPA-NONOate is a zwitterion capable of rapidly releasing 2 M NO per mole of parent compound. The half-life of PAPA-NONOate at room temperature is ~76.6 min in PBS and culture medium (20). We found that very low (50 nM-3 µM) concentrations of PAPA-NONOate were effective in decreasing Isc values in both A6 and M1 cell lines within 1 min, and persisted for at least 10 min without significant change. Figure 1 shows the effect of on Isc 5 min after applying 1.5 µM PAPA-NONOate to the apical membrane of both A6 and M1 cell lines. As a control for the NO donor compound, we allowed the same concentration of PAPA-NONOate to expire at room temperature overnight and then applied the expired PAPA-NONOate to similarly maintained A6 and M1 cells. This inactivated compound is not capable of donating NO. In this way, we could determine whether an effect on Na+ transport was due to the release of NO or to the metabolites of the parent compound. The data shown in Fig. 1, A and B, left, are expressed as %Isc decrease (from pretreatment Isc measurements) after 1.5 µM active PAPA-NONOate or inactivated compound was added to the apical membrane. Active PAPA-NONOate significantly decreased the Isc in A6 cells from an average value of 8.23 ± 0.68 µA/cm2 to 5.91 ± 0.62 µA/cm2, an ~29% decrease in Isc, n = 20 (Fig. 1A, left). Application of the inactivated PAPA-NONOate did not substantially affect Isc values of A6 cells. The ~3% decrease (n = 12) after applying the control compound did not cause a significant decrease in Na+ current compared with pretreatment Isc values (Fig. 1A, right). However, in A6 cells, the percent current decrease in 1.5 µM PAPA-NONOate vs. inactive compound was statistically significant (P < 0.0005). Similarly, 1.5 µM PAPA-NONOate significantly decreased amiloride-sensitive Isc of M1 cells by 44%, from 10.63 ± 0.60 µA/cm2 to 6.0 ± 0.34 µA/cm2, n = 12 (Fig. 1B, left). The Isc values of M1 cells declined by 19% in the control studies, n = 12 (Fig. 1B, right). Again, statistical comparison between the 1.5 µM NONOate-treated M1 group vs. control compound showed a significant decrease in the changes in current (P < 0.0005).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Nitric oxide (NO) donor drug L-propanamine, 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) release of NO decreases amiloride-sensitive, equivalent short-circuit current (Isc) in A6 and M1 epithelial cell monolayers. A and B: 1.5 µM active PAPA-NONOate decreased Isc by 29% in A6 cells (n = 20; A) and by 44% in M1 cells (n = 12; B). This %Isc decrease from initial untreated values differed significantly from the lower 3% Isc decrease in A6 cells (n = 12) and 19% in M1 cells (n = 12) caused by inactivated NO donor compound in A6 cells (right; control). C and D: amiloride substantially decreased Isc in untreated (no NONOate) A6 cells 67% (n = 12; C) and 76% in M1 cells (n = 36; D). Subsequent application of 1.5 µM PAPA-NONOate to amiloride-inhibited A6 and M1 cells did not further decrease Isc values. *P < 0.0005 (AD).

 
Figure 1, C and D, right, shows that the PAPA-NONOate-inhibited current consists largely of an amiloride-sensitive component. Application of amiloride significantly decreased A6 Isc values from 9.25 ± 1.09 µA/cm2 to 3.08 ± 0.04 µA/cm2 in A6 cells and from 6.1 ± 0.34 µA/cm2 to 1.46 ± 0.10 µA/cm2 in M1 cells (P < 0.0005). Furthermore, application of 1.5 µM PAPA-NONOate to amiloride-inhibited A6 and M1 cells did not further decrease Isc, also shown in Fig. 1, C and D. Together, these data suggest that NO release from PAPA-NONOate compound specifically inhibits ENaC activity as measured by transepithelial Isc recordings.

NO decreases the Po of ENaC in A6 cells. When applied to the apical surface of A6 cells, 1.5 µM PAPA-NONOate decreased the Po of ENaC from 0.186 ± 0.043 to 0.045 ± 0.009 (P < 0.05) without significantly changing the unitary current of the channel (Fig. 2, A and B). The top trace in Fig. 2A is a representative cell-attached single-channel recording from a renal A6 cell before application of PAPA-NONOate showing typical ENaC activity. The bottom trace shown in Fig. 2A shows the same single-channel recording 3 min after application of NO donor, with an apparent decrease in ENaC activity. The Po was calculated from seven independently performed patch-clamp studies, and average values are shown in Fig. 2B. In a separate study, similar to the transepithelial Isc studies described above, we also added 1.5 µM inactivated PAPA-NONOate after an initial control recording period. Again, we found that the parent NO donor and its metabolites were not responsible for the NO-induced decrease in ENaC Po. Inactivated PAPA-NONOate did not substantially decrease Po values in A6 cells (Fig. 2C).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 2. NO decreases the open probability (Po) of epithelial Na+ channel (ENaC) in A6 cells. A: after a control recording period (top trace), 1.5 µM NONOate was added to the apical surface of A6 cells and recording continued for an additional 5 min (bottom trace). Arrows indicate closed state, and downward deflections represent channel activity. B: results of seven independent patch-clamp studies show that NO significantly decreased Po in A6 cells. *P < 0.02. C: inactivated PAPA-NONOate did not substantially decrease Po values in A6 single-channel measurements (n = 4).

 
Our present findings in M1 and A6 renal epithelial cells are consistent with those described in our previous report that NO inhibits lung Na+ transport (24) and provides further evidence for NO's inhibitory role on ENaC activity in kidney epithelial cells.

SGK1 associates with iNOS in coimmunoprecipitation studies and in vivo. The lung and kidney express high levels of iNOS mRNA and protein in response to cell injury (25). Consequently, under some circumstances, very high levels of NO can be produced. Even in unstimulated conditions, however, measurable quantities of NO are produced from iNOS (16). This implies that in the absence of any NOS inhibitor, ENaC Po will always be reduced to a greater or lesser extent by NO. Often, when it is necessary to increase Na+ transport, new channels are inserted in the apical membrane of Na+-transporting epithelial cells. If the Po of the newly inserted channels is reduced by endogenous NO, then Na+ transport will be limited even though there are new apical transporters. Aldosterone increases Na+ transport, and one mechanism by which it produces this increase is by increasing the number of apical channels. The increase is produced by an activation of SGK1. After promoting the insertion of new channels, however, it makes sense that the Po of the new channels would not be inhibited by NO. Therefore, we examined whether SGK1 might also increase Po by inhibiting NOS and NO production while promoting an increase in the number of channels. To test this hypothesis, we first studied whether SGK1, an important regulator of Na+ transport, is associated with iNOS in Na+-transporting epithelia. Our model for SGK1 regulation of ENaC activity presumed that SGK1 decreases iNOS activity via direct phosphorylation of iNOS to maintain low levels of NO. Inhibition of iNOS is a plausible mechanism for normal ENaC function because pharmacologically inhibiting iNOS with the use of N{omega}-nitro-L-arginine methyl ester (37) and iNOS–/– mice (22) renders these mice iNOS deficient and hypertensive.

We first showed that iNOS is easily detectable in Na+-transporting epithelia and coimmunoprecipitates with SGK1. In each 7.5% PAGE Western blot analysis assay, the left bands in Fig. 3A show iNOS immunoprecipitated from A6 cell lysate (Fig. 3A, 1), M1 lysate (Fig. 3A, 2), and primary ATII cell lysate (Fig. 3A, 3) using anti-iNOS antibody. These immunoreactive bands serve as the positive signal control for the coimmunoprecipitation of iNOS with SGK1 in the right lanes of each respective blot in Fig. 3A. With the use of the same experimental protocol used for 13 in Fig. 3A, iNOS protein did not coimmunoprecipitate with rabbit polyclonal anti-GAPDH antibody from A6, M1, and ATII cell lysate (Fig. 3A, negative control).



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 3. Serum- and glucocorticoid-inducible kinase (SGK1) and inducible nitric oxide synthase (iNOS) are closely associated in alveolar Na+-transporting epithelium. A: 7.5% SDS-PAGE gel showing iNOS immunoprecipitated with anti-iNOS antibody (positive control) and coimmunoprecipitated with anti-SGK1 antibody in A6 cells (1), M1 cells (2), and primary cultures of alveolar type II (ATII) epithelial cells (3). iNOS protein did not coimmunoprecipitate with rabbit polyclonal anti-GAPDH antibody (Chemicon, Temecula, CA) in the negative control (right column). All immunoblot analysis was performed with mouse monoclonal anti-iNOS antibody. C: immunofluorescent staining of paraformaldehyde-fixed ATII cells showing colocalization of endogenous SGK1 and iNOS. In the left and middle columns, confocal images show the same field of cells grown in the presence of serum and steroid hormone, costained for SGK1 (green) and iNOS (red), respectively. The images in the right column show the composite images of SGK1 and iNOS and demonstrate that the two proteins colocalize in ATII cells. Columns show 1-µm-thick sections through the cells.

 
The coimmunoprecipitation of SGK1 and iNOS in lung ATII primary cells and renal epithelia strongly suggests that these proteins are associated and exist in proximity to each other in the cells. Therefore, using immunofluorescence microscopy, we next investigated the subcellular localization of SGK1 and iNOS in ATII cells. ATII cells were costained with Alexa 488-conjugated anti-rabbit antibodies subsequent to polyclonal SGK1 antibody labeling (green in Fig. 3B), as well as Alexa 568-conjugated anti-mouse antibody after mouse monoclonal iNOS binding (red in Fig. 3B). The left and middle columns in Fig. 3B show that SGK1 and iNOS, respectively, are predominantly cytoplasmic or at the cell membrane in lung cells. The composite image of SGK1 and iNOS staining patterns (Fig. 3B, right column) shows an overlap of the spectral signals and strongly suggests that SGK1 and iNOS are closely associated in ATII cells. In control studies, no red or green emission was detected in ATII cells stained with Alexa 568 and Alexa 488 secondary antibodies only (data not shown). Our data are in agreement with findings in previous reports of cytoplasmic SGK1 (2, 3) and iNOS (7) expression in mammary and gastric tumor cells, respectively, and demonstrate that SGK1 and iNOS are present together in ATII cells.

SGK1 phosphorylates iNOS in in vitro kinase assays. To demonstrate that SGK1 is capable of phosphorylating iNOS, we performed in vitro kinase assays in which active SGK1 enzyme was incubated with iNOS protein in the presence of radioactively labeled [{gamma}-32P]ATP. Figure 4A shows that iNOS is phosphorylated by SGK1 at high levels similar to those of the positive SGK-tide control (28, 36). Because SGK1 is a Ser/Thr kinase, we next identified which amino acid residues on iNOS are specifically phosphorylated by SGK1.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. SGK1 phosphorylates iNOS in vitro. A: SGK1 phosphorylates hiNOS at levels comparable to the synthetic SGK-tide control. B and C: amino acids surrounding miNOS S733 and S903 are highly conserved in human and murine isoforms. Oligopeptides HAKNVFTMRLKSQQNLQSEK and KPRYYSISSSQDH are both phosphorylated in vitro by the SGK1 enzyme. CPM, counts per minute.

 
Figure 4B shows the amino acids surrounding miNOS serine residues S733 and S903 (which share high sequence identity with the human isoform of iNOS) that were predicted by Net Phos software (Technical University of Denmark) to be potential sites of phosphorylation. Therefore, we generated the oligopeptides HAKNVFTMRLKSQQNLQSEK (peptide 1; S733) and KPRYYSISSSQDH (peptide 2; S903) and performed additional kinase assays to test these peptides as SGK1-phosphorylated substrates. Figure 4C shows that 6.4 ± 2.8% and 12.88 ± 5.28% of 80 µM peptides 1 and 2, respectively, had incorporated [{gamma}-32P]ATP compared with 18.30 ± 8.34% of 80 µM SGK-tide (data not shown). SGK1 did not phosphorylate the control synthetic peptide CFVRSVSGFQLPED, which contained a Ser and a –3 Arg residue in the same context as peptides 1 and 2 above background levels (data not shown). Compared with this finding, the percent [{gamma}-32P]ATP incorporation into peptides 1 and 2, respectively, in Fig. 4 is significant.

Our current data show that SGK1 is capable of phosphorylating iNOS and that the two proteins are closely associated in ATII cells freshly obtained from mouse alveolar epithelium. Below, we describe a novel aldosterone-dependent, posttranslational modification of iNOS in M1 CCD cells.

Aldosterone alters in vivo phosphorylation of iNOS and decreases NO production in Na+-transporting epithelia. First, we metabolically labeled M1 cells with 32P Pi in the presence or absence of 1.5 µM aldosterone. Subsequently, we immunoprecipitated iNOS from 32P-labeled cells and subjected the immunoprecipitate to SDS-PAGE using a 7.5% gel. The outlined region in Fig. 5A highlights 32P-labeled iNOS protein (left) and its corresponding pixel intensity profile (middle). Figure 5A, right, shows the results of Western blot analysis performed to confirm that the 32P-labeled band in the left column was indeed immunoreactive with anti-iNOS antibody. The bands in the autoradiogram and Western blot analysis overlap precisely when the images are superimposed. Similarly, Fig. 5B shows that iNOS protein, which coimmunoprecipitated with anti-SGK1 antibody, from cells grown without aldosterone (left) exhibited less phosphorylated iNOS protein compared with M1 cells grown with aldosterone (right) aldosterone. The average pixel intensities of phosphorylated iNOS from the autoradiogram are expressed as percent control in Fig. 5C. Aldosterone increased the level of 32P-labeled iNOS in M1 cells ~300% above control levels, regardless of whether iNOS protein was directly immunoprecipitated using anti-iNOS antibody (Fig. 5A) or coimmunoprecipitated with polyclonal SGK1 antibody (Fig. 5B).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5. Aldosterone increases 32P labeling of iNOS in M1 cortical collecting duct (CCD) cells. A: iNOS was immunoprecipitated with rabbit polyclonal anti-iNOS antibody. B: iNOS was coimmunoprecipitated with SGK1 using rabbit polyclonal anti-SGK1 antibody. In both A and B, phosphorimaging shows an increase in the intensity of 32P-labeled iNOS proteins in response to 1.5 µM aldosterone (left). The same 32P-labeled membrane was examined using Western blot analysis and probed with mouse monoclonal anti-iNOS antibody to confirm that the phosphorylated band in A and B was immunoreactive with iNOS antibody. C: %control of [32P]ATP incorporation was quantified on the basis of the band intensity profiles above background levels in the intensity profiles. Aldosterone increased the level of phosphorylated iNOS in M1 cells ~3-fold. Values are shown as %control, with samples without aldosterone treatment (open bars) set as 100%, the closed bar representing immunoprecipitated iNOS using anti-iNOS antibody, and the shaded bar representing iNOS that coimmunoprecipitated with SGK1.

 
We also showed that aldosterone decreases NO synthesis in primary alveolar epithelial cells (Fig. 6). Our studies show that 1.5 µM aldosterone decreases the level of NO release in ATII cells by 50% after 4 h of treatment. Together, our studies strongly suggest that aldosterone-mediated phosphorylation of inducible NOS leads to the subsequent decrease in NO production and may be important in regulating normal ENaC activity.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6. Aldosterone decreases NO production in Na+-transporting epithelia. Aldosterone treatment (1.5 µM) significantly decreased NO release in ATII cells. NO levels in aldosterone-treated cells were 50% below control (no aldosterone) values. n = 4; *P < 0.0005.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several types of NO-releasing compounds, such as S-nitrosothiols, organic nitrates, and secondary amine/NO complex ions, are commercially available and reportedly have different effects on Na+ channel activity. Our present studies show that NO released from 1.5 µM PAPA-NONOate donor is very effective in decreasing amiloride-sensitive Na+ current in M1 and A6 cells within minutes. NO generated from PAPA-NONOate is spontaneously released, and 2 M NO per mol of parent compound is effectively produced. The high amounts of NO released from PAPA-NONOate drug are similar to the micromolar amounts produced in response to iNOS activation and may account for the immediate effect of PAPA-NONOate on inhibiting ENaC activity.

Our findings in the M1 CCD and A6 distal nephron cell lines are similar to findings in two other independent groups. Stoos and colleagues (40, 41) showed that acetylcholine-induced NO release from endothelial cells, as well as addition of the NO donor spermine NONOate, inhibited Na+ reabsorption in CCD cells. Rückes-Nilges et al. (38) reported that 1 mM of sodium nitroprusside clearly inhibited amiloride-sensitive Na+ reabsorption in Xenopus kidney A6 distal nephron cell lines. However, in their study, the NO donors sodium nitroprusside and spermine NONOate did not alter either the amiloride-sensitive or the amiloride-insensitive portions of Isc in primary cultures of human nasal epithelial cells. It is becoming apparent that perhaps the efficiency of NO release from different NO donors may effect ion transport differently. The effect of NO also may depend on the cell type examined. For example, our research to date also includes the effect of NO donors S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) on ENaC activity in primary cultured adult rat ATII cells. Application of 100 µM GSNO and SNAP to patch-clamp recording solution did not significantly alter ENaC Po in ATII cells. This finding is similar to the results obtained by Rückes-Nilges et al. (38), who showed that NO released from spermine NONOate had no inhibitory potency on the human nasal epithelium. The different effects of NO donors in renal epithelial cells and in the lung epithelium may pertain to the slow half-lives of the donors used in the studies. GSNO and SNAP have slow half-lives of ~38 ± 5 h, and the half-life of NO release at room temperature is ~4 h for spermine NONOate. Furthermore, the total amount of NO release from SNAP donor corresponds to <2% of the total SNAP present (12). It has been shown that slow release of NO over a long period of time has less potent biological effects than the same amount of NO released rapidly (26). Perhaps the slow half-lives and inefficient release of NO may account for the inability of GSNO, SNAP, and spermine NONOate to alter Na+ transport properties in lung epithelial cells.

SGK1, which is important in mediating both early and late phases of aldosterone activity (1, 6, 33), also may be the key regulator of NO production and iNOS activity after corticosteroid stimulation. Our in vitro kinase assays identified iNOS as a novel SGK1 substrate. Specifically, miNOS oligopeptides that were 20 (peptide 1; S733) and 13 (peptide 2; S903) amino acid residues long were phosphorylated by SGK1. Shorter, nine-amino acid peptide sequences surrounding hiNOS S114, S749, S909, S917, and S965 were not phosphorylated effectively by SGK1 in our kinase assays. Our results suggest that longer peptide sequences may be required for appropriate protein folding and kinase phosphorylation. Interestingly, S903 in peptide 2 is preceded by a Pro residue at the –4 position. Prolines can act as structural disruptors for {alpha}-helices or as a turning point for {beta}-sheets. This may explain the 12.88 ± 5.28% [{gamma}-32P]ATP incorporation into peptide 2. Because iNOS does not fully express the traditionally conserved SGK1 phosphorylation sequence (RxRxxS/T), perhaps protein folding is particularly crucial for appropriate Ser phosphorylation by SGK1.

Figure 7 summarizes and illustrates our model of aldosterone-regulated Na+ transport, involving the SGK1, iNOS, and NO components. In the absence of aldosterone or after direct application of PAPA-NONOate, NO immediately inhibits ENaC by reducing ENaC Po without altering the unitary current or apparent channel density. However, aldosterone-induced increases in the expression of SGK1 may lead to the phosphorylation and hence inactivation of iNOS protein. Indeed, our studies have shown that local production of NO decreases after aldosterone treatment, which may be an important mechanism involved in controlling ENaC activity.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 7. Model for the role of NO in aldosterone-mediated Na+ transport. Aldosterone increases the level of phosphorylated iNOS protein in Na+-transporting epithelia. Our studies implicate SGK1 in this pathway because SGK1 and iNOS coimmunoprecipitate and are closely associated in epithelial cells. Presumably, the phosphorylation of iNOS decreases the conversion of Arg to NO and allows Na+ entry through ENaCs.

 
Although the specific mechanism behind NO inhibition of Na+ channel activity is not identified in this study and remains unclear, we found that NO modification of ENaCs, or ENaC-regulatory proteins, is not immediately reversible by sequestering endogenous NO levels. We performed single-channel patch-clamp studies in the presence of carboxy-2-phenyl-4,4,5,5-tetramethylimldazoline-3-oxide-1-oxyl (cPTIO; Wako Chemicals, Richmond, VA), a specific NO scavenger. Application of 300 nM cPTIO to A6 cells did not acutely increase ENaC activity (data not shown). Other studies have similarly demonstrated that NO inhibits amiloride-sensitive current in lung cells. These studies also put forth convincing evidence that NO may regulate Na+ transport via both cGMP-dependent and cGMP-independent pathways. First, Jain et al. (24) demonstrated that NO donors GSNO and SNAP acted via a cGMP pathway. Pretreating cells with methylene blue, an inhibitor of guanylyl cyclase, blocked the inhibitory effects of the NO donors. Jain et al. further implicated cGMP in the NO-regulatory pathway by demonstrating that the permeable analog of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), decreased the Po of Na+ channels in alveolar lung cells. Next, by pretreating cells with the PKG inhibitor, KT-5823, and blocking the inhibitory effects of GSNO, Jain et al. demonstrated that PKG may be positioned between cGMP and the effector. Lazrak and colleagues (24, 31) also showed that PAPA-NONOate and spermine NONOate decreased 8.6-pS amiloride-sensitive ENaC via cGMP-dependent mechanisms using both whole cell and single-cell patch-clamp techniques in A549 human alveolar cell lines. Similarly to the aforementioned study by Jain et al., Lazrak and colleagues observed a decrease in inward Na+ current when A549 cells were perfused with 8-BrcGMP.

Contrary to these findings, Guo et al. (15) reported a cGMP-independent mechanism of ENaC regulation by NO donors. Although they, too, reported that NO generated by PAPA-NONOate inhibited 60% of the amiloride-sensitive Isc in cultured ATII monolayers, they also reported that the NO-induced decrease in alveolar Isc was not accompanied by an increase in intracellular cGMP levels. Alternatively, the inhibitory effect of NO on ENaC may occur through direct interaction of NO with the channel or with other ENaC-regulatory proteins. DuVall et al. (11) recently suggested that direct nitration or nitrosylation of key Tyr residues on the outer borders of the transmembrane domain (TM) of {alpha}-ENaC subunit (Y134 and Y137 in TM1; Y482, Y484, and Y485 in TM2) may alter ENaC activity.

Overall, the present data support an inhibitory effect of NO on ENaC activity in both M1 CCD and A6 epithelial cell lines. In addition, we have shown that the mechanisms by which aldosterone regulates ENaC function include phosphorylation of iNOS and decreased synthesis of NO, possibly through the SGK1 signaling pathway.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This research was supported by National Institutes of Health Grants R01-HL-071621 and R37-DK-37963.


    ACKNOWLEDGMENTS
 
We thank B. J. Duke for maintaining M1 and A6 cell cultures. Meral Ciblak and Julie Self provided excellent technical assistance in isolating primary cultures of ATII cells.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. C. Eaton, Dept. of Physiology, Emory Univ. of School of Medicine, Whitehead Biomedical Research Bldg., 615 Michael St., Atlanta, GA 30322 (e-mail:deaton{at}physio.emory.edu)

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


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Boyd C and Náray-Fejes-Tóth A. Gene regulation of ENaC subunits by serum- and glucocorticoid-inducible kinase-1. Am J Physiol Renal Physiol 288: F505–F512, 2005.[Abstract/Free Full Text]

2. Brickley DR, Mikosz CA, Hagan CR, and Conzen SD. Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1). J Biol Chem 277: 43064–43070, 2002.[Abstract/Free Full Text]

3. Buse P, Tran SH, Luther E, Phu PT, Aponte GW, and Firestone GL. Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells: a novel convergence point of anti-proliferative and proliferative cell signaling pathways. J Biol Chem 274: 7253–7263, 1999.[Abstract/Free Full Text]

4. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nat Genet 367: 463–467, 1994.[CrossRef]

5. Cetkovic-Cvrlje M, Sandler S, and Eizirik DL. Nicotinamide and dexamethasone inhibit interleukin-1-induced nitric oxide production by RINm5F cells without decreasing messenger ribonucleic acid expression for nitric oxide synthase. Endocrinology 133: 1739–1743, 1993.[Abstract]

6. Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96: 2514–2519, 1999.[Abstract/Free Full Text]

7. Chinje EC, Cowen RL, Feng J, Sharma SP, Wind NS, Harris AL, and Stratford IJ. Non-nuclear localized human NOSII enhances the bioactivation and toxicity of tirapazamine (SR4233) in vitro. Mol Pharmacol 63: 1248–1255, 2003.[Abstract/Free Full Text]

8. Chun TY, Bloem LJ, and Pratt JH. Aldosterone inhibits inducible nitric oxide synthase in neonatal rat cardiomyocytes. Endocrinology 144: 1712–1717, 2003.[Abstract/Free Full Text]

9. Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, Münster C, Chraïbi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, and Staub O. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cell surface expression. EMBO J 20: 7052–7059, 2001.[Abstract/Free Full Text]

10. Dobbs LG, Gonzalez R, and Williams MC. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis 134: 141–145, 1986.[ISI][Medline]

11. DuVall MD, Zhu S, Fuller CM, and Matalon S. Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing {alpha}{beta}{gamma}-rENaC. Am J Physiol Cell Physiol 274: C1417–C1423, 1998.[Abstract/Free Full Text]

12. Ferrero R, Rodríguez-Pascual F, Miras-Portugal MT, and Torres M. Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production. Br J Pharmacol 127: 779–787, 1999.[CrossRef][ISI][Medline]

13. García NH, Plato CF, Stoos BA, and Garvin JL. Nitric oxide-induced inhibition of transport by thick ascending limbs from Dahl salt-sensitive rats. Hypertension 34: 508–513, 1999.[Abstract/Free Full Text]

14. García NH, Stoos BA, Carretero OA, and Garvin JL. Mechanism of the nitric oxide-induced blockade of collecting duct water permeability. Hypertension 27: 679–683, 1996.[Abstract/Free Full Text]

15. Guo Y, Duvall MD, Crow JP, and Matalon S. Nitric oxide inhibits Na+ absorption across cultured alveolar type II monolayers. Am J Physiol Lung Cell Mol Physiol 274: L369–L377, 1998.[Abstract/Free Full Text]

16. Hardiman KM, McNicholas-Bevensee CM, Fortenberry J, Myles CT, Malik B, Eaton DC, and Matalon S. Regulation of amiloride-sensitive Na+ transport by basal nitric oxide. Am J Respir Cell Mol Biol 30: 720–728, 2004.[Abstract/Free Full Text]

17. Hayashi Y, Nishio M, Naito Y, Yokokura H, Nimura Y, Kidaka H, and Watanabe Y. Regulation of neuronal nitric-oxide synthase by calmodulin kinases. J Biol Chem 274: 20597–20602, 1999.[Abstract/Free Full Text]

18. Helms MN, Fejes-Tóth G, and Náray-Fejes-Tóth A. Hormone-regulated transepithelial Na+ transport in mammalian CCD cells requires SGK1 expression. Am J Physiol Renal Physiol 284: F480–F487, 2003.[Abstract/Free Full Text]

19. Hirsch JR, Cermak R, Forssmann WG, Kleta R, Kruhoffer M, Kuhn M, Schafer JA, Sun D, and Schlatter E. Effects of sodium nitroprusside in the rat cortical collecting duct are independent of the NO pathway. Kidney Int 51: 473–476, 1997.[ISI][Medline]

20. Hrabie JA, Klose JR, Wink DA, and Keefer LK. New nitric oxide-releasing zwitterions derived from polyamines. J Org Chem 58: 1472–1476, 1993.[CrossRef][ISI]

21. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, and Rossier BC. Early death due to defective neonatal lung liquid clearance in {alpha}-ENaC-deficient mice. Nat Genet 12: 325–328, 1996.[CrossRef][ISI][Medline]

22. Ihrig M, Dangler CA, and Fox JG. Mice lacking inducible nitric oxide synthase develop spontaneous hypercholesterolaemia and aortic atheromas. Atherosclerosis 156: 103–107, 2001.[CrossRef][ISI][Medline]

23. Ikeda U, Kanbe T, Nakayama I, Kawahara Y, Yokoyama M, and Shimada K. Aldosterone inhibits nitric oxide synthesis in rat vascular smooth muscle cells induced by interleukin-1{beta}. Eur J Pharmacol 290: 69–73, 1995.[CrossRef][Medline]

24. Jain L, Chen XJ, Brown LA, and Eaton DC. Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels. Am J Physiol Lung Cell Mol Physiol 274: L475–L484, 1998.[Abstract/Free Full Text]

25. Kan W, Zhao KS, Jiang Y, Yan W, Huang Q, Wang J, Qin Q, Huang X, and Wang S. Lung, spleen, and kidney are the major places for inducible nitric oxide synthase expression in endotoxic shock: role of p38 mitogen-activated protein kinase in signal transduction of inducible nitric oxide synthase expression. Shock 21: 281–287, 2004.[CrossRef][ISI][Medline]

26. Keefer LK. Nitric oxide-releasing compounds: from basic research to promising drugs. Chemtech 28: 30–35, 1998.

27. Kleinhenz DJ, Fan X, Rubin J, and Hart CM. Detection of endothelial nitric oxide release with the 2,3-diaminonapthalene assay. Free Radic Biol Med 34: 856–861, 2003.[CrossRef][ISI][Medline]

28. Kobayashi T and Cohen P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J 339: 319–328, 1999.[CrossRef][ISI][Medline]

29. Komeima K, Hayashi Y, Naito Y, and Watanabe Y. Inhibition of neuronal nitric-oxide synthase by calcium/calmodulin-dependent protein kinase II{alpha} through Ser847 phosphorylation in NG108-15 neuronal cells. J Biol Chem 275: 28139–28143, 2000.[Abstract/Free Full Text]

30. Kunz D, Walker G, Eberhardt W, and Pfeilschifter J. Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1{beta}-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation. Proc Natl Acad Sci USA 93: 255–259, 1996.[Abstract/Free Full Text]

31. Lazrak A, Samanta A, and Matalon S. Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells. Am J Physiol Lung Cell Mol Physiol 278: L848–L857, 2000.[Abstract/Free Full Text]

32. Lu M, Giebisch G, and Wang W. Nitric oxide-induced hyperpolarization stimulates low-conductance Na+ channel of rat CCD. Am J Physiol Renal Physiol 272: F498–F504, 1997.[Abstract/Free Full Text]

33. Náray-Fejes-Tóth A, Canessa C, Cleaveland ES, Aldrich G, and Fejes-Tóth G. sgk is an aldosterone-induced kinase in the renal collecting duct: effects on epithelial Na+ channels. J Biol Chem 274: 16973–16978, 1999.[Abstract/Free Full Text]

34. Náray-Fejes-Tóth A, Helms MN, Stokes JB, and Fejes-Tóth G. Regulation of sodium transport in mammalian collecting duct cells by aldosterone-induced kinase, SGK1: structure/function studies. Mol Cell Endocrinol 217: 197–202, 2004.[CrossRef][ISI][Medline]

35. Ortiz PA and Garvin JL. Role of nitric oxide in the regulation of nephron transport. Am J Physiol Renal Physiol 282: F777–F784, 2002.[Abstract/Free Full Text]

36. Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, and Hemmings BA. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J 18: 3024–3033, 1999.[Abstract/Free Full Text]

37. Qiu C and Baylis C. Dexamethasone worsens nitric oxide inhibition-induced hypertension and renal dysfunction. Am J Hypertens 13: 1097–1102, 2000.[CrossRef][ISI][Medline]

38. Rückes-Nilges C, Lindemann H, Klimek T, Glanz H, and Weber WM. Nitric oxide has no beneficial effects on ion transport defects in cystic fibrosis human nasal epithelium. Pflügers Arch 441: 133–137, 2000.[CrossRef][ISI][Medline]

39. Snyder PM, Olson DR, and Thomas BC. Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel. J Biol Chem 277: 5–8, 2002.[Abstract/Free Full Text]

40. Stoos BA, García NH, and Garvin JL. Nitric oxide inhibits sodium reabsorption in the isolated perfused cortical collecting duct. J Am Soc Nephrol 6: 89–94, 1995.[Abstract/Free Full Text]

41. Stoos BA and Garvin JL. Actions of nitric oxide on renal epithelial transport. Clin Exp Pharmacol Physiol 24: 591–594, 1997.[ISI][Medline]

42. Stoos BA, Carretero OA, Farhy RD, Scicli G, and Garvin JL. Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells. J Clin Invest 89: 761–765, 1992.[ISI][Medline]

43. Stoos BA, Carretero OA, and Garvin JL. Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells. J Am Soc Nephrol 4: 1855–1860, 1994.[Abstract/Free Full Text]

44. Webster MK, Goya L, Ge Y, Maiyar AC, and Firestone GL. Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13: 2031–2040, 1993.[Abstract]

45. Wulff P, Vallon V, Huang DY, Völkl H, Yu F, Richter K, Jansen M, Schlünz M, Klingel K, Loffing J, Kauselmann G, Bösl MR, Lang F, and Kuhl D. Impaired renal Na+ retention in the sgk1-knockout mouse. J Clin Invest 110: 1263–1268, 2002.[Abstract/Free Full Text]

46. Zhang L, Cui R, Cheng X, and Du J. Antiapoptotic effect of serum and glucocorticoid-inducible protein kinase is mediated by novel mechanism activating I{kappa}{beta} kinase. Cancer Res 65: 457–464, 2005.[Abstract/Free Full Text]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/3/C717    most recent
00006.2005v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Helms, M. N.
Articles by Eaton, D. C.
Articles citing this Article
PubMed
PubMed Citation
Articles by Helms, M. N.
Articles by Eaton, D. C.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.