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The Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
Submitted 9 November 2006 ; accepted in final form 30 August 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2– alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity. single channel; cell-attached patch clamp; dihydroethidium; reactive oxygen species
-ENaC knockout mice die shortly after birth as a result of their inability to clear lung fluid (16) and fluid accumulates in the lung when ENaC is inhibited by amiloride (28, 35).
Several investigators have shown that ENaC activity depends on alveolar oxygen tension. At birth, the increase in O2 tension may contribute to the increased Na reabsorption that helps clear the newborn lung of excess fluid [reviewed previously (12)]. More to the point, O'Brodovich, Matalon, and colleagues have shown that maintaining rat fetal distal lung cells in high (20%) O2 concentrations increased ENaC mRNA (31) and protein (36) expression. It is believed that the increase in total ENaC protein expression contributes to the observed increases in amiloride-sensitive short-circuit current measured in fetal lung cells when switched from low to high PO2 environments (2, 31, 36). Conversely, hypoxic culture conditions decreased amiloride-sensitive 22Na influx, as well as -, 
-,and 
-ENaC mRNA expression and -ENaC protein levels in adult rat alveolar type II cells (29).
There are mechanisms by which some proteins can directly sense oxygen tension (22, 30) so that ENaC's sensitivity to oxygen tension could be a direct effect of oxygen; however, changes in oxygen tension also produce changes in other signaling molecules. Specifically, an increase in cellular oxygen tension increases mitochondrial activity, and oxidative metabolism (or an increase in endogenous oxidase activity) increases production of reactive oxygen species, such as superoxide (O2–). Hence, the O2– by-product of oxygen metabolism could be responsible for regulating Na transport in pneumocytes and in other Na-transporting epithelia. O'Brodovich and colleagues (31) demonstrated that addition of the cell-permeable O2– scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) inhibited the high PO2-induced increases in amiloride-sensitive short-circuit current in lung cells, suggesting that elevated levels of O2– may mediate the oxygen-induced increases in Na transport. Additionally, in hypertensive animal models, microperfusing TEMPO effectively lowered vascular resistance and blood pressure [reviewed previously (33, 34)], further suggesting an important role for O2– in Na transport.
In our present study, we employed the single-channel patch-clamp technique to better understand the role of O2– in distal renal epithelial cells. First, we showed that aldosterone, the principal regulator of renal ENaC, substantially increases O2– production in the A6 distal kidney cell line. We then examined the interaction between O2– and nitric oxide (NO) in the regulation of renal ENaC. Recently, we have shown that NO-releasing molecules, such as S-nitroglutathione and PAPA-NONOate significantly reduced ENaC activity in both lung and kidney epithelia (15, 17). Because O2– rapidly binds to and reduces the biological effects of NO (reviewed in Ref. 21), we considered the possibility that increasing O2– levels in A6 distal collecting duct cells would scavenge NO and decrease NO inhibition of ENaC function. Conversely, we expected that treating cells with the antioxidant compound TEMPO would remove the permissive effect of O2– on Na transport. Finally, we also studied the role of the cGMP pathway in the regulation of renal ENaC by both O2– and NO radicals. Together, our study presents a novel and plausible role for reactive oxygen species signaling in the regulation of transepithelial Na transport in distal kidney cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The A6 cells are an established renal cell line derived from the distal segment of Xenopus laevis nephron. A6 cells grow into tight monolayers, which separate apical Na reabsorption from basolateral ion transport. Additionally, Kerschbaum et al. (20) have shown that Xenopus kidney cells express NO synthase and that the rate of NO synthesis in Xenopus kidney homogenate is 0.18 nM NO·mg protein–1·min–1. These established properties of A6 cells make the distal nephron cell line an appropriate model for studying aldosterone-mediated O2– regulation of NO's inhibitory effect on ENaC.
Xenopus kidney distal nephron A6 cells (subclone 2F3 from Drs. B. Krahenbul and B. Rossier) were maintained in growth medium consisting of three parts Coon's medium F-12 and seven parts Leibovitz's medium L-15 modified for amphibian cells with 104 mM NaCl, 25 mM NaHCO3, 10% FBS (GIBCO), 1.0% streptomycin, 0.6% penicillin, and 1.5 µM aldosterone, at pH 7.4. A6 cells were grown on permeable supports with growth medium replaced three times per week in a 4% CO2 and 26°C incubator. All studies were carried out in A6 cells between passages 97 and 104.
Detection of O2– in A6 Cells
Dihydroethidium (DHE) is a fluorescent probe that intercalates into DNA and has an excitation wavelength of 520 nm and an emission of 610 nm. Fink et al. (13) have shown that 2-hydroxyethidium production from DHE can be used as a quantitative measure of O2– production; indeed, DHE has been used to measure changes in intracellular O2– levels in cultured cells and frozen tissue section (10, 26). However, it may be possible that other strong oxidants can contribute to the increase in fluorescence. In our studies, DHE fluorescence was easily detected and quantified with a Zeiss LSM 510 NLO META laser scanning confocal microscope and compatible LSM 5 Image Browser software (Carl Zeiss, Thornwood, NY).
A6 cells were grown to confluency and then rinsed three times with PBS before serum and hormone restriction or treatment with 50 nM to 1.5 µM aldosterone, 250 µM TEMPO (Sigma-Aldrich, St. Louis, MO), 1 µM dexamethasone, or a mixture of 45 µM hypoxanthine and 17 mU/ml xanthine oxidase (Sigma-Aldrich). Cells were rinsed with PBS and then incubated with 2 µM DHE (Invitrogen, Carlsbad, CA) in PBS solution for 30 min in a light-protected humidified 5% CO2 chamber maintained at 37°C. Cells were then fixed in 4% paraformaldehyde and sealed between a glass slide and coverslip with Vectashield (Vector Laboratories, Burlingame, CA) mounting medium.
Transepithelial Resistance Measurement
A6 cells were grown on Transwell permeable supports (Corning, Acton, MA) to confluency. After 7–10 days in culture, the transepithelial resistances (TER) across cell monolayers were measured with an epithelial voltohmeter equipped with chopstick electrodes (World Precision Instruments, Sarasota, FL).
Electrophysiological Measurements
Cell-attached patch clamp.
For single-channel patch-clamp experiments, A6 cells were seeded onto collagen-coated permeable support inserts until they reached confluency. With the use of the patch-clamp technique, cell-attached recordings were established on the apical membrane of A6 cells grown in complete growth medium. 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
when filled with pipette solution containing (in mM) 96 NaCl, 3.4 KCl, 0.8 CaCl2, 0.8 MgCl2, and 10 HEPES, at pH 7.4. Under the above culture conditions, a high resistance seal (>20 G) was usually formed after slight negative pressure was applied to the patch membrane. Channel currents were sampled at 5 kHz with an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) and filtered at 1 kHz with a low-pass Bessel filter. Data were recorded by a computer with AxoScope8 software (Molecular Devices).
As a measure of ENaC activity, we first calculated the product of the number of channels times the open probability (NPo) using pCLAMP software (Molecular Devices). This product can be calculated from the single-channel record without any assumptions about the total number of channels in a patch or the open probability of a single channel (Po) using the following relationship
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Chemicals used in patch-clamp analysis.
The superoxide dismutase (SOD) mimetic TEMPO was used to scavenge endogenous O2–. 3-Carbamoyl-proxyl (3-CP), which is structurally related to TEMPO but has no O2–-scavenging ability, was included as a control compound for TEMPO treatment. We pharmacologically increased local concentrations of O2– by treating cells with Ethiolat, a SOD inhibitor, or by combining xanthine oxidase (lyophilized from bovine milk) with hypoxanthine. Combining hypoxanthine and xanthine oxidase univalently and divalently reduces dioxygen to generate O2– and H2O2. 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is a selective inhibitor of NO-sensitive guanylyl cyclase. All chemical reagents described above were purchased from Sigma Aldrich. Peroxynitrite was purchased from Upstate (Lake Placid, NY). Because these agents are cell permeable, we pretreated A6 cells with these agents for 1–10 min to produce a maximal effect (where appropriate) without altering cell viability.
cGMP measurements. cGMP levels were measured in A6 cells with a cGMP enzyme immunoassay kit commercially available from Cayman Chemical (Ann Arbor, MI). Cell samples were acetylated with 4 M KOH and acetic anhydride in quick succession to increase the sensitivity of the assay.
Methods for statistical analysis. ENaC Po values were examined before and after the redox state of the cell had been pharmacologically altered. Hence, the same patch-clamp recording before drug treatment could be used as its own control, and statistical significance could be determined by paired t-test analysis, with P values <0.05 considered significant. When multiple comparisons were necessary, repeated-measures ANOVA was performed followed by the Holm t-test to determine which groups significantly differed.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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DHE is a commonly used fluorescent probe used to measure O2– levels in cells (10, 13, 26). Because there are conflicting reports about the effect of corticosteroids on cellular O2– production (4, 25), we first wanted to determine whether aldosterone (the principal physiological regulator of ENaC in renal epithelia) or dexamethasone (a synthetic glucocorticoid that can also activate ENaC) could stimulate O2– production in A6 distal kidney cells. In Fig. 1, A and B, the effects of corticosteroid treatment on DHE intensity in A6 cells are compared after serum and hormone deprivation, as well after treatment with 250 µM TEMPO, a cell-permeable O2–-scavenging compound. Figure 1A shows DHE-labeled A6 cells grown on permeable supports. In Fig. 1B, we found that serum- and hormone-depleted A6 cells decreased endogenous O2– production to very low levels, which were comparable to sequestering all O2– anion with TEMPO in the presence of 1.5 µM aldosterone. Although in Fig. 1B we were specifically testing the hypothesis that aldosterone regulates O2– production in A6 cells, our results provide further additional support that DHE labeling is an effective compound useful for measuring O2– levels in cultured cells. It may be possible that other strong oxidants can contribute to the change in fluorescence intensity measured. However, in our study, the change in DHE fluorescence is most likely due to changes in cellular O2– production, since DHE fluorescence intensities decreased after the cell-permeable O2– scavenger TEMPO was added to the cells. Thus we can be confident that, in A6 cells, the increase in DHE fluorescence after corticosteroid treatment is due to changes in O2– levels. Indeed, Fig. 1B shows that aldosterone and dexamethasone increased O2– production to the same extent. Figure 1C shows that 50–100 nM aldosterone could also significantly induce O2– production in A6 cells in a dose-dependent manner.
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Sequestering Endogenous O2– Decreases ENaC Po in A6 Cells
O2– are continually produced in the mitochondria or via oxidase activity in all cell types. In Fig. 1, we showed that aldosterone can increase O2– production in A6 distal nephron cells. This finding strongly suggests that one mechanism by which aldosterone could be regulating Na reabsorption in the distal nephron is signaling via reactive oxygen species. Therefore, to investigate whether endogenous O2– is involved in the regulation of Na transport in A6 cells, we measured ENaC activity in cells treated with 250 µM TEMPO. TEMPO is a cell-permeable O2– scavenger, commonly used as a SOD mimetic. To be certain that the cellular response can be associated with decreasing the level of O2– in A6 cells, we subjected the same cells to 250 µM 3-CP, an inactive analog of TEMPO.
Using the cell-attached patch-clamp technique, we continually measured single channel (ENaC) activity in an A6 cell before any drug application (control) and then after 3-CP and TEMPO drug application. Because 3-CP and TEMPO can easily permeate the cell membrane, ENaC Po was quantified 2 min after each drug application. We found that 3-CP treatment did not significantly alter ENaC Po. However, after each application of TEMPO, cells responded with a marked decrease in ENaC activity (Fig. 2A). TEMPO decreased the average Po of ENaC from 0.10 ± 0.03 to 0.03 ± 0.01 in A6 cells. To further illustrate our study, we show in Fig. 2B a representative, continuous recording of an A6 cell that includes a control recording period (no drug treatment) followed by both 3-CP and TEMPO treatment. From the continuous recording, in which downward deflections represent Na channel openings, it can easily be seen that 3-CP (the control substance) did not affect ENaC activity. However, immediately after TEMPO application, Na channel activity decreased. We have extended parts of the continuous trace to display details of channel activity (Fig. 2C). The enlarged recordings in Fig. 2C clearly show that the probability of seeing multiple levels of channel activity under untreated control and 3-CP conditions declines in the presence of O2–-scavenging compound.
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O2– Counters NO Inhibition of ENaC
Increasing endogenous O2–.
Our group has recently shown that NO-releasing compounds can rapidly decrease ENaC activity in both mammalian alveolar (17) and Xenopus kidney epithelial cells (15). Figure 3A2 shows that application of 1.5 µM PAPA NONOate, which rapidly releases 2 mol of NO per mole of reagent, dramatically decreased ENaC activity from control levels. Because O2– anions interact quickly and irreversibly with NO molecules to form peroxynitrite [the reaction rate is close to the diffusion-limited rate of 6.7 x 109 M–1·s–1 (3)], several studies have suggested that the biological activity of NO may be determined by the endogenous amount of O2–. Presumably, the immediate reaction of NO with O2– would consume NO and prevent activation of guanylate cyclase signaling (which has been shown to inhibit ENaC). Thus the level of O2– present in A6 cells could increase ENaC function by limiting NO inhibition of ENaC. This suggestion was consistent with our observations that 1) aldosterone increases O2– generation in A6 cells, 2) increasing exogenous amounts of NO inhibits ENaC NPo, and 3) sequestering O2– with TEMPO inhibited ENaC activity. If O2– indeed buffers the inhibitory effect of NO on ENaC function, then we would expect that an initial increase in the endogenous level of O2– (achieved by treating cells with SOD inhibitor) followed by NO treatment would buffer the inhibitory effect of NO on ENaC activity. Figure 3A shows the effect of adding 5 mM SOD inhibitor followed by 1.5 µM PAPA NONOate treatment to A6 cell-attached patches sequentially. First, Fig. 3A shows that increasing endogenous amounts of O2– by adding SOD inhibitor did not significantly alter ENaC Po. However, in these cells that were pretreated with SOD inhibitor, NO no longer inhibited ENaC activity. Figure 3B shows a representative continuous cell-attached single-channel recording for a control period of 5 min, after which the cell was treated with SOD inhibitor for 5 min, which was followed by an additional treatment with 1.5 µM of the NO donor compound PAPA NONOate. The single-channel activity has been enlarged in Fig. 3C to show the details of multiple open levels that remain unaffected by NO treatment after SOD inhibitor treatment.
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The effect of increasing hypoxanthine and xanthine oxidase 10-fold (17 mU of xanthine oxidase and 45 µM hypoxanthine) in patch-clamp studies was not different; there was no significant increase in ENaC NPo. We used this 10x higher concentration of drug in DHE oxidative labeling studies to estimate the relative amount of O2– released in our patch-clamp studies described above. Figure 4A shows that, in the presence of aldosterone, xanthine oxidase and hypoxanthine did not increase DHE intensities significantly above untreated cells. Together with Fig. 1, these observations suggest that aldosterone-treated A6 cells already generate very high levels of O2– and that a xanthine oxidase and hypoxanthine mix do not generate reactive oxygen species above and beyond what A6 cells are capable of producing in the presence of aldosterone. Therefore, we next tested the amount of O2– that the pharmacological reagents may be generating in the absence of aldosterone. Figure 4B shows that xanthine oxidase and hypoxanthine substrate increase DHE fluorescence intensity 30% from untreated control values. In light of this, we patched A6 cells that were deprived of serum and hormone for 24 h and then applied xanthine oxidase and substrate.
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To fully address whether exogenous production of O2– could increase ENaC function (by limiting NO inhibition of Na channel activity), one must perform a sequence of drug treatments aimed at culturing cells under the appropriate growth condition and designed to prebind a significant fraction of endogenous O2– that is already generated by aldosterone. A summary of such a patch protocol is shown in Fig. 5A, and results from a single-channel patch are shown with NPo values in Fig. 5B. Because it is requisite for functional ENaC expression, A6 cells were first cultured in aldosterone-containing medium, where a control recording period of 5 min was measured. In this particular patch, there are six observable levels of channel activity with a high NPo value of 1.1. In the same cell, endogenous levels of O2– were increased by using SOD inhibitor. Again, application of SOD inhibitor did not change ENaC activity, although it prevented NO inhibition of ENaC (as shown in Figs. 3 and 5B). In this segment of the patch experiment (5–40 min), ENaC NPo values remained at 1.1, and six levels of activity can still be observed (Fig. 5B). Presumably, ENaC function is unaltered by NO in this patch-clamp recording because endogenous reactive oxygen species are quickly binding to NO and limiting its biological effects.
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)-resistance seals after multiple drug applications and to prevent naturally occurring channel rundown. Role of the cGMP Pathway in the Regulation of ENaC by Oxygen Radicals
We have recently shown that NO inhibits lung Na transport through cGMP-mediated inhibition of cation channels on the surface of type II pneumocytes (17). In our present study, we wanted to determine whether cGMP, generated by guanylyl cyclase activity, could also be acting as the main mediator of NO- and O2–-induced effects on ENaC in A6 cells. The NO signal-transduction pathway has not been examined by single-channel recording techniques in kidney epithelia. To determine how NO exerts an inhibitory effect on ENaC, we first recorded for a control period. Then, while maintaining the same cell-attached patch, we treated the cell with an irreversible inhibitor of soluble guanylyl cyclase (ODQ) for 5 min before adding NO donors to the same patch (Fig. 6, A and B).
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5 min of treatment time. Our finding that the SOD mimetic TEMPO also inhibits ENaC, coupled with the data showing that increasing endogenous O2– levels prevented NO inhibition of ENaC activity, strongly suggests that O2– anions regulate ENaC activity by scavenging NO to form peroxynitrite. This model of the effects of O2– on ENaC would be independent of guanylyl cyclase activity, since peroxynitrite formation would occur so rapidly that NO would have no opportunity to interact with guanylyl cyclase. To support this model for the mechanism of reactive oxygen species signaling in Na-transporting epithelia, we inhibited guanylyl cyclase activity in A6 cells with ODQ compound and then treated cells with TEMPO (Fig. 7). Figures 7A shows a typical and representative single-channel recording in which we first obtained a control recording sample and then measured recordings after ODQ treatment for 5 min, before application of 250 µM TEMPO compound. It is clear from Fig. 7 that TEMPO could still inhibit ENaC Po independent of cGMP production. Control Po values averaged 0.13 ± 0.03 and did not significantly change after ODQ treatment (0.12 ± 0.03). However, TEMPO significantly decreased Po values to 0.04 ± 0.001 (P < 0.05). These data are consistent with the idea that aldosterone-induced increases in the production of O2– could act by sequestering NO in A6 cells. Importantly, these results suggest that O2– reacts quickly with NO and that O2– regulation of ENaC precedes NO signaling to cGMP because ODQ compound failed to inhibit TEMPO's effect on Na transport.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Increased O2– production is commonly associated with neutrophils during respiratory burst activity as a host defense to kill invading microbes. However, aldosterone produced no increase in O2– production in human neutrophil granulocytes (4, 5), but aldosterone does appear to have a pro-oxidative effect in other mineralocorticoid-responsive cells. For example, using DHE labeling, Miyata et al. (25) recently showed that aldosterone stimulates O2– production through activation of NADPH oxidase in rat mesangial cells. Similarly, our study also shows that aldosterone can increase O2– production in an A6 distal kidney cell line in a dose-dependent manner. In relation to steroid hormone signaling, Fig. 8 highlights the pathways for the production of reactive oxygen and nitrogen species (as well as the redox reactions examined in our present study). Figure 8 shows that an increase in metabolism, stimulated by steroid hormones, such as aldosterone, can increase NADPH oxidation of molecular O2 to reactive oxygen species. Data from our present study indicate that elevated O2– levels in the cells may limit NO inhibition of ENaC activity. Interestingly, in a previous study, our group (15) showed that aldosterone decreases NO release in alveolar epithelial cells. We speculated in our previous work (on the basis of some evidence) that serum- and glucocorticoid-inducible kinase phosphorylation of inducible nitric oxide synthase (iNOS) could reduce iNOS activity and thereby reduce production of NO. In this study, we suggest that the steroid-induced production of O2– could promote NO degradation to peroxynitrite. Hence, to maximize net Na reabsorption, aldosterone may signal to decrease NO synthesis and at the same time increase NO degradation by increasing O2– production (which binds quickly to NO and forms peroxynitrite) to prevent NO downregulation of ENaC activity. Combined, our results put forth a novel redox-mediated mechanism of ENaC regulation in A6 cells.
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Although the direct effect of O2– on single-channel ENaC activity has not been previously described, O2– accumulation has been associated with hypertension (reviewed in Refs. 33, 34). Specifically, Beswick et al. (7) reported increased reactive oxygen species production in the mineralocorticoid-induced hypertensive rat. Furthermore, in another recent study, Beswick et al. (8) also showed that long-term antioxidant administration in hypertensive rats lowered blood pressure and normalized O2– production. These previous findings in hypertensive animal models, coupled with our new results, support the idea that aldosterone-induced O2– production may increase Na reabsorption in the distal nephron and thereby lead to hypertension.
We pharmacologically decreased and increased O2– levels in A6 cells. Figure 1 shows that TEMPO is an effective O2– scavenger in A6 cells, (as measured by DHE labeling of O2–). As hypothesized, scavenging O2– significantly decreased ENaC Po in A6 cells. This finding was consistent with the previous observations of O'Brodovich and colleagues (31) that showed that TEMPO could block O2–-induced increases in amiloride-sensitive Na current in rat fetal distal lung cells. To further support our finding that sequestering O2– leads to a decrease in ENaC activity, we performed the complement set of experiments, which involved increasing local concentrations of O2– in A6 cells.
The effect of raising local concentrations of O2– on ENaC NPo can be appreciated after SOD-inhibited cells were challenged with NO. SOD inhibitor treatment in A6 cells prevented NO's inhibitory effect on Na channel activity. This is an important observation because NO has been shown to be a fast-acting inhibitor of ENaC and may be a novel mechanism of ENaC regulation. These data also suggest that O2– is necessary for maintaining ENaC activity but is not sufficient by itself to increase Na transport.
We also applied O2–-generating compounds exogenously to A6 cells. However, the hypoxanthine-xanthine oxidase mix by itself did not cause a change in ENaC Po. This may be due to two reasons. First, aldosterone may be already generating very high levels of O2–, as suggested by the data presented in Fig. 1. Second, superoxide anions are not generated stoichometrically in the reaction between xanthine oxidase and hypoxanthine. In fact, uric acid and H2O2 are the major products from combining xanthine oxidase and hypoxanthine in solution. Therefore, it is difficult to ascertain the precise amount of O2– produced, and increasing the amount of xanthine oxidase and hypoxanthine applied to A6 cells by 10-fold did not change ENaC NPo either. To observe the O2–-generating effects of hypoxanthine and xanthine oxidase, we cultured the cells in medium with aldosterone, followed by SOD inhibitor treatment and an initial low concentration of NO. Figure 5B shows that, in this particular patch, there is ample Na transport activity and that increasing endogenous O2– levels with SOD inhibitor prevented 1.5 µM NO inhibition of ENaC, as expected. We then added an excess of NO in the same cell-attached patch to buffer all endogenous O2– generated by aldosterone signaling in the A6 cell. In addition, we reintroduced O2– exogenously using xanthine oxidase-hypoxanthine mix. In this way, we were able to culture A6 cells in medium that was appropriate for Na transport and to test the effects of exogenous O2–.
The precise mechanism in which NO inhibits ENaC is unclear. Our data from Figs. 3–5, however, suggest that an aldosterone-induced increase in O2– production blocks the inhibitory effect of NO in epithelial cells. ODQ inhibition of NO's effect suggests that NO's signaling pathway involves activation of guanylate cyclase and hence is cGMP dependant. Previously, Jain et al. (17) proposed that cGMP action of renal ENaC is mediated via PKG, leading to phosphorylation and the consequent inhibition of ENaC. 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 of -ENaC subunit (Y134 and Y137 in transmembrane domain 1; Y482, Y484, and Y485 in transmembrane domain 2) may alter ENaC activity.
Treatment of cells, however, with ODQ followed by TEMPO compound indicates that O2– works in a cGMP-independent mechanism because sequestering endogenous O2– significantly decreased ENaC Po in the presence of a cGMP inhibitor. Presumably, O2– interaction with NO occurs quickly, preceding NO activation of cGMP and hence ENaC inhibition. Because O2– and NO react quickly to form peroxynitrite, we also tested the effect of 3 µM peroxynitrite on Na channel function. This by-product of O2– and NO interaction did not significantly alter ENaC Po in our studies. A previous publication by DuVall et al. (11), however, described peroxynitrite inhibition of amiloride sensitive Na currents in Xenopus oocytes. Obvious differences in our outcome of peroxynitrite treatment on Na channel function included differences in the cell model system studied, method of peroxynitrite delivery, and especially length of peroxynitrite exposure in the respective cells. We examined the early effect (within minutes) of peroxynitrite application and found no change in ENaC function. This further supports our previous reported finding that NO inhibits ENaC NPo (and not peroxynitrite formation). In the DuVall et al. study, 1 mM 3-morpholinosydnonimine was used to form peroxynitrite in solution over a course of 2 h, and a decrease in whole cell current was observed in oocytes expressing rat -, -, and -ENaC subunits. Taking both accounts of peroxynitrite effects in consideration, it may be possible that immediate short-term exposure of cells to peroxynitrite has no consequence on Na transport, but generation of peroxynitrite over a long period (
2 h) may hinder ENaC function.
Our present finding, that the interaction between O2– and NO works in concert to regulate ENaC, is also indirectly supported by other redox studies performed in Na-transporting epithelia. For example, Hardiman et al. (14) reported that iNOS–/– mice have lower levels of NO2– in the lungs and that the levels of - and -ENaC protein are substantially decreased in the iNOS knockout mice (compared with wild-type controls). From our findings and line of reasoning, the decrease in NO produced in iNOS–/– mice would lead to an increase in the amount of unbound O2– in the animal and create a physiological state of oxidative stress. This might explain the decline in ENaC protein expression in iNOS knockout mice described by Hardiman et al., since oxidative stress has been shown to disrupt transcription of -ENaC subunit in lung epithelial cells (37). In addition, Wang et al. (37) showed that increasing exogenous H2O2 (a by-product of O2– degradation) impairs glucocorticoid hormone-dependent transcription of amiloride-sensitive ENaC by repressing -ENaC glucocorticoid-responsive element. Together, we conclude that the redox state of a cell is dependent on the level of both NO and O2– present. In vivo, an inhibitory effect of ENaC could be caused by either an increase in NO production or a decrease in O2– generation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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-rENaC. Am J Physiol Cell Physiol 274: C1417–C1423, 1998.-ENaC-deficient mice. Nat Genet 12: 325–328, 1996.[CrossRef][Web of Science][Medline]
B activation and blocked by superoxide scavenger. Am J Physiol Lung Cell Mol Physiol 275: L764–L770, 1998.-subunit in lung epithelial cells through ERK-dependent and thioredoxin-sensitive pathways. J Biol Chem 275: 8600–8609, 2000.This article has been cited by other articles:
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  N. Markadieu, R. Crutzen, A. Boom, C. Erneux, and R. Beauwens Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1428 - F1438. [Abstract] [Full Text] [PDF]
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 C. S. Wilcox and A. Pearlman Chemistry and Antihypertensive Effects of Tempol and Other Nitroxides Pharmacol. Rev., December 1, 2008; 60(4): 418 - 469. [Abstract] [Full Text] [PDF]
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