Modulation of aldosterone-induced stimulation of ENaC synthesis by changing the rate of apical Na+ entry

Lisette Dijkink, Anita Hartog, Carel H. Van Os, and René J. M. Bindels

Department of Cell Physiology, University Medical Centre Nijmegen, 6500 HB Nijmegen, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Primary cultures of immunodissected rabbit connecting tubule and cortical collecting duct cells were used to investigate the effect of apical Na+ entry rate on aldosterone-induced transepithelial Na+ transport, which was measured as benzamil-sensitive short-circuit current (Isc). Stimulation of the apical Na+ entry, by long-term short-circuiting of the monolayers, suppressed the aldosterone-stimulated benzamil-sensitive Isc from 320 ± 49 to 117 ± 14%, whereas in the presence of benzamil this inhibitory effect was not observed (335 ± 74%). Immunoprecipitation of [35S]methionine-labeled beta -rabbit epithelial Na+ channel (rbENaC) revealed that the effects of modulation of apical Na+ entry on transepithelial Na+ transport are exactly mirrored by beta -rbENaC protein levels, because short-circuiting the monolayers decreased aldosterone-induced beta -rbENaC protein synthesis from 310 ± 51 to 56 ± 17%. Exposure to benzamil doubled the beta -rbENaC protein level to 281 ± 68% in control cells but had no significant effect on aldosterone-stimulated beta -rbENaC levels (282 ± 68%). In conclusion, stimulation of apical Na+ entry suppresses the aldosterone-induced increase in transepithelial Na+ transport. This negative-feedback inhibition is reflected in a decrease in beta -rbENaC synthesis or in an increase in beta -rbENaC degradation.

rabbit kidney; cortical collecting duct; connecting tubule; epithelial sodium channel; benzamil


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MINERALOCORTICOID HORMONE aldosterone plays a major role in Na+ homeostasis and, consequently, in extracellular volume regulation by controlling epithelial Na+ channel (ENaC) expression in the kidney (14). The ENaC complex, consisting of three subunits, alpha -, beta -, and gamma -ENaC, is present in the apical membrane of epithelial cells in the distal kidney, distal colon, salivary glands, sweat glands, respiratory tract, and taste buds (3, 23). It has been demonstrated that upregulation of ENaC expression by aldosterone differs among mammals (23) and is also tissue specific (30, 32).

There are three possible mechanisms for aldosterone to enhance Na+ transport: first, the synthesis and insertion of ENaC subunits into the apical membrane; second, the activation of existing Na+ channels by regulatory proteins, by so-called "aldosterone-induced proteins"; and third, the increase in the open probability of Na+ channels (34). In primary cultures of rabbit kidney connecting tubule and cortical collecting duct (CNT and CCD, respectively) cells, the first phase of aldosterone-stimulated transepithelial Na+ transport is likely to be mediated by aldosterone-induced proteins. During the late phase of aldosterone action, the threefold increase in apical Na+ transport is accompanied by an increase in rbENaC mRNA for all three subunits, but with only higher alpha - and beta -subunit protein levels (5).

In addition to regulation of ENaC by aldosterone (5, 24, 25), long-term exposure to vasopressin also stimulates ENaC expression (8). Several other studies identified additional mechanisms involved in the regulation of ENaC activity, including changes in pH, ATP, Ca2+ concentrations, Na+ concentrations, and cell swelling (10, 13, 19). Notably, the role of these parameters in the short-term action of ENaC was studied. Furthermore, the role of these factors in aldosterone-induced stimulation of transepithelial Na+ transport has not been investigated. The aim of the present study was, therefore, to investigate the effect of the rate of apical Na+ entry on long-term effects of aldosterone. So far, the mechanisms by which ENaC synthesis is regulated in response to changes in the rate of apical Na+ entry are still poorly understood. Two forms of negative-feedback regulation by increased Na+ concentrations have been described (1, 33), namely, self-inhibition and feedback inhibition. Self-inhibition could be due to a direct interaction of extracellular Na+ with ENaC itself (28). In salivary duct cells it was found that Na+ channel activity does not change with increasing extracellular Na+ (19), whereas in frog skin Na+ channel activity is controlled by extracellular Na+ (11). On the other hand, feedback inhibition could also be mediated by an increase in intracellular Na+ concentration (19).

Primary cultures of rabbit CNT and CCD cells were used to study the effect of changes in the driving force for apical Na+ entry on aldosterone regulation of ENaC activity. In one protocol, primary cultures were short-circuited to stimulate apical Na+ influx. In another protocol, monolayers were incubated overnight with benzamil to block apical Na+ influx. In both protocols, the benzamil-sensitive short-circuit current (Isc) and beta -rbENaC (where rb is rabbit) protein levels were determined.


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

Primary cultures of rabbit kidney "cortical collecting system." Rabbit kidney CNT and CCD cells, hereafter referred to as the cortical collecting system, were immunodissected from kidney cortex of New Zealand White rabbits (±0.5 kg body wt) with the monoclonal antibody R2G9 and set in primary culture on permeable filters (0.33 or 1.13 cm2; Costar, Cambridge, MA) as previous described in detail (2). All experiments were performed with confluent monolayers between 5 and 8 days after the cells were seeded. Sixteen hours before the experiments, the monolayers were short-circuited by flooding the monolayers with culture medium incubated with aldosterone (10-7 M, both sides) or benzamil (10-5 M, apical) and combinations of these treatments.

Ussing chamber experiments. For the measurement of transepithelial Isc, filter cups (area 0.33 cm2) were routinely washed three times with incubation medium containing (in mM) 140 NaCl, 2 KCl, 1 K2HPO4, 1 KH2PO4, 1 MgCl2, 1 CaCl2, 5 glucose, 5 L-alanine, and 10 HEPES-Tris (pH 7.4) and then mounted between two half-chambers and bathed at 37°C with incubation medium. The solutions bathing the monolayers were connected via agar bridges and Ag-AgCl electrodes to a voltage-clamp current amplifier (Physiological Instruments, San Diego, CA), and the Isc was recorded before and after the addition of 10-5 M benzamil (apical side) The benzamil-sensitive component of the Isc was used as an estimate of transcellular sodium transport.

Measurements of extracellular ion concentrations. Confluent monolayers (0.33 cm2) were treated as described in the text. After 16 h of incubation, extracellular Na+, K+, and Ca2+ concentrations were determined by removing duplicate 20-µl samples from the apical and basolateral compartments. The Na+ and K+ contents of the samples were measured by flame photometry (Eppendorf FCM 6343, Hamburg, Germany). The Ca2+ concentration was measured using a colorimetric test kit (Boehringer, Mannheim, Germany).

Immunoprecipitation of beta -ENaC. Confluent monolayers (1.33 cm2) were treated as described in the text. Two hours before the end of the incubation period, filter cups were washed three times for 5 min with DMEM without methionine and subsequently labeled at 37°C for 2 h by apical addition of 0.2 mCi/filter [35S]methionine (ICN Pharmaceuticals, Irvine, CA). After incubation, the cells were washed, scraped, and immunoprecipitated by incubation with affinity-purified beta -ENaC antisera as previous described in detail (5). The immunoprecipitated proteins were resuspended in 25 µl of Laemmli sample buffer and denatured for 5 min at 95°C. Next, 20 µl of the samples were loaded on a 7% (wt/vol) SDS-polyacrylamide gel and electrophoresed. The gel was stained for 10 min at 65°C with 0.25% (wt/vol) Coomassie brilliant blue, 10% (vol/vol) acetic acid, and 50% (vol/vol) methanol; destained twice for 10 min at 65°C with 7% (vol/vol) acetic acid and 25% (vol/vol) methanol; rinsed in water; and incubated twice for 10 min with DMSO and twice for 15 min with 20% (wt/vol) 2,5-diphenyloxazole (Sigma Chemical, St. Louis, MO) in DMSO. After two 5-min rinses in water, the gel was dried and exposed to a film with an intensifying screen at -80°C. The relative amount of 35S incorporation was determined with Molecular Analyst (Bio-Rad, Hercules, CA).

Chemicals. Benzamil was obtained from Research Biochemical International (Natick, MA). All other chemicals were obtained from Sigma. Benzamil and aldosterone were dissolved in ethanol, the final concentration of which never exceeded 0.1% (vol/vol). Aldosterone was added to the apical and basolateral sides, whereas benzamil was added to the apical side only.

Statistics. Results are given as means ± SE. For all experiments, statistical significance was determined by ANOVA; in the case of significance, individual groups were compared by contrast analysis according to Fisher. The level of statistical significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of the rate of apical Na+ influx on the aldosterone-stimulated, benzamil-sensitive Isc. Primary cultures of rabbit CNT and CCD cells were used to determine whether changes in the rate of apical Na+ influx have an effect on transcellular Na+ transport, measured as the benzamil-sensitive Isc. Stimulation of the rate of apical Na+ influx was accomplished by short-circuiting the monolayers for 16 h by flooding the apical and basolateral compartments with culture medium to establish electrical contact. As a result, the apical membrane of the CCD and CNT cells would be hyperpolarized, which would stimulate the apical Na+ entry. Reduction of apical Na+ entry was accomplished by apical exposure of the monolayers to benzamil for 16 h.

Incubation of the monolayers with aldosterone (10-7 M, both sides) for 16 h significantly (P < 0.05) increased the benzamil-sensitive Isc by 320 ± 49%, whereas in chronically short-circuited monolayers no effect of aldosterone was observed (117 ± 14% of control benzamil-sensitive Isc, P > 0.1) (Fig. 1). After short-circuiting was terminated for 1 h, there was no recovery of the aldosterone-stimulated, benzamil-sensitive Isc (data not shown). Furthermore, short-circuiting the monolayers had no effect on basal benzamil-sensitive Isc (72 ± 10% of control levels, P > 0.1). When the apical Na+ influx was blocked by exposure to benzamil for 16 h, the benzamil-sensitive Isc was doubled compared with the control level. However, no significant effect on the aldosterone-induced, benzamil-sensitive Isc was apparent (335 ± 74 and 320 ± 49% for benzamil and aldosterone exposure and aldosterone exposure alone, respectively). In addition, the doubling of the benzamil-sensitive Isc by benzamil also occurred under short-circuit conditions. After 16 h of incubation, the pH of the apical compartment was reduced to 5.6 in untreated monolayers, whereas during short-circuiting of the monolayers the extracellular pH remained at 7.4. To exclude acidification of the apical compartment as the major regulator in the stimulatory effect of aldosterone, we combined chronic short-circuiting with chronic benzamil treatment. In this latter condition, blockage of the apical Na+ entry at pH 7.4, the short-circuiting-induced inhibition of aldosterone-stimulated transcellular Na+ transport was also not realized.


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Fig. 1.   Effect of apical Na+ entry on the benzamil-sensitive short-circuit current (Isc) across primary cultures of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) cells. Unstimulated and aldosterone (Aldo; 10-7M, both sides)-stimulated monolayers were short-circuited (SC) for 16 h, incubated with benzamil (Benz; 10-5 M, apical side), or both short-circuited and incubated with benzamil. After 16 h of exposure, each filter was washed 3 times with normal medium and Isc was measured in an Ussing chamber. Values were normalized by those obtained for the control cells (13 ± 2 µA/cm2). Values are means ± SE of at least 6 filters. *P < 0.05, significantly different from control.

Effect of extracellular ion concentrations on transepithelial Na+ transport. Next, we determined the Na+, K+, and Ca2+ concentrations in the extracellular medium of control and short-circuited monolayers untreated or treated with aldosterone. Table 1 shows that after 16 h of incubation, the apical Na+ concentration of untreated and aldosterone-treated monolayers decreased from 140 to 82 and 59 mM, respectively, whereas apical K+ concentration increased from 5 to 32 and 38 mM, respectively. The apical Ca2+ concentration in unstimulated and stimulated conditions decreased from 1.0 to 0.36 and 0.47 mM, respectively. The influence of these extracellular ion concentrations on the modulation of the aldosterone-induced, benzamil-sensitive Isc was examined by mimicking the described circumstances. Na+ (80 mM) or 30 mM K+ in the medium during short-circuiting and aldosterone treatment had no stimulating effect on benzamil-sensitive Isc (50 ± 5 and 106 ± 27%, respectively). Moreover, when the extracellular Ca2+ concentration was reduced by the Ca2+ chelator EGTA (0.8 mM) or the intracellular Ca2+ concentration by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (10 µM) for 16 h, the Na+ channel activity did not change in aldosterone-stimulated, short-circuited monolayers (117 ± 25 and 54 ± 31%, respectively). Taken together, the extracellular Na+, K+, or Ca2+ concentrations did not influence the aldosterone-induced ENaC activity of rabbit CNT and CCD cells.

                              
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Table 1.   Changes in ion concentrations in the apical medium after influencing of apical Na+ entry

Effect of the rate of apical Na+ influx on aldosterone-stimulated beta -rbENaC protein levels. The aldosterone-stimulated rbENaC proteins were measured after the driving force for apical Na+ entry was changed. The beta -rbENaC protein levels were determined by radioactive immunoprecipitation of this 97-kDa protein. The results of a representative immunoblot of immunoprecipitated beta -rbENaC from [35S]methionine-labeled primary cultures of rabbit CNT and CCD are shown in Fig. 2. Aldosterone treatment for 16 h increased the beta -rbENaC protein level by 310 ± 51% (Fig. 3). After 16-h short-circuiting, the beta -rbENaC protein level remained unaffected. The inhibitory effect of chronically short-circuiting the monolayers on the aldosterone-induced benzamil-sensitive Isc, as shown above, was accompanied by a significant decrease in aldosterone-stimulated beta -rbENaC protein synthesis from 310 ± 51 to 115 ± 12% of control levels. Furthermore, in monolayers exposed to benzamil for 16 h, the beta -rbENaC protein synthesis was increased to a protein level (282 ± 68%) comparable to that found in aldosterone-induced monolayers. In monolayers in the combined condition of short-circuiting, benzamil exposure, and aldosterone exposure, the beta -rbENaC protein synthesis was stimulated to the same level (286 ± 45%). Thus, also on the protein level, benzamil treatment overcame the inhibitory effect on aldosterone stimulation of short-circuiting the monolayers.


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Fig. 2.   Representative immunoprecipitation of beta -rabbit epithelial sodium channel (rbENaC) in SC, Benz (10-5 M, apical side), or SC+Benz primary cultures of rabbit CCD and CNT cells in control and Aldo (10-7M, both sides)-stimulated conditions. At the end of the stimulation period, cells were labeled with [35S]methionine for 2 h.



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Fig. 3.   Effect of apical Na+ entry on the synthesis of beta -ENaC in primary cultures of rabbit CNT and CCD cells. Levels of beta -rbENaC were determined by immunoprecipitation of [35S]methionine-labeled SC, Benz (10-5 M, apical side), or SC+Benz primary cultures of rabbit CCD and CNT cells in control and Aldo (10-7M, both sides)-stimulated conditions. Values were normalized by those obtained for the control cells. Values are means ± SE of at least 4 experiments. *P < 0.05, significantly different from control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study examines the influence of the rate of apical Na+ entry on aldosterone regulation of transepithelial Na+ transport. In primary cultures of rabbit kidney cortical collecting system, aldosterone exposure stimulates an increase in benzamil-sensitive Na+ transport, resulting from an increase in alpha - and beta -ENaC protein synthesis (5). In the present study, we have demonstrated that the aldosterone-stimulated rbENaC transcription or translation process is blocked by a long-term short-circuiting of the monolayers. It is likely that short-circuiting the monolayers increases the intracellular Na+ concentration due to an increase in the apical Na+ influx by hyperpolarization of the apical membrane (6, 9, 17). Our study further shows that the rate of apical Na+ entry has a significant effect on ENaC expression, because benzamil treatment overrides the inhibitory effect of short-circuiting on aldosterone-stimulated Na+ transport. This feedback inhibition is mediated either by a decrease in beta -rbENaC synthesis or by an increase in beta -rbENaC degradation.

The mechanisms by which intracellular Na+ concentration might affect ENaC activity are unclear. Chronically short-circuiting the monolayers, and thereby increasing intracellular Na+ concentration, may inhibit the aldosterone-induced ENaC expression via regulatory elements on the alpha -, beta -, and/or gamma -ENaC gene(s), for example, via an as yet unidentified Na+-responsive element. Many regulatory pathways, controlled by cytosolic Na+ concentration, are potentially involved in the feedback regulation of ENaC. In salivary duct cells, Komwatana et al. (20, 21) have identified a G0 protein as the mediator of a Na+-feedback system. This described model for feedback regulation is as follows: cytosolic Na+ binds to an intracellular Na+ receptor, activating the G0 protein, and the alpha -subunit of the G0 protein causes the ubiquitine-protein ligase Nedd4 to ubiquinate and inactivate ENaC (16). In Xenopus laevis oocytes, Na+ feedback inhibition is present together with Nedd4-dependent regulation of ENaC but does not require G protein function (15). Nedd4 proteins contain WW domains, which can bind to the PY motifs of beta - and gamma -ENaC subunits and ubiquinate the ENaC COOH termini, leading to endocytosis and degradation of ENaC. This feedback-inhibition model could be applied to the present study in the rabbit cortical collecting system. Short-circuiting of the monolayers will result in a higher cytosolic Na+ concentration, and Na+ binds in a concentration-dependent manner to an intracellular Na+ receptor, which activates Nedd4 and leads to degradation of ENaC subunits. Mutations in the PY motifs associated with Liddle syndrome, an inherited form of salt-sensitive hypertension, also interfere with the feedback regulation by intracellular Na+ (7, 13, 18).

Other feedback loops possibly involved in the downregulation of ENaC have also been studied in the past. Chalfant et al. (4) showed that in lipid bilayers rENaC currents were dependent on intracellular pH and were not influenced by changes in extracellular pH. In A6 cells (36) as well as in rat cortical collecting tubule (27), changes in intracellular pH correlate positively with changes in Na+ current and transepithelial conductance. An important finding in our study was that an extracellular pH between 7.4 and 5.6 is not a dominant factor in aldosterone-stimulated ENaC synthesis, because benzamil overcomes the inhibitory effect of short-circuiting on aldosterone-stimulated ENaC expression. In the short-circuiting situation, aldosterone-induced ENaC expression can also be decreased under the control of reduced ATP levels. The rise in intracellular Na+ will increase the energy used by the Na-K pump, leading to a decrease in ATP levels. Downregulation of ENaC as a result of increased demand for ATP by the Na-K pump is described by Frindt et al. (10). In addition, during benzamil exposure a rise in ATP can be involved in the stimulatory effect on ENaC expression. Another proposed factor involved in feedback inhibition is intracellular Ca2+. In the primary cultures of CNT and CCD there is no effect on the benzamil-sensitive Isc after buffering of the intracellular or extracellular Ca2+ concentrations of the short-circuited and aldosterone-exposed monolayers. The literature on experiments with increased cytoplasmic Ca2+ concentrations reports several discrepancies. In rat kidney, there is no direct effect of Ca2+ measured in inside-out patches, whereas in studies with vesicles of toad bladder and in rabbit cortical collecting tubules an increase in Ca2+ leads to a reduced amiloride-sensitive Na+ influx (9, 12).

One conclusion of our data is that an increase in cell volume, induced by increased Na+ influx, prevents the cell from reacting properly to aldosterone. Apparently, cell volume control has a higher priority then a response to aldosterone. Recently, the role of the cell volume-sensitive kinase sgk (serine-threonine kinase) in ENaC regulation has also been described (22, 26, 29). Sgk is rapidly and strongly upregulated by aldosterone in rat cortical collecting duct. In addition, coexpression of sgk with ENaC in X. laevis oocytes stimulated ENaC activity about sevenfold. The sgk transcription level correlates negatively with cell volume (35). Cell swelling-associated degradation of sgk also provides a possible explanation for the inhibiton of aldosterone-induced Na+ transport, which we observed in short-circuited cells. Blocking of Na+ entry by benzamil may lead to cell shrinkage, and this could stimulate the accumulation of sgk and thereby ENaC activity. It is of interest to note that modulation of Na+ influx by short-circuiting A6 cells in the absence of aldosterone has an effect opposite to what we report here for rabbit primary cultures of CNT and CCD cells in the presence of aldosterone. Rokaw et al. (31) showed that decreasing Na+ influx decreases Isc and increasing Na+ influx increases Isc. These observations were made in culutres in the absence of aldosterone. Therefore, intracellular Na+ concentration may have dual effects depending on the absence or presence of aldosterone.

In conclusion, in the present study we have demonstrated that in primary cultures of the rabbit cortical collecting system the aldosterone transcription-translation process can be inhibited by chronically short-circuiting the monolayers. Chronic benzamil treatment can overcome this inhibitory effect. The obvious explanation is feedback inhibition by an increase in the intracellular Na+ concentration as a result of hyperpolarization of the apical membrane. Further experiments are needed to delineate the molecular mechanism behind this feedback inhibition by intracellular Na+.


    ACKNOWLEDGEMENTS

We thank T. Koks for contributing to the Ussing chamber experiments.


    FOOTNOTES

This work was supported by Dutch Kidney Foundation Grant C94.1348.

Address for reprint requests and other correspondence: R. J. M. Bindels, Department of Cell Physiology, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands (E-mail: ReneB{at}sci.kun.nl).

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

Received 13 December 2000; accepted in final form 17 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abriel, H, and Horisberger JD. Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes. J Physiol (Lond) 516: 31-43, 1999[Abstract/Free Full Text].

2.   Bindels, RJM, Hartog A, Timmermans JAH, and Van Os CH. Active Ca2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH. Am J Physiol Renal Fluid Electrolyte Physiol 261: F799-F807, 1991[Abstract/Free Full Text].

3.   Canessa, C, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[ISI][Medline].

4.   Chalfant, ML, Denton JS, Berdiev BK, Ismailov II, Benos DJ, and Stanton BA. Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel. Am J Physiol Cell Physiol 276: C477-C486, 1999[Abstract/Free Full Text].

5.   Dijkink, L, Hartog A, Deen PMT, Van Os CH, and Bindels RJM Time-dependent regulation by aldosterone of the amiloride-sensitive Na+ channel in rabbit kidney. Pflügers Arch 438: 354-360, 1999[ISI][Medline].

6.   Dinno, MA, and Huang KC. Short circuit current in tight and leaky epithelia. Biochim Biophys Acta 509: 318-325, 1978[ISI][Medline].

7.   Dinudom, A, Harvey KF, Komwatana P, Young JA, Kumar S, and Cook DI. Nedd4 mediates control of an epithelial Na+ channel in salvary duct cells by cytosolic Na+. Proc Natl Acad Sci USA 95: 7169-7173, 1998[Abstract/Free Full Text].

8.   Ecelbarger, CA, Kim GH, Terris J, Masilamani S, Mitchell C, Reyes I, Verbalis JG, and Knepper MA. Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney. Am J Physiol Renal Physiol 279: F46-F53, 2000[Abstract/Free Full Text].

9.   Frindt, G, Silver RB, Windhager EE, and Palmer LG. Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry. Am J Physiol Renal Fluid Electrolyte Physiol 264: F565-F574, 1993[Abstract/Free Full Text].

10.   Frindt, G, Silver RB, Windhager EE, and Palmer LG. Feedback regulation of Na channels in rat CCT. III. Response to cAMP. Am J Physiol Renal Fluid Electrolyte Physiol 268: F480-F489, 1995[Abstract/Free Full Text].

11.   Fuchs, W, Larsen EH, and Lindemann B. Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J Physiol (Lond) 267: 137-166, 1977[ISI][Medline].

12.   Garty, H. Molecular properties of epithelial, amiloride-blockable Na+ channels. FASEB J 8: 522-528, 1994[Abstract/Free Full Text].

13.   Harvey, KF, Dinudom A, Komwatana P, Jolliffe CN, Day ML, Parasivam G, Cook DI, and Kumar S. All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+. J Biol Chem 274: 12525-12530, 1999[Abstract/Free Full Text].

14.   Hummler, E, and Rossier BC. Physiological and pathophysiological role of the epithelial sodium channel in the control of blood pressure. Kidney Blood Pres Res 19: 60-165, 1996.

15.   Hybnera, M, Schreibera R, Boucherota A, Sanchez-Perez A, Poronnik P, Cook DI, and Kunzelmann K. Feedback inhibition of epithelial Na+ channels in Xenopus oocytes does not require G0 or Gi2 proteins. FEBS Lett 459: 443-447, 1999[ISI][Medline].

16.   Ishibashi, H, Dinudom A, Harvey KF, Kumar S, Young JA, and Cook DI. Na+-H+ exchange in in salvary secretory cells is controlled by an intracellular Na+ receptor. Proc Natl Acad Sci USA 96: 9949-9953, 1999[Abstract/Free Full Text].

17.   Ishikawa, T, Marunaka Y, and Rotin D. Electrophysiological characterization of rat epithelial Na+ channel (rENaC) expressed in MDCK-cells---effects of Na+ and Ca2+. J Gen Physiol 111: 825-846, 1998[Abstract/Free Full Text].

18.   Kellenberger, S, Gautschi I, Rossier BC, and Schild L. Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system. J Clin Invest 101: 2714-2750, 1998.

19.   Komwatana, P, Dinudom A, Young JA, and Cook DI. Control of the amiloride-sensitive Na+ current in salivary duct cells by extracellular sodium. J Membr Biol 150: 133-141, 1996[ISI][Medline].

20.   Komwatana, P, Dinudom A, Young JA, and Cook DI. Activators of epithelial Na+ channels inhibit cytosolic feedback control. Evidence for the existence of G protein coupled receptor for cytosolic Na+. J Membr Biol 162: 225-232, 1998[ISI][Medline].

21.   Komwatana, P, Dinudom A, Young JA, and Cook DI. Cytosolic Na+ controls an epithelial Na+ channel via the G0 guanine nucleotide-binding regulatory protein. Proc Natl Acad Sci USA 93: 8107-8111, 1998[Abstract/Free Full Text].

22.   Lang, F, Klingel K, Wagner CA, Stegen C, Wärntges S, Friedrich B, Lanzendörfer Melzig J, Moschen I, Steuer S, Waldegger S, Sauter M, Paulmichl M, Gerke V, Risler T, Gamba G, Capasso G, Kandolf R, Hebert SC, Massry SG, and Bröer S. Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc Natl Acad Sci USA 97: 8157-8162, 2000[Abstract/Free Full Text].

23.   Lingueglia, E, Renard S, Waldmann R, Voilley N, Champigny G, Plass H, Lazdunski M, and Barby P. Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone. J Biol Chem 269: 13736-13739, 1994[Abstract/Free Full Text].

24.   Masilamani, S, Kim GH, Mitchell C, Wade JB, and Knepper MA. Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. J Clin Invest 104: R19-R23, 1999[Abstract/Free Full Text].

25.   May, A, Puoti A, Gaeggeler HP, Horisberger JD, and Rossier BC. Early effects of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells. J Am Soc Nephrol 8: 1813-1822, 1997[Abstract].

26.   Náray-Fejes-Tóth, A, and Fejes-Tóth G. The sgk, an aldosterone-induced gene in mineralocorticoid target cells, regulates the epithelial sodium channel. Kidney Int 57: 1290-1294, 2000[ISI][Medline].

27.   Palmer, LG, and Frindt G. Effects of cell Ca and pH on Na channels from rat cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 253: F333-F339, 1987[Abstract/Free Full Text].

28.   Palmer, LG, Sackin H, and Frindt G. Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule. J Physiol (Lond) 509: 151-162, 1998[Abstract/Free Full Text].

29.   Pearce, D, Verrey F, Chen SY, Mastroberardino L, Meijer OC, Wang J, and Bhargava A. Role of SGK in mineralocorticoid-regulated sodium transport. Kidney Int 57: 1283-1289, 2000[ISI][Medline].

30.   Renard, S, Voilley N, Bassilana F, Lazdunski M, and Barby P. Localization and regulation by steroids of the alpha, beta and gamma subunits of the amiloride sensitive Na+ channel in colon, lung and kidney. Pflügers Arch 430: 299-307, 1995[ISI][Medline].

31.   Rokaw, MD, Sarac E, Lechman E, West M, Angeski J, Johnson JP, and Zeidel ML. Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry. Am J Physiol Cell Physiol 270: C600-C607, 1996[Abstract/Free Full Text].

32.   Stokes, JB, and Sigmund RD. Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity. Am J Physiol Cell Physiol 274: C1699-C1707, 1998[Abstract/Free Full Text].

33.   Turnheim, K. Intrinsic regulation of apical sodium entry in epithelial. Physiol Rev 71: 429-445, 1991[Abstract/Free Full Text].

34.   Verrey, F. Transcriptional control of sodium transport in tight epithelia by adrenal steroids. J Membr Biol 144: 93-110, 1995[ISI][Medline].

35.   Waldegger, S, Barth P, Raber G, and Lang F. Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 94: 4440-4445, 1997[Abstract/Free Full Text].

36.   Zeiske, W, Smets I, Ameloot M, Steels P, and Van Driessche W. Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport. Am J Physiol Cell Physiol 277: C469-C479, 1999[Abstract/Free Full Text].


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