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CELLULAR AND MOLECULAR

Evans Blue Is a Specific Antagonist of the Human Epithelial Na⁺ Channel -Subunit

Department of Molecular Morphology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan

Received July 17, 2005; accepted August 15, 2005.

	Abstract

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The epithelial Na⁺ channel (ENaC) regulates Na⁺ homeostasis in cells and across epithelia. Four homologous ENaC subunits (, , , and ) have been isolated in mammals. Combination of -, -, and -subunits or -, -, and -subunits forms fully functional channels. Amiloride is a well known blocker of the ENaC family that inhibits both channel complexes. However, no specific antagonists are currently known that distinguish them. Here, we show that Evans blue, a diagnostic aid for the measurement of blood volume and vascular permeability, inhibits the activity of the -subunit expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in human ENaC-expressing oocytes were inhibited by the application of Evans blue in a concentration-dependent manner with an IC₅₀ value of 143 µM. Evans blue markedly inhibited the -subunit current but did not block the -subunit current. In conclusion, Evans blue is the first known -subunit-specific antagonist of ENaC. This finding provides us with a key compound for elucidating the physiological and pathological functions of ENaC in humans and for drug development in the ENaC family.

In contrast to the relatively well documented -subunit, the pharmacological profile of the -subunit has been poorly investigated. Amiloride, a potassium-sparing diuretic, has been described as a common blocker of the ENaC family (Canessa et al., 1993; McDonald et al., 1994; Waldmann et al., 1995; Ji et al., 2004). Recently, we reported that capsazepine is the first known chemical activator of the -subunit (Yamamura et al., 2004b). In addition to the -subunit-selective agonist, it is necessary to identify the specific inhibitor to explore the physiological and pathological functions of the -subunit of the ENaC.

In this investigation, the effects of Evans blue, which has been widely used as a diagnostic aid for the measurement of blood volume and vascular permeability (Rogers et al., 1989; Patterson et al., 1992), were examined on the human ENaC (hENaC) current using an electrophysiological technique in the Xenopus oocyte expression system. Here, we show that Evans blue inhibits the activity of the -subunit in a concentration-dependent manner, whereas the -subunit current is not blocked but slightly increased by Evans blue. This result indicates that Evans blue is a -subunit-specific antagonist of ENaC.

	Materials and Methods

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Molecular Biology. All experiments were approved by the Ethics Committee of the Nagoya City University Graduate School of Medical Sciences and were conducted in accordance with the Declaration of Helsinki. Full-length hENaC (GenBank accession no. X76180 [GenBank] ), hENaC (X87159 [GenBank] ), and hENaC (U48937 [GenBank] ) were isolated from human skin cDNA, and hENaC (U38254 [GenBank] ) was from human brain cDNA, as described previously (Yamamura et al., 2004b).

Electrophysiology. Electrophysiological studies using a two-electrode voltage-clamp technique were performed in Xenopus oocytes, as described previously (Yamamura et al., 2004b). In brief, cRNA transcript(s) (1 ng for the homomeric channel or each 0.01 ng for coexpression) was injected into Xenopus oocytes, whereas native oocytes were injected with an equal volume of nuclease-free water. After injection, oocytes were incubated at 20°C in a recording solution supplemented with 10 to 100 µM amiloride for 24 to 48 h. The recording solution had an ionic composition of 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5. Oocytes were clamped at a holding potential of -60 mV, and the current-voltage relationship was measured using a ramp protocol from -100 to 50 mV for 15 s. All electrophysiological experiments were carried out at room temperature (25 ± 1°C).

Chemicals. Pharmacological reagents were obtained from Sigma-Aldrich (St. Louis, MO). Evans blue (6,6'-[(3,3'-dimethyl-[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid] tetrasodium salt) and amiloride [3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecarboxamide] were dissolved in dimethyl sulfoxide at the concentration of 100 mM as stock solutions. It was confirmed that up to 1% of dimethyl sulfoxide did not affect the oocyte currents.

Statistics. Pooled data are shown as the mean ± S.E.M. Statistical significance between two groups and among groups was determined by Student's t test and Scheffé's test after one-way analysis of variance, respectively. Significant difference is expressed in the figures (** or ##, p < 0.01). The data of the relationship between drug concentrations and current responses were fitted using the following equation after normalization: relative current = 1 - (1 - C)/{1 + (K/[A])ⁿ}, where C is the component resistant to the drug, K_d is the apparent dissociation constant of the drug, [A] is the drug concentration, and n is the Hill coefficient.

	Results

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Inhibition of -Subunit Current by Evans Blue. The effects of Evans blue on the currents of hENaC and the complexes with - and -subunits were examined using a two-electrode voltage-clamp technique in the Xenopus oocyte expression system. When the hENaC homomer was expressed in Xenopus oocytes, the mean amplitudes of the 100 µM amiloride-sensitive inward current at a holding potential of -60 mV was 59 ± 4 nA (n = 6, p < 0.01 versus native of 2 ± 1 nA, n = 7; Fig. 1). In hENaC-expressing oocytes, the application of Evans blue at 100 or 300 µM blocked the inward current concentration dependently (n = 6; Fig. 1B). Alternatively, the mean amplitudes of the 100 µM amiloride-sensitive inward current at -60 mV was 592 ± 19 nA in hENaC-expressing oocytes (n = 12). A similar current decrease by Evans blue was observed in hENaC-injected oocytes (n = 12; Fig. 1C). The decrease in inward currents in response to Evans blue was gradually recovered to the resting level by the removal of Evans blue in all hENaC- and -expressing oocytes tested (n = 6 and 12, respectively). After washing for a few minutes, the readministration of Evans blue caused a similar current blockage to the first challenge in hENaC-injected oocytes (data not shown). On the contrary, in native oocytes, the application of 100 or 300 µM Evans blue did not induce any current (n = 7; Fig. 1A).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Inhibition of -subunit current by Evans blue. Whole-cell currents were recorded at a holding potential of -60 mV in the Xenopus oocyte expression system using a two-electrode voltage-clamp technique. A to C, typical traces in responses to amiloride (Ami) and Evans blue (EB) in native (A), hENaC-expressed (B), or -expressed (C) oocytes are presented. Note that hENaC- or -expressed oocytes possessed a larger inward current than native oocytes. The larger currents were mostly inhibited by 100 µM amiloride. In hENaC-injected oocytes, the application of Evans blue reduced the inward current in a concentration-dependent manner. Also, amiloride-sensitive hENaC currents were concentration-dependently abolished by Evans blue. These current decreases were recovered by the removal of Evans blue in hENaC- and -expressing oocytes. Neither the application of Evans blue nor amiloride induced any current in native oocytes.

Inhibitory Effects of Evans Blue on -Subunit Current.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Inhibitory effects of Evans blue on -subunit current. The blockage by Evans blue on amiloride-sensitive current at a holding potential of -60 mV was summarized. A, typical current-voltage relationships in the absence and presence of 300 µM Evans blue in a hENaC-injected oocyte is shown. The application of Evans blue blocked hENaC activity at all voltages examined. B, effects of 300 µM Evans blue on the inward currents in hENaC-expressed oocytes are illustrated. C, current amplitudes sensitive to 300 µM Evans blue in native, hENaC-, and -expressed oocytes are summarized. D, amplitudes of 300 µM Evans blue-sensitive current in hENaC- and -expressed oocytes are replotted after normalization by inward current sensitized with 100 µM amiloride. These inhibitory effects of Evans blue on amiloride-sensitive current were similar regardless of the presence of - and -subunits (p > 0.05). Experimental data were obtained from 6 to 12 oocytes. The statistical significance of the difference is expressed as p < 0.01 (**) versus control or native.

Dose-Dependence of Current Blockage by Evans Blue. The concentration-dependence of the Evans blue-sensitive current was analyzed in hENaC-expressed oocytes. Changing the concentration of Evans blue in a range from 1 to 1000 µM showed that the inward current was significantly decreased by Evans blue at a concentration of 100 µM and more (n = 4, p < 0.05 versus control of 774 ± 20 nA), and the current inhibition was in a concentration-dependent manner (Fig. 3). The IC₅₀ value of Evans blue on the inward currents was 143 µM, and the Hill coefficient was 1.7. To compare the binding affinity between Evans blue and amiloride, a common blocker of the ENaC family, the inhibitory effects of amiloride on the inward current through hENaC were examined. The IC₅₀ value of amiloride on the inward currents was 8 µM, and the Hill coefficient was 0.8 (Fig. 3B). There was no further detectable current decrease when 1 mM amiloride was added during the application of 1 mM Evans blue (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Dose-dependence of current blockage by Evans blue. The concentration-dependence of Evans blue on the hENaC current was analyzed in Xenopus oocytes. A, typical current trace of Evans blue (EB) responsiveness at a concentration between 1 and 1000 µM in a hENaC-injected oocyte is represented. The decrease in an inward current in response to Evans blue was in a concentration-dependent manner. Note that there was no further detectable current decrease when 1 mM amiloride (Ami) was added in the presence of 1 mM Evans blue. B, these sensitivities to Evans blue () and amiloride () in the inward currents in hENaC-expressed oocytes are summarized. The inward current was significantly decreased by Evans blue at a concentration of 100 µM and more (p < 0.05). The IC50 value for Evans blue on the inward currents was 143 µM, and the Hill coefficient was 1.7. A dose-response curve for amiloride on the inward current was plotted; the IC50 value and Hill coefficient were 8 µM and 0.8, respectively. Experimental data were obtained from four oocytes for each.

Specific Antagonism of -Subunit, Not -Subunit, by Evans Blue.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Specific antagonism of -subunit, not -subunit, by Evans blue. The effects of Evans blue on another ENaC core unit, the -subunit, were examined in Xenopus oocytes. A, typical current trace in a hENaC-expressed oocyte is represented. The inward currents were sensitive to amiloride at the lower concentration of 10 µM (Ami). Unexpectedly, in contrast to the -subunit, a concentration-dependent current increase by Evans blue (EB) was observed in hENaC-injected oocytes. The current increase evoked by Evans blue was mostly abolished by the addition of 10 µM amiloride. B, effects of 300 µM Evans blue in the absence and presence of 10 µM amiloride on the inward currents in hENaC-expressed oocytes are summarized. Experimental data were obtained from seven oocytes. The statistical significance of the difference is expressed as p < 0.01 versus control (**) and p < 0.01 versus Evans blue alone (##).

	Discussion

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Ion channels in ENaC, members of the degenerin/ENaC superfamily, play pivotal control elements for Na⁺ homeostasis in cells and across epithelia. Since the ENaC complex contributes to the regulations of Na⁺ balance, blood volume, and blood pressure in epithelia such as the kidney, lung, and colon, the physiological and pharmacological properties have been well studied (Alvarez de la Rosa et al., 2000; Kellenberger and Schild, 2002). On the other hand, the physiological and pathological functions of the -subunit had not yet been identified. Recently, we have shown that the -subunit is widely distributed throughout the brain and is activated by protons, indicating that it may act as a pH sensor in the human brain (Yamamura et al., 2004a). Moreover, from a pharmacological aspect, we demonstrated that capsazepine, which was originally developed as a competitive antagonist for the vanilloid receptor (Bevan et al., 1992; Caterina et al., 1997), is a chemical agonist for the -subunit (Yamamura et al., 2004b). In this study, we found that the application of Evans blue inhibits the activity of the -subunit in a concentration-dependent manner. The most interesting finding is that Evans blue blocked the -subunit but not the -subunit.

When the hENaC complex was expressed in Xenopus oocytes, the application of Evans blue at a concentration of 100 µM and more significantly reduced inward current. Because the reversible current decreased by Evans blue was not observed in native oocytes, Evans blue-sensitive currents were resulted from the ENaC expression. In addition to hENaC, the homomeric -subunit was also inhibited by Evans blue, indicating that it acts directly on the -subunit itself. Evans blue failed to decrease the current amplitude of another ENaC core unit, -subunit with 37% amino acid identity to -subunit, and unexpectedly, Evans blue caused a slight increase in hENaC current. It has been reported that there are differences in Na⁺ permeability (/ = 1.6: 0.6 as P_Li/P_Na) and amiloride sensitivity (/ = 0.1:2.6 µM as IC₅₀ values) between the - and -subunits (Kellenberger and Schild, 2002). Although the affinity of Evans blue with the -subunit was approximately 20-fold lower than that of amiloride in this study, Evans blue was a -subunit-specific inhibitor that distinguished - and -subunits. The -subunit is reported to be expressed in the human kidney and lung (McDonald et al., 1994, 1995), whereas the -subunit is expressed in the brain as well as non-neuronal tissues such as the heart, kidney, and pancreas in humans (Waldmann et al., 1995; Yamamura et al., 2004a). Tissue distribution implies that both the - and -subunit proteins may coexpress to play physiological roles in the human kidney. It seems to be difficult for amiloride to separate the component of either the - or -subunit from the amiloride-sensitive current in the human tissues because of the lower selectivity of amiloride. Our screening of the chemical agonists and antagonists for the -subunit revealed capsazepine and icilin as specific agonists as described previously (Yamamura et al., 2004b, 2005) and Evans blue as a specific antagonist in this study. Although the electrophysiological signals mediated by the -subunit are not detected in intact cells or tissues, these findings of chemical compounds strongly influencing the -subunit may lead to the elucidation of the physiological functions of the -subunit in vitro and in vivo in humans.

Evans blue, an azo dye, has been widely used as an indicator in the determination of blood volume and in studies of vascular permeability because of its strong affinity for albumin (Rogers et al., 1989; Patterson et al., 1992). Several recent studies have shown that this compound is able to modulate some receptors and ion channels at submillimolar concentrations that are necessary to examine microvascular or epithelial permeability. It has been described that Evans blue modulates the non-N-methyl-D-aspartate receptor in rat thalamic neurons (Leßmann et al., 1992), blocks the P₂X-purinergic receptor in rat vas deferens (Bultmann and Starke, 1993), and inhibits the glutamate transporter in rat brain synaptic vesicles (Roseth et al., 1995). Furthermore, recent evidence suggests that Evans blue activates large-conductance Ca²⁺-activated K⁺ channels in sheep bladder myocytes (Hollywood et al., 1998) and cultured endothelial cells of human umbilical veins (Wu et al., 1999). In addition to these actions on receptors and channels, in this investigation, we have clarified that Evans blue inhibits the -subunit of the ENaC.

In conclusion, we found that Evans blue acts selectively on the -subunit of the ENaC rather than the -subunit and causes inhibition of the -subunit current, indicating that Evans blue is a specific antagonist for -subunit of the ENaC. In addition to the physiological function of -subunit as a pH sensor in the human brain, this finding in our study provides a starting point for a number of exciting follow-up investigations into the physiological and pathological roles of ENaC in humans.

	Acknowledgements

 
We thank Katsuyuki Tanaka and Kenji Kajita for technical assistance.

	Footnotes

 
This work was supported by a grant-in-aid for scientific research from the Japan Society for the Promotion of Sciences (to H.Y. and S.S.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.092775.

ABBREVIATIONS: ENaC, epithelial Na⁺ channel; hENaC, human epithelial Na⁺ channel.

Address correspondence to: Dr. Hisao Yamamura, Department of Molecular Morphology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi Mizuhocho Mizuhoku, Nagoya 467-8601, Japan. E-mail: yamamura{at}med.nagoya-cu.ac.jp

	References

Top Abstract Materials and Methods Results Discussion References

 

Alvarez de la Rosa D, Canessa CM, Fyfe GK, and Zhang P (2000) Structure and regulation of amiloride-sensitive sodium channels. Annu Rev Physiol 62: 573-594.[CrossRef][Medline]

Bevan S, Hothi S, Hughes G, James IF, Rang HP, Shah K, Walpole CS, and Yeats JC (1992) Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin. Br J Pharmacol 107: 544-552.[Medline]

Bultmann R and Starke K (1993) Evans blue blocks P₂X-purinoceptors in rat vas deferens. Naunyn-Schmiedeberg's Arch Pharmacol 348: 684-687.[CrossRef][Medline]

Canessa CM, Horisberger JD, and Rossier BC (1993) Epithelial sodium channel related to proteins involved in neurodegeneration. Nature (Lond) 361: 467-470.[CrossRef][Medline]

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

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, and Julius D (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature (Lond) 389: 816-824.[CrossRef][Medline]

Hollywood MA, Cotton KD, McHale NG, and Thornbury KD (1998) Enhancement of Ca²⁺-dependent outward current in sheep bladder myocytes by evans blue dye. Pflueg Arch Eur J Physiol 435: 631-636.[Medline]

Ji HL, Bishop LR, Anderson SJ, Fuller CM, and Benos DJ (2004) The role of Pre-H2 domains of - and -epithelial Na⁺ channels in ion permeation, conductance and amiloride sensitivity. J Biol Chem 279: 8428-8440.[Abstract/Free Full Text]

Kellenberger S and Schild L (2002) Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. Physiol Rev 82: 735-767.[Abstract/Free Full Text]

Leßmann V, Gottmann K, and Lux HD (1992) Evans blue reduces macroscopic desensitization of non-NMDA receptor mediated currents and prolongs excitatory postsynaptic currents in cultured rat thalamic neurons. Neurosci Lett 146: 13-16.[Medline]

McDonald FJ, Price MP, Snyder PM, and Welsh MJ (1995) Cloning and expression of the - and -subunits of the human epithelial sodium channel. Am J Physiol 268: C1157-C1163.

McDonald FJ, Snyder PM, McCray PB Jr, and Welsh MJ (1994) Cloning, expression and tissue distribution of a human amiloride-sensitive Na⁺ channel. Am J Physiol 266: L728-L734.

Patterson CE, Rhoades RA, and Garcia JG (1992) Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung. J Appl Physiol 72: 865-873.[Abstract/Free Full Text]

Rogers DF, Boschetto P, and Barnes PJ (1989) Plasma exudation: correlation between Evans blue dye and radiolabeled albumin in guinea pig airways in vivo. J Pharmacol Methods 21: 309-315.[CrossRef][Medline]

Roseth S, Fykse EM, and Fonnum F (1995) Uptake of L-glutamate into rat brain synaptic vesicles: effect of inhibitors that bind specifically to the glutamate transporter. J Neurochem 65: 96-103.[Medline]

Ugawa S, Minami Y, Guo W, Saishin Y, Takatsuji K, Yamamoto T, Tohyama M, and Shimada S (1998) Receptor that leaves a sour taste in the mouth. Nature (Lond) 395: 555-556.[CrossRef][Medline]

Ugawa S, Ueda T, Ishida Y, Nishigaki M, Shibata Y, and Shimada S (2002) Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J Clin Investig 110: 1185-1190.[CrossRef][Medline]

Waldmann R, Champigny G, Bassilana F, Voilley N, and Lazdunski M (1995) Molecular cloning and functional expression of a novel amiloride-sensitive Na⁺ channel. J Biol Chem 270: 27411-27414.[Abstract/Free Full Text]

Welsh MJ, Price MP, and Xie J (2002) Biochemical basis of touch perception: mechanosensory function of degenerin/epithelial Na⁺ channels. J Biol Chem 277: 2369-2372.[Free Full Text]

Wu SN, Jan CR, Li HF, and Chen SA (1999) Stimulation of large-conductance Ca²⁺-activated K⁺ channels by Evans blue in cultured endothelial cells of human umbilical veins. Biochem Biophys Res Commun 254: 666-674.[CrossRef][Medline]

Yamamura H, Ugawa S, Ueda T, Nagao M, and Shimada S (2004a) Protons activate the -subunit of the epithelial Na⁺ channel in humans. J Biol Chem 279: 12529-12534.[Abstract/Free Full Text]

Yamamura H, Ugawa S, Ueda T, Nagao M, and Shimada S (2004b) Capsazepine is a novel activator of the subunit of the human epithelial Na⁺ channel. J Biol Chem 279: 44483-44489.[Abstract/Free Full Text]

Yamamura H, Ugawa S, Ueda T, Nagao M, and Shimada S (2005) Icilin activates the -subunit of the human epithelial Na⁺ channel. Mol Pharmacol 68: 1142-1147.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.092775v1 315/2/965    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yamamura, H. Articles by Shimada, S. Search for Related Content PubMed PubMed Citation Articles by Yamamura, H. Articles by Shimada, S.