Epithelial Na+ channel δ subunit mediates acid-induced ATP release in the human skin

doi:10.1016/j.bbrc.2008.06.008

Biochemical and Biophysical Research Communications

Volume 373, Issue 1, 15 August 2008, Pages 155-158

https://doi.org/10.1016/j.bbrc.2008.06.008 Get rights and content

Abstract

The amiloride-sensitive epithelial Na⁺ channel (ENaC) regulates Na⁺ homeostasis in cells and across epithelia. Although we described that ENaCδ is a candidate molecule for a pH sensor in the human brain, the physiological and pathological roles of ENaCδ in non-neuronal tissues are still unknown. Here we show a novel physiological function of ENaCδ in peripheral tissues in humans. Expression analyses at the level of mRNA clearly revealed that ENaCδ was abundantly expressed in human epidermis and keratinocytes. In addition, ENaCδ protein was detected in there. In cultured keratinocytes, acidic stress (pH 5.0) evoked ATP release, which was significantly reduced in the presence of 100 μM amiloride or 10 μM benzamil. In conclusion, ENaCδ may be involved in the mechanism underlying pH sensing followed by the regulation of cell viability in the human skin.

Section snippets

Materials and methods

Molecular biology. All experiments were approved by the Ethics Committee of Nagoya City University and were conducted in accordance with the Declaration of Helsinki. Human samples were taken from autopsies within 24 h after death with permission. The extraction of total RNA, RT-PCR, electrophoresis, cloning, and subsequent sequencing were performed as described previously [4], [12]. PCR amplification was carried out for 35 cycles. Specific PCR primers for human ENaCs were designed as follows:

Expression of ENaCδ in human skin and keratinocytes

The expression of mRNA encoding ENaCδ in human skin and cultured keratinocytes (PHK16-0b) was examined by RT-PCR using total RNA extracted from human skin, cultured keratinocytes and specific oligonucleotide primers. RT-PCR analysis showed a clear expression of ENaCδ transcript (467 bp) in human skin and keratinocytes (Fig. 1A). Accessory subunits, β and γ (442 and 517 bp, respectively), clearly showed mRNA expression in both types of samples. Another core unit, ENaCα (432 bp), was also detected

Discussion

Amiloride-sensitive ENaCs, members of the degenerin/ENaC superfamily, regulate essential control elements for Na⁺ homeostasis in cells and across epithelia. Because the ENaCαβγ complex is expressed mainly in epithelia such as the kidney, lung, and colon, playing a pathophysiological role, its physiological and pharmacological characterization has been well documented [1], [2]. On the other hand, the physiological function of ENaCδ has not yet been identified. Recently, we described that ENaCδ

Acknowledgments

We thank Katsuyuki Tanaka and Kenji Kajita for technical assistance. This investigation was supported by Grants-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (to H.Y.) and for Scientific Research (B) and Exploratory Research from the Japan Society for the Promotion of Sciences (to S.S.).

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The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin ion channel family, which also includes the bile acid-sensitive ion channel (BASIC). So far little is known about the effects of bile acids on ENaC function. ENaC is probably a heterotrimer consisting of three well characterized subunits (αβγ). In humans, but not in mice and rats, an additional δ-subunit exists. The aim of this study was to investigate the effects of chenodeoxycholic, cholic, and deoxycholic acid in unconjugated (CDCA, CA, and DCA) and tauro-conjugated (t-CDCA, t-CA, t-DCA) form on human ENaC in its αβγ- and δβγ-configuration. We demonstrated that tauro-conjugated bile acids significantly stimulate ENaC in the αβγ- and in the δβγ-configuration. In contrast, non-conjugated bile acids have a robust stimulatory effect only on δβγENaC. Bile acids stimulate ENaC-mediated currents by increasing the open probability of active channels without recruiting additional near-silent channels known to be activated by proteases. Stimulation of ENaC activity by bile acids is accompanied by a significant reduction of the single-channel current amplitude, indicating an interaction of bile acids with a region close to the channel pore. Analysis of the known ASIC1 (acid-sensing ion channel) crystal structure suggested that bile acids may bind to the pore region at the degenerin site of ENaC. Substitution of a single amino acid residue within the degenerin region of βENaC (N521C or N521A) significantly reduced the stimulatory effect of bile acids on ENaC, suggesting that this site is critical for the functional interaction of bile acids with the channel.
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Citation Excerpt :
Although iontophoretic current applications are not painful, it is likely that the sensitivity of CIV to local anesthesia and chronic treatment with capsaicin reflects, at least in part, the stimulation of cutaneous C-fiber nociceptors that trigger vasodilation through an axon reflex (Berliner, 1997; Hamdy et al., 2001). Indeed, the axon reflex mechanism activates capsaicin-sensitive fibers by acting directly on cutaneous nociceptors such as transient receptor potential vanilloid type-1 (TRPV1) and/or acid-sensing ion channels (ASIC), which are abundantly expressed in human epidermis and keratinocytes (Inoue et al., 2002; Jiang et al., 2006; Southall et al., 2003; Yamamura et al., 2008). We thus suggested that the activation of the capsaicin-sensitive fibers could result from the excitation of cutaneous TRPV1 and/or ASIC channels in response to the cathodal current stimulation.
Cutaneous current-induced vasodilation (CIV) in response to galvanic current application is an integrative model of neurovascular interaction that relies on capsaicin-sensitive fiber activation. The upstream and downstream mechanisms related to the activation of the capsaicin-sensitive fibers involved in CIV are not elucidated. In particular, the activation of cutaneous transient receptor potential vanilloid type-1 (TRPV1) channels and/or acid-sensing ion channels (ASIC) (activators mechanisms) and the release of calcitonin gene-related peptide (CGRP) and substance P (SP) (effector mechanisms) have been tested. To assess cathodal CIV, we measured cutaneous blood flow using laser Doppler flowmetry for 20 min following cathodal current application (240 s, 100 μA) on the skin of the thigh in anesthetized healthy rats for 20 min. CIV was studied in rats treated with capsazepine and amiloride to inhibit TRPV1 and ASIC channels, respectively; CGRP8-37 and SR140333 to antagonize CGRP and neurokinin-1 (NK1) receptors, respectively; compared to their respective controls. Cathodal CIV was attenuated by capsazepine (12 ± 2% vs 54 ± 6%, P < 0.001), amiloride (19 ± 8% vs 61 ± 6%, P < 0.01), CGRP8-37 (15 ± 6% vs 61 ± 6%, P < 0.001) and SR140333 (9 ± 5% vs 54 ± 6%, P < 0.001) without changing local acidification. This is the first integrative study performed in healthy rats showing that cutaneous vasodilation in response to cathodal stimulation is initiated by activation of cutaneous TRPV1 and ASIC channels likely through local acidification. The involvement of CGRP and NK1 receptors suggests that cathodal CIV is the result of CGRP and SP released through activated capsaicin-sensitive fibers. Therefore cathodal CIV could be a valuable method to assess sensory neurovascular function in the skin, which would be particularly relevant to evaluate the presence of small nerve fiber disorders and the effectiveness of treatments.
The δ-subunit of the epithelial Sodium channel (ENaC) enhances channel activity and alters proteolytic ENaC activation
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The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (αβγ). In humans an additional δ-subunit (δ-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of αβγ- and δβγ-hENaC and investigated whether δβγ-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (ΔI_ami) was about 11-fold larger in oocytes expressing δβγ-hENaC than in oocytes expressing αβγ-hENaC. The 2-fold larger single-channel Na⁺ conductance of δβγ-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of δβγ-hENaC must be higher than that of αβγ-hENaC. Experiments exploiting the properties of the known βS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on δβγ-hENaC whole-cell currents compared with its effect on αβγ-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for δβγ-hENaC than for αβγ-hENaC. Proteolytic activation of δβγ-hENaC was associated with the appearance of a δ-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human α-ENaC inhibited ΔI_ami in oocytes expressing αβγ-hENaC but not in those expressing δβγ-hENaC. We conclude that the δ-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.
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It has become clear in recent years that serine proteases have an important role in epidermal homeostasis, and the signaling cascades are gradually being identified. For example, matriptase, prostasin and furin are implicated in a cascade that could activate ENaC, leading to epidermal barrier formation and hydration, probably in part through their involvement in filaggrin processing. Kallikreins can form a signaling cascade to coordinate corneocyte desquamation. Knowledge is also emerging about how endogenous inhibitors, calcium and pH control these cascades. It is becoming clear that some skin pathologies are associated with deregulated serine protease activity. Therefore, a deeper knowledge of the regulation of these serine protease cascades could form the basis for development of appropriate treatments for skin disorders such as Netherton syndrome.
Epithelial Na<sup>+</sup> channel δ subunit is an acid sensor in the human oesophagus
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Gastro-oesophageal reflux disease is caused by the reflux of gastric contents into the oesophagus, and thus the oesophageal lumen is damaged by gastric acid. The acid sensor involved in oesophageal epithelial defense is still unclear. Recently, we described that the epithelial Na⁺ channel δ subunit (ENaCδ) is a candidate molecule for a pH sensor in the human brain. Here, using reverse transcription-polymerase chain reaction and in situ hybridization methods, we showed that the proton-sensitive ENaCδ was strongly expressed in the epithelial layer of the human oesophagus, representative peripheral tissue that can be exposed to an acidic environment. Other ENaC subunits (α, β, and γ) were also localized there. Based on the expression pattern, human oesophageal ENaC complex was mimicked in the Xenopus oocyte expression system and the response to acidic pH was recorded using a two-electrode voltage-clamp technique. The human oesophageal-mimicking ENaCδβγα complex generated an amiloride-sensitive inward current at the holding potential of − 60 mV. The ENaCδβγα current was significantly activated by acidic pH (pH 4.0), approximately equal to the luminal value when gastric acid refluxes into the oesophagus. In conclusion, ENaCδ is a candidate molecule for pH sensing in the gastrointestinal system in humans, providing a novel therapeutic target for gastro-oesophageal reflux disease.
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Epithelial Na+ channel δ subunit mediates acid-induced ATP release in the human skin

Abstract

Section snippets

Materials and methods

Expression of ENaCδ in human skin and keratinocytes

Discussion

Acknowledgments

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Invest. Dermatol.

J. Lab. Clin. Med.

Gastroenterology

Structure and regulation of amiloride-sensitive sodium channels

Annu. Rev. Physiol.

Epithelial Na⁺ channel δ subunit mediates acid-induced ATP release in the human skin