Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation

The amiloride-sensitive epithelial sodium channel (ENaC) is a main determinant of sodium absorption in renal and colonic epithelial cells. Surprisingly, it is also expressed in non-transporting epithelia such as the epidermis. To gain insight into the putative role of ENaC in keratinocytes, we have evaluated its expression in human skin and in cultured human keratinocytes. Our results indicate that (1) ENaC is expressed in the epidermis and in cultured keratinocytes, at the mRNA and at the protein levels, (2) the ratio of expression of the different ENaC subunits is drastically modified at the protein level during cell growth and differentiation, with a selective upregulation of the β subunit, (3) no transepithelial sodium transport function is apparent in cultured keratinocytes, but patch-clamp recordings indicate the existence of functional sodium channels with properties similar to those of the cloned ENaC and (4) ENaC inhibition does not alter keratinocyte proliferation, but it significantly decreases the frequency of dome formation in confluent keratinocyte cultures. These results document for the first time the characteristics of ENaC subunit expression in human keratinocytes, and suggest that ENaC may be important during differentiation.

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The epithelial sodium channel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2012.1945 ◽

2012 ◽

Vol 279 (1748) ◽

pp. 4795-4802 ◽

Cited By ~ 12

Author(s):

Minoru Uchiyama ◽

Sho Maejima ◽

Sumio Yoshie ◽

Yoshihiro Kubo ◽

Norifumi Konno ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Transport ◽

Sodium Current ◽

Apical Cell ◽

Epithelial Sodium Channel ◽

Sodium Absorption ◽

Apical Cell Membrane ◽

Rate Limiting ◽

Complementary Rnas ◽

Australian Lungfish

Epithelial sodium channel (ENaC) is a Na + -selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na + absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri , which is the closest living relative of tetrapods. Neoceratodus ENaC ( n ENaC) comprised three subunits: n ENaCα, β and γ proteins. The n ENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the n ENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. n ENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, n ENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.

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Alterations of ultrastructure and elemental composition in cultured human epidermal keratinocytes after treatment with nitrofurazone and silver sulfadiazine

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127803 ◽

1987 ◽

Vol 45 ◽

pp. 676-677

Author(s):

A. R. Crooker ◽

M. C. Myers ◽

T. L. Beard ◽

E. S. Graham

Keyword(s):

Electron Microscopy ◽

Elemental Composition ◽

Pyrolytic Carbon ◽

Human Keratinocytes ◽

Silver Sulfadiazine ◽

Epidermal Keratinocytes ◽

Human Epidermal Keratinocytes ◽

Culture Systems ◽

Cytotoxicity Endpoints

Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.

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Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes

Molecules ◽

10.3390/molecules26082153 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2153

Author(s):

Raffaella Marina Lecci ◽

Isabella D’Antuono ◽

Angela Cardinali ◽

Antonella Garbetta ◽

Vito Linsalata ◽

...

Keyword(s):

Antioxidant Activities ◽

Biological Activities ◽

Human Keratinocytes ◽

Ldl Oxidation ◽

Trolox Equivalent Antioxidant Capacity ◽

Epidermal Keratinocytes ◽

Olive Cultivar ◽

Human Epidermal Keratinocytes ◽

Apoptotic Effect

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.

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Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism

Journal of Cell Science ◽

10.1242/jcs.112.12.1843 ◽

1999 ◽

Vol 112 (12) ◽

pp. 1843-1853 ◽

Cited By ~ 1

Author(s):

N. Maas-Szabowski ◽

A. Shimotoyodome ◽

N.E. Fusenig

Keyword(s):

Neutralizing Antibodies ◽

Growth Regulation ◽

Keratinocyte Growth Factor ◽

Dermal Fibroblasts ◽

Tissue Homeostasis ◽

Epidermal Keratinocytes ◽

Human Epidermal Keratinocytes ◽

Keratinocyte Proliferation ◽

Normal Human ◽

Kinetics Of

Epithelial-mesenchymal interactions play an important role in regulating tissue homeostasis and repair. For skin, the regulatory mechanisms of epidermal-dermal interactions were studied in cocultures of normal human epidermal keratinocytes (NEK) and dermal fibroblasts (HDF) rendered postmitotic by alpha-irradiation (HDFi). The expression kinetics of different cytokines and their receptors with presumed signalling function in skin were determined at the RNA and protein level in mono- and cocultured NEK and HDFi. In cocultured HDFi, mRNA and protein synthesis of keratinocyte growth factor (KGF) (FGF-7) was strongly enhanced, whereas in cocultured keratinocytes interleukin (IL)-1alpha and -1beta mRNA expression increased compared to monocultures. Thus we postulated that IL-1, which had no effect on keratinocyte proliferation, induced in fibroblasts the expression of factors stimulating keratinocyte proliferation, such as KGF. The functional significance of this reciprocal modulation was substantiated by blocking experiments. Both IL-1alpha and -1beta-neutralizing antibodies and IL-1 receptor antagonist significantly reduced keratinocyte proliferation supposedly through abrogation of KGF production, because IL-1 antibodies blocked the induced KGF production. These data indicate a regulation of keratinocyte growth by a double paracrine mechanism through release of IL-1 which induces KGF in cocultured fibroblasts. Thus IL-1, in addition to its proinflammatory function in skin, may play an essential role in regulating tissue homeostasis.

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Persea americanaMill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/391247 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 8

Author(s):

Maria del R. Ramos-Jerz ◽

Socorro Villanueva ◽

Gerold Jerz ◽

Peter Winterhalter ◽

Alexandra M. Deters

Keyword(s):

Chlorogenic Acid ◽

Dermal Fibroblasts ◽

Persea Americana ◽

Epidermal Keratinocytes ◽

Hacat Keratinocytes ◽

Human Epidermal Keratinocytes ◽

Residual Solvent ◽

Keratinocyte Proliferation ◽

Normal Human

Methanolic avocado (Persea americanaMill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Theirin vitroinfluence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fractionM.2composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore,M.2increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fractionM.6increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fractionM.7contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

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Evolution of the epithelial sodium channel and the sodium pump as limiting factors of aldosterone action on sodium transport

Physiological Genomics ◽

10.1152/physiolgenomics.00002.2011 ◽

2011 ◽

Vol 43 (13) ◽

pp. 844-854 ◽

Cited By ~ 29

Author(s):

Romain A. Studer ◽

Emilie Person ◽

Marc Robinson-Rechavi ◽

Bernard C. Rossier

Keyword(s):

Sodium Channel ◽

Sodium Transport ◽

Salt Intake ◽

Epithelial Sodium Channel ◽

Cell Junctions ◽

Limiting Factors ◽

Last Common Ancestor ◽

Β Subunit ◽

Α Subunit ◽

Additional Function

Despite large changes in salt intake, the mammalian kidney is able to maintain the extracellular sodium concentration and osmolarity within very narrow margins, thereby controlling blood volume and blood pressure. In the aldosterone-sensitive distal nephron (ASDN), aldosterone tightly controls the activities of epithelial sodium channel (ENaC) and Na,K-ATPase, the two limiting factors in establishing transepithelial sodium transport. It has been proposed that the ENaC/degenerin gene family is restricted to Metazoans, whereas the α- and β-subunits of Na,K-ATPase have homologous genes in prokaryotes. This raises the question of the emergence of osmolarity control. By exploring recent genomic data of diverse organisms, we found that: 1) ENaC/degenerin exists in all of the Metazoans screened, including nonbilaterians and, by extension, was already present in ancestors of Metazoa; 2) ENaC/degenerin is also present in Naegleria gruberi , an eukaryotic microbe, consistent with either a vertical inheritance from the last common ancestor of Eukaryotes or a lateral transfer between Naegleria and Metazoan ancestors; and 3) The Na,K-ATPase β-subunit is restricted to Holozoa, the taxon that includes animals and their closest single-cell relatives. Since the β-subunit of Na,K-ATPase plays a key role in targeting the α-subunit to the plasma membrane and has an additional function in the formation of cell junctions, we propose that the emergence of Na,K-ATPase, together with ENaC/degenerin, is linked to the development of multicellularity in the Metazoan kingdom. The establishment of multicellularity and the associated extracellular compartment (“internal milieu”) precedes the emergence of other key elements of the aldosterone signaling pathway.

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Intestinal Mineralocorticoid Receptor Contributes to Epithelial Sodium Channel–Mediated Intestinal Sodium Absorption and Blood Pressure Regulation

Journal of the American Heart Association ◽

10.1161/jaha.117.008259 ◽

2018 ◽

Vol 7 (13) ◽

Cited By ~ 4

Author(s):

Toshifumi Nakamura ◽

Isao Kurihara ◽

Sakiko Kobayashi ◽

Kenichi Yokota ◽

Ayano Murai‐Takeda ◽

...

Keyword(s):

Blood Pressure ◽

Sodium Channel ◽

Mineralocorticoid Receptor ◽

Epithelial Sodium Channel ◽

Blood Pressure Regulation ◽

Sodium Absorption ◽

Pressure Regulation

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Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.1.f46 ◽

2000 ◽

Vol 279 (1) ◽

pp. F46-F53 ◽

Cited By ~ 169

Author(s):

Carolyn A. Ecelbarger ◽

Gheun-Ho Kim ◽

James Terris ◽

Shyama Masilamani ◽

Carter Mitchell ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Transport ◽

Collecting Duct ◽

Rat Kidney ◽

Epithelial Sodium Channel ◽

Sodium Reabsorption ◽

Acute Administration ◽

Brattleboro Rats ◽

Water Restriction ◽

Short Term

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (α, β, and γ) in rat kidney. Seven-day infusion of 1-deamino-[8-d-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of β- and γ-ENaC (to 238% and 288% of vehicle, respectively), whereas α-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of β- and γ- but no significant change in α-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.

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