Association of cyclic adenosine monophosphate with permeability barrier homeostasis of murine skin

Activation of Gs protein increases the intracellular cyclic adenosine monophosphate (cAMP) level, and the Gs protein-linked receptor has been implicated in the skin barrier homeostasis. In this study, we investigated the role of cAMP in epidermal barrier function. The barrier was disrupted by tape stripping or treatment with acetone. Immediately after barrier disruption, reagents affecting the cAMP level were topically applied. Topical application of forskolin, which activates cAMP synthesis delayed barrier recovery, whereas application of the antagonist of cAMP, cAMP-Rp, accelerated barrier recovery. Moreover, application of 9-cyclopentyladenine, an inhibitor of cAMP synthesis also accelerated barrier recovery. Tape stripping was found to increase the cAMP in the epidermis. Light and electron microscopic observations showed the delay of lamellar body secretion by forskolin and acceleration of the lamellar body secretion by cAMP-Rp. Application of an inhibitor of protein kinase A did not affect the barrier recovery rate. The delay of barrier recovery induced by forskolin was blocked by the voltage-gated calcium channel blockers, nifedipine and verapamil. In cultured keratinocytes, forskolin increased the intracellular calcium concentration and both nifedipine and verapamil blocked the increase. These results suggest that intracellular cAMP in the epidermis is involved in skin barrier homeostasis.     

Inducible nitric oxide synthase is required for epidermal permeability barrier homeostasis in mice

Nitric oxide (NO) regulates a variety of epidermal functions, including epidermal proliferation, differentiation and cutaneous wound healing. However, whether nitric oxide (NO) and its synthetic enzymes regulate epidermal permeability barrier homeostasis is not clear. In the present study, we employed inducible nitric oxide synthase (iNOS) KO mice to explore the role of iNOS in epidermal permeability barrier homeostasis. Our results showed that iNOS mice displayed a comparable levels of basal transepidermal water loss rates, stratum corneum hydration and skin surface pH to their wild-type mice, but epidermal permeability barrier recovery was significantly delayed both 2 and 4 hours after acute barrier disruption by tape stripping. In parallel, expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes were lower in iNOS KO mice versus wild-type controls. Topical applications of two structurally unrelated NO donors to iNOS KO mice improved permeability barrier recovery kinetics and upregulated expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes. Together, these results indicate that iNOS and its product regulate epidermal permeability barrier homeostasis in mice.     

Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice

Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.     

Influx of calcium and chloride ions into epidermal keratinocytes regulates exocytosis of epidermal lamellar bodies and skin permeability barrier homeostasis

In the nervous system, influx of calcium and chloride ions into neurons regulates the signaling system by excitation and inhibition, respectively. In this study, we demonstrated the effects of the ion influx into epidermal keratinocytes in the permeability barrier repair process of the skin after damage. Topical application of the neurotransmitters glutamate and nicotine, which activate the calcium channel in neurons, delayed the barrier repair after tape stripping. In contrast, the neurotransmitters GABA and glycine, which activate the chloride channel in neurons, accelerated barrier repair. Topical application of the calcium ionophore ionomycin delayed barrier recovery and chloride ionophore 1 accelerated barrier repair after barrier disruption by tape stripping and acetone treatment. Ionomycin increased the intracellular calcium concentration in cultured keratinocytes whereas the chloride ionophore 1 increased the intracellular chloride ion concentration. In vivo light microscopy and electron microscopy observation showed acceleration of the exocytosis of lipid-containing lamellar bodies by the chloride ionophore and delay of the exocytosis by the calcium ionophore. These results suggest that, like the nervous system, influx of calcium and chloride ions into epidermal keratinocytes through ionotropic receptors plays a crucial role in cutaneous barrier homeostasis.     

Olopatadine hydrochloride accelerates the recovery of skin barrier function in mice

BACKGROUND: The skin barrier function in patients with atopic dermatitis is disrupted and prolonged topical steroid therapy produces epidermal barrier disturbance. Olopatadine hydrochloride (olopatadine; Allelock; Kyowa Hakko Kogyo Co., Ltd, Shizuoka, Japan) is an antiallergic drug with histamine H(1) receptor antagonistic action. This drug alleviates skin inflammation and decreases the number of scratching episodes in a murine model of chronic contact dermatitis. OBJECTIVES: To investigate the effects of olopatadine and a steroid on the recovery of skin barrier function after barrier disruption in mice. METHODS: The skin barrier of the ears of mice was disrupted by tape stripping. The recovery of skin barrier function was monitored by measurement of transepidermal water loss (TEWL) after barrier disruption. Epidermal hyperplasia was induced by repeated tape stripping for 7 days. Olopatadine was administered orally once daily from 3 days before the first barrier disruption. Betamethasone 17-valerate (betamethasone) was applied topically once daily from 3 days before barrier disruption. RESULTS: Tape stripping led to a significant increase in TEWL. TEWL decreased with time after tape stripping and the skin barrier function recovered by over 60% within 9 h after tape stripping. The recovery of skin barrier in olopatadine-treated mice was significantly accelerated, compared with that in vehicle-treated mice. In contrast, the skin barrier recovery in mice treated with topical betamethasone was significantly delayed, compared with that in vehicle-treated mice. Combined treatment with olopatadine and betamethasone ameliorated the delay in barrier recovery induced by topical treatment with betamethasone. In addition, olopatadine significantly prevented the increase in epidermal thickness induced by prolonged barrier disruption. CONCLUSIONS: These results suggest that systemic administration of olopatadine accelerates the recovery of skin barrier function and ameliorates the adverse effects of topical steroids on skin barrier recovery.     

gamma-Aminobutyric acid (A) receptor agonists accelerate cutaneous barrier recovery and prevent epidermal hyperplasia induced by barrier disruption

gamma-Aminobutyric acid, is an amino acid transmitter, which mediates rapid inhibition in the central nervous system. gamma-Aminobutyric acid (A) receptor is a ligand-gated chloride ion channel playing an important part in polarizing the cell membrane and reducing neuronal excitability in the neuron. In this study, we demonstrated the effects of gamma-aminobutyric acid (A) receptor agonists on the cutaneous barrier repair process after the barrier disruption of hairless mice. Topical application of gamma-aminobutyric acid and gamma-aminobutyric acid (A) receptor-specific agonists, musimol and isoguvacine, after barrier disruption accelerated the barrier recovery. The gamma-aminobutyric acid (B)-specific agonist, baclofen, did not affect the barrier recovery rate. The effect of gamma-aminobutyric acid on the barrier recovery was blocked by the gamma-aminobutyric acid (A)-receptor antagonist, bicuculline methobromide, but gamma-aminobutyric acid (B) receptor antagonist, saclofen, did not affect the effect of gamma-aminobutyric acid. Topical application of gamma-aminobutyric acid also prevented epidermal hyperplasia, which was induced by the barrier insults under low environmental humidity and bicuculline methobromide blocked the effect of gamma-aminobutyric acid on the epidermal hyperplasia. Immunoreactivity against gamma-aminobutyric acid (A) polyclonal antibody was observed in hairless mouse epidermis. The fluorescent probe of gamma-aminobutyric acid (A) receptor, TXR-musimol showed the localization of gamma-aminobutyric acid (A) receptor in the epidermis of the hairless mice. Elevation of intracellular chloride ion was induced by gamma-aminobutyric acid in cultured human keratinocytes and it was blocked by bicuculline methobromide. These results suggest that the gamma-aminobutyric acid (A)-like receptor is associated with skin barrier homeostasis and regulation of the receptor clinically effective for barrier dysfunctional or epidermal hyperproliferative diseases.     

Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold‐sensitive TRP receptors in epidermal permeability barrier homoeostasis

Please cite this paper as: Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold‐sensitive TRP receptors in epidermal permeability barrier homoeostasis. Experimental Dermatology 2010; 19 : 791–795. Abstract: TRPA1 and TRPM8 receptors are activated at low temperature (A1: below 17°C and M8: below 22°C). Recently, we observed that low temperature (below 22°C) induced elevation of intracellular calcium in keratinocytes. Moreover, we demonstrated that topical application of TRPA1 agonists accelerated the recovery of epidermal permeability barrier function after disruption. In this study, we examined the effect of topical application of TRPM8 modulators on epidermal permeability barrier homoeostasis. Immunohistochemical study and RT‐PCR confirmed the expression of TRPM8 or TRPM8‐like protein in epidermal keratinocytes. Topical application of TRPM8 agonists, menthol and WS 12 accelerated barrier recovery after tape stripping. The effect of WS12 was blocked by a non‐selective TRP antagonist, Ruthenium Red, and a TRPM8‐specific antagonist, BTCT. Topical application of WS12 also reduced epidermal proliferation associated with barrier disruption under low humidity, and this effect was blocked by BTCT. Our results indicate that TRPM8 or a closely related protein in epidermal keratinocytes plays a role in epidermal permeability barrier homoeostasis and epidermal proliferation after barrier insult.     

Histamine H1 and H2 receptor antagonists accelerate skin barrier repair and prevent epidermal hyperplasia induced by barrier disruption in a dry environment

Keratinocytes have histamine H1 and H2 receptors, but their functions are poorly understood. To clarify the role of histamine receptors in the epidermis, we examined the effects of histamine receptor antagonists and agonists applied epicutaneously on the recovery of skin barrier function disrupted by tape stripping in hairless mice. Histamine H2 receptor antagonists famotidine and cimetidine accelerated the recovery of skin barrier function, but histamine and histamine H2 receptor agonist dimaprit delayed the barrier repair. Application of compound 48/80, a histamine releaser, also delayed the recovery. Imidazole, an analog of histamine, had no effect. The histamine H1 receptor antagonists diphenhydramine and tripelennamine accelerated the recovery. Histamine H3 receptor agonist Nalpha-methylhistamine and antagonist thioperamide had no effect. In addition, topical application of famotidine or diphenhydramine prevented epidermal hyperplasia in mice with skin barrier disrupted by acetone treatment in a dry environment (humidity < 10%) for 4 d. In conclusion, both the histamine H1 and H2 receptors in the epidermis are involved in skin barrier function and the cutaneous condition of epidermal hyperplasia.     

Expression of voltage-gated calcium channel subunit alpha1C in epidermal keratinocytes and effects of agonist and antagonists of the channel on skin barrier homeostasis

Recent studies demonstrated that skin surface electric conditions affect epidermal permeability barrier homeostasis. These results suggest the existence of voltage sensor on the keratinocytes of the epidermis. On the contrary, specific blockers of the voltage-gated calcium channel (VGCC) also affect epidermal barrier homeostasis, but the existence and function of the channel has not been determined. We demonstrated here immunohistochemically the expression of the main subunit of the L-type VGCC, alpha1C, which alone has a calcium channel function, in mouse and human epidermis. Immunostaining, RT-PCR, and Western blotting were carried out to detect the channel protein. Messenger RNA of alpha1C was also detected in mouse epidermis and human keratinocyte culture by RT-PCR. We also evaluated the function of the channel in the cultured human keratinocytes. Previously, we demonstrated that influx of calcium ion into epidermal keratinocytes delayed the barrier recovery after barrier disruption and topical application of calcium channel blocker accelerated the barrier recovery. In this study, topical application of nifedipine and R-(+)-BAY K8644 after tape stripping of hairless mice accelerated the barrier repair rate while application of S-(-)-BAY K8644 delayed the barrier recovery. These results suggest that the VGCC exists on epidermal keratinocytes and plays an important role in skin barrier homeostasis.     

Acute cutaneous barrier disruption activates epidermal p44/42 and p38 mitogen-activated protein kinases in human and hairless guinea pig skin

Acute cutaneous barrier disruption of the skin elicits various homeostatic repair responses in the epidermis. Although several candidates for the signaling mechanisms that induce these responses have been reported, e.g. the calcium and ion concentration, peroxisome proliferator-activated receptor-alpha, and TNF-alpha signaling mediated by sphingomyelinases, the exact nature of the signals remains undertermined. Therefore, assuming that an important group of serine/threonine-signaling kinases, mitogen- and SAPK/JNK, might link the barrier disruption to the subsequent homeostatic responses, the activation of three MAPKs in hairless guinea pig or in human skin after barrier disruption was investigated. The epidermal barrier was insulated with tape stripping or organic solvents, and Western blotting, and immune complex kinase assay. In the skin of hairless guinea pigs, p44/42 MAPK and p38 MAPK, but nor SAPK/JNK, were continued to be activated for at least 180 min. The activation of p44/42 which positively correlated with the number of tape strippings, whereas K+ sucrose solution suppressed its activation. The activation of p44/42 MAPK was also induced by treatment of the skin with organic solvents. In similar fashion, p44/42 and p38 MAPKs were found to be activated in human skin after tape stripping. These results for strongly suggest that the activation of p44/42 and p38 MAPKs links the stimuli of barrier disruption to the subsequent homeostatic responses to repair the barrier defect.     

Negative electric potential induces alteration of ion gradient and lamellar body secretion in the epidermis, and accelerates skin barrier recovery after barrier disruption

Previous reports suggested that ion gradients of ions such as calcium and magnesium in the epidermis play a crucial part in skin barrier homeostasis. We hypothesized that external electric potential affects the ionic gradient and skin barrier homeostasis. We demonstrated here that application of a negative electric potential (0.50 V) on hairless mice skin accelerated the barrier recovery approximately 60.7% of the original level within 1 h compared with control (37.5%) after barrier disruption by acetone treatment. Even after the application of a negative potential, the skin showed accelerated repair for 6 h. On the contrary, the skin that was applied a positive potential for 1 h showed a significant delay in barrier recovery (25.3%) than the control. Ultrastructural studies by electron microscopy suggested that the extent of lamellar body exocytosis into the stratum corneum/stratum granulosum interface increased under a negative potential. Magnesium and calcium ion concentrations in the upper epidermis were relatively higher in the negative portion than in the portion where the positive potential was applied. Topical application of these ions on mice skin also accelerated the barrier recovery. These results suggest that the external electric potential affects the ionic gradients in the epidermis and also influences the skin barrier homeostasis.     

Barium sulphate with a negative zeta potential accelerates skin permeability barrier recovery and prevents epidermal hyperplasia induced by barrier disruption

BACKGROUND: Barium sulphate, a stable inorganic material, has been used in contrast media and cosmetic products because of its stability. As a negative external electric potential accelerates the skin barrier repair after barrier disruption, we hypothesized that topical application of barium sulphate may affect the skin barrier recovery rate depending on its zeta potential. OBJECTIVES: To investigate whether barium sulphate particles in aqueous solution have different zeta potentials depending on their surface structure, and to investigate the possible relation between zeta potential and skin barrier recovery rate. METHODS: Mice were subjected to tape stripping to disrupt barrier function, or were treated with acetone and kept in a dry environment to induce epidermal hyperplasia. They were then treated with different forms of barium sulphate, and barrier recovery was monitored by measurements of transepidermal water loss. RESULTS: There was a significant correlation between the barrier recovery rate and zeta potential of barium sulphate applied topically. Barium sulphate with a negative zeta potential significantly accelerated barrier recovery, but barium sulphate with a positive zeta potential did not accelerate or even delayed barrier repair. Barium sulphate with a negative zeta potential had an X-ray diffraction pattern different from that with a positive potential. The distribution of calcium in the epidermis was also influenced by the polarity of zeta potential. CONCLUSIONS: These findings suggest a new pharmacological approach towards altering barrier function or epidermal hyperplasia with inorganic particles in healthy and diseased skin.     

Effects of topical application of aqueous solutions of hexoses on epidermal permeability barrier recovery rate after barrier disruption

Abstract: Previous studies have suggested that hexose molecules influence the stability of phospholipid bilayers. Therefore, the effects of topical application of all 12 stereoisomers of dextro‐hexose on the epidermal barrier recovery rate after barrier disruption were evaluated. Immediately after tape stripping, 0.1 m aqueous solution of each hexose was applied on hairless mouse skin. Among the eight dextro‐aldohexoses, topical application of altose, idose, mannose and talose accelerated the barrier recovery, while allose, galactose, glucose and gulose had no effect. Among the four dextro‐ketohexoses, psicose, fructose, sorbose and tagatose all accelerated the barrier recovery. As the effects of hexoses on the barrier recovery rate appeared within 1 h, the mechanism is unlikely to be genomic. Instead, these hexoses may influence phase transition of the lipid bilayers of lamellar bodies and cell membrane, a crucial step in epidermal permeability barrier homeostasis.     

Effect of endothelial nitric oxide synthase on epidermal permeability barrier recovery after disruption

Background We previously demonstrated that neuronal nitric oxide synthase (nNOS) in epidermal keratinocytes is associated with epidermal permeability barrier homeostasis. Objectives In the present study, we examined the contributions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) to epidermal permeability barrier homeostasis. Methods We measured the barrier recovery rate after tape stripping of the epidermis of iNOS and eNOS knockout mice, and carried out electron‐microscopic observation of the epidermis after acetone treatment. Results The barrier recovery rate of eNOS knockout mice was significantly faster than that of the wild‐type control, while no significant difference was observed between iNOS knockout mice and wild‐type mice. Electron‐microscopic observation at 1 h after acetone treatment indicated that barrier recovery of both nNOS and eNOS mice was faster than that of wild‐type mice, and lamellar body secretion was accelerated in both types of knockout mice. Conclusions These results suggested that both nNOS and eNOS play roles in epidermal barrier homeostasis and lamellar body secretion.     

Topical stratum corneum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment

Summary Topical act-tone treatment extracts lipids from the stratum corneum. and disrupts the permeability barrier, resulting in a homeostatic response in the viable epidermis that ultimately repairs the barrier. Recently, we have developed an optimal lipid mixture (cholesterol, ceramide. palmitate and linoleate 4–3:2.3:1:1.08) that, when applied topically, accelerates barrier repair following extensive disruption of the barrier by acetone. The present study determined if topical treatment with this optimal lipid mixture would have beneticial effects following disruption of the barrier by petroleum ether, tape stripping, or by detergent treatment. Also, we determined if barrier repair was accelerated after moderate disturbances of barrier function. Following moderate or extensive disruption of the barrier by acetone or petroleum ether (solvents), or tape stripping (mechanical), application of the optimal lipid mixture accelerated barrier repair. Additionally, following barrier disruption with V-laurosarcosine free acid or dodecylbenzensulphuric acid (detergents), the optimal lipid mixture similarly accelerated barrier repair. However, following disruption of the barrier with different detergents, sodium dodecyl sulphate and ammonium lauryl sulphosuccinate. the optimal lipid mixture did not improve barrier recovery. Thus, the optimal lipid mixture is capable of accelerating barrier repair following disruption of the barrier by solvent treatment or tape stripping (mechanical), and by certain detergents such as Sarkosyl and dodecylbenzensulphuric acid. The ability of the opiimal lipid mixture to accelerate barrier repair after both moderate and extensive degrees of barrier disruption suggests a potential clinical use for this approach.     

Beta2-adrenergic receptor antagonist accelerates skin barrier recovery and reduces epidermal hyperplasia induced by barrier disruption

Effects of topical application of adrenergic receptor agonists and antagonists on epidermal barrier repair rate after barrier disruption were studied. Agonists and antagonists of beta1-adrenergic receptor did not affect the barrier repair rate. On the other hand, beta2-adrenergic receptor agonists, procaterol and alprenol, delayed barrier recovery and the beta2 receptor antagonist, ICI-118551, blocked the delay. Moreover, topical application of ICI-118551 or beta1,2 receptor antagonist, clenbuterol alone accelerated barrier recovery. Antagonists of alpha1 and alpha2 receptors did not affect barrier recovery. The delay of barrier repair induced by prodaterol hydrochloride was blocked by a voltage-gated calcium channel blocker, verapamil or nifedipine. In cultured human keratinocytes, procaterol increased the intracellular calcium concentration and the increase was blocked by ICI-118551 and also by verapamil or nifedipine. Topical application of ICI-118551 partially blocked the epidermal hyperplasia induced by acetone treatment under low environmental humidity. These results suggest that the beta2-adrenergic receptor is specifically associated with skin barrier homeostasis.     

Impact of anatomical location on barrier recovery,surface pH and stratum corneum hydration after acute barrier disruption

BACKGROUND: It is not known whether distinct anatomical locations will respond with different recovery rates following acute barrier challenges. OBJECTIVES: To investigate whether barrier parameters differ at five body sites during recovery from acute disruption. METHODS: Acute barrier disruption was achieved by tape stripping and by acetone extraction of stratum corneum lipids. Transepidermal water loss (to assess barrier function), capacitance (for stratum corneum hydration) and skin surface pH were measured at each of five different body sites in 14 human volunteers. Individual measurements were obtained every 24 h for 96 h. RESULTS: Lipid-rich skin areas (e.g. the forehead) were the most vulnerable to barrier disruption by either method. While acetone treatment affected barrier function and decreased stratum corneum hydration, tape stripping similarly altered barrier function but increased capacitance values. Although the effect of barrier disruption on surface pH appeared to vary with location, no significant pattern of variation emerged. Independent of the method used for barrier disruption, the pH normalized within 96 h. CONCLUSIONS: Skin at different body sites shows distinct patterns of barrier recovery that are likely to be related to structural and physiological differences. Therefore, 'anatomically specific' regimens appear possible and relevant for the treatment of cutaneous disorders. In addition, adequate statistical analyses are essential to detect real differences in barrier recovery parameters.     

Rottlerin, a specific inhibitor of protein kinase C-delta, impedes barrier repair response by increasing intracellular free calcium

Several signals have been suggested in maintaining skin barrier homeostasis, but epidermal calcium ions are currently thought to be a main signaling factor. It is not clear, however, exactly how an intracellular calcium level decreases in response to the loss of an extracellular calcium gradient. In this study, we investigated the effects of several broad-type and isozyme-specific protein kinase C (PKC) inhibitors on epidermal permeability barrier recovery. Topical application of chelerythrine chloride, a broad-type PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor, significantly impeded the barrier recovery rate at 3 and 6 hours after barrier disruption. A significant decrease in the number and secretion of lamellar bodies was also observed at the inhibitor-treated site. Calcium ion-capture cytochemistry showed that the epidermal calcium gradient was rapidly reformed in inhibitor-treated skin, though recovery of the corresponding barrier function was not observed. In cultured keratinocytes treated with either inhibitor, increased intracellular calcium did not return to the baseline concentration after extracellular calcium decreased. These results suggest that PKC inhibitors, especially a PKCdelta-specific inhibitor, delay barrier recovery by affecting the intracellular calcium concentration after a loss of the extracellular calcium gradient. Furthermore, PKCdelta is important in controlling a decrease in intracellular calcium concentration.