Topical application of neuronal nitric oxide synthase inhibitor accelerates cutaneous barrier recovery and prevents epidermal hyperplasia induced by barrier disruption

The effect of nitric oxide (NO) on skin barrier recovery rate was evaluated in hairless mouse. Topical application of an NO synthase (NOS) inhibitor and a neuronal nitric oxide synthase (nNOS) inhibitor accelerated the barrier recovery after tape stripping, whereas application of an inducible NOS (iNOS) inhibitor had no effect. After tape stripping, the barrier recovery in nNOS-/- mice was significantly faster than in wild type. Topical application of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) delayed the barrier recovery in hairless mice. Immediately after barrier disruption on skin organ culture, NO release from the skin was significantly increased. The increase was blocked by nNOS inhibitor, but not by iNOS inhibitor. Topical application of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) accelerated the barrier recovery, whereas SIN-1 chloride, a guanylyl cyclase activator, delayed the barrier recovery. In cultured human keratinocytes, SNAP increased the intracellular calcium concentration. The increase was blocked by ODQ, but not by the calcium channel-blocker nifedipine. In calcium-free medium, SNAP increased the intracellular calcium concentration. Topical application of both nNOS inhibitor and ODQ also reduced the epidermal hyperplasia induced by barrier disruption under low environmental humidity. These results suggest that NO plays an important signaling role in cutaneous barrier homeostasis and in epidermal hyperplasia induced by barrier disruption.     

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.     

Cutaneous barrier repair and pathophysiology following barrier disruption in IL-1 and TNF type I receptor deficient mice

Disruption of the permeability barrier elicits a homeostatic repair response which rapidly restores barrier function while repeated barrier perturbation results in cutaneous pathology. In response to barrier disruption there is a marked increase in epidermal TNF-alpha and IL-1 production. To determine the potential role of TNF and IL-1 in mediating the cutaneous changes that occur following barrier disruption we compared the kinetics of barrier recovery and the degree of epidermal hyperplasia and cutaneous inflammation in TNF type I (p55) receptor and IL-1 receptor type I (p80) deficient mice. No abnormalities in epidermal morphology were observed with light or electron microscopy in receptor deficient mice. Under baseline conditions epidermal barrier function was unchanged in receptor deficient mice. Following barrier disruption the kinetics of barrier recovery were similar in control vs TNF receptor deficient mice regardless if the barrier was disrupted by acetone treatment, SDS treatment, or tape stripping. In contrast, barrier recovery was slightly but significantly accelerated regardless of the method of barrier disruption in IL-1 receptor deficient mice. The degree of epidermal hyperplasia and cutaneous inflammation following repeated barrier disruption was similar in control, TNF receptor, and IL-1 receptor deficient mice. The present study demonstrates that barrier recovery is not delayed and the degree of epidermal hyperplasia and inflammation are not altered in either TNF receptor or IL-1 receptor deficient mice, indicating that neither TNF nor IL-1 alone are essential for either barrier repair or the cutaneous pathology induced by barrier perturbation. Whereas the increase in IL-1 following barrier disruption may delay components of the repair response, whether either TNF-alpha or IL-1 regulate aspects of the homeostatic response remains unresolved.     

Modulators of the endocannabinoid system influence skin barrier repair,epidermal proliferation,differentiation and inflammation in a mouse model

Endocannabinoids (ECs) are important regulators of cell signalling. Cannabinoid receptors are involved in keratinocyte proliferation/differentiation. Elevation of the endogenous cannabinoid tone leads to strong anti‐inflammatory effects. Here, we explored the influence of endocannabinoid system (ECS) modulators on skin permeability barrier repair, epidermal proliferation, differentiation and inflammation in hairless mice. We used WOBE440, a selective fatty acid amide hydrolase (FAAH) inhibitor, WOL067‐531, an inhibitor of endocannabinoid reuptake with no relevant FAAH activity, which both signal via cannabinoid receptor‐1 and cannabinoid receptor‐2 (CB‐1R and CB‐2R) and compared them to WOBE15 which signals via CB‐2R. Barrier disruption and skin irritation were induced by tape stripping or by sodium dodecyl sulphate (SDS) patch testing. Immediately after barrier disruption, 30 μL of 0.5% WOBE440, WOL067‐531 and WOBE15 solutions or the vehicle was applied topically. Barrier repair was monitored by transepidermal water loss at 1.5, 3, 5 and 7 hours. We found that barrier repair was significantly delayed by WOL067‐531. A tendency for a delay was noticed for WOBE440, whereas for WOBE15, no effect was observed. Immunohistology showed that the tape‐stripping‐induced increase in epidermal proliferation and filaggrin expression was significantly reduced by topical applications of WOL067‐531 and WOBE440, but not by WOBE15. Also, the SDS‐induced inflammation, as determined by the number of inflammatory cells, was reduced by WOL067‐531 and WOBE440. In summary, we showed that WOL067‐531 exhibits a significant effect on skin barrier repair, epidermal proliferation/differentiation and inflammation.     

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.     

Role of PKC-delta as a signal mediator in epidermal barrier homeostasis

The skin shows an important "epidermal permeability barrier homeostasis" in response to barrier disruption. Calcium ion (Ca(2+)), a major regulator in keratinocyte differentiation and proliferation, plays a crucial role in skin barrier homeostasis. Acute barrier disruption induces an immediate depletion of both extra- and intracellular calcium ions in the epidermis, especially in the upper granular layers, and results in the loss of normal epidermal calcium gradient. Currently, we hypothesize that the change in the intracellular calcium ion concentration triggers the barrier repair responses, such as lamellar body (LB) secretion and increased lipid synthesis in the epidermis. In this article, we suggest that PKC-delta is a signaling mediator for the changes in extracellular and intracellular calcium ion concentration.     

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.     

Tape stripping and sodium dodecyl sulfate treatment increase the molecular weight cutoff of polyethylene glycol penetration across murine skin

Prior studies in hairless mice have demonstrated that acute barrier disruption by acetone treatment increases the molecular weight (MW) cutoff of polyethylene glycol (PEG) penetration through the skin. The objective of the present study was to further investigate the dependence of permeability on MW with different forms of barrier disruption. A series of PEGs ranging in MW from near 300 to over 1000 Da were used to study the effects of tape stripping and sodium dodecyl sulfate (SDS) treatment on the MW permeability profiles of mouse skin in vitro. The 12-h percutaneous penetration of all the PEG 300, 600, and 1000 oligomers generally increased as a function of transepidermal water loss (TEWL) of the skin, either tape-stripped or SDS-treated. In addition, the total penetration of PEG oligomers across control skin, and skin tape-stripped and SDS-treated to different degrees of barrier disruption progressively decreased with increasing MW. There were no significant differences in the percutaneous penetration of the PEG oligomers between skin tape-stripped and SDS-treated to the same degree of barrier disruption. The penetration enhancement relative to control skin was more prominent with larger molecules. The MW cutoff for skin penetration increased with the degree of barrier disruption irrespective of the treatment applied, and was 986 Da (tape stripping) and 766 Da (SDS treatment) at TEWL levels in the range 10–20 g/m² per h in comparison with 414 Da for control skin. In accordance with previous findings in acetone-treated mouse skin, the results strongly suggest that, irrespective of the form of barrier disruption applied, not only higher amounts but also more varieties of chemicals (larger molecules) may penetrate skin with a compromised barrier than normal skin.     

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.     

The morphologic changes in lamellar bodies and intercorneocyte lipids after tape stripping and occlusion with a water vapor-impermeable membrane

It has been reported that artificial restoration of barrier function by a water vapor-impermeable membrane after tape stripping induces barrier abrogation in hairless mice, impeding rather than enhancing barrier recovery. To address this issue, we examined the morphologic changes in the epidermis after tape stripping and occlusion with a water vapor-impermeable membrane in murine skin. Male hairless mice were used for all studies of barrier perturbation and occlusion. Barrier disruption was achieved by repeated application of cellophane tape. Immediately after tape stripping the animals were wrapped in a tightly fitting water vapor-impermeable membrane. Transepidermal water loss (TEWL) was measured 20 min after tape stripping and 14, 24, 36, 48 and 60 h after occlusion. For electron microscopy the samples were treated with osmium tetroxide (OsO ₄ ) or ruthenium tetroxide (RuO ₄ ). When tape-stripped animals were wrapped in a water vapor-impermeable membrane, thereby preventing water flux, barrier function did not recover normally. These results demonstrate that an artificial block to TEWL with an impermeable membrane did not enhance barrier recovery. By electron microscopy many transitional cells and lacunae of various sizes were seen within the intercellular spaces of the stratum corneum after occlusion following tape stripping. Occlusion also caused alterations in both lipid lamellar membrane structures in the stratum corneum interstices and the lamellar bodies in the cytosol of granulocytes and transitional cells. Secreted lamellar body contents also appeared to be abnormal in the stratum corneum-stratum granulosum junction. Received: 17 December 1996 / Accepted: 14 November 1997     

Integrity of the permeability barrier regulates epidermal Langerhans cell density

Summary Previous studies have shown that barrier requirements regulate epidermal liquid and DNA synthesis. In the present study, we examined the possibility that the integrity of the permeability barrier influences epidermal Langerhans cells involved with the immune response. Barrier disruption was achieved by treatment of human skin with acetone, sodium dodecylsulphate (SDS), or tape stripping, until a 10–20-fold increase in transepidermal water loss was achieved. Serial biopsies were performed 6–168 after treatment, and Langerhans cells were complexed with anti-CD1a (Leu6) or S-l00 antibodies, and visualized with an immunoperoxidase technique. Acetone treatment resulted in an increase in epidermal Langerhans cell density, reaching a maximum of 94% over control (P < 0.01) by 24 and 48 h post-treatment. Following SDS treatment or tape stripping, epidermal Langerhans cell density was increased by 100 and 175% (P < 0.01), respectively. There was a linear correlation between the degree of barrier disruption and the increase in epidermal Langerhans cell density. Studies with the Ki-S3 proliferation-associated nuclear antigen revealed a two- to threefold increase in epidermal proliferation after barrier disruption. The time curves of the increase in Langerhans cell density and the increase in epidermal proliferation were similar, suggesting that there was a coordinate regulation. In contrast with our previous studies employing patch test reactions to allergens or irritants, disruption of barrier function neither resulted in an increased dermal Langerhans cell density, nor influenced T lymphocytes (CD3⁺. Leu4⁺). Macrophages (KiM8⁺), ICAM-1 or ELAM-1 expression in the skin. In addition, barrier disruption did not result in either dermal inflammation or epidermal spongiosis. In summary these findings support our hypothesis that the permeability barrier influences epidermal Langerhans cell density, which is involved in maintaining an immunological barrier.     

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.     

精神刺激降低裸鼠表皮CD44水平

目的确定精神刺激对表皮CD44的表达是否有影响。方法利用限制裸鼠自由活动的方法建立精神刺激模型,并于造模结束时用胶带破坏其躯干部表皮通透屏障功能。用免疫组化法观察CD44在完整的表皮及屏障功能破坏6h后表皮的表达。结果与对照组相比,在精神刺激后,无论是完整的,还是表皮通透屏障功能破坏的皮肤,其表皮CD44的表达都明显降低;两组之间,透明质酸的表达没有明显差别。结论精神刺激能降低表皮CD44的表达。     

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.     

Epidermal interleukin-1 alpha generation is amplified at low humidity: implications for the pathogenesis of inflammatory dermatoses

BACKGROUND: We have previously reported that low humidity amplifies the hyperproliferative and inflammatory response to barrier disruption. Other reports suggest that epidermal interleukin (IL)-1 alpha is stimulated by various factors related to epidermal inflammation and that it may induce other proinflammatory molecules. OBJECTIVES: To evaluate the generation of IL-1 alpha in the skin of hairless mice kept under various conditions of environmental humidity. METHODS: We carried out an immunohistochemical study, and evaluated epidermal IL-1 alpha mRNA and protein levels, and release of IL-1 alpha from skin after tape stripping, in hairless mice kept under low or high humidity. RESULTS: The immunohistochemical study showed that the amount of IL-1 alpha in the epidermis was higher in animals kept in a low-humidity environment than in a high-humidity one. The epidermal IL-1 alpha mRNA and protein levels increased significantly when the animals were kept under low humidity. Moreover, the release of IL-1 alpha from skin immediately after tape stripping was significantly higher in animals kept in a low-humidity environment than in a high-humidity one. CONCLUSIONS: These results suggest that IL-1 alpha is an important factor in mediating the relationship between environmental humidity and epidermal pathology.     

Skin permeability barrier and occlusion: no delay of repair in irritated human skin *

It has been reported that occlusive treatment of irritated skin results in a reduction of barrier repair activities in hairless mice. In contrast, the clinically observed benefit of occlusion in the treatment of hand eczema and other chronic skin diseases with a perturbed barrier function is well–known. While the beneficial effect of occlusion has been proven for the treatment on psoriasis there are no controlled clinical studies of the effect of occlusion on irritated human skin. We have therefore evaluated the effect of various occlusive treatments on repair of the human skin permeability barrier under controlled experimental conditions. Barrier perturbation was induced either by application of sodium lauryl sulfate (SLS) or by repeated tape stripping. This was followed by treatment with different occlusive and semipermeable dressings, partly alter pre-treatment with petrolatum. Repair of water barrier function was evaluated by daily measurements of transepidermal water loss (TEWL) for 1 week. SLS irritation and tape stripping led to a 6-fold increase in TEWL as a sign of severe water barrier perturbation, followed by a stepwise decrease over the following days. Occlusion did not significantly delay barrier repair as measured by TEWL. Only in tape-stripped skin did TEWL stay at high levels during treatment with self-adhesive dressings. This may be explained by damage of newly formed stratum corneum caused by changing of these membranes. Our results indicate that, in contrast to earlier observations in hairless mouse skin, permeability barrier repair activities are not significantly delayed by occlusive treatment in human skin.     

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.     

Ultraviolet B-induced alterations of the skin barrier and epidermal calcium gradient

Ultraviolet irradiation induces a variety of cutaneous changes, including epidermal permeability barrier disruption. In the present study, we assessed the effects of ultraviolet B (UVB) irradiation in epidermal barrier function and calcium distribution in murine epidermis. Adult hairless mice were exposed to a single dose of UVB (0.15 J/cm(2)). Barrier function was evaluated by transepidermal water loss (TEWL), lanthanum perfusion. The morphological alterations were examined by histology, immunohistochemistry and electron microscopy using ruthenium tetroxide (RuO(4)) postfixation. For evaluation of the effect on epidermal calcium distribution, the ion-capture cytochemistry was employed. UVB irradiation caused a significant increase in TEWL, which peaked at day 4. In parallel, the increased number of sunburn cells and the changes in epidermal hyperplasia and proliferation were observed. Electron microscopic observation demonstrated that the water-soluble lanthanum tracer was present in the extracellular stratum corneum domains, and the increased intercellular permeability was correlated with defective organization of the extracellular lipid lamellar bilayers of the stratum corneum. Moreover, UVB irradiation also caused an appearance of calcium precipitates in the stratum corneum and transitional cell layers as well as the increased cytosolic calcium in the lower epidermis, reflecting the alterations of the epidermal calcium gradient. These results suggest that the changes of the epidermal calcium distribution pattern may correlate with the perturbation of the epidermal barrier induced by UVB irradiation.