**2.1.2 Exposure to low temperature for one minute**

198 Atopic Dermatitis – Disease Etiology and Clinical Management

TRPV4 (Chung et al. 2003), both of which are activated by high temperature (around 30oC),

Fig. 1. Schematic diagram of skin (top) and photomicrograph illustrating the skin structures

We have shown that changes of calcium dynamics are associated with epidermal permeability barrier homeostasis [Denda 2003a]. TRPs are cation-permeable channels. Thus, we hypothesized that activation of TRPs might influence barrier homeostasis. To evaluate the influence of these receptors on barrier homeostasis, we incubated hairless mouse skin and human skin at various temperatures immediately after tape stripping

(bottom).

were also found to be expressed in keratinocytes.

Previous studies have identified cold-sensitive proteins, TRPA1 and TRPM8, that are activated by low temperature (<22oC) in peripheral nerve cells [Story 2003][Peier 2002b]. Recently, TRPA1 was also found in epidermal cells, in which it is activated by lower temperature (<17oC) [Atoyan 2009]. We demonstrated that exposure of cultured human keratinocytes to low temperature induced elevation of intracellular calcium [Tsutsumi 2010]. When the temperature of the medium was reduced to 17~22oC, elevation of intracellular calcium was observed. The extent of elevation was greater in nondifferentiated cells than in differentiated cells. Application of Ruthenium Red (a nonselective TRP blocker) and HC030031 (a specific antagonist of TRPA1) reduced the elevation. These results suggest that functional cold-sensitive calcium channels, TRPA1and/or TRPM8, are present in human epidermal keratinocytes. Thus, we hypothesized that modulation of TRPA1 and/or TRPM8 might influence epidermal permeability barrier homeostasis.

To test this idea, we first examined the effects of topical application of agonists of TRPA1 and brief cold exposure on the barrier recovery rate after barrier disruption [Denda 2010a]. Topical application of a TRPA1 agonist, allyl isothiocyanate or cinnamaldehyde, accelerated the barrier recovery after tape stripping. The effect of both agonists was blocked by HC030031, an antagonist of TRPA1. Brief exposure (1 minute) to cold (10-15oC) also accelerated barrier recovery and this acceleration was also blocked by HC030031. Electronmicroscopic studies indicated that brief cold exposure accelerated lamellar body secretion between stratum corneum and stratum granulosum, while pre-treatment with HC030031 inhibited the secretion. These results support the hypothesis that TRPA1 is associated with epidermal permeability barrier homeostasis.

We next examined the effect of topical application of TRPM8 modulators on epidermal permeability barrier homeostasis [Denda 2010b]. 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 homeostasis and epidermal proliferation after barrier insult.

Physical and Chemical Factors that Improve Epidermal Permeability Barrier Homeostasis 201

function. Again, red light accelerated barrier recovery, while blue light delayed it. An electronmicroscopic study suggested that red light accelerated lamellar body secretion, while blue light blocked it. These results indicate that visible radiation affects skin barrier homeostasis.

Rhodopsin is a well-known photosensitive protein found in rod cells of the retina and detects light/dark contrast. Cone opsins are also photosensitive receptors in the cone cells of the retina and detect color. We have reported immunochemical studies using antirhodopsin and anti-opsin antibodies on human skin (Tsutsumi 2009). Both mouse retina and human epidermis showed clear immunoreactivity with each antibody. Interestingly, immunoreactivity against longer-wavelength opsin antibody was observed in the basal layer of the epidermis, while immunoreactivity against rhodopsin and shorterwavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are also

In retina, transducin and phosphodiesterase 6 play key roles in signal transmission. Thus, we hypothesized that these proteins might exist in epidermal keratinocytes and be associated with barrier homeostasis (Goto 2011). Immunohistochemical study and reverse transcription-PCR assays confirmed the expression of both transducin and phosphodiesterase 6 in epidermal keratinocytes. Topical application of 3-isobutyl-1 methylxanthine, a non-specific phosphodiesterase inhibitor, blocked the acceleration of barrier recovery by red light. Topical application of zaprinast, a specific inhibitor of phosphodiesterases 5 and 6, also blocked the acceleration, while T0156, a specific inhibitor of phosphodiesterase 5, had no effect. Red-light exposure reduced the epidermal hyperplasia induced by barrier disruption under low humidity, and the effect was blocked by pretreatment with zaprinast. Our results indicate phosphodiesterase 6 is involved in the recovery-accelerating effect of red light on the disrupted epidermal permeability barrier. Also, epidermal keratinocytes have a similar energy conversion

Fig. 3. Effects of visible radiation on epidermal permeability barrier homeostasis.

That is, epidermal keratinocytes might have a sensory system for visible radiation.

expressed in human epidermis.

system to that of the retina.

Fig. 2. Temperature ranges within which various TRP receptors are activated, and effect of their activation on epidermal permeability barrier homeostasis.
