**2.1 Temperature**

#### **2.1.1 Exposure to high temperature for one hour**

Since the end of the last century, a series of thermo-activated receptors, called the transient receptor potential protein (TRP) superfamily, has been found in the peripheral nervous system and cloned. Julius and his co-workers found TRPV1 (VR1) as a polymodal detector of pain-producing heat (>43oC) or chemicals, such as capsaicin and protons, in primary afferent neurons (Caterina et al. 1997). We showed that TRPV1 is also expressed in human epidermal keratinocytes (Denda et al. 2001), and demonstrated its functional activity in human cultured keratinocytes (Inoue et al. 2002). Subsequently, TRPV3 (Peier 2002a) and

Physical and Chemical Factors that Improve Epidermal Permeability Barrier Homeostasis 199

(Denda 2007). At temperatures from 36°C to 40°C, barrier recovery was accelerated in both species compared with the area kept at 34°C. At 34°C or 42°C, barrier recovery at occluded sites was delayed compared with un-occluded sites. Topical application of 4 phorbol 12,13-didecanone, an activator of TRPV4, accelerated barrier recovery, while ruthenium red, a blocker of TRPV4, delayed it. Capsaicin, an activator of TRPV1, delayed barrier recovery, while capsazepin, an antagonist of TRPV1, blocked this delay. 2- Aminoethoxydiphenyl borate and camphor, TRPV3 activators, did not affect the barrier recovery rate. Since TRPV4 is activated at about 35°C and above, while TRPV1 is activated at about 42°C and above, these results suggest that TRPV1 and TRPV4 both influence skin

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

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

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

permeability barrier homeostasis.

permeability barrier homeostasis.

epidermal permeability barrier homeostasis.

insult.

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

TRPV4 (Chung et al. 2003), both of which are activated by high temperature (around 30oC), were also found to be expressed in keratinocytes.

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

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 (Denda 2007). At temperatures from 36°C to 40°C, barrier recovery was accelerated in both species compared with the area kept at 34°C. At 34°C or 42°C, barrier recovery at occluded sites was delayed compared with un-occluded sites. Topical application of 4 phorbol 12,13-didecanone, an activator of TRPV4, accelerated barrier recovery, while ruthenium red, a blocker of TRPV4, delayed it. Capsaicin, an activator of TRPV1, delayed barrier recovery, while capsazepin, an antagonist of TRPV1, blocked this delay. 2- Aminoethoxydiphenyl borate and camphor, TRPV3 activators, did not affect the barrier recovery rate. Since TRPV4 is activated at about 35°C and above, while TRPV1 is activated at about 42°C and above, these results suggest that TRPV1 and TRPV4 both influence skin permeability barrier homeostasis.
