**4.2 Neurotransmitters**

Epidermal keratinocytes express a series of receptors, which were originally found in the central nervous system as neurotransmitter receptors. These receptors can be categorized two groups: ionotropic receptors and G-protein-coupled receptors.

Among the former group, receptors that act as calcium ion or chloride ion permeable channels plays crucial roles in epidermal permeability barrier homeostasis. Topical application of calcium channel agonists delays barrier recovery, while antagonists accelerate barrier repair (Denda et al. 2002a) (Denda 2003) (Fuziwara 2003). Topical application of chloride ion channel agonists accelerates barrier recovery (Denda 2003) (Denda 2002b).

The G-protein coupled receptors influence intracellular cAMP level, which plays a crucial role in epidermal barrier homeostasis (Denda 2003b). Increase of intracellular cAMP in epidermal keratinocytes by topical application of forskolin delays barrier recovery, while cAMP antagonist treatment accelerates barrier recovery. Activation of dopamine 2-like receptors (Fuziwara 2005), melatonin receptors, and serotonin receptor (type 5-HT1) decreases intracellular cAMP and consequently accelerates barrier recovery, while activation of adrenergic 2 receptors increases the intracellular cAMP and delays barrier repair (Denda 2003b). Barrier disruption induces an increase of intracellular cAMP. Thus, topical application of agonists of receptors that reduce the intracellular cAMP level accelerates barrier repair. (Denda 2004, Denda 2005)

Histamine receptors are related to skin barrier function [Ashida and Denda 2001]. Three different types of histamine receptors, H1, H2, H3, and H4 have been reported. First, topical application of histamine H1 and H2 receptor antagonists accelerated barrier repair. Histamine itself, H2 receptor agonist, and histamine releaser delayed barrier repair. Histamine H3 receptor antagonist and agonist did not affect the barrier recovery rate. Topical application of H1 and H2 receptor antagonists prevented the epidermal hyperplasia induced by barrier disruption under low humidity. The mechanism of the

Physical and Chemical Factors that Improve Epidermal Permeability Barrier Homeostasis 207

dantrolene or DHBP, accelerated the barrier recovery. These results suggest that RyR expressed in epidermal keratinocytes is associated with both differentiation of keratinocytes

Topical application of specific protease inhibitors accelerates barrier recovery after barrier disruption (Denda 1997). Topical application of 4-(aminomethyl)cyclohexane carboxylic acid (tranexamic acid), a well known anti-plasmin reagent, also accelerates barrier recovery. In contrast, inactive analogs of tranexamic acid do not influence barrier recovery. Application of several trypsin-like serine protease inhibitors, e.g., leupeptin, TLCK and PMSF, accelerates barrier recovery, while other protease inhibitors, e.g., EDTA, pepstatin, Nethylmaleimide, chymostatin, and TPCK, have no effect on barrier recovery. Although the mechanism was not clarified, it was shown that protease activated receptor type 2 is

Feingold and his co-workers demonstrated an important influence of nuclear hormone receptors on epidermal differentiation and stratum corneum barrier formation. Activation of PPAR by farnesol stimulated the differentiation of epidermal keratinocytes (Feingold 1999)(Hanley 2000). Cornified envelope formation and involucrin and transglutaminase protein and mRNA levels were also increased by the activation of PPAR(Feingold 2000). Interestingly, the inflammatory response was also inhibited (Sheu 2002). Topical application of PPAR activators accelerated barrier recovery after tape stripping or acetone treatment and prevented the epidermal hyperplasia induced by repeated barrier disruption (Feingold 1999). Regulation of nuclear hormone receptors might therefore be a possible approach for

As described above, psychological stress impairs barrier homeostasis. To study the effects of stress on barrier homeostasis, we used three models of stress, i.e., immobilization, a crowded environment and a change of living place [Denda 1998][Denda 2000]. In each case, the barrier recovery rate was delayed after barrier disruption. The plasma corticosterone level was increased by each stress, and it was reduced by application of a sedative drug [Denda 2000]. The delay of barrier repair induced by psychological stress was also prevented by application of a sedative drug or glucocorticoid receptor antagonist [Denda 2000]. These results suggest that psychological stress stimulates increased production of glucocorticoids, which adversely affect skin barrier homeostasis. The effect of psychological stress on skin barrier homeostasis in humans has also been examined [Garg 2001]. Reduction of psychological stress might accelerate the skin barrier repair process. Several studies have demonstrated that certain odorants can reduce stress, acting like a sedative drug [Tsuchiya 1992]. These odorants prevented the delay in skin barrier recovery induced by psychological stress in both mice and humans [Denda 2000a][Denda 2000b]. These results suggest the feasibility of a new skin care strategy based on

and epidermal barrier homeostasis (Denda S 2011).

associated with barrier homeostasis (Hachem 2006).

**4.4 Nuclear hormone receptor activators** 

improvement of the cutaneous barrier.

inhalation of specific odorants.

**5. Psychological factors that influence barrier function** 

**4.3 Protease inhibitors** 

interactions between the histamine receptors and the barrier repair process have not been elucidated yet

Nitric oxide (NO) is also involved in barrier homeostasis. We first demonstrated that neuronal nitric oxide synthase knockout (nNOS-/-) mice showed a faster barrier recovery rate than did wild-type mice. nNOS is expressed in epidermal keratinocytes [Ormerod 1998]. Thus, NO generated by keratinocytes might delay barrier repair. To examine this possibility, we next evaluated the effects of NO donor and NOS inhibitor on the barrier recovery rate. Topical application of a NO donor, S-nitroso-N-acetyl-D,L-penicillamine delayed barrier recovery. The application of a nNOS inhibitor accelerated barrier recovery, while the application of a inducible nitric oxide synthase (iNOS) inhibitor did not affect it. Moreover, topical application of a guanylyl cyclase inhibitor accelerated barrier recovery. We observed the release of NO from a skin organ culture after barrier disruption. Thus, regulation of nNOS in epidermal keratinocytes might be useful approach to improve barrier homeostasis (Ikeyama 2007).


No effect, or experiment has not been done.

Table 2. Effects of agonists and antagonists of neurotransmitter receptors on skin permeability barrier recovery.

Ryanodine receptors (RyR) play an important role as calcium channels in the regulation of intracellular calcium levels in the nervous system and muscles. We investigated the expression of RyR in human epidermis. (Denda 2011) Immunohistochemical studies and RT-PCR indicated the expression of RyR type 1, 2, and 3 proteins in epidermal keratinocytes. The expression level of each RyR subtype was higher in differentiating keratinocytes than in proliferative cells. We also demonstrated the functional expression of RyR by means of calcium imaging. In cultured human keratinocytes, application of the RyR agonist 4-chloro-*m*-cresol (CMC) induced elevation of the intracellular calcium concentration and co-application of the RyR antagonist 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP) blocked the elevation. Application of CMC accelerated keratinocyte differentiation *in vitro*. On the other hand, topical application of CMC after tape-stripping of hairless mouse skin delayed barrier recovery, while application of an RyR antagonist, dantrolene or DHBP, accelerated the barrier recovery. These results suggest that RyR expressed in epidermal keratinocytes is associated with both differentiation of keratinocytes and epidermal barrier homeostasis (Denda S 2011).
