Expressions of rod and cone photoreceptor-like proteins in human epidermis.

Previous reports have suggested the existence of photoreceptors for visible radiation at the surface of the human body. Rhodopsin is a well-known photosensitive protein found in the 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 colour. Here, we describe immunochemical studies using anti-rhodopsin and anti-opsin antibodies on human skin. 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 shorter-wavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and the skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are expressed in human epidermis.

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.

Dopamine D2-like receptor agonists accelerate barrier repair and inhibit the epidermal hyperplasia induced by barrier disruption.

Two families of G protein-coupled receptors of the neurotransmitter dopamine, i.e., dopamine 1-like family (D1-like receptor) and dopamine 2-like family (D2-like receptor), show dopaminergic activity in nerve cells. The D2-like receptor family, composed of D2, D3, and D4 receptors, downregulates the intracellular cAMP signaling pathway, and dopamine receptor agonists reduce the cAMP level in neurons. We previously demonstrated that the cAMP level in epidermal keratinocytes is related to epidermal barrier homeostasis. Since keratinocytes are known to carry various neurotransmitter receptors, we hypothesized that D2-like receptors on keratinocytes might be related to skin barrier homeostasis. In this study, we examined the effect of topical application of receptor agonists and antagonists on skin barrier recovery after barrier disruption. Application of D2-like receptor agonists accelerated barrier recovery, whereas D2-like receptor antagonists delayed it. D2-like receptor agonists also reduced the epidermal hyperplasia induced by barrier disruption under low environmental humidity. Immunohistochemical study and RT-PCR analysis revealed the expression of the D2 receptor in the basal epidermis and the D4 receptor in the uppermost layer of the epidermis. These results suggest that dopaminergic receptors are involved in epidermal barrier homeostasis.

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.

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.

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.

NMDA-type glutamate receptor is associated with cutaneous barrier homeostasis.

Glutamate receptors play an important role in the excitatory synaptic action of the central nervous system. In this study, effects of glutamate receptor agonists and antagonists on skin barrier homeostasis were studied using hairless mouse. Topical application of L-glutamic acid, L-aspartic acid (non-specific glutamate receptor agonists) and N-methyl-D-aspartate (NMDA, NMDA type receptor agonist) delayed the barrier recovery rate after barrier disruption with tape stripping. On the other hand, topical application of D-glutamic acid (non-specific antagonist of glutamate receptor), MK 801 and D-AP5, (NMDA-type receptor antagonists) accelerated the barrier repair. The non-NMDA type receptor agonist, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), did not affect the barrier recovery. Topical application of MK-801 also promoted the healing of epidermal hyperplasia induced by acetone treatment under low environmental humidity. Immediately after barrier disruption on skin organ culture, secretion of glutamic acid from skin was significantly increased. Immunohistochemistry, reverse transcription polymearse chain reaction (RT-PCR) and in situ hybridization showed an expression of NMDA-type receptor-like protein on hairless mouse epidermis. NMDA increased intercellular calcium in cultured human keratinocytes and the increase was blocked by MK 801. These results suggest that glutamate plays an important role as a signal of cutaneous barrier homeostasis and epidermal hyperplasia induced by barrier disruption.

P2X purinergic receptor antagonist accelerates skin barrier repair and prevents epidermal hyperplasia induced by skin barrier disruption.

The effects of ATP receptor agonists/antagonists on skin barrier recovery rate were evaluated in hairless mice. Topical application of ATP and alpha,beta-methylene ATP (agonist of P2X receptor) delayed barrier recovery. Topical application of suramin (nonspecific ATP receptor antagonist), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (P2X receptor antagonist), and 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) (P2X1, P2X3, P2X2/3 antagonist) after barrier disruption accelerated the barrier repair. The P2Y type receptor antagonist Reactive Blue 2 did not affect the barrier repair process. Moreover, topical application of TNP-ATP prevented epidermal hyperplasia induced by barrier insult under low environmental humidity. ATP was secreted immediately after tape stripping on skin in organ culture. alpha,beta-Methylene ATP increased intercellular calcium in cultured keratinocytes and the increase was blocked by TNP-ATP. Both reverse transcription polymerase chain reaction assay and immunohistochemical study showed the existence of protein that had a structure similar to P2X3 on hairless mouse epidermis. These results suggest that cutaneous barrier homeostasis can be regulated by cation flux through a P2X3-like ATP receptor.

Functional vanilloid receptors in cultured normal human epidermal keratinocytes.

Vanilloid receptor subtype 1, VR1, is an ion channel that serves as a polymodal detector of pain-producing chemicals such as capsaicin and protons in primary afferent neurons. Here we showed that both capsaicin and acidification produced elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured human epidermal keratinocytes. The capsaicin- and acidification-evoked increases in [Ca(2+)](i) were inhibited by capsazepine, an antagonist to VR1. VR1-like immunoreactivity was observed in the cells. These findings suggest that functional VR1-like protein is present and functions as a sensor against noxious chemical stimuli, such as capsaicin or acidification, in epidermal keratinocytes.