Γ-Aminobutyric Acid (A) Receptor Agonists Accelerate Cutaneous Barrier Recovery and Prevent Epidermal Hyperplasia Induced by Barrier Disruption Mitsuhiro.

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γ-Aminobutyric Acid (A) Receptor Agonists Accelerate Cutaneous Barrier Recovery and Prevent Epidermal Hyperplasia Induced by Barrier Disruption Mitsuhiro Denda, PhD, Kaori Inoue, Shinji Inomata, Sumiko Denda Journal of Investigative Dermatology Volume 119, Issue 5, Pages 1041-1047 (November 2002) DOI: 10.1046/j.1523-1747.2002.19504.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 GABA and GABA(A)-type receptor agonists, muscimol and isoguvacine, accelerated barrier repair. On the other hand, the GABA(B)-type receptor agonist, baclofen, did not affect the barrier recovery rate. The same tendency was observed at 1, 3, and 6 h after the treatment. Vertical axes show the recovery percentage of each measurement time point to the level to the original level. Error bars show SD of each average. F=173.440, p<0.001, Two points were measured in one flank skin and four to eight animals were used for each treatment. Journal of Investigative Dermatology 2002 119, 1041-1047DOI: (10.1046/j.1523-1747.2002.19504.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The effect of GABA was blocked by the GABA(A)-type receptor antagonist, bicuculline methobromide, but GABA(B)-type receptor antagonist, saclofen, did not block the effect of GABA application. Topical application of bicuculline methobromide or saclofen alone did not affect the barrier recovery rate. The same tendency was observed at 1, 3, and 6 h after the treatment. Vertical axes show the recovery percentage of each measurement time point to the level to the original level. Error bars show SD of each average. F=119.415, p<0.001, Two points were measured in one flank skin and four to six animals were used for each treatment. Journal of Investigative Dermatology 2002 119, 1041-1047DOI: (10.1046/j.1523-1747.2002.19504.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Topical application of GABA prevented the epidermal hyperplasia induced by acetone treatment under low environmental humidity. (A–D) Dark spots show BrdU-positive cells. (A) Shows the hyperproliferative epidermis treated with water after the acetone treatment under the low humidity. Topical application of 1 mM GABA prevented the epidermal hyperplasia (B). The effect of GABA was blocked by bicucullin methobromide (C). The increase was seen on the epidermal basal layer (A). (D) Shows the untreated control. Bars=20 μm. The quantified results of the epidermal thickness are shown in (E). The topical application of GABA significantly reduced the epidermal thickness and it was blocked by bicucullin methobromide. The number of BrdU-positive cells in the epidermis of each treatment (F) shows the same tendency as in (E). Error bars show SD of each average. Journal of Investigative Dermatology 2002 119, 1041-1047DOI: (10.1046/j.1523-1747.2002.19504.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunoreactivity against GABA(A) receptor polyclonal antibody was observed in the hairless mouse epidermis (A, green color). Merged image with Nomarski microscopic observation and DAPI staining (blue color) is shown in (B). The immunoreactivity against the GABA antibody was blocked by a blocking peptide: (C) merged image with DAPI, and (D) Nomarski. Bars=20 μm. Journal of Investigative Dermatology 2002 119, 1041-1047DOI: (10.1046/j.1523-1747.2002.19504.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The fluorescent GABA(A) probe shows localization of the GABA(A) receptor in the nucleated layer of the hairless mouse epidermis: (A) red color, merged image with DAPI, and (B) Nomarski microscopic observation. It was reduced by preincubation with bicucullin methobromide: (C) merged image with DAPI, and (D) Nomarski. Bars=20 μm. Journal of Investigative Dermatology 2002 119, 1041-1047DOI: (10.1046/j.1523-1747.2002.19504.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 There was a significant decrease in MEQ fluorescence (i.e., elevation in intracellular chloride ion concentration) in cultured human keratinocytes treated with 10 μMGABA. The decrease was completely blocked by 10 μM bicucullin methobromide. One hundred keratinocytes were evaluated for each treatment. The vertical axis shows the ratio of the fluorescence after 5 min incubation to those before the treatment. Mean±SD: blank, 0.894±0.044; GABA, 0.814±0.048; GABA and BMB, 0.908±0.069. F=85.086, p<0.0001, One hundreds cells were measured for each treatment. Journal of Investigative Dermatology 2002 119, 1041-1047DOI: (10.1046/j.1523-1747.2002.19504.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions