Thromboxane A2 Induces Itch-Associated Responses through TP Receptors in the Skin in Mice  Tsugunobu Andoh, Yumi Nishikawa, Tomomi Yamaguchi-Miyamoto,

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Thromboxane A2 Induces Itch-Associated Responses through TP Receptors in the Skin in Mice  Tsugunobu Andoh, Yumi Nishikawa, Tomomi Yamaguchi-Miyamoto, Hiroshi Nojima, Shu Narumiya, Yasushi Kuraishi  Journal of Investigative Dermatology  Volume 127, Issue 8, Pages 2042-2047 (August 2007) DOI: 10.1038/sj.jid.5700810 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Scratching after intradermal injection of the TP receptor agonist U-44619 in ICR mice. (a) Time course of scratching after vehicle (upper panel, 0.2% ethanol in saline) and U-46619 (lower panel, 10nmol per site) injection. (b) Dose–response curves for the scratch-inducing effects of U-46619 and histamine. ICR mice were given an intradermal injection of U-46619, histamine, or vehicle (VH; ○, 0.2% ethanol in saline; □, 2% ethanol in saline; △, saline). Values represent the means±SEM for eight animals. *P<0.05 when compared with VH for each agent (Dunnett's multiple comparisons). Journal of Investigative Dermatology 2007 127, 2042-2047DOI: (10.1038/sj.jid.5700810) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Suppressive effects of the TP receptor antagonist ONO-3708 on U-46619-induced scratching in ICR mice. U-46619 (10nmol per site) was injected intradermally alone (CNT) or together with ONO-3708 into ICR mice. Dashed line shows data obtained from mice injected intradermally with vehicle (0.2% ethanol in saline). Values represent the means±SEM for eight animals. *P<0.05 when compared with CNT (Dunnett's multiple comparisons). Journal of Investigative Dermatology 2007 127, 2042-2047DOI: (10.1038/sj.jid.5700810) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Deficiency in TP thromboxane A2 receptor abolishes scratching induced by intradermal injection of U-46619 in mice. U-46619 (10nmol per site) was injected intradermally into wild-type (C57BL/6 strain) and TP receptor-deficient mice (TP-R−/−). Values represent the means±SEM for eight animals. *P<0.05 when compared with wild type (Student's t-test). Journal of Investigative Dermatology 2007 127, 2042-2047DOI: (10.1038/sj.jid.5700810) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression of thromboxane synthase and TP receptor in the mouse skin and dorsal root ganglion neurons. (a) Thromboxane synthase immunoreactivity in the skin. It was observed in the epidermal keratinocytes. Bar=20μm. (b) TP receptor (TP-R) and PGP9.5 immunoreactivities in the skin. TP-R (green) was observed in the epidermal keratinocytes. Scale bar=20μm. (c) Frequency distribution of TP-R-positive neurons in the dorsal root ganglion. Closed and open columns indicate TP-R-positive (n=597) and negative neurons (n=536), respectively. Journal of Investigative Dermatology 2007 127, 2042-2047DOI: (10.1038/sj.jid.5700810) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 U-46619-induced increase in the concentration of intracellular Ca2+ ions in the cultured mouse dorsal root ganglion neurons and keratinocytes. Typical examples of time-course of U-46619-induced changes in the concentration of intracellular Ca2+ ions (fluorescence) in the dorsal root ganglion neuron (a) and keratinocytes (c) Maximal change in the U-46619-induced increase of intracellular Ca2+ ions in the dorsal root ganglion neurons (b, n=20) and keratinocytes (d, n=33). Primary cultures of mouse dorsal root ganglion neurons and keratinocytes were preloaded with fluo-3. U-46619 was administered to the culture medium at the final concentration of 200μM. ONO-3708 (final concentration of 10μM) and vehicle (VH) were administered 5minutes before U-46619. Values represent the means±SEM. *P<0.05 when compared with VH. Journal of Investigative Dermatology 2007 127, 2042-2047DOI: (10.1038/sj.jid.5700810) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Schema of the mechanisms of thromboxane A2-mediated itch. Thromboxane synthase is expressed in the epidermal keratinocytes (KC) and TP receptor (TP-R) is expressed in the KC and primary sensory neurons. KCs may produce and release thromboxane A2 (TXA2), which may act on primary afferents to produce itch signals. TXA2 may also act on KCs to release itch mediators including TXA2 itself. Journal of Investigative Dermatology 2007 127, 2042-2047DOI: (10.1038/sj.jid.5700810) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions