Shindo Yutaka, Fujita Keigo, Tanaka Mari, Fujio Hiroki, Hotta Kohji, Oka Kotaro
Department of Bioscience and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, 223-8522, Japan.
Department of Bioscience and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, 223-8522, Japan; Waseda Research Institute for Science and Engineering, Waseda University, 2-2 Wakamatsucho, Shinjuku-ku, Tokyo, 162-8480, Japan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City, 80708, Taiwan.
Biochem Biophys Res Commun. 2021 Dec 10;582:131-136. doi: 10.1016/j.bbrc.2021.10.046. Epub 2021 Oct 21.
The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 μM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.
皮肤会受到各种外部刺激。角质形成细胞是表皮中的主要细胞类型，它与外周感觉神经元相互作用并调节神经元活动。最近的研究表明，角质形成细胞在伤害感受中起关键作用，并且三磷酸腺苷（ATP）是从角质形成细胞到感觉神经元信号转导的主要介质之一。然而，尚未进行关于ATP介导的从角质形成细胞到感觉背根神经节（DRG）神经元信息流的定量细胞水平分析。在本研究中，我们使用定位于细胞膜外侧的基因编码ATP探针iATPSnFR和钙探针Fura-red同时成像细胞表面ATP和细胞内钙信号。用玻璃针机械刺激角质形成细胞后，在刺激区域周围观察到钙增加和ATP释放，并且这些现象呈正相关。在培养的DRG神经元和与受刺激角质形成细胞相邻的角质形成细胞中，检测到靠近受刺激细胞一侧的细胞内钙浓度增加和细胞表面ATP水平升高。DRG神经元中钙反应与输入ATP信号的比率明显大于角质形成细胞。我们发现DRG神经元比角质形成细胞对ATP更敏感，因此，当与角质形成细胞共培养时，只有DRG神经元对1μM或更低浓度的ATP有反应。此外，角质形成细胞适度机械刺激引起的信号主要传递给DRG神经元。这些发现对于进一步确定表皮中伤害感受的详细机制很重要。
