Kumamoto Junichi, Goto Makiko, Nagayama Masaharu, Denda Mitsuhiro
Japan Science and Technology Agency, CREST, Kawaguchi, Japan; Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan.
Japan Science and Technology Agency, CREST, Kawaguchi, Japan; Shiseido Global Innovation Center, Yokohama, Japan.
J Dermatol Sci. 2017 Apr;86(1):13-20. doi: 10.1016/j.jdermsci.2017.01.002. Epub 2017 Jan 6.
Changes of epidermal calcium ion concentration are involved in regulation of barrier homeostasis and keratinocyte differentiation. Moreover, intracellular calcium dynamics might play a role in skin sensation. But, although calcium dynamics of cultured keratinocytes in response to mechanical stresses has been well studied, calcium propagation in stimulated human epidermis is still poorly understood.
The aim of this study was to demonstrate a novel method for real-time measurement of calcium dynamics in response to point stimulation of human epidermis at the single-cell level.
We examined calcium propagation in cross-sectional samples of living human epidermis ex vivo, as well as in cultured human keratinocytes, by means of two-photon microscopy after stimulating cells in stratum granulosum with the emission laser of a two-photon microscope.
Cells in different epidermal layers showed different responses, and those in stratum basale showed the greatest elevation of intracellular calcium. Calcium propagation in epidermis was inhibited in the presence of apyrase (which degrades adenosine triphosphate; ATP) or gap-junction blockers. In cultured keratinocytes, on the other hand, calcium propagated in a simple concentric wave-like manner from the stimulation site, and propagation was strongly suppressed by apyrase.
Our results suggested that ATP and gap junctions play important roles in calcium propagation induced by point laser stimulation of the uppermost layer of epidermis. Our method should be broadly useful to study calcium dynamics, epidermal physiological mechanisms, and mechanisms of skin sensation at the single-cell level.
表皮钙离子浓度的变化参与屏障稳态的调节和角质形成细胞的分化。此外，细胞内钙动力学可能在皮肤感觉中发挥作用。但是，尽管对培养的角质形成细胞在机械应力作用下的钙动力学已经进行了充分研究，但对受刺激的人表皮中的钙传播仍知之甚少。
本研究的目的是展示一种在单细胞水平实时测量人表皮点刺激后钙动力学的新方法。
我们通过双光子显微镜用其发射激光刺激颗粒层细胞后，利用双光子显微镜检查了离体活人表皮横截面样本以及培养的人角质形成细胞中的钙传播。
不同表皮层的细胞表现出不同的反应，基底层的细胞细胞内钙升高最为明显。在存在ATP酶（降解三磷酸腺苷；ATP）或缝隙连接阻滞剂的情况下，表皮中的钙传播受到抑制。另一方面，在培养的角质形成细胞中，钙以简单的同心波状方式从刺激部位传播，并且传播受到ATP酶的强烈抑制。
我们的结果表明，ATP和缝隙连接在表皮最上层的点激光刺激诱导的钙传播中起重要作用。我们的方法对于在单细胞水平研究钙动力学、表皮生理机制和皮肤感觉机制应该具有广泛的用途。
