Real-time imaging of human epidermal calcium dynamics in response to point laser stimulation

Abstract

BackgroundChanges 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. ObjectiveThe 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. MethodsWe 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. ResultsCells 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. ConclusionOur 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.