Mechanical stimulus-evoked signal transduction between keratinocytes and sensory neurons via extracellular ATP

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 Ca²⁺ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca²⁺ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca²⁺ 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 Ca²⁺ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca²⁺ 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.