Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

Extracellular Adenosine-5′-triphosphate (ATP) is an important multi-functional molecule which can mediate numerous physiological activities by activating purinergic P2 receptors. The objective of this study was to develop a novel optical ATP sensor for in-situ extracellular ATP measurement in biological tissues. The optical ATP sensor was made by applying two layers of sol-gel coating to the end of an optical fiber probe end. The first layer contained ruthenium complex for sensing changes in oxygen concentration which resulted from oxidation of ATP by glycerol kinase and glycerol 3-phosphate oxidase entrapped in the second layer. It was demonstrated that the optical ATP sensor was capable of detecting ATP concentration at a broad range of 10−3 mM to 1.5 mM. A compensation method was established to enable the optical sensor to determine ATP concentration at different oxygen levels. This study also demonstrated the capability of ATP sensor to measure extracellular ATP content in biological tissues (i.e., porcine intervertebral disc). In addition, it was shown that the optical ATP sensor was not affected by pH and derivatives of extracellular ATP. Therefore, the newly developed optical ATP sensor is a good option for in-situ extracellular ATP measurement.