The extracellular ATP-mediated epidermal keratinocyteto-sensory neuron communication; an involvement of keratinocytic ATP in induction of pain

Abstract

ATP acts as an intercellular messenger in a variety of cells. Here we characterized the Ca 2+ wave propagation mediated by extracellular ATP in cultured normal human epidermal keratinocytes (NHEKs) also co-cultured with mouse dorsal root ganglion (DRG) neurons. We also asked about physiological consequence of the ATP-mediated communication in relation to pain by behavioral analysis. Pharmacological characterization showed that NHEKs express functional metabotropic P2Y2 receptors. When a cell was gently stimulated with a glass pipette, an increase in the intracel-lular Ca 2+ concentration ([Ca 2+ ]i) was observed, followed by propagating Ca 2+ waves in neighboring cells in an extracellular ATP-dependent fashion. Using an ATP-imaging technique, the release and diffusion of ATP among NHEKs were confirmed. DRG neurons are known to innervate the epidermis that is mainly composed of keratinocytes. In the co-culture of NHEKs and DRG neurons, mechanical stimulation-evoked Ca 2+ waves in NHEKs evoked the [Ca 2+ ]i elevation in adjacent DRG neurons, which was also dependent on extracellualr ATP and the activation of P2Y2 receptors. Extracellular ATP is a dominant messenger that forms intercellular Ca 2+ waves in NHEKs. In addition, Ca 2+ waves in NHEKs could produce a [Ca 2+ ]i elevation in DRG neurons, suggesting dynamic cross talk between skin and sensory neurons mediated by extracellular ATP. Next we investigated a physiological consequence of the ATP-mediated communications. Injection of the P2Y2 and P2Y4 receptor agonist uridine 5'-triphosphate (UTP) into plantar surface in rats produced the mechanical allodynia in a concentration-dependent manner. The UTP-induced mechanical allodynia was inhibited by the P2 receptor antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate) or antisense oligonucleotide for P2Y2 receptors. Taken together, ATP is a key molecule that mediates pain signal-ing from skin to sensory neurons.