Shenglan Wang^1,2,3, Yi Dai^2,3, Kimiko Kobayashi^2,3, Wanjun Zhu³, Yoko Kogure^2,3, Hiroki Yamanaka³, You Wan⁴, Wensheng Zhang¹, Koichi Noguchi³

¹ State Key Laboratory of Earth Surface Processes and Resource Ecology, Beijing Normal University, Beijing 100088, China;

² Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Hyogo 650-8530, Japan;

³ Department of Anatomy and Neuroscience, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan;

⁴ School of Basic Medical Sciences, Neuroscience Research Institute, Peking University, Beijing 100083, China.

 

Abstract: Proinflammatory agents trypsin and mast cell tryptase cleave and activate protease-activated receptor-2 (PAR-2), which is expressed on sensory nerves and causes neurogenic inflammation. P2X3 is a subtype of the ionotropic receptors for adenosine 5′-triphosphate (ATP), and is mainly localized on nociceptors. Here, we show that a functional interaction of the PAR-2 and P2X3 in primary sensory neurons could contribute to inflammatory pain. PAR-2 activation increased the P2X3 currents evoked by α, β, methylene ATP in dorsal root ganglia (DRG) neurons. Application of inhibitors of either protein kinase C (PKC) or protein kinase A (PKA) suppressed this potentiation. Consistent with this, a PKC or PKA activator mimicked the PAR-2-mediated potentiation of P2X3 currents. In the in vitro phosphorylation experiments, application of a PAR-2 agonist failed to establish phosphorylation of the P2X3 either on the serine or the threonine site. In contrast, application of a PAR-2 agonist induced trafficking of the P2X3 from the cytoplasm to the plasma membrane. These findings indicate that PAR-2 agonists may potentiate the P2X3, and the mechanism of this potentiation is likely to be a result of translocation, but not phosphorylation. The functional interaction between P2X3 and PAR-2 was also confirmed by detection of the α, β, methylene-ATP-evoked extracellular signal-regulated kinases (ERK) activation, a marker of neuronal signal transduction in DRG neurons, and pain behavior. These results demonstrate a functional interaction of the protease signal with the ATP signal, and a novel mechanism through which protease released in response to tissue inflammation might trigger the sensation to pain through P2X3 activation.

 

Keywords: inflammatory pain; protease-activated receptor 2; protein kinase A; protein kinase C; P2X3.