In this work, we establish a role for the bioactive lipid sphingosine 1-phosphate (S1P) and its
receptor S1PR3 in inflammatory thermal pain and itch. S1P injection elicited robust heat hypersensitivity, acute pain, and itch behaviors in mice, which were wholly dependent on S1PR3. S1P-evoked pain was entirely dependent on TRPV1 ion channels, whereas S1P evoked itch was dependent on TRPA1. Consistent with our in vivo findings, we found that S1P activated subsets of both heat- and itch-sensitive dorsal root ganglion (DRG) neurons, and that activation was solely dependent on S1PR3, and not S1PR1. Our data support a model in which S1P elicits neuronal activation via two pathways in distinct subsets of somatosensory neurons. Gβγ-dependent activation of TRPA1 mediates S1PR3-evoked calcium responses in one subpopulation, whereas PLC-dependent activation of TRPV1 mediates S1PR3-evoked calcium responses in the other subpopulation. In the Complete Freund’s Adjuvant (CFA) -induced inflammatory pain model, S1PR3-deficient mice failed to develop heat hypersensitivity, whereas mechanical hypersensitivity developed normally. Pharmacological blockade of S1PR3 activity or S1P production rapidly and selectively ameliorated heat hypersensitivity in wild-type mice treated with CFA. These data definitively identify S1PR3 as a mediator of thermal pain and itch, and, furthermore, demonstrate for the first time that elevated peripheral S1P is an essential driver of chronic inflammatory pain.