Thermosensation is an essential sensory function that is subserved by a variety of transducer molecules, including those from the Transient Receptor Potential (TRP) ion channel superfamily. One of its members, TRPM8 (CMR1), a ligand-gated, nonselective cation channel, is activated by both cold and chemical stimuli in vitro. However, its roles in cold thermosensation and pain in vivo have not been fully elucidated. Here, we show that sensory neurons derived from TRPM8 null mice lack detectable levels of TRPM8 mRNA and protein and that the number of these neurons responding to cold (18 degrees C) and menthol (100 microM) is greatly decreased. Furthermore, compared with WT mice, TRPM8 null mice display deficiencies in certain behaviors, including icilin-induced jumping and cold sensation, as well as a significant reduction in injury-induced responsiveness to acetone cooling. These results suggest that TRPM8 may play an important role in certain types of cold-induced pain in humans.

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein-Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

Transient receptor potential V2 (TRPV2) has been proposed to be a high-threshold thermosensor. However, further elucidation of the channel properties and physiological role of TRPV2 have been hindered by the lack of selective pharmacological tools as well as by the species-dependent differences in the activation of this channel. In the present study, we have used cell-based calcium mobilization and electrophysiological assays to identify and characterize several novel cannabinoid TRPV2 agonists. Among these, cannabidiol was found to be the most robust and potent (EC 50 ϭ 3.7 M), followed by ⌬ 9 -tetrahydrocannabinol (EC 50 ϭ 14 M) and cannabinol (EC 50 ϭ 77.7 M). We also demonstrated that cannabidiol evoked a concentration-dependent release of calcitonin gene-related peptide (CGRP) from cultured rat dorsal root ganglion neurons in a cannabinoid receptor-and TRPV1-independent manner. Moreover, the cannabidiolevoked CGRP release depended on extracellular calcium and was blocked by the nonselective TRP channel blocker, ruthenium red. We further provide evidence through the use of small interfering RNA knockdown and repetitive stimulation studies, to show that cannabidiol-evoked CGRP release is mediated, at least in part, by TRPV2. Together, these data suggest not only that TRPV2 may comprise a mechanism whereby cannabidiol exerts its clinically beneficial effects in vivo, but also that TRPV2 may constitute a viable, new drug target.