TRPV2 is activated by cannabidiol and mediates CGRP release in cultured rat dorsal root ganglion neurons

Abstract

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) = 3.7 microM), followed by Delta(9)-tetrahydrocannabinol (EC(50) = 14 microM) and cannabinol (EC(50) = 77.7 microM). 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 cannabidiol-evoked 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.

Figure 1.

Activation of rat TRPV2 (rTRPV2) by cannabinoids in calcium mobilization assay. A, Structures of selected cannabinoids. B–G, Representative fluorescence traces of the time course (B, E), concentration–response relationship (C, F), and RR antagonism (D, G) of rTRPV2 activation by 30 μm CBD (B–D) and 100 μm CBN (E–G). In B and E, a robust calcium influx was evoked after addition of CBD or CBN (solid black line); the responses can be completely blocked by 30 μm RR (dotted black line); little fluorescence intensity change was observed in the absence of cannabinoids (dashed black line) or in HEK293 parental cells in the presence of the same concentration of cannabinoids (solid gray line).

Figure 2.

Activation of human TRPV2 (hTRPV2) by CBD in calcium mobilization assay. A–C, Representative fluorescence traces of the time course (A), concentration–response relationship (B), and RR antagonism (C) of hTRPV2 activation by CBD.

Figure 3.

Activation of TRPV2 by CBD in whole-cell patch clamp. A, B, Time course of activation by 30 μm CBD at −87 mV for rat (A) and human (B) TRPV2. C, D, Current–voltage (I–V) relationships of rat (C) and human (D) TRPV2 in response to 30 μm CBD. The currents were elicited with a continuous voltage ramp from −87 mV to +23 mV once every 4 s. The I–V plots are averages of five current traces and are subtracted from responses in the concurrent presence of CBD (30 μm) and RR (10 μm).

Figure 4.

CB₁-independent CBD- and CBN-evoked CGRP release. A, Effect of selected cannabinoids on CGRP release from cultured rat DRG neurons. Shown is CGRP release from neuronal cultures preincubated with buffer (open bar) or 10 μm SR141716A (gray bars) and then challenged with 20 μm CBD, 20 μm CBN, or buffer (BL) as indicated in Materials and Methods. B, CGRP release from rat DRG neurons as a function of CBD concentration.

Figure 5.

TRP channel-mediated CBD-evoked CGRP release. A–C, CGRP release in response to 100 nm CAP (A), 30 μm CBD (B), or 30 μm MO (C), in the presence of 10 μm CPZ, no calcium, 10 μm RR, 10 μm SR141716A, 10 μm SR144528, or buffer (BL). D, CGRP release in response to a second exposure of the same or a different agonist (e.g., MO/CBD indicates first treatment with MO followed by CBD). *p < 0.05; **p < 0.01.

Figure 6.

The effects of TRPV2 or TRPA1 siRNA knockdown on CBD- and MO-evoked CGRP release from rat DRG neurons. A, Relative mRNA levels of TRPV1, TRPV2, and TRPA1 in TRPV2, TRPA1, or nontarget siRNA-transfected DRG neurons. B, CBD (30 μm)-, MO (30 μm)-, or CAP (100 nm)-evoked CGRP release in TRPV2, TRPA1, or nontarget siRNA-transfected DRG neurons. **p < 0.01; ***p < 0.001.