A study of the activity of phytocannabinoids on transient receptor potential A1 and M8 channels

Luciano De Petrocellis1, Aniello Schiano Moriello2, Vincenzo Di Marzo3. 1Endocannabinoid Research Group Institute of Cybernetics, National Research Council, Pozzuoli, Italy, 2Endocannabinoid Research Group, Institute of Cybernetics and Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy, 3Endocannabinoid Research Group Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy.

Apart from Δ9-tetrahydrocannabinol (THC), several other phytocannabinoids exhibit pharmacological activities of potential therapeutic interest in vitro and in vivo. The best studied non-psychotropic phytocannabinoid is certainly cannabidiol (CBD), but cannabichromene (CBC), cannabigerol (CBG), tetrahydrocannabiverin (THCV), cannabidiol-acid (CBD-A) and THC-acid (THC-A) have also been the object of pharmacological studies, which have reported a wide range of activities, particularly against cancer and inflammation. Unlike THC, however, most phytocannabinoids are unable to bind with high affinity and activate the two cloned cannabinoid receptors, CB1 and CB2. THCV was recently shown to act as a CB1/CB2 cannabinoid receptor antagonist (Thomas et al., 2005), whereas CBD, CBG and CBC might activate these receptors indirectly, by inhibiting proteins that participate in endocannabinoid inactivation, namely the putative endocannabinoid membrane transporter and the fatty acid amide hydrolase (Ligresti et al., 2006). CBD is also a full agonist at another target for the endocannabinoid anandamide, i.e. the transient receptor potential vanilloid type-1 (TRPV1) channels (Bisogno et al., 2001), which are gated by the hot chili peppers constituent, capsaicin, as well as by noxious heat. THC activates a distinct TRP channel, the “cold-sensitive” TRPA1, which is gated by mustard oil and cinnamaldehyde (Jordt et al., 2004). Finally, we recently reported (De Petrocellis et al., 2007) that anandamide antagonizes the other “cold-sensitive” TRP channel, TRPM8, which is the target for yet another plant natural product, menthol. Based on these findings, we have undertaken a study to evaluate whether phytocannabinoids activate TRPA1 and TRPM8 channels.

We studied the effect of phytocannabinoids on intracellular Ca2+ levels at room temperature, using Fluo-4 (λEX 488 nm, λEM 516 nm) fluorescence in HEK293 cells stably expressing rat recombinant TRPA1 or TRPM8 cDNAs, compared to untransfected cells.

All the phytocannabinoids tested elevated intracellular Ca2+ in a concentration-dependent manner in TRPA1-expressing cells, but were less effective in cells overexpressing TRPM8 and in non-transfected cells. Their efficacy was comparable to that of mustard oil and cinnamaldehyde, and their potency ranked as follows: CBC>CBD> THC>THC-A>CBD-A=CBG=mustard oil>cinnamaldehyde. CBC and CBD exhibited EC50 values in the 60±10-100±15 nM range of concentrations. Furthermore, pre-treatment of cells with phytocannabinoids (1-10 μM) reduced the effect of subsequent mustard oil administration on TRPA1-mediated intracellular Ca2+ elevation. These data suggest that TRPA1 might be one of the molecular targets underlying some of the pharmacological actions of phytocannabinoids.

Bisogno et al., 2001 Br J Pharmacol, 134, 845-852

De Petrocellis et al., 2007 Exp Cell Res. in press Jordt et al., 2004 Nature, 427, 260-265

Ligresti et al., 2006 J Pharmacol Exp Ther, 318, 1375-1387

Thomas et al., 2005 Br J Pharmacol, 146, 917-926.