Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of CAMP, cannabinoid CB1 receptors and endovanilloids

Aniello Schiano Moriello1, Luciano De Petrocellis1, Katarzyna Starowicz2, Marta Vivese1, Pierangelo Orlando3, Vincenzo Di Marzo2. 1Institute of Cybernetics National Research Council, Pozzuoli, Naples, Italy, 2Institute Biomolecular Chemistry, National Research Council, Pozzuoli, Naples, Italy, 3Institute of Protein Biochemistry National Research Council, Naples, Italy.

The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 °C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. TRPV1 is activated by the endocannabinoids anandamide and N-arachidonoyldopamine (NADA), and is also desensitized or sensitised by cannabinoid CB1 receptor stimulation, depending on whether or not it is being currently sensitised by cAMP-induced protein kinase A (PKA) activation. Therefore, we investigated the effect of: 1) anandamide and NADA; 2) CB1 receptor co-stimulation; and 3) activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of the TRPM8 agonists menthol and icilin in HEK-293 cells stably overexpressing the receptor (TRPM8-HEK-293 cells). The effect of the substances on [Ca2+]i was determined by using Fluo-4, a selective intracellular fluorescent probe for Ca2+.Experiments were carried out by measuring cell fluorescence at room temperature (λEX 488 nm, λEM516 nm) before and after the addition of the test compounds at various concentrations. Both 8-Br-cAMP (100μM) and forskolin (10 μM), given to cells 5 min prior to stimulation with menthol and icilin, right-shifted the dose-response curves for the effect of the two compounds on TRPM8-mediated elevation of intracellular Ca2+. The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation with HU210 of human CB1 receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. The effect was counteracted by SR141716A and was negligible in non-CB1-expressing TRPM8-HEK-293 cells. Finally, the endovanilloids/endocannabinoids, anandamide and NADA, strongly antagonised TRPM8 gating by icilin (IC50=150 and 740 nM, respectively), in a way independent from CB1 and TRPV1 receptors. Although these findings need to be confirmed by the use of techniques directly measuring TRPM8 activity and by experiments in TRPM8-constitutively expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors, and identify two potential ways through which anandamide and NADA can influence TRPM8 activity, i.e.: 1) by acting as the first endogenous direct antagonists of TRPM8 channels; 2) by activating CB1 receptors coupled to inhibition of adenylate cyclase and, hence, by either inhibiting or stimulating TRPM8 gating in the absence or presence of elevated cAMP levels, respectively.