Elsevier

Experimental Cell Research

Volume 313, Issue 9, 15 May 2007, Pages 1911-1920
Experimental Cell Research

Research Article
Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): Effect of cAMP, cannabinoid CB1 receptors and endovanilloids

https://doi.org/10.1016/j.yexcr.2007.01.008Get rights and content

Abstract

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. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB1 receptors. Both 8-Br-cAMP (100 μM) and forskolin (10 μM) right-shifted the dose–response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca2+. The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB1 receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.

Introduction

Transient receptor potential (TRP) channels are the vanguard of animal sensory systems, by responding to temperature, touch, osmolarity, pheromones, taste and other physical and chemical stimuli. Their role, however, is broader than classical sensory transduction. TRP channels are not selective for Ca2+ and are made of six transmembrane polypeptide subunits that are believed to assemble as tetramers to form cation-permeable pores. A number of mammalian TRPs are gated in response to thermal stimuli as well as to chemical imitators of burning and cooling sensations, i.e. capsaicin and menthol, respectively. Both TRP of vanilloid type 1 (TRPV1) and TRP of melastatin type 8 (TRPM8) are Ca2+ permeable, non-specific cation channels abundantly expressed in subpopulations of primary afferent neurons. TRPV1 is activated by heat (> 43 °C) and protons (decreased pH), as well as by endogenous ligands (endovanilloids) such as N-arachidonoyldopamine, anandamide and lipoxygenase products of arachidonic acid, and is involved in the transduction of inflammatory and thermal pain [1], [2]. Both diacylglycerols (DAGs) and cAMP regulate the activity of some TRP channels by activating protein kinase C and A (PKC and PKA), respectively, and hence by catalyzing TRP channel Ser/Thr phosphorylation [3]. TRPV1 is also inhibited by phosphatidylinositol-4,5-bisphosphate (PIP2), which also can be considered as an endogenous ligand. Its sensitivity to heat and chemicals is enhanced by bradykinin and other mediators that act via PLCβ to hydrolyze PIP2 and cause DAG formation. DAGs can further activate TRPV1 via PKC [4], [5], [6]. On the other hand, TRPM8 is activated by cold (< 25 °C) and agents like menthol (an active ingredient of peppermint) and several other chilling compounds such as eucalyptol, and icilin (a synthetic compound), which induce a cool sensation [7], [8], [9]. In heterologous expression systems, TRPM8, unlike TRPV1, is activated by PIP2, which therefore can be considered as an endogenous TRPM8 agonist. Hydrolysis of PIP2 to DAGs by PLCβ downregulates TRPM8, which is then further inhibited by DAGs via PKC activation [10], [11].

TRPM8 not only senses temperature at periphery, but also mediates synaptic transmission at the first sensory synapse between dorsal root ganglion (DRG) and dorsal horn (DH) neurons in the spinal cord [12], [13]. However, TRPV1 and TRPM8 are expressed also in nervous and non-nervous tissues that are not subjected to temperatures changes, thus suggesting other important functional roles beyond that temperature sensors. For example, TRPM8 is upregulated in prostate cancer and in other non-prostate primary human tumors (breast, colon, lung and skin) whereas in the corresponding normal tissue is undetectable [14]. The two best known cooling compounds known to activate TRPM8, i.e. the synthetic icilin and menthol, appear to do so through seemingly different mechanisms. Analogous to the putative capsaicin binding site for TRPV1 [15], residues in the transmembrane TM2–TM3 linker region of TRPM8 are involved in icilin sensitivity [16], but not in sensitivity to cold or menthol [17]. Icilin is structurally distinct from menthol, and its activation of TRPM8 is strictly dependent on the presence of extracellular calcium [16], whereas the activation by menthol is not and depends on residues on the TM2 segment and the C-terminus [17].

We previously showed that an increase in the intracellular concentration of cAMP, with subsequent activation of protein kinase A (PKA), leads to an enhancement of anandamide and capsaicin activity on TRPV1 overexpressed in HEK-293 cells [18] and that activation of co-expressed cannabinoid CB1 receptors, partly by decreasing PIP2 levels, also sensitizes TRPV1 to the action of its ligands [19]. Conversely, indirect evidence exists in sensory neurons for TRPM8 desensitization by PKA [20]. Therefore, we investigated here the effects of PKA activation and CB1 stimulation on TRPM8 ligand sensitivity. Furthermore, we studied the effect on TRPM8 of a series of endogenous lipids and pharmacological tools previously shown to influence TRPV1 activity.

Section snippets

Compounds

Icilin and (−)-Menthol were purchased from Sigma Aldrich; Capsaicin, capsazepine, N-arachidonoyl-dopamine (NADA) and resiniferatoxin (RTX) from Alexis Biochemicals; 5-iodo-RTX (IRTX) from Tocris; SR141716A and SR144528 were a kind gift of Sanofi-Aventis; PEA, anandamide, 6-Iodo-Arvanil [21], 6-Iodo-Olvanil [21], 6-I-nordihydrocapsaicin [22] and 6-Iodo-phenylacetylrinvanil [23] were synthesized as described in the corresponding references. HU-210 was a kind gift of Prof. R. Mechoulam, Hebrew

Effect of 8-Br-cAMP and forskolin on TRPM8 activation by icilin

As previously shown [7], [9], icilin dose-dependently elevated intracellular Ca2+ in TRPM8-HEK-293 cells but not in non-transfected cells (Fig. 1 and data not shown). The EC50 of icilin was 0.19 ± 0.03 μM, similar to that previously reported by others [7], [9], [24], [25]. Pre-incubation (5 min), and then continued incubation, of TRPM8-HEK-293 cells with either 8-Br-cAMP (100 μM) or forskolin (10 μM) caused a right-ward shift in the icilin dose–response curve (Fig. 1A). The EC50 was 0.50 ± 0.04 μM

Discussion

Of the several members of the TRP family of ion channels described to date, TRPM8 and TRPV1 receptors act each as a counterpart of the other because of their sensitivity to opposing thermal stimuli, i.e. cold (< 25 °C) and heat (> 42 °C), respectively, and to natural products, i.e. capsaicin and resiniferatoxin on the one hand and menthol and eucalyptol, on the other hand. Accordingly, it has been shown that these two proteins are co-expressed to some extent in DRG [31], dental [32] and

Acknowledgments

We thank Sven-Eric Jordt and David Julius, Department Cellular and Molecular Pharmacology, University of California, San Francisco, for the gift of the rat TRPM8 cDNA construct. This work was partly supported by a Volkswagenstiftung grant to VDM.

References (45)

  • A. Babes et al.

    Cooling inhibits capsaicin-induced currents in cultured rat dorsal root ganglion neurones

    Neurosci. Lett.

    (2002)
  • L.J. Macpherson et al.

    More than cool: promiscuous relationships of menthol and other sensory compounds

    Mol. Cell. Neurosci.

    (2006)
  • R.J. Stein et al.

    Cool (TRPM8) and hot (TRPV1) receptors in the bladder and male genital tract

    J. Urol.

    (2004)
  • N.R. Gavva et al.

    Molecular determinants of vanilloid sensitivity in TRPV1

    J. Biol. Chem.

    (2004)
  • M.J. Caterina et al.

    The capsaicin receptor: a heat-activated ion channel in the pain pathway

    Nature

    (1997)
  • M. Van Der Stelt et al.

    Endovanilloids. Putative endogenous ligands of transient receptor potential vanilloid 1 channels

    Eur. J. Biochem.

    (2004)
  • H.H. Chuang et al.

    Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition

    Nature

    (2001)
  • L.S. Premkumar et al.

    Induction of vanilloid receptor channel activity by protein kinase C

    Nature

    (2000)
  • V. Vellani et al.

    Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

    J. Physiol.

    (2001)
  • D.D. McKemy et al.

    Identification of a cold receptor reveals a general role for TRP channels in thermosensation

    Nature

    (2002)
  • H.J. Behrendt et al.

    Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay

    Br. J. Pharmacol.

    (2004)
  • B. Liu et al.

    Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate

    J. Neurosci.

    (2005)
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