De Petrocellis Luciano, Starowicz Katarzyna, Moriello Aniello Schiano, Vivese Marta, Orlando Pierangelo, Di Marzo Vincenzo
Endocannabinoid Research Group, Institute of Cybernetics, National Research Council, Via Campi Flegrei 34, Comprensorio Olivetti, 80078 Pozzuoli, Naples, Italy.
Exp Cell Res. 2007 May 15;313(9):1911-20. doi: 10.1016/j.yexcr.2007.01.008. Epub 2007 Jan 18.
The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 degrees 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 CB(1) receptors. Both 8-Br-cAMP (100 microM) and forskolin (10 microM) right-shifted the dose-response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca(2+). The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB(1) 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.
褪黑素8型瞬时受体电位通道（TRPM8）可被低温（<25摄氏度）和化合物激活，其受蛋白激酶C介导的磷酸化调控，方式与香草酸1型瞬时受体电位通道（TRPV1）相反，即脱敏而非敏化。由于TRPV1也可被蛋白激酶A（PKA）介导的磷酸化敏化，我们研究了PKA途径的两种激活剂8-溴环磷酸腺苷（8-Br-cAMP）和福斯可林对稳定过表达该通道的HEK-293细胞（TRPM8-HEK-293细胞）中薄荷醇和异丝氨酸对TRPM8活性的影响。我们还研究了以下因素对TRPM8的影响：（1）一系列先前已证明可激活或拮抗TRPV1的化合物，以及（2）对瞬时共表达的大麻素CB(1)受体的共刺激。8-Br-cAMP（100微摩尔）和福斯可林（10微摩尔）均使异丝氨酸和薄荷醇对TRPM8介导的细胞内钙离子效应的剂量反应曲线右移。8-Br-cAMP和福斯可林的抑制作用被选择性PKA抑制剂Rp-cAMP-S减弱。对TRPM8-HEK-293细胞中瞬时共表达的人CB(1)受体的刺激也抑制了TRPM8对异丝氨酸的反应。最后，一些TRPV1激动剂和拮抗剂（但碘化拮抗剂除外）拮抗异丝氨酸诱导的TRPM8激活，对薄荷醇诱导的TRPM8激活的拮抗作用则弱得多。重要的是，内源性香草酸/内源性大麻素花生四烯乙醇胺和N-花生四烯酰多巴胺在亚微摩尔浓度下也拮抗TRPM8。尽管这些发现需要通过直接测量天然表达TRPM8的细胞中TRPM8活性的实验来证实，但它们支持了相同的调节事件对TRPM8和TRPV1受体具有相反作用这一观点，并确定花生四烯乙醇胺和N-花生四烯酰多巴胺为TRPM8通道的首批潜在内源性功能性拮抗剂。
