Wu Z-Z, Chen S-R, Pan H-L
Department of Anesthesiology and Pain Medicine, University of Texas M. D. Anderson Cancer Center, 1400 Holcombe Boulevard, Unit 409, Houston, TX 77030-4009, USA.
Neuroscience. 2006 Aug 11;141(1):407-19. doi: 10.1016/j.neuroscience.2006.03.023. Epub 2006 May 6.
Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.
奥伐尼尔（(N-香草基)-9-油酰胺）是一种无刺激性的瞬时受体电位香草酸亚型1激动剂，可使伤害感受器脱敏并减轻疼痛。但其分子靶点和信号传导机制鲜为人知。通过电压激活的Ca(2+)通道的钙内流在神经递质释放和突触传递中起重要作用。在此，我们确定了奥伐尼尔对初级感觉神经元中电压激活的Ca(2+)通道电流和信号通路的影响。在急性分离的大鼠背根神经节神经元中进行全细胞膜片钳记录。奥伐尼尔（1 microM）引发延迟但持续的内向电流，并对N型、P/Q型、L型和R型电压激活的Ca(2+)通道电流产生显著抑制（约60%）。用特异性瞬时受体电位香草酸亚型1拮抗剂预处理或细胞内应用1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸可消除奥伐尼尔对电压激活的Ca(2+)通道电流的抑制作用。钙调蛋白拮抗剂（蛇孢菌素-A和钙调蛋白抑制肽）在很大程度上阻断了奥伐尼尔和辣椒素对电压激活的Ca(2+)通道电流的作用。此外，钙调神经磷酸酶（蛋白磷酸酶2B）抑制剂（溴氰菊酯和FK-506）消除了奥伐尼尔对电压激活的Ca(2+)通道电流的作用。值得注意的是，1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸、钙调蛋白拮抗剂和钙调神经磷酸酶抑制剂单独使用时均显著增加了电压激活的Ca(2+)通道电流的幅度。此外，双重免疫荧光标记显示奥伐尼尔诱导背根神经节神经元膜表面Ca(V)2.2免疫反应性迅速内化。总的来说，这项研究表明，对无刺激性的瞬时受体电位香草酸亚型1的刺激通过涉及伤害性神经元内Ca(2+)-钙调蛋白和钙调神经磷酸酶的生化途径抑制电压激活的Ca(2+)通道。这一新信息对于我们理解瞬时受体电位香草酸亚型1类似物使伤害感受器脱敏的信号传导机制以及伤害性感觉神经元中细胞内Ca(2+)和电压激活的Ca(2+)通道的反馈调节具有重要意义。
