TRPC7调控疼痛状态下加压素合成和释放的机制研究

Vasopressin is an important neuroendocrine hormone secreted mainly from hypothalamic supraoptic and paraventricular neurons. It is documented that noxious stimuli enhance the level of this substance that acts as an endogenous analgesic mediator through both peripheral and central signaling pathways. Until now, however, the molecular mechanisms underlying this dynamic regulation of vasopressin synthesis/release remain unclear. Our preliminary study showed that TRPC7, a Ca2+-permeable non-selective cation channel of the canonical transient receptor potential subfamily, was specifically expressed in vasopressin- but not oxytocin-producing neurons in the supraoptic and paraventricular nuclei. Moreover, the expression level of TRPC7 was dramatically enhanced by noxious stimuli, while greatly reduced by hypertonic stimuli. Stereotactic application of TRPC7 inhibitors in formalin-induced animal pain model markedly increased nociceptive behaviors and decreased the plasma vasopressin level. These findings together raises the possibility that upregulation of TRPC7 in these brain areas may be a critical process that relieves pain behaviors through facilitated synthesis and/or release of vasopressin. In this project, to test this hypothesis, we propose to investigate the following plan by a combition of morphology, molecular biology, electrophysiology and optogenetics: (1) spatiotemporal expression pattern of TRPC7 along with the development of chronic pain; (2) intrinsic properties of activating this channel and its functional subunit composition; (3) the contribution of Ca2+-CaM-CaMKII/IV-CREB signaling pathway to TRPC7-mediated synthesis of vasopressin. (4) differential molecular mechanisms underlying the somatic/dendritic and axonal release of vasopressin. The results of this study will not only clarify the mechanisms underlying the dynamic changes of vasopressin synthesis and release under chronic pain conditions, but also disclose a clear pathophysiological role of TRPC7 channel in vivo. Simultaneously, the success of the present project could bring us a novel molecular target for future pain therapy.

加压素是生物体内重要的神经内分泌激素，疼痛刺激诱发其合成和分泌增加发挥外周和中枢镇痛作用。但截至目前，影响加压素动态变化的机制仍不清楚。我们的前期研究显示：TRPC7通道特异性表达于视上核和室旁核的加压素神经元，而不表达于催产素神经元；疼痛刺激诱发其表达升高；脑内给予其抑制剂可增强动物的痛行为反应并下调血浆加压素水平。提示TRPC7可能是调控疼痛状态下加压素合成和释放的关键分子。因此，本项目拟运用形态学、分子生物学、电生理学和光遗传学方法探讨TRPC7：（1）随慢性痛发展的时空表达特点和对动物痛行为的影响；（2）在体激活特点和亚基构成；（3）通过Ca2+/CaM/CaMK/CREB途径促进加压素合成的机制；（4）在胞体/树突和轴突等不同部位促进加压素释放的机制。阐明以上问题将从疼痛的角度揭示加压素生物学合成和释放的机制，揭示TRPC7的在体生理病理功能，也为痛与镇痛的研究提供新的分子靶标。

加压素是生物体内重要的神经内分泌激素，疼痛刺激诱发其合成和分泌增加发挥外周和中枢镇痛作用。但截至目前，影响加压素动态变化的机制仍不清楚。本项目的研究结果显示：TRPC7非选择性阳离子通道特异性表达于视上核和室旁核的加压素神经元，而不表达于催产素神经元，其在细胞内主要分布于胞体膜下区和突触后膜下区。急性痛和慢性痛刺激可诱发其表达升高。脑内定位给予其激动剂OAG可显著抑制动物的痛行为学反应，伴随血浆加压素水平的升高；而给予其抑制剂如flufenamat，SKF96365则能增强动物的痛反应，伴随血浆加压素水平的降低。这些药理学干预并不明显影响动物的血浆渗透压和血钠水平，提示TRPC7可能仅是调控疼痛状态下加压素水平的关键分子。加压素神经元还发出轴突侧支投射向脊髓。运用鞘内给药的方法，我们还检测了新近发现的TRPC通道抑制剂醋酸落叶松酯（LA）对神经病理性痛的功效。结果表明，LA浓度依赖性的抑制动物的机械痛和冷痛，此效应与降低小胶质细胞活化和促炎因子的水平有关，而和加压素无直接关系，表明TRPC7在高位脑中的作用和低位脑不同。进一步的膜片钳研究表明，细胞外高钙状态下，OAG可以在急性分离的室旁核和视上核神经元上诱发缓慢进展的非选择性阳离子电流，其电流幅值较小；但降低细胞外钙水平可以直接诱发缓慢进展的大幅值内向电流，此电流可以被SKF96365所抑制。两种状态下的I-V曲线都呈现反转电位在0 mV附近，整流程度较弱的性质。表明室旁核和视上核神经元上确实表达与离体TRPC7性质非常类似的离子通道。进一步的形态学和Western blot研究表明，TRPC7可能通过pERK-pCREB途径促进加压素的合成和释放。本项目不仅探索了加压素合成和释放的上游分子事件，进一步揭示TRPC7的在体生理病理功能，也为痛与镇痛的研究提供新的分子靶标。