ANO1/TMEM16A在细胞外酸中毒引起冠状动脉收缩中的作用

Acidosis severely jeopardizes cardiovascular system, but its impacts on coronary artery (CA) remain controversial. We recently for the first time demonstrated that extracellular acidosis (EA) exclusively constricts rat CA (RCA). ANO1/TMEN16A is a Ca2+-activated chloride channel and plays an important role in regulating ionic homeostasis, membrane potential and myogenic tone of vascular smooth muscle cell (VSMC). We suppose that ANO1 mediate EA-induced RCA constriction. To validate the supposition, the effects of EA on RCA vascular myogenic tone, RCA VSMC chloride currents and intracellular concentrations of Cl-, H+ and Ca2+ will be studied with myograph, patch clamp and intracellular molecule fluorescence probes, respectively. The mechanisms, especially, involvement of chloride channels, underlying the EA-induced modulations will be explored by inhibitor study with chloride channel blockers and related inhibitors. Gene and protein assay with RT-PCR and Western blot will be performed to reveal the expression phenotype of chloride channels and the effect of EA on the phenotype in RCA VSMC, in comparison with VSMC from other arterioles. The possible role of ANO1/TMEN16A in EA-induced RCA constriction will be further clarified by studying the effects of knockdown of ANO1 with anti-rat TMEM16A siRNA on EA-induced modulations in coronary myogenic tone, RCA VSMC chloride currents and intracellular ionic homeostasis. This project is intended to elucidate the mechanisms underlying EA-induced coronary constriction and is of important theoretical significance and application value to reveal new therapeutic targets for the prevention and treatment of acidosis.

酸中毒严重危害心血管系统，但对冠状动脉（CA）的影响尚存争议。前期我们首次证明，细胞外酸中毒（EA）选择性地收缩大鼠冠状动脉（RCA）。ANO1/TMEN16A为钙激活氯通道，对血管平滑肌（VSMC）离子平衡、膜电位和肌张力起重要调节作用。我们推测，ANO1对EA引起的RCA收缩具有介导作用。本项目拟通过血管环、膜片钳和分子荧光探针实验，研究EA对RCA血管张力、VSMC氯电流及细胞内Cl-、H+和Ca2+浓度的影响；并通过抑制剂实验，探讨EA作用机制及其与氯通道的关系。通过基因和蛋白检测研究RCA VSMC氯通道表达特征及EA对该特征的影响，并与其他小动脉进行比较。用抗大鼠TMEM16A siRNA敲低ANO1，进一步证明ANO1在EA收缩RCA、改变RCA VSMC氯电流和细胞内离子平衡中的作用。本项目旨在阐明EA引起RCA收缩的机制，对揭示防治酸中毒新靶点具有重要理论意义和应用价值。

酸中毒尤其细胞外（extracellular acidosis, EA）是一个最常见、最棘手的临床问题，严重EA对心血管系统有确切的损害作用，甚至威胁人的生命。冠状动脉是维持心脏正常功能、防止损伤和修复损伤的保障。我们前期研究证明大鼠冠状动脉（RCA）对EA独具强烈收缩反应。本课题我们用膜片钳技术进一步证明，EA（本实验细胞外液pH 6.8）对大鼠冠状动脉血管平滑肌（RCASMC）膜电位去极化作用最强。TMEM16A编码的anoctamin 1（ANO1）是一种钙激活的氯离子通道(CaCC)，在调节冠状动脉肌张力中作用重要。ANO1是RCA对EA收缩反应异常敏感的重要机制吗？围绕此科学问题本课题组进行了多层面的研究。RT-PCR和Western blot证实，与其他多处组织脏器小血管比，TMEM16A mRNA和ANO1蛋白的表达在RCA平滑肌层及其急性酶解分离的RCASMC上最丰富。膜片钳结果证实EA独特性地增加RCASMC的Cl-外流，荧光分子探针确证EA降低RCASMC内Cl-水平。细胞内Ca2+是激动CaCC的关键离子，细胞内Ca2+释放和外Ca2+内流为其主要来源。荧光分子探针结果为EA增高RCASMC内Ca2+水平。鉴于以上结果我们进一步采用去氯和抑制剂从多层面探讨了EA对RCA效应的作用环节。结果为，去氯、CaCC抑制剂、L-型电压门控钙通道抑制剂和细胞内Ca2+释放抑制剂对EA所致的RCA收缩、Cl-外流增加和细胞内Ca2+水平增高均有显著性抑制作用。鉴于基因干预技术的限制，我们观察了ANO1抗体的作用，结果表明ANO1抗体减弱RCA和其ASMC对EA的所有反应。这些结果提示，EA通过动员细胞内Ca2+和增加细胞外Ca2+内流而增加细胞内Ca2+浓度，Ca2+激活CaCC通道，增加Cl-外流，使RCASMC细胞膜去极化，后者反过来又增强L-型电压门控钙通道的开放，最终细胞外Ca2+内流进一步增加而导致RCA强烈收缩。总之，本课题明确了EA所致RCA收缩与ANO1介导的Cl-跨细胞膜外流、细胞外Ca2+内流和细胞内Ca2+释放之间的连环网络关系，阐明了CaCC在EA引起RCA收缩中的确切作用，揭示了EA损害冠状动脉的新机制。该研究成果将为精准防治EA所致的冠状动脉功能障碍，保护心脏，改善心脏血液循环，最终保障全身血液供应正常提供了一种全新的防治思路。