Rho激酶抑制剂Fasudil对Kv7钾通道选择性调节作用的研究

发布时间：2018-05-09 09:11

本文选题：法舒地尔 + Kv7钾离子通道　；参考：《河北医科大学》2014年硕士论文

【摘要】：电压门控型Kv7（Kv7.1-7.5）钾通道家族由KCNQ基因编码，在调节心肌动作电位及稳定神经元膜电位方面发挥重要作用。Kv7.1通道主要表达于心肌组织，并与其辅助亚单位KCNE1通道共同编码组成延迟整流钾通道（IKs），在调节心肌细胞动作电位时程中发挥着重要作用，KCNQ1/KCNE1突变体构成遗传性心律失常的基础并导致长QT综合症。Kv7.2和Kv7.3通道以四聚体形式共表达产生的电流已经被证实是M电流分子基础。另有研究发现Kv7.5通道也参与M电流的形成。Kv7.2和Kv7.3通道基因突变或M通道功能失调，可引发许多中枢性疾病，如良性家族性新生儿惊厥症（BFNC）。Kv7.4通道主要表达于内耳的耳蜗和前庭器官以及中枢听觉传导通路。Kv7.4通道突变被证实引发遗传性耳聋症（DFNA2）。近期研究发现Kv7.1、Kv7.4、Kv7.5通道高表达于血管平滑肌等多种平滑肌细胞。药理学研究显示Kv7.1对血管张力无影响，Kv7.4、Kv7.5参与血管张力的调节，Kv7.4、Kv7.5调节剂对高血压存在潜在的治疗价值。但是目前高选择性的Kv7.4、Kv7.5调节剂尚未见报道。本研究发现Rho激酶抑制剂法舒地尔（Fasudil）可选择性激活Kv7.4/Kv7.5通道，而对其它Kv7通道无作用。 Fasudil，又名HA1077，是一种新型异喹啉磺酰胺衍生物类Rho激酶（Rho-kinase）选择性抑制药，能强效扩血管，可以有效缓解脑血管痉挛，临床上主要用于改善和预防蛛网膜下腔出血后引起的脑血管病，脑血管痉挛及肺动脉高压的治疗。研究认为Fasudil的血管舒张作用与抑制Rho激酶有关，而我们认为Fasudil的扩血管作用还与增大Kv7钾电流有关。此外，我们发现Fasudil对Kv7钾离子通道家族的调节作用具有选择性。本论文以Kv7钾离子通道家族为出发点，利用电生理穿孔膜片钳技术及四通道微血管张力测定系统，研究Fasudil对表达于HEK293细胞的Kv7钾离子通道、分离培养的乳大鼠的小直径背根神经节（DRG）神经元细胞M通道的作用以及Fasudil对肠系膜阻力血管的作用。 1. Fasudil对表达于HEK293细胞的Kv7钾离子通道的作用目的：研究Fasudil对Kv7.1/KCNE1、Kv7.2、Kv7.2/Kv7.3、Kv7.4及Kv7.5的选择性调节作用。方法：利用电生理穿孔膜片钳技术，观察Fasudil对表达于HEK293细胞的Kv7钾离子通道的作用。瑞替加滨（Retigabine，RTG，Kv7钾离子通道开放剂）为阳性对照药。结果：(1)30μM Fasudil对Kv7.1/KCNE1电流无明显作用。 (2)1μM、3μM、10μM、30μM及100μM Fasudil对Kv7.2及Kv7.2/7.3电流均无明显作用。 (3) Fasudil可浓度依赖性地增强Kv7.4电流，10μM、30μM、100μMFasudil及10μM RTG分别使尾电流增加0.82、1.38、2.03及3.16倍。 (4) Fasudil可浓度依赖性地增强Kv7.5电流。Fasudil使电导-电压（G-V）激活曲线左移，且左移的幅度随浓度升高而增大。30μM Fasudil使G-V曲线由-53.7mV左移至-62.4mV，左移幅度为9.39±1.10mV；10μM RTG使曲线左移至-68.9mV，左移幅度为13.925±1.80mV。结论：Fasudil对Kv7.4及Kv7.5电流有增大作用，而对Kv7.1/KCNE1、Kv7.2及Kv7.2/7.3电流无明显作用，说明Fasudil对Kv7钾电流的调节作用具有选择性。 2. Fasudil对大鼠的小直径背根神经节（DRG）神经元细胞上M电流的作用目的：研究Fasudil对分离培养的大鼠的DRG神经元M电流的影响。方法：利用电生理穿孔膜片钳技术，观察Fasudil对分离培养的大鼠DRG神经元细胞M型钾电流的作用。结果：(1)1μM、3μM、10μM、30μM及100μM Fasudil对大鼠DRG神经元上表达的M型钾电流无明显作用。 (2)1μM、3μM、10μM、30μM及100μM Fasudil对大鼠DRG神经元的静息膜电位无明显作用。结论：Fasudil对大鼠DRG神经元细胞上M电流和静息膜电位均无明显作用，提示Fasudil对Kv7.2/Kv7.3无明显作用。 3. Fasudil对离体大鼠肠系膜二级阻力血管的作用目的：研究Fasudil对离体大鼠肠系膜二级阻力血管的作用及机制。方法：利用微血管张力测定技术，观察Fasudil对离体大鼠肠系膜二级阻力血管的作用。每次实验分为两小组。Fasudil组：10μM Phe预收缩血管，待平稳之后，依次加入不同浓度的Fasudil，记录实验结果；Fasudil+XE991组：10μM Phe预收缩平稳之后，加入10μM XE991（Kv7钾离子通道阻断剂）孵育15min，再依次加入不同浓度的Fasudil，记录实验结果。结果：Fasudil可浓度依赖性地舒张Phe预收缩的肠系膜二级阻力血管，EC50为0.83±0.25μM，，最大舒张率（Emax）为96.12%；Fasudil可浓度依赖性地舒张Phe+XE991预收缩的肠系膜二级阻力血管，量效曲线右移，EC50为5.04±0.96μM，Emax为96.46%。结论：Fasudil舒张Phe+XE991预收缩的血管的量效曲线发生了右移，其机制可能是Fasudil增大了血管平滑肌细胞上Kv7.4及Kv7.5钾电流，使血管舒张。提示Fasudil对Kv7.4及Kv7.5的选择性调节作用是其舒张血管作用的机制之一。以上研究结果表明，Fasudil对Kv7钾通道具有选择性调节作用，为选择性的Kv7钾离子通道调节剂的开发提供了新的思路；另外，Fasudil对Kv7.4和Kv7.5钾电流的增大作用可能是其血管调节的作用机制之一，对其临床应用提供了新的理论依据。
[Abstract]:The voltage gated Kv7 (Kv7.1-7.5) potassium channel family is encoded by the KCNQ gene and plays an important role in regulating the cardiac action potential and stabilizing the neuron membrane potential. The.Kv7.1 channel is mainly expressed in the myocardial tissue, and the delayed rectifier potassium channel (IKs) is composed of its auxiliary subunit KCNE1 channel, and the action potential is adjusted to regulate the action potential of the cardiac myocytes. The process plays an important role. KCNQ1/KCNE1 mutants form the basis of hereditary arrhythmia and lead to the current produced by the co expression of the long QT syndrome.Kv7.2 and Kv7.3 channels in the form of four polymer, which has been confirmed as the molecular basis of the M current. Further studies have found that Kv7.5 channels are also involved in the formation of.Kv7.2 and Kv7.3 channel gene mutations or M of the M current. Dysfunction of the channel can cause many central diseases, such as the benign familial neonatal convulsion (BFNC).Kv7.4 channel is mainly expressed in the inner ear cochlea and vestibule organs, and the.Kv7.4 channel mutation in the central auditory pathway is confirmed to cause hereditary deafness (DFNA2). Recent studies have found that Kv7.1, Kv7.4, Kv7.5 channels are highly expressed in blood vessels. A variety of smooth muscle cells such as smooth muscle cells. Pharmacological studies have shown that Kv7.1 has no effect on vascular tension, Kv7.4, Kv7.5 participates in the regulation of vascular tension, Kv7.4, Kv7.5 regulator has potential therapeutic value for hypertension. However, the high selective Kv7.4, Kv7.5 regulator has not yet been reported. This study found that the Rho kinase inhibitor FDD (Fasudi) L) selectively activates the Kv7.4/Kv7.5 channel, but has no effect on other Kv7 channels.
Fasudil, also known as HA1077, is a new type of ISO quinoline sulfonamide derivative, Rho kinase (Rho-kinase) selective inhibitor, which can effectively expand blood vessels and effectively relieve cerebral vasospasm. It is mainly used to improve and prevent cerebrovascular disease, cerebral vasospasm and pulmonary hypertension after subarachnoid hemorrhage. The study thinks Fas The vasodilatation of udil is associated with the inhibition of Rho kinase, and we believe that the vasodilatation of Fasudil is also related to the increase of the Kv7 potassium current. Furthermore, we found that Fasudil has a selective role in the regulation of the Kv7 potassium channel family.
In this paper, using the Kv7 potassium channel family as the starting point, using the electrophysiological perforation patch clamp technique and the four channel microvascular tension measurement system, the effect of Fasudil on the Kv7 potassium channel expressed in HEK293 cells, the role of the M channel of the small diameter dorsal root ganglion (DRG) of the rat's small diameter dorsal root ganglion (DRG) and the mesenteric resistance to the mesenteric obstruction were studied. The role of force and blood vessel.
Effect of 1. Fasudil on Kv7 potassium channels expressed in HEK293 cells
Objective: To study the selective regulation of Fasudil on Kv7.1/KCNE1, Kv7.2, Kv7.2/Kv7.3, Kv7.4 and Kv7.5.
Methods: the effect of Fasudil on the Kv7 potassium channel expressed in HEK293 cells was observed by electrophysiological perforation patch clamp technique. Retegine (Retigabine, RTG, Kv7 potassium channel opener) was a positive control drug.
Results: (1) 30 M Fasudil had no significant effect on Kv7.1/KCNE1 current.
(2) 1 M, 3 M, 10 M, 30 M and 100 M Fasudil had no obvious effect on Kv7.2 and Kv7.2/7.3 currents.
(3) Fasudil can enhance Kv7.4 currents in a concentration dependent manner. The 10 M, 30 M, 100 MFasudil and 10 M RTG respectively increase the tail current by 0.82,1.38,2.03 and 3.16 times.
(4) Fasudil can enhance the Kv7.5 current.Fasudil to make the conductance voltage (G-V) activation curve move left, and the amplitude of the left shift increases with the concentration of.30 mu M Fasudil to move the G-V curve from -53.7mV left to -62.4mV, and the left shift is 9.39 + 1.10mV, and the 10 mu M makes the left shift to 13.925 +.
Conclusion: Fasudil has an increasing effect on the current of Kv7.4 and Kv7.5, but has no obvious effect on the current of Kv7.1/KCNE1, Kv7.2 and Kv7.2/7.3, indicating that Fasudil has a selective effect on the regulation of the potassium current of Kv7.
Effect of 2. Fasudil on M currents in rat small diameter dorsal root ganglion (DRG) neurons
Objective: To study the effect of Fasudil on the M currents of DRG neurons isolated from rats.
Methods: electrophysiological perforation patch clamp technique was used to observe the effect of Fasudil on M type potassium currents in cultured rat DRG neurons.
Results: (1) 1 mu M, 3 M, 10 M, 30 M and 100 M Fasudil had no significant effect on M potassium currents in rat DRG neurons.
(2) 1 mu M, 3 M, 10 M, 30 M and 100 M Fasudil had no significant effect on resting membrane potential of DRG neurons in rats.
Conclusion: Fasudil has no significant effect on M current and resting membrane potential of DRG neurons in rats, suggesting that Fasudil has no significant effect on Kv7.2/Kv7.3.
Effect of 3. Fasudil on isolated rat mesenteric two grade resistance vessels
Objective: To study the effect and mechanism of Fasudil on isolated rat mesenteric two grade resistance vessels.
Methods: the effect of Fasudil on the two level resistance vessels of rat mesentery in vitro was observed by microvascular tension measurement. Each experiment was divided into two groups of.Fasudil groups: 10 u M Phe preconstricted blood vessels. After the stabilization, the experimental results were added to different concentrations of Fasudil in order, and the Fasudil+XE991 group: after the pre contraction of 10 mu M Phe was stationary, add The 15min was incubated with 10 M XE991 (Kv7 potassium channel blocker), and then the Fasudil was added in order to record the experimental results.
Results: Fasudil had a concentration dependent relaxation of Phe precontracted mesenteric two resistance vessels, EC50 was 0.83 + 0.25 mu M, and the maximum diastolic rate (Emax) was 96.12%; Fasudil could be concentrated on Phe+XE991 precontracted mesenteric two resistance vessels, the volume effect curve was shifted rightward, EC50 was 5.04 + 0.96 micron, Emax was 96.46%.
Conclusion: the volume effect curve of Fasudil diastolic Phe+XE991 precontracted blood vessels has a right shift. The mechanism may be that Fasudil increases the Kv7.4 and Kv7.5 potassium currents on vascular smooth muscle cells and makes the vasodilatation. The selective regulation of Fasudil on Kv7.4 and Kv7.5 is one of the mechanisms of vasodilatation.
The above results show that Fasudil can selectively regulate the potassium channel of Kv7 and provide a new idea for the development of selective Kv7 potassium channel regulator. In addition, the effect of Fasudil on the potassium current of Kv7.4 and Kv7.5 may be one of the mechanisms of its vascular regulation, and provides a new theoretical basis for its clinical application.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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