探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Summary

测量KV7（KCNQ）钾离子通道活性的孤立动脉细胞（中与测量大蟒/扩张的反应（使用压力myography），同时采用膜片钳电生理技术）可以揭示有关的重要信息的的KV7渠道作用在血管平滑肌生理和药理学。

Abstract

电阻动脉的壁内的平滑肌细胞的收缩或松弛确定动脉直径并由此控制的血液流至容器内，并有助于全身血压。收缩过程中被限制主要是由胞内游离钙浓度（〔Ca ^{2 _+]} cyt的），它是依次由多种离子转运和频道控制。离子通道是常见的中间体，在信号转导通路激活血管活性激素对血管收缩或舒张功能。离子通道往往是有针对性的治疗药物，无论是有意（ 如钙离子通道阻断剂诱导的血管舒张和降低血压）或无意的（ 如引起不必要的心血管副作用）。

KV7（KCNQ）电压激活的钾离子通道最近被牵连的生理和治疗的重要塔尔格ETS调节平滑肌收缩。为了阐明KV7渠道在生理信号转导和治疗药物的行动中的具体作用，我们需要研究他们的活动是如何在细胞水平上调节，以及评估他们的贡献的背景下，完整的动脉。

大鼠肠系膜动脉提供了一个有用的模型系统。动脉可以很容易地解剖，结缔组织清除，并用孤立的动脉肌细胞膜片钳电生理准备，或为血管收缩剂/血管舒张反应的在相对生理条件下测量插管，加压。在这里，我们描述了这两种类型的测量方法，并提供一些例子，实验一体化设计可提供更清楚地了解这些离子通道在调节血管张力的作用。

Protocol

1。手术切除的小肠肠系膜血管商场麻醉300-400克SD大鼠（4％）与异氟醚吸入给药。 执行中线剖腹手术暴露的小肠系膜。 Exteriorize通过腹部切口非常小心，以避免创伤暴露的肠管及肠系膜小肠和大肠。轻轻地扇了的肠系膜超过无菌纱布。 外科手术切除小肠和大肠的一部分，包括盲肠（沿利润率和肠系膜上动脉/静脉）从主分支的起源仔细剪裁血管。 切除的组织转移到烧?…

Discussion

这里所描述的方法和实验方法相当强劲，并且能够产生明确的和可重复的结果时一丝不苟的细节。良好的电生理记录和收缩/扩张动脉段是依赖于细胞和动脉血管的健康。细胞制剂可以改变每一天，甚至使用相同的协议。隔离解决方案，可用于长达2周，但如果细胞制备的质量低，随之而来的两个天应准备新的隔离解决方案。我们已经发现，一批一批，因此隔离条件（时间的培养和酶的浓度）酶活?…

Materials

Name	Company	Catalog Number	Comments
Sodium Chloride	Sigma	S5886	Dissecting Solution: 145 Bath solution for Electrophysiology*: 140 Internal solution for electrophysiology: 10 Isolation solution for myocytes*: 140 Bath solution for pressure myography: 145 Lumen solution for pressure myography: 145
Potassium chloride	Sigma	P5405	Dissecting Solution: 4.7 Bath solution for Electrophysiology*: 5.36 Internal solution for electrophysiology: 135 Isolation solution for myocytes*: 5.36 Bath solution for pressure myography: 4.7 Lumen solution for pressure myography: 4.7
Potassium EGTA	Sigma	E4378	Internal solution for electrophysiology: 0.05
HEPES	Sigma	H9136	Bath solution for Electrophysiology*: 10 Internal solution for electrophysiology: 10 Isolation solution for myocytes*: 10
Disodium hydrogen phosphate	Sigma	S5136	Isolation solution for myocytes*: 0.34
Potassium hydrogen phosphate	Sigma	P5655	Isolation solution for myocytes*: 0.44
Magnesium Chloride	Sigma	M2393	Bath solution for Electrophysiology*: 1.2 Internal solution for electrophysiology: 1 Isolation solution for myocytes*: 1.2
Calcium Chloride	Sigma	C7902	Bath solution for Electrophysiology*: 2 Isolation solution for myocytes*: 0.05
Sodium phosphate	Fisher Scientific	BP331-1	Dissecting Solution: 1.2 Bath solution for pressure myography: 1.2 Lumen solution for pressure myography: 1.2
Magnesium Sulfate	Sigma	M2643	Dissecting Solution: 1.17 Bath solution for pressure myography: 1.17 Lumen solution for pressure myography: 1.17
MOPS	Fisher Scientific	BP308	Dissecting Solution: 3 Bath solution for pressure myography: 3 Lumen solution for pressure myography: 3
Pyruvic acid	Sigma	P4562	Dissecting Solution: 2 Bath solution for pressure myography: 2 Lumen solution for pressure myography: 2
EDTA dihydrate	Research Organics	9572E	Dissecting Solution: 0.02 Bath solution for pressure myography: 0.02 Lumen solution for pressure myography: 0.02
D-Glucose	Sigma	G7021	Dissecting Solution: 5 Bath solution for Electrophysiology*: 10 Internal solution for electrophysiology: 20 Isolation solution for myocytes*: 10 Bath solution for pressure myography: 5 Lumen solution for pressure myography: 5
Bovine serum albumin	Sigma	A3912	Dissecting Solution: 1% Lumen solution for pressure myography: 1%
pH			Dissecting Solution: 7.4 Bath solution for Electrophysiology*: 7.3 Internal solution for electrophysiology: 7.2 Isolation solution for myocytes*: 7.2 Bath solution for pressure myography: 7.4 Lumen solution for pressure myography: 7.4
Osmolarity			Dissecting Solution: 300 Bath solution for Electrophysiology*: 298 Internal solution for electrophysiology: 298 Isolation solution for myocytes*: 298 Bath solution for pressure myography: 300 Lumen solution for pressure myography: 300
			*11
			Table 1. Components of solutions used in the experiment.