A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels
A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels
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DOI：
10.1007/s004240050553
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发表时间：
1998-03-01
影响因子：
4.5
通讯作者：
Beech, DJ
中科院分区：
文献类型：
--
作者：
Quinn, K;Beech, DJ
该项目的目的是开发一种方法，使所有的膜片钳配置能够在平滑肌细胞保持嵌入血管的同时常规应用于它们。用酶法和机械法从兔脑中分离小血管。在显微镜下观察到血管，大多数是小动脉，在含钙1.5 mm的溶液中，平均外径为29微米，长度可变为100微米或更长。小动脉不含台盼蓝，对60 mM K+有收缩反应，对左克罗卡林有扩张反应。膜片钳技术在微动脉内的平滑肌细胞上定期制作膜片钳千兆封口。用含两性霉素B的膜片吸管记录的膜电位平均为-72 mV。短小动脉段可被电压钳制。注入去极化电流或浸浴10 mM Ba2+可引起整个小动脉段收缩。记录细胞贴壁膜片、内向外膜片和外向膜片片，研究K+通道的单位电流。该方法简单易行，与以往研究平滑肌细胞离子通道的方法相比有几个优点。值得注意的是，避免单细胞分离意味着最大限度地减少了酶处理，细胞可以在血管壁的正常环境中进行研究。
The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca2+-containing (1.5 mM) solution, of 29 mu m and variable lengths of 100 mu m or more. Arterioles excluded trypan blue, constricted in response to 60 mM K+ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged -72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba2+ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K+ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall.