Clapp L H, Gurney A M
Department of Pharmacology, United Medical School, St Thomas's Hospital, London.
Exp Physiol. 1991 Sep;76(5):677-93. doi: 10.1113/expphysiol.1991.sp003535.
Single cells from the rabbit pulmonary artery were isolated using a new and convenient procedure. Strips of muscle were incubated overnight in papain at 6 degrees C and dispersed the following morning after warming the tissue for 10 min. This method consistently produced a high yield of relaxed cells, which reversibly responded to vasoconstrictors and remained viable for many hours. The electrophysiological properties of these cells were studied using the patch-clamp technique in the whole-cell configuration. In physiological Ca2+ solution with K(+)-filled pipettes, cells had a high input resistance (approximately 17 G omega) and an average resting potential of -55 mV. In voltage clamp, several components of outward current could be identified. Depolarizing voltage steps revealed a prominent, transient current (Itran), having extremely rapid activation (less than 5 ms) and inactivation (less than 15 ms) kinetics. Itran was followed by a more slowly activating current (IKso) that was sustained over 100 ms. Both currents were essentially abolished by a 4-aminopyridine (4-AP) and sensitive to Ca2+ influx. IKso, but not Itran, was blocked by tetraethylammonium (TEA) and had the properties of a Ca(2+)-activated K+ current. Holding the membrane potential at -40 mV completely inactivated Itran and unmasked a time-independent, background current superimposed on IKso. The background current was also blocked by 4-AP. In addition, when adenosine triphosphate (ATP), but not guanosine triphosphate (GTP), was omitted from the patch-pipette, spontaneous bursts of outward current (SOCs) were superimposed on the voltage-activated currents. However, since SOCs were rarely observed when ATP and GTP were present together, they are unlikely to be active under physiological conditions. Thus at least four types of outward current can be distinguished in isolated rabbit pulmonary artery cells. These include a novel transient current which could be activated from the resting potential. It activates much more rapidly than outward currents previously reported in vascular muscle, and would rapidly oppose action potential firing. This current could therefore be responsible for the inability of large elastic arteries to fire action potentials.
采用一种新的简便方法分离兔肺动脉的单个细胞。将肌条在6℃下用木瓜蛋白酶孵育过夜，次日早晨在组织升温10分钟后进行分散处理。该方法始终能产生高产率的松弛细胞，这些细胞对血管收缩剂有可逆反应，并能存活数小时。使用膜片钳技术的全细胞模式研究了这些细胞的电生理特性。在含有钾离子的移液管的生理钙离子溶液中，细胞具有高输入电阻（约17 GΩ），平均静息电位为 -55 mV。在电压钳实验中，可以识别出外向电流的几个成分。去极化电压阶跃显示出一个突出的瞬态电流（Itran），其激活（小于5毫秒）和失活（小于15毫秒）动力学极快。Itran之后是一个激活较慢的电流（IKso），持续超过100毫秒。两种电流基本上都被4-氨基吡啶（4-AP）消除，并且对钙离子内流敏感。IKso，但不是Itran，被四乙铵（TEA）阻断，具有钙激活钾电流的特性。将膜电位保持在 -40 mV完全使Itran失活，并揭示出叠加在IKso上的与时间无关的背景电流。背景电流也被4-AP阻断。此外，当膜片移液管中省略三磷酸腺苷（ATP）而不是三磷酸鸟苷（GTP）时，外向电流的自发爆发（SOCs）叠加在电压激活电流上。然而，由于当ATP和GTP同时存在时很少观察到SOCs，它们在生理条件下不太可能是活跃的。因此，在分离的兔肺动脉细胞中至少可以区分出四种外向电流类型。其中包括一种新的瞬态电流，它可以从静息电位激活。它的激活比先前在血管平滑肌中报道的外向电流快得多，并且会迅速对抗动作电位发放。因此，这种电流可能是大弹性动脉无法产生动作电位的原因。
