Oyama Y, Evans M L, Akaike N, Carpenter D O
New York State Department of Health, University at Albany, Wadsworth Center for Laboratories and Research, NY.
Neurosci Res. 1988 Dec;6(2):174-80. doi: 10.1016/0168-0102(88)90020-x.
In this study, we have used electrophysiological techniques to evaluate the acetylcholinesterase (AChE) activity on single neurons physically isolated from the pedal ganglia of Aplysia californica and kurodai. Acetylcholine (ACh) was applied to cells having Na-dependent response using a concentration clamp technique which allows a step-like complete change of external medium around the cell (diameter more than 120 micron) within 30 msec. When the neuron was exposed to ACh (50 microM) initially by rapid and brief (400-800 msec) flow and continuously after stopping the perfusion, the induced current rose to a peak and then decayed in the presence of ACh. However, the current increased again when the rapid flow of the same ACh solution around the cell was resumed. The increase in the current by the resumption of the perfusion was not seen when carbachol was substituted for ACh or when extraordinarily high concentration (10 mM) of ACh was used. Furthermore, this increase in the current was blocked by the antiAChE agent, edrophonium, in a dose-dependent manner. These results suggest that acetylcholinesterase (AChE) causes a local depletion of ACh at the membrane surface where the rate of hydrolysis of ACh exceeds the rate of ACh diffusion from bulk solution and the increase in the current by the resumption of the perfusion resulted from the restoration of ACh concentration at the membrane surface. This electrophysiological indication of AChE activity may be a useful tool for the study of AChE by other than biochemical means.
在本研究中，我们运用电生理技术评估了从加州海兔和黑鲷的足神经节物理分离出的单个神经元上的乙酰胆碱酯酶（AChE）活性。使用浓度钳技术将乙酰胆碱（ACh）施加于具有钠依赖性反应的细胞，该技术可在30毫秒内使细胞（直径大于120微米）周围的外部介质呈阶梯状完全改变。当神经元最初通过快速短暂（400 - 800毫秒）流动暴露于ACh（50微摩尔），并在停止灌注后持续暴露时，诱导电流上升至峰值，然后在ACh存在的情况下衰减。然而，当恢复细胞周围相同ACh溶液的快速流动时，电流再次增加。当用卡巴胆碱替代ACh或使用极高浓度（10毫摩尔）的ACh时，未观察到灌注恢复导致的电流增加。此外，电流的这种增加被抗AChE药物依酚氯铵以剂量依赖性方式阻断。这些结果表明，乙酰胆碱酯酶（AChE）在膜表面导致ACh局部消耗，在该膜表面ACh的水解速率超过ACh从本体溶液扩散的速率，而灌注恢复导致的电流增加是由于膜表面ACh浓度的恢复。这种AChE活性的电生理指标可能是一种用于通过非生化手段研究AChE的有用工具。
