Clapham D E, Neher E
J Physiol. 1984 Feb;347:255-77. doi: 10.1113/jphysiol.1984.sp015065.
Patch-clamp techniques were used to examine the effect of substance P on acetylcholine-induced current in bovine chromaffin cells. Cells had been enzymatically isolated and kept in short-term culture. Experiments were performed at 22 degrees C. Under whole-cell voltage-clamp conditions substance P alone (2-10 microM) did not induce ionic currents. Acetylcholine (ACh, 20 microM) at -60 mV induced an inward current that desensitized in the continued presence of ACh. The time course of desensitization was somewhat variable from cell to cell. In most cases it could be fitted by a single exponential with time constant of 8-10 s. Substance P (2-50 microM) applied simultaneously with ACh induced what appeared to be an acceleration of the desensitization process. The time course in the presence of 10 microM-substance P (20 microM-ACh) was best fitted by the sum of two exponentials with time constants of 0.6 s and 5 s respectively. The effect was reversible. The recovery of ACh-induced current from desensitization was not affected by substance P. The time constant for recovery was approximately 7 s in the presence or absence of substance P. Single-channel records showed that the conductance of individual channels was not changed by substance P. The mean open time of single channels was shortened by substance P both at high (20 microM) and at low (0.5 microM) concentrations of ACh. The inverse mean open time varied linearly with substance P concentration. Single-channel responses appeared in bursts and clusters after almost complete desensitization at 20 microM-ACh, as was previously observed in frog skeletal muscle. Substance P dramatically reduced ACh current by increasing interburst intervals while decreasing burst duration and the number of openings per burst. We conclude that substance P inhibits ACh-induced depolarization of chromaffin cells either by increasing the rate of desensitization or by inducing channel blockade, which indirectly enhances desensitization. Possible models of desensitization in the absence and presence of substance P are discussed.
采用膜片钳技术研究了P物质对牛嗜铬细胞中乙酰胆碱诱导电流的影响。细胞经酶解分离并进行短期培养。实验在22℃下进行。在全细胞电压钳条件下，单独的P物质（2 - 10微摩尔）不诱导离子电流。-60 mV时，乙酰胆碱（ACh，20微摩尔）诱导内向电流，在持续存在ACh的情况下该电流会脱敏。脱敏的时间进程在细胞之间略有不同。在大多数情况下，它可以用单个指数拟合，时间常数为8 - 10秒。与ACh同时施加的P物质（2 - 50微摩尔）似乎诱导了脱敏过程的加速。在存在10微摩尔P物质（20微摩尔ACh）的情况下，时间进程最好用两个指数之和拟合，时间常数分别为0.6秒和5秒。该效应是可逆的。P物质不影响ACh诱导电流从脱敏状态的恢复。在有或没有P物质的情况下，恢复的时间常数约为7秒。单通道记录显示，单个通道的电导不受P物质的影响。在高（20微摩尔）和低（0.5微摩尔）浓度的ACh下，P物质均缩短了单通道的平均开放时间。平均开放时间的倒数与P物质浓度呈线性变化。在20微摩尔ACh几乎完全脱敏后，单通道反应以爆发和簇状出现，这与之前在青蛙骨骼肌中观察到的情况相同。P物质通过增加爆发间隔、减少爆发持续时间和每次爆发的开放次数，显著降低了ACh电流。我们得出结论，P物质通过增加脱敏速率或诱导通道阻断来抑制ACh诱导的嗜铬细胞去极化，这间接增强了脱敏作用。讨论了在不存在和存在P物质时脱敏的可能模型。
