Interactions of endocannabinoids and nicotinic receptors

nAChR在从R. Lucas（Phoenix，AZ）. 在使用100%串联电阻补偿的全电池电压钳位条件下对电池进行了研究。 ACh的快速灌流产生的电流迅速上升到一个峰值（约80毫秒）和脱敏作为一个双指数函数的时间在持续存在的ACh（5秒）。 这种衰减被建模为两个连续的脱敏状态，前向速率常数k1和k2和后向速率常数k-1和k-2。 速率常数k1随ACh反应的增加而增加，符合序贯机制，而其他参数保持不变。 当将AEA灌流到SH-EP 1细胞上时，对ACh的峰值反应减弱，并随着时间的推移持续降低至约50分钟。对于AEA等亲脂性化合物来说，这种缓慢的时间过程是预期的。 恢复也需要几十分钟，但通过使用脂质清除剂牛血清白蛋白加速到10分钟以下。 更引人注目的是，AEA增加K1成正比的AEA浓度没有饱和的证据。 增加k1，高达25倍，在2微摩尔AEA浓度，产生的尖峰样反应乙酰胆碱。 模拟结果表明，非常迅速进入脱敏引起的AEA可以占约80%的峰值幅度的下降，因为响应更接近扩展ACh前端脱敏之前，在进一步的扩散距离的响应求和。 其余的减少被假定发生通过脱敏的非活性状态的乙酰胆碱受体。 荧光Ca++指示剂fluro-3的共聚焦显微镜显示ACh引起Ca++浓度的瞬时增加。 为了测试亲脂性AEA是否通过细胞内Ca++依赖性机制加速脱敏，我们首先表明通过膜片移液管透析到细胞中的AEA对ACh电流没有影响。 接下来，我们在贴片移液管中使用快速作用的Ca++螯合剂BAPTA，并表明它不能对抗AEA的作用。 为了探索AEA作用的药理学特异性，我们发现大麻素激动剂δ-9-四氢大麻酚（THC）对nAChR没有任何影响。 CB_1受体拮抗剂SR-141716 A与AEA合用时，虽能轻微拮抗K_1的增加，但不能拮抗AEA对K_1峰幅的抑制。 最后，在AEA效应的结构要求的初步测试中，我们测试了八氢类似物，花生酰乙醇酰胺（H-8-AEA）。 尽管与AEA的结构相似，但1微摩尔浓度的H-8-AEA在nAChR处没有活性。 我们的结论是，AEA，在生理相关的浓度，直接阻断乙酰胆碱反应的nAChR主要是通过大大增加其脱敏率。 由于该速率随AEA浓度线性增加，我们怀疑它通过膜的分级改变起作用。 实验来测试这一假设的计划。

The nAChR was stably expressed in the cell line SH-EP1, obtained from R. Lucas (Phoenix, AZ). The cells were studied under whole cell voltage clamp conditions using 100% series resistance compensation. Rapid superfusion of ACh generated currents that rose rapidly to a peak (in about 80 ms) and desensitized as a double exponential function of time in the continued presence of ACh (5 s). This decay was modeled as two sequential desensitized states with forward rate constants k1 and k2 and backward rate constants k-1 and k-2. The rate constant k1 increased with the ACh response, in conformity with the sequential mechanism, while the others remained unchanged. When AEA was superfused onto the SH-EP1 cells, the peak response to ACh was diminished and continued to decrease with time up to about 50 min. This slow time course is expected for a lipophilic compound such as AEA. The recovery also required tens of minutes, but was accelerated to under 10 min by using the lipid scavenger bovine serum albumin. More strikingly, AEA increased k1 in direct proportion to the AEA concentration without evidence of saturation. The increase in k1, up to 25-fold at 2 micromolar AEA concentration, generated the spike-like responses to ACh. Simulations showed that the very rapid entry into desensitization caused by AEA could account for about 80% of the decrease in the peak amplitude because responses closer to the spreading ACh front desensitized before summating with the responses at a further diffusional distance. The rest of the decrease was postulated to occur through desensitization of the inactive state of the AChR. Confocal microscopy of the fluorescent Ca++ indicator fluro-3 showed that ACh caused transient increases in Ca++ concentration. To test if lipophilic AEA accelerated desensitization through an intracellular Ca++ dependent mechanism, we first showed that AEA dialyzed into the cell through the patch pipette had no effect on ACh currents. Next we used the fast acting Ca++ chelator BAPTA in the patch pipette and showed that it failed to oppose the AEA effects. To explore pharmacological specificity of the AEA effects, we showed that the cannabinoid agonist delta-9-tetrahydrocannabinol (THC) had no effects whatever at the nAChR. The CB1 antagonist SR-141716A, co-administered with AEA, failed to antagonize the AEA depression of peak amplitude, though it did antagonize the increase in k1 slightly. Finally, in a preliminary test of the structural requirements of the AEA effect, we tested the octahydro analogue, arachidoyl ethanolamide (H-8-AEA). Despite the structural similarity to AEA, H-8-AEA at 1 micromolar concentration was devoid of activity at the nAChR. We conclude that AEA, at physiologically relevant concentrations, directly blocks ACh responses at the nAChR primarily by greatly increasing its rate of desensitization. As this rate increased linearly with the AEA concentration, we suspect that it acts through graded alterations of the membrane. Experiments to test this hypothesis are planned.