Interactions of endocannabinoids and nicotinic receptors

在从R.Lucas(菲尼克斯，亚利桑那州)获得的SH-EP1细胞中，nAChR稳定表达。在全电池电压钳位条件下，采用100%串联电阻补偿对电池进行了研究。ACh的快速灌流产生的电流迅速上升到一个峰值(约80ms)，并在ACh的持续存在下作为时间的双指数函数减敏(5 S)。这种衰变被模拟为具有正向速率常数k1和k2以及反向速率常数k-1和k-2的两个连续的钝化态。速率常数K1随ACh反应的增加而增大，符合序贯机制，其余各参数不变。 AEA在SH-EP1细胞上灌流后，对ACh的峰值反应减弱，并随着时间的延长而下降，直到约50min。对于像AEA这样的亲脂化合物来说，这种缓慢的时间进程是可望的。恢复也需要数十分钟，但通过使用脂质清除剂牛血清白蛋白，恢复速度加快到10分钟以下。更引人注目的是，AEA增加了K1，与AEA浓度成正比，而没有饱和迹象。在2微摩尔的AEA浓度下，K1增加了25倍，产生了对ACh的棘波样反应。模拟表明，AEA引起的非常迅速的进入减敏可以解释大约80%的峰值幅度的下降，因为靠近扩散ACh波阵面的反应在与更远的扩散距离的反应相加之前减敏。其余的下降被认为是通过AChR的非活动状态的脱敏而发生的。 荧光钙指示剂Fluro-3的共聚焦显微镜显示，ACh引起一过性钙离子浓度升高。为了测试亲脂性AEA是否通过细胞内钙依赖机制加速脱敏，我们首先证明AEA通过膜片移液管透析到细胞内对ACh电流没有影响。接下来，我们在贴片移液管中使用了快速作用的钙离子螯合剂BAPTA，结果表明它不能对抗AEA的作用。 为了探索AEA效应的药理学特异性，我们发现大麻素激动剂Delta-9-四氢大麻酚(THC)在nAChR上没有任何作用。CB1拮抗剂SR-141716A与AEA合用，虽能轻微拮抗K1的升高，但不能拮抗AEA的波幅峰值降低。最后，在对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四烯基乙醇胺(H-8-AEA)。尽管H-8-AEA与AEA结构相似，但1微摩尔浓度的H-8-AEA在nAChR上没有活性。 我们得出结论，在生理相关的浓度下，AEA主要通过显著增加其脱敏速度来直接阻断nAChR的ACh反应。由于这一比率随着AEA浓度的线性增加，我们怀疑它是通过膜的渐进性改变而起作用的。检验这一假说的实验正在计划中。

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.