Biochemistry Of Ligand Gated Ion Channels Important To D

尼古丁乙酰胆碱受体（nAChR），尼古丁作用的配体门控离子通道，在尼古丁滥用中起着重要作用，了解其神经生物学可能是开发更合理的尼古丁滥用治疗方法的核心。受体-配体相互作用的体外表征是理解配体体内作用的重要步骤（即，用于解释来自受体的功能研究的结果）。为了分析在生理条件下的配体-受体相互作用，开发了在37 ° C下的竞争受体测定。本试验使用[I-125]-5-碘-A-85380（5IA），由神经影像学研究分支的工作人员开发并表征为α 4 β 2 nAChR的高亲和力、选择性放射性配体，以测定23 ℃和37 ℃下28种烟碱激动剂和拮抗剂（9种已建立，8种以前和11种最近在我们的化学实验室开发）的亲和力。这些配体的Kd值范围为0.01至25，000 nM。将孵育温度从23 ℃增加到37 ℃导致观察到的Kd值可变增加（从1.0倍增加到3.3倍），并对受体-配体相互作用的动力学产生可变影响。例如，5IA的受体-配体复合物的结合和解离在37 ℃下比在23 ℃下快约10倍，但[H-3]-野靛碱结合动力学速率仅增加3.5倍。温度对受体结合特性的影响的这种可变性表明，在生理温度下的配体-受体相互作用的表征可以更好地预测配体的体内特性，并且在解释α 4 β 2 nAChRs的功能研究结果中可能是重要的。Abbott实验室开发了Me-p-PVC（5-（2-（4-吡啶基）乙烯基）-6-氯-3-（1-甲基-2-（S）-吡咯烷甲氧基）吡啶），该实验室的先前研究表明，该化合物对α 4 β 2烟碱乙酰胆碱受体（nAChR）具有高亲和力，并减弱了尼古丁诱导的86 Rb从IMR-32细胞（含有α 3 β 4 nAChR）的流出。用Me-p-PVC进行的体外结合研究表明，其对α 4 β 2 nAChR的亲和力超过对α 3 β 4受体亚型的亲和力3，000倍（Kd约为1.5）。0.025和90 nM）。 大量轶事证据表明，尼古丁可能是非法使用毒品的入门药物。因此，我们利用药物奖励条件性位置偏好（CPP）的动物行为模型来检验尼古丁对不同类别的滥用药物（特别是阿片类药物、兴奋剂和大麻）产生行为交叉致敏的假设。在大鼠中进行的这些实验表明，尼古丁预处理可在停用尼古丁后至少3至5天内增强苯丙胺的奖励效应，该效应在19天内消失。潜在的机制涉及α 4 β 2 nAChR，因为竞争性α 4 β 2拮抗剂二氢β赤藓定有效地阻断了尼古丁诱导的交叉致敏的发展。有趣的是，在大鼠中，α 7烟碱拮抗剂methylylycaconitine在不阻断尼古丁自我给药的剂量下也可拮抗交叉致敏作用。本研究和已发表的报告明确表明，尼古丁对阿片类药物和精神兴奋剂的奖励作用产生交叉致敏作用，并使用CPP进行测量。对这些药物的奖励作用的交叉致敏的发展涉及尼古丁与α 4 β 2和α 7 nAChR的相互作用，这与尼古丁自我给药不同，尼古丁自我给药主要由α 4 β 2受体介导。

Nicotinic acetylcholine receptors (nAChRs), the ligand gated ion channels at which nicotine acts, play an important role in nicotine abuse and understanding its neurobiology may be central to developing more rational approaches for nicotine abuse treatments. The in vitro characterization of the receptor-ligand interaction is an important step in understanding the action of a ligand in vivo (i.e., for interpretation of the results from the functional studies of receptors). To analyze the ligand-receptor interaction at physiological conditions, a competition receptor assay at 37 degrees C was developed. This assay used [I-125]-5-iodo-A-85380 (5IA), which was developed and characterized by staff of the Neuroimaging Research Branch as a high affinity, selective radioligand for alpha4 beta2 nAChRs, to determine the affinities of 28 nicotinic agonists and antagonists (9 established, 8 previously and 11 recently developed in our chemistry lab) at 23 degrees C and 37 degrees C. The Kd values for these ligands ranged from 0.01 to 25,000 nM. Increasing incubation temperature from 23 to 37 degrees C resulted in variable increases in the observed Kd values (from 1.0 to 3.3-fold) and variable effects on the kinetics of receptor-ligand interaction. For example, the association and dissociation of the receptor-ligand complex for 5IA were approximately 10 times faster at 37 degrees C than that at 23 degrees C, but the rate of [H-3]-cytisine binding kinetics increased only 3.5 fold. This variability in effects of temperature on receptor binding properties suggests that characterization of ligand-receptor interaction at physiological temperature may better predict the in vivo properties of the ligands and may be important in the interpretation of results from the functional studies of alpha4 beta2 nAChRs. Previous studies from Abbott Laboratories, where Me-p-PVC (5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-methyl-2-(S)-pyrrolidinylmethoxy)pyridine) was developed, demonstrated that this compound had high affinity for alpha4beta2 nicotinic acetylcholine receptors (nAChRs) and attenuated nicotine-induced efflux of 86Rb from IMR-32 cells, contained alpha3beta4 nAChRs. In vitro binding studies with Me-p-PVC showed that its affinity for alpha4beta2 nAChRs exceeded that for the alpha3beta4 receptor subtype by a factor of 3,000 (Kd ca. 0.025 and 90 nM, respectively). Substantial anecdotal evidence suggests that nicotine may function as a gateway drug to illicit drug use. As such we utilized an animal behavorial model of drug rewards conditioned place preference (CPP) to test the hypothesis that nicotine produces behavioral cross-sensitization to different classes of abused drugs, specifically opiates, stimulants and marihuana. These experiments in rats demonstrated that nicotine pretreatment enhances the rewarding effects of amphetamine for at least 3 to 5 days following the cessation of nicotine, with this effect dissipating within 19 days. The underlying mechanism involves alpha4beta2 nAChRs as the competitive alpha4beta2 antagonist dihydro beta erythroidine effectively blocked the development of nicotine-induced cross sensitization. Interestingly, the alpha7 nicotinic antagonist methyllycaconitine also antagonized cross-sensitization at doses that do not block nicotine self-administration in rats. This study and published report have clearly demonstrated that nicotine produces cross-sensitization to the rewarding effects of both opiates and psychostimulants measured with CPP. The development of cross-sensitization to the rewarding effects of these drugs involves the interaction of nicotine with both alpha4beta2 and alpha7 nAChRs, which differs from nicotine self-administration, which is primarily mediated by alpha4beta2 receptors.