Schweri M M, Thurkauf A, Mattson M V, Rice K C
Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia.
J Pharmacol Exp Ther. 1992 Jun;261(3):936-42.
Fourphit, a phencyclidine derivative containing an isothiocyanate substitution at the 4-position of the piperidine ring, inhibits the binding of the radiolabeled psychomotor stimulant, [3H]methylphenidate, to sites on the dopamine transport complex in membranes prepared from the crude synaptosomal fraction of rat striatal tissue with an IC50 of 7.1 microM. The inhibition caused by Fourphit is irreversible and is associated with a decrease in the Bmax, but not the KD, of [3H]methylphenidate binding. Pretreatment with saturating concentrations of unlabeled methylphenidate effected a modest (but statistically significant) protection of the stimulant binding site from inactivation by Fourphit, indicating that the acylating phencyclidine derivative may act directly at this site. Preincubation with Fourphit rather than vehicle did not alter the dissociation rate of [3H]methylphenidate when measured in the presence of excess amfonelic acid, nor was any difference detected in the off-rate of [3H]methylphenidate when excess Fourphit was substituted for excess unlabeled methylphenidate as the displacing agent. This lack of effect on the dissociation kinetics of [3H]methylphenidate provides further evidence that Fourphit does not act allosterically at the methylphenidate binding site. Unlike Metaphit (an isomer of Fourphit containing the isothiocyanate moiety at the meta position of the aromatic ring), Fourphit can discriminate between the methylphenidate binding site and the phencyclidine binding site associated with the N-methyl-D-aspartate receptor: Metaphit irreversibly inactivates both binding sites, whereas Fourphit binds reversibly to the phencyclidine binding site. The data suggest that Fourphit may be useful as a relatively selective affinity label for the site on the dopamine transport complex recognized by methylphenidate and other psychomotor stimulants.
Fourphit是一种哌啶环4位含有异硫氰酸酯取代基的苯环己哌啶衍生物，它能抑制放射性标记的精神运动兴奋剂[3H]哌甲酯与大鼠纹状体组织粗突触体部分制备的膜中多巴胺转运复合物位点的结合，IC50为7.1微摩尔。Fourphit引起的抑制是不可逆的，且与[3H]哌甲酯结合的Bmax降低有关，但与KD无关。用饱和浓度的未标记哌甲酯预处理对兴奋剂结合位点免受Fourphit失活有适度（但具有统计学意义）的保护作用，表明酰化苯环己哌啶衍生物可能直接作用于该位点。在存在过量氨苯酸的情况下测量时，用Fourphit而非溶剂预孵育不会改变[3H]哌甲酯的解离速率，当用过量Fourphit替代过量未标记哌甲酯作为置换剂时，[3H]哌甲酯的解离速率也未检测到任何差异。对[3H]哌甲酯解离动力学缺乏影响提供了进一步证据，表明Fourphit不在哌甲酯结合位点起变构作用。与Metaphit（Fourphit的一种异构体，其异硫氰酸酯部分位于芳香环的间位）不同，Fourphit可以区分哌甲酯结合位点和与N-甲基-D-天冬氨酸受体相关的苯环己哌啶结合位点：Metaphit不可逆地使两个结合位点失活，而Fourphit可逆地结合到苯环己哌啶结合位点。数据表明，Fourphit可能作为一种相对选择性的亲和标记物，用于标记哌甲酯和其他精神运动兴奋剂识别的多巴胺转运复合物位点。
