Nobrega José N, Raymond Roger J, Pollock Bruce G
Behavioural Neurobiology Laboratory, Research Imaging Centre, Centre for Addiction and Mental Health, Toronto, ON, Canada; Department of Psychiatry, University of Toronto, Toronto, ON, Canada; Departments of Psychology and Pharmacology & Toxicology, University of Toronto, Canada; Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.
Behavioural Neurobiology Laboratory, Research Imaging Centre, Centre for Addiction and Mental Health, Toronto, ON, Canada.
J Pharmacol Toxicol Methods. 2017 Jul;86:28-33. doi: 10.1016/j.vascn.2017.03.001. Epub 2017 Mar 6.
Assessments of total anticholinergic activity (SAA) in serum are of considerable interest for its potential involvement in cognitive impairment associated with polydrug states in the elderly and other populations. Such estimations have been based on the displacement of radioligand binding in rat brain tissues. The validity of such measurements has been questioned, as a potentially distorting effect of large serum proteins was identified. We sought to develop a modified assay that would be more efficient and free of this potential confound. Cultured CHO cells stably expressing M1 receptors M1WT3 were used. Binding of H-radioligands was conducted in 96-well plates and tested in serum containing known amounts of anticholinergic medications. Effects of endogenous serum proteins were assessed by pre-assay filtration and also by deproteinization with perchloric acid (PCA). Binding of [H]quinuclidinyl benzilate ([H]QNB) or [H]N-methyl-scopolamine ([H]NMS) to M1WT3 cells proved reliable and equally sensitive to varying concentrations of anticholinergic agents. In agreement with previous findings (Cox, Kwatra, Shetty, & Kwatra, 2009), filtration of proteins heavier than 50kDa essentially reduced SAA values to zero. In contrast, PCA preserved more than 70% of the binding seen untreated cell membranes. Cell-based assays also showed significant signal increases compared to the conventional rat brain-based protocol. Further advantages of the cell-based protocol described here include increased sensitivity and reliability, smaller amounts of radioligand needed, and higher throughput. PCA pretreatment eliminates potential artifacts attributable to serum proteins. This step, together with improvements in efficiency, should contribute significantly to the usefulness of the assay.
血清中总抗胆碱能活性（SAA）的评估备受关注，因为它可能与老年人及其他人群中多药状态相关的认知障碍有关。此类评估基于大鼠脑组织中放射性配体结合的置换情况。由于发现了大血清蛋白的潜在干扰作用，此类测量的有效性受到了质疑。我们试图开发一种改进的检测方法，该方法将更高效且无此潜在干扰因素。使用稳定表达M1受体M1WT3的培养CHO细胞。在96孔板中进行H放射性配体的结合，并在含有已知量抗胆碱能药物的血清中进行测试。通过检测前过滤以及用高氯酸（PCA）进行脱蛋白来评估内源性血清蛋白的影响。[H]喹核醇基苯甲酸酯（[H]QNB）或[H]N-甲基东莨菪碱（[H]NMS）与M1WT3细胞的结合被证明是可靠的，并且对不同浓度的抗胆碱能药物同样敏感。与先前的研究结果一致（考克斯、夸特拉、谢蒂和夸特拉，2009年），过滤分子量大于50kDa的蛋白质基本上可将SAA值降至零。相比之下，PCA保留了未经处理细胞膜中超过70%的结合。与传统的基于大鼠脑的实验方案相比，基于细胞的检测方法也显示出显著的信号增加。此处所述基于细胞的实验方案的其他优点包括更高的灵敏度和可靠性、所需放射性配体的量更少以及更高的通量。PCA预处理消除了可归因于血清蛋白的潜在假象。这一步骤以及效率的提高应会显著提升该检测方法的实用性。
