title: An improved, high-efficiency assay for assessing serum anticholinergic activity using cultured cells stably expressing M1 receptors.
abstract: 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 of3H-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 [3H]quinuclidinyl benzilate ([3H]QNB) or [3H]N-methyl-scopolamine ([3H]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.
authors.affiliation: 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. Electronic address: jose.nobrega@camh.ca.
authors.forename: José N
authors.initials: JN
authors.lastname: Nobrega
authors.affiliation: Behavioural Neurobiology Laboratory, Research Imaging Centre, Centre for Addiction and Mental Health, Toronto, ON, Canada.
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Raymond
authors.affiliation: Department of Psychiatry, University of Toronto, Toronto, ON, Canada; Institute of Medical Sciences, University of Toronto, Canada; Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Pollock
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D018680:Cholinergic Antagonists / Q000097:blood* / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D066298:In Vitro Techniques; D018727:Muscarinic Antagonists / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D043643:Receptor, Muscarinic M1 / Q000096:biosynthesis / Q000187:drug effects*; D015203:Reproducibility of Results
chemical_list: D001798:Blood Proteins; D018680:Cholinergic Antagonists; D018727:Muscarinic Antagonists; D043643:Receptor, Muscarinic M1; D011813:Quinuclidinyl Benzilate
keywords: Anticholinergic burden; M1 receptor; M1WT3 cell line; SAA; [(3)H]NMS; [(3)H]QNB