Abstract
A pheochromocytoma of the rat adrenal medulla derived (a.k.a. PC12) cell-based assay for dopamine measurement by luminescence detection was customized for the qualitative evaluation of agonists and antagonists of nicotinic acetylcholine receptors (nAChRs). The assay mechanism begins with ligand binding to transmembrane nAChRs, altering ion flow into the cell and inducing dopamine release from the cell. Following release, dopamine is oxidized by monoamine oxidase generating hydrogen peroxide that catalyzes a chemiluminescence reaction involving luminol and horseradish peroxidase, thus producing a detectable response. Results are presented for the action of nAChR agonists (acetylcholine, nicotine, and cytisine), and antagonists (α-conotoxins (α-CTxs) MII, ImI, LvIA, and PeIA) that demonstrate a luminescence response correlating to the increase or decrease of dopamine release. A survey of cell growth and treatment conditions, including nerve growth factor, nicotine, ethanol, and temperature, led to optimal assay requirements to achieve maximal signal intensity and consistent response to ligand treatment. It was determined that PC12 cells treated with a combination of nerve growth factor and nicotine, and incubated at 37 °C, provided favorable results for a reduction in luminescence signal upon treatment of cells with α-CTxs. The PC12 assay is intended for use as a fast, efficient, and economic qualitative method to assess the bioactivity of molecules that act on nAChRs, in which testing of ligand–nAChR binding hypotheses and computational predictions can be validated. As a screening method for nAChR bioactivity, lead compounds can be assessed for their likelihood of exhibiting desired bioactivity prior to being subjected to more complex quantitative methods, such as electrophysiology or live animal studies.
Highlights
Nicotinic acetylcholine receptors are pentameric transmembrane proteins present throughout the nervous system, brain, and muscle
Studies utilizing acetylcholine binding proteins (AChBPs) allow assessment of ligand binding, but not the ion flow across a membrane required to trigger signaling mechanisms within a cell that results in dopamine release, because the AChBP lacks a transmembrane component
Stimulation and selective Nicotinic acetylcholine receptors (nAChRs) suppression in order to provide the clearest sensitivity of the α-CTx effect on nAChR-induced dopamine release
Summary
Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane proteins present throughout the nervous system, brain, and muscle. Cell-based assays using fluorescent indicators, such as the fluorometric imaging plate reader(FLIPR) fluorescent membrane potential assay or the Flou-4AM calcium indicator assay, are being used with increasing frequency to study nAChR activation [35]. Each of these strategies to study nAChR bioactivity are challenging to implement in their own unique ways. Studies utilizing AChBPs allow assessment of ligand binding, but not the ion flow across a membrane required to trigger signaling mechanisms within a cell that results in dopamine release, because the AChBP lacks a transmembrane component. Time and cost efficient, and reliable PC12 cell method to qualitatively assess the ligand effect on nicotinic acetylcholine receptor-mediated control of dopamine release is presented
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