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Article

Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors

1
Department of Chemistry and Biochemistry, Boise State University, Boise, ID 83725, USA
2
Biomolecular Sciences PhD Program, Boise State University, Boise, ID 83725, USA
*
Author to whom correspondence should be addressed.
Toxins 2019, 11(12), 682; https://doi.org/10.3390/toxins11120682
Received: 14 August 2019 / Revised: 14 November 2019 / Accepted: 18 November 2019 / Published: 20 November 2019
(This article belongs to the Special Issue Conotoxins: Characterization, Pharmacology, and Therapeutics)
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. View Full-Text
Keywords: PC12 cells; alpha-conotoxin; nicotinic acetylcholine receptor; dopamine; luminescence assay PC12 cells; alpha-conotoxin; nicotinic acetylcholine receptor; dopamine; luminescence assay
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MDPI and ACS Style

Marquart, L.A.; Turner, M.W.; McDougal, O.M. Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors. Toxins 2019, 11, 682. https://doi.org/10.3390/toxins11120682

AMA Style

Marquart LA, Turner MW, McDougal OM. Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors. Toxins. 2019; 11(12):682. https://doi.org/10.3390/toxins11120682

Chicago/Turabian Style

Marquart, Leanna A., Matthew W. Turner, and Owen M. McDougal. 2019. "Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors" Toxins 11, no. 12: 682. https://doi.org/10.3390/toxins11120682

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