Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors
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 bind...
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| Format: | Article |
| Language: | English |
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MDPI AG
2019-11-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/11/12/682 |
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| author | Leanna A. Marquart Matthew W. Turner Owen M. McDougal |
| author_facet | Leanna A. Marquart Matthew W. Turner Owen M. McDougal |
| author_sort | Leanna A. Marquart |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T20:52:20Z |
| format | Article |
| id | doaj.art-ea56a2a5eba54c98bfe451f5a84fca8d |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-04-11T20:52:20Z |
| publishDate | 2019-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-ea56a2a5eba54c98bfe451f5a84fca8d2022-12-22T04:03:49ZengMDPI AGToxins2072-66512019-11-01111268210.3390/toxins11120682toxins11120682Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine ReceptorsLeanna A. Marquart0Matthew W. Turner1Owen M. McDougal2Department of Chemistry and Biochemistry, Boise State University, Boise, ID 83725, USABiomolecular Sciences PhD Program, Boise State University, Boise, ID 83725, USABiomolecular Sciences PhD Program, Boise State University, Boise, ID 83725, USAA 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.https://www.mdpi.com/2072-6651/11/12/682pc12 cellsalpha-conotoxinnicotinic acetylcholine receptordopamineluminescence assay |
| spellingShingle | Leanna A. Marquart Matthew W. Turner Owen M. McDougal Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors Toxins pc12 cells alpha-conotoxin nicotinic acetylcholine receptor dopamine luminescence assay |
| title | Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors |
| title_full | Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors |
| title_fullStr | Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors |
| title_full_unstemmed | Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors |
| title_short | Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors |
| title_sort | qualitative assay to detect dopamine release by ligand action on nicotinic acetylcholine receptors |
| topic | pc12 cells alpha-conotoxin nicotinic acetylcholine receptor dopamine luminescence assay |
| url | https://www.mdpi.com/2072-6651/11/12/682 |
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