Dataset Information

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

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 (?-conotoxins (?-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 ?-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.

SUBMITTER: Marquart LA

PROVIDER: S-EPMC6949981 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Marquart Leanna A LA Turner Matthew W MW McDougal Owen M OM

Toxins 20191120 12

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 ...[more]

PMID: 31757080

Similar Datasets

Project description:IntroductionCigarette smoking continues to be one of the most important behavioral causes of morbidity and mortality in the world. Varenicline, an α4β2 nicotinic acetylcholine receptor (nAChR) partial agonist, has been shown to increase smoking quit rates compared with nicotine-based products. This human laboratory, double-blind, placebo-controlled study examined varenicline and placebo effects on α4β2-nAChRs occupancy, nicotine-induced change in [11C]raclopride non-displaceable binding potential (BPND), and behavioral measures of cigarette smoking, nicotine craving, and withdrawal.MethodsCurrent nicotine dependent daily smokers (N = 17) were randomized to varenicline 1 mg twice daily or placebo for 13 days. Using positron emission tomography), we characterized α4β2-nAChRs occupancy using [18F]AZAN and dopamine receptor binding using [11C]raclopride as well as behavioral measures of cigarettes smoked, craving, and nicotine withdrawal.ResultsVarenicline compared with placebo resulted in significant reductions in [18F]AZAN BPND in multiple brain regions including thalamus, midbrain, putamen, and ventral striatum. Following administration of a controlled-dose nicotine cigarette, dopamine release was significantly suppressed in the ventral striatum in the varenicline-treated compared with the placebo group. There was a significant relationship between α4β2-nAChRs BPND measured in thalamus during the [18F]AZAN scan and nicotine-induced change in raclopride BPND in the ventral striatum.ConclusionThis is the first human study to demonstrate a direct relationship between the extent of varenicline occupancy of α4β2-nAChRs and the magnitude of dopamine release following nicotine use.ImplicationsIt has remained unclear how nicotinic receptor blockade through partial agonist medications such as varenicline promotes smoking cessation. One hypothesized mechanism is downstream dampening of the mesolimbic reward dopamine system. For the first time in human smokers, we observed a direct relationship between the extent of varenicline blockade of α4β2-nACh nicotinic receptors and the magnitude of dopamine release following smoking. This has mechanistic and therapeutic implications for improving smoking cessation interventions.

Project description:ObjectiveThe objective of this study is to evaluate the expression of different nicotinic acetylcholine receptors (nAChRs), programmed death ligand-1 (PD-L1), and dopamine receptor D2 (DRD2) as prognostic factors in lung cancer and any correlation among them. Since all of the above genes are typically upregulated in response to smoking, we hypothesized that a correlation might exist between DRD2, PD-L1, and nAChR expression in NSCLC patients with a smoking history and a prediction model may be developed to assess the clinical outcome.MethodsWe retrospectively analyzed samples from 46 patients with primary lung adenocarcinoma who underwent surgical resection at Mayo Clinic Rochester from June 2000 to October 2008. The expression of PD-L1, DRD2, CHRNA5, CHRNA7, and CHRNA9 were analyzed by quantitative PCR and correlated amongst themselves and with age, stage and grade, smoking status, overall survival (OS), and relapse-free survival (RFS).ResultsOnly PD-L1 showed a statistically significant increase in expression in patients older than 65. All the above genes showed higher expression in stage IIIB than IIIA, but none reached statistical significance. Interestingly, we did not observe significant differences among never, former, and current smokers, but patients with pack years greater than 30 showed significantly higher expression of CHRNA9. We observed a strong positive correlation between PD-L1/DRD2, PD-L1/CHRNA5, and CHRNA5/CHRNA7 and a weak positive correlation between DRD2/CHRNA5 and DRD2/CHRNA7. Older age was independently associated with poor OS, whereas lower CHRNA7 expression was independently associated with better OS.ConclusionsWe observed strong positive correlations among PD-L1, DRD2, and some of the nAChRs. We investigated their prognostic significance in lung cancer patients and found CHRNA7 to be an independent prognostic factor. Overall, the results obtained from this preliminary study warrant a large cohort-based analysis that may ultimately lead to potential patient-specific stratification biomarkers predicting cancer-treatment outcomes.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets