Cell-based reporter release assay to determine the activity of calcium-dependent neurotoxins and neuroactive pharmaceuticals
- The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNAThe suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.…
Verfasserangaben: | Andrea Pathe-Neuschäfer-RubeGND, Frank Neuschäfer-RubeGND, Gerhard Paul PüschelORCiDGND |
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DOI: | https://doi.org/10.3390/toxins13040247 |
ISSN: | 2072-6651 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/33808507 |
Titel des übergeordneten Werks (Englisch): | Toxins / Molecular Diversity Preservation International (MDPI) |
Verlag: | MDPI |
Verlagsort: | Basel |
Publikationstyp: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Erstveröffentlichung: | 17.02.2021 |
Erscheinungsjahr: | 2021 |
Datum der Freischaltung: | 14.04.2021 |
Freies Schlagwort / Tag: | VGCC; cell-based assay; muscarinic acetylcholine receptor; neurotoxins; voltage-dependent calcium channels |
Band: | 13 |
Ausgabe: | 4 |
Aufsatznummer: | 247 |
Seitenanzahl: | 13 |
Fördernde Institution: | Universität Potsdam |
Fördernummer: | PA 2021_026 |
Organisationseinheiten: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft |
DDC-Klassifikation: | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
Peer Review: | Referiert |
Fördermittelquelle: | Publikationsfonds der Universität Potsdam |
Publikationsweg: | Open Access / Gold Open-Access |
Lizenz (Deutsch): | CC-BY - Namensnennung 4.0 International |
Externe Anmerkung: | Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 1139 |