Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals.

Szczegóły
Abstrakt

Tytuł:: Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals.
Autorzy:: Pathe-Neuschäfer-Rube A; Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, 14469 Potsdam, Germany.
Neuschäfer-Rube F; Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, 14469 Potsdam, Germany.
Püschel GP; Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, 14469 Potsdam, Germany.
Źródło:: Toxins [Toxins (Basel)] 2021 Mar 30; Vol. 13 (4). Date of Electronic Publication: 2021 Mar 30.
Typ publikacji:: Journal Article; Research Support, Non-U.S. Gov't
Język:: English
Imprint Name(s):: Original Publication: Basel : MDPI
MeSH Terms:: Genes, Reporter*
Calcium/*metabolism
Calcium Channel Blockers/*pharmacology
Calcium Channels/*drug effects
Muscarinic Agonists/*pharmacology
Muscarinic Antagonists/*pharmacology
Neuroblastoma/*metabolism
Neurotoxins/*pharmacology
Secretory Vesicles/*drug effects
Calcium Channels/genetics ; Calcium Channels/metabolism ; Calcium Signaling/drug effects ; Cell Line, Tumor ; Humans ; Luciferases/genetics ; Luciferases/metabolism ; Neuroblastoma/genetics ; Neuroblastoma/pathology ; Pro-Opiomelanocortin/genetics ; Pro-Opiomelanocortin/metabolism ; Secretory Vesicles/genetics ; Secretory Vesicles/metabolism ; Spider Venoms/pharmacology
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Grant Information:: 013L0132A Bundesministerium für Bildung und Forschung; 85000915 European Regional Development Fund
Contributed Indexing:: Keywords: VGCC; cell-based assay; muscarinic acetylcholine receptor; neurotoxins; voltage-dependent calcium channels
Substance Nomenclature:: 0 (Calcium Channel Blockers)
0 (Calcium Channels)
0 (Muscarinic Agonists)
0 (Muscarinic Antagonists)
0 (Neurotoxins)
0 (Spider Venoms)
65988-34-3 (alpha-latrotoxin)
66796-54-1 (Pro-Opiomelanocortin)
EC 1.13.12.- (Luciferases)
SY7Q814VUP (Calcium)
Entry Date(s):: Date Created: 20210403 Date Completed: 20210629 Latest Revision: 20210629
Update Code:: 20240105
PubMed Central ID:: PMC8066854
DOI:: 10.3390/toxins13040247
PMID:: 33808507
: Czasopismo naukowe

Pełny tekst

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 Ca 2+ -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 Ca 2+ -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.

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