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

Abstract

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 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.

A review for cell-based screening methods in drug discovery

“…In order to reduce the response time, the monitoring of its first activation step can be treated as alternative approach by using fluorescence and bioluminescence resonance energy transfer (Michelini et al 2010 ). Cell-based assays are used to identify the best drug candidate (Capula et al 2019 ), measure proliferation (Adan et al 2016 ), toxicity (Li et al 2006 ), motility (Sanookpan et al 2021 ), analyze cell signaling pathways (Pathe-Neuschafer-Rube et al 2021 ), and changes in morphology (Rajalingham 2016 ). Among the cell-based assays, 2D versus 3D culture might also contribute to the results obtained.…”