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.
Highlights
Disorders in neurotransmitter release are key features of severe neuronal diseases like Parkinson, chronic pain, and depression [1,2,3]
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
Key Contribution: The current study provides evidence that a new assay is suitable to measure the activity of muscarinic acetylcholine agonists and antagonists, α-latrotoxin and inhibitors of voltagedependent calcium-channels
Summary
Disorders in neurotransmitter release are key features of severe neuronal diseases like Parkinson, chronic pain, and depression [1,2,3]. Neurotoxins and neuroactive pharmaceutical substances which affect neurotransmitter release are highly interesting tools in the treatment of neuronal diseases. Bacterial neurotoxins such as botulinum toxins and tetanus toxins block neurotransmitter release by the highly specific proteolytic inactivation of target snare proteins SNAP-25, syntaxin and synaptobrevin (VAMP), which are essential for the fusion of neurosecretory vesicles with the plasma membrane [4]. The key step in neurotransmitter release is the surge in the intracellular Ca2+ concentration that triggers the fusion of neurosecretory vesicles with the plasma membrane of the presynaptic cell [5,6,7]. Activation of Gq-coupled GPCR on the surface of the presynaptic cell will lead to activation of phospholipase C, the formation of second messenger IP3 and release of calcium from intracellular stores
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