Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins

Abstract

The trafficking of transient receptor potential (TRP) channels to the plasma membrane and the release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) are implicated in some aspects of chronic migraines. These exocytotic processes are inhibited by cleavage of SNAREs with botulinum neurotoxins (BoNTs); moreover, type A toxin (/A) clinically reduces the frequency and severity of migraine attacks but not in all patients for unknown reasons. Herein, neonatal rat TGNs were stimulated with allyl isothiocyanate (AITC), a TRPA1 agonist, and dose relationships were established to link the resultant exocytosis of CGRP with Ca2+ influx. The CGRP release, quantified by ELISA, was best fit by a two-site model (EC50 of 6 and 93 µM) that correlates with elevations in intracellular Ca2+ [Ca2+]i revealed by time-lapse confocal microscopy of fluo-4-acetoxymethyl ester (Fluo-4 AM) loaded cells. These signals were all blocked by two TRPA1 antagonists, HC-030031 and A967079. At low [AITC], [Ca2+]i was limited because of desensitisation to the agonist but rose for concentrations > 0.1 mM due to a deduced non-desensitising second phase of Ca2+ influx. A recombinant BoNT chimera (/DA), which cleaves VAMP1/2/3, inhibited AITC-elicited CGRP release to a greater extent than SNAP-25-cleaving BoNT/A. /DA also proved more efficacious against CGRP efflux evoked by a TRPV1 agonist, capsaicin. Nerve growth factor (NGF), a pain-inducing sensitiser of TGNs, enhanced the CGRP exocytosis induced by low [AITC] only. Both toxins blocked NGF-induced neuropeptide secretion and its enhancement of the response to AITC. In conclusion, NGF sensitisation of sensory neurons involves TRPA1, elevated Ca2+ influx, and CGRP exocytosis, mediated by VAMP1/2/3 and SNAP-25 which can be attenuated by the BoNTs.

Spatial and temporal coordination of Duox/TrpA1/Dh31 and IMD pathways is required for the efficient elimination of pathogenic bacteria in the intestine ofDrosophilalarvae

“…The activation of TrpA1, potentially leading to the release of Dh31, ((Belinskaia et al, 2023; Kondo et al, 2010; Kunst et al, 2014) could be a consequence of ROS production in the host larval midgut in response to Ecc or Bt . The larval intestine comprises two main cell populations: enterocytes (ECs) and enteroendocrine cells (EECs).…”

Combination of anti-CGRP/CGRP-R mAbs with onabotulinumtoxin A as a novel therapeutic approach for refractory chronic migraine: a retrospective study of real-world clinical evidence and a protocol for a double-blind, randomized clinical trial to establish the efficacy and safety

“…As an alternative to mAbs targeting CGRP signalling, onabotulinumtoxin A has been approved for use in chronic migraine since 2010 ( Simpson et al, 2016 ). It proteolyses SNAP-25, one of the proteins required for the membrane fusion reaction that mediates the exocytosis of CGRP, as well as other neuropeptides and various neurotransmitters ( Meng et al, 2009 ; Cernuda-Morollón et al, 2015 ; Belinskaia et al, 2023 ). Among these neurotransmitters, it is possible to find acetylcholine, glutamate, pituitary adenylate cyclase activating peptide 38 (PACAP 38) and substance P, inhibition of the trafficking of transient receptor potential cation channel subfamily V member 1 [TRPV1], transient receptor potential cation channel subfamily A member 1 [TRPA1] and purinergic receptor P2X ligand-gated ion channel 3 [P2X3] ( Devesa et al, 2014 ; Meng et al, 2016 ) is also implicated in the mechanism of action of onabotulinumtoxin A ( Burstein et al, 2020 ).…”