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

• Published in: International journal of molecular sciences 2023-01, Vol.24 (2), p.1338
• Main Authors: Belinskaia, Mariia, Wang, Jiafu, Kaza, Seshu Kumar, Antoniazzi, Caren, Zurawski, Tomas, Dolly, J. Oliver, Lawrence, Gary W.
• Format: Article
• Language: English
• Subjects: Agonists; AITC; Allyl isothiocyanate; botulinum neurotoxins; Calcitonin; Calcitonin gene-related peptide; Calcium (intracellular); Calcium influx; Calcium ions; Capsaicin; Capsaicin receptors; Confocal microscopy; Desensitization; Efflux; Exocytosis; Growth factors; Headache; Headaches; Isothiocyanate; Migraine; Neonates; Nerve growth factor; Neurons; Neuropeptides; Neurotoxins; Pain; Peptides; Proteins; Sensory neurons; SNAP-25; SNAP-25 protein; Toxins; Transient receptor potential proteins; Trigeminal ganglion; trigeminal ganglion neurons
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24021338

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.