Figure 5.

Capsaicin concentration-dependency for stimulation of CGRP exocytosis from trigeminal ganglion neurons (TGNs) and its inhibition by BoNT/A, BoNT/EA, and LC/E-BoNT/A; only the two latter SNAP-25_E-producing variants retained efficacy against high capsaicin concentration, despite cleaving no more SNAP-25 than BoNT/A. (A) TGNs were exposed to various capsaicin concentrations ([CAP]) for 30 min, and the amount of CGRP released into the bathing solution was quantified and expressed as a % of the total CGRP content (i.e., amount released plus quantity retained inside the cells). The inclining part of the relationship (0.001 to 0.1 μM) was fit with a four-parameter logistic function with R² = 0.884, yielding a half-maximal effective [CAP] (EC₅₀) = 0.02 μM. Individual values for the declining part (0.1 to 10 μM) are connected by a broken line. (B) Western blot of detergent-solubilized lysates of cells that had been pre-incubated for 48 h in the absence (control) or presence of 100 nM of the indicated BoNTs, using an antibody recognizing intact SNAP-25 and cleavage products of BoNT/A (SNAP-25_A) or chimeras containing /E protease (SNAP-25_E). The blot was additionally probed with an antibody to syntaxin-1A/B. Black lines to the left indicate the migration of 37 and 25 kDa molecular weight standards. (C) The amounts of CGRP released, evoked during 30 min by [CAP] from TGNs pre-treated with the toxins as above, are expressed as % of requisite control values for this experiment (7.3 ± 1.2, 33.6 ± 2.8, 24 ± 3.3, and 13.3 ± 2.1% of total CGRP for 0.02, 0.1, 0.3 and 1 μM CAP, respectively). (D) Spontaneous CGRP release over 30 min exposure to HEPES buffered saline lacking capsaicin. (E,F) The mean amounts of total CGRP in cells treated as above. As a lower seeding density of cells was used for the experiments in (F) than in (E), smaller CGRP amounts were detected in the former. Data are presented as mean + SEM. For (D,E), one-way ANOVA was used followed by Bonferroni’s post hoc test, and significance of the latter is indicated with asterisks; **** p < 0.0001, n ≥ 16, N ≥ 4. For (F) unpaired two-tailed t-test with Welch correction was used, *** p < 0.001, n = 15, N = 2.