Figure 7
Effects of TAT-CBD3A6K on evoked transmitter release and TAT-CBD3 and TAT-CBD3A6K on T- and R-type calcium currents. (A, C) Adult rat DRG neurons were maintained in culture for 12–14 days prior to the release experiments. Bar graph of immunoreactive calcitonin gene-related peptide (iCGRP) release expressed as mean percent total iCGRP content of cells in each well ± s.e.m. (n = 9-12 wells/condition as indicated). Neuropeptide release was measured from cells treated with normal HEPES buffer containing 3.5 mM KCl, HEPES buffer containing 10 μM TAT-CBD3A6K, HEPES buffer containing 50 mM KCl or 30 nM Capsaicin (Cap), followed by a final HEPES buffer containing 3.5 mMKCl again. DRGs were exposed to vehicle or TAT-CBD3A6K peptide, at 10 μM, included in the 10 minutes prior to and throughout the high (50 mM) potassium (A) or 30 nM capsaicin (C) challenges. The resulting total TAT peptide exposure time was for 30 min. There was no significant difference in the high potassium-evoked iCGRP release (A) between TAT-CBD3A6K and the control (no treatment) using an ANOVA with Dunnett’s post-hoc test (p > 0.05). The Cap-evoked iCGRP release significantly less in neurons treated with TAT-CBD3A6K compared to control neurons (C, *, p < 0.05; ANOVA with Dunnett’s post-hoc test). The evoked release was obtained by subtracting iCGRP release during the two initial basal fractions from that during the potassium or capsaicin-stimulated fractions and expressing it as percent of total iCGRP content in each group. In all cases, release stimulated by high potassium or capsaicin was significantly higher than basal release. (B, D) The total content of iCGRP measured at the end of the release experiments for either high potassium (A) or 30 nM capsaicin (C) challenges. There were no significant differences in iCGRP content between the conditions tested (p > 0.05). (E) Bar graphs illustrating the average T- and R-type calcium current densities (pA pF-1) ± SEM values for DRGs treated with vehicle (control; open white and open blue bars) or 10 μM TAT-CBD3 (yellow) TAT-CBD3A6K (blue) bath-applied for at least 10 min. Maximal current inhibition for both T- and R-type currents was observed at ~5 min and plateaued thereafter. The number of cells are indicated in parentheses. The asterisk denote statistical significance (p < 0.05; one-way analysis of variance with Dunnett’s post-hoc test) compared to the untreated control cells