Fig. 3. CGRP release evoked by K⁺, capsaicin or bradykinin from TGNs is Ca²⁺-dependent. (A-C) After 7 DIV, neurons were incubated for 30 minutes at 37°C in BR-HBS alone or with vehicle added (see Materials and Methods) for measurement of basal release of CGRP (white bars), and then incubated in BR-HBS in the presence of varying concentrations of (A) K⁺, (B) capsaicin or (C) bradykinin to determine stimulated release of CGRP (hatched bars). Release stimulated by K⁺ (60 mM), capsaicin (1 µM) or bradykinin (0.1 µM) represents 30, 20 and 15% of the total content, respectively. Likewise, Ca²⁺-independent release was determined, except in Ca²⁺-free BR-HBS containing 2 mM EGTA. Buffer in which cells were bathed was carefully removed and the concentration of CGRP determined by enzyme immuno-assay (mean ± s.e.m., n=4). Note that the basal efflux is higher when either 0.1% ethanol or dimethyl sulphoxide was included (Fig. 3B,C) than when absent (Fig. 3A). Note that each stimulus gave significant increments of evoked release over their basal level.