Calcium requirements for exocytosis do not delimit the releasable neuropeptide pool

Recently, it was proposed that secretory vesicles have widely varying Ca²⁺ thresholds for exocytosis. This model can explain adaptation of secretory responses and predicts that incomplete release is a consequence of insufficient Ca²⁺. However, membrane capacitance-based measurements have not supported varying Ca²⁺ thresholds. Here, Green Fluorescent Protein (GFP) imaging is used to test whether a Ca²⁺ limitation determines the size of the releasable neuropeptide pool in differentiated PC12 cells. We show that depolarization-evoked release correlates with failure to sustain fully elevated [Ca²⁺]_i. However, this is coincidental because release remains incomplete when [Ca²⁺]_i is maintained at a relatively high level by application of an ionophore or by dialysis with a buffered Ca²⁺ solution. Furthermore, in contradiction with the existence of high threshold vesicles, stimulating maximal release with moderate [Ca²⁺]_i prevents secretory responses to large increases in [Ca²⁺]_i induced by photolysis of the caged dimethoxynitrophenyl-EGTA-4 (DMNPE-4). Thus, optical measurements show that limited capacity for neuropeptide release in response to depolarization is not caused by an insufficient duration of [Ca²⁺]_i elevation or by variation among vesicles in Ca²⁺ sensitivity for exocytosis.