title: Calcium requirements for exocytosis do not delimit the releasable neuropeptide pool.
abstract: Recently, it was proposed that secretory vesicles have widely varying Ca(2+) thresholds for exocytosis. This model can explain adaptation of secretory responses and predicts that incomplete release is a consequence of insufficient Ca(2+). However, membrane capacitance-based measurements have not supported varying Ca(2+) thresholds. Here, Green Fluorescent Protein (GFP) imaging is used to test whether a Ca(2+) 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 [Ca(2+)](i). However, this is coincidental because release remains incomplete when [Ca(2+)](i) is maintained at a relatively high level by application of an ionophore or by dialysis with a buffered Ca(2+) solution. Furthermore, in contradiction with the existence of high threshold vesicles, stimulating maximal release with moderate [Ca(2+)](i) prevents secretory responses to large increases in [Ca(2+)](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 [Ca(2+)](i) elevation or by variation among vesicles in Ca(2+) sensitivity for exocytosis.
authors.affiliation: Department of Pharmacology, E1351 Biomedical Science Tower, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Lu
authors.forename: Graham C R
authors.initials: GC
authors.lastname: Ellis-Davies
authors.forename: Edwin S
authors.initials: ES
authors.lastname: Levitan
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D004533:Egtazic Acid / Q000031:analogs & derivatives*; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins; D009474:Neurons / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D009489:Neurosecretion / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*
chemical_list: D007476:Ionophores; D008164:Luminescent Proteins; D009479:Neuropeptides; C117677:dimethoxynitrophenyl-EGTA-4; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D002118:Calcium