title: Voltage-gated Ca2+ influx and mitochondrial Ca2+ initiate secretion from Aplysia neuroendocrine cells.
abstract: Neuroendocrine secretion often requires prolonged voltage-gated Ca(2+) entry; however, the ability of Ca(2+) from intracellular stores, such as endoplasmic reticulum or mitochondria, to elicit secretion is less clear. We examined this using the bag cell neurons, which trigger ovulation in Aplysia by releasing egg-laying hormone (ELH) peptide. Secretion from cultured bag cell neurons was observed as an increase in plasma membrane capacitance following Ca(2+) influx evoked by a 5-Hz, 1-min train of depolarizing steps under voltage-clamp. The response was similar for step durations of ≥ 50 ms, but fell off sharply with shorter stimuli. The capacitance change was attenuated by replacing external Ca(2+) with Ba(2+), blocking Ca(2+) channels, buffering intracellular Ca(2+) with EGTA, disrupting synaptic protein recycling, or genetic knock-down of ELH. Regarding intracellular stores, liberating mitochondrial Ca(2+) with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), brought about an EGTA-sensitive elevation of capacitance. Conversely, no change was observed to Ca(2+) released from the endoplasmic reticulum or acidic stores. Prior exposure to FCCP lessened the train-induced capacitance increase, suggesting overlap in the pool of releasable vesicles. Employing GTP-γ-S to interfere with endocytosis delayed recovery (presumed membrane retrieval) of the capacitance change following FCCP, but not the train. Finally, secretion was correlated with reproductive behavior, in that neurons isolated from animals engaged in egg-laying presented a greater train-induced capacitance elevation vs quiescent animals. The bag cell neuron capacitance increase is consistent with peptide secretion requiring high Ca(2+), either from influx or stores, and may reflect the all-or-none nature of reproduction.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
authors.forename: C M
authors.initials: CM
authors.lastname: Hickey
authors.forename: C J
authors.initials: CJ
authors.lastname: Groten
authors.forename: L
authors.initials: L
authors.lastname: Sham
authors.forename: C J
authors.initials: CJ
authors.lastname: Carter
authors.forename: N S
authors.initials: NS
authors.lastname: Magoski
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D001048:Aplysia / Q000502:physiology*; D001522:Behavior, Animal / Q000502:physiology; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D004531:Eggs; D025802:Electric Capacitance; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017952:Ganglia, Invertebrate / Q000166:cytology / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology*; D055099:Neuroendocrine Cells / Q000502:physiology*; D009479:Neuropeptides / Q000096:biosynthesis; D018408:Patch-Clamp Techniques; D012330:RNA, Double-Stranded / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D000477:Alkylating Agents; D015220:Calcium Channels; D009479:Neuropeptides; D012330:RNA, Double-Stranded; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002118:Calcium
keywords: 2-amino-2-hydroxymethyl-propane-1,3-diol; ATP; Ba(2+), Cs(+) tetraethylammonium artificial sea water; Ba(2+)-Cs(+)-TEA ASW; Baf; C(m); CPA; Ca(2+), Cs(+) tetraethylammonium artificial sea water; Ca(2+)-Cs(+)-TEA ASW; Ca(2+)-free artificial salt water; Cntl; DMSO; EGTA; ELH; EtOH; FCCP; GTP; GTP-γ-S; HEPES; I(b); I(ss); IgG; MEM; N-2-hydroxyethlpiperazine-N′-2-ethanesulphonic acid; N-ethylmaleimide; NEM; PBS; Q(cf); Q(t); Q(tc); R(a); R(m); SEM; TEA; TFA; TRIS; access resistance; adenosine triphosphate; bafilomycin A; bag cell neurons; baseline current; calcium channel; capacitance; carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone; cfASW; charge during transient current; control; correction factor charge; current change to a voltage step; cyclopiazonic acid; dimethyl sulfoxide; double-stranded ribonucleic acid; dsRNA; egg-laying hormone; ethanol; ethyleneglycol bis (aminoethylether) tetraacetic acid; guanosine triphosphate; guanosine-5′-[γ-thio]triphosphate; immunogammaglobulin; membrane capacitance; membrane resistance; membrane time constant; minimum essential medium; nASW; normal artificial salt water; peptide release; phosphate-buffered saline; standard error of the mean; steady-state current; step voltage; tcASW; tetraethylammonium; tissue culture artificial salt water; total charge; trifluoroacetic acid; ΔI; ΔV; τ