title: Ca2+ dynamics in the secretory vesicles of neurosecretory PC12 and INS1 cells.
abstract: We have investigated the dynamics of the free [Ca(2+)] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca(2+)-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca(2+)] ([Ca(2+)](SG)] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca(2+) pumps with thapsigargin largely blocked Ca(2+) uptake by the granules in Ca(2+)-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca(2+) pump inhibitor benzohydroquinone induced a rapid release of Ca(2+) from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca(2+) pumps is essential to maintain the steady-state [Ca(2+)](SG). Both inositol 1,4,5-trisphosphate (InsP(3)) and caffeine produced a rapid Ca(2+) release from the granules, suggesting the presence of InsP(3) and ryanodine receptors in the granules. The response to high-K(+) depolarization was different in both cell types, a decrease in [Ca(2+)](SG) in PC12 cells and an increase in [Ca(2+)](SG) in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca(2+)](SG) in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca(2+) through SERCA-type Ca(2+) pumps and can release it through InsP(3) and ryanodine receptors, supporting the hypothesis that secretory granule Ca(2+) may be released during cell stimulation and contribute to secretion.
authors.affiliation: Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Ramón y Cajal, 7, 47005 Valladolid, Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: SantoDomingo
authors.forename: Rosalba I
authors.initials: RI
authors.lastname: Fonteriz
authors.forename: Carmen D
authors.initials: CD
authors.lastname: Lobatón
authors.forename: Mayte
authors.initials: M
authors.lastname: Montero
authors.forename: Alfredo
authors.initials: A
authors.lastname: Moreno
authors.forename: Javier
authors.initials: J
authors.lastname: Alvarez
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium; D020013:Calcium Signaling / Q000187:drug effects; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D009489:Neurosecretion / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*
chemical_list: D002110:Caffeine; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium