title: Calcium dynamics in catecholamine-containing secretory vesicles.
abstract: We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Moreno
authors.forename: Carmen D
authors.initials: CD
authors.lastname: Lobatón
authors.forename: Jaime
authors.initials: J
authors.lastname: Santodomingo
authors.forename: Laura
authors.initials: L
authors.lastname: Vay
authors.forename: Esther
authors.initials: E
authors.lastname: Hernández-SanMiguel
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
authors.forename: Mayte
authors.initials: M
authors.lastname: Montero
authors.forename: Javier
authors.initials: J
authors.lastname: Alvarez
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002395:Catecholamines / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D021381:Protein Transport; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*
chemical_list: D002395:Catecholamines; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D002118:Calcium