title: Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.
abstract: The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.
authors.affiliation: Department of Biochemistry, University of Bristol, BS8 1TD Bristol, United Kingdom.
authors.forename: K J
authors.initials: KJ
authors.lastname: Mitchell
authors.forename: P
authors.initials: P
authors.lastname: Pinton
authors.forename: A
authors.initials: A
authors.lastname: Varadi
authors.forename: C
authors.initials: C
authors.lastname: Tacchetti
authors.forename: E K
authors.initials: EK
authors.lastname: Ainscow
authors.forename: T
authors.initials: T
authors.lastname: Pozzan
authors.forename: R
authors.initials: R
authors.lastname: Rizzuto
authors.forename: G A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000502:physiology; D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D007093:Imidazoles; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000697:Central Nervous System Stimulants; D002614:Chelating Agents; D007093:Imidazoles; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011719:Pyrazines; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; C017144:coelenterazine; D000331:Aequorin; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium