title: Involvement of ryanodine-operated channels in tert-butylhydroperoxide-evoked Ca2+ mobilisation in pancreatic acinar cells.
abstract: Reactive oxygen species and related oxidative damage have been implicated in the initiation of acute pancreatitis, a disease characterised in its earliest stages by disruption of intracellular Ca2+ homeostasis. The present study was carried out in order to establish the effect of the organic pro-oxidant, tert-butylhydroperoxide (tBHP), on the mobilisation of intracellular Ca2+ stores in isolated rat pancreatic acinar cells and the mechanisms underlying this effect. Cytosolic free Ca2+ concentrations ([Ca2+]c) were monitored using a digital microspectrofluorimetric system in fura-2 loaded cells. In the presence of normal extracellular Ca2+ concentrations ([Ca2+]o), perfusion of pancreatic acinar cells with 1 mmol l-1 tBHP caused a slow sustained increase in [Ca2+]c. This increase was also observed in a nominally Ca2+-free medium, indicating a release of Ca2+ from intracellular stores. Pretreatment of cells with tBHP abolished the typical Ca2+ response of both the physiological agonist CCK-8 (1 nmol l-1) and thapsigargin (TPS, 1 micromol l-1), an inhibitor of the SERCA pump, in the absence of extracellular Ca2+. Similar results were observed with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.5 micromol l-1), a mitochondrial uncoupler. In addition, depletion of either agonist-sensitive Ca2+ pools by CCK-8 or TPS or mitochondrial Ca2+ pools by FCCP were unable to prevent the tBHP-induced Ca2+ release. By contrast, simultaneous administration of TPS and FCCP clearly abolished the tBHP-induced Ca2+ release. These results show that tBHP releases Ca2+ from agonist-sensitive intracellular stores and from mitochondria. On the other hand, simultaneous application of FCCP and of 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3-mediated Ca2+ release, was unable to suppress the increase in [Ca2+]c induced by tBHP, while the application of 50 micromol l-1 of ryanodine (which is able to block the ryanodine channels) inhibits tBHP-evoked Ca2+ mobilisation. These findings indicate that tBHP releases Ca2+ from non-mitochondrial Ca2+ pools through ryanodine channels.
authors.affiliation: Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, C/Ramón y Cajal, 4. 18071, Granada, Spain. malbam@ugr.es
authors.forename: María A
authors.initials: MA
authors.lastname: Martínez-Burgos
authors.forename: María P
authors.initials: MP
authors.lastname: Granados
authors.forename: Antonio
authors.initials: A
authors.lastname: González
authors.forename: Juan A
authors.initials: JA
authors.lastname: Rosado
authors.forename: María D
authors.initials: MD
authors.lastname: Yago
authors.forename: Ginés M
authors.initials: GM
authors.lastname: Salido
authors.forename: Emilio
authors.initials: E
authors.lastname: Martínez-Victoria
authors.forename: Mariano
authors.initials: M
authors.lastname: Mañas
authors.forename: José A
authors.initials: JA
authors.lastname: Pariente
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D002478:Cells, Cultured; D015544:Inositol 1,4,5-Trisphosphate; D008297:Male; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012433:Ryanodine / Q000494:pharmacology*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D012433:Ryanodine; D015544:Inositol 1,4,5-Trisphosphate; D020122:tert-Butylhydroperoxide; D002118:Calcium