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First published online May 18, 2006
Journal of Experimental Biology 209, 2156-2164 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02250
Involvement of ryanodine-operated channels in tert-butylhydroperoxide-evoked Ca2+ mobilisation in pancreatic acinar cells
1 Institute of Nutrition and Food Technology, Department of Physiology,
University of Granada, C/Ramón y Cajal, 4. 18071, Granada,
Spain
2 Department of Physiology, Faculty of Veterinary Sciences, University of
Extremadura, Cáceres, Spain
* Author for correspondence at address 1 (e-mail: malbam{at}ugr.es)
Accepted 3 April 2006
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 µmol 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 µmol 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 µmol 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.
Key words: tert-butylhydroperoxide (tBHP), cytosolic Ca2+, reactive oxygen species, pancreatic acinar cell, mitochondria, endoplasmic reticulum (ER), IP3 (inositol-1,4,5-triphosphate), ryanodine channel
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