Monitoring of Ca2+ release from intracellular stores in permeabilized rat parotid acinar cells using the fluorescent indicators Mag-fura-2 and calcium green C18.
The operation of intracellular Ca2+ stores in saponin-permeabilized rat parotid acinar cells was studied by monitoring the Ca2+ concentration within organelles loaded with the low affinity Ca2+ indicator Mag-fura-2. Inositol 1, 4, 5-trisphosphate (InsP3) caused a decrease in the Mag-fura-2 ratio in a dose-dependent manner, and this effect was reversed by a removal of InsP3 or by an addition of the InsP3 receptor antagonist heparin. The changes in Mag-fura-2 ratio indicate the Ca2+ release from InsP3-sensitive Ca2+ stores and Ca2+ re-uptake into the stores in permeabilized acinar cells. The decrease in Mag-fura-2 ratio induced by InsP3 was observed at all regions of the acinar cells, suggesting that the InsP3-sensitive Ca2+ stores are located throughout the cells. The InsP3-induced Ca2+ release was also monitored using the membrane-bound Ca2+ indicator Calcium Green C18 which is sensitive to the changes in Ca2+ concentration immediately adjacent to the membrane of intracellular Ca2+ stores. InsP3 caused a large increase in the Calcium Green C18 fluorescence reflecting Ca2+ release from the stores. The Ca2+ pump inhibitor thapsigargin (ThG) itself had little or no effect on the Mag-fura-2 ratio or Calcium Green C18 fluorescence, but combined application of ThG with a low concentration of InsP3 evoked a significant decrease in the Mag-fura-2 ratio. This result supports the hypothesis that the ThG-induced Ca2+ release is due to InsP3-sensitive Ca2+ release which is mediated by the resting levels of InsP3. Further, none of cyclic ADP-ribose, caffeine or ryanodine changed the Mag-fura-2 ratio and Calcium Green C18 fluorescence, leading to the assumption that the ryanodine-sensitive Ca2+ stores are minor in rat parotid acinar cells.
Tojyo
Y
Y
Department of Dental Pharmacology, School of Dentistry, Health Sciences, University of Hokkaido, Japan. tojyo@hucc.hokudai.ac.jp
Tanimura
A
A
Matsumoto
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D009930:Organic Chemicals; D010306:Parotid Gland / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012503:Saponins
D005456:Fluorescent Dyes; D009930:Organic Chemicals; D012503:Saponins; C095636:calcium green C18; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D002118:Calcium; D016257:Fura-2