TY - JOUR
T1 - Visualization and quantification of endoplasmic reticulum Ca2+ in renal cells using confocal microscopy and Fluo5F
AU - Eaddy, Andre C.
AU - Schnellmann, Rick G.
N1 - Funding Information:
This work was supported by an NIH fellowship ( F30 ES015964 ) to AE and NIH grant ( DK-062028 ) to RGS, and by the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs. Animal facilities were funded by NIH grant C06 RR-015455 and Cell and Molecular Imaging (CMI) facility was supported, in part, by grant P30 CA138313 to the Hollings Cancer Center, Medical University of South Carolina. The authors also thank Dr. Venkat Ramshesh and Dr. John Lemasters (Medical University of South Carolina) for their advice and constructive discussions during the execution of these experiments.
PY - 2011/1/7
Y1 - 2011/1/7
N2 - Sarcoplasmic/endoplasmic reticulum (ER) Ca2+ is the most abundant store of intracellular Ca2+, and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca2+ in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca2+ indicator, to directly monitor changes in RPTC ER Ca2+. Fluo5F staining reflected ER Ca2+, resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca2+ pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15min, respectively, whereas A23187, a Ca2+ ionophore caused more rapid ER Ca2+ release (55% and 75% decrease in fluorescence at 5 and 15min).Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca2+. In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca2+ release. ER Ca2+ release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca2+ in live cells.
AB - Sarcoplasmic/endoplasmic reticulum (ER) Ca2+ is the most abundant store of intracellular Ca2+, and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca2+ in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca2+ indicator, to directly monitor changes in RPTC ER Ca2+. Fluo5F staining reflected ER Ca2+, resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca2+ pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15min, respectively, whereas A23187, a Ca2+ ionophore caused more rapid ER Ca2+ release (55% and 75% decrease in fluorescence at 5 and 15min).Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca2+. In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca2+ release. ER Ca2+ release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca2+ in live cells.
KW - Calcium
KW - Confocal microscopy
KW - Endoplasmic reticulum
KW - Fluo5f
KW - Kidney
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U2 - 10.1016/j.bbrc.2010.11.137
DO - 10.1016/j.bbrc.2010.11.137
M3 - Article
C2 - 21130732
AN - SCOPUS:78650893158
SN - 0006-291X
VL - 404
SP - 424
EP - 427
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -