Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 404, Issue 1, Pages 424-427Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2010.11.137
Keywords
Calcium; Endoplasmic reticulum; Confocal microscopy; Kidney; Fluo5f
Categories
Funding
- NIH [F30 ES015964, DK-062028, C06 RR-015455]
- Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs
- [P30 CA138313]
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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 15 min, respectively, whereas A23187, a Ca2+ ionophore caused more rapid ER Ca2+ release (55% and 75% decrease in fluorescence at 5 and 15 min). 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. (C) 2010 Elsevier Inc. All rights reserved.
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