Visualization and quantification of endoplasmic reticulum Ca2+ in renal cells using confocal microscopy and Fluo5F

Andre C. Eaddy, Rick G. Schnellmann

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)424-427
Number of pages4
JournalBiochemical and Biophysical Research Communications
Volume404
Issue number1
DOIs
StatePublished - Jan 7 2011
Externally publishedYes

Keywords

  • Calcium
  • Confocal microscopy
  • Endoplasmic reticulum
  • Fluo5f
  • Kidney

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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