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

Sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) is the most abundant store of intracellular Ca(2+), and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca(2+) 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 Ca(2+) indicator, to directly monitor changes in RPTC ER Ca(2+). Fluo5F staining reflected ER Ca(2+), resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca(2+) pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca(2+) ionophore caused more rapid ER Ca(2+) 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 Ca(2+). In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca(2+) release. ER Ca(2+) 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 Ca(2+) in live cells.