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

• Published in: Biochemical and biophysical research communications Vol. 404; no. 1; pp. 424 - 427
• Main Authors: Eaddy, Andre C., Schnellmann, Rick G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.01.2011
• Subjects: Aniline Compounds - chemistry; Animals; Calcimycin - pharmacology; Calcium; Calcium - analysis; Calcium - metabolism; Calcium Channel Blockers - pharmacology; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone - pharmacology; cell death; Cells, Cultured; Confocal microscopy; Endoplasmic reticulum; Endoplasmic Reticulum - chemistry; Endoplasmic Reticulum - metabolism; Female; Fluo5f; fluorescence; Fluorescent Dyes - chemistry; ion transport; Ionophores - pharmacology; Kidney; kidney cells; Kidney Tubules, Proximal - chemistry; Kidney Tubules, Proximal - drug effects; Kidney Tubules, Proximal - metabolism; membrane potential; microscopy; Microscopy, Confocal - methods; mitochondrial membrane; monitoring; proximal tubules; Rabbits; Thapsigargin - pharmacology; Xanthenes - chemistry; Calcium; DMSO; Confocal microscopy; Fluo5F; FCCP; RPTC; DAG; SERCA; ER; IP3; Kidney; EGTA; LSCM; TMRM; Endoplasmic reticulum
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2010.11.137

► A new method of monitoring ER Ca2+ directly in live cells. ► Confocal microscopy and low affinity Ca2+ indicator, Fluo5F, reveals ER Ca2+ morphology. ► Fluo5F fluorescence shows no co-localization with mitochondria or cytosol. ► Stimulation of ER Ca2+ release reveal spatiotemporal differences in ER Ca2+ release dynamics. 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.