Visualization and quantification of endoplasmic reticulum Ca in renal cells using confocal microscopy and Fluo5F |
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Authors: | Andre C. Eaddy Rick G. Schnellmann |
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Affiliation: | aCenter for Cell Death, Injury, and Regeneration, Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States;bRalph H. Johnson VA Medical Center Charleston, SC 29401, United States |
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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 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. |
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Keywords: | Abbreviations: ER, endoplasmic reticulum EGTA, ethylene glycol tetraacetic acid RPTC, renal proximal tubular cell TMRM, tetramethyl rhodamine methyl ester Fluo5F, Fluo-5F-AM LSCM, laser scanning confocal microscopy FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone SERCA, sarcoplamic/endoplasmic reticulum ATPase DAG, diacylglycerol IP3, inositol triphosphate DMSO, dimethyl sulfoxide |
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