Previous studies have shown that cytosolic-free Ca2+ (Ca(f)) increases to at least low micromolar concentrations during ATP depletion of isolated kidney proximal tubules. However, peak levels could not be determined precisely with the Ca2+ -sensitive fluorophore, fura-2, because of its high affinity for Ca2+. Now, we have used two low affinity Ca2+ fluorophores, mag-fura-2 (furaptra) and fura-2FF, to quantitate the full magnitude of Ca(f) increase. Between 30 and 60 min after treatment with antimycin to deplete ATP in the presence of glycine to prevent lyric plasma membrane damage, Ca(f) measured with mag-fura-2 exceeded 10 μM in 91% of tubules studied and 68% had increases to greater than 100 μM. Ca(f) increases of similar magnitude that were dependent on influx of medium Ca2+ were also seen using the new low Ca2+ affinity, Mg2+ -insensitive, fluorophore fura-2FF in tubules depleted of ATP by hypoxia, and these increases were reversed by reoxygenation. Total cell Ca2+ levels in antimycin-treated or hypoxic tubules did not change, suggesting that mitochondria were not buffering the increased Ca(f) during ATP depletion. Considered in the context of the high degree of structural preservation of glycine-treated tubule cells during ATP depletion and the commonly assumed Ca2+ requirements for phospholipid hydrolysis, actin disassembly, and ca2+ -mediated structural damage, the remarkable elevations of Ca(f) demonstrated here suggest an unexpected resistance to the deleterious effects of increased Ca(f) during energy deprivation in the presence of glycine.
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