Cytosolic-free calcium increases to greater than 100 micromolar in ATP- depleted proximal tubules

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.