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 lytic plasma membrane damage, Ca-f measured with mag-fura-2 exceeded 10 mu M in 91% of tubules studied and 68% had increases to greater than 100 mu M. 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.