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 isol ated kidney proximal tubules. However, peak levels could not be determ ined precisely with the Ca2+-sensitive fluorophore, fura-2, because of its high affinity for Ca2+. Now, we have used two low affinity Ca2+ f luorophores, mag-fura-2 (furaptra) and fura-2FF, to quantitate the ful l magnitude of Ca-f increase, Between 30 and 60 min after treatment wi th antimycin to deplete ATP in the presence of glycine to prevent lyti c 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 infl ux of medium Ca2+ were also seen using the new low Ca2+ affinity, Mg2-insensitive, fluorophore fura-2FF in tubules depleted of ATP by hypox ia, and these increases were reversed by reoxygenation. Total cell Ca2 + levels in antimycin-treated or hypoxic tubules did not change, sugge sting that mitochondria were not buffering the increased Ca-f during A TP depletion. Considered in the context of the high degree of structur al preservation of glycine-treated tubule cells during ATP depletion a nd the commonly assumed Ca2+ requirements for phospholipid hydrolysis, actin disassembly, and Ca2+-mediated structural damage, the remarkabl e elevations of Ca-f demonstrated here suggest an unexpected resistanc e to the deleterious effects of increased Ca-f during energy deprivati on in the presence of glycine.