Jm. Weinberg et al., CYTOSOLIC-FREE CALCIUM INCREASES TO GREATER-THAN-100 MICROMOLAR IN ATP-DEPLETED PROXIMAL TUBULES, The Journal of clinical investigation, 100(3), 1997, pp. 713-722
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.