Cytosolic free Ca (Caf) was measured in three different preparations of freshly prepared proximal tubules from the rabbit kidney during energy deprivation using
fura-2. Isolated perfused tubules, tubules immobilized on glass cover slips, and tubules in
suspension were subjected to inhibitors of oxidative phosphorylation ("chemical
hypoxia"); the latter two preparations were also subjected to 40 min of
anoxia. During normoxia, Caf ranged from 100 to 180 nM in all three preparations, and chemical
hypoxia caused either no change or a small (30-100%) increase in Caf values. Subsequent addition of Ca
ionophores increased Caf to 300-500 nM in the first 2 min and to greater than 1 microM after 15 min. In individual experiments,
anoxia produced similar responses to those of chemical
hypoxia, eliciting no average significant change in Caf, despite clear evidence for impaired respiration and plasma membrane damage after 40 min of
anoxia. This lack of change in Caf was unrelated to "Ca buffering" by
fura-2 or inactivation of the
dye, since Caf increased to 666 +/- 59 nM upon addition of Ca
ionophore during
anoxia. These data suggest that increased Caf is not a prerequisite for cellular damage during
anoxia in proximal renal tubules. Furthermore, no apparent alteration in plasma membrane permeability to Ca occurs before membrane disruption. Decreased
ATP seems to initiate a series of Caf-independent events that cause irreversible injury.