Studies of mitochondrial calcium movements using chlorotetracycline

1. The association of calcium with isolated rat liver mitochondrial membranes under various metabolic conditions was monitored using the fluorescent chelate probe, chlorotetracycline. Chlorotetracycline fluorescence increased markedly during energized calcium uptake in the absence of a permeant anion. Uncoupler and a respiratory chain inhibitor caused a rapid decrease in chlorotetracycline fluorescence when added either before or after calcium. During calcium uptake experiments concentrations of calcium exceeding 100 μM caused a transient fluorescence increase followed by an extensive decrease in fluorescence. 2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous ⁴⁵Ca. A positive correlation was observed between fluorescence and energized ⁴⁵Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chlorotetracycline fluorescence but an enhanced uptake of exogenous ⁴⁵Ca. 3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with ⁴⁵Ca. Endogenous ⁴⁵Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the ⁴⁵Ca content of the mitochondria or the fluorescence of the probe. 4. Mitochondria treated with antimycin A lost most of their endogenous ⁴⁵Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released ⁴⁵Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the ⁴⁵Ca previously released. 5. Following this energized uptake of ⁴⁵Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the ⁴⁵Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane.