Substrate-dependent effects of calcium on rat retinal mitochondrial respiration: physiological and toxicological studies.

Abstract Cytosolic Ca 2+ overload may play a key role in the process of lead-induced retinal injury and degeneration. We report that retinal calcium content was elevated following developmental and in vitro lead exposure. To determine the concentration-dependent effects of Ca 2+ (5-1000 nM) on retinal mitochondrial bioenergetics an isolation procedure was developed. Isolated mitochondria were efficiently coupled; had good respiratory control ratios with the NAD-linked substrates, glutamate or pyruvate plus malate (G/M or P/M), and the FAD-linked substrate, succinate plus rotenone (S/R); and possessed a Na + /Ca 2+ exchanger. The major finding was that at equimolar [Ca 2+ ] ≥35 nM, mitochondria were more sensitive to and exhibited a greater degree of inhibition of coupled and uncoupled respiration with NAD-linked substrates compared to S/R. At all [Ca 2+ ], decreases in State 3 and uncoupled respiration were similar, thereby eliminating the ATP synthase and ADP/ATP translocase as sites of inhibition and suggesting that opening the mitochondrial permeability transition pore (MTP) did not contribute to the inhibition. The effects of toxicological [Ca 2+ ] were: (1) blocked by ruthenium red, (2) blocked by dibucaine only in the presence of NAD-linked substrates, and (3) partially reversed by NAD + with G/M after opening the MTP. Results with G/M suggest that Ca 2+ acts on the inner membrane phospholipase A 2 to decrease NADH CoQ reductase activity and/or produce a NAD + leak, whereas with S/R, Ca 2+ may inhibit succinate dehydrogenase. In conclusion, Ca 2+ inhibits retinal mitochondrial ATP production, which may contribute to the retinal cell injury and death observed in developmentally lead-exposed rats.