FIGURE 8.

O₂ consumption by mitochondria isolated from mouse retinas and titrated with Ca²⁺. To confirm that Ca²⁺ can influence AGC1 activity in rodent retinas we isolated mitochondria from rat retinas and measured their O₂ consumption over a range of free Ca²⁺ concentrations using either glutamate and malate (Glu/Mal) or pyruvate and malate (Pyr/Mal) as fuel in a buffer containing EGTA. Oxidation of the Glu/Mal mix depends on AGC activity, whereas oxidation of the Pyr/Mal mix occurs independently of AGC1 (67). A, ADP was added at time 0 on this graph followed at 5 min (short dashed line) by either Pyr/Mal (black) or Glu/Mal (red) and then 20-nmol stepwise additions of CaCl₂ (longer dashed lines). B, Ca²⁺ dependence of O₂ consumption rate when Glu/Mal is used as fuel. The K_½ was determined from the curve that best fit the data from the experiments shown in panel A. n = 3; error bars show S.E.). Consistent with previous studies of mitochondria from other tissues (68), we found that Ca²⁺ stimulates O₂ consumption. With mouse retina mitochondria we found a K_½ of 165 nm only when the Glu/Mal mixture is used as a fuel. This confirms that Ca²⁺ can stimulate Asp/Glu exchange in the mouse retina. Note that the millimolar concentrations of Pyr/Mal and Glu/Mal in these experiments are so high that any influence of Ca²⁺ on the K_m values of matrix dehydrogenases would not affect respiration.