Maintenance of Energy-Linked Functions in Rat-Liver Mitochondria Aged in the Presence of Nupercaine

The present study has been designed to examine whether endogenous phospholipase A2 of rat liver mitochondria is involved in the mitochondrial aging process during which time there is a gradual loss of energy-linked functions. Freshly-isolated rat liver mitochondria have a respiratory control ratio of 4 with succinate. During storage at 0—4 °C the respiratory control ratio decreased progressively to 1.0 after 90 h fromthe preparation. During storage there was also an increase in the conversion of mitochondrial phos phatidylethanolamine and phosphatidylcholine to their corresponding lysoderivatives. The local anesthetic nupercaine (recently shown to be a powerful inhibitor of phospholipase A2 in a variety of systems) was effective in both stabilizing energy-linked functions of isolated mitochondria and maintaining the integrity of mitochondrial phospholipid. After 90 h aging at 0—4 °C, the respiratory control ratio of mitochondria treated with 400 μM nupercaine was nearly unchanged and the breakdown of mitochondrial phospholipid markedly reduced. Nupercaine alone can effectively substitute for either EDTA or bovine serum albumin, which are routinely used to improve the stability of isolated mitochondria. As the mitochondrial phospholipase A2 is activated by Ca2+ and free fatty acids, we suggest that the presence of these agents improves mitochondrial stability by preventing activation of the enzyme. We conclude that endogenous phospholipase activity is largely responsible for the damage to aged mitochondria, which results in a loss of their energy-linked functions. The addition of nupercaine during storage makes it possible to maintain rat liver mitochondria tightly coupled for long periods of time. We also suggest that the use of nupercaine should prove generally useful in preserving the structure and functions of other biological membranes containing phospholipase A2 during their preparation and storage.