MITOCHONDRIAL DECAY IN HEPATOCYTES FROM OLD RATS - MEMBRANE-POTENTIALDECLINES, HETEROGENEITY AND OXIDANTS INCREASE

Mitochondrial function during aging was assessed in isolated rat hepat ocytes to avoid the problem of differential lysis when old, fragile mi tochondria are isolated, Rhodamine 123, a fluorescent dye that accumul ates in mitochondria on the basis of their membrane potential, was use d as a probe to determine whether this key function is affected by agi ng. A marked fluorescent heterogeneity was observed in hepatocytes fro m old (20-28 months) but not young (3-5 months) rats, suggesting age-a ssociated alterations in mitochondrial membrane potential, the driving force for,lTP synthesis, Three distinct cell subpopulations were sepa rated by centrifugal elutriation; each exhibited a unique rhodamine 12 3 fluorescence pattern, with the largest population from old rats havi ng significantly lower fluorescence than that seen in young rats. This apparent age-associated alteration in mitochondrial membrane potentia l was confirmed by measurements with radioactive tetraphenylphosphoniu m bromide. Cells from young rats had a calculated membrane potential o f - 154 mV, in cent rast to that of the three subpopulations from old rats of -70 mV (the largest population), -93 mV, and - 154 mV, Product ion of oxidants was examined using 2',7'-dichlorofluorescin, a dye tha t forms a fluorescent product upon oxidation. The largest cell subpopu lation and a minor one from old animals produced significantly more ox idants than cells from young rats. To investigate the molecular cause( s) for the heterogeneity, we determined tile levels of an age-associat ed mtDNA deletion, No significant differences were seen in the three s ubpopulations, Indicating that the mitochondrial decay is due to other mutations, epigenetic changes, or both.