Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon

Abstract

Basal (substrate alone) and maximum rates of H₂O₂ production, oxygen consumption and free radical leak in the respiratory chain were higher in heart mitochondria of the short-lived rat (4 years) than in the long-lived pigeon (35 years). This suggests that the low free radical production of pigeon heart mitochondria is due in part to both a low electron flow and a low percent leak of electrons out of sequence in the respiratory chain. Thenoyltrifluoroacetone did not increase H₂O₂ production with succinate either in rats or pigeons. Mitochondrial H₂O₂ production was higher with pyruvate/malate than with succinate in both animal species. Rotenone and antimycin A increased H₂O₂ production with pyruvate/malate to the maximum levels observed in each species. Addition of myxothiazol to antimycin A-treated mitochondria supplemented with pyruvate/malate decreased H₂O₂ production in both species. All the combinations of inhibitors added with pyruvate/malate resulted in higher rates of H₂O₂ production in rats than in pigeons. When succinate instead of pyruvate/malate was used as substrate, rotenone and thenoyltrifluoroacetone decreased mitochondrial H₂O₂ production in the rat and did not change it in the pigeon. The results indicate that Complexes I and III are the main H₂O₂ generators of heart mitochondria in rats and pigeons and that both Complexes are responsible for the low H₂O₂ production of the bird. p-Chloromercuribenzoate and ethoxyformic anhydride strongly inhibited the H₂O₂ production induced by rotenone with pyruvate/malate in both species. This suggests that the free radical generator of Complex I is located after the ferricyanide reduction site, between the ethoxyformic and the rotenone-sensitive sites.

Introduction

There is increasing evidence that free radicals are important determinants of aging and maximum life span (MLSP) of animals [1]. In healthy tissues the most important source of free radicals is the leak of electrons out of sequence in the mitochondrial respiratory chain 2, 3. It has been found that the rate of succinate-supported mitochondrial oxygen radical generation decreases as MLSP increases in heart, brain and kidney mitochondria from five mammalian species which follow the inverse relationship between basal metabolic rate and MLSP and ranging in body size from that of rat to that of cow 4, 5, 6. The low rate of mitochondrial H₂O₂ production of the species with higher body size and MLSP (like the cow) used in these studies would be due to their low metabolic rate and thus to a low mitochondrial oxygen consumption and electron flow [6]. Thus, free radicals of mitochondrial origin could be the causal link for the well known inverse relationship between metabolic rate and MLSP present in most animals [7]. Nevertheless, this inverse relationship could be also due in principle to other unknown factors causing aging and positively associated with metabolic rate. In order to clarify if the relationship between mitochondrial free radical production and MLSP is more than circumstantial, animal species in which the MLSP is higher than expected from their metabolic rates must be studied. This characteristic occurs in two homeothermic animal groups, primates and birds. In these two cases, the MLSP is between two and four times greater than in the majority of non-primate mammals of the same size or metabolic rate 8, 9. The bird case is specially striking since they are the only vertebrate animals simultaneously showing a high basal metabolic rate and a high MLSP. This indicates that their high MLSP can not be simply due to a slowing of metabolism.

Recent studies in our and other laboratories have compared succinate-supported mitochondrial H₂O₂ production between rats and pigeons. Even though body size and basal metabolic rate are similar in adult rats and pigeons, the former species has a MLSP of 4 years, whereas pigeons show, like birds in general, an extraordinarily high longevity (MLSP=35 years). This 8.75-fold difference in MLSP can not be obtained by comparing short- and long-lived rats strains (diverging in MLSP at best by 1.4). The results showed that pigeon mitochondria produce oxygen radicals at rates lower than those of rats 10, 11. All the data available suggest that a low mitochondrial oxygen radical production is a characteristic of longevous species (mammals or birds), no matter if they have low or high basal metabolic rates. Nevertheless, in all previous works concerning mitochondrial free radical production and MLSP in mammals 4, 5, 6or birds 10, 11only a Complex II-linked substrate (succinate), which by-passes Complex I, was used, and the effects of specific inhibitors of the respiratory chain inhibitors were not studied.

The potential sites of mitochondrial oxygen radical production have been identified at respiratory Complexes I or III 12, 13in studies frequently using rat heart mitochondria or submitochondrial particles. However, neither the mechanisms or the sites responsible for the low rate of free radical production have been studied in any longevous species. In the present paper, the rate of mitochondrial oxygen radical production is simultaneously studied in male adult rats and pigeons using various substrates and inhibitors specific for different segments of the respiratory chain. The study is conducted in mitochondria from a post-mitotic tissue highly relevant for aging, the heart. The results show two mechanisms responsible to a large extent for the low rate of H₂O₂ production of pigeon heart mitochondria and point to Complexes I and III as the main sites of evolutionary decrease of oxygen radical production in the bird. Additional experiments, stimulated by these results, further localize the Complex I site of free radical production within this large multisubunit Complex in both species.