Mise en évidence du mécanisme d'action de l'éthacrynate de sodium sur des mitochondries de foie de rat

Abstract

The competition of ethacrynate with phosphate shown during oxidation of succinate by rat liver mitochondria [2] is confirmed by measuring [ 14 C]ethacrynate incorporation into mitochondria: on increasing the phosphate present during preincubation, less ethacrynate is incorporated into the mitochondria and there is a decrease in the inhibition of succinate oxidation. Ethacrynate exhibits different effects according to the substrate used: a) with NAD‐linked substrates, ethacrynate inhibits the oxidation, this inhibition being insensitive to phosphate and accompanied by complete oxidation of NADH to NAD + ; b) with succinate, as already mentionned, the inhibition is sensitive to phosphate; if rotenone is present during preincubation with ethacrynate, there is oxidation, but it is an uncoupled oxidation, while, with phosphate plus ethacrynate, the oxidation is still coupled. Methylene blue added during preincubation with ethacrynate and rotenone prevents the uncoupling and reestablishes the inhibition of oxidation. As with NAD‐linked substrates, the inhibition of oxidation by ethacrynate is accompanied by complete oxidation of NADH to NAD + ; c) with TMPD + ascorbate (plus rotenone), ethacrynate completely uncouples oxidative phosphorylation; again phosphate prevents this effect. Ethacrynate inhibits the [ 32 P]P i ‐ATP exchange reaction. The presence of Mg ++ ions is required in order to get maximal effect of ethacrynate: the more Mg ++ ions present, the greater is the inhibition (succinate oxidation measurement). The results presented in this paper are discussed on the basis that the primary action of ethacrynate is a phosphate‐sensitive uncoupling action. The fact that inhibition of NAD‐linked substrate oxidation is not sensitive to phosphate suggests that ethacrynate interferes in another way with these substrates. However, by depleting mitochondria of energy‐rich compounds, the penetration of these substrates (assumed to be an energy requiring process) would be inhibited by ethacrynate and, consequently, the oxidation of these substrates would be impossible. As a thiol reagent, ethacrynate is believed to react with specific proteic thiol groups implicated in the formation of an energy‐rich intermediate, and to compete with phosphate in the formation of a phosphorylated energy‐rich compound from a non‐phosphorylated energy‐rich compound.