Inhibition of cardiac mitochondrial respiration by salicylic acid and acetylsalicylate.

Acetylsalicylate, the active ingredient in aspirin, has been shown to be beneficial in the treatment and prevention of cardiovascular disease. Because of the increasing frequency with which salicylates are used, it is important to more fully characterize extra- and intracellular processes that are altered by these compounds. Evidence is provided that treatment of isolated cardiac mitochondria with salicylic acid and to a lesser extent acetylsalicylate resulted in an increase in the rate of uncoupled respiration. In contrast, both compounds inhibited ADP-dependent NADH-linked (state 3) respiration to similar degrees. Under the conditions of our experiments, loss in state 3 respiration resulted from inhibition of the Krebs cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH). Kinetic analysis indicates that salicylic acid acts as a competitive inhibitor at the alpha-ketoglutarate binding site. In contrast, acetylsalicylate inhibited the enzyme in a noncompetitive fashion consistent with interaction with the alpha-ketoglutarate binding site followed by enzyme-catalyzed acetylation. The effects of salicylic acid and acetylsalicylate on cardiac mitochondrial function may contribute to the known cardioprotective effects of therapeutic doses of aspirin, as well as to the toxicity associated with salicylate overdose.

Reversible inactivation of alpha-ketoglutarate dehydrogenase in response to alterations in the mitochondrial glutathione status.

In a previous study, we found that treatment of rat heart mitochondria with H(2)O(2) resulted in a decline and subsequent recovery in the rate of state 3 NADH-linked respiration. These effects were shown to be mediated by reversible alterations in NAD(P)H utilization and in the activities of specific Krebs cycle enzymes alpha-ketoglutarate dehydrogenase (KGDH) and succinate dehydrogenase. The purpose of the current study was to examine potential mechanism(s) by which H(2)O(2) reversibly alters KGDH activity. We report here that inactivation is not simply due to direct interaction of H(2)O(2) with KGDH. In addition, incubation of mitochondria with deferroxamine, an iron chelator, or 1,3-dimethyl-2-thiourea, an oxygen radical scavenger, prior to addition of H(2)O(2) did not alter the rate or extent of inactivation. Thus, inactivation does not appear to involve a more potent oxygen radical formed upon metal-catalyzed oxidation. Inactive KGDH from H(2)O(2)-treated mitochondria was reactivated with dithiothreitol, implicating oxidation of a protein sulfhydryl(s). However, the thioredoxin system had no effect, indicating that enzyme inactivation is not due to the formation of intra- or intermolecular disulfide(s) or a sulfenic acid. Upon incubation of mitochondria with H(2)O(2), reduced GSH levels fell rapidly prior to enzyme inactivation but recovered at the same time as enzyme activity. Importantly, treatment of inactive KGDH with glutaredoxin facilitated the GSH-dependent recovery of KGDH activity. Glutaredoxin is characterized as a specific and efficient catalyst of protein deglutathionylation. Thus, the results of the current study indicate that KGDH activity appears to be modulated through enzymatic glutathionylation and deglutathionylation. These studies demonstrate a novel mechanism by which KGDH activity and mitochondrial function can be modulated by redox status.