Because of the extensive utilization of salicylates in the treatment and prevention of cardiovascular disease, we investigated the effect of salicylic acid and acetylsalicylate on cardiac mitochondrial function. The results of this study indicate that salicylic acid caused both uncoupling of cardiac mitochondrial respiration and inhibition of mitochondrial state 3 respiration. Acetylsalicylate exhibited similar effects on state 3 respiration. However, stimulation of uncoupled respiration by acetylsalicylate was reduced relative to effects observed with salicylic acid. In the case of acetylsalicylate, the acetylated hydroxyl residue likely increases the pKa of the carboxyl group resulting in decreased rates of proton translocation and uncoupled respiration relative to those observed for salicylic acid. Under the conditions of our experiments, salicylic acid and acetylsalicylate reduced state 3 respiration through inhibition of α-ketoglutarate dehydrogenase. Kinetic analyses indicated that salicylic acid acts as a competitive inhibitor at the α-ketoglutarate binding site. In contrast, acetylsalicylate acted as a noncompetitive inhibitor consistent with interaction with the α-ketoglutarate binding site followed by enzyme-catalyzed acetylation. Thus, we have identified novel mechanisms by which salicylates inhibit KGDH activity and mitochondrial function in vitro. KGDH is a key regulatory enzyme in the Krebs cycle, and loss in activity would be expected to have significant effects on NADH production and utilization.
It has been estimated that total plasma salicylate concentrations are 0.5, 1.5 to 2.5, and 3.0 to 10 mM in humans taking analgesic doses, taking therapeutic doses for rheumatoid arthritis, or exposed to acute poisoning, respectively.22 Under these 3 scenarios, based on binding to albumin and other plasma proteins, the concentration of free circulating salicylate is estimated to be 0.005, 0.15 to 0.6, and 1.0 to 5.0 mM. Salicylic acid and acetylsalicylate were found to exert significant and immediate effects on mitochondrial function at low millimolar concentrations (Fig. 2). These alterations would therefore be expected to occur in vivo during acute poisoning. Importantly, noncompetitive inhibitors, such as acetylsalicylate, can exert significant inhibitory effects even at low concentrations if exposure is prolonged through irreversible and thus progressive increase in enzyme inactivation. Additionally, because salicylic acid acts as a proton ionophore uncoupling electron transport from oxidative phosphorylation,37,46 this compound would be expected to accumulate within the mitochondria because of differences in pH between the inner membrane and matrix space. Thus, alterations in cardiac mitochondrial function induced by salicylates may be relevant to prolonged exposure to clinical doses of these compounds or under conditions in which binding to albumin is diminished.
Although numerous beneficial effects of salicylates have been attributed to inhibition of prostaglandin synthesis through the inactivation of cyclooxygenase-1,3-5 there are likely to be other mechanisms that also contribute. The mitochondrial respiratory chain is a major source of free radicals during myocardial ischemia/reperfusion.41 Salicylic acid or acetylsalicylate may play a protective role not simply by scavenging free radicals directly but, through the inhibition of KGDH, by diminishing reducing equivalents (NADH) available for electron transport, and thus free radical generation. Alternatively, inhibition of cardiac mitochondrial respiration by cumulative doses of aspirin could potentially play a role in modulation of myocardial metabolism by creating a state of chemical hibernation. This could profoundly impact myocardial viability during conditions of energy-supply mismatch such as ischemia. The results of the current study provide information and direction for future in vivo investigations necessary to further define mechanisms responsible for the toxic as well as favorable effects of salicylates on the cardiovascular system.
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