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.
1. Awtry EH, Loscalzo J. Aspirin. Circulation.
2. Wu KK. Aspirin and salicylate: An old remedy with a new twist. Circulation.
3. Loll PJ, Picot D, Garavito RM. The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2
synthase. Nat Struct Biol.
4. Roth GJ, Majerus PW. The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein. J Clin Invest.
5. Roth GJ, Stanford N, Majerus PW. Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA.
6. Steering Committee of the Physicians Health Study Research Group. Final report on the aspirin component of the ongoing Physicians' Health Study. N Engl J Med.
7. Peto R, Gray R, Collins R, et al. Randomised trial of prophylactic daily aspirin in British male doctors. Br Med J (Clin Res Ed).
8. The Coronary Drug Project Research Group. Aspirin in coronary heart
disease. J Chronic Dis.
9. The Persantine-Aspirin Reinfarction Study Research Group. Persantine and aspirin in coronary heart
10. Breddin K, Loew D, Lechner K, et al. The German-Austrian aspirin trial: a comparison of acetylsalicylic acid, placebo and phenprocoumon in secondary prevention of myocardial infarction. On behalf of the German-Austrian Study Group. Circulation.
11. Elwood PC, Cochrane AL, Burr ML, et al. A randomized controlled trial of acetyl salicylic acid
in the secondary prevention of mortality from myocardial infarction. BMJ.
12. Elwood PC, Sweetnam PM. Aspirin and secondary mortality after myocardial infarction. Lancet.
13. Cairns JA, Gent M, Singer J, et al. Aspirin, sulfinpyrazone, or both in unstable angina. Results of a Canadian multicenter trial. N Engl J Med.
14. Lewis HD Jr, Davis JW, Archibald DG, et al. Protective effects of aspirin against acute myocardial infarction and death in men with unstable angina. Results of a Veterans Administration Cooperative Study. N Engl J Med.
15. Theroux P, Ouimet H, McCans J, et al. Aspirin, heparin, or both to treat acute unstable angina. N Engl J Med.
16. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico. GISSI-2: a factorial randomised trial of alteplase versus streptokinase and heparin versus no heparin among 12,490 patients with acute myocardial infarction. Lancet.
17. ISIS-3 (Third International Study of Infarct Survival) Collaborative Group. ISIS-3: a randomised comparison of streptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among 41,299 cases of suspected acute myocardial infarction. Lancet.
18. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet.
19. Baigent C, Collins R, Appleby P, et al. ISIS-2: 10 year survival among patients with suspected acute myocardial infarction in randomised comparison of intravenous streptokinase, oral aspirin, both, or neither. The ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. BMJ.
20. Betts WH, Whitehouse MW, Cleland LG, et al. In vitro antioxidant properties of potential biotransformation products of salicylate, sulphasalazine and amidopyrine. J Free Radic Biol Med.
21. Ghiselli A, Laurenti O, De Mattia G, et al. Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients. Free Radic Biol Med.
22. Smith MJ, Dawkins PD. Salicylate and enzymes. J Pharm Pharmacol.
23. Martens ME, Lee CP. Reye's syndrome: salicylates and mitochondrial functions. Biochem Pharmacol.
24. Tomoda T, Takeda K, Kurashige T, et al. Acetylsalicylate
(ASA)-induced mitochondrial dysfunction and its potentiation by Ca2+
25. Tonsgard JH, Getz GS. Effect of Reye's syndrome serum on isolated chinchilla liver mitochondria
. Clin Invest.
26. You K. Salicylate and mitochondrial injury in Reye's syndrome. Science.
27. Rainsford KD. Aspirin and the Salicylates.
London: Butterworths, 1984.
28. Beeley L, Kendall MJ. Effect of aspirin on renal clearance of 125
29. Berg KJ. Acute effects of acetylsalicylic acid on renal function in normal man. Eur J Clin Pharmacol.
30. McIntire SC, Rubenstein RC, Gartner JC Jr, et al. Acute flank pain and reversible renal dysfunction associated with nonsteroidal anti-inflammatory drug use. Pediatrics.
31. Brody TM. Action of sodium salicylate and related compounds on tissue metabolism in vitro. J Pharmacol Exp Ther.
32. Whitehouse MW. Biochemical properties of anti-inflammatory drugs-III. Uncoupling of oxidative phosphorylation in a connective tissue (cartilage) and liver mitochondria
by salicylate analogues: relationship of structure to activity. Biochem Pharmacol.
33. Adams SS, Cobb R. A possible basis for the anti-inflammatory activity of salicylates and other non-hormonal anti-rheumatic drugs. Nature.
34. Mehlman MA, Tobin RB, Sporn EM. Oxidative phosphorylation and respiration
by rat liver mitochondria
from aspirin-treated rats. Biochem Pharmacol.
35. Pocwiardowska E. The effects of antipyretics on metabolic processes in rat liver mitochondria
. Part II. The action of sodium salicylate, and pyrazolones on oxidation of alpha-ketoglutarate. Pol J Pharmacol Pharm.
36. Pocwiardowska E. The effects of antipyretics on metabolism processes in rat liver mitochondria
. Part I. The action of sodium salicylate, and pyrazolones on the reaction of respiratory chain. Pol J Pharmacol Pharm.
37. Haas R, Parker WD Jr, Stumpf D, et al. Salicylate-induced loose coupling: protonmotive force measurements. Biochem Pharmacol.
38. Tomoda T, Takeda K, Kurashige T, et al. Experimental study on Reye's syndrome: inhibitory effect of interferon alfa on acetylsalicylate
-induced injury to rat liver mitochondria
39. Keller BJ, Yamanaka H, Thurman RG. Inhibition of mitochondrial respiration
and oxygen-dependent hepatotoxicity by six structurally dissimilar peroxisomal proliferating agents. Toxicology.
40. Mingatto FE, Santos AC, Uyemura SA, et al. In vitro interaction of nonsteroidal anti-inflammatory drugs on oxidative phosphorylation of rat kidney mitochondria
and ATP synthesis. Arch Biochem Biophys.
41. Sadek HA, Nulton-Persson AC, Szweda PA, et al. Cardiac ischemia/reperfusion, aging, and redox-dependent alterations in mitochondrial function. Arch Biochem Biophys.
42. Schwartz A, Lee KS. Study of heart mitochondria
and glycolytic metabolism in experimentally induced cardiac failure. Circ Res.
43. Sharov VG, Goussev A, Lesch M, et al. Abnormal mitochondrial function in myocardium of dogs with chronic heart
failure. J Mol Cell Cardiol.
44. Sharov VG, Todor AV, Silverman N, et al. Abnormal mitochondrial respiration
in failed human myocardium. J Mol Cell Cardiol.
45. Humphries KM, Yoo Y, Szweda LI. Inhibition of NADH-linked mitochondrial respiration
by 4-hydroxy-2-nonenal. Biochemistry.
46. Gutknecht J. Salicylates and proton transport through lipid bilayer membranes: a model for salicylate-induced uncoupling and swelling in mitochondria
. J Membr Biol.