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Inhibition of Cardiac Mitochondrial Respiration by Salicylic Acid and Acetylsalicylate

Nulton-Persson, Amy C. PhD*; Szweda, Luke I. PhD*; Sadek, Hesham A. MD, PhD†

Journal of Cardiovascular Pharmacology: November 2004 - Volume 44 - Issue 5 - pp 591-595
Original Article

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 α-ketoglutarate dehydrogenase (KGDH). Kinetic analysis indicates that salicylic acid acts as a competitive inhibitor at the α-ketoglutarate binding site. In contrast, acetylsalicylate inhibited the enzyme in a noncompetitive fashion consistent with interaction with the α-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.

From the *Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio; and †Division of Cardiology, University Hospitals of Cleveland, Cleveland, Ohio.

Received for publication July 2, 2004; accepted August 19, 2004.

Reprints: Hesham A. Sadek, MD, PhD, Division of Cardiology, University Hospitals of Cleveland and Case Western Reserve University, 11100 Euclid Avenue, Mail Stop 5038, Cleveland, OH 44106 (e-mail: Has4@po.cwru.edu).

Salicylates, which fall under the general classification of nonsteroidal antiinflammatory drugs (NSAIDs), are the most widely used medications. The effectiveness of acetylsalicylate (aspirin) derives from its known analgesic, antipyretic, antiinflammatory, and antithrombotic properties.1,2 The antithrombotic effects of acetylsalicylate have been attributed to irreversible inhibition of platelet cyclooxygenase-1 (COX-1),3-5 which blocks prostaglandin production and subsequently platelet activation, thrombus formation, and inflammation. The extensive clinical use of acetylsalicylate in the last 2 decades is primarily because of its proven beneficial effects in cardiovascular medicine.1 In recent years, salicylates have been shown to be effective in primary6,7 and secondary prevention of cardiovascular events8-12 and treatment of unstable angina13-15 as well as treatment of acute myocardial infarction.16-19 Inhibition of platelet aggregation and the subsequent decrease in coronary thrombosis are believed to be the primary mechanism of action in prevention and treatment of cardiovascular events.1 Other beneficial effects of salicylates on the ischemic myocardium as well as on coronary vasculature have been attributed to their ability to act as antiinflammatory agents and free radical scavengers.20,21

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. It has previously been reported that salicylic acid can alter mitochondrial metabolism.22 These effects have been studied primarily from the standpoint of salicylate toxicity, particularly with regard to metabolic acidosis associated with salicylic acid overdose22 as well as complications arising from therapeutic doses of salicylates observed in Reye syndrome,23-26 gastric mucosal ulceration,27 and analgesic nephropathy.28-30 In isolated liver and kidney mitochondria, salicylic acid has been shown to act as an uncoupler of oxidative phosphorylation31-38 and can inhibit ADP-dependent mitochondrial respiration.23,34-36,38-40 Although a wide variety of enzymes, including dehydrogenases, decarboxylases, and aminotransferases, can be inhibited by salicylic acid in vitro,22 specific mechanisms responsible for inhibition of mitochondrial respiration have not been determined.

Despite the widespread use of salicylates in the treatment and prevention of cardiovascular disease, the effects of these compounds on cardiac mitochondria have not been investigated. The need for such studies is underscored by findings that mitochondrial function is compromised during cardiac ischemia and reperfusion41 and in heart failure.42-44 In addition, declines in mitochondrial respiration may contribute to cardiac myocyte acidosis and necrosis induced by salicylic acid overdose. In the present study, the effects of salicylic acid and acetylsalicylate on isolated cardiac mitochondria were investigated. We report a novel mechanism of inhibition of mitochondrial function by salicylates and discuss clinical and research implications.

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MATERIALS AND METHODS

Isolation of Subsarcolemmal Mitochondria from Rat Heart

Protocols for maintenance and utilization of rats were approved by the Case Western Reserve University Animal Care and Use Committee. Sprague Dawley rats (250-300 g) obtained from Zivic Miller Laboratories were anesthetized with sodium pentobarbital (∼250 mg/kg) and decapitated. Hearts were removed and immediately immersed and rinsed in ice-cold homogenization buffer containing 180 mM KCl, 5.0 mM MOPS, and 2.0 mM EDTA at pH 7.4. Hearts (0.8-1.0 g) were then minced and homogenized in 20 mL of homogenization buffer with a Polytron homogenizer (low setting, 2 seconds). The homogenate was centrifuged at 500 × g for 5 minutes (5°C), and the supernatant was filtered through cheesecloth. The mitochondrial pellet was obtained on centrifugation of the supernatant at 5000 × g for 10 minutes (5°C). After 2 rinses with ice-cold buffer, the mitochondria were resuspended into homogenization buffer to a final concentration of approximately 35.0 mg/mL. Protein determinations were made using the bicinchoninic acid method (Pierce), using BSA as a standard.

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Electron Transport Chain Assay

Mitochondria were diluted to a protein concentration of 0.5 mg/mL in a hypotonic buffer containing 25 mM KH2PO4 and 0.5 mM EDTA at pH 7.4 to induce swelling. Respiration was initiated by the addition of 1.0 mM NADH, and rates of O2 consumption were measured using a Clark-style oxygen electrode (Instech) in the presence and absence of 10 mM salicylic acid.

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Glutamate-Linked Respiration

Mitochondria were diluted to a protein concentration of 0.5 mg/mL in buffer containing 5.0 mM KH2PO4, 180 mM KCl, and 15 mM glutamate at pH 7.25. State 3 respiration was initiated with the addition of 0.5 mM ADP. Respiratory rates were recorded in the presence of 0 to 10.0 mM salicylic acid or acetylsalicylate as indicated. The rate of state 4 respiration was acquired following consumption of ADP. O2 consumption was measured using a Clark-style oxygen electrode (Instech)

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Uncoupled Respiration

Mitochondria were diluted to 0.5 mg/mL in buffer containing 5.0 mM KH2PO4, and 180 mM KCl. Uncoupled respiration was initiated with the addition of 60 μM dinitrophenol. Salicylic acid or acetylsalicylate was added at concentrations of 0 to 10 mM as indicated. Respiration was measured using a Clark-style oxygen electrode (Instech). All respiratory measurements were performed at room temperature.

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Krebs Cycle Enzyme Activities

Mitochondria were diluted to 0.05 mg/mL in 25.0 mM KH2PO4 and 0.5 mM EDTA at pH 7.25 containing 0.01% Triton X-100 and placed in a sonicating water bath for 30 seconds (Fisher Scientific). KGDH activity was assayed as the rate of NAD+ reduction (340 nm, ϵ = 6200 m−1·cm−1) on addition of 5.0 mM MgCl2, 40.0 μM rotenone, 2.5 mM α-ketoglutarate, 0.1 mM CoASH, 0.2 mM thymine pyrophosphate, and 1.0 mM NAD+ to sonicated mitochondria (0.05 mg/mL mitochondrial protein). Salicylic acid or acetylsalicylate was added just before enzyme analysis to determine its effects on KGDH activity. The effect of salicylates on the kinetic parameters of KGDH were determined for each of the enzyme cofactors and substrates by measuring enzyme activity in the presence of maximal and varying submaximal concentrations of these species.

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Data Analysis

Each figure is representative of at least 5 separate experiments utilizing mitochondria isolated from different rat hearts.

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RESULTS

The Effect of Salicylic Acid and Acetylsalicylate on Mitochondrial Respiration

Incubation of mitochondria with salicylic acid resulted in an increase in state 4 (ADP-independent) respiration and a decrease in state 3 (ADP-dependent) respiration (Fig. 1). As shown in Figure 2, the uncoupling effect (increase in state 4 respiration) reached a maximum at concentrations of salicylic acid between 2.0 and 4.0 mM and resulted in a 5-fold increase in state 4 respiration. Although these observations are consistent with previous reports,31-38 it is of interest that maximum rates of uncoupled respiration are not achieved with increasing concentrations of salicylic acid. Instead, we observed a submaximal peak and a subsequent decline in respiratory rates at concentrations of salicylic acid greater than 4.0 mM (Fig. 2). This was because of salicylic acid-induced inhibition of NADH-linked ADP-dependent (state 3) respiration (IC50 ∼5.0 mM salicylic acid) (Fig. 2). The acetylated form of salicylic acid (acetylsalicylate) was used to gain insight into mechanisms responsible for uncoupling and inhibition of respiration. In contrast to salicylic acid, incubation of mitochondria with acetylsalicylate resulted in only a 2-fold increase in state 4 respiration (Figs. 1 and 2). Nevertheless, the concentration-dependent inhibition of state 3 respiration was similar for salicylic acid and acetylsalicylate (Fig. 1).

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The Effect of Salicylic Acid on Uncoupled Respiration and the Electron Transport Chain

To determine whether inhibition of state 3 respiration is caused by inactivation of either the ATPase and/or the adenine nucleotide translocase (ANT), mitochondria were uncoupled using dinitrophenol (DNP). This protocol stimulates maximum rates of electron transport and oxygen consumption independent of ATP synthesis or ADP transport. Thus, inhibition of DNP-induced uncoupled respiration would indicate effects on mitochondrial components other than ATP synthetic machinery. Salicylic acid inhibited uncoupled respiration in a concentration-dependent fashion with relative loss in activity (not shown) similar to that observed for ADP-dependent respiration (state 3), indicating that salicylic acid inhibits the electron transport chain and/or limits the supply of NADH. To determine if the electron transport chain was affected by salicylic acid, mitochondria were diluted in a hypotonic buffer, and NADH was added as the electron donor, thus bypassing Krebs-cycle-dependent synthesis of NADH. Oxygen consumption was monitored as an indicator of the rate of electron transport. The resulting rates reflected little to no inhibition of electron transport (not shown). These results indicate that salicylic acid inhibits state 3 respiration by limiting the supply of NADH to the electron transport chain.

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Effect of Salicylic Acid on Krebs Cycle Enzyme Activity

We have previously shown that α-ketoglutarate dehydrogenase (KGDH) is the rate-limiting enzyme for glutamate-supported mitochondrial state 3 respiration.45 To further elucidate the mechanism by which salicylic acid and acetylsalicylate inhibit mitochondrial respiration, the impact of these compounds on KGDH activity was assessed. Inclusion of salicylic acid and acetylsalicylate in the assay mixture resulted in a concentration-dependent decrease in KGDH activity (Fig. 3). Increasing the concentration of CoASH, Mg2+, TPP, or NAD+ resulted in no significant reduction in the relative level of KGDH inhibition (not shown). In contrast, increases in α-ketoglutarate concentration relieved inhibition observed for salicylic acid but not acetylsalicylate. Kinetic analysis was performed to determine the effect of salicylic acid and acetylsalicylate on the maximum rate (Vmax) of KGDH and the KM for α-ketoglutarate. This result demonstrates that salicylic acid acts as a competitive inhibitor of KGDH, significantly increasing the KM for α-ketoglutarate with no effect on Vmax (Fig. 4). On the other hand, acetylsalicylate inhibited KGDH activity in a noncompetitive fashion, resulting in no appreciable change in the KM for α-ketoglutarate but a significant decrease in Vmax (Fig. 4). This result is in keeping with irreversible inhibition of cyclooxygenase-1 by acetylsalicylate that is the result of enzyme acetylation.3-5

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DISCUSSION

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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Keywords:

salicylic acid; acetylsalicylate; mitochondria; heart; respiration; α-ketoglutarate dehydrogenase

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