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Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria
FEBS Letters 579 (2005) 4724–4728
FEBS 29844
Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria Michał Wasilewski, Lech Wojtczak* Nencki Institute of Experimental Biology, Pasteura 3, PL-02-093 Warsaw, Poland Received 27 May 2005; revised 27 June 2005; accepted 20 July 2005 Available online 8 August 2005 Edited by Maurice Montal
Abstract Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wie˛ckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151–163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death. Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: N-Acylethanolamine; Anandamide; N-Arachidonoylethanolamine; Reactive oxygen species; Respiratory chain complexes; Heart mitochondria
1. Introduction N-Acylethanolamines (NAEs) are derivatives of fatty acids in which the carboxylic group of the fatty acid is bound by an amide linkage to the amino group of ethanolamine. NAEs and their precursors N-acylethanolamine phospholipids are present in various mammalian tissues at the amounts ranging from about 0.1 to over 20 nmol/g (for reviews see [1–3]), but
* Corresponding author. Fax: +48 22 822 5342. E-mail address: [email protected] (L. Wojtczak).
Abbreviations: DCFH2, 2 0 ,7 0 -dichlorodihydrofluorescein; DCFH2-DA, 2 0 ,7 0 -dichlorodihydrofluorescein diacetate; DCPIP, 2,6-dichlorophenolindophenol; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; NAE, N-acylethanolamine; N-Ara, N-arachidonoylethanolamine; N-Ole, N-oleoylethanolamine; N-Pal, N-palmitoylethanolamine; ROS, reactive oxygen species; Dw, mitochondrial transmembrane potential
their content enormously increases under ischemic conditions, e.g., to 500 nmol/g tissue in canine heart [4] and several fold in rat brain [5]. N-Arachidonoylethanolamine (N-Ara), named anandamide, appeared to be an endogenous ligand of the brain cannabinoid receptor [6,7]. This compound and other NAEs have been found to promote apoptosis and/or inhibit cell proliferation in various cell types [8]. In contrast to a vast literature on the action of NAEs through cell surface receptors (reviewed in [9,10]) much less is known on their interaction with intracellular structures [11]. In a previous investigation [12] we characterized NAEs as moderate mitochondrial uncouplers and openers of the permeability transition pore. We also confirmed the observation of Epps et al. [11] that NAEs exert an inhibitory effect on the mitochondrial uncoupled respiration. The aim of the present study was to specify more precisely the site(s) of attack of N-oleoylethanolamine (N-Ole) and N-Ara on the respiratory chain. We also observed that these NAEs decreased the production of reactive oxygen species (ROS) by respiring mitochondria, but partly prevented the decrease of ROS production caused by chemical protonophores.
2. Materials and methods Rat heart mitochondria were isolated using digestion with nagarse according to Idell-Wenger et al. [13]. Inside-out submitochondrial particles were obtained by brief sonication of mitochondria in 10 mM Tris/HCl (pH 7.4) and subsequent isolation of the particles by ultracentrifugation. Disrupted right-sided mitochondria were obtained by freezing and thawing three times. Except where stated otherwise, the standard incubation medium contained 150 mM KCl and 10 mM Mops/KOH (pH 7.4). Oxygen uptake was measured with a Clark type oxygen electrode (YSI, Yellow Springs, OH, USA) at 25 °C. Spectrophotometric and spectrofluorimetric measurements were made at room temperature that was close to 25 °C using double beam/dual wavelength Shimadzu spectrophotometer model UV-3000 and Shimadzu spectrofluorimeter model RF-5000, respectively. Decylubiquinol used as electron donor for complex III was obtained from decylubiquinone by reduction with NaBH4 [14]. A small crystal of NaBH4 was added to 10 mM decylubiquinone in ethanol. When the liquid became colourless, it was transferred to another tube, carefully omitting the crystal. It was then acidified by addition of 0.1 volume of 1 M HCl and stored under nitrogen. For measurement of H2O2 the fluorescent probe 2 0 ,7 0 -dichlorodihydrofluorescein (DCFH2) was employed [15]. This compound was obtained from its commercially available diacetate ester by alkaline hydrolysis immediately before experiment. Methanol, 5 mM 2 0 ,7 0 dichlorodihydrofluorescein diacetate (DCFH2-DA) and 10 mM KOH were mixed in a ratio of 4:1:20 and this mixture was kept in darkness at room temperature for 30 min. Then, a 1.5-fold volume of 100 mM Tris/HCl (pH 7.4) was added to neutralize the mixture. The probe was then kept on ice in darkness. The assay was performed in 3.0 ml
0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.047
M. Wasilewski, L. Wojtczak / FEBS Letters 579 (2005) 4724–4728 of the standard medium supplemented with 5 lM DCFH2 thus obtained, 1 mM EGTA, 5 mM phosphate and the respiratory substrates 5 mM glutamate + 5 mM malate or 5 mM succinate and containing mitochondria corresponding to 1.0 mg protein. The reaction was started by addition of 10 U of horseradish peroxidase and the fluorescence was measured for 5 min at 500 nm excitation and 530 emission wavelengths. N-Ara (anandamide), N-Ole, horseradish peroxidase, coenzyme Q1, decylubiquinone, rotenone, 2,6-dichlorophenolindophenol (DCPIP), cytochrome c and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) were from Sigma (St. Louis, MO, USA). N-Palmitoylethanolamine (N-Pal) was from Biomol (Hamburg, Germany). DCFH2-DA was from Molecular Probes (Eugene, OR, USA). Antimycin A was from Boehringer (Mannheim, Germany). Stock 60 mM solutions of NAEs were made in ethanol and kept in dark at 20 °C. Those of N-Ole and N-Ara were, in addition, tightly stoppered under nitrogen. In all experiments control samples were always run with same amounts of the solvent alone.
3. Results 3.1. Respiratory chain complexes In the previous investigation [12] we have observed lowering by N-Ara of mitochondrial respiration with NAD-linked substrates. Such effect might have been due to inhibition of primary dehydrogenases, of the respiratory chain, or of substrate transport across the mitochondrial membrane, or to a combination of these factors. To get a closer insight into the mechanism of this inhibitory action, we now examined the effect of three different species of NAE on the oxidation of NADH by insideout submitochondrial particles. In such a system the observed effect was limited to the action on the respiratory chain only. It was found that all three species of NAE inhibited the respiration of the particles, though to a different degree. N-Ara and N-Ole acted in a similar manner showing half inhibition at about 30 nmol/mg protein. N-Pal was much less active and did not reach 50% inhibition (I50) at the highest concentration used in this study, i.e., 360 nmol/mg protein (Fig. 1). To localize more precisely the site of inhibition at the respiratory chain level, we used artificial electron donors and/or acceptors specific for the individual complexes. In these experiments mitochondria disrupted by repeated freezing and thaw-
Fig. 1. Effect of NAEs on NADH oxidation in submitochondrial particles. The standard incubation medium was supplemented with 1 mM NADH and 1 mM EGTA. The amount of submitochondrial particles corresponded to 0.25–1.0 mg protein/ml. Under these conditions oxygen uptake could be completely inhibited by 1.7 lM rotenone. The data are expressed in percentage of oxygen uptake rate in the absence of NAE that amounted to 255 ± 106 ng atom oxygen/ min per mg protein (n = 5). The symbols indicate: j, N-Ara; d, N-Ole; m, N-Pal.
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ing were used, except for complex II that was investigated in intact mitochondria. Activity of complex I was measured using NADH and CoQ1 as electron donor and acceptor, respectively. Antimycin A was used to block electron transfer to complex III. We found that the inhibitory patterns of NAEs were in this case similar to those for the entire respiratory chain. N-Ara at the concentration of 200 nmol/mg protein inhibited complex I by 80% with I50 at about 50 nmol/mg and N-Ole was only slightly less active (Fig. 2A). N-Pal was little active at that concentration. In contrast, non-esterified arachidonic acid inhibited complex I to a small extent only. Neither reduced glutathione nor dithiothreitol had any protective effect against the inhibition of complex I by N-Ara (not shown). As mentioned, complex II was measured in intact mitochondria because its activity became strongly reduced by freezing– thawing or sonication. With succinate as electron donor and DCPIP as artificial electron acceptor, this complex was inhibited by N-Ara and N-Ole at concentrations of 200 nmol/mg protein by 40% and 20%, respectively, and was practically unaffected by N-Pal (Fig. 2B). Complex III was measured with decylubiquinol and cytochrome c as electron donor and electron acceptor, respectively. It appeared to be inhibited by about 50% by either N-Ara or N-Ole at 200 nmol/mg protein (Fig. 2C). No inhibition of complex IV could be observed with either of the NAE species used (Fig. 2D). 3.2. Reactive oxygen species The primary product of single electron reduction of molecular oxygen in the mitochondrial respiratory chain, the so-called electron leak, is the superoxide anion ðO2 Þ. This molecule is usually rapidly converted by mitochondrial superoxide dismutase to H2O2 that can leave mitochondria and can be detected in the medium as an indicator of ROS formation. Since it has been found previously [12] that NAEs increase proton conductance of the inner mitochondrial membrane and have inhibitory effects on the respiratory chain (see also Section 3.1), in this investigation we compared their effects on ROS production with those of typical protonophores and respiratory inhibitors. With glutamate + malate as respiratory substrates, respiratory chain inhibitors rotenone and antimycin A stimulated ROS production several fold, whereas FCCP was inhibitory (Fig. 3A). However, FCCP did not significantly diminish ROS production in mitochondria inhibited by rotenone (Fig. 3A) or antimycin A (not shown). Under these conditions N-Ara appeared to be significantly inhibitory when added alone and was without effect added on top of FCCP (Fig. 3A), which shows that it acted as an uncoupler rather than a respiratory chain blocker. A similar effect was observed for N-Ole (not shown). With succinate as the respiratory substrate, ROS production was also maintained at high level because of a high reduction state of nicotinamide nucleotides due to the reversed electron flux. As expected, under these conditions ROS generation was strongly depressed by FCCP (Fig. 3B). Rotenone was much less inhibitory, although at the concentration used it completely blocked the reversed electron flow. This was apparently due to the fact that NAD reduction was also maintained by endogenous respiratory substrates and oxidation of NADH thus formed was blocked by rotenone. Such explanation is corroborated by the
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M. Wasilewski, L. Wojtczak / FEBS Letters 579 (2005) 4724–4728
Fig. 2. Effect of NAEs and arachidonic acid on the activities of individual respiratory chain complexes. (A) Complex I was measured using CoQ1 as the artificial electron acceptor as described by Estronell et al. [14]. The incubation medium contained 50 mM KCl, 10 mM Tris/HCl (pH 7.4), 75 lM NADH, 2 mM KCN, 3 lM antimycin A and 1 mM EGTA. The amount of disrupted mitochondria corresponded to 20 lg/ml. The rate of NADH oxidation was recorded at 340–380 nm and was started by addition of 50 lM CoQ1. Activity of complex I was calculated by subtracting the rate after addition of 1.7 lM rotenone from the total rate of NADH oxidation. It amounted for the control to 297 ± 65 nmol NADH/min per mg protein (n = 7). (B) Complex II. This was measured in intact mitochondria according to King [16] with modification. As electron acceptor DCPIP alone, without phenazine methosulphate, was used. The standard medium was supplemented with 1.7 lM rotenone, 3 lM antimycin A, 2 mM KCN, 5 mM succinate, 5 mM phosphate and 1 mM EGTA. Concentration of mitochondrial protein was 0.33 mg/ml. The reaction was started by addition of 100 lM DCPIP and the rate of its reduction was recorded at 600 nm. Complex II activity was calculated by subtracting the reaction rate after inhibition of succinate dehydrogenase by 1 mM thenoyltrifluoroacetone. It amounted to 27 ± 11 nmol DCPIP/min per mg protein (n = 4). (C) Complex III was measured in disrupted mitochondria using decylubiquinol as the artificial electron donor and cytochrome c as electron acceptor according to Trounce et al. [17]. The reaction medium contained 250 mM sucrose, 50 mM Tris/HCl (pH 7.4), 1 mM EGTA, 50 lM cytochrome c and 2 mM KCN. Disrupted mitochondria (10 lg protein/ml) were added, followed 10 min later by 50 lM decylubiquinol and the rate of cytochrome c reduction during the first minute was recorded at 550–540 nm. Complex III activity was calculated by subtraction the rate of cytochrome c reduction in the presence of 3 lM antimycin A. It amounted for the control to 219 ± 104 nmol cytochrome c/min per mg protein (n = 7). (D) Complex IV. This was determined by measuring oxygen uptake in a system containing the standard incubation medium supplemented with 100 lM cytochrome c and 5 mM ascorbate. The amount of disrupted mitochondria corresponded to 50 lg protein/ml. Each assay was terminated by addition of 4 mM NaN3 and the activity of complex IV was calculated as difference between the initial O2 uptake rate and that after inhibition by azide. The control activity amounted to 543 ± 145 ng atom oxygen/min per mg protein (n = 3). Symbols: h, arachidonic acid; other symbols are the same as in Fig. 1.
Fig. 3. Effect of N-Ara on generation of ROS by mitochondria. The assay was performed in 3.0 ml of the standard medium supplemented with 5 lM DCFH2, 1 mM EGTA and 5 mM phosphate and the respiratory substrates 5 mM glutamate + 5 mM malate (A) or 5 mM succinate (B). The samples contained mitochondria corresponding to 330 lg/ml protein (A) or 67 lg/ml protein (B). Other additions were as follows: N-arachidonoylethanolamine (N-Ara), 100 nmol/mg protein; rotenone (Rot), 1.7 lM; antimycin A (AntA), 3 lM; FCCP, 1.7 lM. The reaction was started by addition of 10 U of horseradish peroxidase. Fluorescence (excitation, 500 nm; emission, 530 nm) was measured 5 min after addition of peroxidase. The results are means ± S.D. for 5–11 experiments in A and 3–6 experiments in B. Statistical significance: **P < 0.01 with respect to the control (A); *P < 0.05 with respect to FCCP alone (B).
observation that rotenone counteracted the inhibition by FCCP (Fig. 3B). In this rather complex situation N-Ara appeared to be more inhibitory than rotenone but significantly less than FCCP. Moreover, N-Ara partly but also significantly abolished the inhibitory action of FCCP (Fig. 3B).
4. Discussion The present results confirm previous observations [12] on the inhibitory effect of NAEs on mitochondrial respiration. We also show that the inhibition was due to the action on the
M. Wasilewski, L. Wojtczak / FEBS Letters 579 (2005) 4724–4728
respiratory chain rather than on primary dehydrogenases or substrate transport. In the present paper, we show that the most affected point of the respiratory chain is complex I, followed by weaker effects on complexes II and III and no inhibition of complex IV. The action of NAEs thus resembles qualitatively but not quantitatively that of arachidonic acid [18] and ceramides (except that ceramides also affected complex IV) [19]. It has to be mentioned, however, that, in contrast to the results of Cocco et al. [18], in our hands the inhibitory effect of arachidonic acid on complex I was negligible (Fig. 2A). This can be explained by the fact that the amounts of this fatty acid used by these authors, calculated per milligram of submitochondrial particles, were at least three times higher. This shows again that NAEs are stronger inhibitors of complex I than corresponding non-esterified fatty acids. The nature of the inhibition remains, however, to be further elucidated. As found, it was not related to a possible effect on functional SH groups. The unspecific and of little physiological importance membrane-perturbing action of NAEs in their micellar form can also be excluded. Although the critical micelle concentration (CMC) of NAEs has not been, to our knowledge, determined, we can infer from their chemical structure that it must be higher than that for corresponding fatty acids. CMC for arachidonic acid in Ca2+free medium has been determined as 20 lM [20], whereas the absolute concentration of N-Ara in the present experiments with respiratory chain complexes never exceeded that value. Moreover, CMC values for N-Ole and N-Pal should be lower. Yet, their inhibitory effects were always weaker than those of N-Ara. Thus, it can be concluded that all the observed effects of NAEs were due to their monomeric forms. At the present state it can only be speculated that certain NAEs, in particular those with unsaturated fatty acid moieties, can re-arrange the molecular assembly of particular respiratory chain complexes by inserting within the phospholipid core of the inner membrane and thus altering its physicochemical integrity. A similar mechanism may be responsible for increasing proton permeability described previously [12]. In contrast to nonesterified fatty acids, NAEs have no dissociable protons and therefore it is rather unlikely that they function as protonophores. In this context it is interesting to note that ceramides and NAEs are characterized by three common structural features: (i) long hydrocarbon chain(s), (ii) amide linkage and (iii) free alcoholic OH group. It seems therefore likely that they may perturb the inner mitochondrial membrane in a similar way [19]. It is generally assumed that the main sites of oxygen free radical generation at the mitochondrial level are complexes I and III of the respiratory chain and that the rate of ROS production is controlled by factors such as the redox state of respiratory complexes, the rate of electron flow and the magnitude of Dw [21–24]. Thus, uncouplers of oxidative phosphorylation that increase the electron flow through the respiratory chain and collapse Dw drastically decrease ROS production, whereas respiratory inhibitors of the electron transport have usually the opposite effect. Moreover, respiratory inhibitors increase ROS production also in uncoupled mitochondria if they keep the respiratory chain, especially its complex I, in the reduced form. In this context, N-Ara (Fig. 3A) and N-Ole (not shown) behaved as typical uncouplers rather than respiratory inhibitors. Apparently, in iso-
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lated heart mitochondria the degree of complex I reduction is more strongly decreased due to the uncoupling action of NAEs than it could be increased by their inhibitory effect on the electron flow. This is compatible with the action of N-Ara on the respiration of these mitochondria with NAD-linked substrates as illustrated in Fig. 6 of the previous paper [12] where activation rather than inhibition of oxygen uptake could be observed. On the other hand, however, with succinate as the substrate, N-Ara exerted a slight protection against the inhibitory action of chemical protonophores (Fig. 3B) that could be due to its weak inhibitory effect on the respiratory chain. These results on isolated mitochondria do not correlate well with some observations on intact cells and whole organs. For example, ROS production was elevated in PC-12 cells cultured in the presence of micromolar concentrations of N-Ara [25] and in brain mitochondria isolated from rats acutely treated with this compound [26]. The explanation of these conflicting results and the action of NAEs on in vivo free radical reactions await further investigation. It has to be remembered, however, that mitochondria within the cell are not in state 4 but, usually, rather in an active state approaching state 3. Perhaps, only under such conditions the inhibitory effects of NAEs on the respiratory chain and, hence, their activatory action on ROS production could be manifested. The effects of NAEs on isolated mitochondria, as described in the present work, may contribute to our better understanding of the mechanism(s) by which endocannabinoids protect cerebral neurons [27] and isolated rat heart [28] against ischaemia, but may also elicit apoptotic cell death [2,29]. Acknowledgment: This research was partly supported by the State Committee for Scientific Research (Grant No. 3 P04 041 23) and Ministry for Science and Informatics (Grant No. 2 P04C 066 28).
References [1] Schmid, H.H.O., Schmid, P.C. and Natarajan, V. (1990) NAcylated glycerophospholipids and their derivatives. Progr. Lipid Res. 29, 1–43. [2] Hansen, H.S., Moesgaard, B., Hansen, H.H. and Petersen, G. (2000) N-Acylethanolamines and precursor phospholipids – relation to cell injury. Chem. Phys. Lipids 108, 135–150. [3] Maccarrone, M. and Finazzi-Agro`, A. (2002) Endocannabinoids and their actions. Vitam. Horm. 65, 225–255. [4] Epps, D.E., Schmid, P.C., Natarajan, V. and Schmid, H.H.O. (1979) N-Acylethanolamine accumulation in infarcted myocardium. Biochem. Biophys. Res. Commun. 90, 628–633. [5] Moesgaard, B., Jaroszewski, J.W. and Hansen, H.S. (1999) Accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31p NMR study. J. Lipid Res. 40, 515–521. [6] Devane, W.A., Hanusˇ, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. and Mechoulam, R. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946–1949. [7] Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J.-C. and Piomelli, D. (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686–691. [8] Maccarrone, M. and Finazzi-Agro`, A. (2003) The endocannabinoid system, anandamide and the regulation of mammalian cell apoptosis. Cell Death Differ. 10, 946–955. [9] Di Marzo, V., De Petrocellis, L., Fezza, F., Ligresti, A. and Bisogno, T. (2002) Anandamide receptors. Prostag. Leukotr. Ess. Fatty Acids 66, 377–391.
4728 [10] Fride, E. (2002) Endocannabinoids in the central nervous system – an overview. Prostag. Leukotr. Ess. Fatty Acids 66, 221–233. [11] Epps, D.E., Palmer, J.W., Schmid, H.H.O. and Pfeiffer, D.R. (1982) Inhibition of permeability-dependent Ca2+ release from mitochondria by N-acylethanolamines, a class of lipids synthesized in ischemic heart tissue. J. Biol. Chem. 257, 1383–1391. [12] Wasilewski, M., Wieckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Effects of N-acylethanolamines on mitochondrial energetics and permeability transition. Biochim. Biophys. Acta 1657, 151–163. [13] Idell-Wenger, J.A., Grotyohann, L.W. and Neely, J.R. (1982) An improved method for isolation of mitochondria in high yields from normal, ischemic and autolysed rat hearts. Anal. Biochem. 125, 269–276. [14] Estronell, E., Fato, R., Pallotti, F. and Lenaz, G. (1993) Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase. FEBS Lett. 332, 127–131. [15] Rota, C., Chignell, C.F. and Mason, R.P. (1999) Evidence for free radical formation during the oxidation of 2 0 ,7 0 -dichlorofluorescin to the fluorescent dye 2 0 ,7 0 -dichlorofluorescein by horseradish peroxidase: Possible implications for oxidative stress measurements. Free Radic. Biol. Med. 27, 873–881. [16] King, T.E. (1963) Reconstitution of respiratory chain enzyme systems. XI. Use of artificial electron acceptors in the assay of succinate-dehydrogenating enzymes. J. Biol. Chem. 238, 4032– 4036. [17] Trounce, I.A., Kim, Y.L., Jun, A.S. and Wallace, D.C. (1996) Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol. 264, 484–509. [18] Cocco, T., Di Paola, M., Papa, S. and Lorusso, M. (1999) Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation. Free Radic. Biol. Med. 27, 51–59.
M. Wasilewski, L. Wojtczak / FEBS Letters 579 (2005) 4724–4728 [19] Di Paola, M., Cocco, T. and Lorusso, M. (2000) Ceramide interaction with respiratory chain of heart mitochondria. Biochemistry 39, 6660–6668. [20] Richieri, G.V., Ogata, R.T. and Kleinfeld, A.M. (1992) A fluorescently labeled intestinal fatty acid binding protein. Interactions with fatty acids and its use in monitoring free fatty acids. J. Biol. Chem. 267, 23495–23501. [21] Skulachev, V.P. (1996) Role of uncoupled and non-coupled oxidation in maintenance of safety low levels of oxygen and its one-electron reductants. Q. Rev. Biophys. 29, 169–202. [22] Korshunov, S.S., Skulachev, V.P. and Starkov, A.A. (1997) High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416, 15–18. [23] Lenaz, G. (2001) The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology. IUBMB Life 52, 159–164. [24] St-Pierre, J., Buckingham, J.A., Roebuck, S.J. and Brand, M.D. (2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain. J. Biol. Chem. 277, 44784–44790. [25] Sarker, K.P., Obara, S., Nakata, M., Kitajima, I. and Maruyama, I. (2000) Anandamide induces apoptosis of PC-12 cells: involvement of superoxide and caspase-3. FEBS Lett. 472, 39–44. [26] Costa, B. and Colleoni, M. (2000) Changes in rat brain energetic metabolism after exposure to anandamide or D9-tetrahydrocannabinol. Eur. J. Pharmacol. 395, 1–7. [27] Sinor, A.D., Irvin, S.M. and Greenberg, D.A. (2000) Endocannabinoids protect cerebral cortical neurons from in vitro ischemia in rats. Neurosci. Lett. 278, 157–160. [28] Le´picier, P., Bouchard, J.-F., Lagneux, C. and Lamontagne, D. (2003) Endocannabinoids protect the rat isolated heart against ischaemia. Br. J. Pharmacol. 139, 805–815. [29] Maccarrone, M. and Finazzi-Agro`, A. (2003) The endocannabinoid system, anandamide and the regulation of mammalian cell apoptosis. Cell Death Differ. 10, 946–955.
FEBS 29844
Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria Michał Wasilewski, Lech Wojtczak* Nencki Institute of Experimental Biology, Pasteura 3, PL-02-093 Warsaw, Poland Received 27 May 2005; revised 27 June 2005; accepted 20 July 2005 Available online 8 August 2005 Edited by Maurice Montal
Abstract Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wie˛ckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151–163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death. Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: N-Acylethanolamine; Anandamide; N-Arachidonoylethanolamine; Reactive oxygen species; Respiratory chain complexes; Heart mitochondria
1. Introduction N-Acylethanolamines (NAEs) are derivatives of fatty acids in which the carboxylic group of the fatty acid is bound by an amide linkage to the amino group of ethanolamine. NAEs and their precursors N-acylethanolamine phospholipids are present in various mammalian tissues at the amounts ranging from about 0.1 to over 20 nmol/g (for reviews see [1–3]), but
* Corresponding author. Fax: +48 22 822 5342. E-mail address: [email protected] (L. Wojtczak).
Abbreviations: DCFH2, 2 0 ,7 0 -dichlorodihydrofluorescein; DCFH2-DA, 2 0 ,7 0 -dichlorodihydrofluorescein diacetate; DCPIP, 2,6-dichlorophenolindophenol; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; NAE, N-acylethanolamine; N-Ara, N-arachidonoylethanolamine; N-Ole, N-oleoylethanolamine; N-Pal, N-palmitoylethanolamine; ROS, reactive oxygen species; Dw, mitochondrial transmembrane potential
their content enormously increases under ischemic conditions, e.g., to 500 nmol/g tissue in canine heart [4] and several fold in rat brain [5]. N-Arachidonoylethanolamine (N-Ara), named anandamide, appeared to be an endogenous ligand of the brain cannabinoid receptor [6,7]. This compound and other NAEs have been found to promote apoptosis and/or inhibit cell proliferation in various cell types [8]. In contrast to a vast literature on the action of NAEs through cell surface receptors (reviewed in [9,10]) much less is known on their interaction with intracellular structures [11]. In a previous investigation [12] we characterized NAEs as moderate mitochondrial uncouplers and openers of the permeability transition pore. We also confirmed the observation of Epps et al. [11] that NAEs exert an inhibitory effect on the mitochondrial uncoupled respiration. The aim of the present study was to specify more precisely the site(s) of attack of N-oleoylethanolamine (N-Ole) and N-Ara on the respiratory chain. We also observed that these NAEs decreased the production of reactive oxygen species (ROS) by respiring mitochondria, but partly prevented the decrease of ROS production caused by chemical protonophores.
2. Materials and methods Rat heart mitochondria were isolated using digestion with nagarse according to Idell-Wenger et al. [13]. Inside-out submitochondrial particles were obtained by brief sonication of mitochondria in 10 mM Tris/HCl (pH 7.4) and subsequent isolation of the particles by ultracentrifugation. Disrupted right-sided mitochondria were obtained by freezing and thawing three times. Except where stated otherwise, the standard incubation medium contained 150 mM KCl and 10 mM Mops/KOH (pH 7.4). Oxygen uptake was measured with a Clark type oxygen electrode (YSI, Yellow Springs, OH, USA) at 25 °C. Spectrophotometric and spectrofluorimetric measurements were made at room temperature that was close to 25 °C using double beam/dual wavelength Shimadzu spectrophotometer model UV-3000 and Shimadzu spectrofluorimeter model RF-5000, respectively. Decylubiquinol used as electron donor for complex III was obtained from decylubiquinone by reduction with NaBH4 [14]. A small crystal of NaBH4 was added to 10 mM decylubiquinone in ethanol. When the liquid became colourless, it was transferred to another tube, carefully omitting the crystal. It was then acidified by addition of 0.1 volume of 1 M HCl and stored under nitrogen. For measurement of H2O2 the fluorescent probe 2 0 ,7 0 -dichlorodihydrofluorescein (DCFH2) was employed [15]. This compound was obtained from its commercially available diacetate ester by alkaline hydrolysis immediately before experiment. Methanol, 5 mM 2 0 ,7 0 dichlorodihydrofluorescein diacetate (DCFH2-DA) and 10 mM KOH were mixed in a ratio of 4:1:20 and this mixture was kept in darkness at room temperature for 30 min. Then, a 1.5-fold volume of 100 mM Tris/HCl (pH 7.4) was added to neutralize the mixture. The probe was then kept on ice in darkness. The assay was performed in 3.0 ml
0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.047
M. Wasilewski, L. Wojtczak / FEBS Letters 579 (2005) 4724–4728 of the standard medium supplemented with 5 lM DCFH2 thus obtained, 1 mM EGTA, 5 mM phosphate and the respiratory substrates 5 mM glutamate + 5 mM malate or 5 mM succinate and containing mitochondria corresponding to 1.0 mg protein. The reaction was started by addition of 10 U of horseradish peroxidase and the fluorescence was measured for 5 min at 500 nm excitation and 530 emission wavelengths. N-Ara (anandamide), N-Ole, horseradish peroxidase, coenzyme Q1, decylubiquinone, rotenone, 2,6-dichlorophenolindophenol (DCPIP), cytochrome c and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) were from Sigma (St. Louis, MO, USA). N-Palmitoylethanolamine (N-Pal) was from Biomol (Hamburg, Germany). DCFH2-DA was from Molecular Probes (Eugene, OR, USA). Antimycin A was from Boehringer (Mannheim, Germany). Stock 60 mM solutions of NAEs were made in ethanol and kept in dark at 20 °C. Those of N-Ole and N-Ara were, in addition, tightly stoppered under nitrogen. In all experiments control samples were always run with same amounts of the solvent alone.
3. Results 3.1. Respiratory chain complexes In the previous investigation [12] we have observed lowering by N-Ara of mitochondrial respiration with NAD-linked substrates. Such effect might have been due to inhibition of primary dehydrogenases, of the respiratory chain, or of substrate transport across the mitochondrial membrane, or to a combination of these factors. To get a closer insight into the mechanism of this inhibitory action, we now examined the effect of three different species of NAE on the oxidation of NADH by insideout submitochondrial particles. In such a system the observed effect was limited to the action on the respiratory chain only. It was found that all three species of NAE inhibited the respiration of the particles, though to a different degree. N-Ara and N-Ole acted in a similar manner showing half inhibition at about 30 nmol/mg protein. N-Pal was much less active and did not reach 50% inhibition (I50) at the highest concentration used in this study, i.e., 360 nmol/mg protein (Fig. 1). To localize more precisely the site of inhibition at the respiratory chain level, we used artificial electron donors and/or acceptors specific for the individual complexes. In these experiments mitochondria disrupted by repeated freezing and thaw-
Fig. 1. Effect of NAEs on NADH oxidation in submitochondrial particles. The standard incubation medium was supplemented with 1 mM NADH and 1 mM EGTA. The amount of submitochondrial particles corresponded to 0.25–1.0 mg protein/ml. Under these conditions oxygen uptake could be completely inhibited by 1.7 lM rotenone. The data are expressed in percentage of oxygen uptake rate in the absence of NAE that amounted to 255 ± 106 ng atom oxygen/ min per mg protein (n = 5). The symbols indicate: j, N-Ara; d, N-Ole; m, N-Pal.
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ing were used, except for complex II that was investigated in intact mitochondria. Activity of complex I was measured using NADH and CoQ1 as electron donor and acceptor, respectively. Antimycin A was used to block electron transfer to complex III. We found that the inhibitory patterns of NAEs were in this case similar to those for the entire respiratory chain. N-Ara at the concentration of 200 nmol/mg protein inhibited complex I by 80% with I50 at about 50 nmol/mg and N-Ole was only slightly less active (Fig. 2A). N-Pal was little active at that concentration. In contrast, non-esterified arachidonic acid inhibited complex I to a small extent only. Neither reduced glutathione nor dithiothreitol had any protective effect against the inhibition of complex I by N-Ara (not shown). As mentioned, complex II was measured in intact mitochondria because its activity became strongly reduced by freezing– thawing or sonication. With succinate as electron donor and DCPIP as artificial electron acceptor, this complex was inhibited by N-Ara and N-Ole at concentrations of 200 nmol/mg protein by 40% and 20%, respectively, and was practically unaffected by N-Pal (Fig. 2B). Complex III was measured with decylubiquinol and cytochrome c as electron donor and electron acceptor, respectively. It appeared to be inhibited by about 50% by either N-Ara or N-Ole at 200 nmol/mg protein (Fig. 2C). No inhibition of complex IV could be observed with either of the NAE species used (Fig. 2D). 3.2. Reactive oxygen species The primary product of single electron reduction of molecular oxygen in the mitochondrial respiratory chain, the so-called electron leak, is the superoxide anion ðO2 Þ. This molecule is usually rapidly converted by mitochondrial superoxide dismutase to H2O2 that can leave mitochondria and can be detected in the medium as an indicator of ROS formation. Since it has been found previously [12] that NAEs increase proton conductance of the inner mitochondrial membrane and have inhibitory effects on the respiratory chain (see also Section 3.1), in this investigation we compared their effects on ROS production with those of typical protonophores and respiratory inhibitors. With glutamate + malate as respiratory substrates, respiratory chain inhibitors rotenone and antimycin A stimulated ROS production several fold, whereas FCCP was inhibitory (Fig. 3A). However, FCCP did not significantly diminish ROS production in mitochondria inhibited by rotenone (Fig. 3A) or antimycin A (not shown). Under these conditions N-Ara appeared to be significantly inhibitory when added alone and was without effect added on top of FCCP (Fig. 3A), which shows that it acted as an uncoupler rather than a respiratory chain blocker. A similar effect was observed for N-Ole (not shown). With succinate as the respiratory substrate, ROS production was also maintained at high level because of a high reduction state of nicotinamide nucleotides due to the reversed electron flux. As expected, under these conditions ROS generation was strongly depressed by FCCP (Fig. 3B). Rotenone was much less inhibitory, although at the concentration used it completely blocked the reversed electron flow. This was apparently due to the fact that NAD reduction was also maintained by endogenous respiratory substrates and oxidation of NADH thus formed was blocked by rotenone. Such explanation is corroborated by the
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Fig. 2. Effect of NAEs and arachidonic acid on the activities of individual respiratory chain complexes. (A) Complex I was measured using CoQ1 as the artificial electron acceptor as described by Estronell et al. [14]. The incubation medium contained 50 mM KCl, 10 mM Tris/HCl (pH 7.4), 75 lM NADH, 2 mM KCN, 3 lM antimycin A and 1 mM EGTA. The amount of disrupted mitochondria corresponded to 20 lg/ml. The rate of NADH oxidation was recorded at 340–380 nm and was started by addition of 50 lM CoQ1. Activity of complex I was calculated by subtracting the rate after addition of 1.7 lM rotenone from the total rate of NADH oxidation. It amounted for the control to 297 ± 65 nmol NADH/min per mg protein (n = 7). (B) Complex II. This was measured in intact mitochondria according to King [16] with modification. As electron acceptor DCPIP alone, without phenazine methosulphate, was used. The standard medium was supplemented with 1.7 lM rotenone, 3 lM antimycin A, 2 mM KCN, 5 mM succinate, 5 mM phosphate and 1 mM EGTA. Concentration of mitochondrial protein was 0.33 mg/ml. The reaction was started by addition of 100 lM DCPIP and the rate of its reduction was recorded at 600 nm. Complex II activity was calculated by subtracting the reaction rate after inhibition of succinate dehydrogenase by 1 mM thenoyltrifluoroacetone. It amounted to 27 ± 11 nmol DCPIP/min per mg protein (n = 4). (C) Complex III was measured in disrupted mitochondria using decylubiquinol as the artificial electron donor and cytochrome c as electron acceptor according to Trounce et al. [17]. The reaction medium contained 250 mM sucrose, 50 mM Tris/HCl (pH 7.4), 1 mM EGTA, 50 lM cytochrome c and 2 mM KCN. Disrupted mitochondria (10 lg protein/ml) were added, followed 10 min later by 50 lM decylubiquinol and the rate of cytochrome c reduction during the first minute was recorded at 550–540 nm. Complex III activity was calculated by subtraction the rate of cytochrome c reduction in the presence of 3 lM antimycin A. It amounted for the control to 219 ± 104 nmol cytochrome c/min per mg protein (n = 7). (D) Complex IV. This was determined by measuring oxygen uptake in a system containing the standard incubation medium supplemented with 100 lM cytochrome c and 5 mM ascorbate. The amount of disrupted mitochondria corresponded to 50 lg protein/ml. Each assay was terminated by addition of 4 mM NaN3 and the activity of complex IV was calculated as difference between the initial O2 uptake rate and that after inhibition by azide. The control activity amounted to 543 ± 145 ng atom oxygen/min per mg protein (n = 3). Symbols: h, arachidonic acid; other symbols are the same as in Fig. 1.
Fig. 3. Effect of N-Ara on generation of ROS by mitochondria. The assay was performed in 3.0 ml of the standard medium supplemented with 5 lM DCFH2, 1 mM EGTA and 5 mM phosphate and the respiratory substrates 5 mM glutamate + 5 mM malate (A) or 5 mM succinate (B). The samples contained mitochondria corresponding to 330 lg/ml protein (A) or 67 lg/ml protein (B). Other additions were as follows: N-arachidonoylethanolamine (N-Ara), 100 nmol/mg protein; rotenone (Rot), 1.7 lM; antimycin A (AntA), 3 lM; FCCP, 1.7 lM. The reaction was started by addition of 10 U of horseradish peroxidase. Fluorescence (excitation, 500 nm; emission, 530 nm) was measured 5 min after addition of peroxidase. The results are means ± S.D. for 5–11 experiments in A and 3–6 experiments in B. Statistical significance: **P < 0.01 with respect to the control (A); *P < 0.05 with respect to FCCP alone (B).
observation that rotenone counteracted the inhibition by FCCP (Fig. 3B). In this rather complex situation N-Ara appeared to be more inhibitory than rotenone but significantly less than FCCP. Moreover, N-Ara partly but also significantly abolished the inhibitory action of FCCP (Fig. 3B).
4. Discussion The present results confirm previous observations [12] on the inhibitory effect of NAEs on mitochondrial respiration. We also show that the inhibition was due to the action on the
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respiratory chain rather than on primary dehydrogenases or substrate transport. In the present paper, we show that the most affected point of the respiratory chain is complex I, followed by weaker effects on complexes II and III and no inhibition of complex IV. The action of NAEs thus resembles qualitatively but not quantitatively that of arachidonic acid [18] and ceramides (except that ceramides also affected complex IV) [19]. It has to be mentioned, however, that, in contrast to the results of Cocco et al. [18], in our hands the inhibitory effect of arachidonic acid on complex I was negligible (Fig. 2A). This can be explained by the fact that the amounts of this fatty acid used by these authors, calculated per milligram of submitochondrial particles, were at least three times higher. This shows again that NAEs are stronger inhibitors of complex I than corresponding non-esterified fatty acids. The nature of the inhibition remains, however, to be further elucidated. As found, it was not related to a possible effect on functional SH groups. The unspecific and of little physiological importance membrane-perturbing action of NAEs in their micellar form can also be excluded. Although the critical micelle concentration (CMC) of NAEs has not been, to our knowledge, determined, we can infer from their chemical structure that it must be higher than that for corresponding fatty acids. CMC for arachidonic acid in Ca2+free medium has been determined as 20 lM [20], whereas the absolute concentration of N-Ara in the present experiments with respiratory chain complexes never exceeded that value. Moreover, CMC values for N-Ole and N-Pal should be lower. Yet, their inhibitory effects were always weaker than those of N-Ara. Thus, it can be concluded that all the observed effects of NAEs were due to their monomeric forms. At the present state it can only be speculated that certain NAEs, in particular those with unsaturated fatty acid moieties, can re-arrange the molecular assembly of particular respiratory chain complexes by inserting within the phospholipid core of the inner membrane and thus altering its physicochemical integrity. A similar mechanism may be responsible for increasing proton permeability described previously [12]. In contrast to nonesterified fatty acids, NAEs have no dissociable protons and therefore it is rather unlikely that they function as protonophores. In this context it is interesting to note that ceramides and NAEs are characterized by three common structural features: (i) long hydrocarbon chain(s), (ii) amide linkage and (iii) free alcoholic OH group. It seems therefore likely that they may perturb the inner mitochondrial membrane in a similar way [19]. It is generally assumed that the main sites of oxygen free radical generation at the mitochondrial level are complexes I and III of the respiratory chain and that the rate of ROS production is controlled by factors such as the redox state of respiratory complexes, the rate of electron flow and the magnitude of Dw [21–24]. Thus, uncouplers of oxidative phosphorylation that increase the electron flow through the respiratory chain and collapse Dw drastically decrease ROS production, whereas respiratory inhibitors of the electron transport have usually the opposite effect. Moreover, respiratory inhibitors increase ROS production also in uncoupled mitochondria if they keep the respiratory chain, especially its complex I, in the reduced form. In this context, N-Ara (Fig. 3A) and N-Ole (not shown) behaved as typical uncouplers rather than respiratory inhibitors. Apparently, in iso-
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lated heart mitochondria the degree of complex I reduction is more strongly decreased due to the uncoupling action of NAEs than it could be increased by their inhibitory effect on the electron flow. This is compatible with the action of N-Ara on the respiration of these mitochondria with NAD-linked substrates as illustrated in Fig. 6 of the previous paper [12] where activation rather than inhibition of oxygen uptake could be observed. On the other hand, however, with succinate as the substrate, N-Ara exerted a slight protection against the inhibitory action of chemical protonophores (Fig. 3B) that could be due to its weak inhibitory effect on the respiratory chain. These results on isolated mitochondria do not correlate well with some observations on intact cells and whole organs. For example, ROS production was elevated in PC-12 cells cultured in the presence of micromolar concentrations of N-Ara [25] and in brain mitochondria isolated from rats acutely treated with this compound [26]. The explanation of these conflicting results and the action of NAEs on in vivo free radical reactions await further investigation. It has to be remembered, however, that mitochondria within the cell are not in state 4 but, usually, rather in an active state approaching state 3. Perhaps, only under such conditions the inhibitory effects of NAEs on the respiratory chain and, hence, their activatory action on ROS production could be manifested. The effects of NAEs on isolated mitochondria, as described in the present work, may contribute to our better understanding of the mechanism(s) by which endocannabinoids protect cerebral neurons [27] and isolated rat heart [28] against ischaemia, but may also elicit apoptotic cell death [2,29]. Acknowledgment: This research was partly supported by the State Committee for Scientific Research (Grant No. 3 P04 041 23) and Ministry for Science and Informatics (Grant No. 2 P04C 066 28).
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