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Modulation of brain mitochondrial function by deprenyl
Neurochemistry International 48 (2006) 235–241 www.elsevier.com/locate/neuint
Modulation of brain mitochondrial function by deprenyl Analı´a Czerniczyniec, Juanita Bustamante, Silvia Lores-Arnaiz * Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina Received 27 May 2005; received in revised form 7 September 2005; accepted 26 September 2005 Available online 9 November 2005
Abstract The present study shows that deprenyl, a known inhibitor of monoamine oxidase B (MAO B), may generate changes in mitochondrial function. Brain submitochondrial membranes (SMP), synaptosomes and cytosolic fractions were incubated with different deprenyl concentrations and nitric oxide synthase (NOS) activity was measured. The effect of deprenyl on oxygen consumption, calcium-induced permeability transition and hydrogen peroxide (H2O2) production rates was studied in intact mitochondria. Respiratory complexes and monoamine oxidase activities were also measured in submitochondrial membranes. Incubation of brain submitochondrial membranes with deprenyl 10, 25 and 50 mM inhibited nitric oxide synthase activity in a concentrationdependent manner. The same effect was observed in cytosolic fractions and synaptosomes. Monoamine oxidase activity was inhibited at lower deprenyl concentrations (from 0.5 mM). Cytochrome oxidase (complex IV) activity was found 42% increased in the presence of 25 mM deprenyl in a condition of maximal nitric oxide synthase activity. Incubation of brain mitochondria with deprenyl 25 mM produced a 60% increase in oxygen uptake in state 3, but no significant changes were observed in state 4. Pre-incubation of brain mitochondria with deprenyl 0.5 and 1 mM inhibited calcium-induced mitochondrial permeability transition and decreased hydrogen peroxide production rates. Our results suggest that in vitro effects of deprenyl on mitochondrial function can occur through two different mechanisms, involving nitric oxide synthase inhibition and decreased hydrogen peroxide production. # 2005 Elsevier Ltd. All rights reserved. Keywords: Deprenyl; Brain mitochondria; Nitric oxide; Hydrogen peroxide
1. Introduction Deprenyl is an antidepressant used for the treatment of Parkinson and Alzheimer diseases (Olanow and Tatton, 1999). It is known that this drug inhibits monoamine oxidase B (MAO B), therefore, increasing dopamine levels and consequently decreasing reactive oxygen species (Knoll and Magyar, 1972; Laux et al., 1995). These properties contribute to the neuroprotective effects of the drug. It has been reported that MAO B inhibitors are able to increase nitric oxide (NO) production in brain tissue and blood vessels (Thomas, 2000). Also, dopamine agonists increase nitric oxide synthase (NOS) activity in the cells bodies of paraventricular nucleus of the hypothalamus (Melis et al., 1996). Nitric oxide can be produced by intact rat liver mitochondria by a mitochondrial NOS (mtNOS) (Ghafourifar and Richter, * Corresponding author. Tel.: +54 11 4508 3646; fax: +54 11 4508 3646. E-mail address: [email protected] (S. Lores-Arnaiz). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.09.006
1997; Giulivi et al., 1998; Tatoyan and Giulivi, 1998), which has also been described in rat and mouse brain (Lacza et al., 2001; Lores Arnaiz et al., 1999). Physiological situations were reported to affect brain mtNOS activity, e.g., it was upregulated during brain development (Riobo´ et al., 2002) and neuronal plasticity (Lores Arnaiz et al., 2004a). Furthermore, different pharmacological treatments were able to modify brain mtNOS activity (Boveris et al., 2002). Previous results from this laboratory have shown that antipsychotic drugs such as haloperidol and chlorpromazine which act by blocking dopamine receptors, are able to inhibit mtNOS activity in mouse brain (Lores Arnaiz et al., 1999, 2004b). Mitochondrial function appears to be regulated by NO. It is well known that NO exerts a modulatory effect on mitochondrial respiration through the reversible inhibition of cytochrome oxidase (Brown, 2001). In addition, it has been described the ability of this molecule to participate in the regulation of the mitochondrial membrane permeability transition (MPT) (Balakirev et al., 1997). Growing evidence indicates that changes in
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membrane permeability are involved in several forms of neuronal death including apoptosis, excitotoxicity and ischemia (Schinder et al., 1996; Uchino et al., 1995). The aim of this work was to evaluate the mechanisms involved in deprenyl effects on mitochondrial function through the measurements of MAO activity, NO production, respiratory complex activities, oxygen consumption, mitochondrial permeability transition and hydrogen peroxide (H2O2) production. 2. Materials and methods
2.5. Nitric oxide synthase activity Nitric oxide production was measured in submitochondrial membranes using a spectrophotometric method by following the oxidation of oxyhemoglobin to methemoglobin at 37 8C, in a reaction medium containing 50 mM phosphate buffer (pH 5.8 for mitochondrial preparations and pH 7.4 for the cytosolic fractions and synaptosomes), 1 mM CaCl2, 50 mM L-arginine, 100 mM NADPH, 10 mM dithiothreitol, 4 mM Cu–Zn SOD (to avoid interference by O2 ), 0.1 mM catalase (to avoid oxyhemoglobin oxidation by H2O2), 0.5–1.0 mg submitochondrial protein/ml and 25 mM oxyhemoglobin (expressed per heme group). Kinetics were followed at 577–591 nm (e = 11.2 mM 1 cm 1) in a double-beam doublewavelength spectrophotometer (Beckman–Coulter Serie DU) (Boveris et al., 2002).
2.1. Animals 2.6. Respiratory complexes activity Female Swiss mice (20–25 g) from the animal facility of the School of Pharmacy and Biochemistry were used. Animals were housed in an environmentally controlled room and allowed free access to food and water. Animal treatment was carried out in accordance with the guidelines of the National Institute of Health, USA, for the care and use of laboratory animals (NIH Publ. 80-23, 1978).
2.2. Isolation of brain mitochondria Female Swiss mice (20–25 g) were killed by cervical dislocation and the brains were immediately excised. Experimental groups of six mice were used. Brains were weighed and homogenized in a medium consisting of 0.23 M mannitol, 0.07 M sucrose, 10 mM Tris–HCl and 1 mM EDTA (pH 7.4), and homogenized at a ratio of 1 g brain/5 ml homogenization medium. Homogenates were centrifuged at 700 g for 10 min to discard nuclei and cell debris and the pellet was washed to enrich the supernatant that was centrifuged at 8000 g for 10 min. The obtained pellet, washed and resuspended in the same buffer, consisted of intact mitochondria able to carry out oxidative phosphorylation (Lores Arnaiz et al., 1999). The supernatant obtained after 8000 g centrifugation was called ‘‘cytosolic fraction’’. Further mitochondrial purification and synaptosomal fraction separation were performed by Ficoll gradient (Clark and Nicklas, 1970). All the procedure was carried out at 0–2 8C. Submitochondrial membranes (SMP) were obtained from mitochondria by twice freezing and thawing (Boveris et al., 2002). Protein content was assayed by using the Folin phenol reagent and bovine serum albumin as standard (Lowry et al., 1951). For MPT determinations, brains were removed and resuspended in 50 ml of MSH buffer (0.21 M mannitol, 0.07 M sucrose and 5 mM Hepes (pH 7.4)) supplemented with 1 mM EDTA. Homogenates were centrifuged at 600 g for 8 min at 4 8C. The supernatant was decanted and recentrifugated at 5500 g for 15 min to form a mitochondrial pellet that was resuspended in MSH buffer without EDTA and centrifuged again at 5500 g for 15 min (Tyler and Gonze, 1967). The final pellet was resuspended in MSH buffer at a protein concentration of 80–100 mg/ml.
2.3. Western blots SMP, cytosolic fractions and synaptosomes (80 mg) were separated by SDSPAGE (7.5%) and blotted onto nitrocellulose membranes. Membranes were probed with a rabbit polyclonal antibody against the neuronal isoform of nitric oxide synthase (amino terminus, H-299, Santa Cruz Biotechnology, Santa Cruz, CA, USA: dilution 1:500). The nitrocellulose membrane was subsequently incubated with a secondary goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP; dilution 1:1000), and revealed by chemiluminescence with ECL reagent (Bustamante et al., 2002; Boveris et al., 2002). Densitometric analysis of the nNOS bands was performed using the NIH Image 1.54 software. All experiments were performed in duplicate.
2.4. MAO activity MAO activity was measured in SMP by following spectrophotometrically the oxidation of kynuramine at 30 8C, in a reaction medium containing 50 mM phosphate buffer, pH 7.4. Kinetics were followed at 360 nm (e = 4.28 mM 1 cm 1) (Weissbach et al., 1960).
For the determination of NADH–cytochrome c reductase activity (complex I + III), submitochondrial membranes were added with 100 mM phosphate buffer (pH 7.4), 0.2 mM NADH, 0.1 mM cytochrome c and 0.5 mM KCN and followed spectrophotometrically at 30 8C at 550 nm (e = 19.6 mM 1 cm 1). Enzyme activity was expressed in nanomoles cytochrome c reduced per minute per milligram of protein. Succinate cytochrome c reductase activity (complex II + III) was similarly determined and expressed, except that NADH was substituted by 20 mM succinate. Cytochrome oxidase activity (complex IV) was assayed spectrophotometrically at 550 nm by following the rate of oxidation of 50 mM ferrocytochrome c (Yonetani, 1967). The activity was expressed as nanomoles ferrocytochrome c oxidized per minute per milligram of protein. Respiratory complexes activities were also determined in a condition of maximal NOS activity, in the presence of L-arginine, NADPH, DTT, SOD and catalase, in similar concentrations to measure NOS activity.
2.7. Mitochondrial respiration A two-channel respirometer for high-resolution respirometry (Oroboros Oxygraph, Paar KG, Graz, Austria) was used. Mitochondrial respiratory rates were measured in a reaction medium containing 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 4 mM MgCl2, 5 mM phosphate and 0.2% bovine serum albumin at 37 8C. Succinate (7 mM) was used as substrate to measure state 4 respiration; 1 mM ADP was added to measure state 3 respiration (Boveris et al., 1999). Arginine (0.1 mM) or Nv-nitro-L-arginine (LNNA) (0.5 mM) were added to brain mitochondria in order to assay the functional NO effects on mitochondrial respiration.
2.8. Membrane permeability transition Brain mitochondrial suspensions (0.5 mg/ml) were incubated in MSH buffer supplemented with 5 mM succinate, 1 mM rotenone and 1 mM phosphate. MPT was determined by the decrease in absorbance at 540 nm (Azonne et al., 1984; Bustamante et al., 2005). The experiments were started by the addition of calcium 90 nmol/mg protein. Five minutes pre-treatment with 2 mM cyclosporin A (CsA) before calcium addition was assayed in order to inhibit MPT. The effect of L-NNA (1 mM) and different deprenyl concentrations (0.5 and 1 mM) were also studied.
2.9. Hydrogen peroxide production Hydrogen peroxide generation rate was determined in intact brain mitochondria by the scopoletin–horseradish peroxidase method, following the decrease in fluorescence intensity at 365–450 nm (lexc lem) at 37 8C (Boveris, 1984). The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 0.8 mM HRP, 1 mM scopoletin, 6 mM malate, 6 mM glutamate and 0.3 mM SOD. Mitochondria (0.1–0.3 mg protein/ ml) were either incubated for 5 min with 0.5 and 1 mM deprenyl. Antimycin (3 mM) was used as specific inhibitor to obtain maximal H2O2 production. Calibration was made using H2O2 (0.05–0.35 mM) as standard to express the fluorescence changes as nmol H2O2/min mg protein.
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2.10. Drugs and chemicals Deprenyl, kynuramine, ADP, L-arginine, catalase, dithiothreitol, EDTA, glutamic acid, malic acid, mannitol, NADPH, Nv-nitro-L-arginine, hemoglobin, scopoletin, horseradish peroxidase, succinate, sucrose, superoxide dismutase, cytochrome c and trizma base, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other reagents were of analytical grade.
2.11. Statistics Results are expressed as mean values S.E.M. Student’s t-test was used to analyze the significance of differences between paired groups. ANOVA, followed by Dunnett tests, was used to analyze differences between mean values of more than two groups.
3. Results 3.1. Western blot analysis of NOS Western blot analysis identified a protein of 157 kDa reacting with anti-nNOS antibodies (amino terminus) in brain submitochondrial membranes, cytosolic fractions and synaptosomes (Fig. 1). An enriched band corresponding to cytosolic fractions compared with the other subcellular fractions was observed. In agreement, NO production levels were higher in cytosolic fractions than in mitochondria or synaptosomes. 4. Enzyme activities 4.1. Monoamine oxidase In vitro, deprenyl inhibited MAO activity in brain SMP at 0.5–10 mM, although at deprenyl levels greater than 5 mM, the amount of the inhibition remained constant (Fig. 2). 4.2. Nitric oxide synthase Nitric oxide production was measured in different brain subcellular fractions obtaining the following values: for cytosol, 1.11 0.01; for SMP, 0.84 0.06; and for synaptosomes, 0.49 0.06 (in nmol NO/min mg protein). Incubation of brain SMP with deprenyl 10, 25 and 50 mM inhibited NOS activity in a concentration-dependent manner. The same effect was observed in cytosolic fractions and synaptosomes (Fig. 3). Lower concentrations of deprenyl (0.5– 1 mM) were not able to inhibit NOS activity in the studied fractions (data not shown).
Fig. 1. Western blot analysis of nNOS from control brain SMP, synaptosomes and cytosolic fractions. Chemiluminescence ECL technique was used for final detection. NO production values are shown for each of the different subcellular fractions.
Fig. 2. In vitro effect of deprenyl on SMP MAO activity. SMP were incubated with different deprenyl concentrations (0.5–10 mM) during 5 min at 30 8C before the determinations. Values represent the mean S.E.M. of four experiments. ‘‘*’’ p < 0.05.
4.3. Respiratory complexes Deprenyl incubation (25 mM) did not affect brain NADH– cytochrome c reductase (complexes I–III), succinate–cytochrome c reductase (complexes II–III) and cytochrome oxidase (complex IV) activities (data not shown). When the activity of the respiratory complexes were measured in the condition of maximal NOS activity, in the presence of L-arginine, NADPH, DTT, SOD and catalase, deprenyl incubation did not modify brain NADH–cytochrome c reductase (complexes I–III) and succinate–cytochrome c reductase (complexes II–III) activities (Table 1). Cytochrome oxidase (complex IV) activity was 42% increased in the presence of 25 mM deprenyl in the condition of maximal NOS activity (Table 1). The addition of L-NNA to control brain SMP produced an increase of 42% in cytochrome
Fig. 3. Effect of deprenyl incubation on NOS activity in brain SMP, synaptosomes and cytosolic fractions. Subcellular fractions were incubated with different deprenyl concentrations (10–50 mM) during 5 min at 37 8C before the determinations. Values represent the mean S.E.M. of four experiments. ‘‘*’’ p < 0.05.
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Table 1 Effect of deprenyl on the activity of mitochondrial respiratory chain complexes in brain submitochondrial membranes Activity (nmol/min mg protein)
Control
Deprenyl
NADH–cytochrome c reductase Succinate–cytochrome c reductase Cytochrome oxidase Cytochrome oxidase + L-NNA
318 21 116 1 214 15 308 10*
343 17 125 5 304 22* 318 6
SMP were incubated with 25 mM deprenyl during 5 min at 30 8C before the determinations. Experiments were carried out in the condition of maximal NOS activity, in the presence of L-arginine, NADPH, DTT, SOD and catalase. Values represent the mean of three to four experiments. * p < 0.05, significantly different from control (non-incubated) submitochondrial membranes.
oxidase activity, but this effect was not observed in deprenylincubated SMP (Table 1). 5. Mitochondrial function 5.1. Mitochondrial respiration Succinate-dependent oxygen consumption rate was measured in state 4 (resting or controlled respiration) and in state 3 (active respiration, the maximal physiological rate of O2 uptake and ATP synthesis) and the respiratory control ratios were calculated (Boveris et al., 1999). Incubation of brain mitochondria with deprenyl 25 mM produced a 60% increase in the oxygen uptake in state 3, but no significant changes were observed in state 4 (Table 2). The addition of the NOS inhibitor L-NNA markedly increased the respiratory rate in state 3 in control brain mitochondria by 47%, meanwhile this effect was not observed in deprenyl-incubated mitochondria (Table 2). The addition of L-arginine to control and deprenyl-incubated mitochondria in state 3 did not modify oxygen uptake (Table 2). 5.2. Mitochondrial permeability transition Incubation of brain mitochondria with 20 nmol calcium/mg protein was not able to induce MPT, however, 90 nmol calcium/ mg protein induced a partial MPT similar to the permeability induced in liver mitochondria (Bustamante et al., 2005). This Table 2 Effect of deprenyl on brain mitochondrial respiratory rates Condition
Succinate (state 4) + ADP (state 3) Respiratory control State 3 + L-arginine State 3 + L-NNA
Respiration rates (ng-atom O/min mg protein) Control
Deprenyl
13 4 56 4 4.3 0.3 68 3 89 1#
14.5 0.2 90 7* 6.19 0.08* 102 4 98 2
Intact mitochondria were incubated with 25 mM deprenyl during 5 min at 37 8C before the determinations. Values represent the mean of four experiments. * p < 0.05, as compared to control mitochondria. # p < 0.05, as compared to basal state 3 control value.
Fig. 4. Deprenyl inhibition of the calcium-induced permeability transition. (A) Typical example of trace mitochondrial swelling. (B) MPT assessed by changes in light absorbance at 540 nm/min mg protein of mouse brain mitochondria treated as described.
process was classically inhibited by short pre-incubation with CsA (2 mM) (Fig. 4A). Incubation of brain mitochondria with L-NNA significantly inhibits the induction of MPT after addition of 90 nmol calcium/mg protein (Fig. 4B). Brain mitochondria pre-incubation with deprenyl 0.5 and 1 mM inhibited the calcium-induced MPT (90 nmol/mg protein) by 36% and 44%, respectively (Fig. 4B). 5.3. Hydrogen peroxide production Hydrogen peroxide production rate was 0.33 0.06 nmol/ min mg protein in control mouse brain mitochondria and increased by 70% after antimycin addition. The incubation of mitochondria with 0.5 and 1 mM deprenyl was able to decrease H2O2 production rates by 36% and 51%, respectively (Table 3). Antimycin addition to deprenyl-incubated mitochondria led to maximal H2O2 production rates similar to those obtained for control mitochondria (Table 3). 6. Discussion Deprenyl, a MAO B inhibitor, has been used in the treatment of Parkinson disease due to its effects of reducing the catabolism of dopamine and suppressing the generation of reactive oxygen species (Knoll and Magyar, 1972; Laux et al., 1995). This study gives evidence of the involvement of nitric
A. Czerniczyniec et al. / Neurochemistry International 48 (2006) 235–241 Table 3 Effect of deprenyl on brain mitochondrial H2O2 production rate H2O2 production (nmol/min mg protein) Mouse brain mitochondria (control) 0.33 0.06 + 3 mM antimycin 0.56 0.06* Id. + 0.5 mM deprenyl + 3 mM antimycin
0.21 0.02# 0.5 0.1 *
Id. + 1 mM deprenyl + 3 mM antimycin
0.16 0.05# 0.57 0.04*
Mouse brain mitochondria were supplemented with 6 mM malate and 6 mM glutamate, as described. Values represent the mean of three experiments. * p < 0.05, significantly different from its corresponding control value. # p < 0.05, significant differences between deprenyl-incubated mitochondria and control mitochondria.
oxide and hydrogen peroxide in deprenyl effects on mitochondrial function. The presence of NOS was analyzed in different brain subcellular fractions from control animals by Western blot assays. A protein of 157 kDa in submitochondrial membranes, cytosolic fraction and synaptosomes was recognized through the use of a specific anti-nNOS antibody directed against the amino end of the protein. The amounts of nNOS protein detected and of NO production were higher in cytosol than in the other subcellular fractions. Incubation of submitochondrial membranes with deprenyl inhibits NOS activity in a concentration-dependent manner. The same pattern of NOS inhibition was observed after incubation of synaptosomes and cytosolic fractions with deprenyl. Taking into account that MAO is not present in cytosol, deprenyl effects on NOS activity seem to be independent of MAO inhibition. Mitochondrial NO modulates oxygen consumption by a reversible and O2-competitive inhibition of cytochrome oxidase (complex IV) that slows down electron flow, substrate oxidation and stores of chemical energy (Brown, 2001). Furthermore, mitochondrial NO regulates respiratory chain by the inhibition of ubiquinol–cytochrome c reductase activity (complex III) (Poderoso et al., 1996). Deprenyl-incubation inhibited mitochondrial NO production and increased O2 consumption in state 3 in intact mitochondria. This effect can reflect a functional regulatory activity of NO on brain mitochondrial respiration, as previously reported for chlorpromazine treatment (Lores Arnaiz et al., 2004b). Moreover, the addition of LNNA markedly increased the respiratory rate in state 3 in control brain mitochondria, but this effect was not observed in deprenyl-incubated mitochondria. The lack of effect of L-NNA on state 3 respiration rate of deprenyl-incubated mitochondria could be due to the previous inhibition of the enzyme by the drug, providing evidence of the involvement of NO in the increase in mitochondrial state 3 respiratory rate observed after deprenyl incubation. The addition of L-arginine to control and deprenyl-incubated mitochondria in state 3 did not significantly modify respiratory rates. This lack of effect of L-arginine indicates the existence of an important intramitochondrial arginine pool as previously reported (Lores Arnaiz et al., 2004b).
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Inhibition of cytochrome oxidase (complex IV) by NO is considered as a phenomenon of relevant physiological importance (Moncada and Erusalimsky, 2002; Brown, 2003). Due to the fact that in this study, mitochondrial respiratory rates were affected by deprenyl, we decided to evaluate the effect of this drug on mitochondrial respiratory complexes activity. No changes were observed in NADH–cytochrome c reductase (complexes I–III) and succinate–cytochrome c reductase (complexes II–III) activities after deprenyl incubation. These results are consistent with the concept that NO exerts its modulatory effect on respiration mainly by acting on cytochrome oxidase. Deprenyl per se did not affect complex IV activity, but cytochrome oxidase activity was increased after incubation of brain SMP with 25 mM deprenyl, when measured in the condition of maximal NOS activity. In agreement, and as observed in oxygen consumption, the addition of L-NNA increased cytochrome oxidase activity in control SMP but was not able to exert a similar effect in deprenyl-incubated SMP, giving support to the idea that the effect of deprenyl on mitochondrial respiration occurs through NOS inhibition. Mitochondrial permeability transition is a non-selective permeabilization of the inner mitochondrial membrane promoted by the accumulation of excessive quantities of calcium ions and stimulated by a variety of compounds and conditions (Zoratti and Szabo´, 1995). The inner membrane permeabilization caused by mitochondrial permeability transition results in loss of matrix components, impairment of mitochondrial functionality and substantial swelling of the organelle, with consequent outer membrane rupture and cytochrome c release (Zoratti and Szabo´, 1995; Green and Reed, 1998; Kowaltowski and Vercesi, 1999). Evidence was provided that deprenyl attenuated alteration of swelling, membrane potential, cytochrome c release and calcium transport in mitochondria treated with dopamine (Lee et al., 2002). In our study, calcium concentration higher than 90 nmol/ mg protein was able to induce partial mitochondrial permeability transition, which was inhibited by short pre-incubation with CsA. Incubation of brain mitochondria with L-NNA decreases mitochondrial permeability transition induction, indicating a possible effect of NO on mitochondrial permeability transition as suggested by different laboratories (Balakirev et al., 1997). Our results show that deprenyl protects brain calcium-induced mitochondrial permeability transition, at low concentrations (0.5–1 mM). In this range of concentrations, deprenyl was able to inhibit MAO but not NOS; these results suggests that deprenyl protective effect on calciuminduced mitochondrial permeability transition would be mediated by NO independent mechanisms. As oxygen free radicals are capable of inducing mitochondrial permeability transition (Bernardi et al., 1999), it seemed important to evaluate the possible participation of H2O2 on deprenyl effects on mitochondrial permeability transition. Hydrogen peroxide production rates were decreased by deprenyl incubation at low concentrations. Maximal H2O2 production rates (in the presence of antimycin) were not modified by deprenyl incubation, suggesting that deprenyl effect on hydrogen
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peroxide may be mediated mainly by MAO inhibition and not through a direct effect on respiratory chain. Therefore, it seems more probable that the observed decrease in H2O2 production by deprenyl would be responsible for the inhibitory effect of low concentrations of deprenyl on calcium-induced mitochondrial permeability transition. It has been reported that deprenyl provides neuroprotection by stabilizing the mitochondrial membrane potential in PC 12 cultures (Wadia et al., 1998), and by regulating molecular markers of apoptosis such as reactive oxygen species generation, lipid peroxidation, glutathionine and ATP synthesis and cytochrome c release in human neuroblastoma cell lines (Sharma et al., 2003). In summary, we show that in vitro effects of deprenyl have beneficial effects on mitochondrial function such as an increase in mitochondrial respiration and inhibition of calcium-induced mitochondrial permeability transition. Comparing the effects of deprenyl at different concentrations used in this study, we can infer that two different mechanisms are involved. The increase in oxygen consumption by deprenyl would be mediated by NOS inhibition, while mitochondrial permeability transition protective effect may occur as a consequence of MAO inhibition and decreased H2O2 production. Acknowledgements This research was supported by grants from Universidad de Buenos Aires (B131), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (PIP 02272), and Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (PICT 01-8710), Argentina. References Azonne, G.F., Pietrobon, D., Zoratti, M., 1984. Determination of the proton electrochemical gradient across biological membranes. Curr. Top. Bioenerg. 13, 1–77. Balakirev, M.Y., Khramtsov, V.V., Zimmer, G., 1997. Modulation of the mitochondrial permeability transition by nitric oxide. Eur. J. Biochem. 246, 710–718. Bernardi, P., Scorrano, L., Colonia, R., Petronilli, V., Di Lisa, F., 1999. Mitochondrial and cell death: mechanistic aspects and methodological issues. Eur. J. Biochem. 264, 687–701. Boveris, A., 1984. Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods Enzymol. 105, 429– 435. Boveris, A., Costa, L.E., Cadenas, E., Poderoso, J.J., 1999. Regulation of mitochondrial respiration by adenosine diphosphate, oxygen and nitric oxide. Methods Enzymol. 301, 188–198. Boveris, A., Lores Arnaiz, S., Bustamante, J., Alvarez, S., Valdez, L., Boveris, A.D., Navarro, A., 2002. Pharmacological regulation of mitochondrial nitric oxide synthase. Methods Enzymol. 359, 328–339. Brown, G.C., 2001. Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome oxidase. Biochim. Biophys. Acta 1504, 46– 57. Brown, G.C., 2003. NO says yes to mitochondria. Science 299, 838–839. Bustamante, J., Bersier, G., Aron-Badin, R., Cymeryng, C., Parodi, A., Boveris, A., 2002. Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis. Nitric Oxide 6, 333–341. Bustamante, J., Nutt, L., Orrenius, S., Gogvadze, V., 2005. Arsenic stimulates release of cytochrome c from isolated mitochondria via induction of mitochondrial permeability transition. Toxicol. Appl. Pharmacol. 207 (2 Suppl.), 110–116.
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Modulation of brain mitochondrial function by deprenyl Analı´a Czerniczyniec, Juanita Bustamante, Silvia Lores-Arnaiz * Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina Received 27 May 2005; received in revised form 7 September 2005; accepted 26 September 2005 Available online 9 November 2005
Abstract The present study shows that deprenyl, a known inhibitor of monoamine oxidase B (MAO B), may generate changes in mitochondrial function. Brain submitochondrial membranes (SMP), synaptosomes and cytosolic fractions were incubated with different deprenyl concentrations and nitric oxide synthase (NOS) activity was measured. The effect of deprenyl on oxygen consumption, calcium-induced permeability transition and hydrogen peroxide (H2O2) production rates was studied in intact mitochondria. Respiratory complexes and monoamine oxidase activities were also measured in submitochondrial membranes. Incubation of brain submitochondrial membranes with deprenyl 10, 25 and 50 mM inhibited nitric oxide synthase activity in a concentrationdependent manner. The same effect was observed in cytosolic fractions and synaptosomes. Monoamine oxidase activity was inhibited at lower deprenyl concentrations (from 0.5 mM). Cytochrome oxidase (complex IV) activity was found 42% increased in the presence of 25 mM deprenyl in a condition of maximal nitric oxide synthase activity. Incubation of brain mitochondria with deprenyl 25 mM produced a 60% increase in oxygen uptake in state 3, but no significant changes were observed in state 4. Pre-incubation of brain mitochondria with deprenyl 0.5 and 1 mM inhibited calcium-induced mitochondrial permeability transition and decreased hydrogen peroxide production rates. Our results suggest that in vitro effects of deprenyl on mitochondrial function can occur through two different mechanisms, involving nitric oxide synthase inhibition and decreased hydrogen peroxide production. # 2005 Elsevier Ltd. All rights reserved. Keywords: Deprenyl; Brain mitochondria; Nitric oxide; Hydrogen peroxide
1. Introduction Deprenyl is an antidepressant used for the treatment of Parkinson and Alzheimer diseases (Olanow and Tatton, 1999). It is known that this drug inhibits monoamine oxidase B (MAO B), therefore, increasing dopamine levels and consequently decreasing reactive oxygen species (Knoll and Magyar, 1972; Laux et al., 1995). These properties contribute to the neuroprotective effects of the drug. It has been reported that MAO B inhibitors are able to increase nitric oxide (NO) production in brain tissue and blood vessels (Thomas, 2000). Also, dopamine agonists increase nitric oxide synthase (NOS) activity in the cells bodies of paraventricular nucleus of the hypothalamus (Melis et al., 1996). Nitric oxide can be produced by intact rat liver mitochondria by a mitochondrial NOS (mtNOS) (Ghafourifar and Richter, * Corresponding author. Tel.: +54 11 4508 3646; fax: +54 11 4508 3646. E-mail address: [email protected] (S. Lores-Arnaiz). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.09.006
1997; Giulivi et al., 1998; Tatoyan and Giulivi, 1998), which has also been described in rat and mouse brain (Lacza et al., 2001; Lores Arnaiz et al., 1999). Physiological situations were reported to affect brain mtNOS activity, e.g., it was upregulated during brain development (Riobo´ et al., 2002) and neuronal plasticity (Lores Arnaiz et al., 2004a). Furthermore, different pharmacological treatments were able to modify brain mtNOS activity (Boveris et al., 2002). Previous results from this laboratory have shown that antipsychotic drugs such as haloperidol and chlorpromazine which act by blocking dopamine receptors, are able to inhibit mtNOS activity in mouse brain (Lores Arnaiz et al., 1999, 2004b). Mitochondrial function appears to be regulated by NO. It is well known that NO exerts a modulatory effect on mitochondrial respiration through the reversible inhibition of cytochrome oxidase (Brown, 2001). In addition, it has been described the ability of this molecule to participate in the regulation of the mitochondrial membrane permeability transition (MPT) (Balakirev et al., 1997). Growing evidence indicates that changes in
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membrane permeability are involved in several forms of neuronal death including apoptosis, excitotoxicity and ischemia (Schinder et al., 1996; Uchino et al., 1995). The aim of this work was to evaluate the mechanisms involved in deprenyl effects on mitochondrial function through the measurements of MAO activity, NO production, respiratory complex activities, oxygen consumption, mitochondrial permeability transition and hydrogen peroxide (H2O2) production. 2. Materials and methods
2.5. Nitric oxide synthase activity Nitric oxide production was measured in submitochondrial membranes using a spectrophotometric method by following the oxidation of oxyhemoglobin to methemoglobin at 37 8C, in a reaction medium containing 50 mM phosphate buffer (pH 5.8 for mitochondrial preparations and pH 7.4 for the cytosolic fractions and synaptosomes), 1 mM CaCl2, 50 mM L-arginine, 100 mM NADPH, 10 mM dithiothreitol, 4 mM Cu–Zn SOD (to avoid interference by O2 ), 0.1 mM catalase (to avoid oxyhemoglobin oxidation by H2O2), 0.5–1.0 mg submitochondrial protein/ml and 25 mM oxyhemoglobin (expressed per heme group). Kinetics were followed at 577–591 nm (e = 11.2 mM 1 cm 1) in a double-beam doublewavelength spectrophotometer (Beckman–Coulter Serie DU) (Boveris et al., 2002).
2.1. Animals 2.6. Respiratory complexes activity Female Swiss mice (20–25 g) from the animal facility of the School of Pharmacy and Biochemistry were used. Animals were housed in an environmentally controlled room and allowed free access to food and water. Animal treatment was carried out in accordance with the guidelines of the National Institute of Health, USA, for the care and use of laboratory animals (NIH Publ. 80-23, 1978).
2.2. Isolation of brain mitochondria Female Swiss mice (20–25 g) were killed by cervical dislocation and the brains were immediately excised. Experimental groups of six mice were used. Brains were weighed and homogenized in a medium consisting of 0.23 M mannitol, 0.07 M sucrose, 10 mM Tris–HCl and 1 mM EDTA (pH 7.4), and homogenized at a ratio of 1 g brain/5 ml homogenization medium. Homogenates were centrifuged at 700 g for 10 min to discard nuclei and cell debris and the pellet was washed to enrich the supernatant that was centrifuged at 8000 g for 10 min. The obtained pellet, washed and resuspended in the same buffer, consisted of intact mitochondria able to carry out oxidative phosphorylation (Lores Arnaiz et al., 1999). The supernatant obtained after 8000 g centrifugation was called ‘‘cytosolic fraction’’. Further mitochondrial purification and synaptosomal fraction separation were performed by Ficoll gradient (Clark and Nicklas, 1970). All the procedure was carried out at 0–2 8C. Submitochondrial membranes (SMP) were obtained from mitochondria by twice freezing and thawing (Boveris et al., 2002). Protein content was assayed by using the Folin phenol reagent and bovine serum albumin as standard (Lowry et al., 1951). For MPT determinations, brains were removed and resuspended in 50 ml of MSH buffer (0.21 M mannitol, 0.07 M sucrose and 5 mM Hepes (pH 7.4)) supplemented with 1 mM EDTA. Homogenates were centrifuged at 600 g for 8 min at 4 8C. The supernatant was decanted and recentrifugated at 5500 g for 15 min to form a mitochondrial pellet that was resuspended in MSH buffer without EDTA and centrifuged again at 5500 g for 15 min (Tyler and Gonze, 1967). The final pellet was resuspended in MSH buffer at a protein concentration of 80–100 mg/ml.
2.3. Western blots SMP, cytosolic fractions and synaptosomes (80 mg) were separated by SDSPAGE (7.5%) and blotted onto nitrocellulose membranes. Membranes were probed with a rabbit polyclonal antibody against the neuronal isoform of nitric oxide synthase (amino terminus, H-299, Santa Cruz Biotechnology, Santa Cruz, CA, USA: dilution 1:500). The nitrocellulose membrane was subsequently incubated with a secondary goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP; dilution 1:1000), and revealed by chemiluminescence with ECL reagent (Bustamante et al., 2002; Boveris et al., 2002). Densitometric analysis of the nNOS bands was performed using the NIH Image 1.54 software. All experiments were performed in duplicate.
2.4. MAO activity MAO activity was measured in SMP by following spectrophotometrically the oxidation of kynuramine at 30 8C, in a reaction medium containing 50 mM phosphate buffer, pH 7.4. Kinetics were followed at 360 nm (e = 4.28 mM 1 cm 1) (Weissbach et al., 1960).
For the determination of NADH–cytochrome c reductase activity (complex I + III), submitochondrial membranes were added with 100 mM phosphate buffer (pH 7.4), 0.2 mM NADH, 0.1 mM cytochrome c and 0.5 mM KCN and followed spectrophotometrically at 30 8C at 550 nm (e = 19.6 mM 1 cm 1). Enzyme activity was expressed in nanomoles cytochrome c reduced per minute per milligram of protein. Succinate cytochrome c reductase activity (complex II + III) was similarly determined and expressed, except that NADH was substituted by 20 mM succinate. Cytochrome oxidase activity (complex IV) was assayed spectrophotometrically at 550 nm by following the rate of oxidation of 50 mM ferrocytochrome c (Yonetani, 1967). The activity was expressed as nanomoles ferrocytochrome c oxidized per minute per milligram of protein. Respiratory complexes activities were also determined in a condition of maximal NOS activity, in the presence of L-arginine, NADPH, DTT, SOD and catalase, in similar concentrations to measure NOS activity.
2.7. Mitochondrial respiration A two-channel respirometer for high-resolution respirometry (Oroboros Oxygraph, Paar KG, Graz, Austria) was used. Mitochondrial respiratory rates were measured in a reaction medium containing 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 4 mM MgCl2, 5 mM phosphate and 0.2% bovine serum albumin at 37 8C. Succinate (7 mM) was used as substrate to measure state 4 respiration; 1 mM ADP was added to measure state 3 respiration (Boveris et al., 1999). Arginine (0.1 mM) or Nv-nitro-L-arginine (LNNA) (0.5 mM) were added to brain mitochondria in order to assay the functional NO effects on mitochondrial respiration.
2.8. Membrane permeability transition Brain mitochondrial suspensions (0.5 mg/ml) were incubated in MSH buffer supplemented with 5 mM succinate, 1 mM rotenone and 1 mM phosphate. MPT was determined by the decrease in absorbance at 540 nm (Azonne et al., 1984; Bustamante et al., 2005). The experiments were started by the addition of calcium 90 nmol/mg protein. Five minutes pre-treatment with 2 mM cyclosporin A (CsA) before calcium addition was assayed in order to inhibit MPT. The effect of L-NNA (1 mM) and different deprenyl concentrations (0.5 and 1 mM) were also studied.
2.9. Hydrogen peroxide production Hydrogen peroxide generation rate was determined in intact brain mitochondria by the scopoletin–horseradish peroxidase method, following the decrease in fluorescence intensity at 365–450 nm (lexc lem) at 37 8C (Boveris, 1984). The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 0.8 mM HRP, 1 mM scopoletin, 6 mM malate, 6 mM glutamate and 0.3 mM SOD. Mitochondria (0.1–0.3 mg protein/ ml) were either incubated for 5 min with 0.5 and 1 mM deprenyl. Antimycin (3 mM) was used as specific inhibitor to obtain maximal H2O2 production. Calibration was made using H2O2 (0.05–0.35 mM) as standard to express the fluorescence changes as nmol H2O2/min mg protein.
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2.10. Drugs and chemicals Deprenyl, kynuramine, ADP, L-arginine, catalase, dithiothreitol, EDTA, glutamic acid, malic acid, mannitol, NADPH, Nv-nitro-L-arginine, hemoglobin, scopoletin, horseradish peroxidase, succinate, sucrose, superoxide dismutase, cytochrome c and trizma base, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other reagents were of analytical grade.
2.11. Statistics Results are expressed as mean values S.E.M. Student’s t-test was used to analyze the significance of differences between paired groups. ANOVA, followed by Dunnett tests, was used to analyze differences between mean values of more than two groups.
3. Results 3.1. Western blot analysis of NOS Western blot analysis identified a protein of 157 kDa reacting with anti-nNOS antibodies (amino terminus) in brain submitochondrial membranes, cytosolic fractions and synaptosomes (Fig. 1). An enriched band corresponding to cytosolic fractions compared with the other subcellular fractions was observed. In agreement, NO production levels were higher in cytosolic fractions than in mitochondria or synaptosomes. 4. Enzyme activities 4.1. Monoamine oxidase In vitro, deprenyl inhibited MAO activity in brain SMP at 0.5–10 mM, although at deprenyl levels greater than 5 mM, the amount of the inhibition remained constant (Fig. 2). 4.2. Nitric oxide synthase Nitric oxide production was measured in different brain subcellular fractions obtaining the following values: for cytosol, 1.11 0.01; for SMP, 0.84 0.06; and for synaptosomes, 0.49 0.06 (in nmol NO/min mg protein). Incubation of brain SMP with deprenyl 10, 25 and 50 mM inhibited NOS activity in a concentration-dependent manner. The same effect was observed in cytosolic fractions and synaptosomes (Fig. 3). Lower concentrations of deprenyl (0.5– 1 mM) were not able to inhibit NOS activity in the studied fractions (data not shown).
Fig. 1. Western blot analysis of nNOS from control brain SMP, synaptosomes and cytosolic fractions. Chemiluminescence ECL technique was used for final detection. NO production values are shown for each of the different subcellular fractions.
Fig. 2. In vitro effect of deprenyl on SMP MAO activity. SMP were incubated with different deprenyl concentrations (0.5–10 mM) during 5 min at 30 8C before the determinations. Values represent the mean S.E.M. of four experiments. ‘‘*’’ p < 0.05.
4.3. Respiratory complexes Deprenyl incubation (25 mM) did not affect brain NADH– cytochrome c reductase (complexes I–III), succinate–cytochrome c reductase (complexes II–III) and cytochrome oxidase (complex IV) activities (data not shown). When the activity of the respiratory complexes were measured in the condition of maximal NOS activity, in the presence of L-arginine, NADPH, DTT, SOD and catalase, deprenyl incubation did not modify brain NADH–cytochrome c reductase (complexes I–III) and succinate–cytochrome c reductase (complexes II–III) activities (Table 1). Cytochrome oxidase (complex IV) activity was 42% increased in the presence of 25 mM deprenyl in the condition of maximal NOS activity (Table 1). The addition of L-NNA to control brain SMP produced an increase of 42% in cytochrome
Fig. 3. Effect of deprenyl incubation on NOS activity in brain SMP, synaptosomes and cytosolic fractions. Subcellular fractions were incubated with different deprenyl concentrations (10–50 mM) during 5 min at 37 8C before the determinations. Values represent the mean S.E.M. of four experiments. ‘‘*’’ p < 0.05.
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Table 1 Effect of deprenyl on the activity of mitochondrial respiratory chain complexes in brain submitochondrial membranes Activity (nmol/min mg protein)
Control
Deprenyl
NADH–cytochrome c reductase Succinate–cytochrome c reductase Cytochrome oxidase Cytochrome oxidase + L-NNA
318 21 116 1 214 15 308 10*
343 17 125 5 304 22* 318 6
SMP were incubated with 25 mM deprenyl during 5 min at 30 8C before the determinations. Experiments were carried out in the condition of maximal NOS activity, in the presence of L-arginine, NADPH, DTT, SOD and catalase. Values represent the mean of three to four experiments. * p < 0.05, significantly different from control (non-incubated) submitochondrial membranes.
oxidase activity, but this effect was not observed in deprenylincubated SMP (Table 1). 5. Mitochondrial function 5.1. Mitochondrial respiration Succinate-dependent oxygen consumption rate was measured in state 4 (resting or controlled respiration) and in state 3 (active respiration, the maximal physiological rate of O2 uptake and ATP synthesis) and the respiratory control ratios were calculated (Boveris et al., 1999). Incubation of brain mitochondria with deprenyl 25 mM produced a 60% increase in the oxygen uptake in state 3, but no significant changes were observed in state 4 (Table 2). The addition of the NOS inhibitor L-NNA markedly increased the respiratory rate in state 3 in control brain mitochondria by 47%, meanwhile this effect was not observed in deprenyl-incubated mitochondria (Table 2). The addition of L-arginine to control and deprenyl-incubated mitochondria in state 3 did not modify oxygen uptake (Table 2). 5.2. Mitochondrial permeability transition Incubation of brain mitochondria with 20 nmol calcium/mg protein was not able to induce MPT, however, 90 nmol calcium/ mg protein induced a partial MPT similar to the permeability induced in liver mitochondria (Bustamante et al., 2005). This Table 2 Effect of deprenyl on brain mitochondrial respiratory rates Condition
Succinate (state 4) + ADP (state 3) Respiratory control State 3 + L-arginine State 3 + L-NNA
Respiration rates (ng-atom O/min mg protein) Control
Deprenyl
13 4 56 4 4.3 0.3 68 3 89 1#
14.5 0.2 90 7* 6.19 0.08* 102 4 98 2
Intact mitochondria were incubated with 25 mM deprenyl during 5 min at 37 8C before the determinations. Values represent the mean of four experiments. * p < 0.05, as compared to control mitochondria. # p < 0.05, as compared to basal state 3 control value.
Fig. 4. Deprenyl inhibition of the calcium-induced permeability transition. (A) Typical example of trace mitochondrial swelling. (B) MPT assessed by changes in light absorbance at 540 nm/min mg protein of mouse brain mitochondria treated as described.
process was classically inhibited by short pre-incubation with CsA (2 mM) (Fig. 4A). Incubation of brain mitochondria with L-NNA significantly inhibits the induction of MPT after addition of 90 nmol calcium/mg protein (Fig. 4B). Brain mitochondria pre-incubation with deprenyl 0.5 and 1 mM inhibited the calcium-induced MPT (90 nmol/mg protein) by 36% and 44%, respectively (Fig. 4B). 5.3. Hydrogen peroxide production Hydrogen peroxide production rate was 0.33 0.06 nmol/ min mg protein in control mouse brain mitochondria and increased by 70% after antimycin addition. The incubation of mitochondria with 0.5 and 1 mM deprenyl was able to decrease H2O2 production rates by 36% and 51%, respectively (Table 3). Antimycin addition to deprenyl-incubated mitochondria led to maximal H2O2 production rates similar to those obtained for control mitochondria (Table 3). 6. Discussion Deprenyl, a MAO B inhibitor, has been used in the treatment of Parkinson disease due to its effects of reducing the catabolism of dopamine and suppressing the generation of reactive oxygen species (Knoll and Magyar, 1972; Laux et al., 1995). This study gives evidence of the involvement of nitric
A. Czerniczyniec et al. / Neurochemistry International 48 (2006) 235–241 Table 3 Effect of deprenyl on brain mitochondrial H2O2 production rate H2O2 production (nmol/min mg protein) Mouse brain mitochondria (control) 0.33 0.06 + 3 mM antimycin 0.56 0.06* Id. + 0.5 mM deprenyl + 3 mM antimycin
0.21 0.02# 0.5 0.1 *
Id. + 1 mM deprenyl + 3 mM antimycin
0.16 0.05# 0.57 0.04*
Mouse brain mitochondria were supplemented with 6 mM malate and 6 mM glutamate, as described. Values represent the mean of three experiments. * p < 0.05, significantly different from its corresponding control value. # p < 0.05, significant differences between deprenyl-incubated mitochondria and control mitochondria.
oxide and hydrogen peroxide in deprenyl effects on mitochondrial function. The presence of NOS was analyzed in different brain subcellular fractions from control animals by Western blot assays. A protein of 157 kDa in submitochondrial membranes, cytosolic fraction and synaptosomes was recognized through the use of a specific anti-nNOS antibody directed against the amino end of the protein. The amounts of nNOS protein detected and of NO production were higher in cytosol than in the other subcellular fractions. Incubation of submitochondrial membranes with deprenyl inhibits NOS activity in a concentration-dependent manner. The same pattern of NOS inhibition was observed after incubation of synaptosomes and cytosolic fractions with deprenyl. Taking into account that MAO is not present in cytosol, deprenyl effects on NOS activity seem to be independent of MAO inhibition. Mitochondrial NO modulates oxygen consumption by a reversible and O2-competitive inhibition of cytochrome oxidase (complex IV) that slows down electron flow, substrate oxidation and stores of chemical energy (Brown, 2001). Furthermore, mitochondrial NO regulates respiratory chain by the inhibition of ubiquinol–cytochrome c reductase activity (complex III) (Poderoso et al., 1996). Deprenyl-incubation inhibited mitochondrial NO production and increased O2 consumption in state 3 in intact mitochondria. This effect can reflect a functional regulatory activity of NO on brain mitochondrial respiration, as previously reported for chlorpromazine treatment (Lores Arnaiz et al., 2004b). Moreover, the addition of LNNA markedly increased the respiratory rate in state 3 in control brain mitochondria, but this effect was not observed in deprenyl-incubated mitochondria. The lack of effect of L-NNA on state 3 respiration rate of deprenyl-incubated mitochondria could be due to the previous inhibition of the enzyme by the drug, providing evidence of the involvement of NO in the increase in mitochondrial state 3 respiratory rate observed after deprenyl incubation. The addition of L-arginine to control and deprenyl-incubated mitochondria in state 3 did not significantly modify respiratory rates. This lack of effect of L-arginine indicates the existence of an important intramitochondrial arginine pool as previously reported (Lores Arnaiz et al., 2004b).
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Inhibition of cytochrome oxidase (complex IV) by NO is considered as a phenomenon of relevant physiological importance (Moncada and Erusalimsky, 2002; Brown, 2003). Due to the fact that in this study, mitochondrial respiratory rates were affected by deprenyl, we decided to evaluate the effect of this drug on mitochondrial respiratory complexes activity. No changes were observed in NADH–cytochrome c reductase (complexes I–III) and succinate–cytochrome c reductase (complexes II–III) activities after deprenyl incubation. These results are consistent with the concept that NO exerts its modulatory effect on respiration mainly by acting on cytochrome oxidase. Deprenyl per se did not affect complex IV activity, but cytochrome oxidase activity was increased after incubation of brain SMP with 25 mM deprenyl, when measured in the condition of maximal NOS activity. In agreement, and as observed in oxygen consumption, the addition of L-NNA increased cytochrome oxidase activity in control SMP but was not able to exert a similar effect in deprenyl-incubated SMP, giving support to the idea that the effect of deprenyl on mitochondrial respiration occurs through NOS inhibition. Mitochondrial permeability transition is a non-selective permeabilization of the inner mitochondrial membrane promoted by the accumulation of excessive quantities of calcium ions and stimulated by a variety of compounds and conditions (Zoratti and Szabo´, 1995). The inner membrane permeabilization caused by mitochondrial permeability transition results in loss of matrix components, impairment of mitochondrial functionality and substantial swelling of the organelle, with consequent outer membrane rupture and cytochrome c release (Zoratti and Szabo´, 1995; Green and Reed, 1998; Kowaltowski and Vercesi, 1999). Evidence was provided that deprenyl attenuated alteration of swelling, membrane potential, cytochrome c release and calcium transport in mitochondria treated with dopamine (Lee et al., 2002). In our study, calcium concentration higher than 90 nmol/ mg protein was able to induce partial mitochondrial permeability transition, which was inhibited by short pre-incubation with CsA. Incubation of brain mitochondria with L-NNA decreases mitochondrial permeability transition induction, indicating a possible effect of NO on mitochondrial permeability transition as suggested by different laboratories (Balakirev et al., 1997). Our results show that deprenyl protects brain calcium-induced mitochondrial permeability transition, at low concentrations (0.5–1 mM). In this range of concentrations, deprenyl was able to inhibit MAO but not NOS; these results suggests that deprenyl protective effect on calciuminduced mitochondrial permeability transition would be mediated by NO independent mechanisms. As oxygen free radicals are capable of inducing mitochondrial permeability transition (Bernardi et al., 1999), it seemed important to evaluate the possible participation of H2O2 on deprenyl effects on mitochondrial permeability transition. Hydrogen peroxide production rates were decreased by deprenyl incubation at low concentrations. Maximal H2O2 production rates (in the presence of antimycin) were not modified by deprenyl incubation, suggesting that deprenyl effect on hydrogen
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peroxide may be mediated mainly by MAO inhibition and not through a direct effect on respiratory chain. Therefore, it seems more probable that the observed decrease in H2O2 production by deprenyl would be responsible for the inhibitory effect of low concentrations of deprenyl on calcium-induced mitochondrial permeability transition. It has been reported that deprenyl provides neuroprotection by stabilizing the mitochondrial membrane potential in PC 12 cultures (Wadia et al., 1998), and by regulating molecular markers of apoptosis such as reactive oxygen species generation, lipid peroxidation, glutathionine and ATP synthesis and cytochrome c release in human neuroblastoma cell lines (Sharma et al., 2003). In summary, we show that in vitro effects of deprenyl have beneficial effects on mitochondrial function such as an increase in mitochondrial respiration and inhibition of calcium-induced mitochondrial permeability transition. Comparing the effects of deprenyl at different concentrations used in this study, we can infer that two different mechanisms are involved. The increase in oxygen consumption by deprenyl would be mediated by NOS inhibition, while mitochondrial permeability transition protective effect may occur as a consequence of MAO inhibition and decreased H2O2 production. Acknowledgements This research was supported by grants from Universidad de Buenos Aires (B131), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (PIP 02272), and Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (PICT 01-8710), Argentina. References Azonne, G.F., Pietrobon, D., Zoratti, M., 1984. Determination of the proton electrochemical gradient across biological membranes. Curr. Top. Bioenerg. 13, 1–77. Balakirev, M.Y., Khramtsov, V.V., Zimmer, G., 1997. Modulation of the mitochondrial permeability transition by nitric oxide. Eur. J. Biochem. 246, 710–718. Bernardi, P., Scorrano, L., Colonia, R., Petronilli, V., Di Lisa, F., 1999. Mitochondrial and cell death: mechanistic aspects and methodological issues. Eur. J. Biochem. 264, 687–701. Boveris, A., 1984. Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods Enzymol. 105, 429– 435. Boveris, A., Costa, L.E., Cadenas, E., Poderoso, J.J., 1999. Regulation of mitochondrial respiration by adenosine diphosphate, oxygen and nitric oxide. Methods Enzymol. 301, 188–198. Boveris, A., Lores Arnaiz, S., Bustamante, J., Alvarez, S., Valdez, L., Boveris, A.D., Navarro, A., 2002. Pharmacological regulation of mitochondrial nitric oxide synthase. Methods Enzymol. 359, 328–339. Brown, G.C., 2001. Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome oxidase. Biochim. Biophys. Acta 1504, 46– 57. Brown, G.C., 2003. NO says yes to mitochondria. Science 299, 838–839. Bustamante, J., Bersier, G., Aron-Badin, R., Cymeryng, C., Parodi, A., Boveris, A., 2002. Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis. Nitric Oxide 6, 333–341. Bustamante, J., Nutt, L., Orrenius, S., Gogvadze, V., 2005. Arsenic stimulates release of cytochrome c from isolated mitochondria via induction of mitochondrial permeability transition. Toxicol. Appl. Pharmacol. 207 (2 Suppl.), 110–116.
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