300
Biochimica et Biophysica Acta, 1035 (1990) 300-305
Elsevier BBAGEN 23382
Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria" its effect on glutathione levels and Ca 2+ efflux Gabriella Sandri 1, Enrico Panfili 1 and Lars Ernster 2 I Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Trieste (Italy) and 2 Department of Biochemistry, A rrhenius Laboratories, University of Stockholm, Stockholm (Sweden)
(Received 30 January 1990)
Key words: Mitochondria; Hydrogen peroxide; Monoamine oxidase; Glutathione; Calcium efflux; (Rat brain)
H202 production
and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H202 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial N A D P H to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H 202 generated by momoamine oxidase. The H202 accumulation was also inhibited by externally added reduced glutathione or N A D P H but not NADH. External N A D P H was oxidized by added oxidized glutathione but not a-ketoglutarate + N H ~ . These results suggest that the removal of H202 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and N A D P H , possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H 202 generated by monoamine oxidase enhances Ca 2 + release from mitochondria and may thus function as a regulator of mitochondrial Ca 2 + efflux.
Introduction The toxicity of H202 and other reactive species of oxygen is a biological problem of great importance under both physiological and pathological conditions [1]. It is now well established that mitochondria constitute a major cellular site for the formation of superoxide radical [2], which is converted to U202 (and 02) through superoxide dismutase [3]. Although the precise site of mitochondrial superoxide-radical generation is not yet established [4-6], it is probably located in Complex I a n d / o r III of the respiratory chain [7-9]. U202 is also a reaction product of monoamine oxidase (MAO), which is associated with the outer mito-
Abbreviations: MPTP, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; BCNU, 1,3-bis(2 chloroetbyl)-l-nitrosourea; HRP, horse radish peroxidase (E.C. 1.11.1.7); MAO, monoamine oxidase (E.C. 1.4.3.4); GSH peroxidase, glutathione peroxidase (E.C. 1.11.1.9); GSSG reductase, glutathione reductase (E.C. 1.6.4.2). Correspondence: G. Sandri, Department of Biochemistry, University of Trieste, Via Valerio 32, 1-34127 Trieste, Italy.
chondrial membrane [10-12]. H 2 0 2 generated by mitochondrial enzymes is thought to be removed by glutathione peroxidase, located in both the mitochondria and the cytosolic space, or by the extra-mitochondrial enzyme catalase and various peroxidases (cf. Re/. 1). Removal of H 2 0 e by way of glutathione peroxidase may lead to a depletion of glutathione and thereby an oxidation of protein-thiol groups, with a disturbance of cellular Ca 2+ homeostasis as a consequence [13-17]. If not removed, H 2 0 2 may inhibit superoxide dismutase [3] and, in the presence of Fe 2+, it may be converted to the perferryl and hydroxyl radicals, giving rise to lipid peroxidation and oxidative damage to membranes, proteins and nucleic acids (cf. Re/. 1). These circumstances may be especially critical in brain tissue, which possesses relatively poor catalase and peroxidase activities, and where monoamine oxidase plays an important role in neurotransmitter metabolism [18-21]. Despite growing interest in recent years in both the role of monoamine oxidase and the possible involvement of oxidative stress in cerebral pathophysiology, relatively little is known about the mechanisms responsible for the removal of H 2 0 2 generated by monoamine oxidase in
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
301 brain mitochondria. The present study is concerned with this particular problem, using isolated mitochondria from rat brain. Materials and Methods
Non-synaptosomal rat brain mitochondria were prepared from fed young male albino rats according to Rehncrona et al. [22]. H202 production and accumulation in isolated rat brain mitochondria were monitored fluorometrically (JASCO FP-770 spectrofluorometer) by the scopoletin (7-hydroxy-6-methoxy-coumarin; Sigma, U.S.A.) and horse radish peroxidase (Boehringer, F.R.G.) method, according to Boveris [23]. Benzylamine (Erba, Italy), tyramine (Aldrich, U.S.A.), fl-phenylethylamine (Sigma, U.S.A.) and M P T P (kindly supplied by Dr. C. Richter, ETH, Zurich) were used as monoamine oxidase substrates. Pargyline (N-methyl-N-benzyl-2-propynylamine; Sigma, U.S.A.) and clorgyline (N-methyl-N-prop argyl-3-(2,4-dichlorophenoxy)propylamine); Sigma, U.S.A.) were used as specific inhibitors of monoamine oxidase B and A activities, respectively. Ca 2+ uptake and release were recorded spectrophotometrically at 665-685 nm (SIGMA ZWS II dualwavelength spectrophotometer) using purified [24] arsenazo III (2,2'-(1,8-dihydroxy-3,6-disulfo-2,7-naphthalene-bis(azo))dibenzenarsonic acid; Merck, F.R.G.) according to the method of Vallirres et al. [25]. Mitochondrial reduced glutathione content was estimated at 340 nm according to Crowley et al. [26] using glutathione S-transferase (2.5.1.18; Sigma, U.S.A.) and 1-chloro-2,4-dinitrobenzene. BCNU was a generous gift of Bristol Myers, U.S.A. Diamide (1,1'-azobis(N, N-dimethylformamide); Aldrich, U.S.A.) was used in order to oxidize GSH. 1.0-
N A D P H was monitored fluorometrically at 340 nm (excitation wavelength) and 460 nm (emission wavelength). Protein content of rat brain mitochondria was measured according to Gornall et al. [27]. Water and other chemicals were of the highest purity grade. Results and Discussion MA 0 activities and strates
H202 accumulation
with various sub-
Figs. 1A and B show fluorometric recordings used for the measurements of MAO activity and resulting H202 accumulation, respectively, in isolated rat brain mitochondria. In Fig. 1A, scopoletin and horse radish peroxidase (HRP) were added to a buffered suspension of mitochondria, and the monoamine oxidase reaction was then initiated by the addition of benzylamine. After a short lag there was a linear decrease of fluorescence, due to the formation of H202. The slope of this hnear portion of the trace was used for the estimation of the MAO activity. Addition of pargyline, a specific inhibitor of MAO B, blocked the reaction as expected. In Fig. 1B, the mitochondria were incubated in the absence of scopoletin and HRP. MAO reaction was initiated by the addition of benzylamine, and the reaction was allowed to proceed for a desired period of time, in the present case 5 rain, after which it was arrested by the addition of pargyline. The amount of H202 accumulated was determined by adding scopoletin and HRP, and measuring the ensuing decrease of fluorescence. Table I summarizes the MAO A and B activities of rat brain mitochondria as measured with different substrates. Benzylamine and MPTP are substrates for MAO B, tyramine for MAO A and B, and /3-phenylethylB
A
'E
BA
T
1 nmol H202
0.5 ~" 1 nmol H202
1
Parg.
,
/
5 min
1 rain I ....
o.o
mit ÷ medium
I
I
mit
Scop.
mediom *
-
,0.5 min I
t BA
i I'~--I i
l
Parg. Scop.
Fig. 1. MAO-induced H202 production and accumulation in isolated rat-brain mitochondria. Mitochondria (approx. 1.5 mg protein) were incubated in a medium containing 0.225 M mannitol, 0.075 M sucrose, and 0.025 M Tris-HCl, pH 7.5 at 30 ° C. Final volume, 2 ml. When indicated, 1 ~M scopoletin(Scop.), 25/zg horse radish peroxidase(HRP), 200 ~M benzylamine(BA) and 50 ~M pargyline(Parg.) were added. (A) H202 production and (B)H202 accumulation.
302 TABLE I
TABLE II
MA 0 A and B activity of rat-brain mitochondria
MA O-induced H20e accumulation under different respiratory conditions
Substrate
MAO involved
Benzylamine Tyramine fl-Phenylethylamine MPTP
with pargyline + clorgyline
Conditions as in Fig. lB. Incubation times for H202 accumulation: 10 min with benzylamine; and 5 rain with tyramine as substrate. The incubation mixture contained; mitochondria, approx. 1.5 mg protein, 0.225 M mannitol, 0.25 M sucrose, 0.025 M Tris-HCl (pH 7.5), 3 mM potassium phosphate and, when indicated, 5 mM pyruvate, 5 mM malate, 5 mM isocitrate, 15 /xM rotenone, and 1 /~M CCCP. Final volume, 2 ml. Temp. 30 ° C.
0.00
Additions
H202 formed (nmol/min per mg protein) without inhibitor
with pargyline
B A and B
0.27 + 0.07 (6) 0.46 5:0.10 (3)
0.00 0.11
mainlyB B
0.625:0.03 (4) 0.28
0.06 0.00
Conditions as in Fig. 1A. Mean values and standard deviations, with the number of experiments in parentheses.
amine mainly for MAO B. The reaction was inhibited by pargyline in the case of MAO B, and by clorgyline in the case of MAO A. From the data it can be deduced that the MAO A and B activities were approximately equal. The specific activities and the proportion between MAO A and B are in good agreement with those earlier reported for brain cortex [28,29]. Fig. 2 shows the time course of H202 accumulation with different MAO substrates. With tyramine, oxidized by way of both MAO A and B, H202 accumulation proceeded at an initial rate 3- to 4-times higher than with substrates oxidized exclusively or mainly through MAO B, such as benzylamine, MPTP or fl-phenylethylamine. This was in contrast to the rates of MAO A and B activities (of. Table I) which were approximately equal. However, when the mitochondria were aged at 0 ° C for about 1 h, the rate of HzO 2 accumulation with
4-
'~ •~
°0.~_
3
o~2
./
o
0
5
10
minutes
Fig. 2. Time-course of MAO-induced H202 accumulation in isolated rat-brain mitochondria with various substrates. Substrates, added in
concentrations of 200 #M, were: O, tyramine; D, fl-phenylethylamine; ,% benzylamine; o, MPTP; zx, benzylamine, 2 h aged mitochondria (insert). Other conditions as in Fig. 1.
None Pyruvate, malate Pyruvate, malate, rotenone Pyruvate, malate, CCCP Pyruvate, malate, rotenone, CCCP Isocitrate, malate Isocitrate, malate, CCCP
H202 accumulation (nmol/mg protein) benzylamine
tyramine
1.50 0.08 0.21 0.03 0.15 0.32 0.45
2.00 0.13 0.28 0.00 0.25
benzylamine as substrates, i.e., via M A O B, increased (insert of Fig. 2); the rate of H 2 0 2 accumulation with tyramine did not increase appreciably upon aging (data not shown). These findings indicate that the activity of M A O B, but not that of MAO A, decreases as H 2 0 2 accumulates. In addition, H 2 0 2 accumulation showed a short - about 30 s - lag with benzylamine as substrate (cf. Fig. 1 and insert of Fig. 2). It would thus appear that the accumulation of H 2 0 2 via MAO B, in contrast to that via M A O A, is dependent on the presence of a component that is diminished in the mitochondria upon aging. The difference in substrate and inhibitor specificities between M A O A and B has been discussed extensively, including the role of thiol groups and phospholipids [21]. It may be of interest, in this connection, that aging has been shown to result in a depletion of mitochondrial glutathione [30]. Another component that is known to diminish in mitochondria upon aging are endogenous substrates (e.g., fatty acids). M A O-induced 1t202 accumulation and glutathione steady state As shown in Table II, the accumulation of H202 with either benzylamine or tyramine as substrate for MAO was strongly inhibited by the addition of pyruvate + malate, i.e., by conditions that provide reducing power for the maintenance of glutathione in the reduced state through N A D P H and glutathione reductase. Addition of rotenone or CCCP, alone or in combination, affected the inhibition only marginally, indicating that the energy-linked nicotinamide nucleotide dehydrogenase was not a major pathway for providing the reducing equivalents from pyruvate oxidation for the generation of N A D P H . In fact, as shown in Table II, isocitrate was also effective (although to a somewhat lesser extent) in
303
,-
x . , t ~
- 1.5
1004
.E Q.
~
E
~.
-1.0
'~
a-
,,
o ~a 2. ® "6 E 1 e-
E o~
-t-0.5
_
o E c
% "I"
0
i
i
i
f
2
4
6
8
0
, 100
minutes
' 600
Fig. 4. Effect of added GSH on the MAO-induced accumulation of H202 in isolated rat-brain mitochondria. GSH was added to the mitochondria 1 min before the addition of tyramine (200/~M). H202 accumulation was determined after 5 min as described in Fig. lB.
100
inhibiting H202 accumulation, suggesting that the pyruvate effect was at least partly due to the generation of N A D P H by w a y of isocitrate and the N A D P + - l i n k e d isocitrate dehydrogenase. Determination of the mitochondrial G S H content in the course of H202 accumulation resulting from tyramine oxidation by M A O A and B gave the somewhat unexpected result that after an initial decrease b y about 30% the G S H level diminished only slowly, despite the continuing H202 production (Fig. 3). Inhibition of the H202 accumulation by pyruvate + malate prevented the decrease in G S H level. A d d i t i o n of diamide resulted, as expected, in an extensive decrease in G S H content. It has been reported [31] that glutathione peroxidase and glutathione reductase occur in mitochondria in b o t h the intermembrane space and the matrix. Based on the present findings the possibility was considered that the partial decrease in G S H shown in F i g . 3 might be explained in terms of a c o m p a r t m e n t a t i o n of G S H between the intermembrane space and the matrix, and that only the G S H present in the intermembrane space might be readily available for the removal of the micromolar concentrations of H202 generated by M A O in the outer membrane. Support for this explanation was obtained b y showing that externally added G S H efficiently inhibited H202 accumulation originating from M A O (Fig. 4). Moreover, it was found that added N A D P H , but not N A D H , also inhibited H 2 0 2 accumulation (Fig. 5), which is consistent with the conclusion that glutathione reductase also is present in the interm e m b r a n e space. This conclusion could be further substantiated by the finding that added N A D P H was oxidized by added G S S G (Fig. 6) and the reaction was inhibited by B C N U , an inhibitor of glutathione reductase. N o oxidation of added N A D P H was found
H
GSH ~uM]
Fig. 3. GSH content of isolated rat-brain mitochondria during tyr-
amine-induced H202 accumulation. The symbols represent: o, GSH content in absence of tyramine; t,, GSH content during oxidation of tyramine (200 FM); X, GSH content during tyramine oxidation in the presence of pyruvate (5 mM), malate (5 mM) and phosphate (3 mM); =, GSH content after 5 rain incubation of the mitochondria with 100 FM diamide; o, H202 accumulation in the presence of tyramine (200 #M). Other conditions as in Fig. lB.
, 200
•¢
¢
v
E
50
o o
£
k---o ,
,
,
10
20
30
I[
,
100
NADPH orNADH [pM]
Fig. 5. Effects of added NADH and NADPH on MAO-induced H202 accumulation in rat brain mitochondria. NADH (e) or NADPH (0) were added to the mitochondfia 1 min before the addition of tyramine. H202 accumulation was determined after 5 min of incubation as described in Fig. lB.
mit + NADPH
l
GSSG 1 min GSS
t
h
-[ 10 n m ° l r s
NADPH
BCNU
~KG 4-
NH~
Fig. 6. Oxidation of added NADPH by isolated rat-brain mitochondria in the presence of GSSG or a-ketoglutarate+NH2. The incubation mixture contained rat-brain mitochondria (1.5 mg protein), 0.225 M mannitoi, 0.075 M sucrose and 0.025 M Tris-HC1 (pH 7.4). When indicated 100 #M NADPH, 200 vM GSSG, 5 mM a-ketoglutarate (a-KG)+5 mM NH~', and 400 /tM BCNU were added. Final volume, 2 ml, temp. 30 o C. NADPH oxidation was monitored fluorometricaUy as indicated in Materials and Methods.
304 Succ. r o t .
T
10 n m o l e s Ca ~+ ; / / /
l, ec mit
, Ca 2+
/ /
~"~" = ~ ' ¢ - - - ' - ' - - - ~ ' Parg.
RR
/ BA
Fig. 7. Ca 2+ uptake and MAO-induced Ca 2+ release by isolated rat-brain mitoehondria. The incubation mixture contained rat-brain mitochondria (1.5 mg protein), 0.1 M mannitol, 0.05 M KC1, 0.025 M Tris-HCl (pH 7.4), 3 mM potassium phosphate (pH 7.4) and 0.1 mM purified arsenazo II1. Ca 2+ load, 57 nmol/mg mitochondrial protein. When indicated, 5 mM potassium succinate (Succ.)+ 15 #M rotenone, 50 /~M pargyline (Parg.), 200 /xM benzylamine (BA) and 0.05 /~M ruthenium red (RR) were added. Final volume, 2 ml, temp. 30 o C. Ca 2+ uptake and release were spectrophotometrically monitored as indicated in Materials and Methods.
with added a-ketoglutarate + NH~-, a potent oxidant of intra-mitochondrial NADPH by way of glutamate dehydrogenase, showing that the added NADPH did not penetrate the inner membrane, i.e., that the rnitochondria are not damaged. These conclusions regarding glutathione reductase may appear paradoxical, since the data indicate that the fraction of GSH oxidized by MAO-generated H202 via glutathione peroxidase can be reduced by both extraand intra-mitochondrial NADPH (the latter produced, e.g., by isocitrate oxidation). However, a similar behaviour holds for the mitochondrial hexokinase, found mainly in tumours and brain [32], where it is bound to the outer membrane but reacts with both extra- and
r,
M A O-induced 11202 accumulation and Ca 2 + efflux
Mitochondrial Ca 2+ efflux has been shown to take place upon oxidation of reduced nicotinamide nucleotides by various oxidants including acetoacetate, oxaloacetate, menadione and organic hydroperoxides [36-41]. As shown by Richter and his colleagues, this Ca 2÷ efflux probably proceeds by way of a specific protein that is activated by ADP-ribosylation [42], the ADP-ribose being generated through a cleavage of mitochondrial NAD ÷ (but not NADH) through a glycohydrolase [43]. NADPH oxidation is less efficient in stimulating Ca 2÷ efflux and is probably acting through a dephosphorylation of NADP ÷ and NAD ÷ [44]. Baumhiiter and Richter [37] have shown that t-butylhydroperoxide stimulates Ca 2÷ efflux from mitochondria at a rate which is dependent on the Ca 2+ load and which is maximal when ruthenium red is added to
Tyr
BA
'$
intra-mitochondrial ATP (in fact preferentially with the latter). It has been suggested [33] that the enzyme may be located in the contact sites between the inner and outer membranes. A similar location has been proposed for the high-affinity Ca2+-binding protein of brain mitochondria [34]. The possible location of part of the mitochondrial glutathione reductase and glutathione peroxidase in these membrane junctions may be investigated in the future by isolation of the junction fraction as described earlier (cf. Refs. 33 and 34). In addition to the compartmentation of glutathione, discussed above, the present findings regarding the partial depletion of GSH by H202 produced via MAO A and B may also be explained by heterogeneous distribution of the latter enzyme in different parts of the brain [35]. This probably would not, however, obviate our conclusion that at least parts of the mitochondrial glutathione peroxidase and glutathione reductase are accessible to substrates from both the extra- and intra-mitochondrial space.
Pha
MPTP
50
5O
~5-
E
~
lo-
0
E ¢=
5
,.$ o 0
50
0
50
0
Ca2*load (nmoles/mg protein)
Fig. 8. Dependence of MAO-induced Ca 2+ release on Ca 2+ load in the presence of various substrates. Benzylamine (BA), tyramine (Tyr), fl-phenylethylamine (Pha) and MPTP were added in concentrations of 200 #M. Mitochondria were loaded with different amounts of Ca 2÷ 30 s before the initiation of MAO activity by the addition of the various substrates. The data refer to initial rates after the addition of ruthenium red (see Fig. 7), o, in the presence of ruthenium red; e, in the presence of ruthenium red + amine.
305 inhibit the reuptake of Ca 2+. As shown in Fig. 7, a release of Ca 2+ from brain mitochondria is promoted by the substrates of MAO as well. Fig. 8 compares effects of Ca 2+ load on the rate of Ca 2+ efflux in the presence of benzylamine, tyrarnine, fl-phenylethylamine and MPTP in the presence of ruthenium ref. These results are in line with those earlier reported by Baumhiiter and Richter [37] and demonstrate that MAO may play an important role in regulating mitochondrial Ca 2+ efflux.
Acknowledgements The Authors are grateful to Dr. C. Richter for the generous gift of MPTP and his advice for its use, to Mr. B. Gazzin for his skilful technical assistance, to the Fondo per lo Studio e la Ricerca Scientifica delle Malattie del Fegato (Regione Friuli-Venezia Giulia) for the use of the spectrofluorometer, to the Ministero dell'Universit/t e della Ricerca Scientifica e Tecnologica (Rome), to Consiglio Nazionale delle Ricerche (Rome) for financial support, and to Ms. A Nielsen and Ms. K. Nordenbrand for their help in the preparation of the manuscript.
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14 Richter, C. and Frei, B. (1985) in Oxidative Stress (Sies, H., ed.), pp. 211-241, Academic Press, London. 15 Richter, C. and Frei, B. (1988) in Free Radicals in Biology and Medicine, Vol. 4 (Pryor, W.A., ed.), pp. 365-375, Pergamon Press, U.S.A. 16 Orrenius, S., McConkey, D.J., Bellomo, G. and Nieotera, P. (1989) Trends Pharmacol. Sci. 10, 281-285. 17 SiesjiS, B.K. (1988) Crit. Care Med. 16, 954-963. 18 Watson, B.D., Busto, R., Goldberg, W.J., Santiso, N., Yoshida, S. and Ginsberg, M.D. (1984) J. Neurochem. 42, 268-274. 19 Kontos, H.A. (1985) Circ. Res. 57, 508-516. 20 Oreland, U, Arai, Y., Stenstrom, A. and Fowler, C.J. (1983) MOd. Probl. Pharmacopsychiatry 19, 246-254. 21 Singer, T.P. (1987) J. Neural. Transm. [Suppl.] 23, 1-23. 22 Rehncrona, S., Mela, U and Siesji3, B.K. (1979) Stroke 10, 437-446. 23 Boveris, A. (1984) Methods Enzymol. 105, 429-435. 24 Dipolo, R., Requena, J., Brinley, Jr., F.L., Muffins, L.J., Scarpa, A. and Tippert, T. (1976) J. Gen. Physiol. 67, 433-467. 25 Valli6res, J., Scarpa, A. and Somlyo, A.P. (1975) Arch. Biochem. Biophys. 170, 659-669. 26 Crowley, C., Gillham, B. and Thorn, M.B. (1975) Biochem. Med. 13, 287-292. 27 Gornall, A.G., Bardawill, C.G. and David, N.N. (1949) J. Biol. Chem. 177, 751-766. 28 Fowler, C.J., Oreland, L., Wiberg, A., Carlsson, A. and Magnusson, T. (1979) Med. Biol. 57, 406-411. 29 Fowler, C.J. (1982) Drugs of the Future, VIII, 501-517. 30 Olafsdottir, K. and Reed, D.J. (1988) Biochim. Biophys. Acta 964, 377-382. 31 Floh6, L. and Sehlegel, W. (1971) Hoppe-Seyler's Z. Physiol. Chem. 352, 1401-1410. 32 Arora, K.K. and Pedersen, P.L. (1985) J. Biol. Chem. 263, 1742217428. 33 Ohlendieck, K., Riesinger, I., Adams, V., Krause, J. and Brdiczka, D. (1986) Biochim. Biophys. Acta 860, 672-689. 34 Sandri, G., Siagri, M. and Panfili, E. (1988) Cell Calcium 9, 159-165. 35 StenstrSm, A., Hardy, J. and Oreland, L. (1987) Biochem. Pharmaeol. 36, 2031-2035. 36 Lehninger, A.U, Vercesi, A. and Bababunmi, E.A. (1978) Proc. Natl. Acad. SOl. USA 75, 1690-1694. 37 Baumhiiter, C. and Richter, C. (1982) FEBS Lett. 148, 271-275. 38 Richter, C., Winterhalter, K.H., Baumhiiter, S. and LStscher, M.R. and Moser, B. (1983) Proc. Natl. Acad. Sci. USA 80, 3188-3192. 39 Frei, B., Wimerhalter, K.H. and Richter, C. (1985) Eur. J. Biochem. 149, 633-639. 40 Frei, B., Winterhalter, K.H. and Richter, C. (1986) Biochemistry 25, 4438-4443. 41 Ernster, L., Konji, V., Montag, A., Nordenbrand, K. and Sandri, G. (1986) Symp. Biol. Hung. 30, 27-39. 42 Frei, B. and Richter, C. (1988) Biochemistry 27, 529-535. 43 Moser, B., Winterhalter, K.H. and Richter, C. (1983) Arch. Biochem. Biophys. 224, 358-364. 44 Richter, C. (1987) Biochem. Biophys. Res. Commun. 146, 253-257.
Biochimica et Biophysica Acta, 1035 (1990) 300-305
Elsevier BBAGEN 23382
Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria" its effect on glutathione levels and Ca 2+ efflux Gabriella Sandri 1, Enrico Panfili 1 and Lars Ernster 2 I Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Trieste (Italy) and 2 Department of Biochemistry, A rrhenius Laboratories, University of Stockholm, Stockholm (Sweden)
(Received 30 January 1990)
Key words: Mitochondria; Hydrogen peroxide; Monoamine oxidase; Glutathione; Calcium efflux; (Rat brain)
H202 production
and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H202 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial N A D P H to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H 202 generated by momoamine oxidase. The H202 accumulation was also inhibited by externally added reduced glutathione or N A D P H but not NADH. External N A D P H was oxidized by added oxidized glutathione but not a-ketoglutarate + N H ~ . These results suggest that the removal of H202 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and N A D P H , possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H 202 generated by monoamine oxidase enhances Ca 2 + release from mitochondria and may thus function as a regulator of mitochondrial Ca 2 + efflux.
Introduction The toxicity of H202 and other reactive species of oxygen is a biological problem of great importance under both physiological and pathological conditions [1]. It is now well established that mitochondria constitute a major cellular site for the formation of superoxide radical [2], which is converted to U202 (and 02) through superoxide dismutase [3]. Although the precise site of mitochondrial superoxide-radical generation is not yet established [4-6], it is probably located in Complex I a n d / o r III of the respiratory chain [7-9]. U202 is also a reaction product of monoamine oxidase (MAO), which is associated with the outer mito-
Abbreviations: MPTP, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; BCNU, 1,3-bis(2 chloroetbyl)-l-nitrosourea; HRP, horse radish peroxidase (E.C. 1.11.1.7); MAO, monoamine oxidase (E.C. 1.4.3.4); GSH peroxidase, glutathione peroxidase (E.C. 1.11.1.9); GSSG reductase, glutathione reductase (E.C. 1.6.4.2). Correspondence: G. Sandri, Department of Biochemistry, University of Trieste, Via Valerio 32, 1-34127 Trieste, Italy.
chondrial membrane [10-12]. H 2 0 2 generated by mitochondrial enzymes is thought to be removed by glutathione peroxidase, located in both the mitochondria and the cytosolic space, or by the extra-mitochondrial enzyme catalase and various peroxidases (cf. Re/. 1). Removal of H 2 0 e by way of glutathione peroxidase may lead to a depletion of glutathione and thereby an oxidation of protein-thiol groups, with a disturbance of cellular Ca 2+ homeostasis as a consequence [13-17]. If not removed, H 2 0 2 may inhibit superoxide dismutase [3] and, in the presence of Fe 2+, it may be converted to the perferryl and hydroxyl radicals, giving rise to lipid peroxidation and oxidative damage to membranes, proteins and nucleic acids (cf. Re/. 1). These circumstances may be especially critical in brain tissue, which possesses relatively poor catalase and peroxidase activities, and where monoamine oxidase plays an important role in neurotransmitter metabolism [18-21]. Despite growing interest in recent years in both the role of monoamine oxidase and the possible involvement of oxidative stress in cerebral pathophysiology, relatively little is known about the mechanisms responsible for the removal of H 2 0 2 generated by monoamine oxidase in
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
301 brain mitochondria. The present study is concerned with this particular problem, using isolated mitochondria from rat brain. Materials and Methods
Non-synaptosomal rat brain mitochondria were prepared from fed young male albino rats according to Rehncrona et al. [22]. H202 production and accumulation in isolated rat brain mitochondria were monitored fluorometrically (JASCO FP-770 spectrofluorometer) by the scopoletin (7-hydroxy-6-methoxy-coumarin; Sigma, U.S.A.) and horse radish peroxidase (Boehringer, F.R.G.) method, according to Boveris [23]. Benzylamine (Erba, Italy), tyramine (Aldrich, U.S.A.), fl-phenylethylamine (Sigma, U.S.A.) and M P T P (kindly supplied by Dr. C. Richter, ETH, Zurich) were used as monoamine oxidase substrates. Pargyline (N-methyl-N-benzyl-2-propynylamine; Sigma, U.S.A.) and clorgyline (N-methyl-N-prop argyl-3-(2,4-dichlorophenoxy)propylamine); Sigma, U.S.A.) were used as specific inhibitors of monoamine oxidase B and A activities, respectively. Ca 2+ uptake and release were recorded spectrophotometrically at 665-685 nm (SIGMA ZWS II dualwavelength spectrophotometer) using purified [24] arsenazo III (2,2'-(1,8-dihydroxy-3,6-disulfo-2,7-naphthalene-bis(azo))dibenzenarsonic acid; Merck, F.R.G.) according to the method of Vallirres et al. [25]. Mitochondrial reduced glutathione content was estimated at 340 nm according to Crowley et al. [26] using glutathione S-transferase (2.5.1.18; Sigma, U.S.A.) and 1-chloro-2,4-dinitrobenzene. BCNU was a generous gift of Bristol Myers, U.S.A. Diamide (1,1'-azobis(N, N-dimethylformamide); Aldrich, U.S.A.) was used in order to oxidize GSH. 1.0-
N A D P H was monitored fluorometrically at 340 nm (excitation wavelength) and 460 nm (emission wavelength). Protein content of rat brain mitochondria was measured according to Gornall et al. [27]. Water and other chemicals were of the highest purity grade. Results and Discussion MA 0 activities and strates
H202 accumulation
with various sub-
Figs. 1A and B show fluorometric recordings used for the measurements of MAO activity and resulting H202 accumulation, respectively, in isolated rat brain mitochondria. In Fig. 1A, scopoletin and horse radish peroxidase (HRP) were added to a buffered suspension of mitochondria, and the monoamine oxidase reaction was then initiated by the addition of benzylamine. After a short lag there was a linear decrease of fluorescence, due to the formation of H202. The slope of this hnear portion of the trace was used for the estimation of the MAO activity. Addition of pargyline, a specific inhibitor of MAO B, blocked the reaction as expected. In Fig. 1B, the mitochondria were incubated in the absence of scopoletin and HRP. MAO reaction was initiated by the addition of benzylamine, and the reaction was allowed to proceed for a desired period of time, in the present case 5 rain, after which it was arrested by the addition of pargyline. The amount of H202 accumulated was determined by adding scopoletin and HRP, and measuring the ensuing decrease of fluorescence. Table I summarizes the MAO A and B activities of rat brain mitochondria as measured with different substrates. Benzylamine and MPTP are substrates for MAO B, tyramine for MAO A and B, and /3-phenylethylB
A
'E
BA
T
1 nmol H202
0.5 ~" 1 nmol H202
1
Parg.
,
/
5 min
1 rain I ....
o.o
mit ÷ medium
I
I
mit
Scop.
mediom *
-
,0.5 min I
t BA
i I'~--I i
l
Parg. Scop.
Fig. 1. MAO-induced H202 production and accumulation in isolated rat-brain mitochondria. Mitochondria (approx. 1.5 mg protein) were incubated in a medium containing 0.225 M mannitol, 0.075 M sucrose, and 0.025 M Tris-HCl, pH 7.5 at 30 ° C. Final volume, 2 ml. When indicated, 1 ~M scopoletin(Scop.), 25/zg horse radish peroxidase(HRP), 200 ~M benzylamine(BA) and 50 ~M pargyline(Parg.) were added. (A) H202 production and (B)H202 accumulation.
302 TABLE I
TABLE II
MA 0 A and B activity of rat-brain mitochondria
MA O-induced H20e accumulation under different respiratory conditions
Substrate
MAO involved
Benzylamine Tyramine fl-Phenylethylamine MPTP
with pargyline + clorgyline
Conditions as in Fig. lB. Incubation times for H202 accumulation: 10 min with benzylamine; and 5 rain with tyramine as substrate. The incubation mixture contained; mitochondria, approx. 1.5 mg protein, 0.225 M mannitol, 0.25 M sucrose, 0.025 M Tris-HCl (pH 7.5), 3 mM potassium phosphate and, when indicated, 5 mM pyruvate, 5 mM malate, 5 mM isocitrate, 15 /xM rotenone, and 1 /~M CCCP. Final volume, 2 ml. Temp. 30 ° C.
0.00
Additions
H202 formed (nmol/min per mg protein) without inhibitor
with pargyline
B A and B
0.27 + 0.07 (6) 0.46 5:0.10 (3)
0.00 0.11
mainlyB B
0.625:0.03 (4) 0.28
0.06 0.00
Conditions as in Fig. 1A. Mean values and standard deviations, with the number of experiments in parentheses.
amine mainly for MAO B. The reaction was inhibited by pargyline in the case of MAO B, and by clorgyline in the case of MAO A. From the data it can be deduced that the MAO A and B activities were approximately equal. The specific activities and the proportion between MAO A and B are in good agreement with those earlier reported for brain cortex [28,29]. Fig. 2 shows the time course of H202 accumulation with different MAO substrates. With tyramine, oxidized by way of both MAO A and B, H202 accumulation proceeded at an initial rate 3- to 4-times higher than with substrates oxidized exclusively or mainly through MAO B, such as benzylamine, MPTP or fl-phenylethylamine. This was in contrast to the rates of MAO A and B activities (of. Table I) which were approximately equal. However, when the mitochondria were aged at 0 ° C for about 1 h, the rate of HzO 2 accumulation with
4-
'~ •~
°0.~_
3
o~2
./
o
0
5
10
minutes
Fig. 2. Time-course of MAO-induced H202 accumulation in isolated rat-brain mitochondria with various substrates. Substrates, added in
concentrations of 200 #M, were: O, tyramine; D, fl-phenylethylamine; ,% benzylamine; o, MPTP; zx, benzylamine, 2 h aged mitochondria (insert). Other conditions as in Fig. 1.
None Pyruvate, malate Pyruvate, malate, rotenone Pyruvate, malate, CCCP Pyruvate, malate, rotenone, CCCP Isocitrate, malate Isocitrate, malate, CCCP
H202 accumulation (nmol/mg protein) benzylamine
tyramine
1.50 0.08 0.21 0.03 0.15 0.32 0.45
2.00 0.13 0.28 0.00 0.25
benzylamine as substrates, i.e., via M A O B, increased (insert of Fig. 2); the rate of H 2 0 2 accumulation with tyramine did not increase appreciably upon aging (data not shown). These findings indicate that the activity of M A O B, but not that of MAO A, decreases as H 2 0 2 accumulates. In addition, H 2 0 2 accumulation showed a short - about 30 s - lag with benzylamine as substrate (cf. Fig. 1 and insert of Fig. 2). It would thus appear that the accumulation of H 2 0 2 via MAO B, in contrast to that via M A O A, is dependent on the presence of a component that is diminished in the mitochondria upon aging. The difference in substrate and inhibitor specificities between M A O A and B has been discussed extensively, including the role of thiol groups and phospholipids [21]. It may be of interest, in this connection, that aging has been shown to result in a depletion of mitochondrial glutathione [30]. Another component that is known to diminish in mitochondria upon aging are endogenous substrates (e.g., fatty acids). M A O-induced 1t202 accumulation and glutathione steady state As shown in Table II, the accumulation of H202 with either benzylamine or tyramine as substrate for MAO was strongly inhibited by the addition of pyruvate + malate, i.e., by conditions that provide reducing power for the maintenance of glutathione in the reduced state through N A D P H and glutathione reductase. Addition of rotenone or CCCP, alone or in combination, affected the inhibition only marginally, indicating that the energy-linked nicotinamide nucleotide dehydrogenase was not a major pathway for providing the reducing equivalents from pyruvate oxidation for the generation of N A D P H . In fact, as shown in Table II, isocitrate was also effective (although to a somewhat lesser extent) in
303
,-
x . , t ~
- 1.5
1004
.E Q.
~
E
~.
-1.0
'~
a-
,,
o ~a 2. ® "6 E 1 e-
E o~
-t-0.5
_
o E c
% "I"
0
i
i
i
f
2
4
6
8
0
, 100
minutes
' 600
Fig. 4. Effect of added GSH on the MAO-induced accumulation of H202 in isolated rat-brain mitochondria. GSH was added to the mitochondria 1 min before the addition of tyramine (200/~M). H202 accumulation was determined after 5 min as described in Fig. lB.
100
inhibiting H202 accumulation, suggesting that the pyruvate effect was at least partly due to the generation of N A D P H by w a y of isocitrate and the N A D P + - l i n k e d isocitrate dehydrogenase. Determination of the mitochondrial G S H content in the course of H202 accumulation resulting from tyramine oxidation by M A O A and B gave the somewhat unexpected result that after an initial decrease b y about 30% the G S H level diminished only slowly, despite the continuing H202 production (Fig. 3). Inhibition of the H202 accumulation by pyruvate + malate prevented the decrease in G S H level. A d d i t i o n of diamide resulted, as expected, in an extensive decrease in G S H content. It has been reported [31] that glutathione peroxidase and glutathione reductase occur in mitochondria in b o t h the intermembrane space and the matrix. Based on the present findings the possibility was considered that the partial decrease in G S H shown in F i g . 3 might be explained in terms of a c o m p a r t m e n t a t i o n of G S H between the intermembrane space and the matrix, and that only the G S H present in the intermembrane space might be readily available for the removal of the micromolar concentrations of H202 generated by M A O in the outer membrane. Support for this explanation was obtained b y showing that externally added G S H efficiently inhibited H202 accumulation originating from M A O (Fig. 4). Moreover, it was found that added N A D P H , but not N A D H , also inhibited H 2 0 2 accumulation (Fig. 5), which is consistent with the conclusion that glutathione reductase also is present in the interm e m b r a n e space. This conclusion could be further substantiated by the finding that added N A D P H was oxidized by added G S S G (Fig. 6) and the reaction was inhibited by B C N U , an inhibitor of glutathione reductase. N o oxidation of added N A D P H was found
H
GSH ~uM]
Fig. 3. GSH content of isolated rat-brain mitochondria during tyr-
amine-induced H202 accumulation. The symbols represent: o, GSH content in absence of tyramine; t,, GSH content during oxidation of tyramine (200 FM); X, GSH content during tyramine oxidation in the presence of pyruvate (5 mM), malate (5 mM) and phosphate (3 mM); =, GSH content after 5 rain incubation of the mitochondria with 100 FM diamide; o, H202 accumulation in the presence of tyramine (200 #M). Other conditions as in Fig. lB.
, 200
•¢
¢
v
E
50
o o
£
k---o ,
,
,
10
20
30
I[
,
100
NADPH orNADH [pM]
Fig. 5. Effects of added NADH and NADPH on MAO-induced H202 accumulation in rat brain mitochondria. NADH (e) or NADPH (0) were added to the mitochondfia 1 min before the addition of tyramine. H202 accumulation was determined after 5 min of incubation as described in Fig. lB.
mit + NADPH
l
GSSG 1 min GSS
t
h
-[ 10 n m ° l r s
NADPH
BCNU
~KG 4-
NH~
Fig. 6. Oxidation of added NADPH by isolated rat-brain mitochondria in the presence of GSSG or a-ketoglutarate+NH2. The incubation mixture contained rat-brain mitochondria (1.5 mg protein), 0.225 M mannitoi, 0.075 M sucrose and 0.025 M Tris-HC1 (pH 7.4). When indicated 100 #M NADPH, 200 vM GSSG, 5 mM a-ketoglutarate (a-KG)+5 mM NH~', and 400 /tM BCNU were added. Final volume, 2 ml, temp. 30 o C. NADPH oxidation was monitored fluorometricaUy as indicated in Materials and Methods.
304 Succ. r o t .
T
10 n m o l e s Ca ~+ ; / / /
l, ec mit
, Ca 2+
/ /
~"~" = ~ ' ¢ - - - ' - ' - - - ~ ' Parg.
RR
/ BA
Fig. 7. Ca 2+ uptake and MAO-induced Ca 2+ release by isolated rat-brain mitoehondria. The incubation mixture contained rat-brain mitochondria (1.5 mg protein), 0.1 M mannitol, 0.05 M KC1, 0.025 M Tris-HCl (pH 7.4), 3 mM potassium phosphate (pH 7.4) and 0.1 mM purified arsenazo II1. Ca 2+ load, 57 nmol/mg mitochondrial protein. When indicated, 5 mM potassium succinate (Succ.)+ 15 #M rotenone, 50 /~M pargyline (Parg.), 200 /xM benzylamine (BA) and 0.05 /~M ruthenium red (RR) were added. Final volume, 2 ml, temp. 30 o C. Ca 2+ uptake and release were spectrophotometrically monitored as indicated in Materials and Methods.
with added a-ketoglutarate + NH~-, a potent oxidant of intra-mitochondrial NADPH by way of glutamate dehydrogenase, showing that the added NADPH did not penetrate the inner membrane, i.e., that the rnitochondria are not damaged. These conclusions regarding glutathione reductase may appear paradoxical, since the data indicate that the fraction of GSH oxidized by MAO-generated H202 via glutathione peroxidase can be reduced by both extraand intra-mitochondrial NADPH (the latter produced, e.g., by isocitrate oxidation). However, a similar behaviour holds for the mitochondrial hexokinase, found mainly in tumours and brain [32], where it is bound to the outer membrane but reacts with both extra- and
r,
M A O-induced 11202 accumulation and Ca 2 + efflux
Mitochondrial Ca 2+ efflux has been shown to take place upon oxidation of reduced nicotinamide nucleotides by various oxidants including acetoacetate, oxaloacetate, menadione and organic hydroperoxides [36-41]. As shown by Richter and his colleagues, this Ca 2÷ efflux probably proceeds by way of a specific protein that is activated by ADP-ribosylation [42], the ADP-ribose being generated through a cleavage of mitochondrial NAD ÷ (but not NADH) through a glycohydrolase [43]. NADPH oxidation is less efficient in stimulating Ca 2÷ efflux and is probably acting through a dephosphorylation of NADP ÷ and NAD ÷ [44]. Baumhiiter and Richter [37] have shown that t-butylhydroperoxide stimulates Ca 2÷ efflux from mitochondria at a rate which is dependent on the Ca 2+ load and which is maximal when ruthenium red is added to
Tyr
BA
'$
intra-mitochondrial ATP (in fact preferentially with the latter). It has been suggested [33] that the enzyme may be located in the contact sites between the inner and outer membranes. A similar location has been proposed for the high-affinity Ca2+-binding protein of brain mitochondria [34]. The possible location of part of the mitochondrial glutathione reductase and glutathione peroxidase in these membrane junctions may be investigated in the future by isolation of the junction fraction as described earlier (cf. Refs. 33 and 34). In addition to the compartmentation of glutathione, discussed above, the present findings regarding the partial depletion of GSH by H202 produced via MAO A and B may also be explained by heterogeneous distribution of the latter enzyme in different parts of the brain [35]. This probably would not, however, obviate our conclusion that at least parts of the mitochondrial glutathione peroxidase and glutathione reductase are accessible to substrates from both the extra- and intra-mitochondrial space.
Pha
MPTP
50
5O
~5-
E
~
lo-
0
E ¢=
5
,.$ o 0
50
0
50
0
Ca2*load (nmoles/mg protein)
Fig. 8. Dependence of MAO-induced Ca 2+ release on Ca 2+ load in the presence of various substrates. Benzylamine (BA), tyramine (Tyr), fl-phenylethylamine (Pha) and MPTP were added in concentrations of 200 #M. Mitochondria were loaded with different amounts of Ca 2÷ 30 s before the initiation of MAO activity by the addition of the various substrates. The data refer to initial rates after the addition of ruthenium red (see Fig. 7), o, in the presence of ruthenium red; e, in the presence of ruthenium red + amine.
305 inhibit the reuptake of Ca 2+. As shown in Fig. 7, a release of Ca 2+ from brain mitochondria is promoted by the substrates of MAO as well. Fig. 8 compares effects of Ca 2+ load on the rate of Ca 2+ efflux in the presence of benzylamine, tyrarnine, fl-phenylethylamine and MPTP in the presence of ruthenium ref. These results are in line with those earlier reported by Baumhiiter and Richter [37] and demonstrate that MAO may play an important role in regulating mitochondrial Ca 2+ efflux.
Acknowledgements The Authors are grateful to Dr. C. Richter for the generous gift of MPTP and his advice for its use, to Mr. B. Gazzin for his skilful technical assistance, to the Fondo per lo Studio e la Ricerca Scientifica delle Malattie del Fegato (Regione Friuli-Venezia Giulia) for the use of the spectrofluorometer, to the Ministero dell'Universit/t e della Ricerca Scientifica e Tecnologica (Rome), to Consiglio Nazionale delle Ricerche (Rome) for financial support, and to Ms. A Nielsen and Ms. K. Nordenbrand for their help in the preparation of the manuscript.
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