Superoxide production by mitochondria in the ... - Wiley Online Library

Vol. 40, No. 3, October 1996

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 527-534

SUPEROXIDE PRODUCTION BY MITOCHONDRIA IN THE PRESENCE OF NITRIC OXIDE FORMS PEROXYNITRITE

Michael A. Packer, Carolyn M. Porteous and Michael P. Murphy 1 Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand Phone +64-3-479-7871 Facsimile +64-3-479-7866 Email [email protected] Received June 21, 1996 Summary The mitochondrial respiratory chain continually produces superoxide leading to high levels of mitochondrial oxidative stress. This oxidative damage has been attributed to the formation of hydroxyl radicals and hydrogen peroxide from superoxide. Alternatively, mitochondrial superoxide may react with nitric oxide forming the potent oxidant peroxynitrite, thus damaging mitochondrial protein, lipid and DNA. To test this hypothesis we induced mitochondrial superoxide formation in the presence of nitric oxide. Here we demonstrate that mitochondrial superoxide reacts with nitric oxide to tbrm peroxynitfite, suggesting that mitochondria may be a significant intracellular source of peroxynitrite. Key words: Peroxynitrite, Nitric oxide, Superoxide, Mitochondria, Oxidative stress

Introduction About 1-2% of the oxygen consumed by mitochondria forms superoxide (02"') by direct transfer of electrons to oxygen (1-3). This 02"" formation, which increases during hyperoxia, ischaemia-reperfusion or exposure to neurotoxins (4-8), is why mitochondria suffer high levels of oxidative stress. Superoxide itself is not a particularly reactive free radical, therefore much of this oxidative damage to DNA, protein and lipid has been attributed to the secondary formation of the hydroxyl radical and hydrogen peroxide from superoxide. Another possibility is that mitochondrial oxidative damage is caused by peroxynitrite (ONOO-) formed by the reaction of O2"" with nitric oxide ('NO). Nitric oxide is widespread in biological systems (9-11) and reacts close to the diffusion limit (k = 6.7 x 109 M-Is-I) with O2"- to form ONOO" (12, 13). The pKa of ONOO- is 6.8 (14, 15) and peroxynitrous acid decays with a half-life of less than 1 s at physiological pH giving similar reaction products to the powerful oxidants nitrogen dioxide and the hydroxyl radical (16-18). The ONOO" anion is itself a powerful oxidant of protein and non-protein thiol groups (15), in the presence of metals it reacts with protein tyrosyl residues to form 3-nitrotyrosyl adducts (19) and it also reacts with lipids and nucleic acids (15, 20). Therefore, as "NO is found in most tissues and mitochondria continually produce 02"', ONOO- may be formed at a constant rate by mitochondria and thus cause oxidative damage to mitochondrial protein, lipid and DNA. The formation of

Abbreviations used: DHR, dihydrorhodamine-123; "NO, nitric oxide; NO-SP, nitric oxide spermine; ONOO', peroxynitrite; 02"', superoxide; SNAP, S-nitroso-Nacetylpenicillamine; SOD, CuZn superoxide dismutase (EC 1.15.1.1). 1Corresponding author

1039-9712/96/030527--08505.00/0 527

Copyright © 1996 by Academic" Press Australia. All rights of reprtMuction in any form reserved.

Vol. 40, No. 3, 1996

BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

ONOO- would increase whenever the "NO concentration was elevated or mitochondrial O2"" production increased. Formation of ONOO- by cells has been demonstrated (20-23) and is thought to contribute to oxidative tissue injury in a number of human diseases (11, 16, 24), but ONOO" formation by mitochondria has not been reported. To mimic mitochondrial O2"" production we incubated mitochondrial membrane fi-agments with respiratory substrates and antimycin to induce 02 °- formation (2, 25). The membranes were then exposed to either authentic °NO or to "NO-donors and the formation of ONOO" measured. In this paper we show that mitochondrial superoxide production in the presence of °NO leads to ONOO- formation, therefore ONOO" may contribute to the cumulative oxidative damage to mitochondrial DNA, lipid and protein~ Materials and Methods

Materials-The "NO donors, nitric oxide spermine (NO-SP) and S-nitroso-Nacetylpenicillamine (SNAP) were from Research Biochemicals Limited, Natick, MA, USA. NO-SP stock solutions (50 mM) were prepared in 10 mM NaOH and used within 3 hours of preparation (26) and SNAP stock solutions (50 mM) were prepared in argonsparged DMSO immediately before experiments. Dihydrorhodamine-123 (Molecuhtr Probes, OR, USA) and rhodamine-123 (Sigma) stock solutions were prepared in argon sparged dimethylformamide (AR grade) at 28.9 nLM and 1 mM respectively and stored in the dark at -20°C. Type III CuZnSOD (bovine liver) was from Sigma. Preparation of mitochondrial membrane fragments-Beef heart mitochondria, isolated by a standard procedure (27), were suspended at 15 mg/ml in ice cold medium containing 250 mM sucrose, 1 mM EGTA and 5 mM Tris-HCl (pH 7.4) and 10 ml batches were sonicated (6 x 15 s of a microprobe Branson 'sonifler' on setting 4, with 30 s intervals). Medium (10 ml) was then added and the suspension centrifuged at 10,000 x g for 10 min at 4oC and the supernatant was then centrifuged at 100,000 x g for 60 min at 4oc. The pellet was resuspended and centrifuged twice and the mitochondrial membrane fragments suspended at 20 nag protein/ml and stored at -80oc. Protein concentration was measured by the biuret assay using bovine serum albumin as a standard (28). Experiments were carried out by incubating membrane fragments (0.75 mg/ml) in 0.5 ml experimental medium continuing 250 mM sucrose, 5 mM Tris-HC1 (pH 7.4), 1 m_M EGTA with 5 n-LMsuccinate and 13 [aM rotenone at 25oc. Measurement of superoxide formation-The formation of O2 °- was measured as the SOD-sensitive lucigenin-enhanced chemiluminescence of a suspension of mitochondrial membranes (0.75 mg/ml) in 0.5 ml experimental medium containing 100 laM lucigenin (bis-N-methylacridinium nitrate) in a Berthold LB953 luminometer (29). Chemiluminescence was integrated over 10 min mad the background due to spontaneous lucigenin chemiluminescence was subtracted. Measurement and synthesis of nitric oxide-Nitric oxide concentration was measured using a nitric oxide electrode (World Precision Instruments Inc., Sarasota FL., U.S.A.). The "NO electrode was inserted into a stirred 1 ml chamber thermostatted at 25oC to which was added mitochondrial membranes in 0.5 ml experimental medium. The output of the electrode, acquired with a MacLab system, was calibrated by addition of known amounts of authentic "NO to argon-sparged water in the electrode chamber. Nitric oxide was synthesised by the reaction of 1 M NaNO2 with 1 M FeSO4 in 1 M H2SO4 in an enclosed, argon-sparged reaction vessel (30). After passage through 5 M NaOH the "NO was bubbled through argon-sparged water to form a "NO stock solution the concentration of which was determined by the oxidation of oxyhemoglobin to methemoglobin (31) measured at the wavelength pair 578-592 nm using an extinction coefficient of 12.1 mM-I.cm-l (32). Measurement of peroxynitrite formation-The formation of ONOO- was determined from the oxidation of non-fluorescent dihydrorhodamine- 123 (DHR) to fluorescent rhodamine-123 (33). Rhodamine-123 fluorescence was measured in a PerkinElmer MPF-3L fluorescence spectrophotometer using an excitation wavelength of 500 nm and an emission wavelength of 536 nm (slit widths of 2.5 and 3.0 nm, respectively) and data were acquired with a MacLab system. Experiments were carried

528

Vol. 40, No. 3, 1996


out in 0.5 ml of experimental medium thermostatted at 25oc, to which was added 50 laM DHR. The amount of rhodamine-123 formed by oxidation of DHR was calibrated by adding known amounts of rhodamine-123 in the presence of mitochondrial membrane fragments (0.75 mg/ml). This gave a linear relationship between fluorescence intensity and rhodamine concentration up to 1 I.tM. To calibrate the relationship between ONOOformation and the oxidation of DHR to rhodamine-123, known amounts of authentic ONOO- were added to incubations and the increase in rhodamine-123 fluorescence measured. Peroxynitrite was prepared at 4oc by a variation of published procedures (16, 34). Briefly, 5 ml 0.6 M sodium nitrite was mixed with 5 ml acidified hydrogen peroxide (0.7 M H202 in 0.6 M HCI) in a simple flow reactor and the product was run into 5 ml rapidly stirred 1.5 M NaOH to quench the reaction. Residual hydrogen peroxide was degraded by incubation with MnO2 (2 g) for 30 min, which was subsequently removed by filtration. Freeze fractionation of this solution at -20oc formed an upper, concentrated yellow band of ONOO- which was retained. These solutions, containing about 130-150 mM ONOO" (E302 = 1670 M-Icm-I, [ref 35]), were frozen at -80°C and used within 4 days of preparation. Results Mitochondrial superoxide reacts with nitric oxide Mitochondrial membranes in the presence of antimycin and a respiratory substrate produce 02"-. This SOD-sensitive production of 02 °- was measured by reduction of acetylated cytochrome c and by the formation of adrenochrome from adrenaline, however the presence of "NO or °NO donors interferes with these assays (data not shown). Therefore lucigenin chemiluminescence, which is unaffected by "NO, was used to measure 02"- formation by mitochondrial membranes (Fig. l). Addition of the "NO donor SNAP led to a concentration dependent decrease in lucigenin chemiluminescence (Fig. l), presumably because "NO competes with lucigenin for the 02 °- produced by the mitochondnal membranes. It is probable that this interaction of °NO with O2 °- is due to the formation ONOO-, alternatively it could be due to the prevention of mitochondrial O2 °- production by °NO. To eliminate this possibility mitochondrial membrane fragments were incubated in the chamber of a "NO-sensitive electrode, which enabled the °NO concentration to be measured continually (Fig. 2). When a pulse of authentic °NO (fi'om a solution of °NO gas dissolved in water) was added the initial concentration of "NO decayed rapidly due to the reaction of °NO with oxygen (Fig 2A, trace a). In the presence of antimycin, to induce mitochondrial O2 °- formation, the added °NO pulse decayed away more rapidly (Fig. 2A, trace c). When superoxide formation was initiated by addition of antimycin after the addition of °NO, the °NO pulse again decayed more rapidly than in its absence (Fig. 2A, trace b). In the presence of SOD (50 U/ml), which will dismutate any 02 °- formed, addition of antimycin did not lead to a stimulation of the decay of the °NO pulse, giving a result similar to trace a (data not shown). In the experiments described by Fig. 2B NO-SP, which slowly decomposes to gradually release °NO (36), was used to generate a stable °NO concentration for the duration of the incubation. After addition of NO-SP to an incubation of mitochondrial membrane fragments, a stable concentration of °NO was reached within 10 min (Fig. 2B, trace a). When this experiment was repeated in the presence of antimycin, to induce mitochondrial O2 °" formation, the steady-state °NO concentration was substantially decreased (Fig. 2B, trace c). If antimycin was added to the incubation after a stable °NO concentration had been established there was a rapid decrease in the concentration of °NO (Fig. 2B, trace b). When these experiments were repeated in the presence of SOD the decrease in °NO concentration caused by antimycin was substantially decreased (data not shown). 529

Vol. 40, No. 3, 1996

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

0

t.X

C :3 0

T :::+::+:

6-

:i:i:i:!:i:!:i:! ..-,............ :.:.:.:.:.:,:.:,

o

4 °

"~

Z"

5 i

Mito

Anti

I

I

+SOD +SNAP +SNAP (Z00tJM) (SOOuM)

Fig. 1. The °NO donor SNAP decreases SOD-sensitive lucigenin chemiluminescence. Mitochondrial membranes were incubated as described in the materials and methods section with lucigenin (Mito). For other incubations 2 I-tM antimycin on its own (+anti), o1" to g ether with 250 U/ml SOD were + (+SOD) or SNAP (+SNAP"' 200 I.tM. or 500$tM) ,, included. Data m'e the means - SEM for at least three separate experiments, p < 0.0001 by Student's t-test.

Formation of peroxynitrite by mitochondria The experiments described by Figs. 1 and 2 show that "NO and mitochondriallyproduced O2 °- react together; the probable product of this reaction is ONOO'. To test this hypothesis mitochondrial membrane fragments were incubated with DHR and the rate of oxidation of DHR to rhodarnine by ONOO- was measured (Fig. 3). Incubation of membranes with antimycin and subsequent addition of authentic °NO led to the oxidation of DHR to rhodamine (Fig. 3, trace a). Incubation of membranes on their own (data not shown), with antimycin in the absence o f ' N O (Fig. 3. trace c), with antimycin and SOD (50 U/ml, data not shown) or addition of authentic "NO to membranes in the absence of antimycin (Fig. 3, trace b) did not lead to DHR oxidation. To investigate the formation of ONOO" by mitochondria further, mitochondrial membranes were incubated with the °NO donor NO-SP and the rate of ONOO- formation was measured (Fig. 4A). Incubation of membrane fragments with NO-SP and antimycin (Fig. 4A, trace a) led to a stable rate of DHR oxidation. This oxidation of DHR did not occur when membranes were incubated with NO-SP in the absence of antimycin (Fig. 4A, trace c) or with antimycin in the absence of NO-SP (Fig. 4A, trace d). When SOD was included in the incubation the rate of DHR oxidation was decreased substantially (Fig 4A, traces b). The formation of rhodamine by ONOO" was calibrated by addition of authentic ONOO" (Fig. 4B). From this calibration the initial rate of formation of ONOO" by mitochondrial membranes in the experiment described by Fig. 4A was about 860 pmol/min/mg. Discussion

We have shown that mitochondrial 0 2 °- formation in the presence o f ' N O leads to the formation of ONOO °. The induction of mitochondrial 0 2 °- production by antimycin mimics the increased production of O2 °" by mitochondria in pathological situations such

530


Vol. 40, No. 3, 1996

1.5-

A

0

o.s c-

a

c nitric} oxide

- __

i

I

I

1

z

3

time (min)

B

0

antimycin (b only)

O.S

U ¢-

. . . . . O"

t

o

n,trlc

---

--C

oxide s p e r m l n e

/o

2'0

3'0

time (min) Fig. 2. Consumption of authentic nitric oxide by mitochondrial superoxMe fornuaion. Mitochondrial membranes were incubated in the chamber of a "NO sensitive electrode as described in the materials and methods section and the concentration of "NO in the medium was monitored. In panel A, a pulse of authentic "NO ( 10 pM) was added for trace a. For trace c, antimycin (2 gM) was present and an identical addition of authentic "NO was made. For trace b, antimycin (2 I.tM) was added where indicated, after the addition of "NO. For panel B, trace a, NO-SP (100 gM) was added where indicated. For trace b, the experiment described by trace a was repeated and antimycin (2 p.M) was added where indicated. For trace c, the experiment described by trace a was repeated in the presence of antimycin from the start of the incubation. Traces shown are representative of experiments repeated on at least three occasions.

531

Vol. 40, No. 3, 1996


nitric oxide ~

(a andb) !

/

~

~

[rhodamine!T =

onMl

C

Fig. 3. Peroxynitrite formation by membranes caused by addition of authentic nitric oxMe. Mitochondrial membranes were incubated with DHR (50 laM) in the chamber of a fluorimeter as described in the materials and methods section and the rate of oxidation of DHR to rhodamine was monitored. For trace a antimycin (2 t.tM ) was present in the incubation and a pulse of authentic "NO (10 ~ M) was added where indicated. For trace b this experiment was repeated in the absence of antimycin. For trace c antimycin was incubated with mitochondrial membranes and "NO was not added. Traces shown are representative of experiments repeated on at least three occasions.

I [rhodaminel

A

/

a

= o.s .M

b

600 ~,soo~_~,400•~ 300"

~ 200. ~100Ol

o

;

;.

s

[peroxynitrite] (pM) Fig. 4. Peroxynitrite formation by mitochondrial membranes in the presence of a "nitric oxide donor. Mitochondrial membranes were incubated with DHR (50 l.tM) in the chamber of a fluorimeter as described in the materials and methods section and the rate of oxidation of DHR to rhodamine was monitored. For panel A, trace a, antimycin (2 ~tM) and NO-SP (100 ~tM) were present. This experiment was repeated in the presence of 500 U/ml SOD (traceb). Further incubations were carried out with antimycin in the absence of NO-SP (trace c) and with SP-NO in the absence of antimycin (trace d). Traces shown are representative of experiments repeated on at least three occasions. For panel B mitochondrial membranes were incubated with DHR as described for panel A and sequential additions of authentic ONOO" were made to calibrate rhodamine fluorescence. From this calibration 8 . 6 0 N O O - molecules are required to oxidise one DHR molecule to rhodamine. 532

Vol. 40, No. 3, 1996


as ischaemia-reperfusion. In the experiments reported here "NO concentrations of up to 1 ~tM were generated; the resting tissue concentration of NO is about 10 nM (37), but concentrations of 450 nM occur after stimulation of endothelial cells with bradykinin (38) and concentrations of up to 4 gM have been reported during ischaemia-reperfusion (37). Theretore it is probable that mitochondrial ONOO- production similar to that reported here will occur in pathological situations when both "NO and 02 °- concentrations are elevated. This ONOO- may damage mitochondria and cells through non-specific reactions, inhibition of mitochondrial respiration (39) or by inducing mitochondrial calcium efflux and depohu'isation (34). In addition to acute ONOO- production in pathological situations, mitochondria continually produce O2 °- and many tissues are exposed to low levels of °NO all of the time. This may result in the continual production of ONOO- which will cause mitochondrial oxidative stress and contribute to the cumulative damage sustained by mitochondrial DNA, protein and lipid over the lifetime of an organism. In summary, we have shown that mitochondrial 02 °- production in the presence of °NO forms ONOOwhich may play a significant role in mitochondrial oxidative stress.

Acknowledgments: This research was supported by grants fi'om the Health Reseamh Council of New Zealand, the Neurological Foundation of New Zealand and the Anderson and Telford Charitable Trust. MAP is grateful to the University of Otago for the award of a targeted studentship. References 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Turrens, J.F. and Boveris, A. (1980) Biochem. J. 191,421-427. Turrens, J.F., Alexandre, A. and Lehninger, A.L. (1985) Arch. Biochem. Biophys. 237, 408-414. Shigenaga, M.K., Hagen, T.M. and Ames, B.N. (1994) Proc. Natl. Acad. Sci. USA 91, 19771-10778. Freeman, B.A. and Crapo, J.D. (1981) J. Biol. Chem. 256, 10986-10992. Cleeter, M.W.J., Cooper, J.M. and Schapira, A.H.V. (1992) J. Neurochem. 58, 786-789. Hasegawa, E., Takeshige, K., Oishi, T., Murai, Y. and Minakami, S. (1990) Biochem. Biophys. Res. Commun. 170, 1049-1055. Darley-Usmar, V.M., Stone, D., Smith, D. and Martin, J.F. (1991) Ann. Med. 23, 583-588. Turrens, J.F., Beconi, M., Barilla, J., Chavez, U.B. and McCord, J.M. (1991) Free Rad. Res. Comms. 12-13, 681-689. Lowenstein, C.J. and Snyder, S.H. (1992) Cell 70, 705-707. Bredt, D.S. and Snyder, S.H. (.1994) Annu. Rev. Biochem. 63, 175-195. Gross, S.S. and Wolin, M.S, (1995) Annu. Rev. Physiol. 57, 737-769. Saran, M., Michel, C. and Bors, W. (1990) Free Rad. Res. Comms. I0, 221226. Huie, R.E. and Padmaja, S. (1993) Free Rad. Res. Comms. 18, 195-199. Edwards, J. O. and Plumb, R. C. (1994) Prog. lnorg. Chem. 41,599-635. Radi, R., Beckman, J. S., Bush, K. M. and Freeman, B. A. (1991) J. Biol. Chem. 266, 4244-4250. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. and Freeman, B.A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620-1624. Hogg, N., Darley-Usmar, V.M., Wilson, M.T. and Moncada, S. (1992) Biochem. J. 281,419-424. Koppenol, W.H., Moreno, J.J., Pryor, W.A., Ischiropoulos, H. and Beckman, J.S. (1992) Chem. Res. Toxicol. 5, 834-842. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J.C., Smith, C.D. and Beckman, J.S. (1992) Arch. Biochem. Biophys. 298, 431-437.

533

Vol. 40, No. 3, 1996

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39


King, P.A., Anderson, V.E., Edwards, J.O., Gustafson, G., Plumb, R.C. and Suggs, J.W. (1992) J. Am. Chem. Soc. 114, 5430-5432. lschiropoulos, H., Zhu, L. and Beckman, J.S. (1992) Arch. Biochem. Biophys. 298, 446-451. Carreras, M.C., Pargament, G.A., Catz, S.D., Poderoso, J.J. and Boveris, A. (1994) FEBS Lett. 341, 65-68. Kooy, N.W. and Royall, J.A. (1994) Arch. Biochem. Biophys. 310, 352-359. Beckman, J.S., Ye, Y.Z., Anderson, P.G., Chen, J., Accavitti, M.A., Tarpey, M.M. and White, C.R. (1994) Biol. Chem. Hoppe-Seyler 375, 81-88. Boveris, A. and Cadenas, E. (1975) FEBS Lett. 54, 311-314. Diodati, J.G., Quyyumi, A.A. and Keefer, L. K. (1993) J. Cardiovasc. Pharm. 22, 287-292. Smith, A.L. (1967) Meth. Enzymol. 10, 81-86. Gornall, A.G., Bardawill, C.J. and David, M.M. (1949) J. Biol. Chem. 177, 751-766. Gyllenhammar, H. (1987) J. Immunol. Meth. 97, 209-213. Blanchard, A.A. (1946) Inorg. Synth. 2, 126-128. Feelisch, M. (1991) J. Cardiovasc. Pharmacol. 17, $25-$33. loannidis, I. and de Groot, H. (1993) Biochem. J. 296, 341-345. Kooy, N.W., Royall, J.A., Ischiropoulos, H. and Beckman, J.S. (1994) Free Rad. Biol. Med. 16, 149-156. Packer, M. A. and Murphy, M. P. (1994) FEBS Lett. 345, 237-240. Hughes, M.N. and Nicklin, H.G. (1968) J. Am. Chem. Soc. 450-452. Maragos, C.M., Morley, D., Wink, D.A., Dunams, T.M., Saaveda, S.E., Hoffman, A., Bove, A.A., Isaac, L. and Hrabie, J.A., Keefer, L.K. (1991) J. Med. Chem. 34, 3242 - 3247. Malinski, T., Bailey, F., Zhang, Z.G. and Chopp, M. C. (1993) J. Cereb. Blood Flow Metab. 13, 355-358. Malinski, T. and Ziad, T. (1992) Nature 358, 676-678. Radi, R., Rodriguez, M., Castro, L. and Tellefi, R. (1994) Arch. Biochem. Biophys. 308, 89-95.

534