The effect of hyperoxia on superoxide production by lung submitochondrial particles

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 217, No. 2, September, pp. 401-410, 1982

The Effect of Hyperoxia on Superoxide Production Lung Submitochondrial Particles’ JULIO

F. TURRENS: Department

BRUCE A. FREEMAN, JENNIFER JAMES D. CRAP0 of Medicine, Duke University, Durham,

North

by

G. LEVITT, Carolina

AND

27710

Received March 2, 1982, and in revised form April 20, 1982

Hyperoxia increases CN--resistant respiration in rat lung mitochondria, rat lung submitochondrial particles, and porcine lung submitochondrial particles. Cyanide-resistant respiration increases from 0 at normal lung tissue oxygen tensions to 1.2 + 0.06 nmol OZ. min-’ * mg protein-’ (Xk SD) in rat lung mitochondria and 2.7 + 0.2 nmol Oz - mine1 . mg protein-’ in rat lung submitochondrial particles, when assayed at an oxygen concentration of 85%. Superoxide production by submitochondrial particles washed free of superoxide dismutase was directly measured by quantitating superoxide dismutase-inhibitable epinephrine oxidation or cytochrome c reduction, and compared to parallel measurements of CN- resistant respiration. Succinate or NADH was used as substrate and rotenone, antimycin, or CN- were used as respiratory chain inhibitors. At least two components of the respiratory chain produced O;, the ubiquinone-cytochrome b region and the NADH dehydrogenase complex. Superoxide generation by the NADH dehydrogenase is 1.5-fold greater than superoxide production by the ubiquinonecytochrome b segment. Oxidation of NADH by porcine lung mitochondria, treated with either rotenone, antimycin, or CN-, parallels rates of 0; formation and increases directly with oxygen tension, providing another indirect measurement of superoxide production by mitochondrial membranes. These findings support the hypothesis that oxygen toxicity is in part a consequence of increased rates of intracellular 0; and HzOz production.

The lung is the primary target organ for hyperoxic induced injury during exposure at normal barometric pressure. Microscopic examination of lung cells from animals exposed to hyperoxia show several injuries such as chromatin condensation, vacuolization, and mitochondrial swelling (1, 2). After exposure of rats to high concentrations of oxygen, there are mitochondrial changes in lung capillary endothelial cells and alveolar type II cells

which include unusual shapes, increased size, abnormal cristae, and inclusion bodies (1, 3). Superoxide dismutase (SOD): catalase, and glutathione peroxidase evolved to maintain low steady-state levels of intracellular 0; and HzOz under normoxic conditions (4). We propose that during hyperoxia, cellular H202 and 0, production increases as a function of oxygen tension. Superoxide anion and Hz02 can undergo a variety of reactions, some of them cat-

i This study was supported in part by NIH Grants HL-25044 and HL-23805. x To whom all correspondence should be addressed: Box 3177, Duke University Medical Center, Durham, N. C. 27710.

3 Abbreviations used: ADP, adenosine diphosphate; SOD, superoxide dismutase; FCCP, carbonyl cyanide - p - trifluoromethoxyphenylhydrazone; TMPD, N,N,iV’,N’,-tetramethyl-p-phenylenediamine. 401

0003-9861/82/100401-10$02.00/0 Copyright All rights

Q 1982 by Academic Press. Inc. of reproduction in any form reserved.

402

TURRENS

alyzed by metal salts, ultimately yielding hydroxyl radical (OH * ) (5, 6) one of the most potent oxidants known. Recent data support the idea that this reaction can occur in a cellular milieu (7), generating highly toxic secondary products of 0;. Mitochondria have been identified as one of the main sources of 0; and HzOz in a variety of tissues including pigeon heart (8, 9), rat liver, and pigeon lung (4, 10). At least two components of the mitochondrial respiratory chain can reduce oxygen by transfer of one electron, yielding 0;. The first site of 0, formation is located near NADH dehydrogenase, the first component of the respiratory chain (11, 12). The other component of the mitochondrial respiratory chain responsible for 0; formation is located in the ubiquinone-cytochrome b area. Boveris et al. (13) and Cadenas et al. (14) have proposed ubisemiquinone as the autooxidizable molecule responsible for a majority of mitochondrial 0, production. Since the lung is the primary target organ for injury from hyperoxic exposure at normal barometric pressure it is essential to determine the major sites of 02 production in lung cells. Enhanced 0; production by lung mitochondria may correlate with the abnormal appearance of mitochondria in hyperoxic lungs and the associated extensive lung tissue injury. Lung mitochondria are always relatively “hyperoxic,” normally existing at a POz of 100-110 mm Hg, since their oxygen is supplied directly from alveolar gas. In addition, during exposures to 100% oxygen atmospheres (1 ATA), lung mitochondria have oxygen tensions approaching 670 mm Hg. In contrast, other organs are protected against these levels of tissue hyperoxia by the characteristics of the binding of oxygen to hemoglobin. When arterial oxygen tensions are 600-700 mm Hg (1 ATA) the mixed venous POz remains between 40 and 50 mm Hg suggesting that substantial exposure of peripheral body tissues to hyperoxia does not occur (X), when animals are exposed to normobaric 100 % oxygen. Currently, only indirect evidence supports the hypothesis that hyperoxia in-

ET AL.

creases 0; formation in lung cells. Crap0 and Tierney (16) showed that prior exposure of rats to 85% oxygen induces lung SOD and prevents the lethal effects of subsequent exposure of rats to 100% oxygen. Freeman et al. (1’7) have shown a specific induction of both Mn and CuZn SOD in alveolar type II epithelial cells isolated from rats exposed to 85% oxygen. Also, CN--insensitive respiration, an indirect measure of cellular 02 and HzOz production (18, 19), increases as a function of oxygen tension in lung slices and lung mitochondria (20). No direct measurements of hyperoxia-induced 0; formation by lung parenchymal cells or organelles have been reported. In this report, the 0; production by porcine lung submitochondrial particles is examined as a function of oxygen tension, and compared with the rates of NADH oxidation and CN--resistant respiration. EXPERIMENTAL

PROCEDURES

Isolation of mitochoudrin. Rat lung mitochondria were isolated according to Freeman and Crapo (20). Porcine lung mitochondria were isolated from approximately 1 kg tissue. Lungs were diced into cubes 2 cm on a side, and suspended in an isolation buffer containing 0.23 M mannitol, 70 mM sucrose, 1 mbf EDTA, and 5 rnbf Tris-HCl, pH 7.4, in a ratio of 100 g tissue per liter buffer. The tissue was then homogenized in a Sears blender using two 10-s bursts at high speed. The homogenate was filtered through two layers of gauze and pH adjusted to 7.3 with 1 M KOH. The filtrate was then centrifuged at 1500g X 20 min at 4°C. The supernatant was retained and recentrifuged at 10,OOOg X 20 min. The mitochondrial pellet was resuspended in the isolation buffer and recentrifuged at 8500g X 10 min. The pellet usually had a small amount of fibrous material that was discarded. Mitochondria were washed until the pellet was free of visible hemoglobin. Two mitochondrial layers of different appearance were present in this pellet, with the upper layer downy white and the densest layer, darker. The bottom or denser layer of mitochondria had a respiratory control ratio 20% greater than that of the upper layer and was retained for further experiments requiring intact mitochondria. Preparation of submitociwndrid particles. Freshly isolated porcine or rat lung mitochondria were frozen overnight at -70°C. After thawing, the suspension was centrifuged at 3OOOg X 10 min and the pellet was resuspended in a buffer consisting of 0.25 M sucrose, 15 mM MgClr, and 40 mM Tris-HCl, pH 7.4, to a final

HYPEROXIA

AND

concentration of 15 mg protein/ml. The suspension was sonicated at 70 W using a Branson Sonifier [Model W 185 (Heat Systems Ultrasonic, Plainview, N. Y.) equipped with a small probe]. The sonicated mitochondrial suspension was then centrifuged at 25,000~ X 10 min. The pellet was discarded and the supernatant recentrifuged at 105,000~ X 40 min. The submitochondrial particle pellet was resuspended in a buffer consisting of 0.23 M mannitol, 70 mM sucrose, 1 mM EDTA, 5 mM Tris-HCl, pH 7.4, centrifuged at 105,OOOg X 40 min and resuspended in the same buffer. All operations were performed at 0-4°C. Biochemical measurements. Superoxide production was measured by the SOD-inhibitable oxidation of 1 rnhi epinephrine to adrenochrome according to Misra and Fridovich (21) or by the Mn SOD-sensitive reduction of 10 PM acetylated cytochrome c (22). Mn SOD was purified from human liver according to McCord et al. (23). Adrenochrome formation or acetylated cytochrome c reduction was monitored in a split-beam Cary 118 spectrophotometer at 480 nm (E = 4 mM-' - cm-‘) (24) or 550 nm (E = 21 mM-’ * cm-‘) (25), respectively. Acetylation of cytochrome c avoids reoxidation of this hemeprotein by the mitochondrial cytochrome c oxidase and prevents reduction by NADHor succinate-dependent cytochrome c reductases (22). NADH oxidation was measured at 340 nm (E = 6.22 mM-'* cm-‘). Submitochondrial particles were suspended in 50 mM KHzPOl buffer, pH 7.4, at 30°C for assay of 0; production and NADH oxidation. Different oxygen tensions were established in solution by bubbling with gas containing nitrogen and oxygen mixed in proper ratios. Dissolved oxygen concentrations were confirmed by polarographic analysis. Catalase and SOD activities were determined as described by Bergmeyer (26) and McCord and Fridovich (27), respectively. Rates of oxygen consumption were measured using a Clark oxygen electrode in an incubation medium consisting of 145 mM KCl, 20 mM KH2POI, 0.1 mM EDTA, 30 mM Tris-HCI, pH 7.4, and different concentrations of MgCla at 30°C. Protein concentration was quantitated by the method of Lowry et al. (28). Chemicals. Epinephrine, ADP, NADH, rotenone, antimycin A, FCCP, cytochrome c (type III), malic acid, succinic acid, glutamic acid, and malonic acid were purchased from Sigma Chemical Company (St. Louis, MO.). RESULTS

Oxygen Consumption Mitochondria

by Porcine

Lung

For experiments requiring large amounts of lung mitochondria, porcine lungs were

0;

PRODUCTION

403

used. A yield of 0.36 k 0.03 mg mitochondrial protein per gram wet weight of porcine lung (n = 4, a+ SD) was obtained. From measurements of cytochrome oxidase activity in both mitochondria and homogenates (29), a 7.5% recovery was calculated. Three different substrates were used to study characteristics of oxygen consumption by porcine lung mitochondria; malate, which generates intramitochondrial NADH (30), succinate, providing electrons at the ubiquinone level (31), and ascorbate plus TMPD, which reduces the respiratory chain at the cytochrome c-cytochrome c oxidase level (32). Table I summarizes oxygen consumption before (State 4) and after (State 3) ADP addition (33), and the respiratory control ratios of porcine lung mitochondria when either malate, succinate, or ascorbate were used as substrates.4 Malate was used in combination with glutamate (which avoids high intramitochondrial oxaloacetate concentrations) and malonate (which inhibits succinate dehydrogenasedependent electron flow to ubiquinone). Addition of an uncoupler (1 I.IM FCCP) to mitochondria incubated with either malate or succinate (State 3U) resulted in maximal rates of oxygen consumption of 58 and 65 nmol * min-’ . mg mitochondrial protein-‘, respectively. Oxygen consumption and respiratory control ratios of porcine lung mitochondria were measured in the presence of 3 mM MS’, since the respiratory control ratio increased as a function of Me, up to 1 mM (Fig. 1). Mitochondrial Respiration and NADH Oxidation in the Presence of Inhibitors Figure 2 presents a scheme of the respiratory chain, showing sites of inhibition, substrate entry, and sites where 0; ’ Definitions of mitochondrial respiratory states are: State 1, respiration in the absence of added ADP and substrate; State 3, respiration in the presence of added ADP and substrate; State 3U, respiration when substrate and uncoupler are added, and State 4, respiration in the absence of ADP when substrate is added.

404

TURRENS ET AL. TABLE OXYGEN

CONSUWTION

AND RESPIRATORY

I

CONTROL

Addition

12

State 4 respirationb

Malate (5 mM) Glutamate (5 mM) Malonate (2 mM)

10

9.1 f 2.0

Succinate (5 mM)

10 5

Ascorbate (10 mM) TMPD (0.1 mM)

RATIO

OF PORCINE

LUNG

MITOCHONDRIA~

State 3 respirationb

Respiratory control ratio

29.2 ix 9.2

3.1 + 0.7

18.2 f 9.1

41.8 + 10

2.5 f 0.3

13.6 2 3.3

18.9 + 4.5

1.4 + 0.1

‘Values represent 8 + SD. Mitochondrial protein in incubations b Oxygen consumption represents nmol . mixi-‘. mg protein-‘.

formation has been demonstrated to occur in beef heart mitochondria (12, 14). Cyanide-resistant respiration has been previously used as an indirect method for evaluating oxygen radical production of rat lung slices and rat lung mitochondria (20). Similar measurements performed in rat lung mitochondria and submitochondrial particles as a function of oxygen tension (Fig. 3), allowed comparisons to be made between indirect measurements of 0, production by mitochondria (using CN--resistant respiration) and direct measurements of 0; produced by submitochondrial particles reported herein. Cyanide-resistant respiration increased with oxygen concentrations greater than 20% in rat lung mitochondria or greater than 40% in submitochondrial particles, becoming 1.2 f 0.06 and 2.7 f 0.2 nmol. min-’ * mg protein-‘, respectively, at 85% oxygen. NADH oxidation in the presence of inhibitors provided information about both the remaining activity of the respiratory chain and 0; formation by different components of the electron transport chain. In rat lung submitochondrial particles, NADH oxidase activity was 30.1 + 5.8 nmol * min-l * mg protein-’ (n = 3, Xk SD). Following addition of 1 mM KCN or 2 PM antimycin, NADH oxidation by submitochondrial particles was inhibited 67 and 63%) respectively. The remaining activity, more than 30%, is unusually high when compared with antimycin and CN--insen-

was 0.4 mg/ml.

sitive NADH oxidation by submitochondrial particles derived from other tissues. Indeed, NADH oxidation can be inhibited more than 90% when antimycin or CNare added to beef heart submitochondrial particles (34,35). The large fraction of rat lung submitochondrial particle NADH consumption insensitive to CN- and antimycin inhibition could be due to artifactual rearrangements of mitochondrial respiratory chain components during isolation from lung tissue or as a result of sonication, modifying the sensitivity of the respiratory chain to inhibitors or in-

“k-Y?-?

J

MgC12(mM1

FIG. 1. Effect of Mge+ on porcine lung mitochondrial respiratory control ratio. Substrates used were: malate (5 mM), glutamate (5 mM), and malonate (2 mM) (solid circles); succinate (6 mM) (open circles) and ascorbate (10 mM) and TMPD (0.1 mnn) (solid triangles). 0.3 mg/ml mitochondrial protein was used for experiments when either malate or succinate were substrates and 0.6 mg/ml when the substrate was ascorbate. The temperature was 30°C. Other conditions are described under Experimental Procedures.

HYPEROXIA

AND

FIG. 2. Scheme of the respiratory chain showing sites of substrate entry, inhibitor action and potential sites of superoxide anion formation.

creasing the reactivity of its components with oxygen. Porcine lungs were used as a mitochondrial source for subsequent experiments, not only because of similar respiratory characteristics when compared with rat lung mitochondria [Table I, Fig. 1, (20)], but because of the ease of preparation of larger numbers of mitochondria from a single lung. Cyanide-insensitive respiration of porcine lung submitochondrial particles was 0 under normoxic conditions and increased to 1.2 nmol. mine1 * mg protein-’ at 85% oxygen, about half that of rat lung submitochondrial particles. Table II shows rates of NADH oxidation by submitochondrial particles prepared from porcine lung mitochondria before and after addition of inhibitors. NADH oxidase activity, at 21% oxygen, was reduced 95 to 98% following addition of rotenone, antimycin, or CN-.

405

0, PRODUCTION

donors. Results of a representative experiment are shown in Table III. This reaction was antimycin or rotenone insensitive and inhibited by CN- (Table III). Adrenochrome formation by submitochondrial particles in the absence of substrate was also totally inhibited by addition of 150 U/ ml catalase (free of SOD). Subsequent addition of NADH resulted in adrenochrome formation which was completely inhibited by SOD. It has been demonstrated that NADH-dependent adrenochrome formation by beef heart submitochondrial particles was complicated by an autocatalytic reaction, especially at pH 7.5 or higher (12). In pig lung submitochondrial particles, a lag phase of 1 or 2.min in the rate of adrenochrome formation occurred after NADH addition (Fig. 4). Then, the rate of adrenochrome formation indicative of 0; production by submitochondrial particles was linearly related to time for at least 7 min (Fig. 4). The linear rate of adrenochrome formation was used as a measure of 0; production. Inhibitor-insensitive NADH oxidation and 0; generation by porcine lung submitochondrial particles following addition of either rotenone, antimycin or CN-, were studied as a function of oxygen concentration. When NADH was used as substrate, addition of rotenone resulted in NADH dehydrogenase reduction, leaving other components of the respiratory chain highly

Superoxide Production by SubwGtochmdrial Particles Cytochrome c and acetylated cytochrome c were not useful as 0, scavengers when NADH was used as substrate. Addition of NADH to porcine lung submitochondrial particles in the presence of rotenone reduced cytochrome c at rates up to 81.9 nmol * min-’ . m.g protein-‘. This cytochrome c reduction was not inhibited by SOD and may have been directly catalyzed by the NADH dehydrogenase (36). Thus, SOD-sensitive oxidation of epinephrine to adrenochrome was used for subsequent quantitation of 0, production. Submitochondrial particles oxidized epinephrine, even in the absence of electron

5

1520

40 60 Pertent Oxygen

75

85

FIG. 3. Effect of oxygen on CN- resistant respiration by intact rat lung mitochondria (open circles) and submitochondrial particles (solid circles). All data are 8 + SD. Experiments are performed using 0.6 mg/ml mitochondrial protein or 0.2 mg/ml submitochondrial particles protein. The temperature was 25°C.

TURRENS ET AL. TABLE

II

EFFECT OF ADDITION OF DIFFERENT INHIBITORS ON THE RATE OF NADH CONSUMPTION BY PORCINE LUNG SUBMITOCHONDRIAL PARTICLES

Addition

n

NADH oxidase’ (nmol . min-’ * mg protein-‘)

None 2 p~ rotenone 2 PM antimycin 1 mru KCN

3 8 7 10

104 +-31 2.5 k 0.7 3.1 + 0.8 1.6 f 0.5

Activity (WI 100 2.4 3.0 1.5

’ Values represent 8 + SD. Experiments were carried out using a protein concentration of 0.2 mg/ml.

oxidized (37). Thus, in the presence of rotenone, only 02 produced by NADH dehydrogenase can be detected. Addition of NADH to submitochondrial particles in the presence of antimycin reduces both NADH dehydrogenase and ubiquinone, stimulating 0; production by both components of the respiratory chain (12). Cyanide inhibits 0; production by the ubiquinone-cytochrome b area (12). Thus, even though all the components of the respiratory chain were reduced following addition of NADH and CN-, only NADH dehydrogenase could generate 0;. Superoxide production and NADH oxidation by submitochondrial particles treated with rotenone, antimycin, or CNwere parallel and directly related to oxygen concentration (Fig. 5). When antimycin was used (Fig. 5b), the rate of 0, production under hyperoxic conditions was higher than when rotenone blocked the respiratory chain (Fig. 5a). When submitochondrial particle suspensions were saturated with 100% oxygen, NADH dehydrogenase produced 2.5 f 0.4 nmol 0; * min-’ . mg protein-’ (Fig. 5a) while NADH dehydrogenase and ubiquinone assayed together produced 3.4 + 0.4 nmol . min-’ . mg protein-’ (Fig. 5b). However, when submitochondrial particles were treated with 1 MM KCN (Fig. 5c), 0; production in the presence of 100% oxygen was 3.3 f 0.5 nmol * min-’ * mg protein-l, close to the rate of 0; production by submito-

chondrial particles when antimycin is added. Near 0% oxygen, no 0; formation was detected in the presence of all inhibitors. Low rates of NADH oxidation under these conditions can be the consequence of slight oxygen contamination in the samples, since cytochrome c oxidase is saturated with oxygen at tensions as low as 0.5% (38). Superoxide production at the ubiquinone-cytochrome b region was also studied following addition of succinate (6 mM) to antimycin-inhibited submitochondrial particles. This represents 0; formation when NADH dehydrogenase is not reduced. Succinate-dependent 0; formation in the presence of antimycin was 0 at 0% oxygen and increased from 0.7 f 0.2 nmol * min-’ amg protein-’ (n = 4, 8? SD) at 21% oxygen to 1.7 f 0.1 nmol. mini-. mg protein-’ when the submitochondrial particle suspension was equilibrated with 100% oxygen. These rates of 0; production were similar when either acetylated cytochrome c or epinephrine were used for quantitation, validating the quantitative accuracy of epinephrine oxidation as an 0; detector. CN- (1 mM) totally inhibited 0; production in the ubiquinone-cytochrome b area at either 21 or 100% oxygen.

TABLE

III

EFFECT OF DIFFERENT ADDITIONS ON THE RATE OF ADRENOCHROME FORMATION BY SUBMITOCHONDRIAL PARTICLES INCUBATED WITH 2 PM ANTIMYCIN

Addition

Adrenochrome formation (nmol . min-’ * mg protein-‘)

Epinephrine (1 mM) + SMP (0.2 mg/ml) + catalase (450 U/ml) + NADH (0.1 mM) + SOD (150 U/ml)

0.6 6.5 0.5 2.3 0.3

Epinephrine (1 m&i) + SMP (0.3 mg/ml) + CN- (1 mM)

0.4 7.1 0.2

a Oxidation of epinephrine was measured using 0.3 mg/ml suhmitochondrial particle protein.

HYPEROXIA

407

AND 0; PRODUCTION

NADH

2pY

T

Antimycin

AA=.002 1

1.2

i-\ SOD -

\

I min

p--

FIG. 4. NADH-dependent adrenochrome formation by porcine lung submitochondrial particles (SMP). The reaction mixture contained 150 U/ml catalase and 1 mM epinephrine. The reaction was started by addition of 0.1 mM NADH and was inhibited by 150 U/ml SOD. The values adjacent to the trace indicate nmol0;. min-‘. mg protein -I. Other conditions are described under Experimental Procedures.

pleted of Mg2+ during isolation procedures, and may recover more normal metabolic functions upon Mga+ addition. Low respiratory control ratios found in rat lung mitochondria (40) may be due to depletion of Mga+. When oxygen consumption was measured with added M$+, State 3 respiration accounts for 80% of State 3U respiration in the presence of uncouplers, demonstrating that addition of Mga+ restored almost completely the maximal respiratory control ratio. The rate of intracellular oxygen radical production depends on both tissue oxygen tension and the redox state of autooxidiz-

DISCUSSION

Isolation of coupled lung mitochondria is difficult, due to the fibrous nature of lung tissue, the low mitochondrial content per gram lung, and the high lipid content in lungs, which causes uncoupling due to a detergent effect (39). Suitable yields of coupled mitochondria from porcine lung can be obtained following the procedure described herein. The respiratory control ratio of porcine lung mitochondria was increased 40 to 100% by Mga+, depending on which electron donor was used (Fig. 1, Table I). Mitochondria may become de-

‘II;{/: b.

t Antimycin

'

- - c.

l

t

KCN

.

.

0

.

0

0

.

0

I 0

1

I

I

1

20

40

60

80

Percent

I

100 0

20

40

60

80

100

Oaygen

FIG. 5. Superoxide production (open circles) and NADH oxidation (solid circles) by porcine lung submitochondrial particles incubated with 0.1 mM NADH and either 2 PM rotenone (a), 2 CM antimycin (b), or 1 mM CN- (c). Superoxide formation was measured in the presence of 1 mM epinephrine. 0.2 to 0.4 mg/ml submitochondrial particle protein was used.

408

TURRENSETAL.

able cell components, such as the respiratory chain of mitochondria (10). Since mitochondrial cytochrome c oxidase is saturated above oxygen tensions of 0.5%, there is a relatively constant respiratory chain redox state over a wide range of tissue oxygen tensions (38). Thus, increased oxygen concentration in lung tissue during hyperoxia, should enhance the rate of oxygen radical production by mitochondria. Since mitochondria contain SOD, submitochondrial particles freed of SOD were prepared for detecting sites and maximal amounts of 0, production. No SOD or catalase activity could be measured in up to 1 mg of submitochondrial particle protein. Since the respiratory chain is oxidized in submitochondrial particles, rotenone, antimycin, and CN- were used for increasing the reduction of specific sections of the respiratory chain, permitting measurements of 0, production by individual components. Cyanide-insensitive respiration has been used as an indirect measure of 0, formation in lung slices, mitochondria (20) and bacteria (18, 19). It was estimated from CN--resistant respiration measurements that rat lung mitochondria generated 2.6 nmol 0; * min-’ * mg mitochondrial protein-l when maintained under hyperoxic conditions and accounted for 15% of whole lung tissue CN--resistant respiration at 85% oxygen (20). In the present study, both intact mitochondria and submitochondrial particles isolated from rat lungs increased CN--resistant respiration as a function of oxygen tension. At 85% oxygen, CN--resistant respiration in submitochondrial particles was about two times higher than that of intact lung mitochondria (Fig. 3). This may be in part due to a greater concentration of respiratory chain components per milligram protein in submitochondrial particles compared with intact mitochondria. Porcine lung submitochondrial particles maintained at 85% oxygen had a CN--resistant respiration rate of 1.2 nmol . min-’ . mg protein-‘, which is 50% of the CN-resistant respiration rate measured in rat lung submitochondrial particles.

Dionisi et al. (41) and Boveris et al. (13) have shown that 0; is the direct precursor of all mitochondrial HzOz. If it is assumed that submitochondrial particles have no residual peroxidase activity and that 0, dismutes spontaneously with a very high rate constant [lo5 M-‘. s-l, (42)] the rate of superoxide production of these particles will be two times the CN--resistant respiration rate reported herein, since 1 mol oxygen is returned to solution by dismutation for every 2 mol0; generated. Thus, it is predicted from CN--resistant respiration measurements that rat and porcine lung submitochondrial particles generate 0; at rates of 5.0 and 2.4 nmol . min-’ . mg protein-‘, respectively, at 85% Oz. The rate of 0, production by porcine lung submitochondrial particles at 85% oxygen was 2.9 nmol. min-‘a mg protein-’ (Fig. 5~) confirming that in lung mitochondria, CN-resistant respiration is a close measure of 0, production. Porcine lung submitochondrial particles inhibited with antimycin and supplemented with succinate in order to detect 0, production at the ubiquinone-cytochrome b level, produced 0.7 f 0.2 nmol 0,. min-’ . mg protein-l at 21% oxygen. This occurred when either 10 pM acetylated cytochrome c or 1 IIIM epinephrine were used for 0, quantitation. At concentrations of oxygen approaching O%, no 0; formation was detected. When submitochondrial particle suspensions were saturated with 100% oxygen, 0; production increased 2.4-fold in comparison with the rate at 21% oxygen. Addition of CN- inhibited 0; production at the ubiquinonecytochrome b level, showing this a common characteristic among mitochondria from different tissues (12). Beef heart submitochondrial particles, NADH and epinephrine undergo an autocatalytic reaction (12). Porcine lung submitochondrial particles did not undergo this autocatalytic reaction; their epinephrine oxidation was directly related to particle concentration and time. A substrateindependent oxidation of epinephrine by submitochondrial particles was eliminated by catalase or CN- (Table III), sug-

HYPEROXIA

AND

gesting the involvement of a peroxidase activity. The sites of respiratory chain 0; production and the effect of hyperoxia were characterized by supplying submitochondrial particles with NADH or succinate as substrate, and inhibiting the respiratory chain using either rotenone, antimycin, or CN- (Fig. 3). NADH and CN--supplemented porcine lung submitochondrial particles produced 0, at rates which increased from 0 to 3.2 nmol * min-1 * mg protein-’ when oxygen concentration varies from 0 to 100%. When submitochondrial particles were inhibited with antimycin, 0, production reached 3.4 + 0.4 nmol . mine1 * mg protein-l at 100% oxygen, representing 0; formation at both the NADH dehydrogenase and ubiquinone-cytochrome b areas. A lower rate of 0; production (2.4 f 1.1 nmol . min-’ . mg protein-‘) can be detected in submitochondrial particles incubated with rotenone at 100% oxygen, demonstrating again that the lung mitochondrial respiratory chain contains at least two different components capable of reducing oxygen to 0;. One is the NADH dehydrogenase complex, and the other is located at the ubiquinone-cytochrome b area. Although the rate of 0, formation by porcine lung submitochondrial particles incubated with NADH and antimycin (3.4 nmol. min-’ . mg protein-’ at 100% oxygen) is higher than the rate of 0; formation by NADH and rotenone-supplemented particles (2.5 nmol . min-’ . mg protein at 100% oxygen), it was not equal to the rate expected from adding the rates of 0, production by both sites of the respiratory chain when measured independently (4.2 nmol . min-’ * mg protein-‘). The redox state of the respiratory chain components responsible for 0; generation could be affected differently by the use of different inhibitors, accounting for this discrepancy. NADH-supported 0; formation in the presence of CN- was almost as high as the maximal activity reported when particles were inhibited with antimycin (Fig. 5~). Since it was demonstrated that succinate-

0;

PRODUCTION

409

supported 0, production is totally inhibited by CN-, the high rate of 0; production in CN--treated submitochondrial particles may indicate a small CN--sensitive CuZn SOD contamination. It has been demonstrated that beef heart submitochondrial particle NADH dehydrogenase-mediated 0; production was about 50% of the rate of generation at the ubiquinone-cytochrome b region (12). In porcine lung mitochondrial membranes, 0; production by NADH dehydrogenase accounts for a larger proportion of mitochondrial 0; production, reaching 2.4 nmol . min-’ . mg protein-’ at 100% O2 (Fig. 5a) while the ubiquinone-cytochrome b region generated only 1.7 nmol. min-‘a mg protein-’ under similar conditions. Hyperoxia enhanced rotenone, antimytin, and CN--insensitive NADH oxidation by submitochondrial particles to the same degree as 0, production, but at a higher rate for all oxygen tensions. This is the consequence of not only 0; production, but also electron shunting around the sites of antimycin, rotenone, and CN- inhibition, ultimately reducing oxygen by cytochrome c oxidase. Any NADH oxidation measured at a putative oxygen concentration of 0% must be due to electron shunting to oxygen via cytochrome c oxidase, implying slight oxygen contamination under these conditions. Not many methods are available for measuring 0; production by mitochondrial membranes. Epinephrine may react with NADH in a NADH-dehydrogenasemediated reaction (12). Cytochrome c can be oxidized and reduced by the respiratory chain in reactions not involving 0, production. Nitroblue tetrazolium can be reduced by NADH dehydrogenase and succinate dehydrogenase. This study demonstrates that NADH oxidation measured in the presence of either rotenone, antimytin, or CN- is a close estimation of the rate of 0; production, and can be used as an alternative method for estimating oxygen radical production. Crapo et al. (43) showed that aerosolized SOD fails to prevent lung damage in rats exposed to 100% oxygen. Since instilled SOD scavenges 0; only in extracellular

410

TURRENS

spaces, the lack of protection by aerosolized enzyme suggests a major site of 0; formation during hyperoxia must be inside lung cells. This report supports this concept. REFERENCES 1. ROSENBAUM, R. M., WIT~NER, M., AND LENGER, M. (1969) Lab. Invest 20,516-Q%. 2. CRAPO, J. D., MARSH-SALIN, J., INGRAM, P., AND PRATT, P. C. (1978) Lab. Invest. 39,640~653. 3. CRAPO, J. D., BARRY, B. E., FOSCUE, H. A., AND SHEXBURNE, J. (1980) Amer. Rev. Resp. Dis. 122,X3-143. 4. CHANCE, B., SIES, H., AND BOVERIS, A. (1979)

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