Regulation of Hydrogen Peroxide Generation in Cultured Endothelial Cells Vuokko L. Kinnula, A. Richard Whorton, Ling-Yl Chang, and James D. Crapo Departments of Medicine, Pharmacology, Cell Biology, and Pathology, Duke University Medical Center, Durham, North Carolina, and Department of Pulmonary Medicine, Helsinki University Central Hospital, Helsinki, Finland

Endogenous hydrogen peroxide (H20 2) release from aortic endothelial cells was studied in the presence of antioxidant enzyme inhibitors, mitochondrial inhibitors, a microsomal cytochrome P-450 inhibitor, and after oxidative stress induced with H20 2 or menadione. Extracellular H20 2 generation was determined spectrofluorometrically using 3-methoxy-4-hydroxy phenylacetic acid, and intracellular H 20 2 production (in or near peroxisomes) was measured indirectly using aminotriazole, which inactivates catalase in the presence of H20 2 • Extracellular H20 2 release was 0.079 ± 0.005 nmol/min/mg protein in Hanks' balanced salt solution, was constant during a 120-min incubation period, and was not affected by the cell passage number. The half-life for catalase inactivation with aminotriazole was 23 min. Inhibition of catalase, glutathione reductase, or ')'-glutamylcysteine synthetase did not change the rate of extracellular release of H20 2 • Furthermore, inhibition of the mitochondrial respiratory chain (rotenone, antimycin A) or microsomal cytochrome P-450 (8-methoxypsoralen) did not change extracellular H2 0 2 release or intracellular H20 2 production (at peroxisomes) by endothelial cells or cells in which glutathione reductase was inactivated. When the cells were exposed to exogenous H20 2 (30 j.tM), extracellular H20 2 was scavenged primarily by the glutathione redox pathway. Exogenously added H20 2 (100 j.tM) changed intracellular H20 2 production (in or near peroxisomes) only when the glutathione redox cycle was inactivated. Menadione (20 j.tM), which undergoes intracellular redox cycling, increased extracellular H20 2 release almost 4-fold to 0.3 nmol/min/mg protein. Furthermore, menadione increased peroxisomal H20 2 levels and decreased the half-life for catalase inactivation in the presence of aminotriazole to 13 min. Catalase inhibition increased extracellular H20 2 release during menadione treatment, indicating that H20 2 can diffuse across the plasma membrane during oxidant stress. The results suggest that in cultured endothelial cells, reactive oxygen species generated at the mitochondrial or microsomal levels can be scavenged locally without the involvement of peroxisomal catalase and that reactive oxygen species that are released extracellularly are generated by these cells at a site that is inaccessible to catalase or glutathione reductase.

Regulation of reactive oxygen species formation and degradation in tissues is important not only to our understanding of normal oxidative metabolism but also to our understanding of pathologic conditions in which reactive oxygen species are overproduced. Reactive oxygen species are generated and released during respiration by a variety of cell types. Vascular endothelial cells are well recognized as being extremely sensitive to oxidative injury (1-4). The extent of injury in endothelial cells is dependent on the degree of oxidative stress and the status of antioxidant defense systems (5). Although endothelial cells contain both catalase and su-

(Received in original form March 15. 1991 and in final form July 30. 1991) Address correspondence to: James D. Crapo, M.D., Box 3177, Duke University Medical Center, Durham, NC 27710. Abbreviations: aminotriazole, ATZ; 1,3-bis(2-chloroethyl)-I-nitrosourea, BCNU; buthionine sulfoximine, BSO; Hanks' balanced salt solution, HBSS; hydrogen peroxide, H202 ; lactate dehydrogenase, LDH. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 175-182, 1992

peroxide dismutase, their major mechanism to detoxify reactive oxygen species is the glutathione redox cycle (3,6). In earlier studies, it has been demonstrated that endothelial cells can generate significant amounts of superoxide (7-9) and hydrogen peroxide (H 20 2 ) (10). Several previous studies have focused on mechanisms of stimulation of reactive oxygen species generation in these cells. Superoxide released from endothelial cells can be stimulated by a protein kinase C activator or by mobilization of CaH (8). Furthermore, superoxide release from human umbilical endothelial cells can be stimulated by bradykinin (9). In the latter study, it was suggested that bradykinin-induced superoxide release was related to arachidonic acid metabolism through cyclooxygenase. Although it is known that subcellular organelles like mitochondria and microsomes produce reactive oxygen species at rates that are proportional to their local oxygen tension (11, 12), it is not known to what extent reactive oxygen species generated intracellularly in intact cells are scavenged locally and to what extent they are able to diffuse into distant

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subcellular organelles within the cells. Intracellularly generated reactive oxygen species may also be able to diffuse to the adjacent extracellular spaces. Whether or not the reactive oxygen species released into the extracellular spaces by endothelial cells are produced on the cell surface (similar to phagocytic cells) or are produced intracellularly and are therefore susceptible to being scavenged by intracellular antioxidants is not known. In this study, we investigated the effects of inhibition of two important antioxidant enzymes, catalase and glutathione reductase, on the rate of H20 2 production by endothelial cells and the rate of H2 0 2 release into the extracellular spaces. Catalase was inhibited with 3-amino 1:2,4 triazole (aminotriazole [ATZ]), and glutathione reductase was inhibited with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCND). We also studied the significance of subcellular organelles, including mitochondria and endoplasmic reticulum, for H20 2 production in these cells using inhibitors of the mitochondrial electron transport chain and an inhibitor of cytochrome P-450. Experiments were carried out in unstimulated cells and in cells in which oxidant production was increased by incubation of the cells with menadione (7, 13).

Materials and Methods Cell Culture Endothelial cells were collected from bovine aorta and cultured as previously described (5). They were seeded into 25ern- flasks and grown in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. At confluence, the cells were subcultured by 1:5 splits and confluent cells were used between passage numbers 2 to 13. All experiments were done following removal of the culture medium and rinsing 3 times with HEPES-buffered Hanks' balanced salt solution (HBSS), pH 7.4. For those experiments in which cell respiration was investigated, the cells were grown to confluence on Cytodex 1 beads. Culture of cells on beads was essentially as described before (13). Cells were plated in 75-cm 2 flasks and allowed to grow to confluence. After addition of the beads, the cells were cultured for an additional 2 days. The beads were removed by gentle agitation. The medium was exchanged every 3 days, and more cells were produced by adding new aliquots of beads. H20 2 Release from Cells H20 2 release was assayed using a modification of the method of Ruch and co-workers (14). The washed cells were incubated for 15 to 120 min with homovanillic acid (3-methoxy-4-hydroxy phenylacetic acid) and horseradish-peroxidase at pH 7.4, and the fluorescence of the supernatant was measured after adjusting the pH to 10.0 with 0.1 M glycineNaOH buffer (excitation wavelength, 321 nm; emission wavelength, 425 nm). Emission spectra from samples and H20 2 standards were identical. The exact H20 2 concentration of the H20 2 solutions used to establish standard curves was determined spectrophotometrically at 240 nm using the extinction value, 43.6 M -1 cm'. HzOz Production in or near Peroxisomes H20 2 production at peroxisomes was measured indirectly by using the specific catalase inhibitor ATZ, which inhibits

catalase in the presence of H20 2 (15). Earlier studies have shown that ATZ plus catalase in a cell-free system is sensitive to H20 2 and that this system can be used to detect the production of low concentrations of H20 2 over a relatively long time (15). In our preliminary experiments, catalase was inactivated in a cell-free system with 20 mM ATZ using H20 2 concentrations between 10 to 100 ~M. In these experiments, catalase was incubated for 0 to 30 min with ATZ and H20 2 • After the incubation, ATZ was separated from catalase using a Sephadex G75 column and the remaining catalase activity was assayed. The results of these experiments confirmed earlier published data showing that in cellfree systems catalase inactivation follows first-order kinetics during the first 30 min. Washed endothelial cells were incubated in HBSS at 37° C in the presence of 20 mM ATZ for 0 to 30 min. After the incubation period, cells were washed 3 times, scraped, and sonicated in 0.05 M Tris-O.1 mM EDTA (pH 7.4) for the catalase assay. Catalase activity was determined polarographically as an oxygen production rate using a Clark electrode fitted into stirred chamber. The reaction was started by adding the cells into the buffer containing 0.5 mM H20 2 equilibrated with nitrogen. The activity was expressed as nmol O2 produced/min/mg protein. Absolute H20 2 production was not calculated because the method underestimates intracellular H20 2 production in intact cells. Inhibition of Catalase and Glutathione Reductase Washed cells were incubated in HBSS solution with 20 mM ATZ for 60 min to inhibit catalase (15), with 100 ~g1ml BCND for 15 min to inhibit glutathione reductase (16) or with ATZ (20 mM for 75 min) + BCND (100 ~g/ml for 15 min). BCND was dissolved in dimethylsulfoxide because the inhibition by ATZ is reversible in an ethanol-containing medium (15). After the incubation, ATZ and BCND were removed by washing 3 times, since BCND was observed to interfere with the fluorescence determination. H20 2 release from pretreated cells was determined after incubating the cells as described above. In other experiments, confluent cells in Dulbecco's modified Eagle's medium containing 2 % fetal bovine serum were incubated 16 h with 0.5 mM DLbuthionine-s,R-sulfoximine (BSO) to inhibit ')'-glutamylcysteine synthetase (17), and thereby lower glutathione levels (6). In all cases, controls were incubated in vehicle-treated HBSS solution. Inhibition of Mitochondrial Electron Transport and Microsomal Cytochrome P-450 Antimycin A, rotenone, and methoxypsoralen were used. Mitochondrial electron transport was inhibited by incubating the cells for 15 min in HBSS using 10 ~M antimycin or 100 ~M rotenone A dissolved in ethanol. Antimycin had a slight effect on the fluorescence with homovanillic acid and horseradish peroxidase. Because of this, antimycin A was removed by washing the cells 3 times before determining H20 2 release. Rotenone was added 5 min before incubation with homovanillic acid and horseradish peroxidase. Microsomal cytochrome P-450 was inhibited by addition of 250 ~M methoxypsoralen (18) 5 min before incubation. Rotenone or methoxypsoralen had no effect on the H20 2 release assay. For indirect measurements of intracellular H20 2 production

Kinnula, Whorton, Chang et al.: H,O, Generation by Endothelial Cells

at peroxisomes, cells were incubated in the presence of rotenone (100 J.LM), antimycin A (10 J.LM), or 8-methoxypsoralen (250 J.LM) for 5 min and then followed by ATZ incubation for 30 min. These experiments were also conducted in cells in which glutathione reductase had been inactivated by pretreatment with BCND (100 J.Lg/rnl for 15 min). Oxygen consumption by endothelial cells grown on Cytodex beads was measured at 37° C by adding cells to a chamber fitted with a Clark electrode. The electrode was calibrated using HBSS. Oxygen consumption by the cells was determined without and with 10 J.LM antimycin A, 100 J.LM rotenone, and I mM KCN. H,O, Treatment Cells were incubated for 60 min with 10 J.LM, 100 J.LM, and 1 mM H,O,. Previous studies have shown that 1 mM H,O, is toxic to endothelial cells, resulting in a small but detectable lactate dehydrogenase (LDH) release (5). After exposure to H,O" cells were washed and subsequent H,O, release from the cells was determined as described above. In other experiments, cells were incubated for 30 min with a sublethal concentration (30 J.LM) of H,O, (5) to investigate mechanisms involved in the metabolism of exogenous oxidants. To inhibit catalase, cells were pretreated with 20 mM ATZ for 60 min. To inhibit glutathione reductase or deplete glutathione levels, cells were incubated with 100 J.Lg/rnl BCND for 15 min, with 0.5 mM BSO for 16 h, and with 100 J.Lg/rnl BCND for 15 min. To inhibit catalase and glutathione reductase, cells were incubated with ATZ (75 min) + BCND (15min). Aliquots from the supernatant were taken for H,O, measurements to calculate the H,O, consumption from the extracellular medium. In additional experiments, cells were exposed to H,O, (10 and 100 J.LM) for 15 min, and catalase inactivation by these cells was measured. These experiments were also conducted in cells in which glutathione reductase was inactivated with BCND. Menadione Treatment Cells were incubated with menadione (2-methylnaphtoquinone, 20 and 100 J.LM) in DMSo. These concentrations of menadione do not damage endothelial cells if the exposure is less than 2 h (13). In some of these experiments, the cells were pretreated with ATZ or BCND as described above. Menadione alone had a slight fluorescence, which was determined and used to correct sample values.

177

(diluted 1 to 500) and with 9 nm protein A gold for immunolocalization of catalase according to Slot and Geuze (23). Statistical Analysis Data from various groups were expressed as mean ± SE. Two-tailed Student's t tests were used to compare two groups. To compare the significance between multiple experimental groups, two-way variance (ANOVA) in combination with the post-hoc Scheffe's test was used. Statistical significance was defined as P < 0.05.

Results Extracellular H,O, release was measured using homovanillac acid and horseradish peroxidase. Endothelial cells released H,O, into the incubation medium at a constant rate (0.079 ± 0.005 nmol/min/mg protein) over 2 h (Figure I). H,O, release was 0.03 nmol/min/Hr cells. The rate of H,O, release was independent of cell passage number (Table 1). Intracellular H,O, production in or near peroxisomes was estimated by following the rate of inactivation of catalase. In other studies, it has been shown that half-life for catalase inactivation in the presence of ATZ is proportional to the rate of H,O, production (15). Because other antioxidant systems participate in H,O, degradation, this value underestimates the true rate of H,O, generation but is useful as an index of the relative importance of peroxisomal catalase in the overall antioxidant defenses of the cell (see below). The half-life for catalase inactivation in our experiments was 23 min (Figure 2). The absolute rate of intracellular H,O, production cannot be determined using the ATZ method; however, a crude estimate of the amount of H,O, reacting with catalase can be calculated as follows. Catalase activity in our cells was 11.4 mD/mg protein. Pure catalase has a molecular weight of 240,000 and it contains about 50,000 Uzmg enzyme protein. In our experimental conditions, the half-life for catalase inactivation with ATZ was 23 min. Total inactivation required about four half-lives (90 min). In these conditions, H,O, generation calculated from catalase activity and from the above data would give an estimated rate of peroxisomal H,O, generation of 1.0 nmol/mg protein in 90 min. We consider this value to be a rough underestimation of intracellular H,O, production in or near peroxisomes in these cells. Bovine aortic endothelial cells are small cells with irregu-

9-r------------, Other Biochemical Assays LDH activity, which is commonly used as a marker of cellular injury, was determined using conventional spectrophotometric methods (19). Glutathione reductase activity was determined spectrophotometrically (20). Protein was assayed by the method of Lowry and associates (21).

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o Electron Microscopy and Immunocytochemistry Cultured endothelial cells were fixed using 2 % paraformaldehyde in 0.1 M phosphate buffer in the culture flask and subsequently prepared for immunocytochemical labeling and electron microscopic analysis as described by Geuze and colleages (22). Cryo-ultrathin sections of the endothelial cell pellet were incubated with rabbit anti-bovine catalase antisera

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Figure 1. Extracellular hydrogen peroxide (H,O,) production by endothelial cells. The cells were incubated in Hanks' balanced salt solution (HBSS). H,O, release is expressed as nrnol/mg protein (mean ± SE; n = 7).

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TABLE 1

Effect of passage number on H202 release from endothelial cells* H,O, Passage No.

(nmol/min/mg protein)

2 3 4 5 8 9 11 14

0.081 (2) 0.059 (2) 0.082 ± 0.006 0.089 ± 0.008 0.063 ± 0.008 0.077 ± 0.004 0.D75 ± 0.005 0.072 ± 0.007

(4) (4)

(3) (6) (5) (4)

Definition of abbreviation: H,O, = hydrogen peroxide. * Values are mean ± SE, with total number in parentheses.

lar apical surfaces in vivo. The cytoplasm on either side of the nuclei of these cells drops abruptly, giving the vessellumen lining layer a knobby appearance. Our cultured bovine aortic endothelial cells had smooth apical surfaces, attenuated cytoplasm, and showed a typical "cobblestone" morphology. The cells also expressed factor VIII antigen and synthesis of prostacyclin. Electron microscopic examination of ultrathin cryosections of the cultured bovine aortic endothelial cells showed the cytosolic matrix to be less compact than that found in endothelial cells in vivo. All major cell organelles were found in the cultured cells. An abundant amount of mitochondria and endoplasmic reticulum was present. A small number of peroxisomes were localized throughout the cell cytoplasm. The average cell profile had 10 to 20 mitochondrial profiles but only zero to three peroxisomal profiles. The peroxisomes observed did not appear to be preferentially paranuclear or localized adjacent to mitochondria. Immunocytochemical labeling of catalase showed that the enzyme was restricted to peroxisomes (Figure 3). Using specific antisera, background labeling of noncellular material (gelatin, in which cells were embedded) was the same as the labeling density on the cellular compartments other than peroxisomes. Furthermore, when nonimmune serum was used, the same nonspecific cellular labeling pattern

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was observed and there was no peroxisomal labeling. These results confirm the above conclusion that in these cells catalase is located only in peroxisomes. To study the relative importance of different cellular sites on the rate of intracellular H202 generation, cells were incubated in the presence of mitochondrial and microsomal inhibitors. Oxygen consumption measurements using cells grown on beads showed that most of the oxygen consumption was related to mitochondrial respiration in these cells. Cellular respiration decreased 89% in the presence of 10 JlM antimycin A, 91% in the presence of 100 JlM rotenone, and 92 % with 1 mM KCN. Preincubation of the cells with 10 JlM antimycin A, 100 JlM rotenone, or 250 JlM methoxypsoralen had no effect on the rate of H202 production in or near peroxisomes when assessed as catalase inactivation or extracellular H202 release by using homovanillic acid (Table 2). Because others have shown that the glutathione redox cycle is more important than catalase in scavenging extracellularly added H202 (3, 6), H202 production at peroxisomes was also studied using cells in which glutathione reductase was inactivated with BCNU. When these cells were exposed to antimycin A, rotenone, or methyoxypsoralen, catalase in activation was 1.08, 1.04, and 0.96, respectively, when compared with cells that had been pretreated with BCNU only. To further investigate sites of H202 release and degradation, H,02 generation was measured in cells in which catalase or glutathione reductase or both of these enzymes had been inhibited. In control cells, catalase activity was 11.4 ± 2.7 (n = 5) nmol O2 produced/min/mg protein and glutathione reductase activity was 6.9 ± 0.8 (n = 4) mU/mg protein. Catalase activity after 60 min of incubation with ATZ was 14% and after 75 min of incubation was 8% compared with the controls. Glutathione reductase activity after 15 min of incubation with BCNU was decreased to 7 % of the controls. ATZ had no effect on glutathione reductase activity; BeNU had no effect on catalase activity. In our experiments, neither catalase nor glutathione reductase inactivation had any effect on the extracellular H202 release from endothelial cells (Table 3). Preincubation of cells with either BSO alone or with BSO plus BCNU did not change H202 release. H,O, release from BSO-treated cells was 95 ± 2.1% (n = 3) and from BSO + BCNU-treated cells was 93 ± 1.6% (n = 4) compared with the controls. To examine the effect of exogenous H202 on endothelial cells, cells were preincubated for 60 min with 10, 100, and 1,000 Jlm H202 and washed 3 times. While preincubation with 10 JlM H20, produced no change in the H202 release from pretreated cells, preincubation with 100 JlM H202 decreased H202 production by 36 % and preincubation with 1 mM H 202 reduced H 202 production by 86% when compared with controls (Figure 4). This suggests that a component of the pathway leading to H202 generation is oxidant sensitive. Previous results from our laboratory have shown that a 60-min incubation with 100 JlM H202 does not change LDH release from the cells, whereas I mM H202 results in a minimal LDH release (5). These results were reconfirmed in the current studies. When endothelial cells were exposed to 10 or 100 JlM H20 " no change in catalase inactivation with ATZ was found during a 15-min incubation. In the presence of 10 JlM exogenous H 20 h 28.0 ± 3.0% (n = 4) oftotal catalase was

Kinnula, Whorton, Chang et al.: H,O, Generation by Endothelial Cells

179

Figure 3. Immunocytochemical labeling of catalase in cultured endothelial cells. Catalase labeling is found only over peroxisomes, and there are relatively few peroxisomes in these cells. A peroxisome (p) in the middle of the micrograph contains gold particles immunolabeled to catalase. The inset at the upper righthand corner shows an enlarged view of the peroxisome. Nu = nucleus; m = mitochondria; rer = endoplasmic reticulum.

inactivated, and, in the presence of 100 J,tM H,O" 30.7 ± 6.2 % (n = 4) of catalase was inactivated. However, when the cells were pretreated with BCND to inactivate glutathione reductase, catalase inactivation in the presence of 100 J,tM exogenous H,O, was significantly increased (49.7 ± 2.3 %; n = 4; P < 0.05). These results indicate that in endothelial cells extracellularly added H,O, (10 to 100 J,tM) is not normally accessible to peroxisomal catalase, possibly because a significant part of it is scavenged by the glutathione redox pathway. Endothelial cells were also incubated with 30 J,tM H,O" and H,O, consumption from the extracellular medium was calculated. Preincubation of the cells for 60 min with 20 mM ATZ to inactivate catalase had no effect on H,O, degradation, whereas preincubation with BCND (100 J,tg/ml) resulted in a significant decrease in H,O, consumption by the cells (Figure 5A). ATZ had a slight effect on H,O, consumption by these cells only when the glutathione redox cycle was inactivated (Figure 5B). Exogenous H,O, consump-

tion by cells that had been pretreated with BCND or with BSO + BCND did not differ significantly (data not shown). To enhance endogenous H,O, production, cells were incubated with either 20 or 100 J,tM menadione. Menadione resulted in a concentration-dependent accumulation of H,O, in the extracellular medium over 60 min (Figure 6). Incubation for an additional 60 min produced no further accumulation of H,O" perhaps related to the exogenous H,O, effect noted above (Figure 4). Menadione also increased H,O, levels in peroxisomes since incubation of the cells with menadione led to an enhanced ATZ inactivation of catalase (Figure 7). We next studied the role of catalase and the glutathione redox cycle on menadione-stimulated H,O, production by prior inactivation of catalase by preincubation with ATZ or by inhibiting glutathione reductase with BCND. As expected, catalase inactivation increased H,O, release (Figure SA). However, glutathione reductase inhibition decreased H,O, release compared with cells treated with menadione alone (Figure 8B). Other results (Figure 4)

TABLE 2

TABLE 3

Effect of inhibition of the mitochondrial electron transport chain and microsomal cytochrome P-450 on extracellular H2 0 2 release and H2 0 2 production in or near peroxisomes*

Effect of inhibition of catalase (using A1Z) and glutathione reductase (using BeNU) on extracellular H2 0 2 release by endothelial cells

Extracellular H,O, (nmollmin/mg protein)

Peroxisomal H,O, (AIZ inactivation in 30 min)

Control Rotenone

0.056 ± 0.007 (6) 0.068 ± 0.011 (6)

0.56 ± 0.007 (7) 0.56 ± 0.012 (6)

Control Antimycin A

0.076 ± 0.007 (5) 0.073 ± 0.003 (5)

0.56 ± 0.007 (7) 0.57 ± 0.013 (6)

0.048 ± 0.004 (7) Control 8-methoxypsoralen 0.047 ± 0.006 (7)

0.56 ± 0.007 (7) 0.58 ± 0.013 (7)

Definition of abbreviations: H,O, = hydrogen peroxide; ATZ = aminotriazole. * Mitochondrial inhibitors were rotenone (100 I'M). antimycin A (10 I'M), and the microsomal cytochrome P-450 inhibitor 8-methoxypsoralen (250 I'M). Values are mean ± SE, with total number in parentheses.

H,O, (nmollmin/mg protein)

Control ATZt

0.047 ± 0.005 (8) 0.045 ± 0.005 (8)

Control BCNU*

0.048 ± 0.003 (8) 0.055 ± 0.004 (8)

Control ATZ+BCNU§

0.054 ± 0.003 (4) 0.055 ± 0.004 (4)

Definition of abbreviations: ATZ = aminotriazole; BCNU = 1.3-bis(2chloroethyl)-l-nitrosourea; H,O, = hydrogen peroxide. * Values are mean ± SE, with total number in parentheses. t 20 mM for 60 min. t 100 I'g/m1 for 15 min. § ATZ 20 mM for 75 min plus BCNU 100 I'g/m1 for IS min.

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992

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nase C and bradykinin (7-10). This study extends earlier observations focusing on the sites of H 202 generation by these cells by investigating both intracellular and extracellular H 202 generation in these cells under basal conditions and during oxidant stress. Sites for intracellular H 202 production in intact cells are difficult to determine. In this study, the level of H 202 in peroxisomes was assessed by following the inactivation of 80 Menadione

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Kinnula, Whorton, Chang et at.: H20 2 Generation by Endothelial Cells

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Time, min

Figure 8. H202 release by endothelial cells in the presence of 100 ILM menadione. (A) Effect of ATZ (20 mM for 60 min) pretreatment. (B) Effect of BeNU (100 ILg/ml for 15 min) pretreatment (mean ± SE; n = 6). * P < 0.05 versus menadione alone. The difference between the controls and menadione-treated cells was significant at each time point (P < 0.01).

catalase in the presence of ATZ. The outcome of this measurement depends both on the sites of H20 2 production and on the sites for H20 2 detoxification. Importantly, this study found that catalase is specifically localized to peroxisomes, which appear to be randomly distributed within the cell. Using ATZ inactivation of catalase as an index of H20 2 production, others have shown that intracellular H20 2 is readily accessible to peroxisomal catalase in hepatocytes in vitro (24, 25) and in hyperoxic brain in vivo (26). Furthermore, the present data suggest that endogenous H20 2 generation during menadione-induced oxidant stress is accessible to peroxisomal catalase in endothelial cells. It has to be emphasized, however, that when endothelial cells were exposed to low extracellular H2 0 2 concentrations, significant amounts of additional H2 0 2 did not reach peroxisomes unless glutathione reductase was inactivated. These results support the conclusion that peroxisomal catalase may be useful in measuring H 20 2 production in or near peroxisomes during endogenous oxidative stress but that cells primarily scavenge exogenous H20 2 using other cellular antioxidants and peroxidases like the glutathione redox pathway. Earlier studies have shown, using isolated mitochondria and microsomes, that reactive oxygen species are produced both in mitochondrial and microsomal fractions (27) and that mitochondrial H 20 2 production can be markedly enhanced by antimycin A and rotenone (11, 12,27). Lung mito-

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chondrial reactive oxygen species production is associated with both the ubiquinone-cytochrome b region and the NADH dehydrogenase complex in the respiratory chain (11, 12). In the present study, antimycin A and rotenone blocked cellular oxygen consumption by > 89 %. However, mitochondrial and microsomal inhibitors (rotenone, antimycin A, 8-methoxypsoralen) did not change ATZ-induced catalase inactivation in this study even when the glutathione redox cycle was inactivated, suggesting that reactive oxygen species generated in these subcellular organelles under basal conditions are not accessible to peroxisomal catalase. It should be pointed out that the number of peroxisomes in these cells is low and they are not preferentially located close to mitochondria. Thus, it appears that ATZ inactivation of catalase primarily measures H20 2 within or near peroxisomes. Cultured endothelial cells release reactive oxygen species into the adjacent extracellular spaces. The rate of extracellular H20 2 release by these cells is not likely to be influenced by back diffusion of H20 2 into the cells because, in the assay, homovanillic acid in the presence of horseradish peroxidase functions as an efficient trap to scavenge extracellular H20 2 • If the source of the extracellularly released reactive oxygen species was from intracellular metabolism, then the species should be susceptible to being scavenged by intracellular antioxidants. Alternatively, they may be produced near or on the cell surface. In the present study, inhibition of catalase, glutathione reductase, or -y-glutamylcysteine synthetase did not alter H20 2 release into the incubation medium. Catalase and glutathione reductase activities were decreased > 92 % and previous results indicate that most (> 80%) of glutathione would have been depleted (3, 4, 28). Whether the minimal amount of antioxidant enzymes left after inactivation is enough to scavenge all H20 2 produced intracellularly is unclear. It also has to be pointed out that when both catalase and glutathione reductase were inactivated at the same time, these cells consumed exogenous H20 2 from the extracellular medium at a remarkably high rate (Figure 5B). This also suggests that other antioxidants like peroxidases may have a significant role in the scavenging of both endogenous and exogenous H20 2 by endothelial cells. Whether cultured endothelial cells have membrane-bound oxidases or extracellular superoxide/dismutase has not been verified. Recent studies have found binding sites for extracellular xanthine oxidase on endothelial cells (29), though it seems that extracellular reactive oxygen species generation is not associated with xanthine oxidase activity in cultured endothelial cells (30). The results of the present study suggest that H20 2 release from endothelial cells is located at sites inaccessible to glutathione reductase or catalase but the exact mechanism of extracellular H2 0 2 generation in these cells remains unclear. Menadione results in the generation of large amounts of reactive oxygen species (7). It offers one possibility to study the relationship between extracellular and intracellular H20 2 generation and the significance of antioxidant pathways on regulating extracellular H20 2 release during endogenous oxidant stress. In the present study, menadione resulted in augmented H20 2 generation with increased amounts found both intracellularly and extracellularly. These findings show that the methods used in this study to

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detect H20 2 production and release by cells are sufficiently sensitive to detect changes in reactive oxygen species generation in cultured endothelial cells. Quinone (menadione)induced toxicity results from the univalent reduction of the quinone to form a semiquinone radical that can be oxidized regenerating the quinone and producing the superoxide radical (7, 31-33). NADPH cytochrome P-450 reductase and NADH ubiquinone oxidoreductase are the enzymes that catalyze the one-electron reduction of menadione to its semiquinone. In our study, glutathione reductase inhibition before treatment with menadione resulted in a decreased H 20 2 release compared with cells treated only with menadione. We had initially expected glutathione reductase inhibition to increase H20 2 release. The fact that it behaved opposite to our expectations may be related to minimal cell injury and inhibition of NADPH oxidoreductase (31, 33) and/or to a reduced level of NADPH that could act to reduce the rate of cyclical oxidation and reduction of menadione. Interestingly, treatment with menadione and ATZ led to an increased H20 2 release. Elevated H20 2 release in ATZpretreated cells in the presence of menadione suggests that menadione leads to reactive oxygen species production at multiple sites, at least some of which are accessible to peroxisomes where catalase is located. This is in contrast to the pathways for degrading exogenous H202 • Menadionederived H202 is degraded by both catalase and glutathione reductase pathways. Furthermore, because ATZ pretreatment resulted in increased extracellular H202 release in the presence of menadione, it suggests that intracellularly generated H20 2 can diffuse across the plasma membrane to reach the extracellular spaces during oxidant stress. In summary, H20 2 production by endothelial cells was measured focusing on the relationships between antioxidant enzymes and different cellular locations as sites of H202 scavenging and generation by these cells. Extracellular H 20 2 release did not correlate with the antioxidant capacity of the cells, suggesting that there are different locations for the oxygen radical generation that leads to extracellular release of H20 2 and the sites where reactive oxygen species are scavenged in these cells. The results also suggest that reactive oxygen species generated at mitochondrial and microsomal levels in these cells can be scavenged locally without involvement of peroxisomal catalase. Acknowledgments: This work was supported in part by Grants ROi HL-42609 and POI HL-31992 from the National Institutes of Health. Dr. Kinnula was partly supported by the Anti-Tuberculosis Association of Finland.

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