0013-7227/91/1286-2958$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 6 Printed in U.S.A.
Evidence That Hydrogen Peroxide Blocks HormoneSensitive Cholesterol Transport into Mitochondria of Rat Luteal Cells* HAROLD R. BEHRMAN AND RAYMOND F. ATEN Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 tissue. In this paradigm, hydrogen peroxide did not inhibit elevated basal progesterone synthesis in luteal cells produced by in vivo aminoglutethimide treatment, yet LH-stimulated steroidogenesis was blocked. However, treatment of luteal cells with hydrogen peroxide inhibited pregnenolone synthesis in isolated mitochondria, an effect partially reversed by the addition of luteal cell cytosol. In summary, while peroxide inhibited cAMPdependent steroidogenesis, it did not appear to inhibit protein kinase activation or mobilization of cholesterol from intracellular esterified stores. Although peroxide inhibited pregnenolone synthesis, it had no effect on steroidogenesis when substrate was made available by either addition of cholesterol analogs or prior treatment with aminoglutethimide in vivo. We conclude, therefore, that hydrogen peroxide inhibits steroidogenesis by blocking intracellular transport of cholesterol to mitochondria or translocation of cholesterol across the outer mitochondrial membrane. {Endocrinology 128: 2958-2966,1991)
ABSTRACT. In luteal and granulosa cells, hydrogen peroxide abruptly inhibits activation of adenylate cyclase by receptorbound gonadotropin and blocks steroidogenesis. In the present studies a post-cAMP site of peroxide action on inhibition of steroidogenesis was investigated. Steroidogenesis, stimulated by dibutyryl or 8-bromo-cAMP, was inhibited by hydrogen peroxide. Yet, cAMP-dependent protein kinase activation in cytosol or intact cells was unaffected by peroxide treatment. Hydrogen peroxide also did not inhibit the activity of cholesterol esterase and acyl coenzyme-A:acyltransferase. Progesterone synthesis was maximally increased 5- to 50-fold with 25- and 22-hydroxycholesterol, respectively. Unlike that seen with cAMP analogs and LH, however, progestin synthesis stimulated by these celland mitochondria-permeant cholesterol analogs was not inhibited by hydrogen peroxide. Treatment of animals with aminoglutethimide produces a marked accumulation of steroidogenic cholesterol substrate and a large increase in hormone-independent steroidogenesis in subsequently isolated and washed luteal
A
LONG known characteristic of the corpus luteum is the presence of high levels of antioxidants, such as ascorbic acid and lutein, a member of the carotenoid family (1, 2). Both LH and prostaglandin F2« (PGF2«) cause a rapid depletion of luteal ascorbic acid (3, 4), and both agents evoke luteolysis in the rat (5, 6). Direct evidence for the production of reactive oxygen species in the ovary is the generation of hydrogen peroxide (7) and superoxide production (8) during luteolysis. One consequence of the generation of oxygen radicals in tissues is lipid peroxidation, an effect seen in luteal tissue several hours after treatment of rats with PGF2tt (9). We recently proposed that hydrogen peroxide may mediate lytic events involved in the cyclic remodelling of the ovary based on the generation of hydrogen peroxide during luteolysis (7) and the marked antigonadotropic actions evoked by hydrogen peroxide in rat luteal Received January 7,1991. Address requests for reprints to: Dr. Harold Behrman, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. * This work was supported by NIH Grant HD-10718.
(10) and granulosa cells (11). Gonadotropin-sensitive cAMP accumulation and progesterone synthesis are abruptly inhibited by hydrogen peroxide, and cellular ATP is depleted (10, 11), all features of a lytic response. Stimulation of progesterone synthesis in granulosa cells by 8-bromo-cAMP is also inhibited by hydrogen peroxide (11). Thus, peroxide may inhibit gonadotropin-dependent progesterone synthesis not only by inhibiting adenylate cyclase activity, but also at sites beyond cAMP. Rate-limiting steps in steroidogenesis include the mobilization and transport of cholesterol from cholesterol ester depots into mitochondria and the formation of pregnenolone by the mitochondrial cholesterol sidechain cleavage enzyme complex (12). Extracellular cholesterol is not available directly for steroidogenesis because of barriers to diffusion, but cholesterol analogs, such as 22- and 25-hydroxycholesterol, penetrate the cell and mitochondria and produce a prompt stimulation of progesterone synthesis (13, 14). A method was, thus, available to assess whether cAMP-sensitive cholesterol mobilization and transport or cholesterol side-chain cleavage activity are sites of peroxide action in blocking
2958 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
PEROXIDE BLOCKS MITOCHONDRIAL CHOLESTEROL TRANSPORT progesterone synthesis. We show that while peroxide inhibited cAMP-sensitive progesterone synthesis, it had no effect when substrate was made available by the addition of cholesterol analogs or after cholesterol loading by prior aminoglutethimide treatment in vivo (15). We also show that peroxide lacked an effect on the activities of cAMP-dependent protein kinase and the enzymes regulating cholesterol ester turnover, yet it inhibited mitochondrial pregnenolone formation. Thus, based on these findings we conclude that cAMP-dependent transport of cholesterol into mitochondria is inhibited by hydrogen peroxide.
Materials and Methods Hormones, drugs and reagents Ovine LH (NIDDK oLH 24) was obtained from the NIH (Bethesda, MD). PGF2n was purchased from Upjohn Co. (Kalamazoo, MI). Hydrogen peroxide was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). Catalase (2800 U/mg), 3-aminobenzamide, and aminoglutethimide were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and preparation of luteal cells Immature (26-27 days old) female rats (CD strain, Charles Rivers Laboratories, Wilmington, MA) were injected sc with 50 IU PMSG (Gestyl, Organon Pharmaceuticals, West Orange, NJ). Fifty-four hours later, 25 IU hCG (A.P.L., Ayerst Laboratories, Rouses Point, NY) was injected. Luteal cells were isolated 5-6 days after hCG-induced ovulation by enrichment over a Percoll density gradient, as described previously (10). In some experiments animals were treated with aminoglutethimide (15 mg/rat, ip) 2 h before isolation of luteal cells. Aminoglutethimide produces an abrupt reversible inhibition of pregnenolone synthesis and a marked increase in luteal cholesterol levels (15). Isolation and washing of luteal tissue remove the block of steroidogenesis and produce a large increase in progesterone synthesis, independently of LH, in conjunction with cholesterol accumulation (15). Mitochondrial pregnenolone production To assess the effect of hydrogen peroxide on mitochondrial pregnenolone production, luteal cells were isolated and preincubated with hydrogen peroxide (250 fiM; 60 min), followed by treatment with catalase (2800 U/ml) for 10 min. The cells were isolated from medium and homogenized, and mitochondria were isolated according to the method of Tanaka et al. (16). In brief, cells were homogenized in Tris (25 HIM), EGTA (0.5 mM), EDTA (1 mM), and sucrose (25 mM), pH 7.4. After an 800 x g (10 min) centrifugation, the supernatant fraction was centrifuged (5,000 X g; 10 min) to obtain a mitochondrial fraction (pellet). Cytosol was obtained by collection of the supernatant fraction after centrifugation (100,000 X g; 60 min) of the 5,000 X g supernatant fraction of control cells. Pregnenolone production by mitochondria was determined by the method of McNamara and Jefcoate (17). Mitochondria (50 ng protein) were incubated with Tris (25 mM), sucrose (200
2959
mM), sodium phosphate (10 mM), MgCl2 (5 mM), KC1 (20 mM), EDTA (0.2 mM), sodium succinate (10 mM), fatty acid-free BSA (1 mg/ml), and cyanoketone (10 JIM), pH 7.4. Pregnenolone production was determined in the absence and presence of cytosol. The reaction was stopped by the addition of ethanol, and pregnenolone was extracted with hexane and assayed by RIA after evaporation of the hexane. Assay of cAMP, progestins, and pregnenolone cAMP, progesterone, and 20«-dihydroprogesterone levels were determined by RIA, as described previously (18, 19). Pregnenolone was determined by RIA using a commercially available kit (Diagnostic Division, ICN Biomedical, Carson, CA). Assay of cAMP-dependent protein kinase activity cAMP-dependent protein kinase was assayed as previously described (20). In brief, luteal cells were homogenized (108 cells/ ml) in buffer (0.5 M NaCl, 1 mM EDTA, 7 mM mercaptoethanol, and 10 mM Tris-HCl, pH 7.5) that contained 60 tiu ATP. Homogenization was carried out by sonication (Branson cell disruptor, Branson Sonic Power Company, Danbury, CT) on ice with three 10-sec bursts (power setting 4 at 40 duty cycle), with rest intervals of. 10 sec between bursts. The homogenate was centrifuged (15,000 X g; 10 min), and the supernatant fraction (10 n\) was assayed for kinase activity. In experiments to test activation in cytosol, 1 fiM 8-bromo-cAMP was used; for activation in cells, 0.1 and 1 mM 8-bromo-cAMP were used. The reaction mixture contained a final concentration of 20 mM magnesium acetate, 0.5 mM l-methyl-3-isobutylxanthine, 5 mg/ml histone-II-A, 0.3 mM ATP premixed with 5 /nCi [T- 3 2 P] ATP (30 Ci/mmol), and 20 mM potassium phosphate buffer (pH 6.5). The reaction was initiated by the addition of supernatant and incubated at 30 C for 5 min in the absence and presence of cAMP. The reaction was stopped by the addition of 10% trichloroacetic acid, and the precipitated protein was isolated by centrifugation, followed by three washes with trichloroacetic acid. Radioactivity in the protein precipitate was determined by analysis of Cherenkov radiation. The details of each experimental design are given in the figure legends. Assay of cholesterol esterase and acyl coenzymeA:acyltransferase (ACAT) activity The experimental design used to examine the effect of peroxide on cholesterol esterase and ACAT activities is described in Table 3. Cells (106/ml) were isolated by centrifugation (800 X g; 5 min) and stored at -80 C. To obtain sufficient cells for assay of enzyme activities, the cells from 10 separate experiments were combined. Cells were thawed and lysed by a modification of the glycerol-loading method (21). In brief, frozen cell pellets were suspended in 200 ^1 2.4 M glycerol in 5 mM Tris-HCl (pH 7.4) and incubated for 30 min on ice. The cells were diluted with 800 (A homogenization buffer [20 mM TrisHCl (pH 7.4), 5 mM dithiothreitol, 10 mM EDTA, and 250 mM sucrose] and incubated for an additional 30 min. The resulting suspension was homogenized with a glass-Teflon homogenizer and centrifuged at 10,000 X g for 10 min, and the supernatant fraction was retained. The 10,000 x g supernatant fraction was
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Endo • 1991 Vo! 128-No 6
PEROXIDE BLOCKS MITOCHONDRIAL CHOLESTEROL TRANSPORT
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centrifuged at 100,000 X g for 60 min, and the microsomal pellet and cytosol fraction were retained. The microsomal fraction was washed twice in homogenization buffer before assaying for ACAT activity. The cholesterol esterase activity of the cytosol fraction was determined essentially by the method we described previously (22), with slight modification (23). Cytosol protein (250 fig) was diluted with buffer (100 mM potassium phosphate, 1 mM EDTA, and 2.5 mM 2-mercaptoethanol, pH 7.5) and radiolabeled cholesteryl oleate ([4-14C]cholesteryl oleate; 20 mCi/mmole; 60 nM final concentration), and the samples were incubated for 30 min at 37 C. The assay was stopped by addition of chloroformmethanol (2:1) containing cholesterol, cholesteryl oleate, oleic acid, and triolein carrier. The extract was evaporated to dryness and fractionated by TLC, and radioactivity was determined in the free and esterified cholesterol fractions. The total ACAT activity in the microsome fraction was determined essentially as described previously (24), with slight modification (23). Microsomes (100 ng protein) were diluted in ACAT assay buffer (100 mM potassium phosphate, 1 mM glutathione, and 1 mg/ml fatty acid-free BSA, pH 7.4) that contained 100 Mg/ml cholesterol and 600 Mg/ml Tyloxapol and incubated for 30 min at 37 C. Oleolyl CoA ([l-14C]oleolyl-CoA; 6 mCi/mmol; 100 nM final concentration) was added, and the samples were incubated for 15 min at 37 C. The reaction was stopped by extraction, the extracts were fractionated by TLC, and radioactivity associated with cholesteryl oleate was determined. Experimental protocol Cells were incubated in cholesterol-free medium (MEM 2360, Gibco, Grand Island, NY) that contained 1% BSA. For analysis of cAMP and progestin production, the cells and media were heat treated (90 C; 10 min) and stored (-80 C) before assay. For analysis of enzyme activity, the cells were separated from medium by centrifugation (and frozen). The media were heat treated and stored for later analysis of progestin content. The details of each experimental paradigm are shown in the legends of the tables and figures. Except where noted in the text, the cells were preincubated for 10-15 min with 3-aminobenzamide (2.5 mM). We showed previously that this experimental procedure blocks hydrogen peroxide-induced depletion of ATP (10, 11). Thus, the effects seen in the present studies are not confounded by depletion of cellular levels of ATP. To avoid the possibility that hydrogen peroxide may influence the biological activity of the stimulatory agents used in the present studies, the cells were preincubated with H2O2, followed by treatment with excess catalase (2800 U/ml) for 10 min. We previously showed that this treatment effectively removes hydrogen peroxide under identical experimental conditions (10, 11). Control cells received identical treatment, but no hydrogen peroxide. The levels of LH (1 Mg/ml), analogs of cAMP (1 mM), and analogs of cholesterol (10 Mg/ml) were tested deliberately at supramaximal concentrations, except where indicated in the text, to avoid potential confounding of the results due to degradative oxidation.
Statistical analysis Luteal cells from several animals were pooled and aliquoted into incubation tubes. Each treatment was studied in quadruplicate incubations, and each experiment was repeated at least twice. Statistical significance between treatments within each experiment was determined by analysis of variance with a repeated measures design, followed by Duncan's multiple range test (PC Anova, Human Systems Dynamics, Northridge, CA). P< 0.05 was considered significant. Dose-response effects were assessed by regression analysis to determine whether a significant slope was evident. Treatment differences between experiments were determined by analysis of variance, followed by Duncan's multiple range test. P < 0.05 was considered significant. Results We showed previously that hydrogen peroxide inhibits progesterone synthesis in luteal cells in response to LH, with half-maximal and maximal effects at 100 and 300 /xM peroxide, respectively (10). In the present studies, using an identical paradigm, hydrogen peroxide (100 fiM) significantly inhibited progestin synthesis in response to 8-bromo-cAMP and (Bu)2cAMP (Fig. 1 and Tables 1 and 2). Maximal stimulation by both cAMP analogs (~4fold; P < 0.05) was reduced 35-50% by peroxide (Fig. 1 and Table 1). An approximately 2-fold greater inhibition of progesterone compared to 20a-dihydroprogesterone synthesis occurred with peroxide treatment. Since the levels of both progestins were reduced by peroxide and because these are the major steroids of rat luteal cells, net steroidogenesis per se was inhibited by peroxide. Because cAMP is generally accepted to stimulate ste-^
8T
Peroxide (100
O Control
O 6-
4-
c o 0) CO CD CT> O
2-
0.1
0.3
1.0
8-Bromo-Cyclic AMP (mM) FIG. 1. Inhibition of cAMP-stimulated progesterone synthesis by hydrogen peroxide. Cells were preincubated with 3-aminobenzamide (2.5 mM; 10 min) before incubation with hydrogen peroxide (100 MM) for 1 h, followed by incubation with catalase (2800 U/ml) for 10 min. The cells were then incubated for 1 h with 8-bromo-cAMP. Values represent the mean ± SEM of four replicates. Error bars, where not evident, are within the symbol of the figure.
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PEROXIDE BLOCKS MITOCHONDRIAL CHOLESTEROL TRANSPORT
2961
TABLE 1. Effect of hydrogen peroxide on progesterone synthesis in luteal cells Peroxide inhibition
Fold stimulation (treatment/control.)
Treatment LH 8-Bromo-cAMP (1 mM) (Bu)2cAMP (1 mM) 25-Hydroxycholesterol 22-Hydroxycholesterol
3.3 ± 0.4° 4.3 ± 0.8" 4.4 ± 1.0° 4.5 ± 0.6° 28.4 ± 8.3°
42.9 ± 6.1° 34.0 ± 4.7" 39.8 ± 2.8° 11.1 ± 7.1* 11.3 ± 7.2"
Values are the mean ± SEM. n, the number of replicated experiments. Cells were preincubated with 3-aminobenzamide (2.5 mM) for 10 min before incubation with H2O2 (100 /iM) for 10-30 min. The cells were then incubated with catalase (2800 U/ml) for 10 min before treatment with the various stimulators for 60 min. °P
Vol. 128, No. 6 Printed in U.S.A.
Evidence That Hydrogen Peroxide Blocks HormoneSensitive Cholesterol Transport into Mitochondria of Rat Luteal Cells* HAROLD R. BEHRMAN AND RAYMOND F. ATEN Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 tissue. In this paradigm, hydrogen peroxide did not inhibit elevated basal progesterone synthesis in luteal cells produced by in vivo aminoglutethimide treatment, yet LH-stimulated steroidogenesis was blocked. However, treatment of luteal cells with hydrogen peroxide inhibited pregnenolone synthesis in isolated mitochondria, an effect partially reversed by the addition of luteal cell cytosol. In summary, while peroxide inhibited cAMPdependent steroidogenesis, it did not appear to inhibit protein kinase activation or mobilization of cholesterol from intracellular esterified stores. Although peroxide inhibited pregnenolone synthesis, it had no effect on steroidogenesis when substrate was made available by either addition of cholesterol analogs or prior treatment with aminoglutethimide in vivo. We conclude, therefore, that hydrogen peroxide inhibits steroidogenesis by blocking intracellular transport of cholesterol to mitochondria or translocation of cholesterol across the outer mitochondrial membrane. {Endocrinology 128: 2958-2966,1991)
ABSTRACT. In luteal and granulosa cells, hydrogen peroxide abruptly inhibits activation of adenylate cyclase by receptorbound gonadotropin and blocks steroidogenesis. In the present studies a post-cAMP site of peroxide action on inhibition of steroidogenesis was investigated. Steroidogenesis, stimulated by dibutyryl or 8-bromo-cAMP, was inhibited by hydrogen peroxide. Yet, cAMP-dependent protein kinase activation in cytosol or intact cells was unaffected by peroxide treatment. Hydrogen peroxide also did not inhibit the activity of cholesterol esterase and acyl coenzyme-A:acyltransferase. Progesterone synthesis was maximally increased 5- to 50-fold with 25- and 22-hydroxycholesterol, respectively. Unlike that seen with cAMP analogs and LH, however, progestin synthesis stimulated by these celland mitochondria-permeant cholesterol analogs was not inhibited by hydrogen peroxide. Treatment of animals with aminoglutethimide produces a marked accumulation of steroidogenic cholesterol substrate and a large increase in hormone-independent steroidogenesis in subsequently isolated and washed luteal
A
LONG known characteristic of the corpus luteum is the presence of high levels of antioxidants, such as ascorbic acid and lutein, a member of the carotenoid family (1, 2). Both LH and prostaglandin F2« (PGF2«) cause a rapid depletion of luteal ascorbic acid (3, 4), and both agents evoke luteolysis in the rat (5, 6). Direct evidence for the production of reactive oxygen species in the ovary is the generation of hydrogen peroxide (7) and superoxide production (8) during luteolysis. One consequence of the generation of oxygen radicals in tissues is lipid peroxidation, an effect seen in luteal tissue several hours after treatment of rats with PGF2tt (9). We recently proposed that hydrogen peroxide may mediate lytic events involved in the cyclic remodelling of the ovary based on the generation of hydrogen peroxide during luteolysis (7) and the marked antigonadotropic actions evoked by hydrogen peroxide in rat luteal Received January 7,1991. Address requests for reprints to: Dr. Harold Behrman, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. * This work was supported by NIH Grant HD-10718.
(10) and granulosa cells (11). Gonadotropin-sensitive cAMP accumulation and progesterone synthesis are abruptly inhibited by hydrogen peroxide, and cellular ATP is depleted (10, 11), all features of a lytic response. Stimulation of progesterone synthesis in granulosa cells by 8-bromo-cAMP is also inhibited by hydrogen peroxide (11). Thus, peroxide may inhibit gonadotropin-dependent progesterone synthesis not only by inhibiting adenylate cyclase activity, but also at sites beyond cAMP. Rate-limiting steps in steroidogenesis include the mobilization and transport of cholesterol from cholesterol ester depots into mitochondria and the formation of pregnenolone by the mitochondrial cholesterol sidechain cleavage enzyme complex (12). Extracellular cholesterol is not available directly for steroidogenesis because of barriers to diffusion, but cholesterol analogs, such as 22- and 25-hydroxycholesterol, penetrate the cell and mitochondria and produce a prompt stimulation of progesterone synthesis (13, 14). A method was, thus, available to assess whether cAMP-sensitive cholesterol mobilization and transport or cholesterol side-chain cleavage activity are sites of peroxide action in blocking
2958 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
PEROXIDE BLOCKS MITOCHONDRIAL CHOLESTEROL TRANSPORT progesterone synthesis. We show that while peroxide inhibited cAMP-sensitive progesterone synthesis, it had no effect when substrate was made available by the addition of cholesterol analogs or after cholesterol loading by prior aminoglutethimide treatment in vivo (15). We also show that peroxide lacked an effect on the activities of cAMP-dependent protein kinase and the enzymes regulating cholesterol ester turnover, yet it inhibited mitochondrial pregnenolone formation. Thus, based on these findings we conclude that cAMP-dependent transport of cholesterol into mitochondria is inhibited by hydrogen peroxide.
Materials and Methods Hormones, drugs and reagents Ovine LH (NIDDK oLH 24) was obtained from the NIH (Bethesda, MD). PGF2n was purchased from Upjohn Co. (Kalamazoo, MI). Hydrogen peroxide was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). Catalase (2800 U/mg), 3-aminobenzamide, and aminoglutethimide were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and preparation of luteal cells Immature (26-27 days old) female rats (CD strain, Charles Rivers Laboratories, Wilmington, MA) were injected sc with 50 IU PMSG (Gestyl, Organon Pharmaceuticals, West Orange, NJ). Fifty-four hours later, 25 IU hCG (A.P.L., Ayerst Laboratories, Rouses Point, NY) was injected. Luteal cells were isolated 5-6 days after hCG-induced ovulation by enrichment over a Percoll density gradient, as described previously (10). In some experiments animals were treated with aminoglutethimide (15 mg/rat, ip) 2 h before isolation of luteal cells. Aminoglutethimide produces an abrupt reversible inhibition of pregnenolone synthesis and a marked increase in luteal cholesterol levels (15). Isolation and washing of luteal tissue remove the block of steroidogenesis and produce a large increase in progesterone synthesis, independently of LH, in conjunction with cholesterol accumulation (15). Mitochondrial pregnenolone production To assess the effect of hydrogen peroxide on mitochondrial pregnenolone production, luteal cells were isolated and preincubated with hydrogen peroxide (250 fiM; 60 min), followed by treatment with catalase (2800 U/ml) for 10 min. The cells were isolated from medium and homogenized, and mitochondria were isolated according to the method of Tanaka et al. (16). In brief, cells were homogenized in Tris (25 HIM), EGTA (0.5 mM), EDTA (1 mM), and sucrose (25 mM), pH 7.4. After an 800 x g (10 min) centrifugation, the supernatant fraction was centrifuged (5,000 X g; 10 min) to obtain a mitochondrial fraction (pellet). Cytosol was obtained by collection of the supernatant fraction after centrifugation (100,000 X g; 60 min) of the 5,000 X g supernatant fraction of control cells. Pregnenolone production by mitochondria was determined by the method of McNamara and Jefcoate (17). Mitochondria (50 ng protein) were incubated with Tris (25 mM), sucrose (200
2959
mM), sodium phosphate (10 mM), MgCl2 (5 mM), KC1 (20 mM), EDTA (0.2 mM), sodium succinate (10 mM), fatty acid-free BSA (1 mg/ml), and cyanoketone (10 JIM), pH 7.4. Pregnenolone production was determined in the absence and presence of cytosol. The reaction was stopped by the addition of ethanol, and pregnenolone was extracted with hexane and assayed by RIA after evaporation of the hexane. Assay of cAMP, progestins, and pregnenolone cAMP, progesterone, and 20«-dihydroprogesterone levels were determined by RIA, as described previously (18, 19). Pregnenolone was determined by RIA using a commercially available kit (Diagnostic Division, ICN Biomedical, Carson, CA). Assay of cAMP-dependent protein kinase activity cAMP-dependent protein kinase was assayed as previously described (20). In brief, luteal cells were homogenized (108 cells/ ml) in buffer (0.5 M NaCl, 1 mM EDTA, 7 mM mercaptoethanol, and 10 mM Tris-HCl, pH 7.5) that contained 60 tiu ATP. Homogenization was carried out by sonication (Branson cell disruptor, Branson Sonic Power Company, Danbury, CT) on ice with three 10-sec bursts (power setting 4 at 40 duty cycle), with rest intervals of. 10 sec between bursts. The homogenate was centrifuged (15,000 X g; 10 min), and the supernatant fraction (10 n\) was assayed for kinase activity. In experiments to test activation in cytosol, 1 fiM 8-bromo-cAMP was used; for activation in cells, 0.1 and 1 mM 8-bromo-cAMP were used. The reaction mixture contained a final concentration of 20 mM magnesium acetate, 0.5 mM l-methyl-3-isobutylxanthine, 5 mg/ml histone-II-A, 0.3 mM ATP premixed with 5 /nCi [T- 3 2 P] ATP (30 Ci/mmol), and 20 mM potassium phosphate buffer (pH 6.5). The reaction was initiated by the addition of supernatant and incubated at 30 C for 5 min in the absence and presence of cAMP. The reaction was stopped by the addition of 10% trichloroacetic acid, and the precipitated protein was isolated by centrifugation, followed by three washes with trichloroacetic acid. Radioactivity in the protein precipitate was determined by analysis of Cherenkov radiation. The details of each experimental design are given in the figure legends. Assay of cholesterol esterase and acyl coenzymeA:acyltransferase (ACAT) activity The experimental design used to examine the effect of peroxide on cholesterol esterase and ACAT activities is described in Table 3. Cells (106/ml) were isolated by centrifugation (800 X g; 5 min) and stored at -80 C. To obtain sufficient cells for assay of enzyme activities, the cells from 10 separate experiments were combined. Cells were thawed and lysed by a modification of the glycerol-loading method (21). In brief, frozen cell pellets were suspended in 200 ^1 2.4 M glycerol in 5 mM Tris-HCl (pH 7.4) and incubated for 30 min on ice. The cells were diluted with 800 (A homogenization buffer [20 mM TrisHCl (pH 7.4), 5 mM dithiothreitol, 10 mM EDTA, and 250 mM sucrose] and incubated for an additional 30 min. The resulting suspension was homogenized with a glass-Teflon homogenizer and centrifuged at 10,000 X g for 10 min, and the supernatant fraction was retained. The 10,000 x g supernatant fraction was
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Endo • 1991 Vo! 128-No 6
PEROXIDE BLOCKS MITOCHONDRIAL CHOLESTEROL TRANSPORT
2960
centrifuged at 100,000 X g for 60 min, and the microsomal pellet and cytosol fraction were retained. The microsomal fraction was washed twice in homogenization buffer before assaying for ACAT activity. The cholesterol esterase activity of the cytosol fraction was determined essentially by the method we described previously (22), with slight modification (23). Cytosol protein (250 fig) was diluted with buffer (100 mM potassium phosphate, 1 mM EDTA, and 2.5 mM 2-mercaptoethanol, pH 7.5) and radiolabeled cholesteryl oleate ([4-14C]cholesteryl oleate; 20 mCi/mmole; 60 nM final concentration), and the samples were incubated for 30 min at 37 C. The assay was stopped by addition of chloroformmethanol (2:1) containing cholesterol, cholesteryl oleate, oleic acid, and triolein carrier. The extract was evaporated to dryness and fractionated by TLC, and radioactivity was determined in the free and esterified cholesterol fractions. The total ACAT activity in the microsome fraction was determined essentially as described previously (24), with slight modification (23). Microsomes (100 ng protein) were diluted in ACAT assay buffer (100 mM potassium phosphate, 1 mM glutathione, and 1 mg/ml fatty acid-free BSA, pH 7.4) that contained 100 Mg/ml cholesterol and 600 Mg/ml Tyloxapol and incubated for 30 min at 37 C. Oleolyl CoA ([l-14C]oleolyl-CoA; 6 mCi/mmol; 100 nM final concentration) was added, and the samples were incubated for 15 min at 37 C. The reaction was stopped by extraction, the extracts were fractionated by TLC, and radioactivity associated with cholesteryl oleate was determined. Experimental protocol Cells were incubated in cholesterol-free medium (MEM 2360, Gibco, Grand Island, NY) that contained 1% BSA. For analysis of cAMP and progestin production, the cells and media were heat treated (90 C; 10 min) and stored (-80 C) before assay. For analysis of enzyme activity, the cells were separated from medium by centrifugation (and frozen). The media were heat treated and stored for later analysis of progestin content. The details of each experimental paradigm are shown in the legends of the tables and figures. Except where noted in the text, the cells were preincubated for 10-15 min with 3-aminobenzamide (2.5 mM). We showed previously that this experimental procedure blocks hydrogen peroxide-induced depletion of ATP (10, 11). Thus, the effects seen in the present studies are not confounded by depletion of cellular levels of ATP. To avoid the possibility that hydrogen peroxide may influence the biological activity of the stimulatory agents used in the present studies, the cells were preincubated with H2O2, followed by treatment with excess catalase (2800 U/ml) for 10 min. We previously showed that this treatment effectively removes hydrogen peroxide under identical experimental conditions (10, 11). Control cells received identical treatment, but no hydrogen peroxide. The levels of LH (1 Mg/ml), analogs of cAMP (1 mM), and analogs of cholesterol (10 Mg/ml) were tested deliberately at supramaximal concentrations, except where indicated in the text, to avoid potential confounding of the results due to degradative oxidation.
Statistical analysis Luteal cells from several animals were pooled and aliquoted into incubation tubes. Each treatment was studied in quadruplicate incubations, and each experiment was repeated at least twice. Statistical significance between treatments within each experiment was determined by analysis of variance with a repeated measures design, followed by Duncan's multiple range test (PC Anova, Human Systems Dynamics, Northridge, CA). P< 0.05 was considered significant. Dose-response effects were assessed by regression analysis to determine whether a significant slope was evident. Treatment differences between experiments were determined by analysis of variance, followed by Duncan's multiple range test. P < 0.05 was considered significant. Results We showed previously that hydrogen peroxide inhibits progesterone synthesis in luteal cells in response to LH, with half-maximal and maximal effects at 100 and 300 /xM peroxide, respectively (10). In the present studies, using an identical paradigm, hydrogen peroxide (100 fiM) significantly inhibited progestin synthesis in response to 8-bromo-cAMP and (Bu)2cAMP (Fig. 1 and Tables 1 and 2). Maximal stimulation by both cAMP analogs (~4fold; P < 0.05) was reduced 35-50% by peroxide (Fig. 1 and Table 1). An approximately 2-fold greater inhibition of progesterone compared to 20a-dihydroprogesterone synthesis occurred with peroxide treatment. Since the levels of both progestins were reduced by peroxide and because these are the major steroids of rat luteal cells, net steroidogenesis per se was inhibited by peroxide. Because cAMP is generally accepted to stimulate ste-^
8T
Peroxide (100
O Control
O 6-
4-
c o 0) CO CD CT> O
2-
0.1
0.3
1.0
8-Bromo-Cyclic AMP (mM) FIG. 1. Inhibition of cAMP-stimulated progesterone synthesis by hydrogen peroxide. Cells were preincubated with 3-aminobenzamide (2.5 mM; 10 min) before incubation with hydrogen peroxide (100 MM) for 1 h, followed by incubation with catalase (2800 U/ml) for 10 min. The cells were then incubated for 1 h with 8-bromo-cAMP. Values represent the mean ± SEM of four replicates. Error bars, where not evident, are within the symbol of the figure.
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PEROXIDE BLOCKS MITOCHONDRIAL CHOLESTEROL TRANSPORT
2961
TABLE 1. Effect of hydrogen peroxide on progesterone synthesis in luteal cells Peroxide inhibition
Fold stimulation (treatment/control.)
Treatment LH 8-Bromo-cAMP (1 mM) (Bu)2cAMP (1 mM) 25-Hydroxycholesterol 22-Hydroxycholesterol
3.3 ± 0.4° 4.3 ± 0.8" 4.4 ± 1.0° 4.5 ± 0.6° 28.4 ± 8.3°
42.9 ± 6.1° 34.0 ± 4.7" 39.8 ± 2.8° 11.1 ± 7.1* 11.3 ± 7.2"
Values are the mean ± SEM. n, the number of replicated experiments. Cells were preincubated with 3-aminobenzamide (2.5 mM) for 10 min before incubation with H2O2 (100 /iM) for 10-30 min. The cells were then incubated with catalase (2800 U/ml) for 10 min before treatment with the various stimulators for 60 min. °P
