0013-7227/91/1285-2253$03.00/0 Endocrinology Copyright,© 1991 by The Endocrine Society
Vol. 128, No. 5 Printed in U.S.A.
Inhibition of Gonadotropin Action and Progesterone Synthesis by Xanthine Oxidase in Rat Luteal Cells* EFTHALIA GATZULI, RAYMOND F. ATEN, AND HAROLD R. BEHRMAN Reproductive Biology Section, Departments of Obstetrics/Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510
or catalase alone abolished the actions of xanthine oxidase. While depletion of ATP by xanthine oxidase was prevented by 3-amino-benzamide, an inhibitor of DNA repair, inhibition of cAMP and progesterone production was still evident. Xanthine oxidase also inhibited progesterone synthesis stimulated by 8bromo-cAMP. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, did not reverse the inhibition of cAMP accumulation by xanthine oxidase, and the enzyme had no effect on LH receptor binding activity. Since catalase reversed the effects of xanthine oxidase, we conclude that superoxide was rapidly dismuted to hydrogen peroxide and mediated the antigonadotropic and antisteroidogenic actions of xanthine oxidase in luteal cells. The sensitivity of luteal cells to xanthine oxidase raises the possibility that this enzyme may serve as a significant source of hydrogen peroxide in the corpus luteum. (Endocrinology 128: 2253-2258, 1991)
ABSTRACT. Hydrogen peroxide produces marked antigonadotropic and lytic actions in luteal cells, but the effects of superoxide, the archetypal oxygen radical, are unknown. Xanthine oxidase generates superoxide, and the activity of this enzyme, and purine substrate, are increased under ischemia, such as that seen at luteal regression. We therefore examined the actions of xanthine oxidase on luteal cells to assess the effects of this enzyme and the superoxide anion on luteal function. Xanthine oxidase, in the presence of hypoxanthine (50 /xM), produced marked inhibition of LH-sensitive cAMP and progesterone production with complete inhibition at 25 mU/ml and half-maximal inhibition at about 5 mU/ml. These antigonadotropic actions of xanthine oxidase were rapid with maximal effects within 5 min, followed several minutes later by substantial depletion of ATP. Heat, superoxide dismutase, and catalase
M
AJOR reactive oxygen species include the superoxide anion, hydrogen peroxide, and the hydroxyl radical, and evidence for the generation of reactive oxygen species in the ovary is available. Early studies showed that antioxidant vitamins such as ascorbic acid are present in high concentrations in the ovary and become depleted at luteal regression or in response to gonadotropin (1-3). More recent studies showed that generation of superoxide occurs in the rat ovary (4, 5), peroxidation is seen in membranes from regressing rat luteal tissue (6), and hydrogen peroxide has marked antigonadotropic and antisteroidogenic actions in rat ovarian cells (7, 8). The cellular origin and nature of the reactive oxygen species in the ovary are unknown. Potential sources include phagocytic leukocytes, parenchymal steroidogenic cells, and endothelial cells (9). Several enzymes generate reactive oxygen species. These include a unique plasma membrane NADPH oxidase in phagocytes, oxidases of mitochondrial, microsomal, and peroxisomal origin, and cytosolic xanthine oxidase in endothelial cells (9). Received November 2, 1990. Address requests for reprints to: Dr. Harold Behrman, Department of Obstetrics/Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. * Supported by NIH Grant HD-10718.
The present studies focus on the actions of xanthine oxidase in rat luteal cells. Xanthine oxidase uses oxygen as an electron acceptor to generate the superoxide anion. This enzyme is derived from xanthine dehydrogenase, which uses NADH as an electron acceptor, by oxidation of sulfhydryl groups or proteolytic cleavage (10, 11). Ischemia induces the conversion of xanthine dehydrogenase to xanthine oxidase which in turn induces the production of superoxide and cell damage in some organs (12). Interestingly, blood flow is reduced in the regressing corpus luteum (13). While hydrogen peroxide is known to inhibit steroidogenesis and to block the action of LH, similar information is not available about the superoxide anion. Thus, one objective of the present studies was to determine whether the superoxide anion, produced by xanthine oxidase, influenced luteal cell function. Another objective was to determine the amount of xanthine oxidase necessary to affect luteal function to assess whether endogenous xanthine oxidase could serve as a significant source of reactive oxygen species in the ovary. These studies show that low levels of xanthine oxidase block activation of adenylate cyclase by LH and inhibit cAMP-dependent progesterone synthesis in luteal cells. The luteolytic effects of xanthine oxidase were abolished by catalase,
2253
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
2254
O Control • Heat-Treoted
Is)
3.5
3.5 j
tn
v a* o 10
25
-I 50
Xanthine Oxidase (mil/ml) FIG. 1. Effect of xanthine oxidase on LH-sensitive cAMP and progesterone accumulation in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated (10 min) with 3-aminobenzamide (2.5 mM) before incubation with the indicated concentrations of xanthine oxidase or heat-treated xanthine oxidase (90 C; 10 min) and 50 nM hypoxanthine for 10 min. Catalase (2800 U/ml) and superoxide dismutase (100 U/ ml) were then added to the reaction mixture for 10 min (37 C) to remove superoxide and hydrogen peroxide. LH (1 jig/ml) w a s then added and the reaction mixture incubated for 1 h.
which indicates a hydrogen peroxide-dependent mechanism in isolated luteal cells.
0
10
Xanthine Oxidase (mU/ml) FIG. 2. Reversal of the inhibitory effects of xanthine oxidase by superoxide dismutase and catalase in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated with 3-aminobenzamide (2.5 mM; 10 min). Superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added where indicated 5 min before incubation with xanthine oxidase and 50 nM hypoxanthine (10 min). LH (1 //g/ml) was then added and the reaction mixture incubated for 1 h.
NJ). Fifty-four hours later, 25 IU human CG (hCG) (A.P.L., Ayerst Laboratories, Rouses Point, NY) were injected. Luteal cells were isolated 5-6 days after hCG treatment of the animals by enrichment over a Percoll density gradient as described previously (7). Assays
Materials and Methods Hormones, drugs, and reagents Ovine LH (NIADDK oLH 24) was obtained from the NIH (Bethesda, MD). Catalase (2800 U/mg), 3-aminobenzamide, xanthine oxidase (2 U/mg), and superoxide dismutase (3,000 U/mg) 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 River Laboratories, Wilmington, MA) were injected sc with 50 IU PMSG (Gestyl; Organon Pharmaceuticals, West Orange,
Progesterone and cAMP levels were determined by RIA (7). Receptor binding activity of the LH receptor in membranes and intact cells was determined as described previously (7). Experimental protocol Cells were incubated in media (minimal essential medium 2360; GIBCO, Long Island, NY) which contained 0.1% BSA. For analysis of cAMP and progesterone production, the cells and media were heat-treated (90 C; 10 min) and stored (-80 C) before assay. Details of each experimental paradigm are indicated in the legends of the tables and figures. Except where indicated in the legends, the cells were preincubated for 10-15 min with 3-
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
n
(% of LH control) A. cAMP Control SOD/catalase Catalase Heat B. Progesterone Control SOD/Catalase Catalase Heat
5 4 3 3 5 4 3 3
25.5 ± 77.8 ± 80.7 ± 111.2 ±
4.5 5.9° 16.9° 5.7°
eel m o
aminobenzamide (2.5 mM). We showed previously that this experimental procedure blocks hydrogen peroxide-induced depletion of ATP (7, 8). Cells were then preincubated with xanthine oxidase followed by treatment with excess superoxide dismutase (100 U/ml) and catalase (2800 U/ml) for 10 min to circumvent the possibility that reactive oxygen species may directly degrade the test agents used in the present studies. Control cells were treated identically, but without xanthine oxidase. The levels of LH (1 ^g/ml) and 8-bromo-cAMP (1 mM) were tested deliberately at supramaximal concentrations, except where indicated in the text, to circumvent potential confounding of the results due to degradative oxidation. Statistical analysis Luteal cells from several animals were pooled and aliquoted into incubation tubes in each experiment. Each treatment was studied in quadruplicate incubations, and each experiment was repeated at least three times. 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). A P value less than 0.05 was considered significant. Dose-response effects were assessed by regression analysis to determine whether a significant slope was evident.
Results Xanthine oxidase produced a marked inhibition of LHsensitive cAMP accumulation and progesterone synthesis. The concentration of xanthine oxidase that produced half-maximal inhibition of these responses of LH was about 5 mU/ml (Figs. 1 and 2 and Table 1). Levels of xanthine oxidase of 25 mU/ml blocked completely both cAMP and progesterone synthesis (Figs. 1 and 2). In all
2.0 1.5
o_
1.0
.0
0.5
62.0 ± 6.6 88.4 ± 8.3" 95.9 ± 3.0° 98.7 ± 11.1°
Values are mean ± SEM. SOD, Superoxide dismutase. n = number of replicated experiments. SOD/catalase (100 and 2800 U/ml, respectively) or catalase (2800 U/ml) alone were added to the cells 10 min before addition of xanthine oxidase (25 mU/ml) in an experimental paradigm identical to that described in Fig. 1. Heat treatment of xanthine oxidase was identical to that described in Fig. 1. 0 P < 0.05 from control.
2.5
(pmol.
Treatment
Xanthine oxidase (25 mU/ml)
3.0
Cycl
TABLE 1. Summary of experiments on reversal of the inhibitory effects of xanthine oxidase in luteal cells
2255
O XO (25 mU/ml) • XO + Catalase
0.0
30
O XO (25 mU/ml) • XO + Catalase in
o
o 30
Time (min) FlG. 3. Time course of the antigonadotropic effects of xanthine oxidase and reversal of the inhibitory effects by catalase in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated for 10 min with 3aminobenzamide (2.5 mM; 10 min). Catalase (2800 U/ml) was added where indicated for 10 min before incubation with xanthine oxidase (25 mU/ml) for the indicated time intervals in the presence of 50 fiM hypoxanthine. Catalase (2800 U/ml) was added again after xanthine oxidase treatment before incubation with LH (1 Mg/ml; 1 h).
experiments hypoxanthine was used at a concentration of 50 nM, which is similar to levels in tissues (14). The levels of progesterone and cAMP in the absence of LH were not significantly changed by xanthine oxidase (data not shown). The effects of xanthine oxidase were dependent on enzyme activity because denaturation with heat completely blocked the effects of xanthine oxidase (Fig. 1 and Table 1). We concluded that reactive oxygen species mediated the actions of xanthine oxidase because simultaneous treatment of the cells with xanthine oxidase, superoxide dismutase, and catalase completely blocked the effects of xanthine oxidase (Fig. 2 and Table 1). While the superoxide anion is the major product of xanthine oxidase, hydrogen peroxide can also be produced either by spontaneous dismutation of superoxide or via endogenous superoxide dismutase. Thus, additional studies were
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
2256 70
in
O Control
O XO (25 mU/ml) • 3-ABA (2.5 mM)
60
Endo«1991 Voll28«No5
•
XO (25 mU/ml)
m o
50
o
o
40
o
E Q.
30 o
20
o
10 30
10
Time (min) FIG. 4. Time course of effect of xanthine oxidase on the levels of ATP in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated in the absence and presence of 3-aminobenzamide (3-ABA; 2.5 mM) for 10 min before addition of xanthine oxidase (25 mU/ml) and hypoxanthine (50 HM) for the indicated intervals. Excess superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added to remove superoxide and hydrogen peroxide 10 min before incubation with LH (1 Mg/ml) for 1 h. TABLE 2. Effect of xanthine oxidase on LH receptor binding activity in luteal cells A. Effect of incubation time with xanthine oxidase Incubation time (min)
Specific binding
0 10 60
18.1 ± 0.7 17.8 ± 0.2 19.2 ± 0.5
B. Effect of xanthine oxidase concentration Xanthine oxidase (mU/ml)
Specific binding {%)
0 10 25 50
17.1 ± 0.8 18.0 ± 0.1 18.6 ± 0.3 18.7 ± 0.2
Values are mean ± SEM of 4 replicates within each experiment. In (A) 106 cells were incubated with xanthine oxidase (25 mU/ml) and hypoxanthine (50 nm) for the indicated times after a preincubation (10 min) with 3-aminobenzamide (2.5 mM). Before incubation (2 h, 37 C) with [125I]hCG (100,000 cpm; 10"10 M), the cells were incubated (10 min) with catalase (2800 U/ml) and superoxide dismutase (100 U/ml). In (B), the cells were treated similarly but with variable levels of xanthine oxidase for 10 min. Nonspecific binding was 0.6%.
carried out to determine whether hydrogen peroxide contributed to the antigonadotropic actions of xanthine oxidase. Catalase completely blocked the inhibitory actions of xanthine oxidase in luteal cells, which indicates that the major, if not the only, mediator of the antigonadotropic actions of xanthine oxidase was hydrogen peroxide (Fig. 3 and Table 1). The antigonadotropic responses to xanthine oxidase
1.0
0.3
IBMX (mM) FIG. 5. Effect of xanthine oxidase on LH-sensitive cAMP accumulation in the absence and presence of IBMX. Cells were preincubated for 10 min with 3-aminobenzamide (2.5 mM) before the incubation with xanthine oxidase (25 mU/ml) and hypoxanthine (50 MM) for 10 min. Superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added to remove excess superoxide and hydrogen peroxide 10 min before incubation with LH (1 /xg/ml) and the indicated concentrations of IBMX.
0)
5
•
O Control • XO (25 mU/ml)
o
m o
43-
c o
2-
0) •*->
(0
!-•
o 0.1
0.3
8-Bromo-cAMP (mM) FIG. 6. Effect of xanthine oxidase on progesterone production stimulated by 8-bromo-cAMP. Cells were preincubated for 10 min with 3aminobenzamide (2.5 mM) before incubation with xanthine oxidase (25 mU/ml) and hypoxanthine (50 ^M) for 10 min. Superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added to remove excess superoxide and hydrogen peroxide 10 min before incubation with the indicated concentrations of 8-bromo-cAMP for 1 h.
were extremely rapid with almost complete inhibition of cAMP accumulation and progesterone synthesis within 5 min (Fig. 3). Depletion of cellular ATP levels was also produced by xanthine oxidase, but this response was slower than abrogation of the responses to LH; maximal depletion (62 %; P < 0.05) occurred at 10 min (Fig. 4). Cell depletion of ATP by xanthine oxidase was blocked by 3-aminobenzamide, an inhibitor of DNA repair (15). However, the antigonadotropic actions of xanthine oxidase in these studies were not due to ATP depletion because the cells were pretreated with 3-aminobenzamide
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
with a paradigm identical to that shown in Fig. 3. Xanthine oxidase had no effect on LH receptor binding activity in luteal cells. Table 2 shows that exposure of the cells for up to 60 min with 25 mU/ml xanthine oxidase, a level that maximally inhibited the action of LH, had no effect on the specific binding of radiolabeled hCG. Similarly, supramaximal levels of xanthine oxidase were also without effect on LH receptor binding activity in luteal cells. Xanthine oxidase treatment of luteal membranes also had no effect on LH receptor binding activity (data not shown). The biological activity of LH was also not impaired because the cells were incubated with an excess of superoxide dismutase and catalase before incubation with a supramaximal level of LH. Thus, the antigonadotropic actions of xanthine oxidase were probably not due to impaired binding of the LH receptor. Isobutylmethylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, amplified the cAMP response to LH in luteal cells by about 2-fold (P < 0.05, three experiments) (Fig. 5). However, IBMX did not reverse the antigonadotropic actions of xanthine oxidase. Thus, inhibition of LH-sensitive cAMP accumulation by xanthine oxidase does not appear to be due to an increase in cAMP degradation. Taken together the results indicate that xanthine oxidase prevents activation of adenylate cyclase by LH. Progesterone synthesis was dose dependently stimulated by 8-bromo-cAMP by about 5-fold (P < 0.05, three experiments) (Fig. 6). Xanthine oxidase (25 mU/ml) produced almost complete inhibition of cAMP-dependent progesterone synthesis at all doses of 8-bromocAMP. Thus, progesterone synthesis is inhibited by xanthine oxidase by at least two mechanisms, first by inhibition of cAMP production by LH and second by inhibition cAMP-dependent steroidogenesis.
Discussion The present studies show that reactive oxygen species generated by xanthine oxidase markedly influence endocrine and functional activities of rat luteal cells. Although the major reactive oxygen species produced by xanthine oxidase is the superoxide anion (16), hydrogen peroxide was the biologically relevant product found to influence luteal cell function. The effect xanthine oxidase evoked in luteal cells was rapid inhibition of gonadotropin-sensitive cAMP and progesterone synthesis, and depletion of ATP. While depletion of ATP was prevented by 3-aminobenzamide, the antigonadotropic actions of xanthine oxidase were not. Thus, interruption of cAMP and progesterone production was not due to ATP depletion, but to a selective action of xanthine oxidase on these parameters of luteal cell function.
2257
These findings raise the possibility that xanthine oxidase could play a role in the loss of luteal function at luteolysis because abrogation of gonadotropin action is an early feature of functional luteolysis (17). In fact, the response of luteal cells to xanthine oxidase is like that seen with the natural luteolysin prostaglandin F2a from the standpoint of gonadotropin-dependent responses (17). Another feature of luteolysis is the recent finding that ATP levels, and adenine purines in general, are depleted in regressing luteal tissue (18). Depletion of ATP and adenine-derived purines leads to an increase in levels of extracellular purines that are rapidly converted by ectoenzymes to hypoxanthine and xanthine, the substrates of xanthine oxidase (13,14,16). Thus, if xanthine oxidase is present, the conditions for generation of reactive oxygen species in the regressing corpus luteum would be extant and could be of biological significance. Incubation of luteal cells with hypoxanthine or xanthine, substrates of xanthine oxidase, does not impair cellular responses to gonadotropin (19). Indeed, hypoxanthine at levels used in the present studies amplifies the response to LH in luteal cells (19). Thus, it appears that xanthine oxidase of luteal cell origin is not a significant source of the enzyme from the standpoint of production of reactive oxygen species and impairment of cell function. Evidence points to endothelial cells in other tissues as a major source of xanthine oxidase, particularly after ischemia (20-22). The marked vascularization of the corpus luteum and the fact that blood flow is reduced in the regressing corpus luteum make xanthine oxidase a potentially important candidate for generation of reactive oxygen species in this gland. Interestingly, hydrogen peroxide was the major mediator of the antigonadotropic and ATP-depleting actions of xanthine oxidase in luteal cells because catalase reversed the actions of xanthine oxidase. In addition, the effects seen with xanthine oxidase in the present studies are identical to those seen with direct addition of hydrogen peroxide to luteal cells (7). Since the major oxygen species produced by xanthine oxidase is the superoxide anion (16), it is evident that luteal cells effectively dismute superoxide into hydrogen peroxide. While spontaneous dismutation of superoxide occurs (9), rat luteal tissues contain superoxide dismutase (4), but of unknown nature. Three different forms of superoxide dismutase have been identified in other tissues, and each is a separate gene product. They include a mitochondrial Mn-metalloenzyme, a cytosolic Cu-Zn enzyme, and an extracellular Cu-Zn enzyme (23-25). It is probable that superoxide, generated by xanthine oxidase, was dismuted to hydrogen peroxide extracellularly since the superoxide anion poorly penetrates cells, in contrast to hydrogen peroxide (9). Notwithstanding the nature of the dismutation process, it is evident from the present studies that
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2258
XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
hydrogen peroxide mediated the effects of extracellular superoxide, derived from xanthine oxidase.
References 1. Hoch-Ligeti C, Bourne GH 1948 Changes in the concentration and histological distribution of ascorbic acid in ovaries, adrenals and livers of rats during oestrus cycles. Br J Pathol 29:400-407 2. Deane HW 1952 Histochemical observations on the ovary and oviduct of the albino rat during the estrous cycle. Am J Anat 91:363-393 3. Parlow AF 1972 Influence of differences in the persistence of luteinizing hormone in blood on their potency in the ovarian ascorbic acid depletion bioassay. Endocrinology 91:1109-1112 4. Laloraya M, Kumar GP, Laloraya MM 1988 Changes in the levels of superoxide anion radical and superoxide dismutase during the estrous cycle of rattus norvegicus and induction of superoxide dismutase in rat ovary by lutropin. Biochem Biophys Res Commun 157:146-153 5. Sawada M, Carlson JC 1989 Superoxide radical production in plasma membrane samples from regressing corpora lutea. Can J Physiol Pharmacol 67:465-471 6. Sawada M, Carlson JC 1985 Association of lipid peroxidation during luteal regression in the rat and natural aging in the rotifer. Exp Gerontol 20:179-186 7. Behrman HR, Preston SL 1989 Luteolytic actions of peroxide in rat ovarian cells. Endocrinology 124:2895-2900 8. Margolin YM, Aten RF, Behrman HR 1990 Antigonadotropic and antisteroidogenic actions of hydrogen peroxide in rat granulosa cells. Endocrinology 127:245-250 9. Halliwell B, Gutteridge JMC 1989 Free Radicals in Biology and Medicine, ed 2. Clarendon Press, Oxford, U.K. 10. Corte ED, Stirpe F 1972 The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme. Biochem J 126:739-745 11. Giulia M, Lorenzoni BE, Stirpe F 1973 Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties. Biochem J 131:191-198
Endo• 1991 Vo)128«No5
12. McCord JM 1985 Oxygen-derived free radicals in postischemic tissue injury. Mech Dis 312:159-163 13. Pang CY, Behrman HR 1979 Relationship of luteal blood flow and corpus luteum function in pseudopregnant rats. Am J Physiol 237:E30-E34 14. Arch JRS, Newsholme EA 1980 The control of the metabolism and the hormonal role of adenosine. In: Campbell PN, Dickens S (eds) Biochemical Society. Academic Press, New York, pp 82-123 15. Schraufstatter IU, Hyslop PA, Hinshaw DB, Spragg RG, Sklar LA, Cochrane CG 1986 Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase. Proc Natl Acad Sci USA 83:4908-4912 16. McCord JM, Fridovich I 1968 The reduction of cytochrome C by milk xanthine oxidase. J Biol Chem 243:5753-5760 17. Behrman HR 1979 Prostaglandins in hypothalamo-pituitary and ovarian function. Annu Rev Physiol 41:685-700 18. Soodak LK, MacDonald GJ, Behrman HR 1988 Luteolysis is linked to LH-induced depletion of ATP in vivo. Endocrinology 122:187193 19. Brennan T, Ohkawa R, Gore SD, Behrman HR 1983 Adeninederived purines increase ATP levels in the luteal cell: evidence that cell levels of ATP may limit the stimulation of cyclic AMP by LH. Endocrinology 112:499-508 20. Friedl HP, Till GO, Ryan US, Ward PA 1989 Mediator-induced activation of xanthine oxidase in endothelial cells. FASEB J 3:2512-2518 21. Ratych RE, Chuknyiska RS, Bulkley GB 1987 The primary localization of free radical generation after anoxia/reoxygenation in in isolated endothelial cells. Surgery 102:122-131 22. Jarasch E-D, Grund C, Bruder G, Heid HW, Keenan TW, Franke WW 1981 Localization of xanthine oxidase in mammary gland epithelium and capillary endothelium. Cell 25:67-82 23. Weisinger RA, Fridovich I 1973 Mitochondrial superoxide dismutase: site of synthesis and intramitochondrial localization. J Biol Chem 248:4793-4796 24. Marklund SL 1982 Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci USA 79:76347638 25. Karlsson K, Marklund SL 1988 Extracellular superoxide dismutase in the vascular system of mammals. Biochem J 255:223-228
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Vol. 128, No. 5 Printed in U.S.A.
Inhibition of Gonadotropin Action and Progesterone Synthesis by Xanthine Oxidase in Rat Luteal Cells* EFTHALIA GATZULI, RAYMOND F. ATEN, AND HAROLD R. BEHRMAN Reproductive Biology Section, Departments of Obstetrics/Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510
or catalase alone abolished the actions of xanthine oxidase. While depletion of ATP by xanthine oxidase was prevented by 3-amino-benzamide, an inhibitor of DNA repair, inhibition of cAMP and progesterone production was still evident. Xanthine oxidase also inhibited progesterone synthesis stimulated by 8bromo-cAMP. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, did not reverse the inhibition of cAMP accumulation by xanthine oxidase, and the enzyme had no effect on LH receptor binding activity. Since catalase reversed the effects of xanthine oxidase, we conclude that superoxide was rapidly dismuted to hydrogen peroxide and mediated the antigonadotropic and antisteroidogenic actions of xanthine oxidase in luteal cells. The sensitivity of luteal cells to xanthine oxidase raises the possibility that this enzyme may serve as a significant source of hydrogen peroxide in the corpus luteum. (Endocrinology 128: 2253-2258, 1991)
ABSTRACT. Hydrogen peroxide produces marked antigonadotropic and lytic actions in luteal cells, but the effects of superoxide, the archetypal oxygen radical, are unknown. Xanthine oxidase generates superoxide, and the activity of this enzyme, and purine substrate, are increased under ischemia, such as that seen at luteal regression. We therefore examined the actions of xanthine oxidase on luteal cells to assess the effects of this enzyme and the superoxide anion on luteal function. Xanthine oxidase, in the presence of hypoxanthine (50 /xM), produced marked inhibition of LH-sensitive cAMP and progesterone production with complete inhibition at 25 mU/ml and half-maximal inhibition at about 5 mU/ml. These antigonadotropic actions of xanthine oxidase were rapid with maximal effects within 5 min, followed several minutes later by substantial depletion of ATP. Heat, superoxide dismutase, and catalase
M
AJOR reactive oxygen species include the superoxide anion, hydrogen peroxide, and the hydroxyl radical, and evidence for the generation of reactive oxygen species in the ovary is available. Early studies showed that antioxidant vitamins such as ascorbic acid are present in high concentrations in the ovary and become depleted at luteal regression or in response to gonadotropin (1-3). More recent studies showed that generation of superoxide occurs in the rat ovary (4, 5), peroxidation is seen in membranes from regressing rat luteal tissue (6), and hydrogen peroxide has marked antigonadotropic and antisteroidogenic actions in rat ovarian cells (7, 8). The cellular origin and nature of the reactive oxygen species in the ovary are unknown. Potential sources include phagocytic leukocytes, parenchymal steroidogenic cells, and endothelial cells (9). Several enzymes generate reactive oxygen species. These include a unique plasma membrane NADPH oxidase in phagocytes, oxidases of mitochondrial, microsomal, and peroxisomal origin, and cytosolic xanthine oxidase in endothelial cells (9). Received November 2, 1990. Address requests for reprints to: Dr. Harold Behrman, Department of Obstetrics/Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. * Supported by NIH Grant HD-10718.
The present studies focus on the actions of xanthine oxidase in rat luteal cells. Xanthine oxidase uses oxygen as an electron acceptor to generate the superoxide anion. This enzyme is derived from xanthine dehydrogenase, which uses NADH as an electron acceptor, by oxidation of sulfhydryl groups or proteolytic cleavage (10, 11). Ischemia induces the conversion of xanthine dehydrogenase to xanthine oxidase which in turn induces the production of superoxide and cell damage in some organs (12). Interestingly, blood flow is reduced in the regressing corpus luteum (13). While hydrogen peroxide is known to inhibit steroidogenesis and to block the action of LH, similar information is not available about the superoxide anion. Thus, one objective of the present studies was to determine whether the superoxide anion, produced by xanthine oxidase, influenced luteal cell function. Another objective was to determine the amount of xanthine oxidase necessary to affect luteal function to assess whether endogenous xanthine oxidase could serve as a significant source of reactive oxygen species in the ovary. These studies show that low levels of xanthine oxidase block activation of adenylate cyclase by LH and inhibit cAMP-dependent progesterone synthesis in luteal cells. The luteolytic effects of xanthine oxidase were abolished by catalase,
2253
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
2254
O Control • Heat-Treoted
Is)
3.5
3.5 j
tn
v a* o 10
25
-I 50
Xanthine Oxidase (mil/ml) FIG. 1. Effect of xanthine oxidase on LH-sensitive cAMP and progesterone accumulation in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated (10 min) with 3-aminobenzamide (2.5 mM) before incubation with the indicated concentrations of xanthine oxidase or heat-treated xanthine oxidase (90 C; 10 min) and 50 nM hypoxanthine for 10 min. Catalase (2800 U/ml) and superoxide dismutase (100 U/ ml) were then added to the reaction mixture for 10 min (37 C) to remove superoxide and hydrogen peroxide. LH (1 jig/ml) w a s then added and the reaction mixture incubated for 1 h.
which indicates a hydrogen peroxide-dependent mechanism in isolated luteal cells.
0
10
Xanthine Oxidase (mU/ml) FIG. 2. Reversal of the inhibitory effects of xanthine oxidase by superoxide dismutase and catalase in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated with 3-aminobenzamide (2.5 mM; 10 min). Superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added where indicated 5 min before incubation with xanthine oxidase and 50 nM hypoxanthine (10 min). LH (1 //g/ml) was then added and the reaction mixture incubated for 1 h.
NJ). Fifty-four hours later, 25 IU human CG (hCG) (A.P.L., Ayerst Laboratories, Rouses Point, NY) were injected. Luteal cells were isolated 5-6 days after hCG treatment of the animals by enrichment over a Percoll density gradient as described previously (7). Assays
Materials and Methods Hormones, drugs, and reagents Ovine LH (NIADDK oLH 24) was obtained from the NIH (Bethesda, MD). Catalase (2800 U/mg), 3-aminobenzamide, xanthine oxidase (2 U/mg), and superoxide dismutase (3,000 U/mg) 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 River Laboratories, Wilmington, MA) were injected sc with 50 IU PMSG (Gestyl; Organon Pharmaceuticals, West Orange,
Progesterone and cAMP levels were determined by RIA (7). Receptor binding activity of the LH receptor in membranes and intact cells was determined as described previously (7). Experimental protocol Cells were incubated in media (minimal essential medium 2360; GIBCO, Long Island, NY) which contained 0.1% BSA. For analysis of cAMP and progesterone production, the cells and media were heat-treated (90 C; 10 min) and stored (-80 C) before assay. Details of each experimental paradigm are indicated in the legends of the tables and figures. Except where indicated in the legends, the cells were preincubated for 10-15 min with 3-
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
n
(% of LH control) A. cAMP Control SOD/catalase Catalase Heat B. Progesterone Control SOD/Catalase Catalase Heat
5 4 3 3 5 4 3 3
25.5 ± 77.8 ± 80.7 ± 111.2 ±
4.5 5.9° 16.9° 5.7°
eel m o
aminobenzamide (2.5 mM). We showed previously that this experimental procedure blocks hydrogen peroxide-induced depletion of ATP (7, 8). Cells were then preincubated with xanthine oxidase followed by treatment with excess superoxide dismutase (100 U/ml) and catalase (2800 U/ml) for 10 min to circumvent the possibility that reactive oxygen species may directly degrade the test agents used in the present studies. Control cells were treated identically, but without xanthine oxidase. The levels of LH (1 ^g/ml) and 8-bromo-cAMP (1 mM) were tested deliberately at supramaximal concentrations, except where indicated in the text, to circumvent potential confounding of the results due to degradative oxidation. Statistical analysis Luteal cells from several animals were pooled and aliquoted into incubation tubes in each experiment. Each treatment was studied in quadruplicate incubations, and each experiment was repeated at least three times. 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). A P value less than 0.05 was considered significant. Dose-response effects were assessed by regression analysis to determine whether a significant slope was evident.
Results Xanthine oxidase produced a marked inhibition of LHsensitive cAMP accumulation and progesterone synthesis. The concentration of xanthine oxidase that produced half-maximal inhibition of these responses of LH was about 5 mU/ml (Figs. 1 and 2 and Table 1). Levels of xanthine oxidase of 25 mU/ml blocked completely both cAMP and progesterone synthesis (Figs. 1 and 2). In all
2.0 1.5
o_
1.0
.0
0.5
62.0 ± 6.6 88.4 ± 8.3" 95.9 ± 3.0° 98.7 ± 11.1°
Values are mean ± SEM. SOD, Superoxide dismutase. n = number of replicated experiments. SOD/catalase (100 and 2800 U/ml, respectively) or catalase (2800 U/ml) alone were added to the cells 10 min before addition of xanthine oxidase (25 mU/ml) in an experimental paradigm identical to that described in Fig. 1. Heat treatment of xanthine oxidase was identical to that described in Fig. 1. 0 P < 0.05 from control.
2.5
(pmol.
Treatment
Xanthine oxidase (25 mU/ml)
3.0
Cycl
TABLE 1. Summary of experiments on reversal of the inhibitory effects of xanthine oxidase in luteal cells
2255
O XO (25 mU/ml) • XO + Catalase
0.0
30
O XO (25 mU/ml) • XO + Catalase in
o
o 30
Time (min) FlG. 3. Time course of the antigonadotropic effects of xanthine oxidase and reversal of the inhibitory effects by catalase in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated for 10 min with 3aminobenzamide (2.5 mM; 10 min). Catalase (2800 U/ml) was added where indicated for 10 min before incubation with xanthine oxidase (25 mU/ml) for the indicated time intervals in the presence of 50 fiM hypoxanthine. Catalase (2800 U/ml) was added again after xanthine oxidase treatment before incubation with LH (1 Mg/ml; 1 h).
experiments hypoxanthine was used at a concentration of 50 nM, which is similar to levels in tissues (14). The levels of progesterone and cAMP in the absence of LH were not significantly changed by xanthine oxidase (data not shown). The effects of xanthine oxidase were dependent on enzyme activity because denaturation with heat completely blocked the effects of xanthine oxidase (Fig. 1 and Table 1). We concluded that reactive oxygen species mediated the actions of xanthine oxidase because simultaneous treatment of the cells with xanthine oxidase, superoxide dismutase, and catalase completely blocked the effects of xanthine oxidase (Fig. 2 and Table 1). While the superoxide anion is the major product of xanthine oxidase, hydrogen peroxide can also be produced either by spontaneous dismutation of superoxide or via endogenous superoxide dismutase. Thus, additional studies were
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
2256 70
in
O Control
O XO (25 mU/ml) • 3-ABA (2.5 mM)
60
Endo«1991 Voll28«No5
•
XO (25 mU/ml)
m o
50
o
o
40
o
E Q.
30 o
20
o
10 30
10
Time (min) FIG. 4. Time course of effect of xanthine oxidase on the levels of ATP in isolated luteal cells. Mean ± SEM (n = 4). Cells were preincubated in the absence and presence of 3-aminobenzamide (3-ABA; 2.5 mM) for 10 min before addition of xanthine oxidase (25 mU/ml) and hypoxanthine (50 HM) for the indicated intervals. Excess superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added to remove superoxide and hydrogen peroxide 10 min before incubation with LH (1 Mg/ml) for 1 h. TABLE 2. Effect of xanthine oxidase on LH receptor binding activity in luteal cells A. Effect of incubation time with xanthine oxidase Incubation time (min)
Specific binding
0 10 60
18.1 ± 0.7 17.8 ± 0.2 19.2 ± 0.5
B. Effect of xanthine oxidase concentration Xanthine oxidase (mU/ml)
Specific binding {%)
0 10 25 50
17.1 ± 0.8 18.0 ± 0.1 18.6 ± 0.3 18.7 ± 0.2
Values are mean ± SEM of 4 replicates within each experiment. In (A) 106 cells were incubated with xanthine oxidase (25 mU/ml) and hypoxanthine (50 nm) for the indicated times after a preincubation (10 min) with 3-aminobenzamide (2.5 mM). Before incubation (2 h, 37 C) with [125I]hCG (100,000 cpm; 10"10 M), the cells were incubated (10 min) with catalase (2800 U/ml) and superoxide dismutase (100 U/ml). In (B), the cells were treated similarly but with variable levels of xanthine oxidase for 10 min. Nonspecific binding was 0.6%.
carried out to determine whether hydrogen peroxide contributed to the antigonadotropic actions of xanthine oxidase. Catalase completely blocked the inhibitory actions of xanthine oxidase in luteal cells, which indicates that the major, if not the only, mediator of the antigonadotropic actions of xanthine oxidase was hydrogen peroxide (Fig. 3 and Table 1). The antigonadotropic responses to xanthine oxidase
1.0
0.3
IBMX (mM) FIG. 5. Effect of xanthine oxidase on LH-sensitive cAMP accumulation in the absence and presence of IBMX. Cells were preincubated for 10 min with 3-aminobenzamide (2.5 mM) before the incubation with xanthine oxidase (25 mU/ml) and hypoxanthine (50 MM) for 10 min. Superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added to remove excess superoxide and hydrogen peroxide 10 min before incubation with LH (1 /xg/ml) and the indicated concentrations of IBMX.
0)
5
•
O Control • XO (25 mU/ml)
o
m o
43-
c o
2-
0) •*->
(0
!-•
o 0.1
0.3
8-Bromo-cAMP (mM) FIG. 6. Effect of xanthine oxidase on progesterone production stimulated by 8-bromo-cAMP. Cells were preincubated for 10 min with 3aminobenzamide (2.5 mM) before incubation with xanthine oxidase (25 mU/ml) and hypoxanthine (50 ^M) for 10 min. Superoxide dismutase (100 U/ml) and catalase (2800 U/ml) were then added to remove excess superoxide and hydrogen peroxide 10 min before incubation with the indicated concentrations of 8-bromo-cAMP for 1 h.
were extremely rapid with almost complete inhibition of cAMP accumulation and progesterone synthesis within 5 min (Fig. 3). Depletion of cellular ATP levels was also produced by xanthine oxidase, but this response was slower than abrogation of the responses to LH; maximal depletion (62 %; P < 0.05) occurred at 10 min (Fig. 4). Cell depletion of ATP by xanthine oxidase was blocked by 3-aminobenzamide, an inhibitor of DNA repair (15). However, the antigonadotropic actions of xanthine oxidase in these studies were not due to ATP depletion because the cells were pretreated with 3-aminobenzamide
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XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
with a paradigm identical to that shown in Fig. 3. Xanthine oxidase had no effect on LH receptor binding activity in luteal cells. Table 2 shows that exposure of the cells for up to 60 min with 25 mU/ml xanthine oxidase, a level that maximally inhibited the action of LH, had no effect on the specific binding of radiolabeled hCG. Similarly, supramaximal levels of xanthine oxidase were also without effect on LH receptor binding activity in luteal cells. Xanthine oxidase treatment of luteal membranes also had no effect on LH receptor binding activity (data not shown). The biological activity of LH was also not impaired because the cells were incubated with an excess of superoxide dismutase and catalase before incubation with a supramaximal level of LH. Thus, the antigonadotropic actions of xanthine oxidase were probably not due to impaired binding of the LH receptor. Isobutylmethylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, amplified the cAMP response to LH in luteal cells by about 2-fold (P < 0.05, three experiments) (Fig. 5). However, IBMX did not reverse the antigonadotropic actions of xanthine oxidase. Thus, inhibition of LH-sensitive cAMP accumulation by xanthine oxidase does not appear to be due to an increase in cAMP degradation. Taken together the results indicate that xanthine oxidase prevents activation of adenylate cyclase by LH. Progesterone synthesis was dose dependently stimulated by 8-bromo-cAMP by about 5-fold (P < 0.05, three experiments) (Fig. 6). Xanthine oxidase (25 mU/ml) produced almost complete inhibition of cAMP-dependent progesterone synthesis at all doses of 8-bromocAMP. Thus, progesterone synthesis is inhibited by xanthine oxidase by at least two mechanisms, first by inhibition of cAMP production by LH and second by inhibition cAMP-dependent steroidogenesis.
Discussion The present studies show that reactive oxygen species generated by xanthine oxidase markedly influence endocrine and functional activities of rat luteal cells. Although the major reactive oxygen species produced by xanthine oxidase is the superoxide anion (16), hydrogen peroxide was the biologically relevant product found to influence luteal cell function. The effect xanthine oxidase evoked in luteal cells was rapid inhibition of gonadotropin-sensitive cAMP and progesterone synthesis, and depletion of ATP. While depletion of ATP was prevented by 3-aminobenzamide, the antigonadotropic actions of xanthine oxidase were not. Thus, interruption of cAMP and progesterone production was not due to ATP depletion, but to a selective action of xanthine oxidase on these parameters of luteal cell function.
2257
These findings raise the possibility that xanthine oxidase could play a role in the loss of luteal function at luteolysis because abrogation of gonadotropin action is an early feature of functional luteolysis (17). In fact, the response of luteal cells to xanthine oxidase is like that seen with the natural luteolysin prostaglandin F2a from the standpoint of gonadotropin-dependent responses (17). Another feature of luteolysis is the recent finding that ATP levels, and adenine purines in general, are depleted in regressing luteal tissue (18). Depletion of ATP and adenine-derived purines leads to an increase in levels of extracellular purines that are rapidly converted by ectoenzymes to hypoxanthine and xanthine, the substrates of xanthine oxidase (13,14,16). Thus, if xanthine oxidase is present, the conditions for generation of reactive oxygen species in the regressing corpus luteum would be extant and could be of biological significance. Incubation of luteal cells with hypoxanthine or xanthine, substrates of xanthine oxidase, does not impair cellular responses to gonadotropin (19). Indeed, hypoxanthine at levels used in the present studies amplifies the response to LH in luteal cells (19). Thus, it appears that xanthine oxidase of luteal cell origin is not a significant source of the enzyme from the standpoint of production of reactive oxygen species and impairment of cell function. Evidence points to endothelial cells in other tissues as a major source of xanthine oxidase, particularly after ischemia (20-22). The marked vascularization of the corpus luteum and the fact that blood flow is reduced in the regressing corpus luteum make xanthine oxidase a potentially important candidate for generation of reactive oxygen species in this gland. Interestingly, hydrogen peroxide was the major mediator of the antigonadotropic and ATP-depleting actions of xanthine oxidase in luteal cells because catalase reversed the actions of xanthine oxidase. In addition, the effects seen with xanthine oxidase in the present studies are identical to those seen with direct addition of hydrogen peroxide to luteal cells (7). Since the major oxygen species produced by xanthine oxidase is the superoxide anion (16), it is evident that luteal cells effectively dismute superoxide into hydrogen peroxide. While spontaneous dismutation of superoxide occurs (9), rat luteal tissues contain superoxide dismutase (4), but of unknown nature. Three different forms of superoxide dismutase have been identified in other tissues, and each is a separate gene product. They include a mitochondrial Mn-metalloenzyme, a cytosolic Cu-Zn enzyme, and an extracellular Cu-Zn enzyme (23-25). It is probable that superoxide, generated by xanthine oxidase, was dismuted to hydrogen peroxide extracellularly since the superoxide anion poorly penetrates cells, in contrast to hydrogen peroxide (9). Notwithstanding the nature of the dismutation process, it is evident from the present studies that
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2258
XANTHINE OXIDASE ACTION IN RAT LUTEAL CELLS
hydrogen peroxide mediated the effects of extracellular superoxide, derived from xanthine oxidase.
References 1. Hoch-Ligeti C, Bourne GH 1948 Changes in the concentration and histological distribution of ascorbic acid in ovaries, adrenals and livers of rats during oestrus cycles. Br J Pathol 29:400-407 2. Deane HW 1952 Histochemical observations on the ovary and oviduct of the albino rat during the estrous cycle. Am J Anat 91:363-393 3. Parlow AF 1972 Influence of differences in the persistence of luteinizing hormone in blood on their potency in the ovarian ascorbic acid depletion bioassay. Endocrinology 91:1109-1112 4. Laloraya M, Kumar GP, Laloraya MM 1988 Changes in the levels of superoxide anion radical and superoxide dismutase during the estrous cycle of rattus norvegicus and induction of superoxide dismutase in rat ovary by lutropin. Biochem Biophys Res Commun 157:146-153 5. Sawada M, Carlson JC 1989 Superoxide radical production in plasma membrane samples from regressing corpora lutea. Can J Physiol Pharmacol 67:465-471 6. Sawada M, Carlson JC 1985 Association of lipid peroxidation during luteal regression in the rat and natural aging in the rotifer. Exp Gerontol 20:179-186 7. Behrman HR, Preston SL 1989 Luteolytic actions of peroxide in rat ovarian cells. Endocrinology 124:2895-2900 8. Margolin YM, Aten RF, Behrman HR 1990 Antigonadotropic and antisteroidogenic actions of hydrogen peroxide in rat granulosa cells. Endocrinology 127:245-250 9. Halliwell B, Gutteridge JMC 1989 Free Radicals in Biology and Medicine, ed 2. Clarendon Press, Oxford, U.K. 10. Corte ED, Stirpe F 1972 The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme. Biochem J 126:739-745 11. Giulia M, Lorenzoni BE, Stirpe F 1973 Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties. Biochem J 131:191-198
Endo• 1991 Vo)128«No5
12. McCord JM 1985 Oxygen-derived free radicals in postischemic tissue injury. Mech Dis 312:159-163 13. Pang CY, Behrman HR 1979 Relationship of luteal blood flow and corpus luteum function in pseudopregnant rats. Am J Physiol 237:E30-E34 14. Arch JRS, Newsholme EA 1980 The control of the metabolism and the hormonal role of adenosine. In: Campbell PN, Dickens S (eds) Biochemical Society. Academic Press, New York, pp 82-123 15. Schraufstatter IU, Hyslop PA, Hinshaw DB, Spragg RG, Sklar LA, Cochrane CG 1986 Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase. Proc Natl Acad Sci USA 83:4908-4912 16. McCord JM, Fridovich I 1968 The reduction of cytochrome C by milk xanthine oxidase. J Biol Chem 243:5753-5760 17. Behrman HR 1979 Prostaglandins in hypothalamo-pituitary and ovarian function. Annu Rev Physiol 41:685-700 18. Soodak LK, MacDonald GJ, Behrman HR 1988 Luteolysis is linked to LH-induced depletion of ATP in vivo. Endocrinology 122:187193 19. Brennan T, Ohkawa R, Gore SD, Behrman HR 1983 Adeninederived purines increase ATP levels in the luteal cell: evidence that cell levels of ATP may limit the stimulation of cyclic AMP by LH. Endocrinology 112:499-508 20. Friedl HP, Till GO, Ryan US, Ward PA 1989 Mediator-induced activation of xanthine oxidase in endothelial cells. FASEB J 3:2512-2518 21. Ratych RE, Chuknyiska RS, Bulkley GB 1987 The primary localization of free radical generation after anoxia/reoxygenation in in isolated endothelial cells. Surgery 102:122-131 22. Jarasch E-D, Grund C, Bruder G, Heid HW, Keenan TW, Franke WW 1981 Localization of xanthine oxidase in mammary gland epithelium and capillary endothelium. Cell 25:67-82 23. Weisinger RA, Fridovich I 1973 Mitochondrial superoxide dismutase: site of synthesis and intramitochondrial localization. J Biol Chem 248:4793-4796 24. Marklund SL 1982 Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci USA 79:76347638 25. Karlsson K, Marklund SL 1988 Extracellular superoxide dismutase in the vascular system of mammals. Biochem J 255:223-228
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