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The effect of acute stress and opioid antagonist on the activity of NADPH-P450 reductase in rat Leydig cells
PII:
J. Steroid Biochem. Molec. Biol. Vol. 66, No. 1-2, pp. 51±54, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0960-0760(98)00003-X 0960-0760/98 $19.00 + 0.00
The Effect of Acute Stress and Opioid Antagonist on the Activity of NADPH-P450 Reductase in Rat Leydig Cells T. KosticÂ*, S. AndricÂ, D. Maric and R. KovacÏevic Institute of Biology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Yugoslavia
Previous studies indicate that acute immobilization stress (IMO; 2 h) impaired testicular steroidogenesis primarily at the testicular level decreasing the activity of certain steroidogenic enzymes. In the present study unstressed rats as well as IMO rats (2 h) were treated by intratesticular injection of naltrexone methobromide (NMB; peripheral opioid receptor antagonist; 36 mg/testis) or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats the activity of P450c17 was signi®cantly reduced as well as the activity of NADPH-P450 reductase (which catalyzes the transfer of electrons from NADPH to cytochrome P450), while the activity of NADH-b5 reductase was not affected. Present data con®rmed previous results that acute IMO reduced testicular P450c17 activity and implicate that decreased activity of NADPH-P450 reductase could be responsible for the inhibition of P450c17 under IMO conditions, while NADH-b5 reductase is probably not involved. NMB treatment antagonized the inhibitory effect of IMO on P450c17 and NADPH-P450 reductase activities. Such results put forward the implication that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO on testicular steroidogenesis, and allow the speculation that NADPH-P450 reductase could be a possible site of such an inhibition. # 1998 Elsevier Science Ltd. All rights reserved. J. Steroid Biochem. Molec. Biol., Vol. 66, No. 1-2, pp. 51±54, 1998
INTRODUCTION
17,20-lyase. To catalyze these two activities, P450c17 must receive reducing equivalents from its obligatory redox partner, NADPH-cytochrome P450 (NADPHP450) reductase. However, in the case of lyase activity in rat testis cytochrome b5 is also required [7± 10]. According to Yanagibashi and Hall [11], the ratio of 17a-hydroxylase to 17,20-lyase activity is regulated by the amount of available NADPH-P450 reductase; the activity of NADPH-P450 reductase in testicular microsomes is 3±4 times higher than that of adrenal microsomes, which may account for the greater activity of lyase in the testes comparing to adrenals. Since the ratio of 17a-hydroxylase to 17,20lyase activity of P450c17 is determined by the rate of ¯ow of electrons to the enzyme, changes in the effective abundance of the electron-donating redox partner could in¯uence the 17,20-lyase activity of P450c17 [11]. The aim of the present study was to investigate the effect of IMO on the activity of NADPH-P450 reductase and NADH-b5 reductase in the postmitochondrial fraction of Leydig cells, as well as the effect of intratesticular injection of NMB on the activity of
Several studies have shown that acute immobilization stress (IMO) impaired testicular steroidogenesis primarily at the testicular level; IMO reduced serum testosterone (T) levels without altering serum luteinizing hormone (LH) values [1±3]. Acute IMO does not affect the binding capacity or af®nity of the LH receptor on Leydig cells, suggesting that the effect of stress occurs, in part, at a postreceptor site [3]. At the same time, IMO rats exhibited a decreased activity of 17a-hydroxylase-17,20-lyase (P450c17) [1, 3, 4], and 3b-hydroxysteroid dehydrogenase (3bHSD) [1, 5] compared to freely moving rats. Naltrexone methobromide (NMB), a peripheral opioid antagonist, attenuated the inhibitory effect of IMO on T production, as well as on the testicular activity of P450c17 [4, 6] and 3bHSD [6]. P450c17 is a single microsomal enzyme that catalyzes two biosynthetic activities: 17a-hydroxylase and *Correspondence to T. KosticÂ. Tel. 350 122; Fax: 450 620; e-mail: [email protected]. Received 31 Jul. 1997; accepted 24 Nov. 1997. 51
T. Kostic et al.
52
these enzymes in IMO and freely moving rats. The activity of P450c17, and serum T levels in these rats were also recorded.
MATERIAL AND METHODS
Chemicals Antitestosterone serum No. 250 was supplied by G. D. Niswender (Colorado State University). Medium 199 (M199) was purchased from GIBCO Laboratories; NADPH, NADH, cytochrome c, bovine serum albumin (BSA, Fraction V), collagenase (Type I), testosterone, progesterone, pregnenolone, androstendione were purchased from Sigma; Dextran T70 was purchased from Pharmacia, Uppsala, Sweden, charcoal Norit A from Serva, Heidelberg, Germany; Tris (hydroxymetil) aminomethane from Bethesda Research Laboratories; [1,2,6,73 H(N)]testosterone was obtained from New England Nuclear. Naltrexone methobromide was a generous gift from Boehringer. All other chemicals were analytical grade. Animals and treatments Adult male Wistar rats bred in our laboratory and raised under controlled environmental conditions (temperature 22 228C; 14 h light/10 h dark) with food and water ad libitum were used for the experiments. Rats were handled daily during a 1-week period of acclimation before experiments and then subjected to acute IMO (2 h) as previously described [13] or left undisturbed (controls). Rats were treated by intratesticular injection with either the vehicle (saline) or peripheral opioid receptor antagonist naltrexone methobromide (NMB; 36 mg/testis). Treatments were administered at the beginning and at 1 h of the stress period. Treatment and control animals were sacri®ced by decapitation at the end of the IMO period, trunk blood was collected and serum samples were stored at ÿ208C until assayed for T by radioimmunoassay (RIA). Preparation of testicular postmitochondrial fractions The testes were decapsulated and homogenized in 50 mM phosphate buffer containing sucrose (0.25 M) pH 7.4 using a glass±glass homogenizer. The homogenates were centrifuged at 48C for 20 min at 1500 g and the pellets were discarded. The resulting supernatants were mixed with dextran-coated charcoal [0.05% Dextran T 70 and 0.5% Norit A in the ratio 1:1 (v/v) in sucrose buffer] and the suspensions were agitated for 10 min at 0±28C and then centrifuged at 1500 g for 10 min in order to remove endogenous steroids [14]. The supernatant fractions were further centrifuged at 12000 g for 20 min, and postmitochondrial testicular fractions were used for determination of enzyme activity. Protein content in
postmitochondrial fractions was estimated by the Bradford method[15] using BSA as the standard. Estimation of P450c17 activity The P450c17 activity in postmitochondrial testicular fractions was estimated by following the conversion of progesterone (P) or 17-hydroxyprogesterone (17OHP) to T and D4-androstenedione (D4A) to T. If the conversion of D4A to T was unchanged comparing to the corresponding control samples, the obtained changes in the conversion of P to T or 17OHP to T was attributed to the activity of P450c17 in investigated samples. The incubation solution contained in the ®nal volume of 0.25 ml the following components: steroid substrates (10 mM), NADPH (1 mM) and phosphate buffer (0.1 M), pH 7.4. Incubations were performed for 15 min at 378C in a shaking water bath in an atmosphere of 95% O2± 5% CO2. All samples were incubated in 4 replicates plus blanks without substrate or without the postmitochondrial fraction. After centrifugation at 1500 g at 48C for 10 min the supernatants were stored at ÿ208C until assayed for T by RIA. Because the antitestosterone serum showed a high cross-reactivity with dihydrotestosterone (DHT), assay values are referred to as T + DHT levels. Preparation of Leydig cell postmitochondrial fractions Isolated Leydig cells were prepared by collagenase dispersion as previously described [16]. Individual interstitial cells were resuspended in 5 ml of 17 mM Tris, 140 mM NH4Cl solution, pH 7.2 and incubated for 10 min at room temperature. This procedure eliminates red blood cells and interference of hemoglobin. The cell pellets were washed twice with M199 containing 0.1% BSA, and twice with saline (0.9% NaCl). Postmitochondrial fractions of Leydig cells were prepared as described in Section 2.3. Estimation of NADPH-P450 and NADH-b5 reductase activity NADPH-P450 reductase activities were measured as the change in absorbancy at 550 nm. The cuvette (®nal volume 0.6 ml) contained 0.2 mM NADPH, 1 mM KCN, 30 mM cytochrome c and the postmitochondrial fraction (0.15 mg) in 0.1 M phosphate buffer. The reactions were initiated by addition of NADPH [17, 18]. The millimolar extinction difference between reduced and oxidized cytochrome c was 21.2 at 550 nm. For the assay of NADH-b5 reductase, the reaction mixture contained 0.1 mM NADH, 0.2 mM potassium ferricyanide and the postmitochondrial fraction of Leydig cells (0.15 mg) in 0.1 M phosphate buffer (®nal volume 0.6 ml). A value of 1.02 mMÿ1 cmÿ1 was used as the molecular extinction decrement of the absorption at 420 nm [18].
Stress and P450c17 system activity
53
Table 1. Effect of IMO and intratesticular injection of NMB on serum T levels and activities of NADPH-P450 and NADH-b5 reductase Serum T + DHT (ng/ml) P450 reductase (nmol/mg min) b5 reductase (nmol/mg min)
Control
IMO
NMB±IMO
4.2520.62 (17) 3.0220.09 (12) 82.023.0 (6)
2.04220.16 (13)a 2.392 0.05 (11)a 87.0 23.0 (6)
3.402 0.30 (11)b 3.252 0.05 (11)b
Data represent mean2SEM, n in parentheses. Signi®cance: a p < 0.005 vs control; b p < 0.005 vs IMO group.
The statistical signi®cance was established by the non-parametric Mann±Whitney test.
RESULTS
Serum T levels were reduced after 2 h IMO. Intratesticular injection of NMB antagonized IMO induced a decrease in serum T level (Table 1), but did not affect serum T levels in freely moving rats (results not shown). The activity of NADPH-P450 reductase was signi®cantly decreased after a 2 h IMO period (p < 0.005), while treatment of stressed rats with NMB induced the full recovery of the enzyme activity (Table 1). The activity of NADH-b5 reductase was not affected by IMO. The effect of IMO and intratesticular injection of NMB on the activity of P450c17 was tested by following the conversion of P or 17OHP to T by the postmitochondrial fraction, since conversion of D4A to T was not affected by any treatment (Fig. 1(A),(B)). In stressed rats, conversion of P to T was reduced by the same degree (040%) as the conversion of 17OHP to T. The animals subjected to IMO and treated with NMB (Fig. 1(B)), exhibited a recovery of P450c17 activity.
DISCUSSION
It is well established[11, 12] that NADPH-P450 reductase is the obligatory redox partner of P450c17, and that the ratio of 17a-hydroxylase to 17,20-lyase activity is determined by the ¯ow rate of electrons to the enzyme. It can be increased by increasing the molar concentration of the electron-donating redox partner, such as P450 oxidoreductase or possibly cytochrome b5. It is also shown that the lyase activity can be increased by Ser±Thr phosphorylation, which increases the af®nity for the redox partner [12]. The present results demonstrated that in IMO rats the activity of NADPH-P450 reductase was signi®cantly reduced, allowing the speculation that it could be one of the possible reasons for a decreased activity of P450c17 in IMO rats observed in this study as well as in previous reports [1, 3, 4]. However these reports did not offer a possible explanation for the reduced lyase activity of P450c17 during stress. As previously shown [1] and con®rmed in present experiments (Fig. 1(A)), androgen production in IMO rats was inhibited by the same degree if either P (43%) or 17OHP (37%) was used as a substrate suggesting that lyase activity of P450c17 is in¯uenced by IMO stress, secondary to the reduced activity of NADPHP450 reductase. On the other hand, as suggested by several authors [8±10], cytochrome b5 is another redox partner speci®c for 17,20-lyase activity. It is
Fig. 1. Effect of IMO (2 h; panel A, B) and treatment with NMB (panel B) on the activity of P450c17. Postmitochondrial testicular fractions were incubated in the presence of steroid substrates (P, 17OHP and D4A; 10 mM) and NADPH (1 mM). NMB (36 mg/testis) was injected into the testes at the beginning of and at 1 h of the IMO period and the animals were sacri®ced at the end of 2 h IMO session. Columns represent mean2 SEM, n in parentheses. Signi®cance: * p < 0.05; ** p < 0.005 vs unstressed control; & p < 0.05 vs IMO rats.
T. Kostic et al.
54
also shown that the cytochrome b5 electron transport system is coupled to D5 3bHSD since NADH formed by conversion of pregnenolone to P reduced cytochrome b5 by the action of NADH-b5 reductase [7, 20]. Our previous results [1, 6] and that of Srivastava et al. [5] clearly demonstrated the decreased activity of 3bHSD in IMO rats. Decreased activity of 3bHSD, allows speculation that it could induce a decrease of the ¯ow rate of electrons from cytochrome b5 to P450c17. Since in the present experiments the level of cytochrome b5 was not measured in IMO rats, the role of this electron transport protein in altering the activity of P450c17 during IMO stress remains open. Cytochrome b5 functions as an electron transfer protein and readily accepts electrons from its native reductase, NADH-cytochrome b5 reductase and also from NADPH-P450 reductase [19]. In the present experiments the activity of NADH-b5 reductase was not altered by IMO stress, implicating that this enzyme is not involved in the stress-induced decrease of the activity of P450c17. Intratesticular administration of NMB normalized the activity of NADPH-P450 reductase (Table 1). It was accompanied by the recovery of P450c17 activity in IMO rats, as well as by the increase of serum T concentration to control values, in accordance with previous results [4, 6]. These results put forward the speculation that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO stress on testicular steroidogenesis and that NADPHP450 reductase could be one of possible sites of such an inhibition. Further studies are required to elucidate the possible way of endogenous opioid peptide induced inhibition of NADPH-P450 reductase. AcknowledgementsÐThe free supplies of the Niswender antisera and naltrexone methobromide from Boehringer Ingelheim KG are greatly acknowledged. This research was supported by a joint grant from the Serbia Research Association.
REFERENCES 1. Maric D., Kostic T. and KovacÏevic R., Effect of acute and chronic immobilization stress on rat Leydig cell steroidogenesis. J. Steroid Biochem. Mol. Biol. 58 (1996) 351±355. 2. Mann D. R. and Orr T. E., Effect of restraint stress on gonadal proopiomelanocortin peptides and the pituitary±testicular axis in rats. Life Sciences 46 (1990) 1601±1609. 3. Orr T. E., Taylor M. F., Bhattacharyya A. K., Collins D. C. and Mann D. R., Acute immobilization stress disrupts testicular steroidogenesis in adult male rats by inhibiting the activities of 17a-hydroxylase and 17,20-lyase without affecting the binding of LH/hCG receptors. J. Androl. 15 (1994) 302±308.
4. Akinbami M. A., Taylor M. F., Collins D. C. and Mann D. R., Effect of a peripheral and a central acting opioid antagonist on the testicular response to stress in rats. Neuroendocrinology 59 (1994) 343±348. 5. Srivastava R. K., Taylor M. F. and Mann D. R., Effect of immobilization stress on plasma luteinizing hormone, testosterone, and corticosterone concentrations and on 3bhydroxysteroid dehydrogenase activity in the testes of adult rats. P.S.E.B.M. 204 (1993) 231±235. 6. KosticÂ, T., AndricÂ, S., KovacÏevicÂ, R. and MaricÂ, D., The effect of opioid antagonists in local regulation of testicular response to acute stress in adult rats. Steroids, 62, in press. 7. Ohba H., Inano H. and Tamaoki B., Contribution of microsomal cytochrome b5 electron transport system coupled with D5-3b-hydroxysteroid dehidrogenase to androgen synthesis from progesterone. Biochem. Biophys. Res. Commun. 103 (1981) 1273±1280. 8. Onoda M. and Hall P. F., Cytochrome b5 stimulates puri®ed testicular microsomal cytochrome P450 (C21 side-chain cleavage). Biochem. Biophys. Res. Commun. 108 (1982) 454±460. 9. Aoyama T., Nagata K., Yamazoe Y., Kato R., Matsunaga E., Gelboin H. V. and Gonzalez F. J., Cytochrome b5 potentiation of cytochrome P-450 catalytic activity demonstrated by a vaccinia virus-mediated in situ reconstitution system. Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 5425±5429. 10. Kominami S., Ogawa N., Morimune R., Huang D. Y. and Takemori S., The role of cytochrome b5 in adrenal microsomal steroidogenesis. J. Steroid Biochem. Mol. Biol. 42 (1992) 57± 64. 11. Yanagibashi K. and Hall P. F., Role of electron transport in the regulation of the lyase activity of C21 side-chain cleavage P-450 from porcine adrenal and testicular microsomes. Journal of Biological Chemistry 261 (1986) 8429±8433. 12. Miller W. L., Auchus R. J. and Geller D. H., The regulation of 17,20-lyase activity. Steroids 62 (1997) 133±142. 13. Kvetnansky R., Weise V. K. and Kopin I. J., Elevation of adrenal tyrosine hydroxylase and phenyletanolamine-N-methyl transferase by repeated immobilization of rats. Endocrinology 87 (1970) 744±749. 14. Luzzani F. and Sof®entini A., Studies on cytosol progestinbinding components in the utero-placental unit of the pregnant hamster. J. Steroid Biochem. 13 (1979) 697±701. 15. Bradford M. M., A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein±dye binding. Anal. Biochem. 72 (1976) 248±254. 16. KovacÏevic R. and SaracÏ M., Bromocriptine-induced inhibition of hydroxylase/lyase activity of adult rat Leydig cells. J. Steroid Biochem. Mol. Biol. 46 (1993) 841±845. 17. Williams C. H. and Kamin H., Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver. J. Biol. Chem. 237 (1962) 587±595. 18. Inano H., Ishii-Ohba H., Suzuki K. and Ikeda K., Reasons for reduced activities of 17a-hydroxylase and C17±C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60 Co. J. Steroid Biochem. 35 (1990) 711±714. 19. Lee-Robichaud P., Kaderbhai M. A., Kaderbhai N., Wright J. N. and Akhtar M., Interaction of human CYP17 (P-45017a, 17a-hydroxylase-17,20-lyase) with cytochrome b5: importance of the orientation of the hydrophobic domain of cytochrome b5. Biochem. J. 321 (1997) 857±863. 20. Lee-Robichaud P., Wright J. N., Akhtar M. E. and Akhtar M., Modulation of the activity of human 17a-hydroxylase-17,20lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications. Biochem. J. 308 (1995) 901±908.
J. Steroid Biochem. Molec. Biol. Vol. 66, No. 1-2, pp. 51±54, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0960-0760(98)00003-X 0960-0760/98 $19.00 + 0.00
The Effect of Acute Stress and Opioid Antagonist on the Activity of NADPH-P450 Reductase in Rat Leydig Cells T. KosticÂ*, S. AndricÂ, D. Maric and R. KovacÏevic Institute of Biology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Yugoslavia
Previous studies indicate that acute immobilization stress (IMO; 2 h) impaired testicular steroidogenesis primarily at the testicular level decreasing the activity of certain steroidogenic enzymes. In the present study unstressed rats as well as IMO rats (2 h) were treated by intratesticular injection of naltrexone methobromide (NMB; peripheral opioid receptor antagonist; 36 mg/testis) or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats the activity of P450c17 was signi®cantly reduced as well as the activity of NADPH-P450 reductase (which catalyzes the transfer of electrons from NADPH to cytochrome P450), while the activity of NADH-b5 reductase was not affected. Present data con®rmed previous results that acute IMO reduced testicular P450c17 activity and implicate that decreased activity of NADPH-P450 reductase could be responsible for the inhibition of P450c17 under IMO conditions, while NADH-b5 reductase is probably not involved. NMB treatment antagonized the inhibitory effect of IMO on P450c17 and NADPH-P450 reductase activities. Such results put forward the implication that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO on testicular steroidogenesis, and allow the speculation that NADPH-P450 reductase could be a possible site of such an inhibition. # 1998 Elsevier Science Ltd. All rights reserved. J. Steroid Biochem. Molec. Biol., Vol. 66, No. 1-2, pp. 51±54, 1998
INTRODUCTION
17,20-lyase. To catalyze these two activities, P450c17 must receive reducing equivalents from its obligatory redox partner, NADPH-cytochrome P450 (NADPHP450) reductase. However, in the case of lyase activity in rat testis cytochrome b5 is also required [7± 10]. According to Yanagibashi and Hall [11], the ratio of 17a-hydroxylase to 17,20-lyase activity is regulated by the amount of available NADPH-P450 reductase; the activity of NADPH-P450 reductase in testicular microsomes is 3±4 times higher than that of adrenal microsomes, which may account for the greater activity of lyase in the testes comparing to adrenals. Since the ratio of 17a-hydroxylase to 17,20lyase activity of P450c17 is determined by the rate of ¯ow of electrons to the enzyme, changes in the effective abundance of the electron-donating redox partner could in¯uence the 17,20-lyase activity of P450c17 [11]. The aim of the present study was to investigate the effect of IMO on the activity of NADPH-P450 reductase and NADH-b5 reductase in the postmitochondrial fraction of Leydig cells, as well as the effect of intratesticular injection of NMB on the activity of
Several studies have shown that acute immobilization stress (IMO) impaired testicular steroidogenesis primarily at the testicular level; IMO reduced serum testosterone (T) levels without altering serum luteinizing hormone (LH) values [1±3]. Acute IMO does not affect the binding capacity or af®nity of the LH receptor on Leydig cells, suggesting that the effect of stress occurs, in part, at a postreceptor site [3]. At the same time, IMO rats exhibited a decreased activity of 17a-hydroxylase-17,20-lyase (P450c17) [1, 3, 4], and 3b-hydroxysteroid dehydrogenase (3bHSD) [1, 5] compared to freely moving rats. Naltrexone methobromide (NMB), a peripheral opioid antagonist, attenuated the inhibitory effect of IMO on T production, as well as on the testicular activity of P450c17 [4, 6] and 3bHSD [6]. P450c17 is a single microsomal enzyme that catalyzes two biosynthetic activities: 17a-hydroxylase and *Correspondence to T. KosticÂ. Tel. 350 122; Fax: 450 620; e-mail: [email protected]. Received 31 Jul. 1997; accepted 24 Nov. 1997. 51
T. Kostic et al.
52
these enzymes in IMO and freely moving rats. The activity of P450c17, and serum T levels in these rats were also recorded.
MATERIAL AND METHODS
Chemicals Antitestosterone serum No. 250 was supplied by G. D. Niswender (Colorado State University). Medium 199 (M199) was purchased from GIBCO Laboratories; NADPH, NADH, cytochrome c, bovine serum albumin (BSA, Fraction V), collagenase (Type I), testosterone, progesterone, pregnenolone, androstendione were purchased from Sigma; Dextran T70 was purchased from Pharmacia, Uppsala, Sweden, charcoal Norit A from Serva, Heidelberg, Germany; Tris (hydroxymetil) aminomethane from Bethesda Research Laboratories; [1,2,6,73 H(N)]testosterone was obtained from New England Nuclear. Naltrexone methobromide was a generous gift from Boehringer. All other chemicals were analytical grade. Animals and treatments Adult male Wistar rats bred in our laboratory and raised under controlled environmental conditions (temperature 22 228C; 14 h light/10 h dark) with food and water ad libitum were used for the experiments. Rats were handled daily during a 1-week period of acclimation before experiments and then subjected to acute IMO (2 h) as previously described [13] or left undisturbed (controls). Rats were treated by intratesticular injection with either the vehicle (saline) or peripheral opioid receptor antagonist naltrexone methobromide (NMB; 36 mg/testis). Treatments were administered at the beginning and at 1 h of the stress period. Treatment and control animals were sacri®ced by decapitation at the end of the IMO period, trunk blood was collected and serum samples were stored at ÿ208C until assayed for T by radioimmunoassay (RIA). Preparation of testicular postmitochondrial fractions The testes were decapsulated and homogenized in 50 mM phosphate buffer containing sucrose (0.25 M) pH 7.4 using a glass±glass homogenizer. The homogenates were centrifuged at 48C for 20 min at 1500 g and the pellets were discarded. The resulting supernatants were mixed with dextran-coated charcoal [0.05% Dextran T 70 and 0.5% Norit A in the ratio 1:1 (v/v) in sucrose buffer] and the suspensions were agitated for 10 min at 0±28C and then centrifuged at 1500 g for 10 min in order to remove endogenous steroids [14]. The supernatant fractions were further centrifuged at 12000 g for 20 min, and postmitochondrial testicular fractions were used for determination of enzyme activity. Protein content in
postmitochondrial fractions was estimated by the Bradford method[15] using BSA as the standard. Estimation of P450c17 activity The P450c17 activity in postmitochondrial testicular fractions was estimated by following the conversion of progesterone (P) or 17-hydroxyprogesterone (17OHP) to T and D4-androstenedione (D4A) to T. If the conversion of D4A to T was unchanged comparing to the corresponding control samples, the obtained changes in the conversion of P to T or 17OHP to T was attributed to the activity of P450c17 in investigated samples. The incubation solution contained in the ®nal volume of 0.25 ml the following components: steroid substrates (10 mM), NADPH (1 mM) and phosphate buffer (0.1 M), pH 7.4. Incubations were performed for 15 min at 378C in a shaking water bath in an atmosphere of 95% O2± 5% CO2. All samples were incubated in 4 replicates plus blanks without substrate or without the postmitochondrial fraction. After centrifugation at 1500 g at 48C for 10 min the supernatants were stored at ÿ208C until assayed for T by RIA. Because the antitestosterone serum showed a high cross-reactivity with dihydrotestosterone (DHT), assay values are referred to as T + DHT levels. Preparation of Leydig cell postmitochondrial fractions Isolated Leydig cells were prepared by collagenase dispersion as previously described [16]. Individual interstitial cells were resuspended in 5 ml of 17 mM Tris, 140 mM NH4Cl solution, pH 7.2 and incubated for 10 min at room temperature. This procedure eliminates red blood cells and interference of hemoglobin. The cell pellets were washed twice with M199 containing 0.1% BSA, and twice with saline (0.9% NaCl). Postmitochondrial fractions of Leydig cells were prepared as described in Section 2.3. Estimation of NADPH-P450 and NADH-b5 reductase activity NADPH-P450 reductase activities were measured as the change in absorbancy at 550 nm. The cuvette (®nal volume 0.6 ml) contained 0.2 mM NADPH, 1 mM KCN, 30 mM cytochrome c and the postmitochondrial fraction (0.15 mg) in 0.1 M phosphate buffer. The reactions were initiated by addition of NADPH [17, 18]. The millimolar extinction difference between reduced and oxidized cytochrome c was 21.2 at 550 nm. For the assay of NADH-b5 reductase, the reaction mixture contained 0.1 mM NADH, 0.2 mM potassium ferricyanide and the postmitochondrial fraction of Leydig cells (0.15 mg) in 0.1 M phosphate buffer (®nal volume 0.6 ml). A value of 1.02 mMÿ1 cmÿ1 was used as the molecular extinction decrement of the absorption at 420 nm [18].
Stress and P450c17 system activity
53
Table 1. Effect of IMO and intratesticular injection of NMB on serum T levels and activities of NADPH-P450 and NADH-b5 reductase Serum T + DHT (ng/ml) P450 reductase (nmol/mg min) b5 reductase (nmol/mg min)
Control
IMO
NMB±IMO
4.2520.62 (17) 3.0220.09 (12) 82.023.0 (6)
2.04220.16 (13)a 2.392 0.05 (11)a 87.0 23.0 (6)
3.402 0.30 (11)b 3.252 0.05 (11)b
Data represent mean2SEM, n in parentheses. Signi®cance: a p < 0.005 vs control; b p < 0.005 vs IMO group.
The statistical signi®cance was established by the non-parametric Mann±Whitney test.
RESULTS
Serum T levels were reduced after 2 h IMO. Intratesticular injection of NMB antagonized IMO induced a decrease in serum T level (Table 1), but did not affect serum T levels in freely moving rats (results not shown). The activity of NADPH-P450 reductase was signi®cantly decreased after a 2 h IMO period (p < 0.005), while treatment of stressed rats with NMB induced the full recovery of the enzyme activity (Table 1). The activity of NADH-b5 reductase was not affected by IMO. The effect of IMO and intratesticular injection of NMB on the activity of P450c17 was tested by following the conversion of P or 17OHP to T by the postmitochondrial fraction, since conversion of D4A to T was not affected by any treatment (Fig. 1(A),(B)). In stressed rats, conversion of P to T was reduced by the same degree (040%) as the conversion of 17OHP to T. The animals subjected to IMO and treated with NMB (Fig. 1(B)), exhibited a recovery of P450c17 activity.
DISCUSSION
It is well established[11, 12] that NADPH-P450 reductase is the obligatory redox partner of P450c17, and that the ratio of 17a-hydroxylase to 17,20-lyase activity is determined by the ¯ow rate of electrons to the enzyme. It can be increased by increasing the molar concentration of the electron-donating redox partner, such as P450 oxidoreductase or possibly cytochrome b5. It is also shown that the lyase activity can be increased by Ser±Thr phosphorylation, which increases the af®nity for the redox partner [12]. The present results demonstrated that in IMO rats the activity of NADPH-P450 reductase was signi®cantly reduced, allowing the speculation that it could be one of the possible reasons for a decreased activity of P450c17 in IMO rats observed in this study as well as in previous reports [1, 3, 4]. However these reports did not offer a possible explanation for the reduced lyase activity of P450c17 during stress. As previously shown [1] and con®rmed in present experiments (Fig. 1(A)), androgen production in IMO rats was inhibited by the same degree if either P (43%) or 17OHP (37%) was used as a substrate suggesting that lyase activity of P450c17 is in¯uenced by IMO stress, secondary to the reduced activity of NADPHP450 reductase. On the other hand, as suggested by several authors [8±10], cytochrome b5 is another redox partner speci®c for 17,20-lyase activity. It is
Fig. 1. Effect of IMO (2 h; panel A, B) and treatment with NMB (panel B) on the activity of P450c17. Postmitochondrial testicular fractions were incubated in the presence of steroid substrates (P, 17OHP and D4A; 10 mM) and NADPH (1 mM). NMB (36 mg/testis) was injected into the testes at the beginning of and at 1 h of the IMO period and the animals were sacri®ced at the end of 2 h IMO session. Columns represent mean2 SEM, n in parentheses. Signi®cance: * p < 0.05; ** p < 0.005 vs unstressed control; & p < 0.05 vs IMO rats.
T. Kostic et al.
54
also shown that the cytochrome b5 electron transport system is coupled to D5 3bHSD since NADH formed by conversion of pregnenolone to P reduced cytochrome b5 by the action of NADH-b5 reductase [7, 20]. Our previous results [1, 6] and that of Srivastava et al. [5] clearly demonstrated the decreased activity of 3bHSD in IMO rats. Decreased activity of 3bHSD, allows speculation that it could induce a decrease of the ¯ow rate of electrons from cytochrome b5 to P450c17. Since in the present experiments the level of cytochrome b5 was not measured in IMO rats, the role of this electron transport protein in altering the activity of P450c17 during IMO stress remains open. Cytochrome b5 functions as an electron transfer protein and readily accepts electrons from its native reductase, NADH-cytochrome b5 reductase and also from NADPH-P450 reductase [19]. In the present experiments the activity of NADH-b5 reductase was not altered by IMO stress, implicating that this enzyme is not involved in the stress-induced decrease of the activity of P450c17. Intratesticular administration of NMB normalized the activity of NADPH-P450 reductase (Table 1). It was accompanied by the recovery of P450c17 activity in IMO rats, as well as by the increase of serum T concentration to control values, in accordance with previous results [4, 6]. These results put forward the speculation that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO stress on testicular steroidogenesis and that NADPHP450 reductase could be one of possible sites of such an inhibition. Further studies are required to elucidate the possible way of endogenous opioid peptide induced inhibition of NADPH-P450 reductase. AcknowledgementsÐThe free supplies of the Niswender antisera and naltrexone methobromide from Boehringer Ingelheim KG are greatly acknowledged. This research was supported by a joint grant from the Serbia Research Association.
REFERENCES 1. Maric D., Kostic T. and KovacÏevic R., Effect of acute and chronic immobilization stress on rat Leydig cell steroidogenesis. J. Steroid Biochem. Mol. Biol. 58 (1996) 351±355. 2. Mann D. R. and Orr T. E., Effect of restraint stress on gonadal proopiomelanocortin peptides and the pituitary±testicular axis in rats. Life Sciences 46 (1990) 1601±1609. 3. Orr T. E., Taylor M. F., Bhattacharyya A. K., Collins D. C. and Mann D. R., Acute immobilization stress disrupts testicular steroidogenesis in adult male rats by inhibiting the activities of 17a-hydroxylase and 17,20-lyase without affecting the binding of LH/hCG receptors. J. Androl. 15 (1994) 302±308.
4. Akinbami M. A., Taylor M. F., Collins D. C. and Mann D. R., Effect of a peripheral and a central acting opioid antagonist on the testicular response to stress in rats. Neuroendocrinology 59 (1994) 343±348. 5. Srivastava R. K., Taylor M. F. and Mann D. R., Effect of immobilization stress on plasma luteinizing hormone, testosterone, and corticosterone concentrations and on 3bhydroxysteroid dehydrogenase activity in the testes of adult rats. P.S.E.B.M. 204 (1993) 231±235. 6. KosticÂ, T., AndricÂ, S., KovacÏevicÂ, R. and MaricÂ, D., The effect of opioid antagonists in local regulation of testicular response to acute stress in adult rats. Steroids, 62, in press. 7. Ohba H., Inano H. and Tamaoki B., Contribution of microsomal cytochrome b5 electron transport system coupled with D5-3b-hydroxysteroid dehidrogenase to androgen synthesis from progesterone. Biochem. Biophys. Res. Commun. 103 (1981) 1273±1280. 8. Onoda M. and Hall P. F., Cytochrome b5 stimulates puri®ed testicular microsomal cytochrome P450 (C21 side-chain cleavage). Biochem. Biophys. Res. Commun. 108 (1982) 454±460. 9. Aoyama T., Nagata K., Yamazoe Y., Kato R., Matsunaga E., Gelboin H. V. and Gonzalez F. J., Cytochrome b5 potentiation of cytochrome P-450 catalytic activity demonstrated by a vaccinia virus-mediated in situ reconstitution system. Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 5425±5429. 10. Kominami S., Ogawa N., Morimune R., Huang D. Y. and Takemori S., The role of cytochrome b5 in adrenal microsomal steroidogenesis. J. Steroid Biochem. Mol. Biol. 42 (1992) 57± 64. 11. Yanagibashi K. and Hall P. F., Role of electron transport in the regulation of the lyase activity of C21 side-chain cleavage P-450 from porcine adrenal and testicular microsomes. Journal of Biological Chemistry 261 (1986) 8429±8433. 12. Miller W. L., Auchus R. J. and Geller D. H., The regulation of 17,20-lyase activity. Steroids 62 (1997) 133±142. 13. Kvetnansky R., Weise V. K. and Kopin I. J., Elevation of adrenal tyrosine hydroxylase and phenyletanolamine-N-methyl transferase by repeated immobilization of rats. Endocrinology 87 (1970) 744±749. 14. Luzzani F. and Sof®entini A., Studies on cytosol progestinbinding components in the utero-placental unit of the pregnant hamster. J. Steroid Biochem. 13 (1979) 697±701. 15. Bradford M. M., A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein±dye binding. Anal. Biochem. 72 (1976) 248±254. 16. KovacÏevic R. and SaracÏ M., Bromocriptine-induced inhibition of hydroxylase/lyase activity of adult rat Leydig cells. J. Steroid Biochem. Mol. Biol. 46 (1993) 841±845. 17. Williams C. H. and Kamin H., Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver. J. Biol. Chem. 237 (1962) 587±595. 18. Inano H., Ishii-Ohba H., Suzuki K. and Ikeda K., Reasons for reduced activities of 17a-hydroxylase and C17±C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60 Co. J. Steroid Biochem. 35 (1990) 711±714. 19. Lee-Robichaud P., Kaderbhai M. A., Kaderbhai N., Wright J. N. and Akhtar M., Interaction of human CYP17 (P-45017a, 17a-hydroxylase-17,20-lyase) with cytochrome b5: importance of the orientation of the hydrophobic domain of cytochrome b5. Biochem. J. 321 (1997) 857±863. 20. Lee-Robichaud P., Wright J. N., Akhtar M. E. and Akhtar M., Modulation of the activity of human 17a-hydroxylase-17,20lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications. Biochem. J. 308 (1995) 901±908.