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Regulation of prolactin secretion by adrenal steroids in oestrogen-treated ovariectomized rats: Participation of endogenous opioid peptides
Neurophamuxology,Vol. 36, No. 10, pp. 1433-1438, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0028-3908/97 $17.00 + 0.00
Pergamon PII: s0028-3908(9700109-3
Regulation of Prolactin Secretion by Adrenal Steroids in Oestrogen-treated Ovariectomized Rats: Participation of Endogenous Opioid Peptides R. W. CARGN, A. M. SALICIONI and R. P. DEIS* Laboratorio de Reproduccidn y Lactancia, LARLAC-CONICET, Argentina
Casilla de Correo 855, 5500 Mendoza,
(Accepted 30 May 1997) Summary-The
purpose of the present study was to determine whether glucocorticoid inhibition of prolactin (PRL) release in oestrogen-treated ovariectomized (OVX) rats is mediated by endogenous opioid peptides (EOPs). All the animals were OVX and given oestradiol benzoate (OB, 20 pg/rat, s.c.) 2 weeks later (day 0). On day 3 they received vehicle, mifepristone (MIF, 10 mg/kg, s.c.) or hydrocortisone (HYD, 2 mg/rat, s.c.), in combination with the opioid antagonist naloxone (NAL, 2 mg/kg, i.p.) or vehicle. Serum PRL concentration was then measured by RIA at 13.00 and 18.00 hr, to include assessment of diurnal variation of PRL secretion. At 13.00 hr either MIF or NAL alone increased PRL secretion with no additional effect when NAL was combined with MIF. HYD had no significant inhibitory effect, but NAL with HYD increased PRL secretion. At 18.00 hr serum PFL concentration was higher than at 13.00 hr, and not affected significantly by MIF or NAL alone, although PRL secretion was increased by treatment with both. HYD inhibited PRL secretion and this inhibition was prevented by NAL. In a second experiment to distinguish antiglucocorticoid and antiprogesterone effects of MIF, we administered progesterone (2 mg/rat, s.c.) or a specific progesterone antiserum. In contrast with MIF, the progesterone antibody had no effect on PRL secretion at 13.00 hr, nor on the stimulation by NAL, while progesterone (unlike HYD) increased PRL secretion and NAL attenuated this response; this was opposite to the effect of NAL with HYD. Similarly, at 18.00 hr the interaction of MIF and NAL was not explained by antagonism of progesterone. Together, these results indicate inhibition of PRL by glucocorticoids but not progesterone, mediated in part by EOPs. At 18.00 hr endogenous glucocorticoids do not regulate oestrogen-stimulated PRL release, although HYD is inhibitory through EOPs. 0 1997 Elsevier Science Ltd. Keywords--Mifepristone,
progesterone, hydrocortisone,
Ovarian steroids are important regulators of prolactin (PRL) secretion (Brown-Grant, 1974; Caligaris et al., 1974). However, accumulated evidence indicates that glucocorticoids can negatively regulate PRL secretion (Deis et al., 1989; Brann et al., 1990; Putnam et al., 1991). Thus, in oestrogen-primed ovariectomized rats, serum PRL increases after adrenalectomy or treatment with mifepristone, a glucocorticoid/progesterone antagonist, while the administration of progesterone antibody has no effect, suggesting that adrenal glucocorticoids negatively regulate PRL release in this experimental model. Moreover, this inhibition is evident at 13.00 hr but not at 18.00 hr (Caron et aE., 1994) suggesting, since glucocorticoid secretion is greater at 18.00 hr than at 13.00 hr due to the diurnal rhythm in end.ogenous secretion of glucocorti*To whom correspondence 273976.
should be addressed. Fax: 54-61-
oestrogen, naloxone.
coids (Spencer et al., 1993), that there are diurnal changes in the sensitivity to glucocorticoids of mechanisms regulating PRL secretion (Kiem et al., 1995). Endogenous opioid peptides (EOPs) are involved in the regulation of PRL secretion during pro-oestrus (Ieiri et al., 1980) and lactation (Ferland et al., 1978; Baumann and Rabii, 1990). Most of these studies show that EOPs and mu or kappa opioids stimulate PRL release (Krulich et al., 1986a,b; Leadem and Yagenova, 1987). However, an inhibitory role of EOPs on PRL release has also been demonstrated under certain conditions (Singh et al., 1992; Soaje and Deis, 1994, Caron and Deis, 1996). Furthermore, glucocorticoids modify opioid-regulated PRL secretion since dexamethasone treatment abolishes PRL release induced by stress or morphine administration (Rossier et al., 1980). The purpose of the present study was to determine whether glucocorticoid inhibition of PRL release ob-
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R. W. Cardn et
served in oestrogen-treated ovariectomized rats at 13.00 hr is mediated by EOPs, by testing effects of the opioid antagonist naloxone (NAL), alone or in combination with the glucocorticoid/progesterone antagonist mifepristone. For comparison, the participation of EOPs in PRL secretion at 18.00 hr, when endogenous glucocorticoids have no inhibitory effects, was also investigated. We further tested whether mifepristone was acting as a glucocorticoid or progesterone antagonist by comparing effects with treatment with a progesterone antiserum.
METHODS
Animals Virgin female rats bred in our laboratory (originally Wistar strain) and weighing 180-220 g were used. They were kept under controlled lighting (lights on 06.0020.00 hr) and environmental temperature (22-24°C); standard rat chow (Nutric, Cordoba, Argentina) and water were available ad Zibitum. Experimental protocol All the animals were ovariectomized and treated 1416 days later at 13.00 hr with oestradiol benzoate (OB) to stimulate PRL secretion. This day was designated as day 0. On day 3 the rats were treated with mifepristone, hydrocortisone, progesterone antiserum or progesterone; respectively to antagonize endogenous glucocorticoids, to test effects of exogenous glucocorticoids, to distinguish antagonism of glucocorticoids and progesterone by mifepristone, and to test effects of exogenous progesterone. Blood samples for PRL RIA were taken at 13.00 hr and 18.00 hr from the same animals (except in group 5). Thirty minutes before blood-sampling, rats were given either 0.9% saline solution or NAL, to antagonize EOPs. Surgical procedures and drug treatment Ovariectomy was performed through two dorsolateral incisions under ether anaesthesia between 08.00 and 09.00 hr. Group 1 was injected S.C. with vehicle at 08.00 hr on day 3 (n = 18). Group 2 was injected S.C.with mifepristone at 08.00 hr on day 3 (n = 18). Group 3 was injected S.C. with hydrocortisone at 08.00 hr on day 3 (n = 17). Group 4 was injected i.p. with 50 ~1 of progesterone antiserum at 08.00 and 13.30 hr on day 3, to distinguish the antiprogesterone from the antiglucocorticoid activity of mifepristone (n = 16). Group 5 received progesterone at 08.00 hr and was sampled at 13.00 hr (n = 15) or received progesterone at 13.00 hr and was killed at 18.00 hr on day 3 (n = 16). Two further groups were ovariectomized and given vehicle on day 0 instead of oestradiol benzoate, then either S.C.vehicle (group 6, n = 16) or mifepristone as
al.
above (group 7, n = 16) on day 3. These were also blood-sampled at 13.00 and 18.00 hr. All the groups were divided into two subgroups, receiving an i.p. injection of either NAL or 0.9% saline 30 min before blood sampling. OB (20 pg/rat: Schering, Buenos Aires, Argentina) was given S.C. in 0.2 ml purified sunflower seed oil. Mifepristone (RU-38486; 17P-hydroxy- 1lb(6dimethylamino-phenyl) 17a-(prop-1-ynyl)oestra-4,9-dien-3-one, kindly provided by M. Gamier, Roussel-Uclaf, Romaineville, France) was dissolved in sunflower seed oil (10 mg/ ml) and injected S.C.(10 mg/kg). Progesterone antiserum was raised in our laboratory (Deis et al., 1989; Caron et al., 1994) and kept frozen (-20°C) in aliquots until administration. In preliminary experiments in vitro we found that 1 ~1 antiserum bound approximately 60 ng (0.19 nmol) [3H]progesterone and only 0.3 ng (0.86 pmol) [3H]corticosterone. Thus, the volume of antiserum injected (100 ~1) was more than enough to neutralize essentially all circulating progesterone (which is about 4.6 ng/ml at 13.00 hr in OB-primed ovariectomized rats, Salicioni et al., 1993) without interfering with the action of glucocorticoids. Progesterone and hydrocortisone (Sigma Co., U.S.A.) were dissolved in sunflower seed oil (10 mg/ml) and injected S.C. (2 mg/rat). Naloxone (NAL, Sigma Co., St Louis, MO.) was dissolved in 0.9% saline and injected i.p. (2 mg/kg) at 12.30 or 17.30 hr on day 3. Blood sampling At 13.00 hr on day 3, blood samples of 600 ~1 were obtained by cardiac puncture with a 21-gauge needle attached to a l-ml syringe. In experienced hands, this simple method takes only a few seconds and does not induce an increase in PRL (Deis et al., 1989). At 18.00 hr, trunk blood was obtained by decapitation. In preliminary experiments we did not find differences in serum PRL levels between samples obtained by cardiac puncture or decapitation. All work was in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985). Blood was allowed to clot at room temperature and serum was separated and stored frozen (-20°C) until assayed for PRL. Determination of PRL PRL was measured by a double-antibody RIA using materials kindly provided by Dr S. Raiti, NIADDK Rat Pituitary Hormone Distribution Program. PRL was radioiodinated using the chloramine-T method. Results are expressed in terms of the rat PRL RP-3 standard preparation. Assay sensitivity was 1 lug/l serum and inter- and intra-assay coefficients of variation were 8 and 3%, respectively. Statistical analysis The statistical analysis of data was performed using two-way analysis of variance (ANOVA-II) followed by Student-Newman-Keuls tests to compare all OB treated
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Regulation of prolactin secretion by adrenal steroids #
# T
T
SJIO B 9
1 I
VEHICLE
0 m
Vehicle Naloxone
* # id #
MIF
HYD
Fig. 1. Effects of mifepristone, hydrocortisone and naloxone on serum PlU levels in oestradiol benzoate-treated ovariectomized rats. (A) 13.00 hr and (B) 18.00 hr. Mifepristone (MlF, 10 mg/kg) or hydrocortisone (HYD, 2 mgkat) was given S.C.at 08.00 hr. Naloxone (2 mgkg i.p., solid bars) or vehicle (open bars) was given 30 min ibefore sampling. *P ~0.05 vs. the respective group without naloxone. #p < 0.05 vs. vehicle only. Values are mean f SEM.
l(B)) serum PRL concentration in rats given vehicle was greater than at 13.00 br (p c 0.01) and was not modified by NAL. Mifepristone did not further increase serum PRL, but NAL increased PRL secretion in rats treated with mifepristone (p < 0.05 vs. vehicle). Hydrocortisone significantly reduced PRL values at 18.00 hr, and NAL reversed this effect. ESfect of progesterone or progesterone antiserum and NAL on serum PRL levels in oestrogen-primed rats
Progesterone greatly increased serum PRL concentration at 13.00 hr @ < 0.001 vs. vehicle). This increase was partially prevented by NAL (Fig. 2). Progesterone antiserum did not modify serum PRL values at 13 .OOhr and did not prevent the increase in PRL secretion after NAL (Fig. l(A), Fig. 2(A)). At 18.00 hr, progesterone antiserum increased @I<0.05) serum PRL concentration and NAL in combination with antiserum, decreased PRL secretion (Fig. 2(B)). At 18.00 hr progesterone increased PRL secretion (p c 0.05) less effectively than at 13.00 hr (p < 0.05), but NAL did not modify this response (Fig. 2(B)). DISCUSSION The present results provide evidence that the inhibition of PRL secretion by adrenal glucocorticoids in oestrogentreated ovariectomized rats (Caron et al., 1994) involves EOPs. The inhibitory action of EOPs on PRL secretion
600
studied at 13.00 or 18.00 hr. To statistically analyse results from the progesterone/progesterone antiserum experiment (groups 4 and 5), logarithmic transformation of data was applied. groups
500
‘-
RESULTS Effect of mifepristone, hydrocortisone and NAL on PRL secretion in oestrogen-primed ovan’ectomized rats In rats not primed with OB the serum PRL values were
low at 13.00 and 18.00 hr (4.9 f 1.3 and 3.0 + 0.5 ng/ ml, respectively), and were not altered by NAL and/or mifepristone (data not shown). In OB-primed rats, serum PRL concentration at 13.00 hr (Fig. l(A)) was significantly enhanced by NAL (p < 0.05). As we have reported previously (Caron et al., 1994), mifepristone treatment of OB-primed ovariectomized rats greatly increased (4-fold) serum PRL at 13.00 hr. This increase in serum PRL was not modified by NAL (Fig. 1). Serum PRL concentration was greater after mifepristone (with or without NAL) than after NAL alone (p < 0.05). Hydrocortisone did not significantly modify serum PRL at 13.00 hr nor the effect of NAL. At 18.00 hr (Fig.
A
##
600
lB 0 -
Vehicle Naloxone
400
z C 6 -m
300
g 5 b m
200
100
0
Fig. 2. Effects of progesterone antiserum, progesterone and naloxone on serum PRL levels in oestradiol benzoate-treated ovariectomized rats. (A) 13.00 hr and (B) 18.00 hr. Progesterone antiserum (Ab, 50 ~1, i.p.) was given at 08.00 and 13.30 hr on day 3 and the rats were blood-sampled at 13.00 hr and 18.00 hr. Progesterone (P, 2 mg/rat, s.c.) was injected at 08.OOhr or at 13.OOhr. Thirty minutes before sampling, the rats were given naloxone (2 mg/kg, i.p., solid bars) or vehicle (open bars). *p < 0.05 and **p c 0.01 vs. the respective group without naloxone. #D c 0.05 and #& < 0.01 vs. vehicle groups (horizontal bars). Values are mean f SEM.
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R. W. Carbn et al.
has been shown under different conditions (Singh et al., 1992; Soaje and Deis, 1994; Caron and Deis, 1996). Neither NAL nor mifepristone altered PRL secretion in rats not primed with oestrogen, in which basal levels of PRL were low, as expected (Cat-on et al., 1994); thus oestrogen stimulation of PRL secretion is necessary for the effects of glucocorticoids and opioids. NAL alone increased PRL secretion at 13.00 hr, demonstrating an inhibitory EOP tone on PRL secretion at this time. This EOP is not likely to be acting at the anterior pituitary gland, since opiates do not influence PRL secretion at this level (Wiesner et al., 1984). Consequently, NAL is concluded to be acting in the brain, either stimulating a PRL releasing factor or interfering with secretion of a PRL inhibiting factor. Mifepristone is a synthetic 19-nor steroid with high affinity for glucocorticoid and progesterone receptors (Philibert, 1984). It is a type II glucocorticoid receptor antagonist, while hydrocortisone and endogenous glucocorticoids have greater affinity for type I receptors (De Kloet et al., 1975; Veldhuis et al., 1982; Reul and De Kloet, 1985). Type II receptors are occupied when corticoid secretion is increased (Reul et al., 1987). Our results indicate endogenous glucocorticoid inhibition of PRL secretion via a type II receptor (at 13.00 hr) and availability of type I receptors, for hydrocortisone action, at 18.00 hr. Both receptors can evidently mediate glucocorticoid action on PRL secretion via EOPs. Since the specific progesterone antibody did not modify PRL secretion at 13.00 hr, while it was enhanced by mifepristone, blockade of progesterone action is unlikely to be responsible for the effect of mifepristone at this time; which can be concluded to be acting as a glucocorticoid antagonist. As expected (Caron et d., 1994), the removal of the action of circulating progesterone by the specific antiserum did not modify serum PRL at 13.00 hr, confirming that circulating progesterone is not active in inhibiting oestrogen-induced PRL secretion at this time, while exogenous progesterone had a potent stimulatory effect. Moreover, this marked stimulation by progesterone was mediated partly by a stimulatory EOP action, since it was reduced significantly by NAL treatment. Thus, glucocorticoids and progesterone can have opposite effects on PRL secretion at 13.00 hr and both effects are partly mediated by EOPs. We propose that progesterone treatment prevents the inhibitory regulation of oestrogen-induced PRL release by glucocorticoids and shifts the action of EOPs from inhibition to stimulation. However, in oestrogen-primed rats the dominant endogenous steroid influence is the inhibitory action of glucocorticoid. Oestrogen induced a larger increase in serum PRL levels at 18.00 hr than at 13.00 hr, as shown by others (Caligaris et al., 1974). The lack of a significant effect of mifepristone or NAL alone, but the stimulation of PRL secretion when given together, suggests that circulating glucocorticoids and EOPs weakly regulate the afternoon
PRL surge induced by oestrogen. Similarly, progesterone was weakly stimulatory at 18.00 hr; however, this action seemed to be independent of EOPs, unlike that at 13.00 hr. Singh et al. (1992) observed a suppression of PRL secretion in the afternoon by morphine in oestrogentreated ovariectomized rats. Our results indicate that EOPs, in the presence of endogenous steroids, do not inhibit PRL secretion in the afternoon. Indeed, following blockade of progesterone action with antiserum, PRL secretion was increased, but decreased by NAL, indicating a stimulatory action of glucocorticoid via EOPs in the absence of progesterone (Fig. 2(B)). The stimulatory effect of mifepristone alone on PRL secretion at 13 .OOhr, and the lack of an effect at 18.00 hr, could be due to a decrease in responsiveness of the system to the inhibitory effect of glucocorticoids, perhaps reflected in the higher PRL values in the vehicle controls at 18.00 hr. This difference is not likely to be due to fading of mifepristone action by 18.00 hr, since repeated injection of mifepristone (at 08.00 and 13.00 hr) did not increase serum PRL at 18.00 hr (results not shown). Furthermore, hydrocortisone injection produced a NAL-reversible inhibition of PRL secretion at 18.00 hr, despite the higher circulating corticosterone level in the afternoon in rats (Kiem et al., 1995). The bidirectional involvement of EOPs in the regulation of PRL release could be related to changes in opioid receptors and/or in brain levels of EOPs or could be a consequence of glucocorticoids and progesterone acting through different EOP mechanisms. However, no consistent effects of adrenalectomy or glucocorticoid replacement on hypothalamic opioid systems have been reported (Krieger et al., 1979; Lee et al., 1980; Lim et al., 1982; Bimberg et d., 1983; Vale et al., 1983; Beaulieu et al., 1988), although there are glucocorticoid receptors in hypothalamic P-endorphin neurones (Chao et al., 1989; Cintra and Bortolotti, 1992) and corticosterone enhances the production of hypothalamic p-endorphin in vitro (Yang et al., 1994). In conclusion, in oestrogen-primed ovariectomized rats the secretion of PRL at 13.00 hr is inhibited by glucocorticoid, partly through EOP action. Endogenous glucocorticoids may not participate in the regulation of the rise of PRL secretion at 18.00 hr, although EOP inhibition can still be demonstrated, particularly after inhibition of PRL secretion by hydrocortisone. In contrast, exogenous progesterone stimulates PRL secretion via EOPs at 13.00 hr, but not at 18.00 hr. At 18.00 hr endogenous progesterone may be masking a stimulatory action of endogenous glucocorticoid, also mediated by EOPs; this requires further investigation. EOPs mediate inhibitory actions of glucocorticoid and inhibitory actions of progesterone. These results indicate that the diurnal variation in PRL in oestrogen-primed ovariectomized rats results, in part, from interactions between changes in glucocorticoid secretion and EOP mechanisms.
Regulation of prolactin secretion by adrenal steroids Acknowledgements-The authors offer sincere thanks to Dr J. A. Russell for valuable discussion on the manuscript and to Mrs F. E. Guinazd de Di Nasso for her expert technical assistance. This work was supported by a Grant (PID 312210088) from Consejo National de Investigaciones Cientificas y TCcnicas (CONICET), Argentina.
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Pergamon PII: s0028-3908(9700109-3
Regulation of Prolactin Secretion by Adrenal Steroids in Oestrogen-treated Ovariectomized Rats: Participation of Endogenous Opioid Peptides R. W. CARGN, A. M. SALICIONI and R. P. DEIS* Laboratorio de Reproduccidn y Lactancia, LARLAC-CONICET, Argentina
Casilla de Correo 855, 5500 Mendoza,
(Accepted 30 May 1997) Summary-The
purpose of the present study was to determine whether glucocorticoid inhibition of prolactin (PRL) release in oestrogen-treated ovariectomized (OVX) rats is mediated by endogenous opioid peptides (EOPs). All the animals were OVX and given oestradiol benzoate (OB, 20 pg/rat, s.c.) 2 weeks later (day 0). On day 3 they received vehicle, mifepristone (MIF, 10 mg/kg, s.c.) or hydrocortisone (HYD, 2 mg/rat, s.c.), in combination with the opioid antagonist naloxone (NAL, 2 mg/kg, i.p.) or vehicle. Serum PRL concentration was then measured by RIA at 13.00 and 18.00 hr, to include assessment of diurnal variation of PRL secretion. At 13.00 hr either MIF or NAL alone increased PRL secretion with no additional effect when NAL was combined with MIF. HYD had no significant inhibitory effect, but NAL with HYD increased PRL secretion. At 18.00 hr serum PFL concentration was higher than at 13.00 hr, and not affected significantly by MIF or NAL alone, although PRL secretion was increased by treatment with both. HYD inhibited PRL secretion and this inhibition was prevented by NAL. In a second experiment to distinguish antiglucocorticoid and antiprogesterone effects of MIF, we administered progesterone (2 mg/rat, s.c.) or a specific progesterone antiserum. In contrast with MIF, the progesterone antibody had no effect on PRL secretion at 13.00 hr, nor on the stimulation by NAL, while progesterone (unlike HYD) increased PRL secretion and NAL attenuated this response; this was opposite to the effect of NAL with HYD. Similarly, at 18.00 hr the interaction of MIF and NAL was not explained by antagonism of progesterone. Together, these results indicate inhibition of PRL by glucocorticoids but not progesterone, mediated in part by EOPs. At 18.00 hr endogenous glucocorticoids do not regulate oestrogen-stimulated PRL release, although HYD is inhibitory through EOPs. 0 1997 Elsevier Science Ltd. Keywords--Mifepristone,
progesterone, hydrocortisone,
Ovarian steroids are important regulators of prolactin (PRL) secretion (Brown-Grant, 1974; Caligaris et al., 1974). However, accumulated evidence indicates that glucocorticoids can negatively regulate PRL secretion (Deis et al., 1989; Brann et al., 1990; Putnam et al., 1991). Thus, in oestrogen-primed ovariectomized rats, serum PRL increases after adrenalectomy or treatment with mifepristone, a glucocorticoid/progesterone antagonist, while the administration of progesterone antibody has no effect, suggesting that adrenal glucocorticoids negatively regulate PRL release in this experimental model. Moreover, this inhibition is evident at 13.00 hr but not at 18.00 hr (Caron et aE., 1994) suggesting, since glucocorticoid secretion is greater at 18.00 hr than at 13.00 hr due to the diurnal rhythm in end.ogenous secretion of glucocorti*To whom correspondence 273976.
should be addressed. Fax: 54-61-
oestrogen, naloxone.
coids (Spencer et al., 1993), that there are diurnal changes in the sensitivity to glucocorticoids of mechanisms regulating PRL secretion (Kiem et al., 1995). Endogenous opioid peptides (EOPs) are involved in the regulation of PRL secretion during pro-oestrus (Ieiri et al., 1980) and lactation (Ferland et al., 1978; Baumann and Rabii, 1990). Most of these studies show that EOPs and mu or kappa opioids stimulate PRL release (Krulich et al., 1986a,b; Leadem and Yagenova, 1987). However, an inhibitory role of EOPs on PRL release has also been demonstrated under certain conditions (Singh et al., 1992; Soaje and Deis, 1994, Caron and Deis, 1996). Furthermore, glucocorticoids modify opioid-regulated PRL secretion since dexamethasone treatment abolishes PRL release induced by stress or morphine administration (Rossier et al., 1980). The purpose of the present study was to determine whether glucocorticoid inhibition of PRL release ob-
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served in oestrogen-treated ovariectomized rats at 13.00 hr is mediated by EOPs, by testing effects of the opioid antagonist naloxone (NAL), alone or in combination with the glucocorticoid/progesterone antagonist mifepristone. For comparison, the participation of EOPs in PRL secretion at 18.00 hr, when endogenous glucocorticoids have no inhibitory effects, was also investigated. We further tested whether mifepristone was acting as a glucocorticoid or progesterone antagonist by comparing effects with treatment with a progesterone antiserum.
METHODS
Animals Virgin female rats bred in our laboratory (originally Wistar strain) and weighing 180-220 g were used. They were kept under controlled lighting (lights on 06.0020.00 hr) and environmental temperature (22-24°C); standard rat chow (Nutric, Cordoba, Argentina) and water were available ad Zibitum. Experimental protocol All the animals were ovariectomized and treated 1416 days later at 13.00 hr with oestradiol benzoate (OB) to stimulate PRL secretion. This day was designated as day 0. On day 3 the rats were treated with mifepristone, hydrocortisone, progesterone antiserum or progesterone; respectively to antagonize endogenous glucocorticoids, to test effects of exogenous glucocorticoids, to distinguish antagonism of glucocorticoids and progesterone by mifepristone, and to test effects of exogenous progesterone. Blood samples for PRL RIA were taken at 13.00 hr and 18.00 hr from the same animals (except in group 5). Thirty minutes before blood-sampling, rats were given either 0.9% saline solution or NAL, to antagonize EOPs. Surgical procedures and drug treatment Ovariectomy was performed through two dorsolateral incisions under ether anaesthesia between 08.00 and 09.00 hr. Group 1 was injected S.C. with vehicle at 08.00 hr on day 3 (n = 18). Group 2 was injected S.C.with mifepristone at 08.00 hr on day 3 (n = 18). Group 3 was injected S.C. with hydrocortisone at 08.00 hr on day 3 (n = 17). Group 4 was injected i.p. with 50 ~1 of progesterone antiserum at 08.00 and 13.30 hr on day 3, to distinguish the antiprogesterone from the antiglucocorticoid activity of mifepristone (n = 16). Group 5 received progesterone at 08.00 hr and was sampled at 13.00 hr (n = 15) or received progesterone at 13.00 hr and was killed at 18.00 hr on day 3 (n = 16). Two further groups were ovariectomized and given vehicle on day 0 instead of oestradiol benzoate, then either S.C.vehicle (group 6, n = 16) or mifepristone as
al.
above (group 7, n = 16) on day 3. These were also blood-sampled at 13.00 and 18.00 hr. All the groups were divided into two subgroups, receiving an i.p. injection of either NAL or 0.9% saline 30 min before blood sampling. OB (20 pg/rat: Schering, Buenos Aires, Argentina) was given S.C. in 0.2 ml purified sunflower seed oil. Mifepristone (RU-38486; 17P-hydroxy- 1lb(6dimethylamino-phenyl) 17a-(prop-1-ynyl)oestra-4,9-dien-3-one, kindly provided by M. Gamier, Roussel-Uclaf, Romaineville, France) was dissolved in sunflower seed oil (10 mg/ ml) and injected S.C.(10 mg/kg). Progesterone antiserum was raised in our laboratory (Deis et al., 1989; Caron et al., 1994) and kept frozen (-20°C) in aliquots until administration. In preliminary experiments in vitro we found that 1 ~1 antiserum bound approximately 60 ng (0.19 nmol) [3H]progesterone and only 0.3 ng (0.86 pmol) [3H]corticosterone. Thus, the volume of antiserum injected (100 ~1) was more than enough to neutralize essentially all circulating progesterone (which is about 4.6 ng/ml at 13.00 hr in OB-primed ovariectomized rats, Salicioni et al., 1993) without interfering with the action of glucocorticoids. Progesterone and hydrocortisone (Sigma Co., U.S.A.) were dissolved in sunflower seed oil (10 mg/ml) and injected S.C. (2 mg/rat). Naloxone (NAL, Sigma Co., St Louis, MO.) was dissolved in 0.9% saline and injected i.p. (2 mg/kg) at 12.30 or 17.30 hr on day 3. Blood sampling At 13.00 hr on day 3, blood samples of 600 ~1 were obtained by cardiac puncture with a 21-gauge needle attached to a l-ml syringe. In experienced hands, this simple method takes only a few seconds and does not induce an increase in PRL (Deis et al., 1989). At 18.00 hr, trunk blood was obtained by decapitation. In preliminary experiments we did not find differences in serum PRL levels between samples obtained by cardiac puncture or decapitation. All work was in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985). Blood was allowed to clot at room temperature and serum was separated and stored frozen (-20°C) until assayed for PRL. Determination of PRL PRL was measured by a double-antibody RIA using materials kindly provided by Dr S. Raiti, NIADDK Rat Pituitary Hormone Distribution Program. PRL was radioiodinated using the chloramine-T method. Results are expressed in terms of the rat PRL RP-3 standard preparation. Assay sensitivity was 1 lug/l serum and inter- and intra-assay coefficients of variation were 8 and 3%, respectively. Statistical analysis The statistical analysis of data was performed using two-way analysis of variance (ANOVA-II) followed by Student-Newman-Keuls tests to compare all OB treated
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Regulation of prolactin secretion by adrenal steroids #
# T
T
SJIO B 9
1 I
VEHICLE
0 m
Vehicle Naloxone
* # id #
MIF
HYD
Fig. 1. Effects of mifepristone, hydrocortisone and naloxone on serum PlU levels in oestradiol benzoate-treated ovariectomized rats. (A) 13.00 hr and (B) 18.00 hr. Mifepristone (MlF, 10 mg/kg) or hydrocortisone (HYD, 2 mgkat) was given S.C.at 08.00 hr. Naloxone (2 mgkg i.p., solid bars) or vehicle (open bars) was given 30 min ibefore sampling. *P ~0.05 vs. the respective group without naloxone. #p < 0.05 vs. vehicle only. Values are mean f SEM.
l(B)) serum PRL concentration in rats given vehicle was greater than at 13.00 br (p c 0.01) and was not modified by NAL. Mifepristone did not further increase serum PRL, but NAL increased PRL secretion in rats treated with mifepristone (p < 0.05 vs. vehicle). Hydrocortisone significantly reduced PRL values at 18.00 hr, and NAL reversed this effect. ESfect of progesterone or progesterone antiserum and NAL on serum PRL levels in oestrogen-primed rats
Progesterone greatly increased serum PRL concentration at 13.00 hr @ < 0.001 vs. vehicle). This increase was partially prevented by NAL (Fig. 2). Progesterone antiserum did not modify serum PRL values at 13 .OOhr and did not prevent the increase in PRL secretion after NAL (Fig. l(A), Fig. 2(A)). At 18.00 hr, progesterone antiserum increased @I<0.05) serum PRL concentration and NAL in combination with antiserum, decreased PRL secretion (Fig. 2(B)). At 18.00 hr progesterone increased PRL secretion (p c 0.05) less effectively than at 13.00 hr (p < 0.05), but NAL did not modify this response (Fig. 2(B)). DISCUSSION The present results provide evidence that the inhibition of PRL secretion by adrenal glucocorticoids in oestrogentreated ovariectomized rats (Caron et al., 1994) involves EOPs. The inhibitory action of EOPs on PRL secretion
600
studied at 13.00 or 18.00 hr. To statistically analyse results from the progesterone/progesterone antiserum experiment (groups 4 and 5), logarithmic transformation of data was applied. groups
500
‘-
RESULTS Effect of mifepristone, hydrocortisone and NAL on PRL secretion in oestrogen-primed ovan’ectomized rats In rats not primed with OB the serum PRL values were
low at 13.00 and 18.00 hr (4.9 f 1.3 and 3.0 + 0.5 ng/ ml, respectively), and were not altered by NAL and/or mifepristone (data not shown). In OB-primed rats, serum PRL concentration at 13.00 hr (Fig. l(A)) was significantly enhanced by NAL (p < 0.05). As we have reported previously (Caron et al., 1994), mifepristone treatment of OB-primed ovariectomized rats greatly increased (4-fold) serum PRL at 13.00 hr. This increase in serum PRL was not modified by NAL (Fig. 1). Serum PRL concentration was greater after mifepristone (with or without NAL) than after NAL alone (p < 0.05). Hydrocortisone did not significantly modify serum PRL at 13.00 hr nor the effect of NAL. At 18.00 hr (Fig.
A
##
600
lB 0 -
Vehicle Naloxone
400
z C 6 -m
300
g 5 b m
200
100
0
Fig. 2. Effects of progesterone antiserum, progesterone and naloxone on serum PRL levels in oestradiol benzoate-treated ovariectomized rats. (A) 13.00 hr and (B) 18.00 hr. Progesterone antiserum (Ab, 50 ~1, i.p.) was given at 08.00 and 13.30 hr on day 3 and the rats were blood-sampled at 13.00 hr and 18.00 hr. Progesterone (P, 2 mg/rat, s.c.) was injected at 08.OOhr or at 13.OOhr. Thirty minutes before sampling, the rats were given naloxone (2 mg/kg, i.p., solid bars) or vehicle (open bars). *p < 0.05 and **p c 0.01 vs. the respective group without naloxone. #D c 0.05 and #& < 0.01 vs. vehicle groups (horizontal bars). Values are mean f SEM.
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R. W. Carbn et al.
has been shown under different conditions (Singh et al., 1992; Soaje and Deis, 1994; Caron and Deis, 1996). Neither NAL nor mifepristone altered PRL secretion in rats not primed with oestrogen, in which basal levels of PRL were low, as expected (Cat-on et al., 1994); thus oestrogen stimulation of PRL secretion is necessary for the effects of glucocorticoids and opioids. NAL alone increased PRL secretion at 13.00 hr, demonstrating an inhibitory EOP tone on PRL secretion at this time. This EOP is not likely to be acting at the anterior pituitary gland, since opiates do not influence PRL secretion at this level (Wiesner et al., 1984). Consequently, NAL is concluded to be acting in the brain, either stimulating a PRL releasing factor or interfering with secretion of a PRL inhibiting factor. Mifepristone is a synthetic 19-nor steroid with high affinity for glucocorticoid and progesterone receptors (Philibert, 1984). It is a type II glucocorticoid receptor antagonist, while hydrocortisone and endogenous glucocorticoids have greater affinity for type I receptors (De Kloet et al., 1975; Veldhuis et al., 1982; Reul and De Kloet, 1985). Type II receptors are occupied when corticoid secretion is increased (Reul et al., 1987). Our results indicate endogenous glucocorticoid inhibition of PRL secretion via a type II receptor (at 13.00 hr) and availability of type I receptors, for hydrocortisone action, at 18.00 hr. Both receptors can evidently mediate glucocorticoid action on PRL secretion via EOPs. Since the specific progesterone antibody did not modify PRL secretion at 13.00 hr, while it was enhanced by mifepristone, blockade of progesterone action is unlikely to be responsible for the effect of mifepristone at this time; which can be concluded to be acting as a glucocorticoid antagonist. As expected (Caron et d., 1994), the removal of the action of circulating progesterone by the specific antiserum did not modify serum PRL at 13.00 hr, confirming that circulating progesterone is not active in inhibiting oestrogen-induced PRL secretion at this time, while exogenous progesterone had a potent stimulatory effect. Moreover, this marked stimulation by progesterone was mediated partly by a stimulatory EOP action, since it was reduced significantly by NAL treatment. Thus, glucocorticoids and progesterone can have opposite effects on PRL secretion at 13.00 hr and both effects are partly mediated by EOPs. We propose that progesterone treatment prevents the inhibitory regulation of oestrogen-induced PRL release by glucocorticoids and shifts the action of EOPs from inhibition to stimulation. However, in oestrogen-primed rats the dominant endogenous steroid influence is the inhibitory action of glucocorticoid. Oestrogen induced a larger increase in serum PRL levels at 18.00 hr than at 13.00 hr, as shown by others (Caligaris et al., 1974). The lack of a significant effect of mifepristone or NAL alone, but the stimulation of PRL secretion when given together, suggests that circulating glucocorticoids and EOPs weakly regulate the afternoon
PRL surge induced by oestrogen. Similarly, progesterone was weakly stimulatory at 18.00 hr; however, this action seemed to be independent of EOPs, unlike that at 13.00 hr. Singh et al. (1992) observed a suppression of PRL secretion in the afternoon by morphine in oestrogentreated ovariectomized rats. Our results indicate that EOPs, in the presence of endogenous steroids, do not inhibit PRL secretion in the afternoon. Indeed, following blockade of progesterone action with antiserum, PRL secretion was increased, but decreased by NAL, indicating a stimulatory action of glucocorticoid via EOPs in the absence of progesterone (Fig. 2(B)). The stimulatory effect of mifepristone alone on PRL secretion at 13 .OOhr, and the lack of an effect at 18.00 hr, could be due to a decrease in responsiveness of the system to the inhibitory effect of glucocorticoids, perhaps reflected in the higher PRL values in the vehicle controls at 18.00 hr. This difference is not likely to be due to fading of mifepristone action by 18.00 hr, since repeated injection of mifepristone (at 08.00 and 13.00 hr) did not increase serum PRL at 18.00 hr (results not shown). Furthermore, hydrocortisone injection produced a NAL-reversible inhibition of PRL secretion at 18.00 hr, despite the higher circulating corticosterone level in the afternoon in rats (Kiem et al., 1995). The bidirectional involvement of EOPs in the regulation of PRL release could be related to changes in opioid receptors and/or in brain levels of EOPs or could be a consequence of glucocorticoids and progesterone acting through different EOP mechanisms. However, no consistent effects of adrenalectomy or glucocorticoid replacement on hypothalamic opioid systems have been reported (Krieger et al., 1979; Lee et al., 1980; Lim et al., 1982; Bimberg et d., 1983; Vale et al., 1983; Beaulieu et al., 1988), although there are glucocorticoid receptors in hypothalamic P-endorphin neurones (Chao et al., 1989; Cintra and Bortolotti, 1992) and corticosterone enhances the production of hypothalamic p-endorphin in vitro (Yang et al., 1994). In conclusion, in oestrogen-primed ovariectomized rats the secretion of PRL at 13.00 hr is inhibited by glucocorticoid, partly through EOP action. Endogenous glucocorticoids may not participate in the regulation of the rise of PRL secretion at 18.00 hr, although EOP inhibition can still be demonstrated, particularly after inhibition of PRL secretion by hydrocortisone. In contrast, exogenous progesterone stimulates PRL secretion via EOPs at 13.00 hr, but not at 18.00 hr. At 18.00 hr endogenous progesterone may be masking a stimulatory action of endogenous glucocorticoid, also mediated by EOPs; this requires further investigation. EOPs mediate inhibitory actions of glucocorticoid and inhibitory actions of progesterone. These results indicate that the diurnal variation in PRL in oestrogen-primed ovariectomized rats results, in part, from interactions between changes in glucocorticoid secretion and EOP mechanisms.
Regulation of prolactin secretion by adrenal steroids Acknowledgements-The authors offer sincere thanks to Dr J. A. Russell for valuable discussion on the manuscript and to Mrs F. E. Guinazd de Di Nasso for her expert technical assistance. This work was supported by a Grant (PID 312210088) from Consejo National de Investigaciones Cientificas y TCcnicas (CONICET), Argentina.
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