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Early ontogeny of κ-opioid receptor regulation of prolactin secretion in the rat
DevelopmentalBrain Research, 37 (1987) 189-196 Elsevier
189
BRD 50646
Early ontogeny of r-opioid receptor regulation of prolactin secretion in the rat Lisa A. Bero, Scott N. Lurie and Cynthia M. Kuhn Department of Pharmacology, Duke UniversityMedical Center, Durham, NC27710 (U.S,A.) (Accepted 19 May 1987)
Key words: Prolactin; Development; Opioid; Opiate/~, 6, r U50488; Serotonin
Although both p- and r-opioid components of prolactin (PRL) secretion have been identified in the adult rat, the neural pathways through which these multiple receptor subtypes modulate PRL secretion have not been thoroughly investigated. The present study utilizes the differential ontogeny of opioid systems which alter PRL release to examine the mechanisms by which p- and r-receptors regulate prolactin. The responses of PRL, corticosterone and growth hormone to opioid receptor subtype-specificagonists were studied in neonatal rats. The PRL response to the r-agonist, U50488, preceded the response to the/.~-agonist, morphiceptin. Like adults, neonates demonstrated a growth hormone, but not a PRL, response to the delta agonist, [D-pen2,penS]enkephalin. U50488-induced PRL secretion was not attenuated by cyproheptadine in adults or neonates, suggesting that the r-opioid mechanism operates independently of serotonin. In contrast, the PRL response to morphine was attenuated in adult rats. In addition, U50488 decreased median eminence dopamine synthesis in both adults and neonates. These findings suggest that the early developing, serotonin-independent opioid regulation of PRL is mediated through r-receptors, while the later-developing mechanism which requires intact serotonergic transmission works through/.t-receptors, r-Receptors appear to regulate PRL secretion by directly inhibiting the activity of tuberoinfundibular dopamine neurons, while/~-receptorsmight regulate the tonic dopaminergic inhibition of PRL through a serotonergic pathway. INTRODUCTION Opioid peptides modulate the secretion of all of the anterior pituitary hormones, including prolactin (PRL). More than one opioid receptor subtype is associated with the regulation of each of the anterior pituitary hormones and both p- and r-receptors appear to modulate PRL secretion in the adult rat./~-and rselective antagonists attenuate morphine-induced PRL secretion 19,24,26, while putative/~- and r-selective opioid agonists stimulate P R L release 2°,25. The neural pathways through which r- and/~-receptors modify PRL secretion have not been thoroughly investigated. In the adult rat, opiates appear to increase PRL release through a hypothalamic mechanism involving both serotonergic and dopaminergic systems. Morphine-induced stimulation of PRL is attenuated by blockade of serotonergic func-
tion 1s'29. Morphine concomitantly increases serotonin turnover and decreases dopamine turnover in the hypothalamus 1'1°A4'16. In fact, decreased dopamine synthesis in the median eminence following morphine can be blocked by pretreatment with a serotonin antagonist or neurotoxin 9. Our laboratory has found that at least two opioid systems which are stimulatory to PRL secretion can be identified in the adult rat: one which matures early and acts independently of serotonin and another which matures later and requires intact serotonergic innervation. The proposed serotonergic mechanism for opiate-induced PRL secretion presents an apparent paradox when the relative ontogeny of opioid and serotonergic control of PRL is considered. Although the PRL response to morphine is present as early as 5 days of age after birth, the PRL response to serotonergic drugs does not develop until around day 15 (ref.
Correspondence: L.A. Bero. Present address: Department of Pharmacology, Box 0050, University of California - - San Francisco, San Francisco, CA 94143, U.S.A. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
190 5). In addition, disruption of serotonergic function attenuates morphine-induced PRL secretion in adult rats, but not neonatal rats 6. One possible explanation for these data is that two opioid receptor-subtype mechanisms exist, one of which is linked to serotonin and matures late. The goal of the present study is to characterize the ontogeny of p- and ~c-components of PRL secretion and to determine the role of serotonin in the PRL response to stimulation of each of these subtypes. Three specific results are demonstrated: (1) the PRL response to a x-selective agonist develops before the PRL response to a p-selective agonist; (2) ~c-induced PRL secretion is mediated through a non-serotonergic mechanism and (3) ~c-receptors act at the level of the hypothalamus to directly modify dopamine release into the pituitary portal circulation. MATERIALS AND METHODS Animals Experiments were performed using 10- or 50-dayold Sprague-Dawley rats obtained from Zivic-Miller Laboratories (Allison Park, PA). These ages were selected because we have shown previously that 10-day-old rats lack a PRL response to serotonin agonists but demonstrate a serotonin-independent opiate-induced PRL response. In contrast, PRL release in 50-day-old (adult) rats increases in response to serotonergic stimulation and disruption of serotonergic function attenuates opiate-induced PRL secretion 5'6. Six-day-old rats, which have a PRL response profile similar to the 10-day-old rats, were used for the p- and 6-agonist experiment to facilitate intracerebral injection of the drugs. Mothers with litters of both males and females were ordered for experiments with 6- or 10-day-old rats because we did not observe any sex differences in the PRL responses examined in this study in rats under 15 days of age. Litters were culled to 10 and all pups were cross-fostered at birth. Data reported for 50-day-old rats were obtained from male rats. The animals were housed in a vivarium with a 12 h light-12 h dark day-night cycle with food and water available ad libitum. To avoid differential maternal treatment, pups injected with drug or vehicle treatments were randomized among litters. Mother rats with litters were housed in individual plastic cages
and were transferred to the laboratory the day before the experiment to control for the stress of being moved from a familiar environment. Adult rats were housed 2 per cage in hanging wire cages and remained in the vivarium throughout the experiment. The 50-day-old rats were injected with saline once daily for two days before the experiment to minimize stress-induced changes in hormone secretion during the experiment. Neonatal rats were handled briefly and remained with their mothers throughout the experiment because even brief separation from the mother has been reported to disrupt hormone secretion in the rat pup 21. All experiments were completed between 08.00 and 11.00 h to control for diurnal variations in hormone secretion. Serum collection Animals were killed by decapitation and trunk blood was collected in polypropylene centrifuge tubes. Blood from two pups was pooled for each sample from rat pups. Fifty day old rats were removed individually from the animal housing room and immediately decapitated. The blood was allowed to clot on ice, then centrifuged under refrigeration at 3000 rpm for 20 min. Serum was stored at -20 °C until assayed. Brain dissection For the neurotransmitter turnover study, brains were dissected on ice immediately following decapitation. The median eminence was identified under a dissecting microscope, removed using iris scissors and immediately frozen on dry ice. The tissue was stored at -40 °C for no more than 2 weeks until assayed. Drug treatments The opiates tested were the putative ~c-agonist, U50488 (Upjohn, Kalamazoo, MI); the/~-selective agonist, [N-MePhe3,D-Pro4]morphiceptin (Peninsula Laboratories, Belmont, CA); the &selective agonist, [D-pen2,penS]enkephalin (Peninsula Laboratories, Inc.), the putative r-agonist, dynorphinl_13 (Sigma Chemicals, St. Louis, MO) and morphine HC1 (Merck and Company, Rahway, NJ). A dose-response curve was obtained for U50488 and a dose which produced a half-maximal PRL response (0.5 mg/kg, s.c.) was selected for the serotonin antagonist study. A dose of morphine (1 mg/kg, s.c.) which has
191 been previously determined in our laboratory to produce half-maximal PRL stimulation was used in the antagonist study. Rats were sacrificed 30 min after administration of morphine or U50488 when PRL secretion is maximal. Both morphine and U50488 were dissolved in saline. Because the peptides morphiceptin, dynorphin and [D-pen2,penS]enkephalin do not readily cross the blood-brain barrier, they were injected intracerebrally through bregma in 6-day-old rats using a Hamilton microliter syringe. The pups did not appear behaviorly stressed by this procedure, nor did basal hormone levels of saline-injected rats differ from those of non-injected pups. Morphiceptin (0.15 pg), [D-penZ,penS]enkephalin (0.19 ktg) and dynorphin (2 /~g) were dissolved in saline and injected in a volume of 10 ~1. Control rats received a 10-pl injection of saline at the same time. Pups were sacrificed at the time of maximal hormone secretion, 20 min following injection for the measurement of PRL and growth hormone, or 60 min after injection when corticosterone was measured. Cyproheptadine was used as a serotonin receptor antagonist, at a dose and time previously shown to selectively block serotonin agonist-induced hormone secretion 4'6. Cyproheptadine HCI was obtained from Sigma Chemicals (St. Louis, MO) and administered as a suspension in distilled water. Water was administered as the vehicle at the appropriate time for all cyproheptadine experiments. Rats were pretreated with cyproheptadine (10 mg/kg, i.p.) 45 min before injection of opiate or serotonergic agonists. Neurotransmitter synthesis studies Dopamine synthesis was calculated by measuring the increase in the accumulation of the precursor, 3,4-dihydroxyphenylalanine (DOPA), following administration of the amino acid decarboxylase inhibitor, m-hydroxybenzylhydrazine (NSD 1015) (75 mg/kg, i.p.). DOPA accumulation was measured in the median eminence, the site of dopamine nerve terminals which regulate PRL release. This method of estimating synthesis was selected because the D O P A accumulation determined correlates with tuberoinfundibular dopaminergic nerve activity8. This method provides a selective estimate of dopamine synthesis because dopamine nerve terminals outnumber norepinephrine nerve terminals in the medi-
an eminence and the turnover of norepinephrine is relatively slow compared to dopamine 7. A time course for the accumulation of DOPA was determined in 10- and 50-day-old rats. Accumulation of precursor was measured 15, 30 and 45 min after injection of NSD 1015 (75 mg/kg, i.p.). All U50488 experiments were done at 30 min, a time point during the linear phase of DOPA accumulation. A submaximal dose of U50488 (0.5 mg/kg, s.c.) or saline was injected at the same time as saline or NSD 1015 (75 mg/kg, s.c.) and rats were sacrificed 30 min later. Hormone assays Serum was analyzed for PRL and growth hormone (GH) by radioimmunoassay using reagents kindly supplied by Dr. A.F. Parlow of the Rat Pituitary Distribution Program of the NIAMDD. Data are expressed as ng/ml serum using PRL RP-3 and G H RP1 as standards. The EDs0s are 0.40 ng and 1.9 ng for the PRL and growth hormone assays, respectively. The limit of sensitivity is 200 pg for the PRL assay and 100 pg for the growth hormone assay. Corticosterone was measured by radioimmunoassay following extraction from serum with ethyl acetate. Antiserum was supplied by Radioassay Systems Laboratories (Carson, CA) and [3H]corticosterone was obtained from New England Nuclear. The corticosterone assay has an EDs0 of 120 pg and sensitivity of 30 Pg. The intra-assay coefficient of variation is 2% for each of the assays and the interassay coefficients of variation are 14%, 9% and 15%, respectively, for the PRL, G H and corticosterone assays. All samples from a single experiment were analyzed in the same assay. DOPA assay The median eminence was homogenized in 1 ml buffer (0.2 M HCIO 4, 0.5 mM EDTA, 0.5 mM sodium metabisulfite) using an ultrasonic cell disrupter and centrifuged in a Beckman microfuge for 3 min. The supernatant was transferred to another tube for extraction and the pellet was retained for later protein determination by the method of Lowry et al. 22. Following alumina extraction by a modification of the method of Anton and Sayre 2, the median eminence was assayed for D O P A by on-line trace enrichment HPLC using a modification of the method of
192 Kilts et al. 17. Standard curves were prepared using varying concentrations of norepinephrine, dopamine, epinephrine, D O P A and the internal standard, 3,4-dihydroxybenzylamine ( D H B A ) (Sigma Chemicals, St. Louis, MO). The extract was adsorbed onto a cation exchange precolumn (Lichrosorb K A T , E. Merck, Darmstadt, F.R.G.) using a mobile phase containing 0.02 M potassium acetate, 0.02 M citrate, 0.05 mM E D T A , pH 2.7. Catecholamines are eluted from the precolumn onto an analytical column (Econosphere C-18, 100 mm × 4.6 mm, 3 # m , Alltech Assoc., Deerfield, IL) using the second mobile phase consisting of 0.2 M potassium acetate, 0.02 M citrate, 0.75 M octane sulfanate, 0.5 mM E D T A , 4% acetonitrile, p H 2.7. Samples were quantitated by electrochemical detection using a TL-5A glassy carbon electrode and LC-4A controller (Bioanalytical Systems, West Lafayette, IN). The detector potential was set at +0.60 mV vs Ag/AgC1 reference electrode, the optimal oxidation potential for D O P A . The concentration of D O P A in an unknown sample was calculated by linear regression analysis of the D O P A standard curve. All values were first normalized for recovery using the peak height measurement of the internal standard, D H B A . The assay sensitivity is 10 pg catecholamine per sample.
TEN DAY OLD RATS
--~3
i
10
MORPHINE (Srng/kg)
T
Z 2 i..-
,5
i T T
o_
SALINE
025
0
5
U50488 (mg/kg, sc )
8
g Z O uJ
6
4 L.) O L) 0
SALINE
025
05
I0
MORPHINE (5mo/kg)
U50488 (rag / kg, s c)
Fig. 1. U50488 (0.25, 0.5, 1.0 mg/kg, s.c.), morphine (5 mg/kg, s.c.) or saline as administered to 10-day-old rats 30 min before blood collection, n = at least 6 for each dose. *, P < 0.05 vs saline (Duncan's Multiple Range Test for U50488 dose-response curve, Student's t-test for morphine).
Statistics
Data are expressed as the mean ___ S.E.M. The data were analyzed by one-way analysis of variance ( A N O V A ) to test for dose-response relationships, or two-way A N O V A to determine the significance of combined treatment effects. The data were then analyzed by Duncan's multiple range test to assess the significance of specific points. W h e n single comparisons were made, data were analyzed by Student's ttest.
RESULTS The ~:-agonist, U50488 produced a dose-related increase in both P R L (F = 12.61, P < 0.01) and corticosterone (F = 8.70, P < 0.01) secretion in 10-dayold rats (Fig. 1). In 50-day-old rats, U50488 also produced dose-related increases in P R L (F = 23.87, P < 0.01) and corticosterone (F = 74.04, P < 0.01) (Fig.
2). Morphine stimulated P R L release at both ages tested (Figs. 1 and 2). Additional opiate receptor subtype selective drugs TABLE I Prolactin responses to opioid receptor subtype agonists
Morphiceptin (0.15/~g), [D-pen:,penS]enkephalin (0.19 #g) or dynorphin (2 gg) was injected i.c. 20 rain before sacrifice. U50488 (1 mg/kg, s.c.) was injected 30 rain before sacrifice. Treatment
n
Prolactin (ng/ml)
Vehicle U50488 Vehicle Dynorphin Vehicle Morphiceptin Vehicle [D-pen2,penS]enkephalin
6 7 6 7 5 5 7 8
1.04 + 0,17 1.73 + 0.24* 0.91 _+0.07 1.42 _+0.21" 0.63 _+0.04 0.68 + 0.04 0.33 + 0.04 0.33 + 0.03
*P < 0.05 vs respective vehicle (Student's t-test).
193 ADULT RATS
ADULT RATS
25
10 DAY OLD RATS
16 14
20
[]Vehicle
[ ] Vehicle
[]Cy~,o
[ ] Cypro
12 A 15
10
u
g
¢-
Z
--8 Z
10
u 6 T
a. 2
SALINE
025
0S
10
50
(Smg/kg)
SALINE
U50488 ( m g / k g , s c )
J_
160
12o o 80 t3 u
40
I
/VORPHINE U504~
MORPHINE
SALINE
U50488
MORPHINE
Fig. 3. Ten- or 50-day-old rats were pretreated with cyproheptadine (10 mg/kg, i.p.) (striped bars) or vehicle (clear bars) 45 min before injection with saline, U50488 (0.5 mg/kg, s.c.) or morphine (1 mg/kg, s.c.). The animals were killed 30 min later and PRL was assayed, n = 10 for each group, except n = 6 for the two 50-day-old morphine groups. *, P < 0.05 vs saline, t = P < 0.05 vs morphine + vehicle (Duncan's Multiple Range Test).
T
SALINE
025
0 5
10
50
MORPHINE (5mg/kg}
U 5 0 4 8 8 (mg/kg, sc )
Fig. 2. U50488 (0.25, 0.5, 1.0, 5.0 mg/kg, s.c.), morphine (5 mg/kg, s.c.) or saline was administered to 50-day-old rats 30 min before blood collection, n = at least 6 for each dose. *, P < 0.05 vs saline (Duncan's Multiple Range Test for U50488 dose-response curve, Student's t-test for morphine).
were studied in the rat pup (Table I). Like U50488 (1 mg/kg, s.c.), dynorphin (2 gg, i.c.) stimulated P R L secretion in 6-day-old rats. In contrast, the/~-selective agonist, morphiceptin (0.15/~g, i.c.) increased serum corticosterone (saline: 5.5 + 0.5 (6), morphiceptin: 18.9 _+ 1.1 (6); P < 0.001) but did not stimulate P R L release (Table I). A 0-selective agonist, [Dpen2,penS]enkephalin (0.19/ag, i.c.) increased only growth h o r m o n e release (saline: 93 ___ 9 (7), enkephalin: 189 __. 16; P < 0.001), but not P R L (Table I) or corticosterone (saline: 4.1 + 0.6 (7), enkephalin: 3.9 + 0.6) release. The effect of the serotonin antagonist, cyproheptadine (10 mg/kg, i.p.) on the P R L response to U50488 (0.5 mg/kg, s.c.) or morphine (1 mg/kg, s.c.) was examined (Fig. 3). Cyproheptadine did not alter the P R L response to morphine (F = 0.27, n.s.) or U50488 (F = 0.002, n.s.) in 10-day-old rats. In con-
trast, cyproheptadine attenuated the P R L response to morphine (F = 11.80, P < 0.01), but not U50488 (F = 0.38, n.s.), in 50-day-old rats. In our earlier studies, cyproheptadine alone did not alter basal P R L levels in 10- or 50-day-old rats so the cyproheptadine plus saline data were not shown in this experiment. A time course of the accumulation of median eminence D O P A following administration of NSD1015 (75 mg/kg, i.p.) was determined in 10- (r = 0.9887)
TEN DAY OLDS-TIME COURSE OF DOPA ACCUMULATION
2
SALINE
l~
3b
~5
MINUTES AFTER NSD1015
Fig. 4. Ten-day-old rats were injected with NSD1015 (75 mg/kg, i.p.) 15, 30 or 45 min before sacrifice and the median eminence was assayed for DOPA as described in the text. n = 5 for each saline or NSD1015 time point.
194 50 DAY OLDS-TIME COURSE OF DOPA ACCUMULATION
_ii g
'I° I
i~
SALIINE
4t5
3o
MLNUTES AFTER NSD1015
Fig. 5. Fifty-day-old rats were injected with NSD1015 (75 mg/kg, i.p.), 15, 30 or 45 min before sacrifice and the median eminence was assayed for DOPA as described in the text. n = 5 for each saline or NSD1015 time point. and 50- (r = 0.9986)-day-old rats (Figs. 4 and 5). U50488 (0.5 mg/kg, s.c.), in a dose at which the P R L response was not attenuated by cyproheptadine, decreased median eminence D O P A accumulation in 10-day-old rats (F = 5.90, P < 0.05) (Table II) without altering basal D O P A levels in the median eminence. In 50-day-old rats, U50488 (0.5 mg/kg, s.c.) also decreased median eminence D O P A accumulation (F = 13.60, P < 0.01) (Table II). U50488 plus saline increased basal D O P A levels, suggesting that the actual decrease in dopamine synthesis following U50488 may be greater than that observed. DISCUSSION The present study demonstrates the regulation of TABLE II Effect of U50488 on dopamine synthesis
Rats were injected with NSD1015 (75 mg/kg, i.p.) and U50488 (0.5 mg/kg, s.c.) 30 min before sacrifice. D-fl-Dihydroxyphenylalanine (DOPA) was measured in the median eminence by HPLC as described in the text. Data are expressed Pg/k~gprotein, mean + S.E.M.
Saline + saline NSD + saline NSD + U50488 Saline + U50488
10 days old
50 days old
n
DOPA
n
DOPA
6 7 7 6
0.75 + 0.08 4.17 +0.26" 3.17 +0.21"** 0.67 + 0.11
6 5 8 4
1.26 + 0.39 7.02 + 0.38* 4.97 +0.84"** 4.62 + 0.80*
*P < 0.05 vs saline + saline. **P < 0.05 vs NSD1015 + saline.
P R L secretion by at least two distinct opioid systems. These two systems show a differential ontogeny and appear to interact with different neurotransmitter systems. In contrast to studies in adults which suggest that mu receptors play a role in P R L regulation 19'z426, the p-agonist morphiceptin did not increase P R L release in 10-day-old rat pups. g-Receptors do appear to play a role in the early regulation of P R L secretion as the ~c-agonist, U50488, stimulates P R L secretion in a dose-dependent manner in both adults and neonates. Increases in P R L secretion in response to U50488 and other ~c-agonists have been previously reported in adult male rats e°'25. Athough we found that dynorphin, a putative ligand for ~c-receptors, stimulates P R L secretion in neonates, other studies from our laboratory suggest that dynorphin is not as selective for 7c-receptors as U50488. Therefore, U50488 was used for detailed characterization of the development of the P R L response to ~c-agonists. In summary, these agonist experiments suggest that the P R L response to ~c-receptor stimulation develops before the P R L response to p-receptor activation. In contrast to the P R L response, the growth hormone and corticosterone responses to opioid receptor subtype agonists are comparable in neonates and adults, suggesting that the opioid regulation of these hormones matures at an early age. Both ~c- and p-receptors have been implicated in the control of corticosterone secretion in the adult r a t l'11'25-27 and in the present study, morphiceptin and U50488, but not [Dpen2,penS]enkephalin, increase corticosterone secretion in neonates. Delta opiate receptors appear to regulate growth hormone, but not PRL, secretion in adult rats 19'24. The current finding that neonatal rats demonstrate increased growth hormone secretion, but not P R L secretion, to [D-pen2,penS]enkephalin suggests that d-receptor control of growth hormone secretion matures early. Furthermore, our previous studies show that opiates stimulate growth h o r m o n e through similar neurotransmitter mechanisms in both pups and adults 4, while they modulate P R L through different mechanisms 6. Our previous demonstration that the early-developing P R L response to opiates is serotonin-independent 6, suggests that the P R L response to kappa receptors might also be serotonin-independent. The finding that cyproheptadine does not block the P R L response to U50488 in neonates or adults supports
195 this hypothesis, as does the finding that morphine-induced PRL secretion was attenuated by cyproheptadine in adult rats, but not neonatal rats. Because morphine is relatively non-selective at different opiate receptor subtypes, we suggest that morphine acts at kappa receptors to stimulate PRL release in 10-day-old rats. The existence of an opiate receptor subtype mechanism which functions in neonates and acts independently of serotonin is consistent with our earlier findings that 10-day-old rats do not demonstrate a PRL response to serotonin agonists 5. In adult rats, the ability of cyproheptadine to attenuate morphine-induced PRL release suggests that serotonin mediates the actions of p-receptors on PRL secretion. The low dose of morphine used in the present study would optimize morphine actions at the preceptor in adult rats. Indeed, we have found that the PRL response to higher doses of morphine, which probably act at both p- and r-receptors, are not as completely blocked by cyproheptadine. The demonstration of a PRL response to U50488 which is not attenuated by cyproheptadine suggests that the earlydeveloping r-component of PRL regulation persists into adulthood, and that an additional serotonin-dependent pathway is superimposed on the neonatal mechanism. To investigate the mechanism of the proposed early developing, serotonin-independent r-opioid component of PRL regulation, we examined the effect of U50488 on tuberoinfundibular dopamine neuron activity. Because tonic dopaminergic regulation of PRL secretion is functional early in development 5,23, modulation of dopamine release represents a potential mechanism for opioid control of neonatal PRL secretion. The median eminence DOPA accumulation following NSD1015 observed in this study was similar in magnitude and time course to that reported by others for adult 7 and neonatal 16 rats. The elevation of basal DOPA levels in adult rats following U50488 suggests that U50488 might inhibit decarboxylase activity. The robust decrease in NSD1015induced D O P A accumulation observed after U50488 treatment suggests that this x-agonist decreases dopamine release into the pituitary portal circula-
tion, thereby removing tonic dopaminergic inhibition and increasing PRL release. These findings do not preclude the possibility that r-agonists act directly at the level of the pituitary to trigger PRL release. However, opiates do not have direct pituitary actions in adult rats 13'28and the similarity of the effect of U50488 on DOPA turnover in both neonates and adults suggests that the predominant r-receptor action occurs at the hypothalamic dopamine neurons. The failure of cyproheptadine to alter the PRL-stimulating effect of U50488 suggests further that serotonin neurons are not involved in xreceptor mediated PRL secretion. In contrast, morphine has been reported to increase serotonin turnover in the adult hypothalamus 15, suggesting a role for serotonin in the later-developing/~-receptor-mediated PRL secretion. In summary, the /~- and x-opiate receptors involved in PRL regulation exhibit a different functional ontogeny and appear to act through different mechanisms, r-Receptors might play an important role in the maintenance of appropriate PRL secretion sufficient for early functions of PRL such as sexual differentiation. The appearance of the later developing opioid mechanism correlates with the development of adult mechanisms which mediate surges of PRL secretion associated with maternal behavior and stress 12. A similar early ontogeny of the antinociceptive response to r-agonists has been reported 3, suggesting that the receptor subtypes mediating a variety of opioid effects may change during development. Furthermore, the present demonstration that x- and p-agonists seem to act through distinct neural pathways shows that opioids also continue to regulate PRL release through multiple mechanisms in the adult. ACKNOWLEDGEMENTS The authors wish to thank Aaron Miller for his valuable technical assistance. L.A.B. supported by Pharmaceutical Manufacturers' Association Predoctoral Fellowship. Research supported by D A 02739 to C.M.K.
196 REFERENCES 1 Alper, R.H., Demarest, K.T. and Moore, K.E., Morphine differentially alters synthesis and turnover of dopamine in central neuronal systems, J. Neural Trans., 48 (1980) 157-165. 2 Anton, A.H. and Sayre, D.F., A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines, J. Pharmacol. Exp. Ther., 138 (1962) 360-375. 3 Barr, G.A., Paredes, W., Erickson, K.L. and Zukin, R.S., Kappa opioid receptor-mediated analgesia in the developing rat, Dev. Brain Res., 29 (1986) 145-152. 4 Bero, L.A. and Kuhn, C.M., Catecholaminergic regulation of opiate-stimulated growth hormone secretion in the developing rat, J. Pharmacol. Exp. Ther., 237 (1986) 137-142. 5 Bero, L.A. and Kuhn, C.M., The differential ontogeny of opioid, dopaminergic and serotonergic regulation of prolactin secretion, J. Pharmacol. Exp. Ther., 240 (1987) 825-830. 6 Bero, L.A. and Kuhn, C.M., The role of serotonin in opiate-induced prolactin secretion and antinociception in the developing rat, J. Pharmacol. Exp. Ther., 240 (1987) 831-836. 7 Demarest, K.T., Alper, R.H. and Moore, K.E., DOPA accumulation is a measure of dopamine synthesis in the median eminence and posterior pituitary, J. Neural Transm., 46 (1979) 183-193. 8 Demarest, K.T. and Moore, K.E., Accumulation of LDOPA in the median eminence: an index of tuberoinfundibular dopaminergic nerve activity, Endocrinology, 106 (1980) 463-468. 9 Demarest, K.T. and Moore, K.E., Disruption of 5-hydroxytryptamine neuronal function blocks the action of morphine on tuberoinfundibular dopamine neurons, Life Sci., 28 (1981) 1345-1351. 10 Deyo, S.N., Swift, R.M. and Miller, R.J., Morphine and endorphins modulate dopamine turnover in rat median eminence, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3006-3009. 11 Eisenberg, R.M., Plasma corticosterone changes in response to central or peripheral administration of kappa and sigma opiate agonists, J~ Pharmacol. Exp. Ther., 233 (1985) 863-869. 12 Fenske, M. and Wuttke, W., Development of stress-induced pituitary prolactin and thyrotropin-stimulating hormone release in male rats, Acta. Endocrinol., 85 (1977) 729-735. 13 Grandison, L. and Guidotti, A., Regulation of prolactin release by endogenous opiates, Nature (London), 270 (1977) 357-359. 14 Gudelsky, G.A. and Porter, J.C., Morphine- and opioid peptide-induced inhibition of the release of dopamine from tuberoinfundibular neurons, Life Sci., 25 (1979) 1697-1702. 15 Johnston, C.A. and Moore, K.E., The effect of morphine on 5-hydroxytryptamine synthesis and metabolism in the
striatum, and several discreet hypothalamic regions of the brain, J. Neural Transm., 57 (1983) 65-73, 16 Johnston, C.A, and Negro-Vilar, A., Maturation of the prolactin and proopiomelanocortin-derived peptide responses to ether stress and morphine: neurochemical analysis, Endocrinology, 118 (1986) 797-804. 17 Kilts, C.D., Anderson, C.M., Ely, T. and Nishita, K., Absence of synthesis-modulating nerve terminal autoreceptors in mesoamygdaloid and other mesolimbic dopamine neuronal populations, submitted. 18 Koenig, J.I., Mayfield, M.A., Coppings, R.J., McCann, S.M. and Krulich, L., Role of central nervous neurotransmitters in mediating the effects of morphine on growth hormone and prolactin secretion in the rat, Brain Res., 197 (1980) 453-468. 19 Koenig, J.I., Mayfield, M.A., McCann, S.M. and Krulich, L., Differential role of the opioid mu and delta receptors in the activation of prolactin (PRL) and growth hormone (GH) secretion by morphine in the male rat, Life Sci., 34 (1984) 1829-1837. 20 Krulich, L., Koenig, J.I., Conway, S., McCann, S.M. and Mayfield, M.A., Opioid kappa receptors and the secretion of prolactin (PRL) and growth hormone (GH) in the rat, Neuroendocrinology, 42 (1986) 75-81. 21 Kuhn, C.M., Butler, S.R. and Schanberg, S.M., Selective depression of serum growth hormone during maternal deprivation in rat pups, Science, 201 (1978) 1034-1036. 22 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 23 Ojeda, S.R. and McCann, S.M., Development of dopaminergic and estrogenic control of prolactin release in the female rat, Endocrinology, 95 (1974) 1499-1505. 24 Panerai, A.E., Petraglia, F., Sacerdote, P. and Genazzani, A.R., Mainly mu-opiate receptors are involved in luteinizing hormone and prolactin secretion, Endocrinology, 117 (1985) 1096-1099. 25 Pechnick, R., George, R. and Poland, R.E., Identification of multiple opiate receptors through neuroendocrine responses. I. Effects of agonists, J. Pharmacol. Exp. Ther., 232 (1985) 163-169. 26 Pechnick, R., George, R. and Poland, R.E., Identification of multiple opiate receptors through neuroendocrine responses. II. Antagonism of mu, kappa and sigma agonists by naloxone and WIN 44,441-3, J. Pharmacol. Exp. Ther., 232 (1985) 170-177. 27 Pfeiffer, A. and Pfeiffer, D.G., Central kappa- and muopiate receptors may mediate the opiate-induced release of GH and ACTH in rats, Acta Endocrinol., 155, Suppl. 264 (1984) 40-41. 28 Rivier, C., Vale, W., Ling, N., Brown, M. and Guillemin, R., Stimulation in vivo of the secretion of prolactin and growth hormone by fl-endorphin, Endocrinology, 100 (1977) 238-241. 29 Spampinato, S., Locatelli, V., Cocchi, D., Vincentini, L., Bajusz, S., Ferri, S. and Muller, E., Involvement of brain serotonin in the prolactin-releasing effect of opioid peptides, Endocrinology, 105 (1979) 163-170.
189
BRD 50646
Early ontogeny of r-opioid receptor regulation of prolactin secretion in the rat Lisa A. Bero, Scott N. Lurie and Cynthia M. Kuhn Department of Pharmacology, Duke UniversityMedical Center, Durham, NC27710 (U.S,A.) (Accepted 19 May 1987)
Key words: Prolactin; Development; Opioid; Opiate/~, 6, r U50488; Serotonin
Although both p- and r-opioid components of prolactin (PRL) secretion have been identified in the adult rat, the neural pathways through which these multiple receptor subtypes modulate PRL secretion have not been thoroughly investigated. The present study utilizes the differential ontogeny of opioid systems which alter PRL release to examine the mechanisms by which p- and r-receptors regulate prolactin. The responses of PRL, corticosterone and growth hormone to opioid receptor subtype-specificagonists were studied in neonatal rats. The PRL response to the r-agonist, U50488, preceded the response to the/.~-agonist, morphiceptin. Like adults, neonates demonstrated a growth hormone, but not a PRL, response to the delta agonist, [D-pen2,penS]enkephalin. U50488-induced PRL secretion was not attenuated by cyproheptadine in adults or neonates, suggesting that the r-opioid mechanism operates independently of serotonin. In contrast, the PRL response to morphine was attenuated in adult rats. In addition, U50488 decreased median eminence dopamine synthesis in both adults and neonates. These findings suggest that the early developing, serotonin-independent opioid regulation of PRL is mediated through r-receptors, while the later-developing mechanism which requires intact serotonergic transmission works through/.t-receptors, r-Receptors appear to regulate PRL secretion by directly inhibiting the activity of tuberoinfundibular dopamine neurons, while/~-receptorsmight regulate the tonic dopaminergic inhibition of PRL through a serotonergic pathway. INTRODUCTION Opioid peptides modulate the secretion of all of the anterior pituitary hormones, including prolactin (PRL). More than one opioid receptor subtype is associated with the regulation of each of the anterior pituitary hormones and both p- and r-receptors appear to modulate PRL secretion in the adult rat./~-and rselective antagonists attenuate morphine-induced PRL secretion 19,24,26, while putative/~- and r-selective opioid agonists stimulate P R L release 2°,25. The neural pathways through which r- and/~-receptors modify PRL secretion have not been thoroughly investigated. In the adult rat, opiates appear to increase PRL release through a hypothalamic mechanism involving both serotonergic and dopaminergic systems. Morphine-induced stimulation of PRL is attenuated by blockade of serotonergic func-
tion 1s'29. Morphine concomitantly increases serotonin turnover and decreases dopamine turnover in the hypothalamus 1'1°A4'16. In fact, decreased dopamine synthesis in the median eminence following morphine can be blocked by pretreatment with a serotonin antagonist or neurotoxin 9. Our laboratory has found that at least two opioid systems which are stimulatory to PRL secretion can be identified in the adult rat: one which matures early and acts independently of serotonin and another which matures later and requires intact serotonergic innervation. The proposed serotonergic mechanism for opiate-induced PRL secretion presents an apparent paradox when the relative ontogeny of opioid and serotonergic control of PRL is considered. Although the PRL response to morphine is present as early as 5 days of age after birth, the PRL response to serotonergic drugs does not develop until around day 15 (ref.
Correspondence: L.A. Bero. Present address: Department of Pharmacology, Box 0050, University of California - - San Francisco, San Francisco, CA 94143, U.S.A. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
190 5). In addition, disruption of serotonergic function attenuates morphine-induced PRL secretion in adult rats, but not neonatal rats 6. One possible explanation for these data is that two opioid receptor-subtype mechanisms exist, one of which is linked to serotonin and matures late. The goal of the present study is to characterize the ontogeny of p- and ~c-components of PRL secretion and to determine the role of serotonin in the PRL response to stimulation of each of these subtypes. Three specific results are demonstrated: (1) the PRL response to a x-selective agonist develops before the PRL response to a p-selective agonist; (2) ~c-induced PRL secretion is mediated through a non-serotonergic mechanism and (3) ~c-receptors act at the level of the hypothalamus to directly modify dopamine release into the pituitary portal circulation. MATERIALS AND METHODS Animals Experiments were performed using 10- or 50-dayold Sprague-Dawley rats obtained from Zivic-Miller Laboratories (Allison Park, PA). These ages were selected because we have shown previously that 10-day-old rats lack a PRL response to serotonin agonists but demonstrate a serotonin-independent opiate-induced PRL response. In contrast, PRL release in 50-day-old (adult) rats increases in response to serotonergic stimulation and disruption of serotonergic function attenuates opiate-induced PRL secretion 5'6. Six-day-old rats, which have a PRL response profile similar to the 10-day-old rats, were used for the p- and 6-agonist experiment to facilitate intracerebral injection of the drugs. Mothers with litters of both males and females were ordered for experiments with 6- or 10-day-old rats because we did not observe any sex differences in the PRL responses examined in this study in rats under 15 days of age. Litters were culled to 10 and all pups were cross-fostered at birth. Data reported for 50-day-old rats were obtained from male rats. The animals were housed in a vivarium with a 12 h light-12 h dark day-night cycle with food and water available ad libitum. To avoid differential maternal treatment, pups injected with drug or vehicle treatments were randomized among litters. Mother rats with litters were housed in individual plastic cages
and were transferred to the laboratory the day before the experiment to control for the stress of being moved from a familiar environment. Adult rats were housed 2 per cage in hanging wire cages and remained in the vivarium throughout the experiment. The 50-day-old rats were injected with saline once daily for two days before the experiment to minimize stress-induced changes in hormone secretion during the experiment. Neonatal rats were handled briefly and remained with their mothers throughout the experiment because even brief separation from the mother has been reported to disrupt hormone secretion in the rat pup 21. All experiments were completed between 08.00 and 11.00 h to control for diurnal variations in hormone secretion. Serum collection Animals were killed by decapitation and trunk blood was collected in polypropylene centrifuge tubes. Blood from two pups was pooled for each sample from rat pups. Fifty day old rats were removed individually from the animal housing room and immediately decapitated. The blood was allowed to clot on ice, then centrifuged under refrigeration at 3000 rpm for 20 min. Serum was stored at -20 °C until assayed. Brain dissection For the neurotransmitter turnover study, brains were dissected on ice immediately following decapitation. The median eminence was identified under a dissecting microscope, removed using iris scissors and immediately frozen on dry ice. The tissue was stored at -40 °C for no more than 2 weeks until assayed. Drug treatments The opiates tested were the putative ~c-agonist, U50488 (Upjohn, Kalamazoo, MI); the/~-selective agonist, [N-MePhe3,D-Pro4]morphiceptin (Peninsula Laboratories, Belmont, CA); the &selective agonist, [D-pen2,penS]enkephalin (Peninsula Laboratories, Inc.), the putative r-agonist, dynorphinl_13 (Sigma Chemicals, St. Louis, MO) and morphine HC1 (Merck and Company, Rahway, NJ). A dose-response curve was obtained for U50488 and a dose which produced a half-maximal PRL response (0.5 mg/kg, s.c.) was selected for the serotonin antagonist study. A dose of morphine (1 mg/kg, s.c.) which has
191 been previously determined in our laboratory to produce half-maximal PRL stimulation was used in the antagonist study. Rats were sacrificed 30 min after administration of morphine or U50488 when PRL secretion is maximal. Both morphine and U50488 were dissolved in saline. Because the peptides morphiceptin, dynorphin and [D-pen2,penS]enkephalin do not readily cross the blood-brain barrier, they were injected intracerebrally through bregma in 6-day-old rats using a Hamilton microliter syringe. The pups did not appear behaviorly stressed by this procedure, nor did basal hormone levels of saline-injected rats differ from those of non-injected pups. Morphiceptin (0.15 pg), [D-penZ,penS]enkephalin (0.19 ktg) and dynorphin (2 /~g) were dissolved in saline and injected in a volume of 10 ~1. Control rats received a 10-pl injection of saline at the same time. Pups were sacrificed at the time of maximal hormone secretion, 20 min following injection for the measurement of PRL and growth hormone, or 60 min after injection when corticosterone was measured. Cyproheptadine was used as a serotonin receptor antagonist, at a dose and time previously shown to selectively block serotonin agonist-induced hormone secretion 4'6. Cyproheptadine HCI was obtained from Sigma Chemicals (St. Louis, MO) and administered as a suspension in distilled water. Water was administered as the vehicle at the appropriate time for all cyproheptadine experiments. Rats were pretreated with cyproheptadine (10 mg/kg, i.p.) 45 min before injection of opiate or serotonergic agonists. Neurotransmitter synthesis studies Dopamine synthesis was calculated by measuring the increase in the accumulation of the precursor, 3,4-dihydroxyphenylalanine (DOPA), following administration of the amino acid decarboxylase inhibitor, m-hydroxybenzylhydrazine (NSD 1015) (75 mg/kg, i.p.). DOPA accumulation was measured in the median eminence, the site of dopamine nerve terminals which regulate PRL release. This method of estimating synthesis was selected because the D O P A accumulation determined correlates with tuberoinfundibular dopaminergic nerve activity8. This method provides a selective estimate of dopamine synthesis because dopamine nerve terminals outnumber norepinephrine nerve terminals in the medi-
an eminence and the turnover of norepinephrine is relatively slow compared to dopamine 7. A time course for the accumulation of DOPA was determined in 10- and 50-day-old rats. Accumulation of precursor was measured 15, 30 and 45 min after injection of NSD 1015 (75 mg/kg, i.p.). All U50488 experiments were done at 30 min, a time point during the linear phase of DOPA accumulation. A submaximal dose of U50488 (0.5 mg/kg, s.c.) or saline was injected at the same time as saline or NSD 1015 (75 mg/kg, s.c.) and rats were sacrificed 30 min later. Hormone assays Serum was analyzed for PRL and growth hormone (GH) by radioimmunoassay using reagents kindly supplied by Dr. A.F. Parlow of the Rat Pituitary Distribution Program of the NIAMDD. Data are expressed as ng/ml serum using PRL RP-3 and G H RP1 as standards. The EDs0s are 0.40 ng and 1.9 ng for the PRL and growth hormone assays, respectively. The limit of sensitivity is 200 pg for the PRL assay and 100 pg for the growth hormone assay. Corticosterone was measured by radioimmunoassay following extraction from serum with ethyl acetate. Antiserum was supplied by Radioassay Systems Laboratories (Carson, CA) and [3H]corticosterone was obtained from New England Nuclear. The corticosterone assay has an EDs0 of 120 pg and sensitivity of 30 Pg. The intra-assay coefficient of variation is 2% for each of the assays and the interassay coefficients of variation are 14%, 9% and 15%, respectively, for the PRL, G H and corticosterone assays. All samples from a single experiment were analyzed in the same assay. DOPA assay The median eminence was homogenized in 1 ml buffer (0.2 M HCIO 4, 0.5 mM EDTA, 0.5 mM sodium metabisulfite) using an ultrasonic cell disrupter and centrifuged in a Beckman microfuge for 3 min. The supernatant was transferred to another tube for extraction and the pellet was retained for later protein determination by the method of Lowry et al. 22. Following alumina extraction by a modification of the method of Anton and Sayre 2, the median eminence was assayed for D O P A by on-line trace enrichment HPLC using a modification of the method of
192 Kilts et al. 17. Standard curves were prepared using varying concentrations of norepinephrine, dopamine, epinephrine, D O P A and the internal standard, 3,4-dihydroxybenzylamine ( D H B A ) (Sigma Chemicals, St. Louis, MO). The extract was adsorbed onto a cation exchange precolumn (Lichrosorb K A T , E. Merck, Darmstadt, F.R.G.) using a mobile phase containing 0.02 M potassium acetate, 0.02 M citrate, 0.05 mM E D T A , pH 2.7. Catecholamines are eluted from the precolumn onto an analytical column (Econosphere C-18, 100 mm × 4.6 mm, 3 # m , Alltech Assoc., Deerfield, IL) using the second mobile phase consisting of 0.2 M potassium acetate, 0.02 M citrate, 0.75 M octane sulfanate, 0.5 mM E D T A , 4% acetonitrile, p H 2.7. Samples were quantitated by electrochemical detection using a TL-5A glassy carbon electrode and LC-4A controller (Bioanalytical Systems, West Lafayette, IN). The detector potential was set at +0.60 mV vs Ag/AgC1 reference electrode, the optimal oxidation potential for D O P A . The concentration of D O P A in an unknown sample was calculated by linear regression analysis of the D O P A standard curve. All values were first normalized for recovery using the peak height measurement of the internal standard, D H B A . The assay sensitivity is 10 pg catecholamine per sample.
TEN DAY OLD RATS
--~3
i
10
MORPHINE (Srng/kg)
T
Z 2 i..-
,5
i T T
o_
SALINE
025
0
5
U50488 (mg/kg, sc )
8
g Z O uJ
6
4 L.) O L) 0
SALINE
025
05
I0
MORPHINE (5mo/kg)
U50488 (rag / kg, s c)
Fig. 1. U50488 (0.25, 0.5, 1.0 mg/kg, s.c.), morphine (5 mg/kg, s.c.) or saline as administered to 10-day-old rats 30 min before blood collection, n = at least 6 for each dose. *, P < 0.05 vs saline (Duncan's Multiple Range Test for U50488 dose-response curve, Student's t-test for morphine).
Statistics
Data are expressed as the mean ___ S.E.M. The data were analyzed by one-way analysis of variance ( A N O V A ) to test for dose-response relationships, or two-way A N O V A to determine the significance of combined treatment effects. The data were then analyzed by Duncan's multiple range test to assess the significance of specific points. W h e n single comparisons were made, data were analyzed by Student's ttest.
RESULTS The ~:-agonist, U50488 produced a dose-related increase in both P R L (F = 12.61, P < 0.01) and corticosterone (F = 8.70, P < 0.01) secretion in 10-dayold rats (Fig. 1). In 50-day-old rats, U50488 also produced dose-related increases in P R L (F = 23.87, P < 0.01) and corticosterone (F = 74.04, P < 0.01) (Fig.
2). Morphine stimulated P R L release at both ages tested (Figs. 1 and 2). Additional opiate receptor subtype selective drugs TABLE I Prolactin responses to opioid receptor subtype agonists
Morphiceptin (0.15/~g), [D-pen:,penS]enkephalin (0.19 #g) or dynorphin (2 gg) was injected i.c. 20 rain before sacrifice. U50488 (1 mg/kg, s.c.) was injected 30 rain before sacrifice. Treatment
n
Prolactin (ng/ml)
Vehicle U50488 Vehicle Dynorphin Vehicle Morphiceptin Vehicle [D-pen2,penS]enkephalin
6 7 6 7 5 5 7 8
1.04 + 0,17 1.73 + 0.24* 0.91 _+0.07 1.42 _+0.21" 0.63 _+0.04 0.68 + 0.04 0.33 + 0.04 0.33 + 0.03
*P < 0.05 vs respective vehicle (Student's t-test).
193 ADULT RATS
ADULT RATS
25
10 DAY OLD RATS
16 14
20
[]Vehicle
[ ] Vehicle
[]Cy~,o
[ ] Cypro
12 A 15
10
u
g
¢-
Z
--8 Z
10
u 6 T
a. 2
SALINE
025
0S
10
50
(Smg/kg)
SALINE
U50488 ( m g / k g , s c )
J_
160
12o o 80 t3 u
40
I
/VORPHINE U504~
MORPHINE
SALINE
U50488
MORPHINE
Fig. 3. Ten- or 50-day-old rats were pretreated with cyproheptadine (10 mg/kg, i.p.) (striped bars) or vehicle (clear bars) 45 min before injection with saline, U50488 (0.5 mg/kg, s.c.) or morphine (1 mg/kg, s.c.). The animals were killed 30 min later and PRL was assayed, n = 10 for each group, except n = 6 for the two 50-day-old morphine groups. *, P < 0.05 vs saline, t = P < 0.05 vs morphine + vehicle (Duncan's Multiple Range Test).
T
SALINE
025
0 5
10
50
MORPHINE (5mg/kg}
U 5 0 4 8 8 (mg/kg, sc )
Fig. 2. U50488 (0.25, 0.5, 1.0, 5.0 mg/kg, s.c.), morphine (5 mg/kg, s.c.) or saline was administered to 50-day-old rats 30 min before blood collection, n = at least 6 for each dose. *, P < 0.05 vs saline (Duncan's Multiple Range Test for U50488 dose-response curve, Student's t-test for morphine).
were studied in the rat pup (Table I). Like U50488 (1 mg/kg, s.c.), dynorphin (2 gg, i.c.) stimulated P R L secretion in 6-day-old rats. In contrast, the/~-selective agonist, morphiceptin (0.15/~g, i.c.) increased serum corticosterone (saline: 5.5 + 0.5 (6), morphiceptin: 18.9 _+ 1.1 (6); P < 0.001) but did not stimulate P R L release (Table I). A 0-selective agonist, [Dpen2,penS]enkephalin (0.19/ag, i.c.) increased only growth h o r m o n e release (saline: 93 ___ 9 (7), enkephalin: 189 __. 16; P < 0.001), but not P R L (Table I) or corticosterone (saline: 4.1 + 0.6 (7), enkephalin: 3.9 + 0.6) release. The effect of the serotonin antagonist, cyproheptadine (10 mg/kg, i.p.) on the P R L response to U50488 (0.5 mg/kg, s.c.) or morphine (1 mg/kg, s.c.) was examined (Fig. 3). Cyproheptadine did not alter the P R L response to morphine (F = 0.27, n.s.) or U50488 (F = 0.002, n.s.) in 10-day-old rats. In con-
trast, cyproheptadine attenuated the P R L response to morphine (F = 11.80, P < 0.01), but not U50488 (F = 0.38, n.s.), in 50-day-old rats. In our earlier studies, cyproheptadine alone did not alter basal P R L levels in 10- or 50-day-old rats so the cyproheptadine plus saline data were not shown in this experiment. A time course of the accumulation of median eminence D O P A following administration of NSD1015 (75 mg/kg, i.p.) was determined in 10- (r = 0.9887)
TEN DAY OLDS-TIME COURSE OF DOPA ACCUMULATION
2
SALINE
l~
3b
~5
MINUTES AFTER NSD1015
Fig. 4. Ten-day-old rats were injected with NSD1015 (75 mg/kg, i.p.) 15, 30 or 45 min before sacrifice and the median eminence was assayed for DOPA as described in the text. n = 5 for each saline or NSD1015 time point.
194 50 DAY OLDS-TIME COURSE OF DOPA ACCUMULATION
_ii g
'I° I
i~
SALIINE
4t5
3o
MLNUTES AFTER NSD1015
Fig. 5. Fifty-day-old rats were injected with NSD1015 (75 mg/kg, i.p.), 15, 30 or 45 min before sacrifice and the median eminence was assayed for DOPA as described in the text. n = 5 for each saline or NSD1015 time point. and 50- (r = 0.9986)-day-old rats (Figs. 4 and 5). U50488 (0.5 mg/kg, s.c.), in a dose at which the P R L response was not attenuated by cyproheptadine, decreased median eminence D O P A accumulation in 10-day-old rats (F = 5.90, P < 0.05) (Table II) without altering basal D O P A levels in the median eminence. In 50-day-old rats, U50488 (0.5 mg/kg, s.c.) also decreased median eminence D O P A accumulation (F = 13.60, P < 0.01) (Table II). U50488 plus saline increased basal D O P A levels, suggesting that the actual decrease in dopamine synthesis following U50488 may be greater than that observed. DISCUSSION The present study demonstrates the regulation of TABLE II Effect of U50488 on dopamine synthesis
Rats were injected with NSD1015 (75 mg/kg, i.p.) and U50488 (0.5 mg/kg, s.c.) 30 min before sacrifice. D-fl-Dihydroxyphenylalanine (DOPA) was measured in the median eminence by HPLC as described in the text. Data are expressed Pg/k~gprotein, mean + S.E.M.
Saline + saline NSD + saline NSD + U50488 Saline + U50488
10 days old
50 days old
n
DOPA
n
DOPA
6 7 7 6
0.75 + 0.08 4.17 +0.26" 3.17 +0.21"** 0.67 + 0.11
6 5 8 4
1.26 + 0.39 7.02 + 0.38* 4.97 +0.84"** 4.62 + 0.80*
*P < 0.05 vs saline + saline. **P < 0.05 vs NSD1015 + saline.
P R L secretion by at least two distinct opioid systems. These two systems show a differential ontogeny and appear to interact with different neurotransmitter systems. In contrast to studies in adults which suggest that mu receptors play a role in P R L regulation 19'z426, the p-agonist morphiceptin did not increase P R L release in 10-day-old rat pups. g-Receptors do appear to play a role in the early regulation of P R L secretion as the ~c-agonist, U50488, stimulates P R L secretion in a dose-dependent manner in both adults and neonates. Increases in P R L secretion in response to U50488 and other ~c-agonists have been previously reported in adult male rats e°'25. Athough we found that dynorphin, a putative ligand for ~c-receptors, stimulates P R L secretion in neonates, other studies from our laboratory suggest that dynorphin is not as selective for 7c-receptors as U50488. Therefore, U50488 was used for detailed characterization of the development of the P R L response to ~c-agonists. In summary, these agonist experiments suggest that the P R L response to ~c-receptor stimulation develops before the P R L response to p-receptor activation. In contrast to the P R L response, the growth hormone and corticosterone responses to opioid receptor subtype agonists are comparable in neonates and adults, suggesting that the opioid regulation of these hormones matures at an early age. Both ~c- and p-receptors have been implicated in the control of corticosterone secretion in the adult r a t l'11'25-27 and in the present study, morphiceptin and U50488, but not [Dpen2,penS]enkephalin, increase corticosterone secretion in neonates. Delta opiate receptors appear to regulate growth hormone, but not PRL, secretion in adult rats 19'24. The current finding that neonatal rats demonstrate increased growth hormone secretion, but not P R L secretion, to [D-pen2,penS]enkephalin suggests that d-receptor control of growth hormone secretion matures early. Furthermore, our previous studies show that opiates stimulate growth h o r m o n e through similar neurotransmitter mechanisms in both pups and adults 4, while they modulate P R L through different mechanisms 6. Our previous demonstration that the early-developing P R L response to opiates is serotonin-independent 6, suggests that the P R L response to kappa receptors might also be serotonin-independent. The finding that cyproheptadine does not block the P R L response to U50488 in neonates or adults supports
195 this hypothesis, as does the finding that morphine-induced PRL secretion was attenuated by cyproheptadine in adult rats, but not neonatal rats. Because morphine is relatively non-selective at different opiate receptor subtypes, we suggest that morphine acts at kappa receptors to stimulate PRL release in 10-day-old rats. The existence of an opiate receptor subtype mechanism which functions in neonates and acts independently of serotonin is consistent with our earlier findings that 10-day-old rats do not demonstrate a PRL response to serotonin agonists 5. In adult rats, the ability of cyproheptadine to attenuate morphine-induced PRL release suggests that serotonin mediates the actions of p-receptors on PRL secretion. The low dose of morphine used in the present study would optimize morphine actions at the preceptor in adult rats. Indeed, we have found that the PRL response to higher doses of morphine, which probably act at both p- and r-receptors, are not as completely blocked by cyproheptadine. The demonstration of a PRL response to U50488 which is not attenuated by cyproheptadine suggests that the earlydeveloping r-component of PRL regulation persists into adulthood, and that an additional serotonin-dependent pathway is superimposed on the neonatal mechanism. To investigate the mechanism of the proposed early developing, serotonin-independent r-opioid component of PRL regulation, we examined the effect of U50488 on tuberoinfundibular dopamine neuron activity. Because tonic dopaminergic regulation of PRL secretion is functional early in development 5,23, modulation of dopamine release represents a potential mechanism for opioid control of neonatal PRL secretion. The median eminence DOPA accumulation following NSD1015 observed in this study was similar in magnitude and time course to that reported by others for adult 7 and neonatal 16 rats. The elevation of basal DOPA levels in adult rats following U50488 suggests that U50488 might inhibit decarboxylase activity. The robust decrease in NSD1015induced D O P A accumulation observed after U50488 treatment suggests that this x-agonist decreases dopamine release into the pituitary portal circula-
tion, thereby removing tonic dopaminergic inhibition and increasing PRL release. These findings do not preclude the possibility that r-agonists act directly at the level of the pituitary to trigger PRL release. However, opiates do not have direct pituitary actions in adult rats 13'28and the similarity of the effect of U50488 on DOPA turnover in both neonates and adults suggests that the predominant r-receptor action occurs at the hypothalamic dopamine neurons. The failure of cyproheptadine to alter the PRL-stimulating effect of U50488 suggests further that serotonin neurons are not involved in xreceptor mediated PRL secretion. In contrast, morphine has been reported to increase serotonin turnover in the adult hypothalamus 15, suggesting a role for serotonin in the later-developing/~-receptor-mediated PRL secretion. In summary, the /~- and x-opiate receptors involved in PRL regulation exhibit a different functional ontogeny and appear to act through different mechanisms, r-Receptors might play an important role in the maintenance of appropriate PRL secretion sufficient for early functions of PRL such as sexual differentiation. The appearance of the later developing opioid mechanism correlates with the development of adult mechanisms which mediate surges of PRL secretion associated with maternal behavior and stress 12. A similar early ontogeny of the antinociceptive response to r-agonists has been reported 3, suggesting that the receptor subtypes mediating a variety of opioid effects may change during development. Furthermore, the present demonstration that x- and p-agonists seem to act through distinct neural pathways shows that opioids also continue to regulate PRL release through multiple mechanisms in the adult. ACKNOWLEDGEMENTS The authors wish to thank Aaron Miller for his valuable technical assistance. L.A.B. supported by Pharmaceutical Manufacturers' Association Predoctoral Fellowship. Research supported by D A 02739 to C.M.K.
196 REFERENCES 1 Alper, R.H., Demarest, K.T. and Moore, K.E., Morphine differentially alters synthesis and turnover of dopamine in central neuronal systems, J. Neural Trans., 48 (1980) 157-165. 2 Anton, A.H. and Sayre, D.F., A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines, J. Pharmacol. Exp. Ther., 138 (1962) 360-375. 3 Barr, G.A., Paredes, W., Erickson, K.L. and Zukin, R.S., Kappa opioid receptor-mediated analgesia in the developing rat, Dev. Brain Res., 29 (1986) 145-152. 4 Bero, L.A. and Kuhn, C.M., Catecholaminergic regulation of opiate-stimulated growth hormone secretion in the developing rat, J. Pharmacol. Exp. Ther., 237 (1986) 137-142. 5 Bero, L.A. and Kuhn, C.M., The differential ontogeny of opioid, dopaminergic and serotonergic regulation of prolactin secretion, J. Pharmacol. Exp. Ther., 240 (1987) 825-830. 6 Bero, L.A. and Kuhn, C.M., The role of serotonin in opiate-induced prolactin secretion and antinociception in the developing rat, J. Pharmacol. Exp. Ther., 240 (1987) 831-836. 7 Demarest, K.T., Alper, R.H. and Moore, K.E., DOPA accumulation is a measure of dopamine synthesis in the median eminence and posterior pituitary, J. Neural Transm., 46 (1979) 183-193. 8 Demarest, K.T. and Moore, K.E., Accumulation of LDOPA in the median eminence: an index of tuberoinfundibular dopaminergic nerve activity, Endocrinology, 106 (1980) 463-468. 9 Demarest, K.T. and Moore, K.E., Disruption of 5-hydroxytryptamine neuronal function blocks the action of morphine on tuberoinfundibular dopamine neurons, Life Sci., 28 (1981) 1345-1351. 10 Deyo, S.N., Swift, R.M. and Miller, R.J., Morphine and endorphins modulate dopamine turnover in rat median eminence, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3006-3009. 11 Eisenberg, R.M., Plasma corticosterone changes in response to central or peripheral administration of kappa and sigma opiate agonists, J~ Pharmacol. Exp. Ther., 233 (1985) 863-869. 12 Fenske, M. and Wuttke, W., Development of stress-induced pituitary prolactin and thyrotropin-stimulating hormone release in male rats, Acta. Endocrinol., 85 (1977) 729-735. 13 Grandison, L. and Guidotti, A., Regulation of prolactin release by endogenous opiates, Nature (London), 270 (1977) 357-359. 14 Gudelsky, G.A. and Porter, J.C., Morphine- and opioid peptide-induced inhibition of the release of dopamine from tuberoinfundibular neurons, Life Sci., 25 (1979) 1697-1702. 15 Johnston, C.A. and Moore, K.E., The effect of morphine on 5-hydroxytryptamine synthesis and metabolism in the
striatum, and several discreet hypothalamic regions of the brain, J. Neural Transm., 57 (1983) 65-73, 16 Johnston, C.A, and Negro-Vilar, A., Maturation of the prolactin and proopiomelanocortin-derived peptide responses to ether stress and morphine: neurochemical analysis, Endocrinology, 118 (1986) 797-804. 17 Kilts, C.D., Anderson, C.M., Ely, T. and Nishita, K., Absence of synthesis-modulating nerve terminal autoreceptors in mesoamygdaloid and other mesolimbic dopamine neuronal populations, submitted. 18 Koenig, J.I., Mayfield, M.A., Coppings, R.J., McCann, S.M. and Krulich, L., Role of central nervous neurotransmitters in mediating the effects of morphine on growth hormone and prolactin secretion in the rat, Brain Res., 197 (1980) 453-468. 19 Koenig, J.I., Mayfield, M.A., McCann, S.M. and Krulich, L., Differential role of the opioid mu and delta receptors in the activation of prolactin (PRL) and growth hormone (GH) secretion by morphine in the male rat, Life Sci., 34 (1984) 1829-1837. 20 Krulich, L., Koenig, J.I., Conway, S., McCann, S.M. and Mayfield, M.A., Opioid kappa receptors and the secretion of prolactin (PRL) and growth hormone (GH) in the rat, Neuroendocrinology, 42 (1986) 75-81. 21 Kuhn, C.M., Butler, S.R. and Schanberg, S.M., Selective depression of serum growth hormone during maternal deprivation in rat pups, Science, 201 (1978) 1034-1036. 22 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 23 Ojeda, S.R. and McCann, S.M., Development of dopaminergic and estrogenic control of prolactin release in the female rat, Endocrinology, 95 (1974) 1499-1505. 24 Panerai, A.E., Petraglia, F., Sacerdote, P. and Genazzani, A.R., Mainly mu-opiate receptors are involved in luteinizing hormone and prolactin secretion, Endocrinology, 117 (1985) 1096-1099. 25 Pechnick, R., George, R. and Poland, R.E., Identification of multiple opiate receptors through neuroendocrine responses. I. Effects of agonists, J. Pharmacol. Exp. Ther., 232 (1985) 163-169. 26 Pechnick, R., George, R. and Poland, R.E., Identification of multiple opiate receptors through neuroendocrine responses. II. Antagonism of mu, kappa and sigma agonists by naloxone and WIN 44,441-3, J. Pharmacol. Exp. Ther., 232 (1985) 170-177. 27 Pfeiffer, A. and Pfeiffer, D.G., Central kappa- and muopiate receptors may mediate the opiate-induced release of GH and ACTH in rats, Acta Endocrinol., 155, Suppl. 264 (1984) 40-41. 28 Rivier, C., Vale, W., Ling, N., Brown, M. and Guillemin, R., Stimulation in vivo of the secretion of prolactin and growth hormone by fl-endorphin, Endocrinology, 100 (1977) 238-241. 29 Spampinato, S., Locatelli, V., Cocchi, D., Vincentini, L., Bajusz, S., Ferri, S. and Muller, E., Involvement of brain serotonin in the prolactin-releasing effect of opioid peptides, Endocrinology, 105 (1979) 163-170.