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Mechanisms for the stimulatory effects of opioidergic and serotonergic input signals on prolactin in pregnant rats
Life Sciences, Vol. 50, pp. 1479-1489 Printed in the USA
Pergamon Press
MECHANISMS FOR THE STIMULATORY EFFECTS OF OPIOIDERGIC AND SEROTONERGIC INPUT SIGNALS ON PROLACTIN IN PREGNANT RATS Cathleen A. Sagrillo and James L. Voogt Department of Physlology The University of Kansas Medical Center Kansas City, Kansas 66103 (Received in final form March 3, 1992) Summary Dopamine (DA) neurons participate in tonic inhibition of prolactin (PRL), whereas B-endorphin (B-End) and serotonin (5-HT) neurons appear to be important stimulatory links for nocturnal PRL surges that occur throughout the first half of pregnancy in the rat. The purpose of this study was to determine how these neuronal components might be organized within the pathway controlling PRL release during gestation. Maximal stimulation of DA receptors with the agonist bromocriptlne mesylate (Bromo) completely blocked the PRL response to B-End (100 ng/~l/min for 15 min) given intracerebroventricularly (i.c.v.) on day 8 of pregnancy. DA receptor blockade, produced by implanting a 25 mg pellet of haloperidol (Hal) on day 7 of pregnancy, resulted in PRL levels of 500-600 ng/ml by the following morning. B-End i.c.v, or 250 mg/ml/kg BW of the D A synthesis inhibitor, a-methyl-p-tyrosine (a-MPT), given during the intersurge period, were equally effective in significantly increasing PRL (p < 0.01} above pretreatment levels. B-End and a-MPT evoked similar increases in rats pretreated with Hal, suggesting the stimulatory effect of B-End on nocturnal PRL surges may primarily be due to DA inhibition. The next objective was to determine how BEnd and 5-HT might interact to stimulate the nocturnal surge. Day 8 pregnant rats were infused continuously with the opioid receptor blocker, naloxone hydrochloride (Nal), at a rate of 2.0 mg/10 min from 1000-1300 h. The PRL response to an injection of 20 mg/kg BW 5-hydroxytryptophan (5-HTP) at 1200 h was greatly attenuated, compared to controls infused with saline instead of Nal. This suggests that 5-HT stimulates PRL, at least in part, by an action at opioid receptors. Distilled H20 or 10 mg/kg BW of the selective S 2 receptor blocker, ketanserin tartrate (Ket), was given intraperitoneally (i.p.) during the intersurge period on day 8 of pregnancy. All animals demonstrated an identical response to B-End given 2 hours later, regardless of the type of pretreatment. It appears that B-End does not stimulate PRL by way of an S 2 receptor. Although B-End induced a significant increase in PRL on day 16 of pregnancy, the response was attuenuated by more than 60% compared to the response on day 8 of pregnancy. This attenuation may involve placental lactogens, shown to be secreted during this time and to inhibit PRL secretion. Overall, the data from this report suggest that 5-HT acts through B-End, which in turn, inhibits DA, resulting in generation of a nocturnal PRL surge during early pregnancy, which is subsequently lost. Mating stimulates a neuroendocrine reflex in the rat w h i c h generates a twice daily pattern of prolactln (PRL) release (1-3). A diurnal and a nocturnal P R L s u r g e occur during the first ten days (4) of pregnancy (5) and pseudopregnancy (3,6). The surges stimulate the corpus luteum to secrete progesterone which is necessary to m a i n t a i n the integrity of these conditions (5,6). James L. Voogt, Department Center, Kansas City, Kansas
of Physiology, 66103
The
University
of
Kansas
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
Medical
1480
~-endorphin
and PRL During Pregnancy
Vol.
50, No. 20, 1992
A number of neuronal factors appear to play a role in the stimulation and inhibition of PRL (7). Studies suggest that endogenous opioid peptides (EOPs) play a significant role in regulating PRL increases that occur in various physiological conditions in the rat (8). Previous work in our laboratory demonstrated that EOPs may have a significant part in regulating the nocturnal PRL surges of pregnancy (9). Dopamine (DA) neurons provide the major tonic inhibition of PRL release (10). Hypothalamic opioids stimulate PRL release by inhibiting DA neuronal activity (11-15), release (14,16,17) and neurotransmitter synthesis (18,19). We have shown that EOPs may stimulate the nocturnal PRL surge in the pregnant rat by inhibiting the tuberoinfundibular DA (TIDA) neurons (9). fl-endorphin (B-End) m a y be the particular EOP involved (C.A. Sagrillo and J.L. Voogt, submitted for publication). Serotonin (5-HT) appears to be another important hypothalamic factor involved in regulating both the diurnal (20-22) and nocturnal (23) PRL surges. Little is known as to how 5-HT stimulates PRL release. Evidence suggests that 5-HT increases PRL by stimulating a PRL releasing factor (PRF) (24,25), as well as inhibiting DA (26). There is no agreement as to whether an EOP is involved (27,28). The goal of the present study was to determine how DA, B-End and 5-HT are involved in stimulating the nocturnal PRL surges of pregnancy. Experiments were designed based on the following objectives: i) to establish whether B-End stimulates the nocturnal PRL surge in the pregnant rat independent of DA; 2) to determine whether 5-HT stimulates the surge by acting through an opioidergic pathway; 3) to ascertain whether B-End stimulates the surge by way of a serotonergic mechanism; and 4) to measure the PRL response to exogenous B-End during late pregnancy, when PRL surges are absent and the PRL response to 5-HT has disappeared. Materials
and Methods
Animals Adult female Sprague-Dawley rats (Sasco Co, Omaha, NE) weighing 200-250 g were housed in a temperature (24°C) - and light (0600-1800 h)- controlled room. Animals had access to food and water ad libitum. Estrous cycles were monitored by daily vaginal lavage using physiological saline (Sal). A female was paired with a male on the day of proestrus for the purpose of mating. Visualization of sperm in the lavage on the following morning was designated day 0 of pregnancy. Surqical
Procedures
Rats were anesthetized with ketamine/l% acepromazine maleate (120 mg/kg BW) intramuscularly (i.m.) on day 1 or day 8 of pregnancy. A 21 gauge stainless steel outer cannula (Plastics One, Inc., Roanoke, VA) was placed chronically into the right lateral ventricle of the brain using a Kopf stereotaxic instrument. The cannula was implanted 1.0 mm posterior and 1.5 E ~ right of bregma according to the atlas of Paxinos and Watson (29). The shaft of the cannula was lowered 4.0 m m below the dorsal surface of the brain and then raised until a vertical column of artificial cerebral spinal fluid (ACSF) within an attached piece of PE 50 tubing began to fall. The cannula was raised another 0.5 m m so the tip was above the floor of the ventricle. The apparatus was cemented into place using dental acrylic adhered to 3 screws in the skull serving as the foundation. A 26 gauge dummy cannula extending 0 . S m m b e l o w the tip of the outer cannula was inserted until the day of the experiment. An injection of 15,000 IU of penicillin was given i.m. following the surgery. Cannulation of the left carotid artery and/or the right jugular vein was p e r f o r m e d under ether anesthesia on day 7 or 15 of pregnancy. Each cannula was exteriorized through the back of the neck and extended outside of the cage. Patency of the cannulas was maintained by flushing with a 20 IU/ml heparin-Sal (Hep-Sal) solution.
Vol. 50, No. 20, 1992
~-endorphin and PRL During Pregnancy
1481
Experimental Procedures Day 8 pregnant rats were connected to the right lateral ventricle infusion apparatus approximately 30 min before the beginning of the infusion. At this time, the dummy cannula was removed and replaced by an inner cannula which extended 0.5 ram below the tip of the outer cannula. An extension of PE 50 tubing approximately 55 cm long attached to the inner cannula contained either human B-End (Sigma Chemical Co., St. Louis, MO} or ACSF. The ACSF consisted of a modified Krebs-Ringer's phosphate solution (30) containing 123 m M N a C l , 10.2 m M Na2HPO4, 4.8 m M KCl, 1.8 m M NaH2PO4, 1.2 m M MgSO 4 and 0.78 m M CaCl2, adjusted to pH 7.4. At the time of the infusion, a 20 G1 Hamilton syringe filled with 15 ~i ACSF was inserted into the free end of the PE 50 tubing. The syringe delivered B-End (100 ng/~l ACSF/min) or ACSF (1 ~i/min) for 15 min from 1145-1200 h. Blood samples of approximately 250 ~i were taken -5, 15, 30, 60, 90 and 150 min relative to the beginning of the infusion. Each sample was replaced with an equal volume of 1:50 Hep-Sal solution in all experiments. In addition, animals were pretreated with one of several compounds preceding the infusion of B-End. One group was treated with 550 ~ g b r o m o c r i p tine m e s y l a t e / 1 0 0 ~i 50% ethanol-Sal intra-arterially (i.a.) (Bromo/EtOH-Sal; Sandoz Research Inst., East Hanover, NJ). A single blood sample was taken and Bromo was administered via the carotid artery at 1045 h, 1 hour before B-End. Control animals were injected with the EtOH-Sal vehicle solution (100 ~i), followed by B-End. In a second group, each animal was implanted subcutaneously (s.c.) with a 25 mg pellet of haloperidal (Hal; Innovative Research of America, Toledo, OH) on day 7 of pregnancy, at least 24 hours before B-End. Control rats were sham-operated and received the same B-End treatment on the following day. A third group was given i0 mg ketanserin tartrate/ml distilled water/kg BW (Ket/dH20; Janssen Pharmaceutica, Beerse, Belgium) or dH20 intraperitoneally (i.p.) at 0945 h, 2 hours preceding B-End. Blood samples were taken -5 and 30 min to monitor the initial effect of Ket on plasma PRL levels. In a separate experiment, day 8 or day 16 pregnant animals received the same regimen of B-End or ACSF treatment as above. In this study, however, the effect of B-End alone was the primary interest. In another experiment, H a l p e l l e t s were implanted on day 7 of pregnancy for at least 24 hours while control animals were sham-operated, as previously described. On the following day, rats were injected with 250 mg a-methyl-ptyrosine methyl ester, free base/ml Sal/kg BW (a-MPT; Sigma) through the carotid artery at 1145 h. Blood samples were taken -5, 15, 30, 60, 90 and 120 min relative to the time of the injection. In a final experiment, day 8 pregnant rats were connected to a peristaltic pump w h i c h delivered Sal (10 ~l/min) via the right Jugular vein beginning at 0930 h. The infusion of Sal was replaced with the infusion of 2.0 mg naloxone hydrochlorlde/10 min (Nal; Sigma) from 1000-1300 h, while controls continued to receive Sal during this time. In conjunction, an injection of 20 m g 5-hydroxyL-tryptophan/ml Sal/kg BW (5-HTP; Sigma) was given through the carotid artery at 1200 h. Blood samples were taken -5 and every 30 mln until the time of 5-HTP treatment. Thereafter, the frequency of sampling was increased to -5, 5, i0, 15, 30 and 60 min with respect to the time of the injection. Assays Blood was sampled via the left carotid artery cannula and collected into heparinized m i c r o c e n t r i f u g e tubes. Following each experiment, plasma was separated by centrifugation (10,000 x g for 2 min) and frozen at -20"C until assayed. Plasma samples were assayed for PRL at 5 and 25 ~i by the method of Niswender et al (31), using a standard double antibody RIA kit with PRL RP-I as the reference (NIDDK) and {125I} PRL (New England Nuclear, Boston MA) as the labeled antigen. The lower limit of sensitivity was 50 pg. The intraassay coefficient of variation was 12.1% and the interassay variation was 9.1%.
1482
~-endorphin
Statistical
and PRL D u r i n g P r e g n a n c y
Vol.
50, No.
20, 1992
Analyses
D a t a are e x p r e s s e d as t h e m e a n ± SE. D i f f e r e n c e s in p l a s m a P R L b e t w e e n t r e a t m e n t s w e r e e v a l u a t e d by 2 - w a y a n a l y s i s of v a r i a n c e f o l l o w e d by N e w m a n - K e u l s m u l t i p l e r a n g e t e s t or an u n p a i r e d S t u d e n t ' s t test. Differences were significant if p < 0.05. Results Bromo
blocks
B-End-induced
PRL release
The o b j e c t i v e of this e x p e r i m e n t was to d e t e r m i n e w h e t h e r B-End can s t i m u l a t e P R L release, e v e n w h e n DA r e c e p t o r s on t h e l a c t o t r o p h are m a x i m a l l y stimulated. F i g u r e 1 s u m m a r i z e s t h e e f f e c t of Bromo, a D 2 D A r e c e p t o r agonist, on B - E n d - i n d u c e d PRL r e l e a s e d u r i n g t h e i n t e r s u r g e p e r i o d in d a y 8 p r e g n a n t rats. C o n t r o l a n i m a l s p r e t r e a t e d w i t h t h e 50% E t O H - S a l v e h i c l e (i00 ~i i.a.) at 1045 h h a d a m i n o r i n c r e a s e in P R L i hour f o l l o w i n g t h e injection. Subsequent i n f u s i o n of B-End (i00 n g / ~ I / m i n for 15 m i n i.c.v.) b e g i n n i n g at 1145 h c a u s e d a d r a m a t i c i n c r e a s e in PRL to 1383 ng/ml by t h e e n d of t h e i n f u s i o n period. S t i m u l a t i o n w a s transient, however, and P R L r e t u r n e d to b a s a l levels by 1415 h. In contrast, p r e t r e a t m e n t w i t h B r o m o (550 ~ g / 1 0 0 ~i i.a.) a b o l i s h e d the s t i m u l a t o r y e f f e c t of B-End in e x p e r i m e n t a l animals. B r o m o treatment, f o l l o w e d by e i t h e r B-End or its A C S F v e h i c l e (i ~ I / m i n for 15 m i n i.c.v.), r e s u l t e d in s i m i l a r PRL v a l u e s that r e m a i n e d at basal l e v e l s t h r o u g h o u t the experiment.
1600 1400, ,~.
1200 -
c ~ c
101~-
~
800
o
~fo ~'
•
8oo -
400
-
200 0
"
I
A
I
" i
~
~
'
I
,
i
.
i
I
i
,
~
FIG.
1
Bromo blocked B-End-stimulated PRL release. D a y 8 p r e g n a n t rats w e r e p r e t r e a t e d w i t h an i.a. i n j e c t i o n of S r o m o (550 /~g) or its v e h i c l e at 1045 h, as d e n o t e d by the arrow. Animals were subseq u e n t l y a d m i n i s t e r e d an i.c.v, i n f u s i o n of e i t h e r B-End (i00 n g / ~ i / min) or A C S F from 1 1 4 5 - 1 2 0 0 h, as r e p r e s e n t e d by t h e line b e l o w t h e graph. T h e n u m b e r of rats in e a c h g r o u p r a n g e d from 5-7. Vertical lines r e p r e s e n t the SE of t h e m e a n s in this and all s u b s e q u e n t graphs. * V e h i c l e / B - E n d > Bromo/ACSF, B r o m o / B - E n d (p < 0.05). Both B-End
and a-MPT
This e x p e r i m e n t release, e v e n w h e n DA i m p l a n t e d s.c. w i t h a m o r n i n g of d a y 7 of
stimulate
FRL in H a l - t r e a t e d
rats
w a s p e r f o r m e d to d e t e r m i n e w h e t h e r B-End can s t i m u l a t e P R L r e c e p t o r s in the p i t u i t a r y are blocked. Each animal was p e l l e t of Hal (25 mg), a D A r e c e p t o r antagonist, on t h e pregnancy. As shown in F i g u r e 2, Hal e l e v a t e d PRL to
Vol. 50, No. 20, 1992
~-endorphin and PRL During Pregnancy
1483
500-600 ng/ml by the m o r n i n g of day 8 of pregnancy. Prl levels are normally less than 20 ng/ml during this time of day. Animals were then given B-End (100 ng/~l/min for 15 min i.c.v.) or the tyrosine hydroxylase inhibitor, ~-MPT (250 mg/ml/kg BW i.a.), at 1145 h and the resultant increases in PRL were compared between the two groups, a-MPT was administered to ascertain the extent of DA receptor blockade by the Hal treatment. Initially, B-End and a-MPT were equally effective in significantly increasing PRL (p < 0.01) during the first 30 min following treatment, compared to pretreatment levels. However, 1 hour after the injections, PRL continued to increase in the ~-MPT group, while levels remained stable or declined slowly in the rats administered B-End (p < 0.01). Thereafter, PRL decreased toward pretreatment levels in all animals, although it remained somewhat elevated in the a-MPT-treated group.
1800 "
1500 i
Hal/B-End al/MPT
~ c .,.., o ~
12~"
9OO.
a. ~
600 -
~
3OO -
,
,
|
,
FIG. 2 Both B-End and a-MPT induced a significant increase in PRL in rats pretreated with Hal. Animals were implanted with a pellet of Hal (25 mg) on the morning of day 7 of pregnancy. On the following day, animals received either a single i.a. injection of ~-MPT (250 m g / m l / k g BW) at 1145 h (arrow) or an i.c.v, infusion of B-End (I00 ng/~l/min from 1145-1200 h (line). PRL was significantly greater than pretreatment levels after 30 min of either treatment (p < 0.01). n = 8/group. * Hal/a-MPT > Hal/B-End (p < 0.01). B-End and a-MPT induce a similar increase in PRL in sham-operated animals The objective of the next experiment was to compare the stimulatory effects of B-End to a-MPT on PRL release in rats that were not pretreated with Hal, and therefore, having less DA receptor blockade. Animals were sham-operated (the control for groups in Figure 2) on the morning of day 7 of pregnancy and treated with B-End (100 ng/~i/min for 15 rain i.c.v. ) or a-MPT (250 mg/ml/kg BW i.a.), as above. Figure 3 shows that pretreatment PRL levels, measured at 1140 h, ranged from 16-35 ng/ml. Subsequent treatment with B-End stimulated a significant increase in PRL (p < 0.001) to 1462 ng/ml by the end of the infusion period. A similar significant increase (p < 0.001) was observed 30 rain following the injection of s-MPT. During the remaining time of the experiment, PRL continued to be significantly higher (p < 0.001) in the s-MPT group compared to B-End, where PRL declined gradually to basal levels.
1484
~-endorphin
and PRL D u r i n g P r e g n a n c y
Vol.
50, No. 20, 1992
211111 -
1800
E 1500 C
1200
Sham/8-End
9oo E
~o-
~ ~"
• 3OO -
0
FIG.
3
Both B-End and a-MPT stimulated PRL r e l e a s e in s h a m - o p e r a t e d c o n t r o l animals. This data p r o v i d e d t h e c o n t r o l for t h e r e s u l t s shown in F i g u r e 2. Rats w e r e s h a m - o p e r a t e d on t h e m o r n i n g of d a y 7 of pregnancy. On t h e next day, e i t h e r a s i n g l e i.a. i n j e c t i o n of a - M P T (250 m g / m l / k g BW) w a s g i v e n at 1145 h (arrow) or an i.c.v. i n f u s i o n of B-End (I00 ng/#I/min) was a d m i n i s t e r e d from 1 1 4 5 - 1 2 0 0 h (line). P l a s m a PRL i n c r e a s e d s i g n i f i c a n t l y a b o v e p r e t r e a t m e n t l e v e l s w i t h i n 30 m i n of e i t h e r i n j e c t i o n (p < 0.001). n = 6/group. * S h a m / a - M P T > S h a m / B - E n d (p < 0.01).
120
~,
100
•
E ol
Sal/5-HTP Nal/5-HTP
80-
C 60-
m
40.
E E.
2o.
0
i
i
i
8 o
.
i
i
i
i
i
A
saline
naloxone
FIG.
4
Nal a t t e n u a t e d t h e i n c r e a s e in PRL by 5-HTP. Experimental rats r e c e i v e d a c o n t i n u o u s i.v. i n f u s i o n of Nal (2.0 m g / 1 0 min) f r o m 1 0 0 0 - 1 3 0 0 h on d a y 8 of pregnancy, w h e r e a s c o n t r o l s r e c e i v e d Sal, as i n d i c a t e d by the line b e l o w t h e graph. In addition, an i.a. i n j e c t i o n of 5-HTP (20 m g / m l / k g BW) was a d m i n i s t e r e d at 1200 h (arrow). n = 6-7/group. * Values significantly different from N a l / 5 - H T P (p < 0.05).
Vol. 50, No. 20, 1992
Nal attenuates
~-endorphin
5-HTP-stimulated
and PRL During Pregnancy
1485
PRL release
The p u r p o s e of the fourth experiment was to determine w h e t h e r the 5-HT precursor, 5-HTP, stimulates PRL release on day 8 of p r e g n a n c y by w a y of opioid receptors. Experimental animals received a continuous infusion of the opioid receptor blocker, Nal (2.0 mg/100 ~i/10 m i n i.v.), w h i l e control animals were given a continuous infusion of Sal (100 ~1/i0 m i n i.v.) from 1000-1300 h. In addition, rats were injected w i t h 5-HTP (20 m g / m l / k g BW i.a.) at 1200 h. As shown in Figure 4, the control group d e m o n s t r a t e d an abrupt increase in PRL in response to the 5-HTP injection. On the other hand, the Nal g r o u p responded to 5-HTP with an increase in PRL that was attenuated by 75% compared to control animals. PRL declined toward pretreatment levels in both groups by 1300 h, 1 hour after treatment w i t h 5-HTP. These results suggest that 5-HT stimulates PRL, at least in part, by an action at opioid receptors. Ket does not b l o c k B-End-induced
PRL release
This experiment was designed to determine w h e t h e r B-End stimulates PRL release on day 8 of p r e g n a n c y by w a y of a serotonin S 2 receptor. Experimental animals were p r e t r e a t e d with Ket (10 mg/ml/kg BW i.p.), a selective S 2 receptor blocker, w h i l e control animals received an injection of dH20 (1 m l / k g BW i.p.) at 0945 h. This dose of Ket is effective in blocking PRL surges during p r e g n a n c y (20,23). Two hours later, an infusion of B-End (100 ng/~I/min for 15 m i n i.c.v.) was given from 1145-1200 h. Control and experimental groups showed an identical response to B-End, regardless of the type of pretreatment received. PRL increased d r a m a t i c a l l y to 1900-2150 ng/ml by the end of the infusion period and d e c r e a s e d to pretreatment levels during the next 2 hours. This suggests that B-End does not stimulate PRL via stimulation of an S 2 receptor. B-End-stimulated preqnanc7
PRL
release
is
qreatl 7
attenuated
on
day
16
vs
day
8
of
The last experiment was performed to compare the stimulatory effects of B-End on PRL release on day 8 vs day 16 of pregnancy. This was of interest because the nocturnal and diurnal PRL surges that occur on day 8, disappear by day 16. Figure 6 illustrates that an infusion of B-End (100 ng/~l/min i.c.v.) from 1145-1200 h on day 8 of pregnancy increased PRL to greater than 1400 ng/ml by the end of the infusion period. However, the response by day 16 pregnant rats to B-End was attenuated by more than 60% (p < 0.001). Differences in PRL were no longer present at 1245 h and both groups approached basal levels, as m e a s u r e d in A C S F - t r e a t e d control animals by 1315 h. Discussion The nocturnal P R L s u r g e s that occur throughout the first half of gestation in the rat appear to be regulated by a complex set of inhibitory and stimulatory neuronal interactions. DA is a major inhibitor of PRL release (10), whereas B-End (C.A. Sagri110 and J.L. Voogt, submitted for publication) and 5-HT (20,23) appear to be important stimulators of the surge. In this report, various p h a r m a c o l o g i c a l tools were employed to determine how DA, B-End and 5-HT neurons might be integrated within the pathway controlling the nocturnal PRL surges of pregnancy. All experiments were conducted during the intersurge period between 0900-1500 h in pregnant rats. During this time, the endogenous PRFs that stimulate PRL during the nocturnal surge do not appear to play an active role (32), resulting in basal levels of PRL release. Therefore, any change in PRL observed during an experiment can be attributed to the treatment in question, and not to an ongoing endogenous rhythmic event. The purpose of the first two experiments was to determine w h e t h e r B-End generates PRL surges in the pregnant rat by, in part, a m e c h a n i s m not involving DA. Day 8 pregnant rats were pretreated during the intersurge period w i t h a dose of Hromo known to m a x i m a l l y stimulate DA receptors. Subsequent treatment with B-End, shown to d r a m a t i c a l l y increase PRL release in controls (Figure 3), did not stimulate PRL above basal levels (Figure 1). This same observation was reported by other investigators (33,34). On the other hand, H a i s e n l e d e r et al (35) showed that TRH, a much weaker stimulator of PRL release, was able to
1486
~-endorphin
2400
A
2OO0
• o
121111
and PRL D u r i n g P r e g n a n c y
Vol.
50, No.
20, 1992
-
• dH20/8-End ,lO---KeUS-End c~ 1600 c n
~
8O0
a.
400
E
,¢: ¢- i"" •
•
i
•
A
i
•
l
,
i
•
J= i
~
FIG.
5
Ket d i d not inhibit B - E n d - s t i m u l a t e d PRL release. Experimental a n i m a l s r e c e i v e d an i.p. i n j e c t i o n of Ket (10 mg/ml/kg BW) w h i l e t h e c o n t r o l g r o u p w a s g i v e n v e h i c l e (dH20) at 0945 h on d a y 8 of pregnancy (arrow). B-End (i00 ng/#i/min) w a s i n f u s e d i.c.v. 2 h o u r s later from 1 1 4 5 - 1 2 0 0 h (line). n = 7-8/group.
1800 A
*
•
1500
¢= 1200 C 900 O 6O0 E U) ¢1 0. 3O00
~ i
ACSF 8-End-Day8
~ d ' D a l
16
._d
i
i FIG.
i
i
6
T h e P R L r e s p o n s e to B-End w a s g r e a t l y a t t e n u a t e d on d a y 16 vs d a y 8 of p r e g n a n c y . B-End (I00 ng/~I/min) or A C S F (i #i/min) w a s infused i.c.v, b e t w e e n 1 1 4 5 - 1 2 0 0 h (line) on d a y 8 or d a y 16 of p r e g nancy. E a c h d a t a p o i n t r e p r e s e n t s 5-6 rats/group. ** B - E n d - D a y 8 > B - E n d - D a y 16 (p < 0.001); * B-End-Day-8, 16 > A C S F (p < 0.05).
Vol.
50, No. 20, 1992
fl-endorphin and PRL During Pregnancy
increase PRL in pregnant rats in the presence of Bromo. B-End does not increase PRL by stimulating TRH.
This
1487
suggests
that
In the second experiment, D A receptors were b l o c k e d by implanting each pregnant rat with a pellet of Hal for 24 hours. This manner of drug delivery was chosen to p r o v i d e continuous blockade of D A receptors, resulting in steadystate levels of P R L b e t w e e n 500-600 ng/ml. Subsequent treatment with B-End, as illustrated in Figure 2, increased PRL even more w i t h i n 30 minutes of the infusion. Initially, the data suggested that the m e c h a n i s m of B-End-induced PRL release was irrespective of DA regulation, assuming that DA receptors were completely b l o c k e d by Hal. However, a-MPT, a DA synthesis inhibitor, stimulated PRL release to a similar m a g n i t u d e in a separate group of H a l - t r e a t e d animals. This suggests that DA receptors were not completely b l o c k e d by Hal and that a number of receptors were still available to bind DA. The observation that PRL continued to increase as the supply of DA was depleted by ~-MPT supports this theory. In this respect, our results in pregnant rats agree w i t h others w h o have found that DA receptor blockade (36) or DA synthesis inhibition (13) t o t a l l y b l o c k e d B-End-induced PRL release. This suggests that B-End-induced PRL release in the pregnant rat m a y be accounted for by inhibition of DA. The initial P R L r e s p o n s e to B-End was nearly identical in m a g n i t u d e to the response to s-MPT in both Hal-treated (Figure 2) and sham-operated control animals (Figure 3). However, the delayed action and longer lasting effects of a-MPT compared to B-End, characteristic of many synthesis inhibitors, suggest that B-End and a-MPT do not inhibit D A by the same mechanism. Also, it is possible that it takes longer for a-MPT to enter the cells, and therefore, its effect is delayed, or that B-End is m e t a b o l i z e d more rapidly. It is unclear whether opioids act directly on DA neurons or w h e t h e r they exert their P R L r e l e a s i n g effect through an interneuronal pathway. The results of Spampinato et al (27) suggest that EOPs interact with a serotonergic system to increase PRL release. On the other hand, Tache et al (28) d e m o n s t r a t e d that an intact serotonergic pathway was not necessary for EOPs to stimulate PRL release. Results from a previous study in our laboratory, using selective S 2 receptor blockers, suggest that 5-HT m a y act as an important neuromodulator of PRL release during p r e g n a n c y (20). In light of these conflicting data, a set of experiments w a s p e r f o r m e d to find whether an interrelationship exists between B-End and 5-HT neurons within the pathway controlling the nocturnal PRL surge. One experiment was designed to determine whether the 5-HT precursor, 5-HTP, stimulates PRL by w a y of an opioid interneuronal pathway. Pretreatment during the intersurge period with the opioid receptor blocker, Nal, greatly attenuated the PRL response to 5-HTP in day 8 pregnant rats (Figure 4). There was little doubt that the majority of opioid receptors was inhibited because the same dose of Nal completely blocked the nocturnal PRL surge (9). This suggests that 5-HT stimulates the nocturnal surge, in part, by way of an opioidergic interneuronal pathway. The alternative approach was taken in a separate experiment to determine w h e t h e r B-End stimulates the nocturnal surge by way of a serotonergic interneuronal pathway, specifically via S 2 receptors. Day 8 pregnant rats were pretreated during the intersurge period with Ket, a selective S 2 receptor antagonist, at a dose shown to inhibit the PRL surges during pregnancy (23). Blocking the 5-HT p a t h w a y in this manner did not impair the stimulation of PRL release by B-End (Figure 5). This data differs from the results of other researchers who have found that less specific 5-HT receptor antagonists b l o c k the PRL stimulatory effect of morphine sulfate in male rats (37-39). In our model of the pregnant rat, it appears that B-End does not increase PRL t h r o u g h a serotonergic pathway, at least not by w a y of S 2 receptors. A l t h o u g h D A neuronal activity was significantly increased by Nal (9), there was no effect on 5-HT neuronal activity (unpublished observations}. Again, these results imply an opioidergic action through DA neurons, and not 5-HT neurons, to stimulate the nocturnal PRL surge. A d m i n i s t r a t i o n of 5-HTP stimulated PRL release in day 8 pregnant rats, however, this stimulatory response disappeared by day 16 of pregnancy, w h e n PRL surges were absent as well (40). This suggested that the portion of the neuronal p a t h w a y controlling PRL release was no longer responsive to serotonergic input during late pregnancy. This also is partially true for B-End. Day
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Vol. 50, No. 20, 1992
16 pregnant rats were treated with B-End using the same regimen employed on day 8 of pregnancy (Figure 6). Even though B-End generated a significant increase in PRL, the response was greatly attenuated compared to the increase on day 8. Although the cause of this attenuation is not known, it may involve a change in the TIDA neurons because of their probable site for B-End's stimulatory effects. Perhaps the secretion of placental lactogens (PLs), thought to inhibit PRL secretion (41-44), stimulate the TIDA neurons and B-End's ability to inhibit these neurons is partially blocked. Precedence for this was shown in a study in which secretion of PL-I by tumor cells completely blocked the PRL response to serotonergic drugs (40). Future experiments will determine whether PL-I also blocks the stimulatory effect of B-End on PRL, and whether this is by way of TIDA neurons. Alternatively, the lactotroph may become unresponsive to changes in hypothalamic input during late pregnancy (35). By using various agonists and antagonists of DA, EOPs and 5-HT, we have provided additional information as to what factors might be involved in controlling the nocturnal PRL surges of pregnancy. It appears that 5-HT acts through B-End, which in turn, inhibits DA, resulting in PRL secretion. However, further research is needed to identify all of the interneuronal components of this intricate pathway. Acknowledqements Supported by NIH grant HD-24190. We thank Janssen Pharmaceutica, Beerse, Belgium, for providing ketanserin and the NIDDK for supplying the PRL RIA material. References i. 2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
R.L. BUTCHER, N.W. FUGO and W.E. COLLINS, Endocrinology 90 1125-1127 (1972). M.E. FREEMAN, M.S. SMITH, S.J. NAZIAN and J.D. NEILL, Endocrinology 94 875-882 (1974). M.S. SMITH, M.E. FREEMAN and J.D. NEILL, Endocrinology 9--6219-226 (1975). M.S. SMITH and J.D. NEILL, Endocrinology 98 696-701 (1976). W.K. MORISHIGE and I. ROTHCHILD, Endocrinology 95 260-274 (1974). M.S. SMITH, B.K. McLEAN and J.D. NEILL, Endocrinology 9__81370-1377 (1976). R.I.WEINER, P.R. FINDELL and C. KORDON, The Physiology of Reproduction, E. Knobil, J.D. Neill, L.L. Ewing, G.S. Greenwald, C.L. Markert and D.W. Pfaff (eds), 1235-1281, Raven Press, New York (1988). W.R. CROWLEY, Peptide Hormones: Effects and Mechanisms of Action, A. Negro-Vilar and P.M. Corm (eds), vol 3, 79-116, CRC Press, Boca Raton (1988). C.A. SAGRILLO and J.L. VOOGT, Endocrinology 1 2 9 9 2 5 - 9 3 0 (1991). N. BEN-JONATHAN, L.A. ARBOGAST and J.F. HYDE, Progress in Neurobiology 3_33 399-447 (1989). L. FERLAND, K. FUXE, P. ENEROTH, J.A. GUSTAFSSON and P. SKETT, Bur J Pharmacol 4__3389-90 (1977). S.N. DEYO, R.M. SWIFT and R.J. MILLER, Proc Natl Acad Sci 7-6 3006-3009 (1979). D.A. van VUGT, J.F. BRUNI, P.W. SYLVESTER, H.T. CHEN, T. IEIRI and J. MEITES, Life Sci 2-4 2361-2368 (1979). M.M. WILKES and S.S.C. YEN, Life Sci 2_/7 1387-1391 (1980). G.R. van LOON, D. HO and C. KIM, Endocrinology 106 76-80 (1980). G.A. GUDELSKY and J.C. PORTER, Life Sci 25 1697-1702 (1979). C.A. LEADEM, W.R. CROWLEY, J.W. SIMPKINS and S.P. KALRA, Neuroendocrinology 40 497-500 (1985). G.R. van LOON, E.B. de SOUZA, D. HO and S.H. SHIN, Can J Physiol Pharmacol 5-8 436-439 (1980). J. ARITA and F. KIMURA, Endocrinology 123 694-699 (1988). A. MISTRY and J.L. VOOGT, Endocrinology 125 2875-2880 (1989). B.J. AREY and M.E. FREEMAN, Endocrinology 126 279-284 (1990). G.A. JAHN and R.P. DEIS, J Endocr 117 415-422 (1988). A.M. MISTRY and J.L. VOOGT, Life Sci 4_/7 693-701 (1990). J.A. CLEMENS, M.E. ROUSH and R.W. FULLER, Life Sci 22 2209-2214 (1978). T.L. GARTHWAITE and T.C. HAGEN, Neuroendocrinology 29 215-220 (1979). N.S. PILOTTE and J.C. PORTER, Endocrinology 108 2137-2141 (1981). S. SPAMPINATO, V. LOCATELLI, D. COCCHI, L. VICENTINI, S. BAJUSZ, S. FERRI and E.E. MULLER, Endocrinology 105 163-170 (1979).
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38. 39. 40. 41. 42. 43. 44.
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and P R L D u r i n g
Pregnancy
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Y. TACHE, G. CHARPENET, M. CHRETIEN and R. COLLU, Central Nervous System Effects of Hypothalamic Hormones and Other Peptides, R. Collu, A. Barbeau, J.R. Ducharme and J.G. Rochefort (eds), 301-313, Raven Press, N e w Y o r k (1979). G. PAXINOS and C. WATSON, The Rat Brain in Stereotaxic Coordinates, ed 2, Academic Press, San Diego (1986). J.C. CHEN, K.K. RHEE, D.M. BEAUDRY and V.D. RAMIREZ, N e u r o e n d o c r i n o l o g y 38 362-370 (1984). G.D. NISWENDER, C.L. CHEN, A.R. MIDGLEY JR, J. MEITES and S. ELLIS, Proc Soc Exp Eiol M e d 130 793-797 (1969). B.J. AREY, R.L.W. A V E R I L L and M.E. FREEMAN, E n d o c r i n o l o g y 124 119-123 (1989). J. TAKAHARA, J. KAGEYAMA, S. YUNOKI, W. YAKUSHIJI, J. YAMAUCHI, N. K A G E Y A M A and T. OFUJI, Life Sci 22 2205-2208 (1978). G.R. van LOON, E.B. de SOUZA and S.H. SHIN, N e u r o e n d o c r i n o l o g y 3_~1292-296 (1980). D.J. HAISENLEDER, J.A. MOY, R.R. G A L A and D.M. LAWSON, E n d o c r i n o l o g y 119 1980-1988 (1986). T. OKAJIMA, T. MOTOMATSU, K. KATO and H. IBAYASHI, Life Sci 26 699-705 (1980). L. FERLAND, P.A. KELLY, F. DENIZEAU and F. LABRIE, C h a r a c t e r i s t i c s and Function of Opioids, J.M. van Ree and L. Terenius (eds), 353-354, E l s e v i e r / N o r t h Holland Biomedical Press, A m s t e r d a m (1978). J.I. KOENIG, M.A. MAYFIELD, S.M. McCANN and L. KRULICH, Life Sci 25 853-864 (1979). J. KOENIG, M.A. MAYFIELD, R.J. COPPINGS, S.M. McCANN and L. KRULICH, Brain Res 197 453-468 (1980). H. TOMOGANE, A.M. MISTRY and J.L. VOOGT, E n d o c r i n o l o g y 130 23-28 (1992). L. Y O G E V and J. TERKEL, J Endocr 84 421-424 (1980). J. VOOGT, M. ROBERTSON and H. FRIESEN, Biol Reprod 2-6 800-805 (1982). P.A. TONKOWICZ and J.L. VOOGT, Endocrinology 113 1314-1318 (1983). J. V O O G T and W.J. de GREEF, Proc Soc Exp Biol Med 191 403-407 (1989).
Pergamon Press
MECHANISMS FOR THE STIMULATORY EFFECTS OF OPIOIDERGIC AND SEROTONERGIC INPUT SIGNALS ON PROLACTIN IN PREGNANT RATS Cathleen A. Sagrillo and James L. Voogt Department of Physlology The University of Kansas Medical Center Kansas City, Kansas 66103 (Received in final form March 3, 1992) Summary Dopamine (DA) neurons participate in tonic inhibition of prolactin (PRL), whereas B-endorphin (B-End) and serotonin (5-HT) neurons appear to be important stimulatory links for nocturnal PRL surges that occur throughout the first half of pregnancy in the rat. The purpose of this study was to determine how these neuronal components might be organized within the pathway controlling PRL release during gestation. Maximal stimulation of DA receptors with the agonist bromocriptlne mesylate (Bromo) completely blocked the PRL response to B-End (100 ng/~l/min for 15 min) given intracerebroventricularly (i.c.v.) on day 8 of pregnancy. DA receptor blockade, produced by implanting a 25 mg pellet of haloperidol (Hal) on day 7 of pregnancy, resulted in PRL levels of 500-600 ng/ml by the following morning. B-End i.c.v, or 250 mg/ml/kg BW of the D A synthesis inhibitor, a-methyl-p-tyrosine (a-MPT), given during the intersurge period, were equally effective in significantly increasing PRL (p < 0.01} above pretreatment levels. B-End and a-MPT evoked similar increases in rats pretreated with Hal, suggesting the stimulatory effect of B-End on nocturnal PRL surges may primarily be due to DA inhibition. The next objective was to determine how BEnd and 5-HT might interact to stimulate the nocturnal surge. Day 8 pregnant rats were infused continuously with the opioid receptor blocker, naloxone hydrochloride (Nal), at a rate of 2.0 mg/10 min from 1000-1300 h. The PRL response to an injection of 20 mg/kg BW 5-hydroxytryptophan (5-HTP) at 1200 h was greatly attenuated, compared to controls infused with saline instead of Nal. This suggests that 5-HT stimulates PRL, at least in part, by an action at opioid receptors. Distilled H20 or 10 mg/kg BW of the selective S 2 receptor blocker, ketanserin tartrate (Ket), was given intraperitoneally (i.p.) during the intersurge period on day 8 of pregnancy. All animals demonstrated an identical response to B-End given 2 hours later, regardless of the type of pretreatment. It appears that B-End does not stimulate PRL by way of an S 2 receptor. Although B-End induced a significant increase in PRL on day 16 of pregnancy, the response was attuenuated by more than 60% compared to the response on day 8 of pregnancy. This attenuation may involve placental lactogens, shown to be secreted during this time and to inhibit PRL secretion. Overall, the data from this report suggest that 5-HT acts through B-End, which in turn, inhibits DA, resulting in generation of a nocturnal PRL surge during early pregnancy, which is subsequently lost. Mating stimulates a neuroendocrine reflex in the rat w h i c h generates a twice daily pattern of prolactln (PRL) release (1-3). A diurnal and a nocturnal P R L s u r g e occur during the first ten days (4) of pregnancy (5) and pseudopregnancy (3,6). The surges stimulate the corpus luteum to secrete progesterone which is necessary to m a i n t a i n the integrity of these conditions (5,6). James L. Voogt, Department Center, Kansas City, Kansas
of Physiology, 66103
The
University
of
Kansas
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
Medical
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A number of neuronal factors appear to play a role in the stimulation and inhibition of PRL (7). Studies suggest that endogenous opioid peptides (EOPs) play a significant role in regulating PRL increases that occur in various physiological conditions in the rat (8). Previous work in our laboratory demonstrated that EOPs may have a significant part in regulating the nocturnal PRL surges of pregnancy (9). Dopamine (DA) neurons provide the major tonic inhibition of PRL release (10). Hypothalamic opioids stimulate PRL release by inhibiting DA neuronal activity (11-15), release (14,16,17) and neurotransmitter synthesis (18,19). We have shown that EOPs may stimulate the nocturnal PRL surge in the pregnant rat by inhibiting the tuberoinfundibular DA (TIDA) neurons (9). fl-endorphin (B-End) m a y be the particular EOP involved (C.A. Sagrillo and J.L. Voogt, submitted for publication). Serotonin (5-HT) appears to be another important hypothalamic factor involved in regulating both the diurnal (20-22) and nocturnal (23) PRL surges. Little is known as to how 5-HT stimulates PRL release. Evidence suggests that 5-HT increases PRL by stimulating a PRL releasing factor (PRF) (24,25), as well as inhibiting DA (26). There is no agreement as to whether an EOP is involved (27,28). The goal of the present study was to determine how DA, B-End and 5-HT are involved in stimulating the nocturnal PRL surges of pregnancy. Experiments were designed based on the following objectives: i) to establish whether B-End stimulates the nocturnal PRL surge in the pregnant rat independent of DA; 2) to determine whether 5-HT stimulates the surge by acting through an opioidergic pathway; 3) to ascertain whether B-End stimulates the surge by way of a serotonergic mechanism; and 4) to measure the PRL response to exogenous B-End during late pregnancy, when PRL surges are absent and the PRL response to 5-HT has disappeared. Materials
and Methods
Animals Adult female Sprague-Dawley rats (Sasco Co, Omaha, NE) weighing 200-250 g were housed in a temperature (24°C) - and light (0600-1800 h)- controlled room. Animals had access to food and water ad libitum. Estrous cycles were monitored by daily vaginal lavage using physiological saline (Sal). A female was paired with a male on the day of proestrus for the purpose of mating. Visualization of sperm in the lavage on the following morning was designated day 0 of pregnancy. Surqical
Procedures
Rats were anesthetized with ketamine/l% acepromazine maleate (120 mg/kg BW) intramuscularly (i.m.) on day 1 or day 8 of pregnancy. A 21 gauge stainless steel outer cannula (Plastics One, Inc., Roanoke, VA) was placed chronically into the right lateral ventricle of the brain using a Kopf stereotaxic instrument. The cannula was implanted 1.0 mm posterior and 1.5 E ~ right of bregma according to the atlas of Paxinos and Watson (29). The shaft of the cannula was lowered 4.0 m m below the dorsal surface of the brain and then raised until a vertical column of artificial cerebral spinal fluid (ACSF) within an attached piece of PE 50 tubing began to fall. The cannula was raised another 0.5 m m so the tip was above the floor of the ventricle. The apparatus was cemented into place using dental acrylic adhered to 3 screws in the skull serving as the foundation. A 26 gauge dummy cannula extending 0 . S m m b e l o w the tip of the outer cannula was inserted until the day of the experiment. An injection of 15,000 IU of penicillin was given i.m. following the surgery. Cannulation of the left carotid artery and/or the right jugular vein was p e r f o r m e d under ether anesthesia on day 7 or 15 of pregnancy. Each cannula was exteriorized through the back of the neck and extended outside of the cage. Patency of the cannulas was maintained by flushing with a 20 IU/ml heparin-Sal (Hep-Sal) solution.
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~-endorphin and PRL During Pregnancy
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Experimental Procedures Day 8 pregnant rats were connected to the right lateral ventricle infusion apparatus approximately 30 min before the beginning of the infusion. At this time, the dummy cannula was removed and replaced by an inner cannula which extended 0.5 ram below the tip of the outer cannula. An extension of PE 50 tubing approximately 55 cm long attached to the inner cannula contained either human B-End (Sigma Chemical Co., St. Louis, MO} or ACSF. The ACSF consisted of a modified Krebs-Ringer's phosphate solution (30) containing 123 m M N a C l , 10.2 m M Na2HPO4, 4.8 m M KCl, 1.8 m M NaH2PO4, 1.2 m M MgSO 4 and 0.78 m M CaCl2, adjusted to pH 7.4. At the time of the infusion, a 20 G1 Hamilton syringe filled with 15 ~i ACSF was inserted into the free end of the PE 50 tubing. The syringe delivered B-End (100 ng/~l ACSF/min) or ACSF (1 ~i/min) for 15 min from 1145-1200 h. Blood samples of approximately 250 ~i were taken -5, 15, 30, 60, 90 and 150 min relative to the beginning of the infusion. Each sample was replaced with an equal volume of 1:50 Hep-Sal solution in all experiments. In addition, animals were pretreated with one of several compounds preceding the infusion of B-End. One group was treated with 550 ~ g b r o m o c r i p tine m e s y l a t e / 1 0 0 ~i 50% ethanol-Sal intra-arterially (i.a.) (Bromo/EtOH-Sal; Sandoz Research Inst., East Hanover, NJ). A single blood sample was taken and Bromo was administered via the carotid artery at 1045 h, 1 hour before B-End. Control animals were injected with the EtOH-Sal vehicle solution (100 ~i), followed by B-End. In a second group, each animal was implanted subcutaneously (s.c.) with a 25 mg pellet of haloperidal (Hal; Innovative Research of America, Toledo, OH) on day 7 of pregnancy, at least 24 hours before B-End. Control rats were sham-operated and received the same B-End treatment on the following day. A third group was given i0 mg ketanserin tartrate/ml distilled water/kg BW (Ket/dH20; Janssen Pharmaceutica, Beerse, Belgium) or dH20 intraperitoneally (i.p.) at 0945 h, 2 hours preceding B-End. Blood samples were taken -5 and 30 min to monitor the initial effect of Ket on plasma PRL levels. In a separate experiment, day 8 or day 16 pregnant animals received the same regimen of B-End or ACSF treatment as above. In this study, however, the effect of B-End alone was the primary interest. In another experiment, H a l p e l l e t s were implanted on day 7 of pregnancy for at least 24 hours while control animals were sham-operated, as previously described. On the following day, rats were injected with 250 mg a-methyl-ptyrosine methyl ester, free base/ml Sal/kg BW (a-MPT; Sigma) through the carotid artery at 1145 h. Blood samples were taken -5, 15, 30, 60, 90 and 120 min relative to the time of the injection. In a final experiment, day 8 pregnant rats were connected to a peristaltic pump w h i c h delivered Sal (10 ~l/min) via the right Jugular vein beginning at 0930 h. The infusion of Sal was replaced with the infusion of 2.0 mg naloxone hydrochlorlde/10 min (Nal; Sigma) from 1000-1300 h, while controls continued to receive Sal during this time. In conjunction, an injection of 20 m g 5-hydroxyL-tryptophan/ml Sal/kg BW (5-HTP; Sigma) was given through the carotid artery at 1200 h. Blood samples were taken -5 and every 30 mln until the time of 5-HTP treatment. Thereafter, the frequency of sampling was increased to -5, 5, i0, 15, 30 and 60 min with respect to the time of the injection. Assays Blood was sampled via the left carotid artery cannula and collected into heparinized m i c r o c e n t r i f u g e tubes. Following each experiment, plasma was separated by centrifugation (10,000 x g for 2 min) and frozen at -20"C until assayed. Plasma samples were assayed for PRL at 5 and 25 ~i by the method of Niswender et al (31), using a standard double antibody RIA kit with PRL RP-I as the reference (NIDDK) and {125I} PRL (New England Nuclear, Boston MA) as the labeled antigen. The lower limit of sensitivity was 50 pg. The intraassay coefficient of variation was 12.1% and the interassay variation was 9.1%.
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Statistical
and PRL D u r i n g P r e g n a n c y
Vol.
50, No.
20, 1992
Analyses
D a t a are e x p r e s s e d as t h e m e a n ± SE. D i f f e r e n c e s in p l a s m a P R L b e t w e e n t r e a t m e n t s w e r e e v a l u a t e d by 2 - w a y a n a l y s i s of v a r i a n c e f o l l o w e d by N e w m a n - K e u l s m u l t i p l e r a n g e t e s t or an u n p a i r e d S t u d e n t ' s t test. Differences were significant if p < 0.05. Results Bromo
blocks
B-End-induced
PRL release
The o b j e c t i v e of this e x p e r i m e n t was to d e t e r m i n e w h e t h e r B-End can s t i m u l a t e P R L release, e v e n w h e n DA r e c e p t o r s on t h e l a c t o t r o p h are m a x i m a l l y stimulated. F i g u r e 1 s u m m a r i z e s t h e e f f e c t of Bromo, a D 2 D A r e c e p t o r agonist, on B - E n d - i n d u c e d PRL r e l e a s e d u r i n g t h e i n t e r s u r g e p e r i o d in d a y 8 p r e g n a n t rats. C o n t r o l a n i m a l s p r e t r e a t e d w i t h t h e 50% E t O H - S a l v e h i c l e (i00 ~i i.a.) at 1045 h h a d a m i n o r i n c r e a s e in P R L i hour f o l l o w i n g t h e injection. Subsequent i n f u s i o n of B-End (i00 n g / ~ I / m i n for 15 m i n i.c.v.) b e g i n n i n g at 1145 h c a u s e d a d r a m a t i c i n c r e a s e in PRL to 1383 ng/ml by t h e e n d of t h e i n f u s i o n period. S t i m u l a t i o n w a s transient, however, and P R L r e t u r n e d to b a s a l levels by 1415 h. In contrast, p r e t r e a t m e n t w i t h B r o m o (550 ~ g / 1 0 0 ~i i.a.) a b o l i s h e d the s t i m u l a t o r y e f f e c t of B-End in e x p e r i m e n t a l animals. B r o m o treatment, f o l l o w e d by e i t h e r B-End or its A C S F v e h i c l e (i ~ I / m i n for 15 m i n i.c.v.), r e s u l t e d in s i m i l a r PRL v a l u e s that r e m a i n e d at basal l e v e l s t h r o u g h o u t the experiment.
1600 1400, ,~.
1200 -
c ~ c
101~-
~
800
o
~fo ~'
•
8oo -
400
-
200 0
"
I
A
I
" i
~
~
'
I
,
i
.
i
I
i
,
~
FIG.
1
Bromo blocked B-End-stimulated PRL release. D a y 8 p r e g n a n t rats w e r e p r e t r e a t e d w i t h an i.a. i n j e c t i o n of S r o m o (550 /~g) or its v e h i c l e at 1045 h, as d e n o t e d by the arrow. Animals were subseq u e n t l y a d m i n i s t e r e d an i.c.v, i n f u s i o n of e i t h e r B-End (i00 n g / ~ i / min) or A C S F from 1 1 4 5 - 1 2 0 0 h, as r e p r e s e n t e d by t h e line b e l o w t h e graph. T h e n u m b e r of rats in e a c h g r o u p r a n g e d from 5-7. Vertical lines r e p r e s e n t the SE of t h e m e a n s in this and all s u b s e q u e n t graphs. * V e h i c l e / B - E n d > Bromo/ACSF, B r o m o / B - E n d (p < 0.05). Both B-End
and a-MPT
This e x p e r i m e n t release, e v e n w h e n DA i m p l a n t e d s.c. w i t h a m o r n i n g of d a y 7 of
stimulate
FRL in H a l - t r e a t e d
rats
w a s p e r f o r m e d to d e t e r m i n e w h e t h e r B-End can s t i m u l a t e P R L r e c e p t o r s in the p i t u i t a r y are blocked. Each animal was p e l l e t of Hal (25 mg), a D A r e c e p t o r antagonist, on t h e pregnancy. As shown in F i g u r e 2, Hal e l e v a t e d PRL to
Vol. 50, No. 20, 1992
~-endorphin and PRL During Pregnancy
1483
500-600 ng/ml by the m o r n i n g of day 8 of pregnancy. Prl levels are normally less than 20 ng/ml during this time of day. Animals were then given B-End (100 ng/~l/min for 15 min i.c.v.) or the tyrosine hydroxylase inhibitor, ~-MPT (250 mg/ml/kg BW i.a.), at 1145 h and the resultant increases in PRL were compared between the two groups, a-MPT was administered to ascertain the extent of DA receptor blockade by the Hal treatment. Initially, B-End and a-MPT were equally effective in significantly increasing PRL (p < 0.01) during the first 30 min following treatment, compared to pretreatment levels. However, 1 hour after the injections, PRL continued to increase in the ~-MPT group, while levels remained stable or declined slowly in the rats administered B-End (p < 0.01). Thereafter, PRL decreased toward pretreatment levels in all animals, although it remained somewhat elevated in the a-MPT-treated group.
1800 "
1500 i
Hal/B-End al/MPT
~ c .,.., o ~
12~"
9OO.
a. ~
600 -
~
3OO -
,
,
|
,
FIG. 2 Both B-End and a-MPT induced a significant increase in PRL in rats pretreated with Hal. Animals were implanted with a pellet of Hal (25 mg) on the morning of day 7 of pregnancy. On the following day, animals received either a single i.a. injection of ~-MPT (250 m g / m l / k g BW) at 1145 h (arrow) or an i.c.v, infusion of B-End (I00 ng/~l/min from 1145-1200 h (line). PRL was significantly greater than pretreatment levels after 30 min of either treatment (p < 0.01). n = 8/group. * Hal/a-MPT > Hal/B-End (p < 0.01). B-End and a-MPT induce a similar increase in PRL in sham-operated animals The objective of the next experiment was to compare the stimulatory effects of B-End to a-MPT on PRL release in rats that were not pretreated with Hal, and therefore, having less DA receptor blockade. Animals were sham-operated (the control for groups in Figure 2) on the morning of day 7 of pregnancy and treated with B-End (100 ng/~i/min for 15 rain i.c.v. ) or a-MPT (250 mg/ml/kg BW i.a.), as above. Figure 3 shows that pretreatment PRL levels, measured at 1140 h, ranged from 16-35 ng/ml. Subsequent treatment with B-End stimulated a significant increase in PRL (p < 0.001) to 1462 ng/ml by the end of the infusion period. A similar significant increase (p < 0.001) was observed 30 rain following the injection of s-MPT. During the remaining time of the experiment, PRL continued to be significantly higher (p < 0.001) in the s-MPT group compared to B-End, where PRL declined gradually to basal levels.
1484
~-endorphin
and PRL D u r i n g P r e g n a n c y
Vol.
50, No. 20, 1992
211111 -
1800
E 1500 C
1200
Sham/8-End
9oo E
~o-
~ ~"
• 3OO -
0
FIG.
3
Both B-End and a-MPT stimulated PRL r e l e a s e in s h a m - o p e r a t e d c o n t r o l animals. This data p r o v i d e d t h e c o n t r o l for t h e r e s u l t s shown in F i g u r e 2. Rats w e r e s h a m - o p e r a t e d on t h e m o r n i n g of d a y 7 of pregnancy. On t h e next day, e i t h e r a s i n g l e i.a. i n j e c t i o n of a - M P T (250 m g / m l / k g BW) w a s g i v e n at 1145 h (arrow) or an i.c.v. i n f u s i o n of B-End (I00 ng/#I/min) was a d m i n i s t e r e d from 1 1 4 5 - 1 2 0 0 h (line). P l a s m a PRL i n c r e a s e d s i g n i f i c a n t l y a b o v e p r e t r e a t m e n t l e v e l s w i t h i n 30 m i n of e i t h e r i n j e c t i o n (p < 0.001). n = 6/group. * S h a m / a - M P T > S h a m / B - E n d (p < 0.01).
120
~,
100
•
E ol
Sal/5-HTP Nal/5-HTP
80-
C 60-
m
40.
E E.
2o.
0
i
i
i
8 o
.
i
i
i
i
i
A
saline
naloxone
FIG.
4
Nal a t t e n u a t e d t h e i n c r e a s e in PRL by 5-HTP. Experimental rats r e c e i v e d a c o n t i n u o u s i.v. i n f u s i o n of Nal (2.0 m g / 1 0 min) f r o m 1 0 0 0 - 1 3 0 0 h on d a y 8 of pregnancy, w h e r e a s c o n t r o l s r e c e i v e d Sal, as i n d i c a t e d by the line b e l o w t h e graph. In addition, an i.a. i n j e c t i o n of 5-HTP (20 m g / m l / k g BW) was a d m i n i s t e r e d at 1200 h (arrow). n = 6-7/group. * Values significantly different from N a l / 5 - H T P (p < 0.05).
Vol. 50, No. 20, 1992
Nal attenuates
~-endorphin
5-HTP-stimulated
and PRL During Pregnancy
1485
PRL release
The p u r p o s e of the fourth experiment was to determine w h e t h e r the 5-HT precursor, 5-HTP, stimulates PRL release on day 8 of p r e g n a n c y by w a y of opioid receptors. Experimental animals received a continuous infusion of the opioid receptor blocker, Nal (2.0 mg/100 ~i/10 m i n i.v.), w h i l e control animals were given a continuous infusion of Sal (100 ~1/i0 m i n i.v.) from 1000-1300 h. In addition, rats were injected w i t h 5-HTP (20 m g / m l / k g BW i.a.) at 1200 h. As shown in Figure 4, the control group d e m o n s t r a t e d an abrupt increase in PRL in response to the 5-HTP injection. On the other hand, the Nal g r o u p responded to 5-HTP with an increase in PRL that was attenuated by 75% compared to control animals. PRL declined toward pretreatment levels in both groups by 1300 h, 1 hour after treatment w i t h 5-HTP. These results suggest that 5-HT stimulates PRL, at least in part, by an action at opioid receptors. Ket does not b l o c k B-End-induced
PRL release
This experiment was designed to determine w h e t h e r B-End stimulates PRL release on day 8 of p r e g n a n c y by w a y of a serotonin S 2 receptor. Experimental animals were p r e t r e a t e d with Ket (10 mg/ml/kg BW i.p.), a selective S 2 receptor blocker, w h i l e control animals received an injection of dH20 (1 m l / k g BW i.p.) at 0945 h. This dose of Ket is effective in blocking PRL surges during p r e g n a n c y (20,23). Two hours later, an infusion of B-End (100 ng/~I/min for 15 m i n i.c.v.) was given from 1145-1200 h. Control and experimental groups showed an identical response to B-End, regardless of the type of pretreatment received. PRL increased d r a m a t i c a l l y to 1900-2150 ng/ml by the end of the infusion period and d e c r e a s e d to pretreatment levels during the next 2 hours. This suggests that B-End does not stimulate PRL via stimulation of an S 2 receptor. B-End-stimulated preqnanc7
PRL
release
is
qreatl 7
attenuated
on
day
16
vs
day
8
of
The last experiment was performed to compare the stimulatory effects of B-End on PRL release on day 8 vs day 16 of pregnancy. This was of interest because the nocturnal and diurnal PRL surges that occur on day 8, disappear by day 16. Figure 6 illustrates that an infusion of B-End (100 ng/~l/min i.c.v.) from 1145-1200 h on day 8 of pregnancy increased PRL to greater than 1400 ng/ml by the end of the infusion period. However, the response by day 16 pregnant rats to B-End was attenuated by more than 60% (p < 0.001). Differences in PRL were no longer present at 1245 h and both groups approached basal levels, as m e a s u r e d in A C S F - t r e a t e d control animals by 1315 h. Discussion The nocturnal P R L s u r g e s that occur throughout the first half of gestation in the rat appear to be regulated by a complex set of inhibitory and stimulatory neuronal interactions. DA is a major inhibitor of PRL release (10), whereas B-End (C.A. Sagri110 and J.L. Voogt, submitted for publication) and 5-HT (20,23) appear to be important stimulators of the surge. In this report, various p h a r m a c o l o g i c a l tools were employed to determine how DA, B-End and 5-HT neurons might be integrated within the pathway controlling the nocturnal PRL surges of pregnancy. All experiments were conducted during the intersurge period between 0900-1500 h in pregnant rats. During this time, the endogenous PRFs that stimulate PRL during the nocturnal surge do not appear to play an active role (32), resulting in basal levels of PRL release. Therefore, any change in PRL observed during an experiment can be attributed to the treatment in question, and not to an ongoing endogenous rhythmic event. The purpose of the first two experiments was to determine w h e t h e r B-End generates PRL surges in the pregnant rat by, in part, a m e c h a n i s m not involving DA. Day 8 pregnant rats were pretreated during the intersurge period w i t h a dose of Hromo known to m a x i m a l l y stimulate DA receptors. Subsequent treatment with B-End, shown to d r a m a t i c a l l y increase PRL release in controls (Figure 3), did not stimulate PRL above basal levels (Figure 1). This same observation was reported by other investigators (33,34). On the other hand, H a i s e n l e d e r et al (35) showed that TRH, a much weaker stimulator of PRL release, was able to
1486
~-endorphin
2400
A
2OO0
• o
121111
and PRL D u r i n g P r e g n a n c y
Vol.
50, No.
20, 1992
-
• dH20/8-End ,lO---KeUS-End c~ 1600 c n
~
8O0
a.
400
E
,¢: ¢- i"" •
•
i
•
A
i
•
l
,
i
•
J= i
~
FIG.
5
Ket d i d not inhibit B - E n d - s t i m u l a t e d PRL release. Experimental a n i m a l s r e c e i v e d an i.p. i n j e c t i o n of Ket (10 mg/ml/kg BW) w h i l e t h e c o n t r o l g r o u p w a s g i v e n v e h i c l e (dH20) at 0945 h on d a y 8 of pregnancy (arrow). B-End (i00 ng/#i/min) w a s i n f u s e d i.c.v. 2 h o u r s later from 1 1 4 5 - 1 2 0 0 h (line). n = 7-8/group.
1800 A
*
•
1500
¢= 1200 C 900 O 6O0 E U) ¢1 0. 3O00
~ i
ACSF 8-End-Day8
~ d ' D a l
16
._d
i
i FIG.
i
i
6
T h e P R L r e s p o n s e to B-End w a s g r e a t l y a t t e n u a t e d on d a y 16 vs d a y 8 of p r e g n a n c y . B-End (I00 ng/~I/min) or A C S F (i #i/min) w a s infused i.c.v, b e t w e e n 1 1 4 5 - 1 2 0 0 h (line) on d a y 8 or d a y 16 of p r e g nancy. E a c h d a t a p o i n t r e p r e s e n t s 5-6 rats/group. ** B - E n d - D a y 8 > B - E n d - D a y 16 (p < 0.001); * B-End-Day-8, 16 > A C S F (p < 0.05).
Vol.
50, No. 20, 1992
fl-endorphin and PRL During Pregnancy
increase PRL in pregnant rats in the presence of Bromo. B-End does not increase PRL by stimulating TRH.
This
1487
suggests
that
In the second experiment, D A receptors were b l o c k e d by implanting each pregnant rat with a pellet of Hal for 24 hours. This manner of drug delivery was chosen to p r o v i d e continuous blockade of D A receptors, resulting in steadystate levels of P R L b e t w e e n 500-600 ng/ml. Subsequent treatment with B-End, as illustrated in Figure 2, increased PRL even more w i t h i n 30 minutes of the infusion. Initially, the data suggested that the m e c h a n i s m of B-End-induced PRL release was irrespective of DA regulation, assuming that DA receptors were completely b l o c k e d by Hal. However, a-MPT, a DA synthesis inhibitor, stimulated PRL release to a similar m a g n i t u d e in a separate group of H a l - t r e a t e d animals. This suggests that DA receptors were not completely b l o c k e d by Hal and that a number of receptors were still available to bind DA. The observation that PRL continued to increase as the supply of DA was depleted by ~-MPT supports this theory. In this respect, our results in pregnant rats agree w i t h others w h o have found that DA receptor blockade (36) or DA synthesis inhibition (13) t o t a l l y b l o c k e d B-End-induced PRL release. This suggests that B-End-induced PRL release in the pregnant rat m a y be accounted for by inhibition of DA. The initial P R L r e s p o n s e to B-End was nearly identical in m a g n i t u d e to the response to s-MPT in both Hal-treated (Figure 2) and sham-operated control animals (Figure 3). However, the delayed action and longer lasting effects of a-MPT compared to B-End, characteristic of many synthesis inhibitors, suggest that B-End and a-MPT do not inhibit D A by the same mechanism. Also, it is possible that it takes longer for a-MPT to enter the cells, and therefore, its effect is delayed, or that B-End is m e t a b o l i z e d more rapidly. It is unclear whether opioids act directly on DA neurons or w h e t h e r they exert their P R L r e l e a s i n g effect through an interneuronal pathway. The results of Spampinato et al (27) suggest that EOPs interact with a serotonergic system to increase PRL release. On the other hand, Tache et al (28) d e m o n s t r a t e d that an intact serotonergic pathway was not necessary for EOPs to stimulate PRL release. Results from a previous study in our laboratory, using selective S 2 receptor blockers, suggest that 5-HT m a y act as an important neuromodulator of PRL release during p r e g n a n c y (20). In light of these conflicting data, a set of experiments w a s p e r f o r m e d to find whether an interrelationship exists between B-End and 5-HT neurons within the pathway controlling the nocturnal PRL surge. One experiment was designed to determine whether the 5-HT precursor, 5-HTP, stimulates PRL by w a y of an opioid interneuronal pathway. Pretreatment during the intersurge period with the opioid receptor blocker, Nal, greatly attenuated the PRL response to 5-HTP in day 8 pregnant rats (Figure 4). There was little doubt that the majority of opioid receptors was inhibited because the same dose of Nal completely blocked the nocturnal PRL surge (9). This suggests that 5-HT stimulates the nocturnal surge, in part, by way of an opioidergic interneuronal pathway. The alternative approach was taken in a separate experiment to determine w h e t h e r B-End stimulates the nocturnal surge by way of a serotonergic interneuronal pathway, specifically via S 2 receptors. Day 8 pregnant rats were pretreated during the intersurge period with Ket, a selective S 2 receptor antagonist, at a dose shown to inhibit the PRL surges during pregnancy (23). Blocking the 5-HT p a t h w a y in this manner did not impair the stimulation of PRL release by B-End (Figure 5). This data differs from the results of other researchers who have found that less specific 5-HT receptor antagonists b l o c k the PRL stimulatory effect of morphine sulfate in male rats (37-39). In our model of the pregnant rat, it appears that B-End does not increase PRL t h r o u g h a serotonergic pathway, at least not by w a y of S 2 receptors. A l t h o u g h D A neuronal activity was significantly increased by Nal (9), there was no effect on 5-HT neuronal activity (unpublished observations}. Again, these results imply an opioidergic action through DA neurons, and not 5-HT neurons, to stimulate the nocturnal PRL surge. A d m i n i s t r a t i o n of 5-HTP stimulated PRL release in day 8 pregnant rats, however, this stimulatory response disappeared by day 16 of pregnancy, w h e n PRL surges were absent as well (40). This suggested that the portion of the neuronal p a t h w a y controlling PRL release was no longer responsive to serotonergic input during late pregnancy. This also is partially true for B-End. Day
1488
~-endorphin and PRL During Pregnancy
Vol. 50, No. 20, 1992
16 pregnant rats were treated with B-End using the same regimen employed on day 8 of pregnancy (Figure 6). Even though B-End generated a significant increase in PRL, the response was greatly attenuated compared to the increase on day 8. Although the cause of this attenuation is not known, it may involve a change in the TIDA neurons because of their probable site for B-End's stimulatory effects. Perhaps the secretion of placental lactogens (PLs), thought to inhibit PRL secretion (41-44), stimulate the TIDA neurons and B-End's ability to inhibit these neurons is partially blocked. Precedence for this was shown in a study in which secretion of PL-I by tumor cells completely blocked the PRL response to serotonergic drugs (40). Future experiments will determine whether PL-I also blocks the stimulatory effect of B-End on PRL, and whether this is by way of TIDA neurons. Alternatively, the lactotroph may become unresponsive to changes in hypothalamic input during late pregnancy (35). By using various agonists and antagonists of DA, EOPs and 5-HT, we have provided additional information as to what factors might be involved in controlling the nocturnal PRL surges of pregnancy. It appears that 5-HT acts through B-End, which in turn, inhibits DA, resulting in PRL secretion. However, further research is needed to identify all of the interneuronal components of this intricate pathway. Acknowledqements Supported by NIH grant HD-24190. We thank Janssen Pharmaceutica, Beerse, Belgium, for providing ketanserin and the NIDDK for supplying the PRL RIA material. References i. 2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
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50, No. 20, 1992
~-endorphin
and P R L D u r i n g
Pregnancy
1489
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