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Dopamine inhibition of prolactin release but not synthesis in the male syrian hamster: In vitro studies
Life Sciences, Vol. 38, pp. 1915-1921 Printed in the U.S.A.
DOPAMINE
Pergamon Press
INHIBITION OF PROLACTIN RELEASE BUT NOT SYNTHESIS IN THE MALE SYRIAN HAMSTER: IN VITRO STUDIES David E. Blask and K. Michael
Orstead
Department of Anatomy, University of Arizona, College Arizona 85724
of Medicine, Tucson,
(Received in final form March 5, 1986) Summary The h y p o t h a l a m i c m e c h a n i s m s c o n t r o l l i n g p r o l a c t i n (PRL) c e l l function in the male Syrian hamster are unclear. Equally unclear is the role of d o p a m i n e (DA) in r e g u l a t i n g l a c t o t r o p h i c c e l l activity in long photoperiod-exposed hamsters p a r t i c u l a r l y with respect to PRL s y n t h e s i s and release. The synthesis of PRL, as measured by the incorporation of 3H-leucine into newly synthesized PRL, by anterior p i t u i t a r y g l a n d s from male hamsters is linear over a five h incubation period. Approximately two-fold more 3HPRL remained in the pituitary glands than in the medium by the end of the i n c u b a t i o n period. The incubation of h a m s t e r hemipituitaries with DA at concentrations of either 5 X 10~7M or 5 X 10-5M, r e s u l t e d in a 77% to 83% i n h i b i t i o n of the r e l e a s e of i m m u n o r e a c t l v e PRL into the m e d i u m as compared with controls. Similarly, the release of 3H-PRL into the medium was inhibited by 71% to 76% as compared with controls; however, the s y n t h e s i s of PRL was v i r t u a l l y the same among the e x p e r i m e n t a l and control groups. These r e s u l t s suggest that DA may be an important regulator of short-term PRL release but not synthesis in the long photoperiod-exposed male hamster. The Syrian hamster ( M e s o c r i c e t u s auratus) is a h i g h l y p h o t o s e n s i t i v e , s e a s o n a l l y - b r e e d i n g species in which the r e p r o d u c t i v e tract regresses in response to either total light deprivation or exposure to short photoperlod for s e v e r a l weeks (I). Furthermore, the decrease in serum PRL l e v e l s that occurs in short p h o t o p e r i o d - e x p o s e d male hamsters is a c r i t i c a l event in mediating gonadal atrophy (2). Recently, we have shown that long-term light deprivation markedly inhibits the synthesis, storage and release of prolactin (PRL) in both male and female hamsters, an effect that appears to be pinealm e d i a t e d (3). H o w e v e r , the precise n e u r o e n d o c r i n e m e c h a n i s m s by which the pineal gland accomplishes this inhibition of FRL cell function are unknown. In fact, very l i t t l e is known about the h y p o t h a l a m i c - n e u r o e n d o c r i n e regulation of PRL in the Syrian hamster. The transplantation of homografts of anterior pituitary tissue beneath the k i d n e y capsule results [n h y p e r p r o l a c t i n e m i a in m a l e hamsters (4) s u g g e s t i n g that PRL s e c r e t i o n is released from hypothalamic inhibition under those circumstances. S u r p r i s i n g l y , u n l i k e in the rat ,medial basal h y p o t h a l a m i c extracts from hamsters do not inhibit the release of immunoreactive (RIA) PRL from hamster pituitaries in vitro; however, these same extracts inhibit the synthesis of PRL in a dose-dependent manner (5). 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
1916
Effects of Dopamine on Hamster Prolactin
Vol. 38, No. 21, 1986
Although bromocriptine has been shown to inhibit PRL secretion in the hamster in v i v o (6,7), it is u n c l e a r what, if any, r o l e d o p a m i n e (DA) p l a y s in the p h y s i o l o g i c a l r e g u l a t i o n of PRL in this species. In the rat, it is w e l l a c c e p t e d that DA is a p h y s i o l o g i c a l P R L - i n h i b i t o r y f a c t o r (PIF) since it i n h i b i t s both the r e l e a s e and s y n t h e s i s of PRL in v i t r o at c o n c e n t r a t i o n s a p p r o x i m a t i n g its p h y s i o l o g i c a l range in h y p o p h y s i a l p o r t a l b l o o d (8,9). ~owever, in the male hamster, concentrations of DA ranging from 10-8M to 10"M have been shown to be virtually ineffective in suppressing PRL secretion in vitro unless the pituitaries are derived from short photoperiod-exposed hamsters (I0, Ii). W h i l e it seems p r o b a b l e that PRL in the S y r i a n h a m s t e r is p r i m a r i l y under tonic i n h i b i t o r y c o n t r o l as it is in the rat, it is u n c l e a r as to the exact nature of its regulation. As pointed out by Dannies (12), the release of PRL is not a simply-regulated process; rather, PRL processing is a complex series of events which include synthesis, storage, degradation and release. Hence, the present investigation was designed to address the following questions: I) Does DA d i r e c t l y inhibit PRL c e l l f u n c t i o n in p i t u i t a r i e s of long p h o t o p e r i o d - e x p o s e d m a l e h a m s t e r s in vitro? 2) W h i c h a s p e c t s of PRL processing does DA affect?
Materials and Methods A d u l t m a l e Syrian h a m s t e r s (90 - I00 g BW) were used as d o n o r s of a n t e r i o r pituitary glands. The animals were maintained on a lighting schedule of 14 h of light and I0 h of d a r k n e s s per day ( l i g h t s on from 0600 - 2000 h). On the day of the experiments, the animals were killed by rapid decapitation between 0900 - I000 h. The p i t u i t a r y of each a n i m a l was d i s s e c t e d free, the p o s t e r i o r lobe r e m o v e d and a n t e r i o r pituitary hemisected. Hemipituitaries (HP) were r a n d o m i z e d among p l a s t i c v i a l s such that there were three HP in each vial containing i ml of leucine-free Minimal Essentials Medium (Gibco Labs) to which was added L-leucine (final concentration of 4.6 X 10-6M). In the time course study the pituitaries were incubated for either 30 min, I, 2, 3 or 5 h after r e m o v a l and p l a c e m e n t into the v i a l s (6 v i a l s per time point ) each of w h i c h c o n t a i n e d lO uCi of 3 H - l e u c i n e ( R e s e a r c h P r o d u c t s I n t e r n a t i o n a l , s p e c i f i c a c t i v i t y = 40 C i / m M ) in m e d i u m c o n t a i n i n g c o l d l e u c i n e as i n d i c a t e d above. In the case of the DA e x p e r i m e n t , the HP were pre-incubated for one h in medium containing cold leucine only. Following preincubation, the medium was removed from each vial and replaced with fresh medium now containing labeled-leucine in addition to cold leucine. Dopamine (Sigma Chem. Co.) was p l a c e d into two grou~s of 6 v i a l s each at a final concentration of either 5 X 10-7M or 5 X i0 JM. Ascorbic acid was added to each of the DA c o n t a i n i n g v ~ a l s as w e l l as the c o n t r o l v i a l s at a final c o n c e n t r a t i o n of 5.6 X IO-~M; the i n c u b a t i o n was c a r r i e d out for an additional four h period. A l l the preincubations and incubations were carried out in a Dubnoff metabolic shaker, at 37°C, with constant shaking (60 cycles/min) and gassing 95%O2:5%C02. Following i n c u b a t i o n for v a r i o u s l e n g t h s of time, the pituitaries were removed from the medium, blotted and weighed (mean wt of 3 HP = 3.08 + 0.i0 mg) and s o n i c a t e d in 0.5 ml of 0.OIM p h o s p h a t e b u f f e r e d s a l i n e (PBS, pH = 7.5). The p i t u i t a r y s o n i c a t e s as w e l l as the i n c u b a t i o n m e d i a were stored at -20 °C u n t i l the time of e l e c t r o p h o r e s i s and radioimmunoassay. The incorporation of 3H-leucine into newly synthesized PRL was measured by subjecting incubation media and pituitary homogenates to polyacrylamide disc
Vol. 38, No. 21, 1986
Effects of Dopamine on Hamster Prolactin
1917
gel electrophoresis as previously described for the rat (13). The validation and adaptation of this technique for use in measuring hamster PRL synthes{s is described in detail elsewhere (3). In all experiments, the samples were analyzed in the same elgctrophoretlc run. Prolactln synthesis was expressed as the total amount of 3H-PRL in the pituitaries plus the medium. Pituitary homogenates and incubation media were assayed for RIA-PRL with a homologous assay system developed by Soares et al. ([4). All samples were run in the same assay; the intra-assay coefficient of variation was less that 10%. All data were analyzed by a one-way analysis of variance followed by Student-Neuman-Keuls muliple range test on an IBM personal computer.
Results Both the synthesis and release of newly five h period (Fig. I). T h r o u g h o u t the amounts of JH-PRL were released into the were a l w a y s a p p r o x i m a t e l y two to three medium.
7'
synthesized PRL were linear over a incubation, a l t h o u g h s i g n i f i c a n t medium, pituitary levels of 3H-PRL times higher in the HP than in the
I/TOTAL (PiT+MEDIUM)
6, A
5-
x
4-
,,t i o
/
,//'t
:E a. a
I
._1
rr a. I It~
PITUITARY
I
.//&.fff~ ' 1
0
TIME
(hrs)
FIG. I Time course of the in vitro synthesis and release of hamster prolactin (PRL) by male hamster hemipituitaries. Each point represents the mean + SEM; there were 6 vials (containing 3 hemipituitaries each) per time point. -Dopamine at r e l e a s e of Similarly, 76% by both
both concentrations caused a marked 77% to 83% inhibition of the R I A - P R L into the m e d i u m as compared with c o n t r o l s (Fig. 2). the r e l e a s e of 3H-PRL into the m e d i u m was i n h i b i t e d from 71% to c o n c e n t r a t i o n s of DA (Fig. 3). The total (pituitary + medium)
1918
Effects of Dopamine on Hamster Prolactin
Vol. 38, No. 21, 1986
l e v e l s of 3H-PRL in the g r o u p t r e a t e d w i t h 5 X I 0 - 7 ~ DA was not d i f f e r e n t from controls; however, the total a m o u n t s of J H - P R L at the h i g h e r c o n c e n t r a t i o n of DA were s l i g h t l y but s i g n i f i c a n t l y h i g h e r than c o n t r o l s (Fig. 3). 7
T 6,
:3.
~E
C) O
I-Z_ UJ
4
O) ,(
uJ .J
3,
uJ 0C
z F-
o J
2,
T
0
CONTROL
DOPAMINE 5 x 10-7M
FIG.
DOPAMINE 5x 10-5M
2
The effects of dopamine on the release of immunoreactive prolactin into the i n c u h a t i o n m e d i u m o v e r a 4 h period. Each bar r e p r e s e n t s the mean + SEM. There were 6 vials (each containing 3 hemipituitaries) per group. Dopamine groups versus the control group, p < 0.001. Although the pituitary levels of R I A - P R L were not s i g n i f i c a n t l y d i f f e r e n t among any of the groups, the levels of 3H-PRL were significantly higher in the DA-treated groups than in the controls (Table 1). Discussion The i n c o r p o r a t i o n of 3 H - l e u c i n e into PRL n e w l y s y n t h e s i z e d by h a m s t e r pituitaries was l i n e a r o v e r a five h i n c u b a t i o n p e r i o d s i m i l a r to that which occurs in the rat (15). Additionally, the release of newly synthesized PRL into the incubation medium was also linear over the entire duration of the incubation. H o w e v e r , u n l i k e in the rat~ most of the PRL r e m a i n s in the p i t u i t a r y r a t h e r than being s e c r e t e d into the medium, c o r r o b o r a t i n g the results of our previous report (3). In this study, we a t t e m p t e d to r e s o l v e the q u e s t i o n of w h e t h e r DA, at concentrations that inhibit PRL synthesis and secretion in the rat, inhibits PRL synthesis and secretion in the male hamster. Both concentrations of DA ~sed in the present study markedly inhibited the release of both RIA-PRL and H-PRL into the medium. An unanticipated finding was that the synthesis of PRL was not i n h i b i t e d by DA in our study. This c o n t r a s t s to r e s u l t s in the rat in w h i c h DA inhibits both PRL r e l e a s e and s y n t h e s i s ([5). To our knowledge, the hamster appears to be the only mammalian species studied thus
Vol. 38, No. 21, 1986
Effects of Dopamine on Hamster
Prolactin
1919
far in which an inhibitory effect of DA on PRL release occurs in the absence of an inhibitory effect on synthesis. Perhaps, if the incubation period had been extended for several more h an inhibitory effect of DA on PRL synthesis might have been observed as well. 9,
-T" 8,
7,
A
[~
T
II
r~]
TOTAL (PIT + MEDIUM)
MEDIUM
6,
0 x '5 o. a
5
4,
,o. -r o3
3-
2,
I.
o DOPAMINE 5 x 10 - 7 M
CONTROL
DOPAMINE 5 x 10-5M
FIG. 3 The effects of d o p a m i n e on the s y n t h e s i s and r e l e a s e of p r o l a c t i n (PRL) in v i t r o o v e r a 4 h period. The bars r e p r e s e n t the mean + SEM. There were 6 vials (each containing 3 hemipituitaries) in each group.-- The levels of 3HPRL in the medium in the dopamine-treated groups ~iffered from the control, p < 0.001. T o t a l 3 H - P R L l e v e l s in the 5 x 1 0 - J M d o p a m i n e - t r e a t e d group differed from the control, p < 0.05. TABLE I The Effects of Dopamine on Anterior Pituitary Levels of 3H-Prolactin and Immunoreactive (RIA)-PRL Following 4 h of Incubation In Vitro.
TREATMENT
3H-PRL (DPM X 10 -4)
(PRL)
RIA-PRL (ug)
Control
5.51
+
0.33
26.72
+
0.57
Dopamine (5 X 10-7M)
7.05
~
0.53*
26.39
+
0.98
Dopamine (5 X Io-SM)
7.71 ~
0.21"
28.48
+
1.07
V a l u e s r e p r e s e n t the mean + SEM. There were 6 v i a l s (each c o n t a i n i n g hemipituitaries) per group. ~ p < 0.05 versus control group.
3
1920
Effects of Dopamine on Hamster Prolactin
Vol. 38, No. 21, 1986
As ~ould be expected in the case of uninhibited PRL synthesis, the inhibition of JH-PRL release by DA resulted in a corresponding accumulation of labeled PRL in the pituitaries. In contrast, the D A - i n d u c e d i n h i b i t i o n of RIA-PRL release did not result in a c o r r e s p o n d i n g increase in pituitary l e v e l s of RIA-PRL. One e x p l a n a t i o n could be that DA caused an increase in the intracellular degradation of RIA-PRL that would otherwise have accumulated in the g l a n d s as a r e s u l t of D A - i n h i b i t e d PRL release. In fact, others have shown that if PRL release is inhibited by either DA or its agonists, the PRL not released from the cell is degraded (12). Apparently, any degradation of unreleased, newly synthesized PRL may have been masked by unrestricted PRL synthesis in the DA-treated groups. It is likely that DA-inhibited PRL release is accomplished via a DA receptormediated mechanism since the DA receptor antagonist pimozide blocks the PRLinhibitory effects of DA on female hamster pituitaries (unpublished results). Furthermore, a recent preliminary report by Burns et al. (16) indicates that male hamster pituitaries contain both high and low affinity DA binding sites. Our r e s u l t s are in marked contrast to those of Steger et al. (10,1[) who observed either no inhibition or a marginal inhibition of PRL release from the pituitaries of long photoperiod-exposed male hamsters in response to DA. These discrepancies might be explicable by the fact that even our lowest dose (5 X 10-7M) was f i v e - f o l d higher than the highest dose (10-7M) examined by Steger et al. (11). Another explanation for the differences in the results may be that single hemi-pituitaries were incubated for only one to two h in the presence of DA in the other investigations ([0,11). As a l l u d e d to p r e v i o u s l y , light d e p r i v a t i o n m a r k e d l y inhibits both the synthesis and r e l e a s e of PRL in m a l e hamsters (3). Furthermore, hamster medial basal hypothalamic extracts inhibit the synthesis of PRL but ,lot the release of RIA-PRL in vitro (5). Conversely, in the present investigation, DA inhibited the r e l e a s e of PRL but not its synthesis. This suggests to us that DA may not be the primary factor r e s p o n s i b l e for the l o n g - t e r m inhibition of PRL cell activity in light-deprived male hamsters. Rather, DA may be more important in the s h o r t - t e r m r e g u l a t i o n of PRL under the conditions of long photoperiod in the male hamster. Acknowledgements This work was supported by NIH B i o m e d i c a l R e s e a r c h Support Grant RR05765. The authors wish to thank Wanda McGill for technical assistance and Dr. Frank Talamantes, Univ. of California, Santa Cruz, for the generous supply of the hamster PRL a n t i s e r u m and purified hamster PRL. Send reprint requests to D a v i d E. Blask, Ph.D., M.D., D e p a r t m e n t of Anatomy, The U n i v e r s i t y of Arizona, College of Medicine, Tucson, Arizona 85724. References 1. 2. 3. 4. 5.
R.J. REITER, Endocr. Rev. ~ 109-131 (1980). A. BARTKE, B.D. GOLDMAN, F.J. BEX, R.P. KELCH, M.S. SMITH, S. D A L T E R I O P.C. DOHERTY, Endocrinology 106 167-172 (1980). D.E. BLASK, C.A. LEADEM, K.M. ORSTEAD and B.R. LARSEN, N e u r o e n d o c r i n o l ogy (in press). F. BEX, A. BARTKE, B.D. G O L D M A N and S. DALTERIO, E n d o c r i n o l o g y 103 2069-2080 (1978). K.M. ORSTEAD and D.E. BLASK, Prolactin~ Basic and Clinical Correlates R.M. MacLeod, M.O. Thorner and U. Scapagnini (eds.), I 113-12[, Livinia Press, Padova (1985).
Vol. 38, No. 21, 1986
6. 7. 8. 9. I0. ii. 12. 13. 14. 15.
16.
Effects of Dopamine on Hamster Prolactin
1921
A. BARTKE, M.P. HOGAN and G.B. CUTTY, J. Androl. 1 115-120 (1980). K . T . BORER, C.S. CAMPBELL, J. TABOR, K. JORGENSON, S. KANDARIAN and L.GORDON, Biol. Reprod. 29 38-47 (1983). R.M. MACLEOD and J.E. LEHYMEYER, Endocrinology 94 1077-1085 (1974). C.J. SHAAR and J.A. CLEMENS, Endocrinology 95 1202-1212 (1974). R.W. STEGER, A. BARTKE, B.D. GOLDMAN, M.J. SOARES and F. TALAMANTES, Biol. Reprod. 29 872-878 (1983). R.W. STEGER, K.S. MATT, H.G. KLEMCKE and A. BARTKE, Neuroendocrinology 41 89-96 (1985). P.S. DANNIES, Biochem. Pharmacol. 31 2845-2849 (1982). R.A. REISFELD, U.J. LEWIS, D.E. WILLIAMS, Nature 195 281-283 (1962). M.J. SOARES, P. COLOSI and F. TALAMANTES, Proc. Soc. Exp. Biol. Med. 172 379-381 (1983). R.M. MACLEOD and J.E. LEHMEYER, Lactogenic Hormones, G.E.W. Wolstenholme and J. Knight (eds.), pp. 53-82, Churchill Livingstone, London (1972). D.M. BURNS, C.A. LEADEM and B. BENSON, Prog. |5th Ann. Mtg. Soc. Neurosci. p. 658, Abst. #196.9, (1985).
DOPAMINE
Pergamon Press
INHIBITION OF PROLACTIN RELEASE BUT NOT SYNTHESIS IN THE MALE SYRIAN HAMSTER: IN VITRO STUDIES David E. Blask and K. Michael
Orstead
Department of Anatomy, University of Arizona, College Arizona 85724
of Medicine, Tucson,
(Received in final form March 5, 1986) Summary The h y p o t h a l a m i c m e c h a n i s m s c o n t r o l l i n g p r o l a c t i n (PRL) c e l l function in the male Syrian hamster are unclear. Equally unclear is the role of d o p a m i n e (DA) in r e g u l a t i n g l a c t o t r o p h i c c e l l activity in long photoperiod-exposed hamsters p a r t i c u l a r l y with respect to PRL s y n t h e s i s and release. The synthesis of PRL, as measured by the incorporation of 3H-leucine into newly synthesized PRL, by anterior p i t u i t a r y g l a n d s from male hamsters is linear over a five h incubation period. Approximately two-fold more 3HPRL remained in the pituitary glands than in the medium by the end of the i n c u b a t i o n period. The incubation of h a m s t e r hemipituitaries with DA at concentrations of either 5 X 10~7M or 5 X 10-5M, r e s u l t e d in a 77% to 83% i n h i b i t i o n of the r e l e a s e of i m m u n o r e a c t l v e PRL into the m e d i u m as compared with controls. Similarly, the release of 3H-PRL into the medium was inhibited by 71% to 76% as compared with controls; however, the s y n t h e s i s of PRL was v i r t u a l l y the same among the e x p e r i m e n t a l and control groups. These r e s u l t s suggest that DA may be an important regulator of short-term PRL release but not synthesis in the long photoperiod-exposed male hamster. The Syrian hamster ( M e s o c r i c e t u s auratus) is a h i g h l y p h o t o s e n s i t i v e , s e a s o n a l l y - b r e e d i n g species in which the r e p r o d u c t i v e tract regresses in response to either total light deprivation or exposure to short photoperlod for s e v e r a l weeks (I). Furthermore, the decrease in serum PRL l e v e l s that occurs in short p h o t o p e r i o d - e x p o s e d male hamsters is a c r i t i c a l event in mediating gonadal atrophy (2). Recently, we have shown that long-term light deprivation markedly inhibits the synthesis, storage and release of prolactin (PRL) in both male and female hamsters, an effect that appears to be pinealm e d i a t e d (3). H o w e v e r , the precise n e u r o e n d o c r i n e m e c h a n i s m s by which the pineal gland accomplishes this inhibition of FRL cell function are unknown. In fact, very l i t t l e is known about the h y p o t h a l a m i c - n e u r o e n d o c r i n e regulation of PRL in the Syrian hamster. The transplantation of homografts of anterior pituitary tissue beneath the k i d n e y capsule results [n h y p e r p r o l a c t i n e m i a in m a l e hamsters (4) s u g g e s t i n g that PRL s e c r e t i o n is released from hypothalamic inhibition under those circumstances. S u r p r i s i n g l y , u n l i k e in the rat ,medial basal h y p o t h a l a m i c extracts from hamsters do not inhibit the release of immunoreactive (RIA) PRL from hamster pituitaries in vitro; however, these same extracts inhibit the synthesis of PRL in a dose-dependent manner (5). 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
1916
Effects of Dopamine on Hamster Prolactin
Vol. 38, No. 21, 1986
Although bromocriptine has been shown to inhibit PRL secretion in the hamster in v i v o (6,7), it is u n c l e a r what, if any, r o l e d o p a m i n e (DA) p l a y s in the p h y s i o l o g i c a l r e g u l a t i o n of PRL in this species. In the rat, it is w e l l a c c e p t e d that DA is a p h y s i o l o g i c a l P R L - i n h i b i t o r y f a c t o r (PIF) since it i n h i b i t s both the r e l e a s e and s y n t h e s i s of PRL in v i t r o at c o n c e n t r a t i o n s a p p r o x i m a t i n g its p h y s i o l o g i c a l range in h y p o p h y s i a l p o r t a l b l o o d (8,9). ~owever, in the male hamster, concentrations of DA ranging from 10-8M to 10"M have been shown to be virtually ineffective in suppressing PRL secretion in vitro unless the pituitaries are derived from short photoperiod-exposed hamsters (I0, Ii). W h i l e it seems p r o b a b l e that PRL in the S y r i a n h a m s t e r is p r i m a r i l y under tonic i n h i b i t o r y c o n t r o l as it is in the rat, it is u n c l e a r as to the exact nature of its regulation. As pointed out by Dannies (12), the release of PRL is not a simply-regulated process; rather, PRL processing is a complex series of events which include synthesis, storage, degradation and release. Hence, the present investigation was designed to address the following questions: I) Does DA d i r e c t l y inhibit PRL c e l l f u n c t i o n in p i t u i t a r i e s of long p h o t o p e r i o d - e x p o s e d m a l e h a m s t e r s in vitro? 2) W h i c h a s p e c t s of PRL processing does DA affect?
Materials and Methods A d u l t m a l e Syrian h a m s t e r s (90 - I00 g BW) were used as d o n o r s of a n t e r i o r pituitary glands. The animals were maintained on a lighting schedule of 14 h of light and I0 h of d a r k n e s s per day ( l i g h t s on from 0600 - 2000 h). On the day of the experiments, the animals were killed by rapid decapitation between 0900 - I000 h. The p i t u i t a r y of each a n i m a l was d i s s e c t e d free, the p o s t e r i o r lobe r e m o v e d and a n t e r i o r pituitary hemisected. Hemipituitaries (HP) were r a n d o m i z e d among p l a s t i c v i a l s such that there were three HP in each vial containing i ml of leucine-free Minimal Essentials Medium (Gibco Labs) to which was added L-leucine (final concentration of 4.6 X 10-6M). In the time course study the pituitaries were incubated for either 30 min, I, 2, 3 or 5 h after r e m o v a l and p l a c e m e n t into the v i a l s (6 v i a l s per time point ) each of w h i c h c o n t a i n e d lO uCi of 3 H - l e u c i n e ( R e s e a r c h P r o d u c t s I n t e r n a t i o n a l , s p e c i f i c a c t i v i t y = 40 C i / m M ) in m e d i u m c o n t a i n i n g c o l d l e u c i n e as i n d i c a t e d above. In the case of the DA e x p e r i m e n t , the HP were pre-incubated for one h in medium containing cold leucine only. Following preincubation, the medium was removed from each vial and replaced with fresh medium now containing labeled-leucine in addition to cold leucine. Dopamine (Sigma Chem. Co.) was p l a c e d into two grou~s of 6 v i a l s each at a final concentration of either 5 X 10-7M or 5 X i0 JM. Ascorbic acid was added to each of the DA c o n t a i n i n g v ~ a l s as w e l l as the c o n t r o l v i a l s at a final c o n c e n t r a t i o n of 5.6 X IO-~M; the i n c u b a t i o n was c a r r i e d out for an additional four h period. A l l the preincubations and incubations were carried out in a Dubnoff metabolic shaker, at 37°C, with constant shaking (60 cycles/min) and gassing 95%O2:5%C02. Following i n c u b a t i o n for v a r i o u s l e n g t h s of time, the pituitaries were removed from the medium, blotted and weighed (mean wt of 3 HP = 3.08 + 0.i0 mg) and s o n i c a t e d in 0.5 ml of 0.OIM p h o s p h a t e b u f f e r e d s a l i n e (PBS, pH = 7.5). The p i t u i t a r y s o n i c a t e s as w e l l as the i n c u b a t i o n m e d i a were stored at -20 °C u n t i l the time of e l e c t r o p h o r e s i s and radioimmunoassay. The incorporation of 3H-leucine into newly synthesized PRL was measured by subjecting incubation media and pituitary homogenates to polyacrylamide disc
Vol. 38, No. 21, 1986
Effects of Dopamine on Hamster Prolactin
1917
gel electrophoresis as previously described for the rat (13). The validation and adaptation of this technique for use in measuring hamster PRL synthes{s is described in detail elsewhere (3). In all experiments, the samples were analyzed in the same elgctrophoretlc run. Prolactln synthesis was expressed as the total amount of 3H-PRL in the pituitaries plus the medium. Pituitary homogenates and incubation media were assayed for RIA-PRL with a homologous assay system developed by Soares et al. ([4). All samples were run in the same assay; the intra-assay coefficient of variation was less that 10%. All data were analyzed by a one-way analysis of variance followed by Student-Neuman-Keuls muliple range test on an IBM personal computer.
Results Both the synthesis and release of newly five h period (Fig. I). T h r o u g h o u t the amounts of JH-PRL were released into the were a l w a y s a p p r o x i m a t e l y two to three medium.
7'
synthesized PRL were linear over a incubation, a l t h o u g h s i g n i f i c a n t medium, pituitary levels of 3H-PRL times higher in the HP than in the
I/TOTAL (PiT+MEDIUM)
6, A
5-
x
4-
,,t i o
/
,//'t
:E a. a
I
._1
rr a. I It~
PITUITARY
I
.//&.fff~ ' 1
0
TIME
(hrs)
FIG. I Time course of the in vitro synthesis and release of hamster prolactin (PRL) by male hamster hemipituitaries. Each point represents the mean + SEM; there were 6 vials (containing 3 hemipituitaries each) per time point. -Dopamine at r e l e a s e of Similarly, 76% by both
both concentrations caused a marked 77% to 83% inhibition of the R I A - P R L into the m e d i u m as compared with c o n t r o l s (Fig. 2). the r e l e a s e of 3H-PRL into the m e d i u m was i n h i b i t e d from 71% to c o n c e n t r a t i o n s of DA (Fig. 3). The total (pituitary + medium)
1918
Effects of Dopamine on Hamster Prolactin
Vol. 38, No. 21, 1986
l e v e l s of 3H-PRL in the g r o u p t r e a t e d w i t h 5 X I 0 - 7 ~ DA was not d i f f e r e n t from controls; however, the total a m o u n t s of J H - P R L at the h i g h e r c o n c e n t r a t i o n of DA were s l i g h t l y but s i g n i f i c a n t l y h i g h e r than c o n t r o l s (Fig. 3). 7
T 6,
:3.
~E
C) O
I-Z_ UJ
4
O) ,(
uJ .J
3,
uJ 0C
z F-
o J
2,
T
0
CONTROL
DOPAMINE 5 x 10-7M
FIG.
DOPAMINE 5x 10-5M
2
The effects of dopamine on the release of immunoreactive prolactin into the i n c u h a t i o n m e d i u m o v e r a 4 h period. Each bar r e p r e s e n t s the mean + SEM. There were 6 vials (each containing 3 hemipituitaries) per group. Dopamine groups versus the control group, p < 0.001. Although the pituitary levels of R I A - P R L were not s i g n i f i c a n t l y d i f f e r e n t among any of the groups, the levels of 3H-PRL were significantly higher in the DA-treated groups than in the controls (Table 1). Discussion The i n c o r p o r a t i o n of 3 H - l e u c i n e into PRL n e w l y s y n t h e s i z e d by h a m s t e r pituitaries was l i n e a r o v e r a five h i n c u b a t i o n p e r i o d s i m i l a r to that which occurs in the rat (15). Additionally, the release of newly synthesized PRL into the incubation medium was also linear over the entire duration of the incubation. H o w e v e r , u n l i k e in the rat~ most of the PRL r e m a i n s in the p i t u i t a r y r a t h e r than being s e c r e t e d into the medium, c o r r o b o r a t i n g the results of our previous report (3). In this study, we a t t e m p t e d to r e s o l v e the q u e s t i o n of w h e t h e r DA, at concentrations that inhibit PRL synthesis and secretion in the rat, inhibits PRL synthesis and secretion in the male hamster. Both concentrations of DA ~sed in the present study markedly inhibited the release of both RIA-PRL and H-PRL into the medium. An unanticipated finding was that the synthesis of PRL was not i n h i b i t e d by DA in our study. This c o n t r a s t s to r e s u l t s in the rat in w h i c h DA inhibits both PRL r e l e a s e and s y n t h e s i s ([5). To our knowledge, the hamster appears to be the only mammalian species studied thus
Vol. 38, No. 21, 1986
Effects of Dopamine on Hamster
Prolactin
1919
far in which an inhibitory effect of DA on PRL release occurs in the absence of an inhibitory effect on synthesis. Perhaps, if the incubation period had been extended for several more h an inhibitory effect of DA on PRL synthesis might have been observed as well. 9,
-T" 8,
7,
A
[~
T
II
r~]
TOTAL (PIT + MEDIUM)
MEDIUM
6,
0 x '5 o. a
5
4,
,o. -r o3
3-
2,
I.
o DOPAMINE 5 x 10 - 7 M
CONTROL
DOPAMINE 5 x 10-5M
FIG. 3 The effects of d o p a m i n e on the s y n t h e s i s and r e l e a s e of p r o l a c t i n (PRL) in v i t r o o v e r a 4 h period. The bars r e p r e s e n t the mean + SEM. There were 6 vials (each containing 3 hemipituitaries) in each group.-- The levels of 3HPRL in the medium in the dopamine-treated groups ~iffered from the control, p < 0.001. T o t a l 3 H - P R L l e v e l s in the 5 x 1 0 - J M d o p a m i n e - t r e a t e d group differed from the control, p < 0.05. TABLE I The Effects of Dopamine on Anterior Pituitary Levels of 3H-Prolactin and Immunoreactive (RIA)-PRL Following 4 h of Incubation In Vitro.
TREATMENT
3H-PRL (DPM X 10 -4)
(PRL)
RIA-PRL (ug)
Control
5.51
+
0.33
26.72
+
0.57
Dopamine (5 X 10-7M)
7.05
~
0.53*
26.39
+
0.98
Dopamine (5 X Io-SM)
7.71 ~
0.21"
28.48
+
1.07
V a l u e s r e p r e s e n t the mean + SEM. There were 6 v i a l s (each c o n t a i n i n g hemipituitaries) per group. ~ p < 0.05 versus control group.
3
1920
Effects of Dopamine on Hamster Prolactin
Vol. 38, No. 21, 1986
As ~ould be expected in the case of uninhibited PRL synthesis, the inhibition of JH-PRL release by DA resulted in a corresponding accumulation of labeled PRL in the pituitaries. In contrast, the D A - i n d u c e d i n h i b i t i o n of RIA-PRL release did not result in a c o r r e s p o n d i n g increase in pituitary l e v e l s of RIA-PRL. One e x p l a n a t i o n could be that DA caused an increase in the intracellular degradation of RIA-PRL that would otherwise have accumulated in the g l a n d s as a r e s u l t of D A - i n h i b i t e d PRL release. In fact, others have shown that if PRL release is inhibited by either DA or its agonists, the PRL not released from the cell is degraded (12). Apparently, any degradation of unreleased, newly synthesized PRL may have been masked by unrestricted PRL synthesis in the DA-treated groups. It is likely that DA-inhibited PRL release is accomplished via a DA receptormediated mechanism since the DA receptor antagonist pimozide blocks the PRLinhibitory effects of DA on female hamster pituitaries (unpublished results). Furthermore, a recent preliminary report by Burns et al. (16) indicates that male hamster pituitaries contain both high and low affinity DA binding sites. Our r e s u l t s are in marked contrast to those of Steger et al. (10,1[) who observed either no inhibition or a marginal inhibition of PRL release from the pituitaries of long photoperiod-exposed male hamsters in response to DA. These discrepancies might be explicable by the fact that even our lowest dose (5 X 10-7M) was f i v e - f o l d higher than the highest dose (10-7M) examined by Steger et al. (11). Another explanation for the differences in the results may be that single hemi-pituitaries were incubated for only one to two h in the presence of DA in the other investigations ([0,11). As a l l u d e d to p r e v i o u s l y , light d e p r i v a t i o n m a r k e d l y inhibits both the synthesis and r e l e a s e of PRL in m a l e hamsters (3). Furthermore, hamster medial basal hypothalamic extracts inhibit the synthesis of PRL but ,lot the release of RIA-PRL in vitro (5). Conversely, in the present investigation, DA inhibited the r e l e a s e of PRL but not its synthesis. This suggests to us that DA may not be the primary factor r e s p o n s i b l e for the l o n g - t e r m inhibition of PRL cell activity in light-deprived male hamsters. Rather, DA may be more important in the s h o r t - t e r m r e g u l a t i o n of PRL under the conditions of long photoperiod in the male hamster. Acknowledgements This work was supported by NIH B i o m e d i c a l R e s e a r c h Support Grant RR05765. The authors wish to thank Wanda McGill for technical assistance and Dr. Frank Talamantes, Univ. of California, Santa Cruz, for the generous supply of the hamster PRL a n t i s e r u m and purified hamster PRL. Send reprint requests to D a v i d E. Blask, Ph.D., M.D., D e p a r t m e n t of Anatomy, The U n i v e r s i t y of Arizona, College of Medicine, Tucson, Arizona 85724. References 1. 2. 3. 4. 5.
R.J. REITER, Endocr. Rev. ~ 109-131 (1980). A. BARTKE, B.D. GOLDMAN, F.J. BEX, R.P. KELCH, M.S. SMITH, S. D A L T E R I O P.C. DOHERTY, Endocrinology 106 167-172 (1980). D.E. BLASK, C.A. LEADEM, K.M. ORSTEAD and B.R. LARSEN, N e u r o e n d o c r i n o l ogy (in press). F. BEX, A. BARTKE, B.D. G O L D M A N and S. DALTERIO, E n d o c r i n o l o g y 103 2069-2080 (1978). K.M. ORSTEAD and D.E. BLASK, Prolactin~ Basic and Clinical Correlates R.M. MacLeod, M.O. Thorner and U. Scapagnini (eds.), I 113-12[, Livinia Press, Padova (1985).
Vol. 38, No. 21, 1986
6. 7. 8. 9. I0. ii. 12. 13. 14. 15.
16.
Effects of Dopamine on Hamster Prolactin
1921
A. BARTKE, M.P. HOGAN and G.B. CUTTY, J. Androl. 1 115-120 (1980). K . T . BORER, C.S. CAMPBELL, J. TABOR, K. JORGENSON, S. KANDARIAN and L.GORDON, Biol. Reprod. 29 38-47 (1983). R.M. MACLEOD and J.E. LEHYMEYER, Endocrinology 94 1077-1085 (1974). C.J. SHAAR and J.A. CLEMENS, Endocrinology 95 1202-1212 (1974). R.W. STEGER, A. BARTKE, B.D. GOLDMAN, M.J. SOARES and F. TALAMANTES, Biol. Reprod. 29 872-878 (1983). R.W. STEGER, K.S. MATT, H.G. KLEMCKE and A. BARTKE, Neuroendocrinology 41 89-96 (1985). P.S. DANNIES, Biochem. Pharmacol. 31 2845-2849 (1982). R.A. REISFELD, U.J. LEWIS, D.E. WILLIAMS, Nature 195 281-283 (1962). M.J. SOARES, P. COLOSI and F. TALAMANTES, Proc. Soc. Exp. Biol. Med. 172 379-381 (1983). R.M. MACLEOD and J.E. LEHMEYER, Lactogenic Hormones, G.E.W. Wolstenholme and J. Knight (eds.), pp. 53-82, Churchill Livingstone, London (1972). D.M. BURNS, C.A. LEADEM and B. BENSON, Prog. |5th Ann. Mtg. Soc. Neurosci. p. 658, Abst. #196.9, (1985).