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Effects of methionine-enkephalin on prolactin release and catecholamine levels and turnover in the median eminence
89 European Journal of Pharmacology, 43 ( 1977 ) 89--90
© Elsevier/North-Holland Biomedical Press Rapid communication EFFECTS OF METHIONINE-ENKEPHALIN ON PROLACTIN RELEASE AND CATECHOLAMINE LEVELS AND TURNOVER IN THE MEDIAN EMINENCE LOUISE FERLAND**, KJELL FUXE*, PETER ENEROTH**, JAN-AKE GUSTAFSSON*** and PAUL SKETT*** *Department of Histology, and ***Department of Chemistry, Karolinska Instituter, S-104 01 Stockholm 60, Sweden, and **Hormone Laboratory, Karolinska Hospital, S-104 01 Stockholm 60, Sweden
Received 7 March 1977, accepted 8 March 1977
Hughes et al. have recently isolated and described the structure of a pentapeptide from pig brain with potent morphine-like acticity. This pentapeptide is named methionineenkephalin (met-enkephalin) and is structurally identical to the 61--65 sequence of ovine ~-lipotropin (~-LPH). Recent findings indicate the presence of enkephalin-like immunoreactive material in nerve terminals of the medial basal hypothalamus of rat (Elde et al., 1976). Furthermore, Lien et al. (1976) and Labrie (personal communication, 1976) have found that met-enkephalin increased prolactin release in the rat as has been observed after morphine treatment. In the present study, the effects of metenkephalin on catecholamine (CA) levels and on CA disappearance after tyrosine hydroxylase inhibition have been studied as well as the effects on prolactin serum levels. Met-enkephalin (Peninsula Laboratories, San Carlos, Calif., U.S.A.) was infused (60 pg in 20 pl of sterile physiological saline) during 1 h into the lateral ventricle of adult male rats under fluothane-air anesthesia. Rats were decapitated at the end of the infusion. The brains were immediately dissected out and the blood was centrifuged and kept at --20°C until radioimmunoassayed for prolactin (NIH kit). By means of a quantititave microfluorimetrical application of the Falck--Hillarp technique *Fellow of the Medical Research Council of Canada.
(LBfstrSm et al., 1976) the CA stores in the median eminence were measured. The subependymal layer (SEL). contains mainly noradrenaline (NA) nerve terminals and the lateral palisade zone (LPZ) mainly dopamine (DA) nerve terminals (LSfstr6m et al., 1976). In the medial palisade zone (MPZ) a mixture of DA and NA nerve terminals is present, where DA nerve terminals predominate (50-75% of total CA nerve terminals) (LSfstrSm et al., 1976). Changes in CA turnover were evaluated by studying the depletion of CA stores following treatment with a tyrosine hydroxylase inhibitor (~-methyltyrosine methylester, H44/68). This decline is, by and large, exponential in the median eminence (LSfstrSm et al., 1976). H44/68 was injected i.p. (250 mg/kg in physiological saline) at the same time as the infusion of met-enkephalin was started. After infusion of met-enkephalin alone (60 pg/1 h), the CA stores in the MPZ of the median eminence were increased by about 35% whereas the CA stores in the SEL were reduced by 28% (fig. 1). A trend towards an increase of CA stores in the LPZ was also noted. A marked increase (400%) in prolactin serun~ levels occurred at the same time. In experiments where H44/68 was used in addition, a significant reduction of the H44/ 68-induced CA disappearance was observed in both the MPZ and LPZ, whereas there were no significant changes in the H44/68-induced
CA
0
150
SEL
FLUORESCENCE MPZ
LRZ
PRL LEVEL
0
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1::3 LU ,~ IOO
]MET-ENK
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i N= 4
75
/
D
~
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u ~L u © Z
:i:!
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+ H44/68
a_ 150
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N=4 100
%
60 z [u
(presumably mainly DA stores) in MPZ can at least partially be explained on the basis that DA release is reduced without a concomitant equivalent of DA synthesis, since met-enkephalin was found to decrease CA turnover in MPZ. Furthermore, the present findings give evidence that met-enkephalin reduces DA turnover in LPZ. Thus, met-enkephalininduced increases of prolactin secretion can at least in part be mediated by a reduction of DA activity in the median eminence. To which extent these CA changes are also directly engaged in the increase of serum growth hormone levels observed after metenkephalin (Dupont et al., 1977) cannot be evaluated from the present data.
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Fig. 1. Effect of a 1-h infusion of met-enkephalin (60 ~g) in the lateral ventriele of adult male rats under fluothane anesthesia on prolactin serum levels and on CA stores and turnover in the median eminence. The mierofluorimetrical data are expressed in per eent of the saline-infused rats or of the salineinfused H44/68-treated rats. H 4 4 / 6 8 was injected i.p. at the dose of 250 mg/kg at the beginning of the infusion.
CA disappearance in the SEL (fig. 1). The serum prolactin levels were markedly increased by H44/68 treatment. A further increase (30%) of the prolactin levels was seen following met-enkephalin treatment. The present results show that met-enkephalin reduces NA concentration in SEL. The relation between this change and the changes in hormonal levels is not clear at the present time. One possible explanation of the fact that no acceleration of H44/68-induced NA disappearance in SEL was observed may be that NA synthesis in this system was inhibited by met-enkephalin. The increase in CA stores
This work has been supported by a grant (04X715) from the Swedish Medical Research Council. The N I A M D D Rat Pituitary H o r m o n e Distribution Program is gratefully acknowledged for the rat prolactin radioimmunoassay kit. P.S. is grateful to the Ciba--Geigy Fellowship Trust for a fellowship.
References D u p o n t , A., L. Cusan, M. Garon, F. Labrie and C.H. Li, 1977, Stimulation of growth h o r m o n e release by beta-endorphin and methionine-enkephalin; Proc. Nat. Acad. Sci (in press). Elde, R., T. HSkfelt, O. Johansson and L. Terenius, 1976, I m m u n o h i s t o c h e m i c a l studies using antibodies to leucine-enkephalin: Initial observations on the nervous system of the rat, Neuroscience 1, 349. Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan and H.R. Morris, 1975, Identification of two related pentapeptides from the brain with p o t e n t opiate agonist activity, Nature, London 258,577. Lien, E.L., R.L. Fenichel, V. Garsky, D. Sarantakis and N.H. Grant, 1976, Enkephalin-stimulated prolactin release, Life Sci. 1 9 , 8 3 7 . L6fstr6m, A., G. Jonsson, F.-A. Wiesel and K. Fuxe, 1976, Microfluorimetric q u a n t i t a t i o n of catecholamines in the rat median eminence. II. Turnover changes in h o r m o n a l states, J. Histochem. Cytochem. 24,430.
© Elsevier/North-Holland Biomedical Press Rapid communication EFFECTS OF METHIONINE-ENKEPHALIN ON PROLACTIN RELEASE AND CATECHOLAMINE LEVELS AND TURNOVER IN THE MEDIAN EMINENCE LOUISE FERLAND**, KJELL FUXE*, PETER ENEROTH**, JAN-AKE GUSTAFSSON*** and PAUL SKETT*** *Department of Histology, and ***Department of Chemistry, Karolinska Instituter, S-104 01 Stockholm 60, Sweden, and **Hormone Laboratory, Karolinska Hospital, S-104 01 Stockholm 60, Sweden
Received 7 March 1977, accepted 8 March 1977
Hughes et al. have recently isolated and described the structure of a pentapeptide from pig brain with potent morphine-like acticity. This pentapeptide is named methionineenkephalin (met-enkephalin) and is structurally identical to the 61--65 sequence of ovine ~-lipotropin (~-LPH). Recent findings indicate the presence of enkephalin-like immunoreactive material in nerve terminals of the medial basal hypothalamus of rat (Elde et al., 1976). Furthermore, Lien et al. (1976) and Labrie (personal communication, 1976) have found that met-enkephalin increased prolactin release in the rat as has been observed after morphine treatment. In the present study, the effects of metenkephalin on catecholamine (CA) levels and on CA disappearance after tyrosine hydroxylase inhibition have been studied as well as the effects on prolactin serum levels. Met-enkephalin (Peninsula Laboratories, San Carlos, Calif., U.S.A.) was infused (60 pg in 20 pl of sterile physiological saline) during 1 h into the lateral ventricle of adult male rats under fluothane-air anesthesia. Rats were decapitated at the end of the infusion. The brains were immediately dissected out and the blood was centrifuged and kept at --20°C until radioimmunoassayed for prolactin (NIH kit). By means of a quantititave microfluorimetrical application of the Falck--Hillarp technique *Fellow of the Medical Research Council of Canada.
(LBfstrSm et al., 1976) the CA stores in the median eminence were measured. The subependymal layer (SEL). contains mainly noradrenaline (NA) nerve terminals and the lateral palisade zone (LPZ) mainly dopamine (DA) nerve terminals (LSfstr6m et al., 1976). In the medial palisade zone (MPZ) a mixture of DA and NA nerve terminals is present, where DA nerve terminals predominate (50-75% of total CA nerve terminals) (LSfstrSm et al., 1976). Changes in CA turnover were evaluated by studying the depletion of CA stores following treatment with a tyrosine hydroxylase inhibitor (~-methyltyrosine methylester, H44/68). This decline is, by and large, exponential in the median eminence (LSfstrSm et al., 1976). H44/68 was injected i.p. (250 mg/kg in physiological saline) at the same time as the infusion of met-enkephalin was started. After infusion of met-enkephalin alone (60 pg/1 h), the CA stores in the MPZ of the median eminence were increased by about 35% whereas the CA stores in the SEL were reduced by 28% (fig. 1). A trend towards an increase of CA stores in the LPZ was also noted. A marked increase (400%) in prolactin serun~ levels occurred at the same time. In experiments where H44/68 was used in addition, a significant reduction of the H44/ 68-induced CA disappearance was observed in both the MPZ and LPZ, whereas there were no significant changes in the H44/68-induced
CA
0
150
SEL
FLUORESCENCE MPZ
LRZ
PRL LEVEL
0
[~C O N T R OL'",
1::3 LU ,~ IOO
]MET-ENK
! 3O
i N= 4
75
/
D
~
5o
u ~L u © Z
:i:!
90 ~_ a:
+ H44/68
a_ 150
g O a w
N=4 100
%
60 z [u
(presumably mainly DA stores) in MPZ can at least partially be explained on the basis that DA release is reduced without a concomitant equivalent of DA synthesis, since met-enkephalin was found to decrease CA turnover in MPZ. Furthermore, the present findings give evidence that met-enkephalin reduces DA turnover in LPZ. Thus, met-enkephalininduced increases of prolactin secretion can at least in part be mediated by a reduction of DA activity in the median eminence. To which extent these CA changes are also directly engaged in the increase of serum growth hormone levels observed after metenkephalin (Dupont et al., 1977) cannot be evaluated from the present data.
:::::
iiili
5
O w
Acknowledgements
3(?
:g-- 5o -?
@
Fig. 1. Effect of a 1-h infusion of met-enkephalin (60 ~g) in the lateral ventriele of adult male rats under fluothane anesthesia on prolactin serum levels and on CA stores and turnover in the median eminence. The mierofluorimetrical data are expressed in per eent of the saline-infused rats or of the salineinfused H44/68-treated rats. H 4 4 / 6 8 was injected i.p. at the dose of 250 mg/kg at the beginning of the infusion.
CA disappearance in the SEL (fig. 1). The serum prolactin levels were markedly increased by H44/68 treatment. A further increase (30%) of the prolactin levels was seen following met-enkephalin treatment. The present results show that met-enkephalin reduces NA concentration in SEL. The relation between this change and the changes in hormonal levels is not clear at the present time. One possible explanation of the fact that no acceleration of H44/68-induced NA disappearance in SEL was observed may be that NA synthesis in this system was inhibited by met-enkephalin. The increase in CA stores
This work has been supported by a grant (04X715) from the Swedish Medical Research Council. The N I A M D D Rat Pituitary H o r m o n e Distribution Program is gratefully acknowledged for the rat prolactin radioimmunoassay kit. P.S. is grateful to the Ciba--Geigy Fellowship Trust for a fellowship.
References D u p o n t , A., L. Cusan, M. Garon, F. Labrie and C.H. Li, 1977, Stimulation of growth h o r m o n e release by beta-endorphin and methionine-enkephalin; Proc. Nat. Acad. Sci (in press). Elde, R., T. HSkfelt, O. Johansson and L. Terenius, 1976, I m m u n o h i s t o c h e m i c a l studies using antibodies to leucine-enkephalin: Initial observations on the nervous system of the rat, Neuroscience 1, 349. Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan and H.R. Morris, 1975, Identification of two related pentapeptides from the brain with p o t e n t opiate agonist activity, Nature, London 258,577. Lien, E.L., R.L. Fenichel, V. Garsky, D. Sarantakis and N.H. Grant, 1976, Enkephalin-stimulated prolactin release, Life Sci. 1 9 , 8 3 7 . L6fstr6m, A., G. Jonsson, F.-A. Wiesel and K. Fuxe, 1976, Microfluorimetric q u a n t i t a t i o n of catecholamines in the rat median eminence. II. Turnover changes in h o r m o n a l states, J. Histochem. Cytochem. 24,430.