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Supression of basal and stress-induced prolactin release and stimulation of luteinizing hormone secretion by α-melanocyte-stimulating hormone
Life Sciences, Vol, 36, pp. 1661-1668 Printed in the U.S.A.
Pergamon Press
SUPPRESSION OF BASALAND STRESS-INDUCI~) PROLACTIN RELEASE AND STIMUIATION OF LUTEINIZING HORMONE SB~RETION BY ~-M~OCYTESTIMULATING HORMONE* Connie B. Newman, Sharon L. Wardlaw, and Andrew G. Frantz, Department of Medicine, ColumbiaUniversity College of Physicians and Surgeons New York, N.Y. 10032 (Received in final form February 18, 1985)
Summary ~-MSH and B-endorphin, both synthesized ~rom a common precursor, have opposite behavioral actions. In order to determine if these peptides have opposite effects on pituitary function, basal LH secretion and basal and stress-induced prolactin release were studied in adult male rats after intraventricular injection of ~-MSH. Each rat also received intraventricular saline in order to serve as its own control. 18 pg ~-MSH stimulated plasma I}I from 16.5 + 2.5 (SEM) ng/ml to a peak of 27.2 + 4.0 and 26.0 + 4.9 ng/ml at 5 and l0 rain, and suppressed prolactin from 3.5 +-0.7 ng/ml to 1.3 + 0.1 and 1.2 + 0.1 ng/ml at 15 and 30 min. Int-raventricular~-MSH-also significantly blunted the prolactin rise associated with the stress of swimming. 10 and 20 rain after the onset of swimming, prolactin levels in rats pretreated with ~-MSH were significantly diminished: 7.4 + 1.5 and 6.5 + 2.0 ng/ml vs 23.8 + 3.6 and 15.2 + 2.8 after normal saline. Similarly, des-acetyl ~-MS~I which is the p'redominantform of ~-M~I in the hypothalamus, diminishedthe stress-induced prolaetln rise from 18.4 + 5.3 and ll.2 + 3.4 ng/ml at I0 and 20 min to 10.0 + 2.4 and 5.5 +-l. 6 ng/ml. We" conclude that centrally administered ~-M~-I stim~ates ~ and suppresses basal and stress-induced prolaetin release in male rats. These actions are opposite to those previously shown for B-endorphin and suggest that a-MSH may antagonizethe effects of B-endorphin on pituitary function. ~-M~-I, N-acetyl ACTH 1-13 amide, is synthesized together with other proopiomelanocortin-derived peptides in the brain and in the anterior and intermediate lobes of the pituitary. The post-translational processing of proopiomelanocortin differs in these regions, with ~-MS~I, CLIP (AC~-I 18-39), and B-endorphinbeing the major cleavage products in the brain and pituitary intermediate lobe, while AC~H and B-lipotropin are the main products of proopiomelanocortin in the anterior pituitary (1,2). Additionalpost-translational processing of these proopiomelanocortin-derived peptides also occurs in pituitary and brain, resulting in acetylated and deacetylated forms of ~-MSH and B-endorphin. In animal species with a pituitary intermediate lobe, the intermediate lobe is the main source of ~-MS~I. However, both rat and human brain contain significant amounts of ~-M~-I (3-9), which is present predominantly in a deaeetylated form (I0-12). *Presented in part at the 65th Annual Meeting of The Endocrine Society, San Antonio, Texas, June 8-10, 1983, Abst. 335. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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~-MSH Suppresses PRL and Stimulates LH
Vol. 36, No. 17, 1985
The pigmentary actions of~-M~I are well recognized (2). Intraventricular administration of ~-MSH has also been shown to produce behavioral effects which are opposite to those seen after intraventricular administration of B-endorphin (2,13-18). In view of the opposing behavioral actions of ~-MSH and B-endorphin, and also in light of the recent observation that ~-MSH has a suppressive effect on prolactin secretion in rats (19), we have questioned whether ~-MSH and B-endorphin might have opposing effects on anterior pituitary hormone secretion. The present study was designed to determine the effect of centrally admininistered ~-MSH on basal LH secretion and on both basal and stress-induced prolactin release in rats. Also, since most brain ~-MSH has been shown to be of the deacetylated form, we have studied the effect of des-acetyl ~-M~I on stress-mediated prolactin secretion.
Materials and Methods Cannulation Adult male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, Mass.) weighing 175-225 grams were housed in a light-controlled environment (lights on 7 A.M. - 7 P.M.), and given free access to water and Purina Rat Chow. Rats were handled daily. One week after arrival, and 3 to 8 days prior to experimentation, under pentobarbital anesthesia, a 21 gauge stainless steel cannula was stereotactically inserted into the right lateral cerebral ventricle of each rat, using a modification of the method described by Hayden et al (20). At the same time, a silastic catheter for blood withdrawal was placed in the right atrium according to a modification of the procedure of Brown and Hedge (21). Tne catheter was led subcutaneously to the top of the animal's head, where it was connected to an L-shaped metal cannula that was fixed to the skull with dental cement. This cannula was connected to polyethylene tubing, protected by a spring coil, which led outside the cage. Animals were caged singly, and were moving freely throughout the experiments. Cages were covered during experimentation for the purpose of minimizing stress. Three experiments were performed between 8 A.M. and 2 P.M., and all rats were tested twice at the same time of day in order to serve as their own controls. In each experiment, the rat received, on separate days, a 13~l intraventricular injection of normal saline as a control, and on another occasion either 18 ~g ~-MSH or 18 ~g des-acetyl ~-MSH (Peninsula Laboratories) dissolved in normal saline. These peptide solutions were stored frozen in small aliquots. Each aliquot was used within one month of preparation, and was thawed only once, on the day of the experiment. During experimentation, blood samples of 0.7 ml were withdrawn at varying intervals, immediately centrifuged in a Beckman microfuge, and the plasma frozen for later assay. Red blood cells were resuspended in heparinized saline and returned to the animal. Experiment h
Effect of ~-MSH on basal prolactin and LH secretion.
3 blood samples were collected from 10 non-stressed, resting rats at 15 minute intervals prior to the intraventricular administration of the test substance, and subsequently blood samples were drawn at 5, 10, 15, 30, 60, 90, and 120 minutes. This experimental protocol was repeated 2 days later. Half the animals received ~-MSH on day l and normal saline on day 3, while in the other half the order of injections was reversed. Experiment 2:
Effect of ~-MSH on stress-induced prolactin secretion.
10 minutes after intraventricular injection of either normal saline or 18 ~ g ~-MSH, as described above, 8 rats were stressed by swimming for 20 minutes in 20 C water. 3 blood samples were taken before swimming, 2 during swimming at 10 and 20 minutes, and 3 afterwards at 35, 50, and 90 minutes.
Vol. 36, No. 17, 1985
Experiment 3:
~-MSH Suppresses PRL and Stimulates LH
1663
Effect of des-aeetyl ct-M~I on stress-induced prolactin secretion.
9 rats received 13 ~l intraventricular injections of either normal saline or 18 pg des-acetyl a-MSH, and were tested according to the same protocol used in experiment 2. Radioimmunoassay Plasma I~I and prolactin were determined by double antibody radioimmunoassay using materials provided by the National Institute of Health (rat LH reference preparation, NIAMDD-rat 12-I-RP-1; rat prolactin reference preparation, NIADDKPRL-RP-3). Statistical Analysis Data on the e f f e c t s of a-MSH on basal prolactin and LH s e c r e t i o n were analyzed by two way analysis of v a r i a n c e . In the s t r e s s studies, prolactin r e l e a s e was determined by calculating the a r e a s under the plasma concentration c u r v e s between 0 and 50 min. The amount of prolactin r e l e a s e d a f t e r a-M~I or d e s - a c e t y l a-MSH t r e a t m e n t , as compared with normal saline t r e at m en t in the same animal, was analyzed by paired t - t e s t . Results Experiment 1:
Effect of a-MSH on basal prolactin and LH s e c r e t i o n .
Figure 1 shows the prolactin r e s p o n s e a f t er i n t r a v e n t r i c u l a r injection of normal saline and 18 p g a-MSH, a-MSH was e f f e c t i v e in s u p p r e s s i n g basal prolactin s e c r e t i o n (p < .001). A f t e r a - M ~ I , prolactin fell from a mean baseline value of 3.5 + 0.7 (SEM) ng/ml to 1.3 + 0.1 and 1.2 + 0.1 n g / m l at 15 and 30 min. Prola-ctin levels a f t e r normal saline injection were not significantly changed. Figure 2 shows the r e s p o n s e o f I ~ in the same animals, a - M ~ I significantly enhanced LH s e c r e t i o n (p <.001). LH l e v e l s r o s e from a mean baseline value of 16.5 + 2.5 ng/ml to a peak of 27.2 + 4 . 0 n g / m l 5 min a f t e r i n t r a v e n t r i c u l a r a-MSH, and r~mained elevated for a 30 rain p e r i o d . Plasma [l-I did not significantly change a f t e r normal saline administration. Experiment 2:
Effect of a-MSH on s t r e s s - i n d u c e d prolactin s e c r e t i o n .
a-MSH significantly blunted swimming s t r e s s - i n d u c e d prolactin r e l e a s e . Mean prolactin r e l e a s e between 0 and 50 min after a-MSH was 39.2% + 5.8 (SEM) that o f the saline controls (p < .001). As shown in figure 3, in the control experiment, plasma prolactin r o s e to 23.8 + 3.6 and 15.2 + 2.8 n g / m l 10 and 20 min a f t e r swimming, as compared with 7:-4 + 1.5 and 6.5"+ 2 .0 ng/ml at the same i n t e r v a l s a f t e r a-MSH. Experiment 3:
Effect of d e s - a c e t y l a-MSH on s t r e s s - i n d u c e d prolactin s e c r e t i o n .
D e s - a c e t y l a-MSH was also e f f e c t i v e in blunting swimming s t r e s s - i n d u c e d prolactin s e c r e t i o n . Mean prolactin r e l e a s e between 0 and 50 rain a f t e r d e s - a c e t y l ~-MIYH was 59.5% + 6.6 (SEM) that of the saline controls ( p < .001). As shown in figure 4, aft er normal saline injection prolactin r o s e to 18.4 + 5.3 and 11.2 + 3.4 ng/m l 10 and 20 min following swimming, as compared with 10.-0 + 2 .4 and 5 .5 + 1.6 ng/ m l at the same i n t e r v a l s a f t e r d e s - a c e t y l a-MS'H.
1664
~-MSH Suppresses
PRL and S t i m u l a t e s
LH
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intraventricular
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Vol. 36, No. 17, 1985
a-MSH Suppresses PRL and Stimulates LH
1665
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Figure 4. Plasma prolactin (_+ SEM) in 9 male rats subjected to swimming stress and pretreated with either 18 ~g des-acetyl ~-MSH or normal saline. Des-acetyl ~-MSH significantly diminished the stress-induced rise in plasma prolactin.
1666
e-MSH Suppresses PRL and Stimulates LH
Vol.
36, No. 17, 1985
Discussion lhese data demonstrate that intraventricular ~-MSH simultaneously stimulates LH and suppresses prolactin secretion, and significantly blunts stress-induced prolactin release in male rats. In addition we have shown that the deacetylated form of ~-MSH which is less potent than ~-M~-I in producing skin darkening and some behavioral changes (10), also diminishes stress-induced prolactin secretion. T h i s observation is significant in view of the evidence that des-acetyl ~-M~I is the major form of ~-MSH in brain (10-12). The effects of ~-MSH on prolactin and LH release are opposite to those previously demonstrated for B-endorphin (22-26). Behavioral studies have shown that ~-MSH and B-endorphin produce opposite behavioral effects after intraventricular administration to rats (2,13-18). ~-M~rl causes hyperalgesia, increased arousal, facilitation of learning, and enhanced sexual activity, while B-endorphin injection results in analgesia, catalepsy, and diminished sexual function. ~-MSH has also been shown to attenuate opioid-induced analgesia, thus suggesting that ~-M~I may be an endogenous opiate antagonist (27,28). Thus, ~-MSH and B-endorphin, both products of a common precursor, have antagonistic actions on behavior, as well as on anterior pituitary function in rats. The effect of ~-MS}I on basal prolactin secretion which we have demonstrated is in agreement with the observation of Khorram and colleagues that injection of 2 ~g ~-MSH into the third ventricle lowered plasma prolactin in ovariectomized, estrogen primed rats (19). The inhibition of prolactin release by ~-M~I may be mediated by the hypothalamic dopaminergic neuronal system. ~-M~I has been shown to increase dopaminergic neuronal activity in the arcuate nucleus of the rat hypothalamus (29), an effect opposite to that of B-endorphin, which has been found to diminish hypothalamic dopamine turnover (30), and decrease dopamine release into rat portal blood (31). Khorram and colleagues (19) observed that ~-MSH had no effect on plasma prolactin in ovariectomized rats when administered intravenously, or when injected intraventricularly one hour after treatment with the dopamine receptor antagonist spiroperidol. They also found that ~-M~{ did not alter prolactin release from rat hemipituitaries of male and ovariectomized female rats, thus indicating that ~-MSH may act at a hypothalamic level to decrease prolactin secretion, perhaps by affecting dopamine turnover. Previous investigations of the effect of ~-MSH on LH secretion have provided variable results. A i d e and Celis (32) observed that in ovariectomized, estrogen-primed female rats, intravenous injection of 30 ~g ~-MSH augmented the l ~ response to LHRH, but had no effect on LH when administered alone. More recently Khorram et al. (33) reported that in chronically ovariectomized unprimed rats, administration of 2 lag ~-MSH into the third ventricle significantly lowered plasma LH for one hour after injection, while the intravenous injection of 5 ~ g ~-Mb~l had no effect. However, they also observed that in ovariectomized rats primed with 50 ~g estradiol 72 hours before the experiment, intraventricular injection of 2 ~g ~-M~-I did not change LH levels. Our finding of enhanced LH secretion after central ~-MSH administration in intact male rats contrasts with the inhibitory effect observed by these investigators in chronically ovariectomized rats, and perhaps can be explained by the different sex steroid environments of the animals studied. The hypothesis that the sex steroid environment is an important determinant of the LH response to ~-MSH is supported by the observations of Reid et al. who found that intravenous c~-MSH stimulated LH in normal men (34) and in women in the luteal phase of the menstrual cycle, but not in women in the early follicular phase (35). In this respect, ~-MSH is similar to the opiate antagonist naloxone which enhances LH secretion in normal men and in late follicular or luteal phase women, but not in women in the early follicular phase of the menstrual cycle (36). The mechanism by which ~-MSH stimulates LH release is not completely understood. Our observation of enhanced LH secretion following intraventricular ~-MSH
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~-MSH Suppresses PRL and Stimulates LH
1667
administration suggests that ~-M~H may act centrally to stimulate LH. ~-M~I has been reported to increase hypothalamic dopamine turnover (29), however the role of dopamine in ~ secretion remains controversial (37,38). The study of Miyake and Yen (39) suggests that ~-M~I acts directly at the pituitary to augment LH secretion. In their hypothalamic-pituitary superfusion system, ~-MS~I induced LH release from pituitaries of male rats, but not female proestrus rats, and did not have any effect on I~IRH release from the medial basal hypothalamus of rats of either species. The actions of ~-M~I that we have described were achieved with pharmacological doses of the peptide, similar to the doses used previously in behavioral studies (15,27). Althoughthe exact physiological significance of our observations remains to be determined, the data presented here together with the finding that a-M~-I is present in high concentrations in rat median eminence (4) suggest that ~-MSH in either its acetylated or de-acetylated form may participate in the modulation of anterior pituitary function, achieving effects opposite to those of B-endorphin. In previous studies in this laboratory, both intravenous naloxone and intraventricular anti-~-endorphin antiserum in rats significantly diminished the rise in prolactin due to swimming stress, thus showing that ~-endorphin is an endogenous regulator of prolactin secretion (40, 41). In the present investigation, a similar effect was achieved with the intraventricular injection of either ~-MS~I or des-acetyl ~-M~-I. Our finding that acetylated and de-acetylated forms of ~-MSH have effects on LH and prolactin that are similar to those of the opiate receptor antagonist naloxone suggests that ~-M~I may antagonize the effects of ~-endorphin on anterior pituitary function.
Acknowledgement This v~rk has been supported by USPHS grants AM 07271, AM 31075, and CA I1704. References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15.
D.T. KRIB3ER, A.S. LIOTrA, M.J. BROWNSTEIN and E.A. ZIMMERMAN, Rec. Prog. Horm. Res. 36 277-344 (1980) T.L. O'DONOHUE and D.M. DORSA, Peptides 3 353-395 (1982). D. DU}~, J.C. LISSITZKY, R. LECLERC and G. PELLETIER, EndocrinologyI02 1283-1291 (1978). T.L. O'DONOHUE, R.L. MILI.ER and D.M. JACOBOWITZ, Brain Res. 176 I01-123 (1979). G. KLEBI~, C. GRAMSCH, V. HOLLT, P. MJ~IRAEIN, A. PASI and A. HERZ, Neuroendocrinol. 31 39-45 (1980). C. O ~ and J.C. PORT~, Endocrinology I02 697-705 (1978). H. VAUDREY, M.C. TONON, C. DELARUE, R. VAIIZANTand J. KRAICI~, Neuroendocrinol. 27 9-24 (1978). Y.P IX)H, L. ZUCKER, H. VERSPAGAET and T.B. VAN WIMI~RSMAGREIDANUS, J. Neurosci. Res. 4 147-156 (1979). T.L. O'DONOHUE, G.E. HOLMQUIS'I'and D.M.JACOBOWITZ, Neurosci. Lett. 14 271-274 (1979). T.L. O'DONOHUE, G.E. HANDELMANN, R.L. MILLER and D.M. JACOBOWITL, Science 215 1125-1127 (1982). A.I. SMI2TI, K.J.A. EDWARDSON, J.A. BIGGINSand J.R. McDERMOTF, JR., Neurosci. Lett. 30 133-138 (1982). A.S. LIOTYA and D.T. KRIBGER, Program of the 65th Annual Meeting of the Endocrine Society, June 8-10, 1983, San Antonio, Texas, Abst. 45. D. DE WII~D and B. BOHUS, Nature 212 1484-1486 (1966) D. DE WII~D, Life Sci. 20 195-204 (I~7-7). C.A. SANDMAN and A.J-~-KASTIN, Peptides 2 231-233 (1981).
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T . B . VAN WIMERSMA GREIDANUS, G. A.A. DE ROTI~, A.J. THODY and A.N. E~RLE, In Peptides of the Pars Intermedia, eds. D. Evered and G. Lawrenson. pp. 277-294, Pittman Medical, London (1981). 17. B.E. BECKWITH and C.A. SANDMAN, Peptides 3 411-420 (1982). 18. P.C. DATFA and M.G. KING, Neurosci. and Biobehav. Rev. 6 297-310 (1982). 19. O. KHORRAM, H. MIZUNUMA and S.M. McCANN, Neuroendoerinol. 34 433-437 ( 1982). 20. J.G. HAYDEN, L.R. JOHNSON and R.P. MAICKEL, Life Sci. 5 1509-1515 ( 1966). 21. M.R. BROWN and G.A. HEDGE, Neuroendocrinol. 9 158-174 (1972). 22. A. DUPONT, L. CUSAN, F. LA~IE, D.H. COY and C.H. LI, Biochem. Biophys. Res. Comm. 75 76-82 (1977). 23. C. RIVIER, W. VALE, N. LING, M. BROWN and R. GUILLEMIN, Endocrinology I00 238-241 (1977). 24. Y. KATO, Y. IWASAKI, H. ABE, S. OHGO and H. IMURA, P r o c . Soc. Exp. Biol. Med. 158 431-436 (1978). 25. F. KINOSHITA, Y. NAKAI, H. KATAKAMI, Y. KATO, H. YAJIMA and H. IMURA, Life Sci. 27 843-846 (1980). 26. F. KINOSHFII~, Y. NAKAI, H. KATAKAMIand H. IMURA, Life Sci. 30 1915-1919 (1982). 27. J.I. S~EKELY, E. MIGLF_~Z, Z. DUNAI-KOVACS, I. TARNAWA, A.Z. RONAI, L. GRAF and S. ~AJUSZ, Life Sci. 24 1931-1938 (1979). 28. P.C. CONTR]~RAS and A.E. TAKEMORI, J. Pharm. Exp. Therap. 229 21-26 ( 1984). 29. W. LICHT]~GER and F. MONNET, Life Sci. 25 2079-2087 (1979). 30. S.N. DEYO, R.M. SWIFFand R.J. MU.I,ER, Pro~].Natl. Acad. Sci. USA 76 3006-3009 (1979). 31. G . A . GUDELSIKY and J.C. PORTER, Life Sci. 25 1697-1702 (1979). 32. S. AIDE and M.E. CELI$, Neuroendocrinol. 31 I---16-120 (1980). 33. O. KHORRAM, L.R.DePALATIS and S.M. McCTF~NN, Endocrinologyll4 227-233 (1984). 34. R.L. REID, N. LING and S.S.C. YEN, J. Clin. Endoerinol. Metab. 52 159-161 ( 1981). 35. R . L . REID, N. LING and S.S.C. YEN, J. Clin. EndocrinolMetab. 58 773-777 ( 1984). 36. M.E. QUIGLEY and S.S.C. YEN, J. Clin. Endocrinol. Metab. 51 179-181 (1980). 37. C.A. BARRACLOUGH and P.M. WISE, Endocr. Rev. 3 91-I19 (1982). 38. S.P. KALRA and P.S. KAIfIA, Endocr. Rev. 4 311-3-51 (1983). 39. A. MIYAKEand S.S.C. YEN, Life Sci. 29 26-37-2640 (1981). 40. V.V. RAGAVAN and A.G. FRANTZ, Life~ i . 28 921-929 (1981). 41. V.V. RAGAVAN and A.G. FRANTZ, Endocrinolo-~I09 1769-1771 (1981).
Pergamon Press
SUPPRESSION OF BASALAND STRESS-INDUCI~) PROLACTIN RELEASE AND STIMUIATION OF LUTEINIZING HORMONE SB~RETION BY ~-M~OCYTESTIMULATING HORMONE* Connie B. Newman, Sharon L. Wardlaw, and Andrew G. Frantz, Department of Medicine, ColumbiaUniversity College of Physicians and Surgeons New York, N.Y. 10032 (Received in final form February 18, 1985)
Summary ~-MSH and B-endorphin, both synthesized ~rom a common precursor, have opposite behavioral actions. In order to determine if these peptides have opposite effects on pituitary function, basal LH secretion and basal and stress-induced prolactin release were studied in adult male rats after intraventricular injection of ~-MSH. Each rat also received intraventricular saline in order to serve as its own control. 18 pg ~-MSH stimulated plasma I}I from 16.5 + 2.5 (SEM) ng/ml to a peak of 27.2 + 4.0 and 26.0 + 4.9 ng/ml at 5 and l0 rain, and suppressed prolactin from 3.5 +-0.7 ng/ml to 1.3 + 0.1 and 1.2 + 0.1 ng/ml at 15 and 30 min. Int-raventricular~-MSH-also significantly blunted the prolactin rise associated with the stress of swimming. 10 and 20 rain after the onset of swimming, prolactin levels in rats pretreated with ~-MSH were significantly diminished: 7.4 + 1.5 and 6.5 + 2.0 ng/ml vs 23.8 + 3.6 and 15.2 + 2.8 after normal saline. Similarly, des-acetyl ~-MS~I which is the p'redominantform of ~-M~I in the hypothalamus, diminishedthe stress-induced prolaetln rise from 18.4 + 5.3 and ll.2 + 3.4 ng/ml at I0 and 20 min to 10.0 + 2.4 and 5.5 +-l. 6 ng/ml. We" conclude that centrally administered ~-M~-I stim~ates ~ and suppresses basal and stress-induced prolaetin release in male rats. These actions are opposite to those previously shown for B-endorphin and suggest that a-MSH may antagonizethe effects of B-endorphin on pituitary function. ~-M~-I, N-acetyl ACTH 1-13 amide, is synthesized together with other proopiomelanocortin-derived peptides in the brain and in the anterior and intermediate lobes of the pituitary. The post-translational processing of proopiomelanocortin differs in these regions, with ~-MS~I, CLIP (AC~-I 18-39), and B-endorphinbeing the major cleavage products in the brain and pituitary intermediate lobe, while AC~H and B-lipotropin are the main products of proopiomelanocortin in the anterior pituitary (1,2). Additionalpost-translational processing of these proopiomelanocortin-derived peptides also occurs in pituitary and brain, resulting in acetylated and deacetylated forms of ~-MSH and B-endorphin. In animal species with a pituitary intermediate lobe, the intermediate lobe is the main source of ~-MS~I. However, both rat and human brain contain significant amounts of ~-M~-I (3-9), which is present predominantly in a deaeetylated form (I0-12). *Presented in part at the 65th Annual Meeting of The Endocrine Society, San Antonio, Texas, June 8-10, 1983, Abst. 335. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
1662
~-MSH Suppresses PRL and Stimulates LH
Vol. 36, No. 17, 1985
The pigmentary actions of~-M~I are well recognized (2). Intraventricular administration of ~-MSH has also been shown to produce behavioral effects which are opposite to those seen after intraventricular administration of B-endorphin (2,13-18). In view of the opposing behavioral actions of ~-MSH and B-endorphin, and also in light of the recent observation that ~-MSH has a suppressive effect on prolactin secretion in rats (19), we have questioned whether ~-MSH and B-endorphin might have opposing effects on anterior pituitary hormone secretion. The present study was designed to determine the effect of centrally admininistered ~-MSH on basal LH secretion and on both basal and stress-induced prolactin release in rats. Also, since most brain ~-MSH has been shown to be of the deacetylated form, we have studied the effect of des-acetyl ~-M~I on stress-mediated prolactin secretion.
Materials and Methods Cannulation Adult male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, Mass.) weighing 175-225 grams were housed in a light-controlled environment (lights on 7 A.M. - 7 P.M.), and given free access to water and Purina Rat Chow. Rats were handled daily. One week after arrival, and 3 to 8 days prior to experimentation, under pentobarbital anesthesia, a 21 gauge stainless steel cannula was stereotactically inserted into the right lateral cerebral ventricle of each rat, using a modification of the method described by Hayden et al (20). At the same time, a silastic catheter for blood withdrawal was placed in the right atrium according to a modification of the procedure of Brown and Hedge (21). Tne catheter was led subcutaneously to the top of the animal's head, where it was connected to an L-shaped metal cannula that was fixed to the skull with dental cement. This cannula was connected to polyethylene tubing, protected by a spring coil, which led outside the cage. Animals were caged singly, and were moving freely throughout the experiments. Cages were covered during experimentation for the purpose of minimizing stress. Three experiments were performed between 8 A.M. and 2 P.M., and all rats were tested twice at the same time of day in order to serve as their own controls. In each experiment, the rat received, on separate days, a 13~l intraventricular injection of normal saline as a control, and on another occasion either 18 ~g ~-MSH or 18 ~g des-acetyl ~-MSH (Peninsula Laboratories) dissolved in normal saline. These peptide solutions were stored frozen in small aliquots. Each aliquot was used within one month of preparation, and was thawed only once, on the day of the experiment. During experimentation, blood samples of 0.7 ml were withdrawn at varying intervals, immediately centrifuged in a Beckman microfuge, and the plasma frozen for later assay. Red blood cells were resuspended in heparinized saline and returned to the animal. Experiment h
Effect of ~-MSH on basal prolactin and LH secretion.
3 blood samples were collected from 10 non-stressed, resting rats at 15 minute intervals prior to the intraventricular administration of the test substance, and subsequently blood samples were drawn at 5, 10, 15, 30, 60, 90, and 120 minutes. This experimental protocol was repeated 2 days later. Half the animals received ~-MSH on day l and normal saline on day 3, while in the other half the order of injections was reversed. Experiment 2:
Effect of ~-MSH on stress-induced prolactin secretion.
10 minutes after intraventricular injection of either normal saline or 18 ~ g ~-MSH, as described above, 8 rats were stressed by swimming for 20 minutes in 20 C water. 3 blood samples were taken before swimming, 2 during swimming at 10 and 20 minutes, and 3 afterwards at 35, 50, and 90 minutes.
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Experiment 3:
~-MSH Suppresses PRL and Stimulates LH
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Effect of des-aeetyl ct-M~I on stress-induced prolactin secretion.
9 rats received 13 ~l intraventricular injections of either normal saline or 18 pg des-acetyl a-MSH, and were tested according to the same protocol used in experiment 2. Radioimmunoassay Plasma I~I and prolactin were determined by double antibody radioimmunoassay using materials provided by the National Institute of Health (rat LH reference preparation, NIAMDD-rat 12-I-RP-1; rat prolactin reference preparation, NIADDKPRL-RP-3). Statistical Analysis Data on the e f f e c t s of a-MSH on basal prolactin and LH s e c r e t i o n were analyzed by two way analysis of v a r i a n c e . In the s t r e s s studies, prolactin r e l e a s e was determined by calculating the a r e a s under the plasma concentration c u r v e s between 0 and 50 min. The amount of prolactin r e l e a s e d a f t e r a-M~I or d e s - a c e t y l a-MSH t r e a t m e n t , as compared with normal saline t r e at m en t in the same animal, was analyzed by paired t - t e s t . Results Experiment 1:
Effect of a-MSH on basal prolactin and LH s e c r e t i o n .
Figure 1 shows the prolactin r e s p o n s e a f t er i n t r a v e n t r i c u l a r injection of normal saline and 18 p g a-MSH, a-MSH was e f f e c t i v e in s u p p r e s s i n g basal prolactin s e c r e t i o n (p < .001). A f t e r a - M ~ I , prolactin fell from a mean baseline value of 3.5 + 0.7 (SEM) ng/ml to 1.3 + 0.1 and 1.2 + 0.1 n g / m l at 15 and 30 min. Prola-ctin levels a f t e r normal saline injection were not significantly changed. Figure 2 shows the r e s p o n s e o f I ~ in the same animals, a - M ~ I significantly enhanced LH s e c r e t i o n (p <.001). LH l e v e l s r o s e from a mean baseline value of 16.5 + 2.5 ng/ml to a peak of 27.2 + 4 . 0 n g / m l 5 min a f t e r i n t r a v e n t r i c u l a r a-MSH, and r~mained elevated for a 30 rain p e r i o d . Plasma [l-I did not significantly change a f t e r normal saline administration. Experiment 2:
Effect of a-MSH on s t r e s s - i n d u c e d prolactin s e c r e t i o n .
a-MSH significantly blunted swimming s t r e s s - i n d u c e d prolactin r e l e a s e . Mean prolactin r e l e a s e between 0 and 50 min after a-MSH was 39.2% + 5.8 (SEM) that o f the saline controls (p < .001). As shown in figure 3, in the control experiment, plasma prolactin r o s e to 23.8 + 3.6 and 15.2 + 2.8 n g / m l 10 and 20 min a f t e r swimming, as compared with 7:-4 + 1.5 and 6.5"+ 2 .0 ng/ml at the same i n t e r v a l s a f t e r a-MSH. Experiment 3:
Effect of d e s - a c e t y l a-MSH on s t r e s s - i n d u c e d prolactin s e c r e t i o n .
D e s - a c e t y l a-MSH was also e f f e c t i v e in blunting swimming s t r e s s - i n d u c e d prolactin s e c r e t i o n . Mean prolactin r e l e a s e between 0 and 50 rain a f t e r d e s - a c e t y l ~-MIYH was 59.5% + 6.6 (SEM) that of the saline controls ( p < .001). As shown in figure 4, aft er normal saline injection prolactin r o s e to 18.4 + 5.3 and 11.2 + 3.4 ng/m l 10 and 20 min following swimming, as compared with 10.-0 + 2 .4 and 5 .5 + 1.6 ng/ m l at the same i n t e r v a l s a f t e r d e s - a c e t y l a-MS'H.
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~-MSH Suppresses
PRL and S t i m u l a t e s
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Vol. 36, No. 17, 1985
a-MSH Suppresses PRL and Stimulates LH
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e-MSH Suppresses PRL and Stimulates LH
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Discussion lhese data demonstrate that intraventricular ~-MSH simultaneously stimulates LH and suppresses prolactin secretion, and significantly blunts stress-induced prolactin release in male rats. In addition we have shown that the deacetylated form of ~-MSH which is less potent than ~-M~-I in producing skin darkening and some behavioral changes (10), also diminishes stress-induced prolactin secretion. T h i s observation is significant in view of the evidence that des-acetyl ~-M~I is the major form of ~-MSH in brain (10-12). The effects of ~-MSH on prolactin and LH release are opposite to those previously demonstrated for B-endorphin (22-26). Behavioral studies have shown that ~-MSH and B-endorphin produce opposite behavioral effects after intraventricular administration to rats (2,13-18). ~-M~rl causes hyperalgesia, increased arousal, facilitation of learning, and enhanced sexual activity, while B-endorphin injection results in analgesia, catalepsy, and diminished sexual function. ~-MSH has also been shown to attenuate opioid-induced analgesia, thus suggesting that ~-M~I may be an endogenous opiate antagonist (27,28). Thus, ~-MSH and B-endorphin, both products of a common precursor, have antagonistic actions on behavior, as well as on anterior pituitary function in rats. The effect of ~-MS}I on basal prolactin secretion which we have demonstrated is in agreement with the observation of Khorram and colleagues that injection of 2 ~g ~-MSH into the third ventricle lowered plasma prolactin in ovariectomized, estrogen primed rats (19). The inhibition of prolactin release by ~-M~I may be mediated by the hypothalamic dopaminergic neuronal system. ~-M~I has been shown to increase dopaminergic neuronal activity in the arcuate nucleus of the rat hypothalamus (29), an effect opposite to that of B-endorphin, which has been found to diminish hypothalamic dopamine turnover (30), and decrease dopamine release into rat portal blood (31). Khorram and colleagues (19) observed that ~-MSH had no effect on plasma prolactin in ovariectomized rats when administered intravenously, or when injected intraventricularly one hour after treatment with the dopamine receptor antagonist spiroperidol. They also found that ~-M~{ did not alter prolactin release from rat hemipituitaries of male and ovariectomized female rats, thus indicating that ~-MSH may act at a hypothalamic level to decrease prolactin secretion, perhaps by affecting dopamine turnover. Previous investigations of the effect of ~-MSH on LH secretion have provided variable results. A i d e and Celis (32) observed that in ovariectomized, estrogen-primed female rats, intravenous injection of 30 ~g ~-MSH augmented the l ~ response to LHRH, but had no effect on LH when administered alone. More recently Khorram et al. (33) reported that in chronically ovariectomized unprimed rats, administration of 2 lag ~-MSH into the third ventricle significantly lowered plasma LH for one hour after injection, while the intravenous injection of 5 ~ g ~-Mb~l had no effect. However, they also observed that in ovariectomized rats primed with 50 ~g estradiol 72 hours before the experiment, intraventricular injection of 2 ~g ~-M~-I did not change LH levels. Our finding of enhanced LH secretion after central ~-MSH administration in intact male rats contrasts with the inhibitory effect observed by these investigators in chronically ovariectomized rats, and perhaps can be explained by the different sex steroid environments of the animals studied. The hypothesis that the sex steroid environment is an important determinant of the LH response to ~-MSH is supported by the observations of Reid et al. who found that intravenous c~-MSH stimulated LH in normal men (34) and in women in the luteal phase of the menstrual cycle, but not in women in the early follicular phase (35). In this respect, ~-MSH is similar to the opiate antagonist naloxone which enhances LH secretion in normal men and in late follicular or luteal phase women, but not in women in the early follicular phase of the menstrual cycle (36). The mechanism by which ~-MSH stimulates LH release is not completely understood. Our observation of enhanced LH secretion following intraventricular ~-MSH
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~-MSH Suppresses PRL and Stimulates LH
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administration suggests that ~-M~H may act centrally to stimulate LH. ~-M~I has been reported to increase hypothalamic dopamine turnover (29), however the role of dopamine in ~ secretion remains controversial (37,38). The study of Miyake and Yen (39) suggests that ~-M~I acts directly at the pituitary to augment LH secretion. In their hypothalamic-pituitary superfusion system, ~-MS~I induced LH release from pituitaries of male rats, but not female proestrus rats, and did not have any effect on I~IRH release from the medial basal hypothalamus of rats of either species. The actions of ~-M~I that we have described were achieved with pharmacological doses of the peptide, similar to the doses used previously in behavioral studies (15,27). Althoughthe exact physiological significance of our observations remains to be determined, the data presented here together with the finding that a-M~-I is present in high concentrations in rat median eminence (4) suggest that ~-MSH in either its acetylated or de-acetylated form may participate in the modulation of anterior pituitary function, achieving effects opposite to those of B-endorphin. In previous studies in this laboratory, both intravenous naloxone and intraventricular anti-~-endorphin antiserum in rats significantly diminished the rise in prolactin due to swimming stress, thus showing that ~-endorphin is an endogenous regulator of prolactin secretion (40, 41). In the present investigation, a similar effect was achieved with the intraventricular injection of either ~-MS~I or des-acetyl ~-M~-I. Our finding that acetylated and de-acetylated forms of ~-MSH have effects on LH and prolactin that are similar to those of the opiate receptor antagonist naloxone suggests that ~-M~I may antagonize the effects of ~-endorphin on anterior pituitary function.
Acknowledgement This v~rk has been supported by USPHS grants AM 07271, AM 31075, and CA I1704. References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15.
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