Endocrinology 100: 238, 1977 STIMULATION IN VIVO OF THE SECRETION OF PROLACTIN AND GROWTH HORMONE BY 3-ENDORPHIN C. Rivier, W. Vale, N. Ling, M. Brown and R. Guillemin Laboratories for Neuroendocrinology The Salk Institute, La Jolla, California
92037
ABSTRACT: Morphine sulfate (MS) and the opioid peptide 8-endorphin 6-LPH-(61-91) stimulate prolactin and growth hormone release in steroid-primed and non-treated male rats when injected intravenously or intracistemally. On a molar basis 8-endorphin is at least 20 times more potent than MS, whereas Met5-enkephalin (B-LPH-(61-65)) and a-endorphin (6-LPH-(61-76)) are devoid of activity at the dose injected (300 yg). The in vivo stimulatory effects of 8-endorphin on prolactin secretion are reversed by the opiate antagonist naloxone. The absence of in vitro effect of MS and e-endorphin on prolactin and growth hormone secretion by cultured rat pituitary cells suggest that they have a central nervous system site of action.
Several laboratories have recently reported the isolation and characterization of morphinomimetic brain oligopeptides. Hughes et al. (1) isolated Leu5-enkephalin and Met5-enkephalin from porcine brain; Met5-enkephalin is identical to the sequence (6165) of 8-lipotropin, while Leu5-enkephalin shows the sequence (61-64). Guillemin and co-workers (2,3) isolated two opiate-like peptides, termed endorphins, from porcine posterior pituitary and hypothalamic extracts; a-endorphin and y~endorphin are respectively identical to the sequence 8-LPH-(61-76) and 6-LPH-(61-77). The entire C-terminal fragment of 8-lipotropin, 6-LPH-(61-91), identified by Bradbury et al. (4) and Li and Chung (5) from whole pituitary extracts, also has morphinomimetic properties. This laboratory has engaged in the isolation of the endogenous ligand of the opiate receptors on the hypothesis (2,9) that the endogenous opiate-like peptides might be involved in the mechanisms of release of pituitary hormones, since the opiate alkaloids are well known to stimulate secretion of prolactin and growth hormone Submitted September 27, 1976
(6-9). We report here on the effects of three opioid peptides on the secretion of prolactin and growth hormone. MATERIALS AND METHODS Cell Culture Assay: Enzymatically dispersed normal rat pituitary cells were maintained in culture for 4 days, then exposed to the peptides for 4 hours (10). Rat Bioassay: Male Sprague-Dawley rats, 44-46 days old, were treated with estrogen (50 yg) and progesterone (25 mg) for 3 days, then anesthetized with urethane (150 mg/100 g body weight)(11). The test materials were injected into the jugular vein (iv) in a volume of 1 ml or into the cisterna magna (ic) in a volume of 25 yl. Blood samples were taken immediately prior to the injection, and 10 min later. Hormone determinations: PRL and GH levels were measured by double antibody methods as previously described (6,11). Drugs and Peptides: Morphine sulfate was purchased from Merck Sharpe and
238 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Dohme. Naloxone hydrochloride (Nallyl-nor-oxymorphone) was a gift from Endo Labs., Garden City, New York. Met5-enkephalin, B-LPH-(61-65) , ctendorphin ( B-LPH-(61-76))and B-endorphin ( B-LPH-(61-91)) were synthesized using solid phase methodology (12). Statistical Analysis: All experiments were carried out in a randomized block design. Following analysis of variance, differences between treatments were determined by the multiple range tests of Dunnett and Duncan.
239
200
•
CONTROL
10/iq
Figure 2: Effect of ic administered B-endorphin on PRL and GH secretion in steroid-primed rats. Details as in Fig. 1. *, p < 0.05.
30p.g OO/ig 300/ig
Figure 1: Effect of several doses of iv administered morphine sulfate (MS) and opioid peptides on PRL secretion in steroid-primed rats. Each bar represents the mean and standard error of the mean for groups of 5 rats. -, p > 0.05; **, p < 0.01. RESULTS AND DISCUSSION Figure 1 illustrates the effect of several doses of iv administered morphine sulfate (MS) and opioid peptides on PRL secretion. Met5-enkephalin and a-endorphin elevate plasma PRL levels. On a molar basis, B-endorphin is at least 20 times more potent than MS, Met5-enkephalin or a-endorphin. These results were obtained in steroid-primed rats; similar observations have been made in non-treated animals. In the myenteric plexus bioassay (3) as well as in studies of
• »
1
100 -
50 -
CONTROL
NALOXONE /3-ENDORPHIN NALOXONE 250/ig 100/ig + /3-ENDORPHIN
Figure 3: Effect of naloxone on Bendorphin induced secretion of PRL in steroid-primed rats. Details as in Figure 1.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
240
RAPID COMMUNICATIONS
TABLE 1:
Endo • 1977 Vol 100 • No 1
Effect of 8-endorphin, Met5-enkephalin and a-endorphin on PRL and GH secretion by cultured rat pituitary cells
Treatment
Experiment 1: Control S-endorphin
Met -enkephalin
Experiment 2: Control a-endorphin
ng GH SEM dish.hr
SEM
0.01 0.1 1.0 10.
yg/ml yg/ml yg/ml yg/ml
0.01 0.1 1.0 10.
yg/ml yg/ml yg/ml yg/ml
0.01 yg/ml 1.0 yg/ml
109 120 118 132 125
2 4 4 5 4
107 121 126
3 3
117
143 141 139
-(a)
792 827 767 940 860
107 159 90 63 84
4
770 797
60 107 128
2
797
196
4
417 474 474
13 23 27
3 6
808
(a) p > 0.05 competition for the opiate receptor in synaptosomal preparations (3,13-15) 6-endorphin is at least 5 times as potent as normorphine or levorphanol, whereas Met5-enkephalin and Leu5-enkephalin have relative affinities of ca. 0.3 (3,13,14). B-endorphin is also a potent stimulator of PRL and GH secretion when injected ic (Fig. 2). Pharmacological effects of MS on several receptors are reversed or prevented by the opiate antagonist naloxone (9,15,16); similarly naloxone, at doses which do not affect PRL secretion, inhibits the stimulatory effect of 6-endorphin on the secretion of PRL (Fig.3). Most effects of the opioid peptides, but not all, have been found to be prevented or reversed by naloxone (17). As is the case for MS (9), all three morphinomimetic peptides are devoid of in vitro activity (Table 1), suggesting a central nervous system site of action of these substances in their releasing GH and PRL jLn vivo. These results are at variance with the recent findings of Lien et al. (18) who reported that as little as 5 ng/ml Met^-enkephalin and Leu5-enkephalin significantly
increases PRL secretion by cultured pituitary cells. Since we have repeatedly failed to observe any stimulatory effect of these two peptides in vitro, the reason for this discrepancy is unknown. We have previously found that a variety of pharmacological stimuli of PRL secretion, including TRF, neurotensin, substance P, histamine and ot-adrenergic agonists, are not affected by concomitant naloxone administration (9). These results would indicate that the stimulatory activity of these latter substances does not involve an opiate-receptor dependent step. Although the significance of opiate-like peptides in the physiological control of PRL and GH secretion remains to be determined, these results indicate one more biological activity shared by MS and the neurotropic brain peptides related to 0lipotropin. ACKNOWLEDGEMENT The authors thank E. Tucker, C. Douglas, A. Wolfe, M. Mercado, L.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Koski, R. Kaiser, and S. Minick for excellent technical assistance, and Jean Rivier for his contribution in assembling the protected 8-endorphin onto the chloromethylated resin. We are indebted to the National Institute of Arthritis, Metabolism and Digestive Diseases, Rat Pituitary Hormone programs, for supplying the rat PRL and GH radioimmunoassay kits, and Endo Labs for the gift of naloxone. Research supported by NIH grants Nos. AM-18811, AM-16707, National Foundation 1-411, and the William Randolph Hearst Foundation. REFERENCES 1. Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan, and H.R. Morris, Nature 258: 577, 1975. 2.
Guillemin, R., N. Ling, and R. Burgus, C R Acad Sci, Series [D] (Paris) 282^:1821, 1976.
3. Lazarus, L.H., N. Ling, and R. Guillemin, Proc Natl Acad Sci USA 23:2156, 1976. 4. Bradbury, A.F., D.G. Smyth, C.R. Shepp, N.J.M. Birdsall, and E.C. Hulme, Nature 206:793, 1976 5. Li, C.H., and D. Chung, Proc Natl Acad Sci USA 22:1145, 1976.
241
Hecht (eds.), Narcotics and the Hypothalamus, Raven Press, New York, 1974, p. 121. 8. Martin, J., Neuroendocrinology 13: 339, 1974. 9. Rivier, C , M. Brown and W. Vale, Program 58th Endocrine Society Meeting, San Francisco, 1976, p. 119 (Abstract). 10. Vale, W., G. Grant, M. Amoss, R. Blackwell, and R. Guillemin, Endocrinology 21 : 562, 1972. 11. Rivier, C , and W. Vale, Endocrinology 95:91%, 1974. 12. Ling, N., and W. Vale, Biochem. Biophys Res Coramun £3_:801, 1975. 13. Cox, B.M., A. Goldstein, and C.H. Li, Proc Natl Acad Sci USA 73.: 1821, 1976. 14. Terenius, L., A. Wahlstrom, G. Lindesbert, S. Karlsson, and U. Ragnasson, Biochem Biophys Res Commun 21:175, 1976. 15. Packman, P.M., J.A. Rothchild, Endocrinology 99jl, 1976. 16. Vale, W., C. Rivier, and M. Brown, Ann Review Physio1 (in press).
6. Brown, M., and W. Vale, Endocrinology 92:1151, 1975.
17. Bloom, F., D. Segal, N. Ling, and R. Guillemin, Science 194_, 1976 (in press).
7. McCann, S.M., S.R. Ojeda, C. Libertun, P.G. Harms, and L. Krulich. In Zimmerman, E., and H.
18. Lien, E.L., R.L. Fenichet, V. Garsky, D. Sarantakis, and N.H. Grant, Life Sci 19_:837, 1976.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
92037
ABSTRACT: Morphine sulfate (MS) and the opioid peptide 8-endorphin 6-LPH-(61-91) stimulate prolactin and growth hormone release in steroid-primed and non-treated male rats when injected intravenously or intracistemally. On a molar basis 8-endorphin is at least 20 times more potent than MS, whereas Met5-enkephalin (B-LPH-(61-65)) and a-endorphin (6-LPH-(61-76)) are devoid of activity at the dose injected (300 yg). The in vivo stimulatory effects of 8-endorphin on prolactin secretion are reversed by the opiate antagonist naloxone. The absence of in vitro effect of MS and e-endorphin on prolactin and growth hormone secretion by cultured rat pituitary cells suggest that they have a central nervous system site of action.
Several laboratories have recently reported the isolation and characterization of morphinomimetic brain oligopeptides. Hughes et al. (1) isolated Leu5-enkephalin and Met5-enkephalin from porcine brain; Met5-enkephalin is identical to the sequence (6165) of 8-lipotropin, while Leu5-enkephalin shows the sequence (61-64). Guillemin and co-workers (2,3) isolated two opiate-like peptides, termed endorphins, from porcine posterior pituitary and hypothalamic extracts; a-endorphin and y~endorphin are respectively identical to the sequence 8-LPH-(61-76) and 6-LPH-(61-77). The entire C-terminal fragment of 8-lipotropin, 6-LPH-(61-91), identified by Bradbury et al. (4) and Li and Chung (5) from whole pituitary extracts, also has morphinomimetic properties. This laboratory has engaged in the isolation of the endogenous ligand of the opiate receptors on the hypothesis (2,9) that the endogenous opiate-like peptides might be involved in the mechanisms of release of pituitary hormones, since the opiate alkaloids are well known to stimulate secretion of prolactin and growth hormone Submitted September 27, 1976
(6-9). We report here on the effects of three opioid peptides on the secretion of prolactin and growth hormone. MATERIALS AND METHODS Cell Culture Assay: Enzymatically dispersed normal rat pituitary cells were maintained in culture for 4 days, then exposed to the peptides for 4 hours (10). Rat Bioassay: Male Sprague-Dawley rats, 44-46 days old, were treated with estrogen (50 yg) and progesterone (25 mg) for 3 days, then anesthetized with urethane (150 mg/100 g body weight)(11). The test materials were injected into the jugular vein (iv) in a volume of 1 ml or into the cisterna magna (ic) in a volume of 25 yl. Blood samples were taken immediately prior to the injection, and 10 min later. Hormone determinations: PRL and GH levels were measured by double antibody methods as previously described (6,11). Drugs and Peptides: Morphine sulfate was purchased from Merck Sharpe and
238 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Dohme. Naloxone hydrochloride (Nallyl-nor-oxymorphone) was a gift from Endo Labs., Garden City, New York. Met5-enkephalin, B-LPH-(61-65) , ctendorphin ( B-LPH-(61-76))and B-endorphin ( B-LPH-(61-91)) were synthesized using solid phase methodology (12). Statistical Analysis: All experiments were carried out in a randomized block design. Following analysis of variance, differences between treatments were determined by the multiple range tests of Dunnett and Duncan.
239
200
•
CONTROL
10/iq
Figure 2: Effect of ic administered B-endorphin on PRL and GH secretion in steroid-primed rats. Details as in Fig. 1. *, p < 0.05.
30p.g OO/ig 300/ig
Figure 1: Effect of several doses of iv administered morphine sulfate (MS) and opioid peptides on PRL secretion in steroid-primed rats. Each bar represents the mean and standard error of the mean for groups of 5 rats. -, p > 0.05; **, p < 0.01. RESULTS AND DISCUSSION Figure 1 illustrates the effect of several doses of iv administered morphine sulfate (MS) and opioid peptides on PRL secretion. Met5-enkephalin and a-endorphin elevate plasma PRL levels. On a molar basis, B-endorphin is at least 20 times more potent than MS, Met5-enkephalin or a-endorphin. These results were obtained in steroid-primed rats; similar observations have been made in non-treated animals. In the myenteric plexus bioassay (3) as well as in studies of
• »
1
100 -
50 -
CONTROL
NALOXONE /3-ENDORPHIN NALOXONE 250/ig 100/ig + /3-ENDORPHIN
Figure 3: Effect of naloxone on Bendorphin induced secretion of PRL in steroid-primed rats. Details as in Figure 1.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
240
RAPID COMMUNICATIONS
TABLE 1:
Endo • 1977 Vol 100 • No 1
Effect of 8-endorphin, Met5-enkephalin and a-endorphin on PRL and GH secretion by cultured rat pituitary cells
Treatment
Experiment 1: Control S-endorphin
Met -enkephalin
Experiment 2: Control a-endorphin
ng GH SEM dish.hr
SEM
0.01 0.1 1.0 10.
yg/ml yg/ml yg/ml yg/ml
0.01 0.1 1.0 10.
yg/ml yg/ml yg/ml yg/ml
0.01 yg/ml 1.0 yg/ml
109 120 118 132 125
2 4 4 5 4
107 121 126
3 3
117
143 141 139
-(a)
792 827 767 940 860
107 159 90 63 84
4
770 797
60 107 128
2
797
196
4
417 474 474
13 23 27
3 6
808
(a) p > 0.05 competition for the opiate receptor in synaptosomal preparations (3,13-15) 6-endorphin is at least 5 times as potent as normorphine or levorphanol, whereas Met5-enkephalin and Leu5-enkephalin have relative affinities of ca. 0.3 (3,13,14). B-endorphin is also a potent stimulator of PRL and GH secretion when injected ic (Fig. 2). Pharmacological effects of MS on several receptors are reversed or prevented by the opiate antagonist naloxone (9,15,16); similarly naloxone, at doses which do not affect PRL secretion, inhibits the stimulatory effect of 6-endorphin on the secretion of PRL (Fig.3). Most effects of the opioid peptides, but not all, have been found to be prevented or reversed by naloxone (17). As is the case for MS (9), all three morphinomimetic peptides are devoid of in vitro activity (Table 1), suggesting a central nervous system site of action of these substances in their releasing GH and PRL jLn vivo. These results are at variance with the recent findings of Lien et al. (18) who reported that as little as 5 ng/ml Met^-enkephalin and Leu5-enkephalin significantly
increases PRL secretion by cultured pituitary cells. Since we have repeatedly failed to observe any stimulatory effect of these two peptides in vitro, the reason for this discrepancy is unknown. We have previously found that a variety of pharmacological stimuli of PRL secretion, including TRF, neurotensin, substance P, histamine and ot-adrenergic agonists, are not affected by concomitant naloxone administration (9). These results would indicate that the stimulatory activity of these latter substances does not involve an opiate-receptor dependent step. Although the significance of opiate-like peptides in the physiological control of PRL and GH secretion remains to be determined, these results indicate one more biological activity shared by MS and the neurotropic brain peptides related to 0lipotropin. ACKNOWLEDGEMENT The authors thank E. Tucker, C. Douglas, A. Wolfe, M. Mercado, L.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Koski, R. Kaiser, and S. Minick for excellent technical assistance, and Jean Rivier for his contribution in assembling the protected 8-endorphin onto the chloromethylated resin. We are indebted to the National Institute of Arthritis, Metabolism and Digestive Diseases, Rat Pituitary Hormone programs, for supplying the rat PRL and GH radioimmunoassay kits, and Endo Labs for the gift of naloxone. Research supported by NIH grants Nos. AM-18811, AM-16707, National Foundation 1-411, and the William Randolph Hearst Foundation. REFERENCES 1. Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan, and H.R. Morris, Nature 258: 577, 1975. 2.
Guillemin, R., N. Ling, and R. Burgus, C R Acad Sci, Series [D] (Paris) 282^:1821, 1976.
3. Lazarus, L.H., N. Ling, and R. Guillemin, Proc Natl Acad Sci USA 23:2156, 1976. 4. Bradbury, A.F., D.G. Smyth, C.R. Shepp, N.J.M. Birdsall, and E.C. Hulme, Nature 206:793, 1976 5. Li, C.H., and D. Chung, Proc Natl Acad Sci USA 22:1145, 1976.
241
Hecht (eds.), Narcotics and the Hypothalamus, Raven Press, New York, 1974, p. 121. 8. Martin, J., Neuroendocrinology 13: 339, 1974. 9. Rivier, C , M. Brown and W. Vale, Program 58th Endocrine Society Meeting, San Francisco, 1976, p. 119 (Abstract). 10. Vale, W., G. Grant, M. Amoss, R. Blackwell, and R. Guillemin, Endocrinology 21 : 562, 1972. 11. Rivier, C , and W. Vale, Endocrinology 95:91%, 1974. 12. Ling, N., and W. Vale, Biochem. Biophys Res Coramun £3_:801, 1975. 13. Cox, B.M., A. Goldstein, and C.H. Li, Proc Natl Acad Sci USA 73.: 1821, 1976. 14. Terenius, L., A. Wahlstrom, G. Lindesbert, S. Karlsson, and U. Ragnasson, Biochem Biophys Res Commun 21:175, 1976. 15. Packman, P.M., J.A. Rothchild, Endocrinology 99jl, 1976. 16. Vale, W., C. Rivier, and M. Brown, Ann Review Physio1 (in press).
6. Brown, M., and W. Vale, Endocrinology 92:1151, 1975.
17. Bloom, F., D. Segal, N. Ling, and R. Guillemin, Science 194_, 1976 (in press).
7. McCann, S.M., S.R. Ojeda, C. Libertun, P.G. Harms, and L. Krulich. In Zimmerman, E., and H.
18. Lien, E.L., R.L. Fenichet, V. Garsky, D. Sarantakis, and N.H. Grant, Life Sci 19_:837, 1976.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 07:57 For personal use only. No other uses without permission. . All rights reserved.
