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The β-endorphin-induced secretion of growth hormone but not of prolactin is inhibited by an endogenous opioid antagonist
European Journal of Pharmacology, 129 (1986) 385-387
385
Elsevier EJP 450SC Short communication
The fl-endorphin-induced secretion of growth hormone but not of prolactin is inhibited by an endogenous opioid antagonist Dolores Collado-Escobar, Lucio C. Rovati, Ilaria Ganzetti, D a n i e l a Cocchi *, Alberto E. Panerai a n d C h o H a o Li * Department of Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy, and * Laboratory of Molecular Endocrinology, University of California, San Francisco, U.S.A.
Received 30 July 1986, accepted12 August 1986
The naturally occurring peptide human fl-endorphin-(1-27) (hfl-EP-(1-27) has been shown to antagonize fl-endorphin (hfl-EP)-induced analgesia. We have evaluated the effects of the fragment on hfl-EP-induced growth hormone (GH) and prolactin (PRL) release. It inhibited fl-EP-induced GH release in a dose-related way but left fl-EP-induced PRL stimulation unchanged. fl-Endorphin-(1-27) (human); Growth hormone release; Prolactin release
1. Introduction
It is known that the presence of the NH 2 terminal [MetS]enkephalin segment of human flendorphin (hfl-EP) is essential for its biological activity (Li, 1981). Substitution or omission analogs modified in the non-enkephalin segment may have a higher binding potency than hfl-EP in vitro, but a lower analgesic potency in vivo, acting as partial antagonists. Analogs may instead be purer agonists than the parent molecule since they possess lower binding activity in vitro and higher analgesic potency in vivo (Li et al., 1982; Yamashiro et al., 1982). The analgesic action of hfl-EP is antagonized by peptides such as hfl-EP-(6-31) (Lee et al., 1980) and hfl-EP-(1-27) (Hammonds et al., 1984) with a high ratio of binding to analgesic potency. In a previous paper we have shown that hfl-EP(6-31) was able also to antagonize the stimulation of prolactin (PRL) elicited by hfl-EP (Panerai et al., 1984). We have now evaluated the ability of * To whom all correspondenceshould be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
another hfl-EP homolog, the naturally occurring hfl-EP-(1-27), to interfere with the hfl-EP-induced growth hormone (GH) and PRL stimulation.
2. Materials and methods
Male Sprague-Dawley rats (Charles River, Calco) weighing 220-250 g were used in the experiments. Laboratory chow and bottled tap water were available at all times. The animals were housed singly and maintained on 14 h light/10 h darkness. 2.1. Surgery
One week before the experiments the animals were implanted with two polyethylene cannulas (PE10) in the lateral cerebral ventricles (i.c.v.). The day before the experiments, silastic indwelling atrial cannulas (0.6 ×0.11 cm) were implanted into the same rats through the right external jugular vein. This was done with the rats under light anaesthesia (tribromoethanol, 200 m g / k g i.p.).
386
2.2. Experimental procedure
GH n~ /ml
T h e d a y of the experiment, p o l y e t h y l e n e t u b i n g extensions (20 cm) were a t t a c h e d to the atrial c a n n u l a s in o r d e r to o b t a i n s a m p l e s while the a n i m a l s (0.3 ml) were moving freely. A f t e r two baseline s a m p l e s h a d been collected, test drugs were s i m u l t a n e o u s l y injected t h r o u g h the two i.c.v. i m p l a n t e d c a n n u l a s (10 ffl of volume). F u r t h e r b l o o d s a m p l e s were collected 0, 10, 20, 30 a n d 60 m i n later. T h e s a m p l e s were centrifuged at 3 500 r.p.m, for 15 min a n d the p l a s m a was s e p a r a t e d a n d stored at - 2 0 ° C until r e a d y for r a d i o i m m u n o a s s a y . Statistical e v a l u a t i o n of the results was d o n e with D u n n e t t ' s t-test for m u l t i p l e c o m parisons. P l a s m a G H a n d P R L were a n a l y z e d with m a t e r i a l s p r o v i d e d b y N I A D D K (Bethesda, M D ) . D a t a are expressed in terms of r G H - R P - 2 and rPRL-RP-3.
30C
2.3. Drugs Synthetic hfl-EP (Li et al., 1977) was a d m i n i s tered i.c.v, at the dose of 0 . 2 5 / ~ g / r a t , dissolved in 10 /~l of sterile saline. Synthetic hfl-EP-(1-27) ( Z a o r a l et al., 1981) was a d m i n i s t e r e d at two dose levels (0.25 a n d 1 f i g / r a t ) dissolved in 10 ffl of sterile saline.
3. Results
200
i_ \
III
IO0
I ///
i I
I /
o
J. .
0
.
.
.
.
.
.
.
'-. ~/ " k.,..
.
.
10
. . . . . . . .
. . . .
20 TIME ( m i n )
30
60
Fig. 1. Effect of simultaneous administration of hfl-EP-(1-27) on hfl-EP-induced growth hormone release. ( ) Saline i.c.v.; ( . . . . ) hfl-EP 0.25 p,g i.c.v.; ( . . . . . ) hfl-EP-(1-27) 0.25 p,g i.c.v. + hfl-EP 0.25 /~g i.c.v.; (. . . . . . ) hfl-EP-(l-27) 1 #g i.c.v. + hfl-EP 0.25 fig i.c.v. Each point represents the mean _+S.E.M. of 5-6 determinations. * P at least < 0.05 vs. hfl-EP alone.
h f l - E P - i n d u c e d analgesia ( H a m m o n d s et al., 1984). T h e presence of this p e p t i d e in some b r a i n areas in a m o u n t s equal to or greater than the p a r e n t
r PRL nc /ml 50
W h e n a d m i n i s t e r e d alone, hfl-EP-(1-27) d i d not affect the basal G H or P R L release (figs. 1 a n d 2) at either of the doses used. C o n c o m i t a n t a d m i n i s t r a t i o n of the p e p t i d e i n d u c e d a d o s e - r e l a t e d significant i n h i b i t i o n of h f l - E P - i n d u c e d G H release that was evident at time 10 min for the lower dose a n d until time 30 min for the higher dose (fig. 1). hfl-EP-(1-27) left the h f l - E P - i n d u c e d P R L release u n c h a n g e d at all time intervals (fig. 2).
/?\~
40
20
/.-¢
l
.f/,,'
T h e f r a g m e n t of the hfl-EP molecule, hfl-EP(1-27) d i s p l a y s stronger b i n d i n g than analgesic p r o p e r t i e s a n d acts as c o m p e t i t i v e a n t a g o n i s t of
7"-"-"~,
Ik
\ " ~ ~'t '\
10
0
10
4. Discussion
/9-...
20 3 60 TIME(rain) Fig. 2. Effect of simultaneous administration of hfl-EP-(l-27) on hfl-EP-induced prolactin release. ( ) Saline i.c.v.; ( - - --) hfl-EP 0.25 p,g i.c.v.; ( . . . . . ) hfl-EP-(1-27) 0.25 #g i.c.v.+hfl-EP 0.25 p,g i.c.v.; (. . . . . . ) hfl-EP-(1-27) 1 /xg i.c.v. + hfl-EP 0.25 /tg i.c.v. Each point represents the mean+ S.E.M. of 5-6 determinations.
387 molecule would suggest that it can act as an endogenous inhibitor of effects of hfl-EP. The results presented herein are at least partially consistent with this concept. As shown for analgesia, hfl-EP-(1-27) reduced the stimulatory effect of hfl-EP on G H release in a dose-related way. Instead, the peptide was without effect on the P R L increase elicited by the parent molecule. These data emphasise once more the notion that opiate receptors (and neurons) controlling G H and P R L are completely different. P R L release seems to be activated through #1 (Spiegel et al., 1982) and ~ receptors while the 8 receptors are p r o b a b l y not involved (Koenig and Kriilich, 1984). Stimulation of G H secretion by opioids however seems to be mediated mainly by the 8 receptors (Koenig and Kfiilich, 1984). The mechanism of the hfl-EP-(1-27) inhibition of h f l - G H release could probalby consist in competition at the receptor level. Binding studies have shown that hfl-EP-(1-27) retained 30% of the p o t e n c y of hfl-EP in displacing [3H]hfl-EP from rat brain membranes ( H a m m o n d s et al., 1984). It can be postulated from our data that hfl-EP-(1-27) is a more potent antagonist of ~ (involved in G H regulation) than o f / x receptors (controlling P R L secretion). Our results are consistent with the increasing evidence that the study of the physiological role of the endogenous opioids must not be approached without taking into consideration that agonist and antagonist peptides are secreted concomitantly, thus introducing a further modulatory step in their effects.
References Hammonds, R.G., Jr., P. Nicolas and C.H. Li, 1984, fl-Endorphin-1(1-27) is an antagonist of fl-endorphin analgesia, Proc. Natl. Acad. Sci. U.S.A. 81, 1389. Koenig, J.I. and L. K~lich, 1984, Differential role of multiple opioid receptors in the regulation of secretion of prolactin and growth hormone in rats, in: Opioid Modulation of Endocrine Function, eds. G. Delitalia, M. Motta and M. Serio (Raven Press, New York) p. 89. Lee, N.M., L. Friedman, T.H. Laybin, H.H. Cho and C.H. Li, 1980, Peptide inhibition of morphine and fl-endorphin-induced analgesia, Proc. Natl. Acad. Sci. U.S.A. 77, 5525. Li, C.H., ed., 1981, in: Hormonal Proteins and Peptides, Vol. 10 (Academic Press, New York) p. 1. Li, C.H., D. Yamashiro and P. Nicolas, 1982, fl-Endorphin: replacement of tyrosine in position 27 by tryptophan increases analgesic potency: preparation and properties of the 2-nitrophenylsulfenyl derivative, Proc. Natl. Acad. Sci. U.S.A. 79, 1042. Li, C.H., D. Yamashiro, L.F. Tseng and H.H. Loh, 1977, Synthesis and analgesic activity of human fl-endorphin, J. Med. Chem. 20, 325. Panerai, A.E., D. Cocchi, M. Parenti, A. Martini, P. Mantegazza and C.H. Li, 1984, Effects of fl-endorphin fragment 6-31 on morphine and fl-endorphin-induced growth hormone and prolactin release, European J. Pharmacol. 99, 341. Spiegel, K., I.A. Kourides and G.W. Pasternak, 1982, Different receptors mediate morphine-induced prolactin and growth hormone release, Life Sci. 31, 2177. Yamashiro, D., P. Nicolas and C.H. Li, 1982, fl-Endorphin: synthesis and properties of analogs modified in position 8 and 31, Int. J. Pept. Prot. Res. 20, 43. Zaoral, M., D. Yamashiro, R.J. Hammonds, Jr. and C.H. Li, 1981, fl-Endorphin: synthesis and radioreceptor binding activity of flh-EP-(1-27) and its analogs, Int. J. Pept. Prot. Res. 17, 292.
385
Elsevier EJP 450SC Short communication
The fl-endorphin-induced secretion of growth hormone but not of prolactin is inhibited by an endogenous opioid antagonist Dolores Collado-Escobar, Lucio C. Rovati, Ilaria Ganzetti, D a n i e l a Cocchi *, Alberto E. Panerai a n d C h o H a o Li * Department of Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy, and * Laboratory of Molecular Endocrinology, University of California, San Francisco, U.S.A.
Received 30 July 1986, accepted12 August 1986
The naturally occurring peptide human fl-endorphin-(1-27) (hfl-EP-(1-27) has been shown to antagonize fl-endorphin (hfl-EP)-induced analgesia. We have evaluated the effects of the fragment on hfl-EP-induced growth hormone (GH) and prolactin (PRL) release. It inhibited fl-EP-induced GH release in a dose-related way but left fl-EP-induced PRL stimulation unchanged. fl-Endorphin-(1-27) (human); Growth hormone release; Prolactin release
1. Introduction
It is known that the presence of the NH 2 terminal [MetS]enkephalin segment of human flendorphin (hfl-EP) is essential for its biological activity (Li, 1981). Substitution or omission analogs modified in the non-enkephalin segment may have a higher binding potency than hfl-EP in vitro, but a lower analgesic potency in vivo, acting as partial antagonists. Analogs may instead be purer agonists than the parent molecule since they possess lower binding activity in vitro and higher analgesic potency in vivo (Li et al., 1982; Yamashiro et al., 1982). The analgesic action of hfl-EP is antagonized by peptides such as hfl-EP-(6-31) (Lee et al., 1980) and hfl-EP-(1-27) (Hammonds et al., 1984) with a high ratio of binding to analgesic potency. In a previous paper we have shown that hfl-EP(6-31) was able also to antagonize the stimulation of prolactin (PRL) elicited by hfl-EP (Panerai et al., 1984). We have now evaluated the ability of * To whom all correspondenceshould be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
another hfl-EP homolog, the naturally occurring hfl-EP-(1-27), to interfere with the hfl-EP-induced growth hormone (GH) and PRL stimulation.
2. Materials and methods
Male Sprague-Dawley rats (Charles River, Calco) weighing 220-250 g were used in the experiments. Laboratory chow and bottled tap water were available at all times. The animals were housed singly and maintained on 14 h light/10 h darkness. 2.1. Surgery
One week before the experiments the animals were implanted with two polyethylene cannulas (PE10) in the lateral cerebral ventricles (i.c.v.). The day before the experiments, silastic indwelling atrial cannulas (0.6 ×0.11 cm) were implanted into the same rats through the right external jugular vein. This was done with the rats under light anaesthesia (tribromoethanol, 200 m g / k g i.p.).
386
2.2. Experimental procedure
GH n~ /ml
T h e d a y of the experiment, p o l y e t h y l e n e t u b i n g extensions (20 cm) were a t t a c h e d to the atrial c a n n u l a s in o r d e r to o b t a i n s a m p l e s while the a n i m a l s (0.3 ml) were moving freely. A f t e r two baseline s a m p l e s h a d been collected, test drugs were s i m u l t a n e o u s l y injected t h r o u g h the two i.c.v. i m p l a n t e d c a n n u l a s (10 ffl of volume). F u r t h e r b l o o d s a m p l e s were collected 0, 10, 20, 30 a n d 60 m i n later. T h e s a m p l e s were centrifuged at 3 500 r.p.m, for 15 min a n d the p l a s m a was s e p a r a t e d a n d stored at - 2 0 ° C until r e a d y for r a d i o i m m u n o a s s a y . Statistical e v a l u a t i o n of the results was d o n e with D u n n e t t ' s t-test for m u l t i p l e c o m parisons. P l a s m a G H a n d P R L were a n a l y z e d with m a t e r i a l s p r o v i d e d b y N I A D D K (Bethesda, M D ) . D a t a are expressed in terms of r G H - R P - 2 and rPRL-RP-3.
30C
2.3. Drugs Synthetic hfl-EP (Li et al., 1977) was a d m i n i s tered i.c.v, at the dose of 0 . 2 5 / ~ g / r a t , dissolved in 10 /~l of sterile saline. Synthetic hfl-EP-(1-27) ( Z a o r a l et al., 1981) was a d m i n i s t e r e d at two dose levels (0.25 a n d 1 f i g / r a t ) dissolved in 10 ffl of sterile saline.
3. Results
200
i_ \
III
IO0
I ///
i I
I /
o
J. .
0
.
.
.
.
.
.
.
'-. ~/ " k.,..
.
.
10
. . . . . . . .
. . . .
20 TIME ( m i n )
30
60
Fig. 1. Effect of simultaneous administration of hfl-EP-(1-27) on hfl-EP-induced growth hormone release. ( ) Saline i.c.v.; ( . . . . ) hfl-EP 0.25 p,g i.c.v.; ( . . . . . ) hfl-EP-(1-27) 0.25 p,g i.c.v. + hfl-EP 0.25 /~g i.c.v.; (. . . . . . ) hfl-EP-(l-27) 1 #g i.c.v. + hfl-EP 0.25 fig i.c.v. Each point represents the mean _+S.E.M. of 5-6 determinations. * P at least < 0.05 vs. hfl-EP alone.
h f l - E P - i n d u c e d analgesia ( H a m m o n d s et al., 1984). T h e presence of this p e p t i d e in some b r a i n areas in a m o u n t s equal to or greater than the p a r e n t
r PRL nc /ml 50
W h e n a d m i n i s t e r e d alone, hfl-EP-(1-27) d i d not affect the basal G H or P R L release (figs. 1 a n d 2) at either of the doses used. C o n c o m i t a n t a d m i n i s t r a t i o n of the p e p t i d e i n d u c e d a d o s e - r e l a t e d significant i n h i b i t i o n of h f l - E P - i n d u c e d G H release that was evident at time 10 min for the lower dose a n d until time 30 min for the higher dose (fig. 1). hfl-EP-(1-27) left the h f l - E P - i n d u c e d P R L release u n c h a n g e d at all time intervals (fig. 2).
/?\~
40
20
/.-¢
l
.f/,,'
T h e f r a g m e n t of the hfl-EP molecule, hfl-EP(1-27) d i s p l a y s stronger b i n d i n g than analgesic p r o p e r t i e s a n d acts as c o m p e t i t i v e a n t a g o n i s t of
7"-"-"~,
Ik
\ " ~ ~'t '\
10
0
10
4. Discussion
/9-...
20 3 60 TIME(rain) Fig. 2. Effect of simultaneous administration of hfl-EP-(l-27) on hfl-EP-induced prolactin release. ( ) Saline i.c.v.; ( - - --) hfl-EP 0.25 p,g i.c.v.; ( . . . . . ) hfl-EP-(1-27) 0.25 #g i.c.v.+hfl-EP 0.25 p,g i.c.v.; (. . . . . . ) hfl-EP-(1-27) 1 /xg i.c.v. + hfl-EP 0.25 /tg i.c.v. Each point represents the mean+ S.E.M. of 5-6 determinations.
387 molecule would suggest that it can act as an endogenous inhibitor of effects of hfl-EP. The results presented herein are at least partially consistent with this concept. As shown for analgesia, hfl-EP-(1-27) reduced the stimulatory effect of hfl-EP on G H release in a dose-related way. Instead, the peptide was without effect on the P R L increase elicited by the parent molecule. These data emphasise once more the notion that opiate receptors (and neurons) controlling G H and P R L are completely different. P R L release seems to be activated through #1 (Spiegel et al., 1982) and ~ receptors while the 8 receptors are p r o b a b l y not involved (Koenig and Kriilich, 1984). Stimulation of G H secretion by opioids however seems to be mediated mainly by the 8 receptors (Koenig and Kfiilich, 1984). The mechanism of the hfl-EP-(1-27) inhibition of h f l - G H release could probalby consist in competition at the receptor level. Binding studies have shown that hfl-EP-(1-27) retained 30% of the p o t e n c y of hfl-EP in displacing [3H]hfl-EP from rat brain membranes ( H a m m o n d s et al., 1984). It can be postulated from our data that hfl-EP-(1-27) is a more potent antagonist of ~ (involved in G H regulation) than o f / x receptors (controlling P R L secretion). Our results are consistent with the increasing evidence that the study of the physiological role of the endogenous opioids must not be approached without taking into consideration that agonist and antagonist peptides are secreted concomitantly, thus introducing a further modulatory step in their effects.
References Hammonds, R.G., Jr., P. Nicolas and C.H. Li, 1984, fl-Endorphin-1(1-27) is an antagonist of fl-endorphin analgesia, Proc. Natl. Acad. Sci. U.S.A. 81, 1389. Koenig, J.I. and L. K~lich, 1984, Differential role of multiple opioid receptors in the regulation of secretion of prolactin and growth hormone in rats, in: Opioid Modulation of Endocrine Function, eds. G. Delitalia, M. Motta and M. Serio (Raven Press, New York) p. 89. Lee, N.M., L. Friedman, T.H. Laybin, H.H. Cho and C.H. Li, 1980, Peptide inhibition of morphine and fl-endorphin-induced analgesia, Proc. Natl. Acad. Sci. U.S.A. 77, 5525. Li, C.H., ed., 1981, in: Hormonal Proteins and Peptides, Vol. 10 (Academic Press, New York) p. 1. Li, C.H., D. Yamashiro and P. Nicolas, 1982, fl-Endorphin: replacement of tyrosine in position 27 by tryptophan increases analgesic potency: preparation and properties of the 2-nitrophenylsulfenyl derivative, Proc. Natl. Acad. Sci. U.S.A. 79, 1042. Li, C.H., D. Yamashiro, L.F. Tseng and H.H. Loh, 1977, Synthesis and analgesic activity of human fl-endorphin, J. Med. Chem. 20, 325. Panerai, A.E., D. Cocchi, M. Parenti, A. Martini, P. Mantegazza and C.H. Li, 1984, Effects of fl-endorphin fragment 6-31 on morphine and fl-endorphin-induced growth hormone and prolactin release, European J. Pharmacol. 99, 341. Spiegel, K., I.A. Kourides and G.W. Pasternak, 1982, Different receptors mediate morphine-induced prolactin and growth hormone release, Life Sci. 31, 2177. Yamashiro, D., P. Nicolas and C.H. Li, 1982, fl-Endorphin: synthesis and properties of analogs modified in position 8 and 31, Int. J. Pept. Prot. Res. 20, 43. Zaoral, M., D. Yamashiro, R.J. Hammonds, Jr. and C.H. Li, 1981, fl-Endorphin: synthesis and radioreceptor binding activity of flh-EP-(1-27) and its analogs, Int. J. Pept. Prot. Res. 17, 292.