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‘γ-MSH’ fragments from ACTH-β-LPH precursor have an affinity for opiate receptors
European Journal of Pharmacology, 64 (1980) 161--164
161
© Elsevier/North-Holland Biomedical Press Short c o m m u n i c a t i o n '7-MSH' F R A G M E N T S FROM ACTH-~-LPH P R E C U R S O R HAVE AN A F F I N I T Y FO R OPIATE RECEPTORS SHOGO OKI *, KAZUWA NAKAO *, YOSHIKATSU NAKAI *, NICHOLAS LING ** and HIROO IMURA * • Second Division, Department of Internal Medicine, Kyoto University Faculty of Medicine, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto, 606, Japan and ** Laboratories for Neuroendocrinology, The Salk Institute for Biological Studies, La Jolla, California 92037, U.S.A.
Received 8 April 1980, accepted 14 April 1980
S. OKI, K. NAKAO, Y. NAKAI, N. LING and H. IMURA. ~-MSH fragments from ACTH-~J-LPHprecursor have an affinity for opiate receptors, European J. Pharmacol. 64 (1980) 161--164. 3,-Melanotropin (7-MSH), a putative peptide residing in the cryptic N-terminal portion of ACTH-~-LPH precursor, shares several amino acid residues with a-MSH or ~-MSH. The present study revealed that ~/-MSH and structurally related peptides had as potent an affinity for rat brain opiate receptors as did ACTH1.24 when 3Hnaloxone was used as a iigand. Thus, 7-MSH and structurally related peptides may have physiological significance in the activities of the CNS. 7-MSH
Opiate receptor
1. I n t r o d u c t i o n T he discovery of a d r e n o c o r t i c o t r o p i n (ACTH)-/3-1ipotropin (LPH) c o m m o n precursor protein (Nakanishi et al., 1977; Mains et al., 1977) has drawn a t t e n t i o n to t he primary structure o f the precursor protein and to possible biological functions o f the remaining p o r t i o n . Nakanishi et al. {1979) r e p o r t e d t he p ri m a r y structure o f t he precursor protein after experiments using c o m p l e m e n t a r y DNA (cDNA) cloning and nucleotide sequence anhlyses. T h e y showed t h a t the remaining Nterminal p o r tio n has an amino acid sequence strikingly similar to those o f a-MSH and ~-MSH and predicted the presence of a n o t h e r h o r m o n e o f physiological significance. The p o r t i o n b e t w e e n Arg-Lys and Arg-Arg, a possible site o f cleavage b y trypsin-like enzymes, was thus t e r m e d 7-melanotropin (7-MSH), although the terminal group of this peptide is n o t know n definitively, The t e rm i n o lo g y o f ~,-MSH is based on t he structural similarities to a-MSH and/3-MSH, b u t its
biological activity is u n k n o w n , a-MSH,/3-MSH and ACTH have c o m m o n sequences, which are active in binding to opiate receptors (Terenius, 1975) and in eliciting c o m p l e x behavioral responses in the central nervous system (CNS) (De Wied, 1974; Gispen et al., 1975). T he present study was an a t t e m p t to det erm i ne w het her or n o t 7-MSH and structurally related peptides have an affinity for rat brain opiate receptors.
2. Materials and m e t h o d s 2.1. R e c e p t o r p r e p a r a t i o n s
Receptor preparations were obtained according to the m e t h o d o f Pasternak et al. (1975). Briefly, male Wistar rats weighing 180-220 g were decapitated and t he brains immediately removed. The cerebellum, 3Hopiate binding t o which is neglibible, was excised and the remainder of the brain was immediately placed in 30 volumes of iced
162 50 mM Tris-HC1 buffer, pH 7.7 and homogenized with a P o tt e r h o m o g e n i z e r at 4 ° C. The homogenates were t hen centrifuged at 4 9 0 0 0 × g for 15 min. The pellet was resuspended in Tris-HC1 buffer, incubated at 37°C for 30 min and recentrifuged at 49 000 × g for 15 min. This pellet was t h e n resuspended in 50 volumes of Tris-HC1 buf f e r and stored at --20°C until assayed.
S. OKI ET AL. acid sequence o f 73-MSH is H-Tyr-SS-Val-MetGly-His-Phe-S°-Arg-Trp-Asp-Arg.Phe-4S.Gly_ Arg-Arg-Asn-Gly-4°-Ser-Ser~Ser~Ser-Gly-3S-ValGly-Gly-Ala-Ala-3°-Gln-OH). ~/,-MSH corresponds to amino acid residues --55 to --45, of which the phenylalanine residue at position --45 is in t he form o f an amide. ACTH,.:4 (Cortrosyn) was obtained from Organon and ~-MSH and methionine-enkephalin (Met-Enk) from Dr. H. Yajima, K y o t o University.
2.2. Assay procedure Reaction mixtures (final volume 1 ml) containing tissue suspension and unlabeled drugs or peptides were incubated at 25°C for 15 min. After t he incubation, 3 nM 3Hn a l o x o n e (New England Nuclear, 1 7 . 4 C i / mmol) was added and the reaction allowed to proceed for 30 min at 25°C. Bacitracin (50 pg/ml) was added to all assay tubes containing 7-MSH and its related peptides. All determinations were p e r f o r m e d in triplicate, T h e incubation was t er m i nat ed by filtering t h e m i x t u r e over Whatman glass filters (GF-B} u n d e r vacuum. The filters were t hen washed with twice 5 ml of cold Tris-HC1 b u f f e r and placed in 10 ml o f T r i t o n - T o l u e n e scintillation fluor ( 3 3 3 m l T r i t on X-100, 6 6 7 m l toluene, 5.5 g 2,5
2.3. Peptides Three peptides, t e r m e d 7 r M S H , 72-MSH and 73-MSH were synthesized by t he solid phase m e t h o d (Ling et al., 1980). 73-MSH and 7:-MSH correspond to amino acid residues --55 to --29 and --55 to --44 o f the c o m m o n precursor molecule, respectively (the amino
3. Results Fig. 1 shows the displacement curves of 71-MSH, 72-MSH, 73-MSH, a-MSH, ACTH, Met-Enk and nal oxone. 7,-MSH, 7:-MSH, 73-MSH, ACTH and Met-Enk apparently displaced 3H-naloxone binding t o rat brain opiate receptors, while a-MSH at concentrations up to 1 0 - S M did n o t displace 3Hnaloxone. The concent rat i ons of peptides which displaced 50% of t he 3H-naloxone binding to rat brain opiate receptors (ICs0)
8~°o ~ ~ ~
(%)
~CO,
~
......
,"~.~, ~
J
2~
0]
0 ~'
.~-E~.~-E~
~
~
" -~ . . . . . . . . -5 c..... ,,~o~:ogM) Fig. 1 . The displacement curves of 3H-naloxone binding to rat brain opiate receptors by various concentrations of 71-MSH, 72-MSH, 73-MSH, ACTHI.:4, (~-MSH, Met-Enk and naloxone. Rat brain opiate receptors were prepared as described in the text and preincubated with 50/~g/ml bacitracin and unlabeled peptides at 25°C for 15 rain and further incubated with 3 nM 3H-naloxone at 25°C for 30 min. Data, expressed as a percent of the control specific 3Hnaloxone binding are plotted on the ordinate against the drug concentration (M) on the abscissa. -9
-8
7-MSH AND OPIATE RECEPTOR
163
BIB
4. D i s c u s s i o n
C'I.)
-~ \
-\~o\
\~ ~:\,,
~ , \\ 'x~<,~ oi\\~
6C
4c
xh-MSH ~'2-MSH
o)3- MSH 2C
.ACTH(I-2Z,)
-7 -6 -5 concentration(iogM) Fig. 2. The displacement curves of 3H-naloxone binding to rat brain opiate receptors by 71-MSH, 72-MSH, ~%-MSH and ACTHI.24 in the absence (solid line) and presence (dotted line) of 100 mM NaC1.
were: naloxone; 4.0 X 10 -~ M, 7,-MSH: 5.9 X 10 -6 M, 73-MSH: 2.3 X 10 -6 M, ACTH,.24: 7.0 X 10 -6 M and Met-Enk: 1.3 X 10 -7 M. The inhibition of 3H-naloxone binding by the addition of 1 X 10 -s M 72-MSH or a-MSH was below 50% and the percent inhibition at 1 × 10 -5 M of 72-MSH was 30% and that of a-MSH was below 5%. Fig. 2 shows the 'sodium effect' on 7,MSH, 7:-MSH, 73-MSH and ACTH,_24 inhibition of 3H-naloxone binding to opiate receptors. Sodium reduced their p o t e n c y in competing for 3H-naloxone binding sites. A 'sodium ratio' (ICs0 in displacing 3H-naloxone binding in the presence of 100 mM NaC1/ICs0 in displacing 3H-naloxone binding in the absence of added sodium) for each peptide was not determined because its ICs0 in the presence of 100 mM NaC1 was greater than 1 X 10 -s M.
The present studies demonstrated that 7MSHs have a binding affinity for opiate receptors from rat brain in vitro. The fact that displacement curves of 7-MSHs were almost comparable to that of ACTH is of interest, since 7-MSHs share amino acid sequences with ACTH. However, when compared on the basis of IC50, 7-MSHs or ACTH are t w e n t y to sixty times less p o t e n t than Met-Enk, although 73-MSH was the most potent of all 7-MSHs and was three times more potent than ACTH,_24. The C-terminal elongation of the amino acid sequence of 72-MSH and the amidation of the C-terminal Phe residue increased the ICs0. Further studies are required to clarify the binding site. The addition of sodium reduced the binding p o t e n c y of 7-MSHs as it did t h a t of ACTH. This suggests agonistic properties for these peptides. However, ACTH is known to counteract morphine-induced analgesia (Gispen et al., 1976). It is still u n k n o w n , therefore, whether 7-MSHs act as opiate agonists or as mixed agonists-antagonists. The activities of a-MSH or ACTH fragments in the CNS have been shown in several experiments, such as inhibition of the extinction of active avoidance behavior (De Wied, 1974) or stimulation of the induction of excessive grooming (Gispen et al., 1975). The observation that excessive grooming can be blocked by pretreatment with opiate antagonists (Gispen and Wiegant, 1976) suggests that these effects are mediated by opiate receptors. The fact that both ACTH and 7-MSHs bind similarly to brain opiate receptors suggests that 7-MSHs are active in the brain, but further studies are required to elucidate such activity of 7-MSHs in the CNS.
Acknowledgements We are greatly indebted to Professor H. Yajima, University of Kyoto, Japan, for the generous supply of a-MSH and Met-Enk, to Professor S. Numa, University of Kyoto, for advice, to Dr. H. Jingami for
164
S. OKI ET AL.
helpful assistance and to M. Ohara for assistance with
Ling, N., S. Ying, S. Minick and R. Guillemin, 1980,
the manuscript.
Synthesis and biological activity of four 3-melanotropin peptides derived from the cryptic region of the adrenocorticotropin/fi-lipotropin precursor, Life Sci. 25, 1773. Mains, R.E., B.A. Eipper and N. Ling, 1977, Common precursor to corticotropins and endorphins, Proc. Nat. Acad. Sci. U.S.A. 74, 3014. Nakanishi, S., A. Inoue, S. Tail and S. Numa, 1977, Cell-free translation product containing corticotropin and fi-endorphin encoded by messenger RNA from anterior lobe and intermediate lobe of bovine pituitary, FEBS. Lett. 84, 105. Nakanishi, S., A. Inoue, T. Kita, M. Nakamura, A.C.Y. Chang, S.N. Cohen and S. Numa, 1979, Nucleotide sequence of cloned cDNA for bovine corticotropin~-lipotropin precursor, Nature 278,423. Pasternak, G.W., H.A. Wilson and S.H. Snyder, 1975, Differential effects of protein-modifying reagents on receptor binding of opiate agonists and antagonists, Mol. Pharmacol. 11,340. Terenius, L., 1975, Effect of peptides and amino acids on dihydromorphine binding to the opiate receptor, J. Pharm. Pharmacol. 27,450.
References De Wied, D., 1974, Pituitary-adrenal system hormones and behavior, in: The Neurosciences, Third Study Program, eds. F.O. Schmitt and F.G. Worden (Rockefeller University Press, New York) p. 653. Gispen, W.H., J. Buitelaar, V.M. Wiegant, L. Terenius and D. De Wied, 1976, Interaction between ACTH fragments, brain opiate receptors and morphineinduced analgesia, European J. Pharmac. 3 9 , 3 9 3 . Gispen, W.H., V.M. Wiegant, H.M. Greven and D. De Wied, 1975, The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: structure activity studies, Life Sci. 17,645. Gispen, W.H. and V.M. Wiegant, 1976, Opiate Antagonists suppress ACTHI.24 induced grooming in the rat, Neurosci. Lett. 2 , 1 5 9 .
161
© Elsevier/North-Holland Biomedical Press Short c o m m u n i c a t i o n '7-MSH' F R A G M E N T S FROM ACTH-~-LPH P R E C U R S O R HAVE AN A F F I N I T Y FO R OPIATE RECEPTORS SHOGO OKI *, KAZUWA NAKAO *, YOSHIKATSU NAKAI *, NICHOLAS LING ** and HIROO IMURA * • Second Division, Department of Internal Medicine, Kyoto University Faculty of Medicine, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto, 606, Japan and ** Laboratories for Neuroendocrinology, The Salk Institute for Biological Studies, La Jolla, California 92037, U.S.A.
Received 8 April 1980, accepted 14 April 1980
S. OKI, K. NAKAO, Y. NAKAI, N. LING and H. IMURA. ~-MSH fragments from ACTH-~J-LPHprecursor have an affinity for opiate receptors, European J. Pharmacol. 64 (1980) 161--164. 3,-Melanotropin (7-MSH), a putative peptide residing in the cryptic N-terminal portion of ACTH-~-LPH precursor, shares several amino acid residues with a-MSH or ~-MSH. The present study revealed that ~/-MSH and structurally related peptides had as potent an affinity for rat brain opiate receptors as did ACTH1.24 when 3Hnaloxone was used as a iigand. Thus, 7-MSH and structurally related peptides may have physiological significance in the activities of the CNS. 7-MSH
Opiate receptor
1. I n t r o d u c t i o n T he discovery of a d r e n o c o r t i c o t r o p i n (ACTH)-/3-1ipotropin (LPH) c o m m o n precursor protein (Nakanishi et al., 1977; Mains et al., 1977) has drawn a t t e n t i o n to t he primary structure o f the precursor protein and to possible biological functions o f the remaining p o r t i o n . Nakanishi et al. {1979) r e p o r t e d t he p ri m a r y structure o f t he precursor protein after experiments using c o m p l e m e n t a r y DNA (cDNA) cloning and nucleotide sequence anhlyses. T h e y showed t h a t the remaining Nterminal p o r tio n has an amino acid sequence strikingly similar to those o f a-MSH and ~-MSH and predicted the presence of a n o t h e r h o r m o n e o f physiological significance. The p o r t i o n b e t w e e n Arg-Lys and Arg-Arg, a possible site o f cleavage b y trypsin-like enzymes, was thus t e r m e d 7-melanotropin (7-MSH), although the terminal group of this peptide is n o t know n definitively, The t e rm i n o lo g y o f ~,-MSH is based on t he structural similarities to a-MSH and/3-MSH, b u t its
biological activity is u n k n o w n , a-MSH,/3-MSH and ACTH have c o m m o n sequences, which are active in binding to opiate receptors (Terenius, 1975) and in eliciting c o m p l e x behavioral responses in the central nervous system (CNS) (De Wied, 1974; Gispen et al., 1975). T he present study was an a t t e m p t to det erm i ne w het her or n o t 7-MSH and structurally related peptides have an affinity for rat brain opiate receptors.
2. Materials and m e t h o d s 2.1. R e c e p t o r p r e p a r a t i o n s
Receptor preparations were obtained according to the m e t h o d o f Pasternak et al. (1975). Briefly, male Wistar rats weighing 180-220 g were decapitated and t he brains immediately removed. The cerebellum, 3Hopiate binding t o which is neglibible, was excised and the remainder of the brain was immediately placed in 30 volumes of iced
162 50 mM Tris-HC1 buffer, pH 7.7 and homogenized with a P o tt e r h o m o g e n i z e r at 4 ° C. The homogenates were t hen centrifuged at 4 9 0 0 0 × g for 15 min. The pellet was resuspended in Tris-HC1 buffer, incubated at 37°C for 30 min and recentrifuged at 49 000 × g for 15 min. This pellet was t h e n resuspended in 50 volumes of Tris-HC1 buf f e r and stored at --20°C until assayed.
S. OKI ET AL. acid sequence o f 73-MSH is H-Tyr-SS-Val-MetGly-His-Phe-S°-Arg-Trp-Asp-Arg.Phe-4S.Gly_ Arg-Arg-Asn-Gly-4°-Ser-Ser~Ser~Ser-Gly-3S-ValGly-Gly-Ala-Ala-3°-Gln-OH). ~/,-MSH corresponds to amino acid residues --55 to --45, of which the phenylalanine residue at position --45 is in t he form o f an amide. ACTH,.:4 (Cortrosyn) was obtained from Organon and ~-MSH and methionine-enkephalin (Met-Enk) from Dr. H. Yajima, K y o t o University.
2.2. Assay procedure Reaction mixtures (final volume 1 ml) containing tissue suspension and unlabeled drugs or peptides were incubated at 25°C for 15 min. After t he incubation, 3 nM 3Hn a l o x o n e (New England Nuclear, 1 7 . 4 C i / mmol) was added and the reaction allowed to proceed for 30 min at 25°C. Bacitracin (50 pg/ml) was added to all assay tubes containing 7-MSH and its related peptides. All determinations were p e r f o r m e d in triplicate, T h e incubation was t er m i nat ed by filtering t h e m i x t u r e over Whatman glass filters (GF-B} u n d e r vacuum. The filters were t hen washed with twice 5 ml of cold Tris-HC1 b u f f e r and placed in 10 ml o f T r i t o n - T o l u e n e scintillation fluor ( 3 3 3 m l T r i t on X-100, 6 6 7 m l toluene, 5.5 g 2,5
2.3. Peptides Three peptides, t e r m e d 7 r M S H , 72-MSH and 73-MSH were synthesized by t he solid phase m e t h o d (Ling et al., 1980). 73-MSH and 7:-MSH correspond to amino acid residues --55 to --29 and --55 to --44 o f the c o m m o n precursor molecule, respectively (the amino
3. Results Fig. 1 shows the displacement curves of 71-MSH, 72-MSH, 73-MSH, a-MSH, ACTH, Met-Enk and nal oxone. 7,-MSH, 7:-MSH, 73-MSH, ACTH and Met-Enk apparently displaced 3H-naloxone binding t o rat brain opiate receptors, while a-MSH at concentrations up to 1 0 - S M did n o t displace 3Hnaloxone. The concent rat i ons of peptides which displaced 50% of t he 3H-naloxone binding to rat brain opiate receptors (ICs0)
8~°o ~ ~ ~
(%)
~CO,
~
......
,"~.~, ~
J
2~
0]
0 ~'
.~-E~.~-E~
~
~
" -~ . . . . . . . . -5 c..... ,,~o~:ogM) Fig. 1 . The displacement curves of 3H-naloxone binding to rat brain opiate receptors by various concentrations of 71-MSH, 72-MSH, 73-MSH, ACTHI.:4, (~-MSH, Met-Enk and naloxone. Rat brain opiate receptors were prepared as described in the text and preincubated with 50/~g/ml bacitracin and unlabeled peptides at 25°C for 15 rain and further incubated with 3 nM 3H-naloxone at 25°C for 30 min. Data, expressed as a percent of the control specific 3Hnaloxone binding are plotted on the ordinate against the drug concentration (M) on the abscissa. -9
-8
7-MSH AND OPIATE RECEPTOR
163
BIB
4. D i s c u s s i o n
C'I.)
-~ \
-\~o\
\~ ~:\,,
~ , \\ 'x~<,~ oi\\~
6C
4c
xh-MSH ~'2-MSH
o)3- MSH 2C
.ACTH(I-2Z,)
-7 -6 -5 concentration(iogM) Fig. 2. The displacement curves of 3H-naloxone binding to rat brain opiate receptors by 71-MSH, 72-MSH, ~%-MSH and ACTHI.24 in the absence (solid line) and presence (dotted line) of 100 mM NaC1.
were: naloxone; 4.0 X 10 -~ M, 7,-MSH: 5.9 X 10 -6 M, 73-MSH: 2.3 X 10 -6 M, ACTH,.24: 7.0 X 10 -6 M and Met-Enk: 1.3 X 10 -7 M. The inhibition of 3H-naloxone binding by the addition of 1 X 10 -s M 72-MSH or a-MSH was below 50% and the percent inhibition at 1 × 10 -5 M of 72-MSH was 30% and that of a-MSH was below 5%. Fig. 2 shows the 'sodium effect' on 7,MSH, 7:-MSH, 73-MSH and ACTH,_24 inhibition of 3H-naloxone binding to opiate receptors. Sodium reduced their p o t e n c y in competing for 3H-naloxone binding sites. A 'sodium ratio' (ICs0 in displacing 3H-naloxone binding in the presence of 100 mM NaC1/ICs0 in displacing 3H-naloxone binding in the absence of added sodium) for each peptide was not determined because its ICs0 in the presence of 100 mM NaC1 was greater than 1 X 10 -s M.
The present studies demonstrated that 7MSHs have a binding affinity for opiate receptors from rat brain in vitro. The fact that displacement curves of 7-MSHs were almost comparable to that of ACTH is of interest, since 7-MSHs share amino acid sequences with ACTH. However, when compared on the basis of IC50, 7-MSHs or ACTH are t w e n t y to sixty times less p o t e n t than Met-Enk, although 73-MSH was the most potent of all 7-MSHs and was three times more potent than ACTH,_24. The C-terminal elongation of the amino acid sequence of 72-MSH and the amidation of the C-terminal Phe residue increased the ICs0. Further studies are required to clarify the binding site. The addition of sodium reduced the binding p o t e n c y of 7-MSHs as it did t h a t of ACTH. This suggests agonistic properties for these peptides. However, ACTH is known to counteract morphine-induced analgesia (Gispen et al., 1976). It is still u n k n o w n , therefore, whether 7-MSHs act as opiate agonists or as mixed agonists-antagonists. The activities of a-MSH or ACTH fragments in the CNS have been shown in several experiments, such as inhibition of the extinction of active avoidance behavior (De Wied, 1974) or stimulation of the induction of excessive grooming (Gispen et al., 1975). The observation that excessive grooming can be blocked by pretreatment with opiate antagonists (Gispen and Wiegant, 1976) suggests that these effects are mediated by opiate receptors. The fact that both ACTH and 7-MSHs bind similarly to brain opiate receptors suggests that 7-MSHs are active in the brain, but further studies are required to elucidate such activity of 7-MSHs in the CNS.
Acknowledgements We are greatly indebted to Professor H. Yajima, University of Kyoto, Japan, for the generous supply of a-MSH and Met-Enk, to Professor S. Numa, University of Kyoto, for advice, to Dr. H. Jingami for
164
S. OKI ET AL.
helpful assistance and to M. Ohara for assistance with
Ling, N., S. Ying, S. Minick and R. Guillemin, 1980,
the manuscript.
Synthesis and biological activity of four 3-melanotropin peptides derived from the cryptic region of the adrenocorticotropin/fi-lipotropin precursor, Life Sci. 25, 1773. Mains, R.E., B.A. Eipper and N. Ling, 1977, Common precursor to corticotropins and endorphins, Proc. Nat. Acad. Sci. U.S.A. 74, 3014. Nakanishi, S., A. Inoue, S. Tail and S. Numa, 1977, Cell-free translation product containing corticotropin and fi-endorphin encoded by messenger RNA from anterior lobe and intermediate lobe of bovine pituitary, FEBS. Lett. 84, 105. Nakanishi, S., A. Inoue, T. Kita, M. Nakamura, A.C.Y. Chang, S.N. Cohen and S. Numa, 1979, Nucleotide sequence of cloned cDNA for bovine corticotropin~-lipotropin precursor, Nature 278,423. Pasternak, G.W., H.A. Wilson and S.H. Snyder, 1975, Differential effects of protein-modifying reagents on receptor binding of opiate agonists and antagonists, Mol. Pharmacol. 11,340. Terenius, L., 1975, Effect of peptides and amino acids on dihydromorphine binding to the opiate receptor, J. Pharm. Pharmacol. 27,450.
References De Wied, D., 1974, Pituitary-adrenal system hormones and behavior, in: The Neurosciences, Third Study Program, eds. F.O. Schmitt and F.G. Worden (Rockefeller University Press, New York) p. 653. Gispen, W.H., J. Buitelaar, V.M. Wiegant, L. Terenius and D. De Wied, 1976, Interaction between ACTH fragments, brain opiate receptors and morphineinduced analgesia, European J. Pharmac. 3 9 , 3 9 3 . Gispen, W.H., V.M. Wiegant, H.M. Greven and D. De Wied, 1975, The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: structure activity studies, Life Sci. 17,645. Gispen, W.H. and V.M. Wiegant, 1976, Opiate Antagonists suppress ACTHI.24 induced grooming in the rat, Neurosci. Lett. 2 , 1 5 9 .