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Characterization of Nα-acetyl-α-endorphin from rat neurointermediate lobe and its distribution in pituitary and brain
Life Sciences, Vol. 33, Sup. I, 1983, pp. 125-128 Printed in the U.S.A.
Pergamon Press
CHARACTERIZATION OF N~-ACETYL-~-ENDORPHIN FROM RAT NEUROINTERMEDIATE LOBE AND ITS DISTRIBUTION IN PITUITARY AND BRAIN
V.M. Wiegant, J. Verhoef, J.P.H. Burbach and A. van Amerongen Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, 3521GD Utrecht, The Netherlands (Received in final form June 26, 1983) Summary
N~-Acetyl-~-endorphin was characterized from rat neurointermediate lobe. The distribution of the acetylated and the non-acetylated form of ~endorphin in dissected areas of pituitary and brain appeared to be uneven. ~-Endorphin appeared to be the main peptide in the anterior pituitary, whereas in the neurointermediate lobe N~-acetyl-~-endorphin accounted for most of the e-endorphin immunoreactivity. In the brain, the highest concentration of ~-endorphin immunoreactivity was found in the hypothalamus. In hypothalamus and thalamus ~-endorphin predominated, whereas in amygdala, hippocampus and septum N~-acetyl-~-endorphin represented most of the ~-endorphin-immunoreactivity. In view of the nonopioid properties of acetylated endorphins, i t is suggested that acetylation represents a mechanism allowing the organism to specifically select the non-opioid behavioral activities enclosed in the endorphin sequence. Introduction ~- And y-endorphin have distinct effects on adaptive behavior in rats (1,2,3). The behavioral activities of these peptides are shared by their non-opioid desTyr I- and des-Enk-congener (2,3) indicating that they do not involve opiate sensitive structures in the brain. ~- And y-endorphin occur in pituitary and brain tissue (4,5). Their distribution in rat brain and pituitary closely parallels that of B-endorphin (5) their putative precursor molecule (6), Recently we have characterized N~-acetyl-y-endorphin as an endogenous neuropeptide in rat brain and pituitary (Wiegant et a l . , submitted). This peptide is devoid of opioid a c t i v i t y , but retains the behavioral activities of Y-type endorphins (Wiegant et a l . ; Van Ree et a l . , submitted). In the present communication we report the characterization of N~-acetyl-a-endorphin from rat neuro-intermediate lobe tissue and its distribution in pituitary and brain. Materials and Methods Male Wistar rats (180 - 200 g) were used. After decapitation of the animals the pituitary and the brain were quickly removed. The pituitary was dissected in anterior and neurointermediate lobe, the brain was dissected in anatomically defined regions (7). Tissues were immediately frozen on dry ice, Extraction of endorphins: Pooled tissues from 18 animals were heated for 10 min IB I M acetic acid (I : 10 w/v) in a boiling water bath and homogenized by ultrasound. The precipitates were removed by centrifugation and used for determination of tissue protein. The supernatants were lyophilized and the resulting residues dissolved in 1.0 ml 0.01M ammoniumacetate (pH 4.15). 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
126
~-endorphin in Pituitary and Brain
Vol. 33, Sup. I, 1983
Fractionation of peptides by HPLC: A f t e r clearing the extracts by c e n t r i f u g a t i o n , the-ywere f r a c t i o n a t e ~ - ~ P L C as described previously (8). In short, a ~Bondapak C18 column was used, with a 45 min concave gradient of 0.01M ammoniumacetate (pH 4.15; solvent A) and methanol ~solvent B). I n i t i a l conditions: A : B = 70 : 30, f i n a l conditions A : B = 25 : 75. Flow rate 2 ml/min. Fractions of 30 sec were collected, dried and reconstituted in 125 mM phosphate buffer (pH 7.5) containing 0.9% sodium chloride, 0.02% sodium azide and 0.25% bovine serum albumine (radioimmunoassay b u f f e r ) . Radioimmunoassay systems: ~-Endorphin immunoreactivity in HPLC-fractions was 'determined using an antiserum (A2) s p e c i f i c a l l y recognizing the free C-terminal Thr16-residue. The antiserum has low c r o s s - r e a c t i v i t y with ~-endorphin (2%) and B-endorphin (1%). lodinated ~-endorphin was used as a tracer. For f u r t h e r details on the procedure and s p e c i f i c i t y of the assay see 8. N~-acetylated-endorphins were assayed with antiserum Nancy Beth # 6 and iodinated N~-acetyl-y-endorphin as a tracer using the same methodology as for the ~endorphin radioimmunoassay (8). The antiserum s p e c i f i c a l l y recognizes the Noa c e t y l - T y r l - r e s i d u e of endorphins. No c r o s s - r e a c t i v i t y was observed with ~-, ~- or B-endorphin. Incubation with aminopeptidase-M: Aliquots of HPLC-fractions containing 5 ng ~-endorphin immunoreactivity were diluted to 112.5 ~I with 0 . 1 M Tris/HCl (pH 8.0). The incubation was started by the addition of 12.5 ul aminopeptidaseM solution (I ~g/10 ~ l l i n 0.9% sodium chloride/t0% g l y c e r o l ) , continued for 90 min at 37 ° C and terminated by d i l u t i o n with I ml 0.01M ammoniumacetate (pH 4.15). Samples were kept frozen u n t i l a second f r a c t i o n a t i o n by HPLC. Synthetic peptides were kindly donated by Drs. H.M. Greven and J. van Nispen (Organon International B.V., Oss, The Netherlands). N~-acetyl-~-endorphin was generated from synthetic N~-acetyl-~-endorphin by incubation with carboxypeptidase A and subsequently p u r i f i e d by HPLC. Purity and a u t h e n t i c i t y of the peptide were confirmed by i t s amino acid composition and resistance to aminopeptidase-M. The antiserum Nancy Beth # 6 was a generous g i f t from Dr. H. A k i l , Ann Arbor, MI, USA. Results The HPLC-chromatogram of rat neurointermediate lobe e x t r a c t showed two main peaks containing ~-endorphin immunoreactivity (Fig. IA). The f i r s t peak (fraction 53) eluted with the same retention time as synthetic ~-endorphin. The retention time of the second peak ( f r a c t i o n 65) was identical to that of synthet i c N~-acetyl-~-endorphin. Aliquots from f r a c t i o n 53 and 65 were assayed in serial d i l u t i o n s for ~-endorphin immunoreactivity. Both d i l u t i o n curves p a r a l l e l l e d the ~-endorphin standard curve (data not shown). Neither of the peaks was recognized by antisera raised against ~- or ~-endorphin (for s p e c i f i c i t y of these antisera see 81. An aliquot of f r a c t i o n 65 (equivalent to 5 ng ~-endorphin immunoreactivity) was incubated with aminopeptidase-M and subsequently fractionated by HPLC (Fig. 2A). Similar to synthetic N~-acetyl-~-endorphin, the ~-endorphin immunoreactivity in the sample appeared to be r e s i s t a n t to the enzyme, as more than 80% eluted with the same retention time as synthetic N~-acetyl-~-endorphin. These data indicate that the peptide in f r a c t i o n 65 containeda blocked N-terminus. The presence of an N~-acetyl-Tyrl is inferred from I) cross-reaction of the peptide with antiserum Nancy Beth, which s p e c i f i c a l l y recognizes N~-acetylated endorphins (sample d i l u t i o n curve p a r a l l e l l e d the standard curve; data not shown), and 2) cochromatography on HPLC with synthetic N~-acetyl-~-endorphin. I t was concluded that f r a c t i o n 65 contained N~-acetyl-~-endorphin. Incubation of an a l i q u o t (5 ng) from f r a c t i o n 53 with aminopeptidase-M resulted in complete disappearance of the ~-endorphin peak on HPLC (Fig. 2B). Only 15% of the immunoreactivity was recovered, e l u t i n g with the retention time of desTyr1-~-endorphin. Based on immunological c h a r a c t e r i s t i c s , co-elution with synt h e t i c ~-endorphin on HPLC and s e n s i t i v i t y f o r aminopeptidase-M i t was conclu-
Vol. 33, Sup. I, 1983
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~-endorphin in Pituitary and Brain
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fraction number FIG. I: HPLC of ~-endorphin immunoreact i v i t y extracted from neurointermediate (A) and anterior lobe (B) of the p i t u i tary. Arrows indicate the elution position of synthetic peptides as determined in consecutive runs.
FIG. 2: HPLC of 5 ng ~-endorphin im-,i munoreactivity from neurointermediate lobe fraction 65 (A; cf. Fig. IA) and fraction 53 (B; cf. Fig IA) after incubation with aminopeptidase-M.
ded that fraction 53 represents non-acetylated ~-endorphin. The regional d i s t r i b u t i o n of the d i f f e r e n t forms of ~-endorphin immunoreactivity in p i t u i t a r y and brain was studied by HPLC fractionation of extracts of dissected areas and quantitation by radioimmunoassay with the A2-antiserum. In the neurointermediate lobe of the p i t u i t a r y ~-endorphin immunoreactivity was almost completely recovered as N~-acetyl -~-endorphin (Fig. I A ) ( r a t i o acetylated:non-acetylated = 10:1),whereas in the anterior lobe (Fig. IB)~-endQrphin appeared to be the predominant peptide (ratio I : 2,5; see also table I ) . Apart from ~- and N -acetyi-~-endorphin a minor peak eluting with retention time similar to desTyr1-~-endorphin was observed in the chromatograms. -Endorphin and NO-acetyl-~-endorphin were also detected in extracts of brain tissue. Marked differences in concentration of ~-endorphin immunoreactivity, as well as in the degree of acetylation, were observed among the various dissected areas of the brain (Table I ) . The highest amount of e-endorphin was found in the hypothalamus. In the hypothalamus and the thalamus, most of the peptides existed in the non-acetylated form (ratio acetylated :no~acetylated i : 5 and I : 2.5 resp.). In the other brain regions studied (septum, hippocampus, amygdala) the acetylated form appeared to be predominant (ratio > I ) . The amygdala showed the highest concentration of N~-acetyl-~-endorphin and the highest r a t i o of all brain areas analysed. Discussion In the present experiments we have i d e n t i f i e d N~-acetyl-m-endorphin as a na-
128
~-endorphin in Pituitary and Brain
Vol. 33, Sup. I, 1983
t u r a l l y occurring peptide in brain and p i t u i t a r y . I t appeared to be the main form of m-endorphin-immunoreactivity in the neuro-intermediate lobe of the pit u i t a r y , and in brain structures remote from hypothalamus such as amygdala, hippocampus and septum. Recently, we have characterized N'~-acetyl-y-endorphin as an endogenous neuropeptide (Wiegant et a l . , subm.). The d i s t r i b u t i o n of the acetylated and non-acetylated forms of m- and y-endorphin in brain and p i t u i t a r y parallels that of acetylated and non-acetylated 6-endorphins (5, 9, Wiegant et a l . , subm.), underscoring the suggested precursor function of 6-endorphin for m- and y-endorphins (6). Nm-acetylation completely destroys the opiate-like properties of y-endorphin and other endorphins (10). In Nm-acetyl-Y-endorphin the residual opioid a c t i v i t y of y-endorphin is l o s t , but the non-opioid behavioral a c t i v i t i e s of the peptide are retained (Wiegant et a l . , Van Ree et a l . , subm.). Preliminary data indicate that also in Nm-acetyl-m-endorphin the nonopioid behavioral properties of m-type endorphins are selectively conserved (Van Ree, unpublished). These obervations suggest that Nm-acetyl-m - and Nmacetyl-y-endorphin represent the naturally occurring congeners of the non-opioid behaviorally active 6-endorphin fragments des-Tyrl-m- and des-Tyr1-y-endorphin. Thus, Nm-acetylation of endorphins represents not merely a mechanism for inactivation of opioid a c t i v i t e s , but may also allow the organism to select spec i f i c sets of biological a c t i v i t i e s present in the endorphins sequence. The biological significance of acetylated 6-endorphins may ly in t h e i r precursor function for these NO-acetylated psychoactive peptides. TABLE I Distribution of m- and N%Acetyl -m-Endorphin tissue protein (mg) anterior lobe neurointermediate lobe hypothalamus thalamus septum hippocampus amygdala
.830 .084 3.72 5.38 1.04 6.26 6.51
m-endorphin (pg)
in Rat P i t u i t a r y and Brain Nm-acetyl-m - approx, r a t i o endorphin(pg) acetyl/non-acetyl
13,000 5,000
4,700 51,200
0.4 10.2
547.0 186.3 26.4 21.5 61.3
99.3 80.0 34.4 31.7 122.8
0.2 0.4 1.3 1.5 2.0
Extracts of dissected areas were fractionated by HPLC. Peptide content of the fractions was determined by ~-endorphin radioimmunoassay. Values represent endorphin content per tissue per animal. References I. D. DE WIED, B. BOHUS, J.M. VAN REE and I. URBAN, J. Pharmacol. exp. Ther. 204 570-580 (1978). 2. D. DE WIED, G.L. KOVACS, B. BOHUS, J.M. VAN REE and H.M. GREVEN, Eur. J. Pharmacol. 49 427-436 (1978). 3. H.M. GREVEN and D. DE WIED, Hormones and the Brain (D. de Wied and P.A. van Keep, edso), pp. 115-127, MTP Press, Lancaster (1980) 4. J. VERHOEF, J.G. LOEBER, J.P.H. BURBACH, W.H. GISPEN, A. WITTER and D. DE WIED, Life Sci. 26 851-859 (1980). 5. J. VERHOEF, V.M.~IEGANT and D. DE WIED, Brain Res. 231 454-460 (1982). 6. J.P.H. BURBACH, E.R. DE KLOET, P. SCHOTMANand D. DE~ED, J. Biol. Chem. 256 12463-12469 (1981). 7. I~TTI~. GISPEN, P. SCHOTMANand E.R. DE KLOET, Neuroendocr. 9 285-296 (1972). 8. J.G. LOEBER, J. VERHOEF, J.P.H. BURBACHand A. WITTER, Bi~chem. Biophys. Res. Commun. 86 1288-1295 (1979). 9. S. ZAKARIAN a N D.G. SMYTH, Nature 296 250-252 (1982). 10. J.F.Wo DEAKIN, J.O. DOSTROVSKYand D--T~.. SMYTH, Biochem. J. 189 501-506 (1980).
Pergamon Press
CHARACTERIZATION OF N~-ACETYL-~-ENDORPHIN FROM RAT NEUROINTERMEDIATE LOBE AND ITS DISTRIBUTION IN PITUITARY AND BRAIN
V.M. Wiegant, J. Verhoef, J.P.H. Burbach and A. van Amerongen Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, 3521GD Utrecht, The Netherlands (Received in final form June 26, 1983) Summary
N~-Acetyl-~-endorphin was characterized from rat neurointermediate lobe. The distribution of the acetylated and the non-acetylated form of ~endorphin in dissected areas of pituitary and brain appeared to be uneven. ~-Endorphin appeared to be the main peptide in the anterior pituitary, whereas in the neurointermediate lobe N~-acetyl-~-endorphin accounted for most of the e-endorphin immunoreactivity. In the brain, the highest concentration of ~-endorphin immunoreactivity was found in the hypothalamus. In hypothalamus and thalamus ~-endorphin predominated, whereas in amygdala, hippocampus and septum N~-acetyl-~-endorphin represented most of the ~-endorphin-immunoreactivity. In view of the nonopioid properties of acetylated endorphins, i t is suggested that acetylation represents a mechanism allowing the organism to specifically select the non-opioid behavioral activities enclosed in the endorphin sequence. Introduction ~- And y-endorphin have distinct effects on adaptive behavior in rats (1,2,3). The behavioral activities of these peptides are shared by their non-opioid desTyr I- and des-Enk-congener (2,3) indicating that they do not involve opiate sensitive structures in the brain. ~- And y-endorphin occur in pituitary and brain tissue (4,5). Their distribution in rat brain and pituitary closely parallels that of B-endorphin (5) their putative precursor molecule (6), Recently we have characterized N~-acetyl-y-endorphin as an endogenous neuropeptide in rat brain and pituitary (Wiegant et a l . , submitted). This peptide is devoid of opioid a c t i v i t y , but retains the behavioral activities of Y-type endorphins (Wiegant et a l . ; Van Ree et a l . , submitted). In the present communication we report the characterization of N~-acetyl-a-endorphin from rat neuro-intermediate lobe tissue and its distribution in pituitary and brain. Materials and Methods Male Wistar rats (180 - 200 g) were used. After decapitation of the animals the pituitary and the brain were quickly removed. The pituitary was dissected in anterior and neurointermediate lobe, the brain was dissected in anatomically defined regions (7). Tissues were immediately frozen on dry ice, Extraction of endorphins: Pooled tissues from 18 animals were heated for 10 min IB I M acetic acid (I : 10 w/v) in a boiling water bath and homogenized by ultrasound. The precipitates were removed by centrifugation and used for determination of tissue protein. The supernatants were lyophilized and the resulting residues dissolved in 1.0 ml 0.01M ammoniumacetate (pH 4.15). 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
126
~-endorphin in Pituitary and Brain
Vol. 33, Sup. I, 1983
Fractionation of peptides by HPLC: A f t e r clearing the extracts by c e n t r i f u g a t i o n , the-ywere f r a c t i o n a t e ~ - ~ P L C as described previously (8). In short, a ~Bondapak C18 column was used, with a 45 min concave gradient of 0.01M ammoniumacetate (pH 4.15; solvent A) and methanol ~solvent B). I n i t i a l conditions: A : B = 70 : 30, f i n a l conditions A : B = 25 : 75. Flow rate 2 ml/min. Fractions of 30 sec were collected, dried and reconstituted in 125 mM phosphate buffer (pH 7.5) containing 0.9% sodium chloride, 0.02% sodium azide and 0.25% bovine serum albumine (radioimmunoassay b u f f e r ) . Radioimmunoassay systems: ~-Endorphin immunoreactivity in HPLC-fractions was 'determined using an antiserum (A2) s p e c i f i c a l l y recognizing the free C-terminal Thr16-residue. The antiserum has low c r o s s - r e a c t i v i t y with ~-endorphin (2%) and B-endorphin (1%). lodinated ~-endorphin was used as a tracer. For f u r t h e r details on the procedure and s p e c i f i c i t y of the assay see 8. N~-acetylated-endorphins were assayed with antiserum Nancy Beth # 6 and iodinated N~-acetyl-y-endorphin as a tracer using the same methodology as for the ~endorphin radioimmunoassay (8). The antiserum s p e c i f i c a l l y recognizes the Noa c e t y l - T y r l - r e s i d u e of endorphins. No c r o s s - r e a c t i v i t y was observed with ~-, ~- or B-endorphin. Incubation with aminopeptidase-M: Aliquots of HPLC-fractions containing 5 ng ~-endorphin immunoreactivity were diluted to 112.5 ~I with 0 . 1 M Tris/HCl (pH 8.0). The incubation was started by the addition of 12.5 ul aminopeptidaseM solution (I ~g/10 ~ l l i n 0.9% sodium chloride/t0% g l y c e r o l ) , continued for 90 min at 37 ° C and terminated by d i l u t i o n with I ml 0.01M ammoniumacetate (pH 4.15). Samples were kept frozen u n t i l a second f r a c t i o n a t i o n by HPLC. Synthetic peptides were kindly donated by Drs. H.M. Greven and J. van Nispen (Organon International B.V., Oss, The Netherlands). N~-acetyl-~-endorphin was generated from synthetic N~-acetyl-~-endorphin by incubation with carboxypeptidase A and subsequently p u r i f i e d by HPLC. Purity and a u t h e n t i c i t y of the peptide were confirmed by i t s amino acid composition and resistance to aminopeptidase-M. The antiserum Nancy Beth # 6 was a generous g i f t from Dr. H. A k i l , Ann Arbor, MI, USA. Results The HPLC-chromatogram of rat neurointermediate lobe e x t r a c t showed two main peaks containing ~-endorphin immunoreactivity (Fig. IA). The f i r s t peak (fraction 53) eluted with the same retention time as synthetic ~-endorphin. The retention time of the second peak ( f r a c t i o n 65) was identical to that of synthet i c N~-acetyl-~-endorphin. Aliquots from f r a c t i o n 53 and 65 were assayed in serial d i l u t i o n s for ~-endorphin immunoreactivity. Both d i l u t i o n curves p a r a l l e l l e d the ~-endorphin standard curve (data not shown). Neither of the peaks was recognized by antisera raised against ~- or ~-endorphin (for s p e c i f i c i t y of these antisera see 81. An aliquot of f r a c t i o n 65 (equivalent to 5 ng ~-endorphin immunoreactivity) was incubated with aminopeptidase-M and subsequently fractionated by HPLC (Fig. 2A). Similar to synthetic N~-acetyl-~-endorphin, the ~-endorphin immunoreactivity in the sample appeared to be r e s i s t a n t to the enzyme, as more than 80% eluted with the same retention time as synthetic N~-acetyl-~-endorphin. These data indicate that the peptide in f r a c t i o n 65 containeda blocked N-terminus. The presence of an N~-acetyl-Tyrl is inferred from I) cross-reaction of the peptide with antiserum Nancy Beth, which s p e c i f i c a l l y recognizes N~-acetylated endorphins (sample d i l u t i o n curve p a r a l l e l l e d the standard curve; data not shown), and 2) cochromatography on HPLC with synthetic N~-acetyl-~-endorphin. I t was concluded that f r a c t i o n 65 contained N~-acetyl-~-endorphin. Incubation of an a l i q u o t (5 ng) from f r a c t i o n 53 with aminopeptidase-M resulted in complete disappearance of the ~-endorphin peak on HPLC (Fig. 2B). Only 15% of the immunoreactivity was recovered, e l u t i n g with the retention time of desTyr1-~-endorphin. Based on immunological c h a r a c t e r i s t i c s , co-elution with synt h e t i c ~-endorphin on HPLC and s e n s i t i v i t y f o r aminopeptidase-M i t was conclu-
Vol. 33, Sup. I, 1983
DTwE
=E
~-endorphin in Pituitary and Brain
127
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20
DT=.E
=,E
AcIE
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fraction number FIG. I: HPLC of ~-endorphin immunoreact i v i t y extracted from neurointermediate (A) and anterior lobe (B) of the p i t u i tary. Arrows indicate the elution position of synthetic peptides as determined in consecutive runs.
FIG. 2: HPLC of 5 ng ~-endorphin im-,i munoreactivity from neurointermediate lobe fraction 65 (A; cf. Fig. IA) and fraction 53 (B; cf. Fig IA) after incubation with aminopeptidase-M.
ded that fraction 53 represents non-acetylated ~-endorphin. The regional d i s t r i b u t i o n of the d i f f e r e n t forms of ~-endorphin immunoreactivity in p i t u i t a r y and brain was studied by HPLC fractionation of extracts of dissected areas and quantitation by radioimmunoassay with the A2-antiserum. In the neurointermediate lobe of the p i t u i t a r y ~-endorphin immunoreactivity was almost completely recovered as N~-acetyl -~-endorphin (Fig. I A ) ( r a t i o acetylated:non-acetylated = 10:1),whereas in the anterior lobe (Fig. IB)~-endQrphin appeared to be the predominant peptide (ratio I : 2,5; see also table I ) . Apart from ~- and N -acetyi-~-endorphin a minor peak eluting with retention time similar to desTyr1-~-endorphin was observed in the chromatograms. -Endorphin and NO-acetyl-~-endorphin were also detected in extracts of brain tissue. Marked differences in concentration of ~-endorphin immunoreactivity, as well as in the degree of acetylation, were observed among the various dissected areas of the brain (Table I ) . The highest amount of e-endorphin was found in the hypothalamus. In the hypothalamus and the thalamus, most of the peptides existed in the non-acetylated form (ratio acetylated :no~acetylated i : 5 and I : 2.5 resp.). In the other brain regions studied (septum, hippocampus, amygdala) the acetylated form appeared to be predominant (ratio > I ) . The amygdala showed the highest concentration of N~-acetyl-~-endorphin and the highest r a t i o of all brain areas analysed. Discussion In the present experiments we have i d e n t i f i e d N~-acetyl-m-endorphin as a na-
128
~-endorphin in Pituitary and Brain
Vol. 33, Sup. I, 1983
t u r a l l y occurring peptide in brain and p i t u i t a r y . I t appeared to be the main form of m-endorphin-immunoreactivity in the neuro-intermediate lobe of the pit u i t a r y , and in brain structures remote from hypothalamus such as amygdala, hippocampus and septum. Recently, we have characterized N'~-acetyl-y-endorphin as an endogenous neuropeptide (Wiegant et a l . , subm.). The d i s t r i b u t i o n of the acetylated and non-acetylated forms of m- and y-endorphin in brain and p i t u i t a r y parallels that of acetylated and non-acetylated 6-endorphins (5, 9, Wiegant et a l . , subm.), underscoring the suggested precursor function of 6-endorphin for m- and y-endorphins (6). Nm-acetylation completely destroys the opiate-like properties of y-endorphin and other endorphins (10). In Nm-acetyl-Y-endorphin the residual opioid a c t i v i t y of y-endorphin is l o s t , but the non-opioid behavioral a c t i v i t i e s of the peptide are retained (Wiegant et a l . , Van Ree et a l . , subm.). Preliminary data indicate that also in Nm-acetyl-m-endorphin the nonopioid behavioral properties of m-type endorphins are selectively conserved (Van Ree, unpublished). These obervations suggest that Nm-acetyl-m - and Nmacetyl-y-endorphin represent the naturally occurring congeners of the non-opioid behaviorally active 6-endorphin fragments des-Tyrl-m- and des-Tyr1-y-endorphin. Thus, Nm-acetylation of endorphins represents not merely a mechanism for inactivation of opioid a c t i v i t e s , but may also allow the organism to select spec i f i c sets of biological a c t i v i t i e s present in the endorphins sequence. The biological significance of acetylated 6-endorphins may ly in t h e i r precursor function for these NO-acetylated psychoactive peptides. TABLE I Distribution of m- and N%Acetyl -m-Endorphin tissue protein (mg) anterior lobe neurointermediate lobe hypothalamus thalamus septum hippocampus amygdala
.830 .084 3.72 5.38 1.04 6.26 6.51
m-endorphin (pg)
in Rat P i t u i t a r y and Brain Nm-acetyl-m - approx, r a t i o endorphin(pg) acetyl/non-acetyl
13,000 5,000
4,700 51,200
0.4 10.2
547.0 186.3 26.4 21.5 61.3
99.3 80.0 34.4 31.7 122.8
0.2 0.4 1.3 1.5 2.0
Extracts of dissected areas were fractionated by HPLC. Peptide content of the fractions was determined by ~-endorphin radioimmunoassay. Values represent endorphin content per tissue per animal. References I. D. DE WIED, B. BOHUS, J.M. VAN REE and I. URBAN, J. Pharmacol. exp. Ther. 204 570-580 (1978). 2. D. DE WIED, G.L. KOVACS, B. BOHUS, J.M. VAN REE and H.M. GREVEN, Eur. J. Pharmacol. 49 427-436 (1978). 3. H.M. GREVEN and D. DE WIED, Hormones and the Brain (D. de Wied and P.A. van Keep, edso), pp. 115-127, MTP Press, Lancaster (1980) 4. J. VERHOEF, J.G. LOEBER, J.P.H. BURBACH, W.H. GISPEN, A. WITTER and D. DE WIED, Life Sci. 26 851-859 (1980). 5. J. VERHOEF, V.M.~IEGANT and D. DE WIED, Brain Res. 231 454-460 (1982). 6. J.P.H. BURBACH, E.R. DE KLOET, P. SCHOTMANand D. DE~ED, J. Biol. Chem. 256 12463-12469 (1981). 7. I~TTI~. GISPEN, P. SCHOTMANand E.R. DE KLOET, Neuroendocr. 9 285-296 (1972). 8. J.G. LOEBER, J. VERHOEF, J.P.H. BURBACHand A. WITTER, Bi~chem. Biophys. Res. Commun. 86 1288-1295 (1979). 9. S. ZAKARIAN a N D.G. SMYTH, Nature 296 250-252 (1982). 10. J.F.Wo DEAKIN, J.O. DOSTROVSKYand D--T~.. SMYTH, Biochem. J. 189 501-506 (1980).