- Email: [email protected]
Opioid peptides and opiates differ in receptor selectivity
P~ychoneuroendocrinology, 1977, Vol. 2, pp. 53-58. Pergamon Press. Printed in Great Britain
OPIOID PEPTIDES A N D OPIATES DIFFER IN RECEPTOR SELECTIVITY LARS TERENIUS Department of Medical Pharmacology, University of Uppsala, Box 573, S-751 23 Uppsala, Sweden SUMMARY (1) The binding properties of opioid receptors in whole rat cerebrum have been studied with tritium-labelled dihydromorphine, naltrexone and Leu-enkephalin as radioindicators and various opioid peptides and opiates as competitors. (2) Dihydromorphine shows high affinity binding to sites which are competitively blocked by various opiate agonists and antagonists (DHM sites). (3) Naltrexone binds to additional sites which also strongly bind other narcotic antagonists (NAL sites). (4) The morphinomimetic enkephalins show high selectivity for DHM sites while ACTH fragments and somatostatin show less selectivity. (5) Leu-enkephalin appears to bind to separate sites (EKN sites) with similar affinity to that for DHM sites. (6) Both dihydromorphine and naltrexone show higher affinity for DHM sites than for EKN sites. (7) In conclusion, at least three kinds of opioid binding sites are observable. These differences in receptor populations may relate to functional differences.
Key Words---Opioid; opioid receptor; morphine; naltrexone; enkephalin.
INTRODUCTION FOLLOWING the original observation that opiates bind to specific receptors in the C N S (Pert & Snyder, 1973; Simon, Hiller & Edelman, 1973; Terenius, 1973) a considerable a m o u n t o f w o r k has been directed to the question o f whether natural ligands for these receptors exist. Such ligands, previously u n k n o w n with regard to interaction with the opiate receptors, have n o w been characterized, and surprisingly found to be low-molecular weight peptides (Hughes, 1975; Terenius & Wahlstr6m, 1975). Hughes, Smith, Kosterlitz, Fothergill, M o r g a n & Morris (1975) recently succeeded in isolating, identifying and synthesizing two related pentapeptides, the enkephalins, with opiate receptor affinity. This p r o m p t e d us to prepare labelled Leu-enkephalin and to c o m p a r e its receptor binding properties with those o f the narcotic agonist, dihydromorphine and the narcotic antagonist, naltrexone. MATERIALS A N D METHODS
Drugs Tritium-labelled dihydromorphine (DHM) at 75 Ci/mmole was obtained from the Radiochemical Centre, Amersham, England. Tritium-labelled naltrexone at 16 Ci/mmole was a gift from Dr. R. Willette, NIMH, Rockville, Md., U.S.A. Leu-enkephalin-3H was prepared in this laboratory from [3,5-diiodo-Tyr(1), leucine(5)]-enkephalin which was obtained from Dr. U. Ragnarsson, Dept. of Biochemistry, University of Uppsala. The iodine atoms were replaced by tritium in a catalysed exchange procedure. Labile tritium was removed and the reaction mixture was chromatographed on a silica gel column (Partisil 10; 20 × 0.45 cm) in a high-pressure liquid chromatography apparatus. The eluant was butanol-acetic acid-water (4:1:1 v/v). Analytical thin-layer chromatography in several systems gave a radiochemical purity of > 98 ~o. The labelled compound also behaved chromatographically as Leu-enkephalin on gel filtration and it was specifically absorbed by antibodies raised against Leu-enkephalin (Elde, HOkfelt, Johansson & Terenius, 1976). Its specific activity was 44 Ci/mmole. 53
54
LARS TERENIUS
Unlabelled dihydromorphine was prepared by the author by catalytic reduction of morphine, unlabelled naloxone was a gift from Endo Labs, and Leu-enkephalin was synthesized here (Terenius, Wahlstr6m, Lindeberg, Karlsson & Ragnarsson, 1976).
Chromatographic identification of radioactivity derived from Leu-enkephalin during incubation Leu-enkephalin was found to be metabolically unstable when incubated with synaptic plasma membrane preparations. Incubation medium (3-5 ml) with labelled peptide which had been exposed to these membrane preparations was heated to 100°C for 15 rain. The medium was cooled to room temperature and run through a Sephadex G10 column (Terenius & Wahlstr6m, 1975) eluted in 0.2 M HAc. The fractions of radioactivity eluted later than the majority of inorganic salts. Thus freeze-drying yielded an essentially salt-free residue to which cold Leu-enkephalin, all possible tyrosine-containing fragments and tyrosine were added in excess before the mixture was spotted onto analytical silica gel thin-layer plates (Fertigplatten, Merck, Darmstadt). The plates were developed in butanol-acetic acid-water (4:1 : 1 v/v) which separates Leu-enkephalin and all possible tyrosine containing peptide fragments completely. After development, the plates were weakly stained with ninhydrin, then the silica layer was scraped down into scintillation vials, eluted and counted for tritium activity.
Tests for receptor affinity The experimental procedure for testing receptor affinity has been described elsewhere (Terenius, 1974). A synaptic plasma membrane fraction of rat cerebrum is incubated with a tritium-labelled opioid. Incubation is terminated by centrifugation in the cold, which separates bound opioid (pellet) from free (supernatant). All incubations are run in a near-physiologic buffer, pH 7.4, at 25°C. The incubation time varied and is given in legends to each separate experiment. Several experiments were aimed at comparing the inhibiting activity of an unlabelled compound against two different labelled drugs (the radioindicators). To maximize the resolving capacity of such analysis, incubations were run in chemically identical solutions, but with different radioindicators. This will result in an identical indicator error with each solution. An example: the competitive affinities of various peptides were tested against dihydromorpbine-aH and naltrexone-3H. Then, two indicator solutions were prepared, (a) dihydromorphine-3H, 0.36 nM + naltrexone, 0.21 riM, (b) dihydromorphine, 0.36 nM + naltrexone-3H, 0.21 riM. Each experimental run included samples without competitor (controls) and those with a large excess (10 -6 M) of nonlabelled counterpart of the radioindicator. Binding in the presence of this excess was not considered and was subtracted from all experimental values. The effect of various competitors was expressed in percent of the corrected control value and the concentrations giving 50 ~o binding inhibition (ICjo) were estimated graphically. Scatchard analysis (Scatchard, 1949) was done using increasing concentrations of labelled Leu-enkephalin. In the calculations of dissociation constants, corrections were made for nonspecific binding remaining in the presence of 10 -6 M carrier (cf. Chamness & McGuire, 1975).
RESULTS When different opioids were tested for competitive affinity against dihydromorphine-3H or naltrexone-3H it became apparent that differences in relative affinities are present. Table TABLE I. SELECTIVITY OF VARIOUS AGENTS FOR OPIOID RECEPTORS
Agent
IC~o (riM) against DHM
Met-enkephalin* Somatostatin ACTHI_2, Dihydromorphine* Naltrexone*
NAL
17 460 10,000 50,000 30,000 60,000 4.0 150 0.20 0.22
ICso ] ICso NAL [ D H M 27 5 2 38 1.1
The incubation solutions were: (DHM), dihydromorphine-3H, 0.36 nM + naltrexone 0.21 riM; (NAL), dihydromorphine, 0.36 n M + naltrexone3H 0.21 riM. Incubations were run at 25°C for 40 rain. * Data from Terenius & Wahlstr6m (1976).
OPIOID RECEPTORSELECTIVITY
55
I summarizes the results with some peptides as competitors and compares them with those obtained with morphine or naltrexone. Apparently, Met-enkephalin is very similar to dihydromorphine in its receptor selectivity, while somatostatin shows lower selectivity, and the ACTH fragment shows very low selectivity approaching that of the almost unselective naloxone. A similar study of relative affinities was attempted comparing the results on incubation with Leu-enkephalin-3H and dihydromorphine-3H. Studies with the labelled enkephalin were obscured by the rapid metabolism. After I0 min of incubation of 0.3 nM Leu-enkephalin with synaptic plasma membrane fractions at 25°C, 45 ~o was intact substance, while after 40 min only 25 ~ remained in the enkephalin fraction. However, the specific fraction of total bound radioactivity remained essentially the same during an incubation period of 5-20 min. A 10-min incubation period was chosen for all subsequent experiments. A Scatchard plot of the binding of Leu-enkephalin to synaptic plasma membranes revealed two components (Fig. 1). Addition of an excess of non-labelled Leu-enkephalin, 10-6M final concentration, saturated the high affinity sites. After correction for the nonspecific binding occurring with this excess of carrier, only the high-affinity site was discernible. The corrected dissociation constants in two experiments were 0.21 × 10-9M and 0.30 × 10-9M, respectively. The following experiments were designed to test whether various opiates could inhibit binding of the labelled Leu-enkephalin (Table II). All active narcotics inhibited binding, albeit at rather high concentrations. The inhibition was stereospecific, since the levo-isomer of each antipodal pair showed the highest inhibitory potency. The high activity of levorphanol and the inactivity of dextrorphan is particularly striking. Finally, an investigation was performed to determine whether various agents would preferentially inhibit the binding of labelled dihydromorphine or labelled Leu-enkephalin. It was found that dihydromorphine or naloxone showed comparatively poor inhibiting activity against Leu-enkephalin-3H, but were much more efficient against dihydromorphine-3H. Leu-enkephalin, on the other hand, was about equally effective against both radioindicators (Fig. 2). Similar results were obtained with Met-enkephalin (not shown). 0.04 0.03 LJ.
o.oz
=_, . . . . . . . . . . . . . .
e....~..~.
0.01
0 0
1000
2OO0
3000
4OOO
I 5O00
! 6000
B (DPM)
FIG. 1. Scatchard analysis of the binding of tritium-labelled Leu-cnkcphalin to rat brain synaptic pl~qma membranes. B = bound, F = free substance. Closed circles were obtained in the presence of 10 - e M nonlabelled substance.
56
LAPS TEREN1US TABLE II. EFFECT OF
OPIATES O N L E U - E N K E P H A L I N - 3 H BINDING TO OPIOID RECEPTORS
Opiate
Concentration (m)
levo-Methadone
~oremaining binding
10 -a 10 -7 10- " 10 -7 10- s 10-7 10- s 10-7
dextro-Methadone Levorphanol Dextrorphan
71 64 99 75 67 37 102 100
Incubation was carried out for 10 min at 25°C. Values are per cent of a control (corrected for nonspecific binding, see Methods) without competitors. There were 3-4 samples in each group. DISCUSSION A current trend in neurobiology is to find biochemical correlates to functional parameters. Such analysis is also being attempted in the field o f natural and synthetic opioids. The first step in the action o f these molecules is to combine with a receptor. I n biochemical terms such a receptor is characterized by its ability to specifically bind the proper agents and no others. Based on their structural selectivity in competition analysis, specific opioid receptors have been f o u n d to exist in the C N S (Pert & Snyder, 1973; Simon et aL, 1973; Terenius, 1973). It soon became apparent, however, that somewhat different results were obtained, dependent u p o n whether a narcotic agonist, such as d i h y d r o m o r p h i n e or etorphine, or a narcotic antagonist, such as naloxone or naltrexone, were used as radioactive indicator drugs (Pert & Snyder, 1974). This is also illustrated in Table I. Studies o f a large n u m b e r o f narcotic agents in the competition assays showed a correlation between binding
I00
Z u.I i
o
80
~ 6o "~ 40 i
8 '~
2O
10
I
40
I
60
% of c o n t r o l
I
80
I00
OHM
FIG. 2. Selectivity of various substances for opioid receptors using dihydromorphine (DHM) or Leu-enkephalin (Leu-EKN) as radioindicators. The incubation solutions were: (DHM) dihydromorphine-3H, 0.24 n M + Leu-enkephalin, 0.30 riM; (Leu-EKN), dihydromorphine, 0.24 n M + Leu-enkephalin-3H, 0.30 riM. The following competitors were tested: © © Dihydromorphine 10-9, 10-s M; /k A Naloxone 3 × 10 -1°, 3 × 10-9 M; • • Leu-enkephalin 3 × 10-9, 3 × 10 -s i.
OPIOID RECEPTORSELECTIVITY
57
properties and pharmacological profile, that is, namotic agonism versus narcotic antagonism. One would, for instance from data in Table I, expect A C T H I _ 2 , to be a narcotic antagonist. In fact, A C T H fragments do show this property (Gispen, Buitelaar, Wiegant, Terenius & de Wied, 1976). It has been postulated that opioid receptors can exist in two forms, one specifically binding narcotic agonists, the other narcotic antagonists (Snyder, 1975). Since the natural opioids occurring in the mammalian nervous system are peptides and consequently chemically very different from natural or synthetic opiates, it became of interest to evaluate their interaction with opioid receptors and to compare them with those of the opioids. Direct incubation with Leu-enkephalin-3H revealed a high-affinity site (Fig. 1). Interestingly, when this binding is analysed by competition techniques and compared with the binding of dihydromorphine-3H, evidence for heterogeneity is present (Fig. 2). Thus, dihydromorphine and naloxone are poor inhibitors of Leu-enkephalin-3H binding. This can only be explained if the receptor population is heterogenous and if Leuenkephalin differs in selectivity between these sites. The functions of these additional sites, revealed by using Leu-enkephalin-aH as the radioindicator, are not known at the present time. In analogy with the results described above on agonist-antagonist discrimination, it is tempting to assume that the difference may play a functional role. One interesting property of the Leu-enkephalin sites will be their relative insensitivity to naloxone. I am grateful to Mr. L. Johansson and Mrs. I. Eriksson for experimental assistance. The work was supported by the Swedish Medical Research Council (B76-25X-03766-05). REFERENCES CnA~,W.SS, G. C. & McG~Rr, W. L. (1975) Scatchard plots: common errors in correction and interpretation. Steroids 26, 538-542. ELDE, R., H6KVELT,T., JOnANSSON,O. & TEmENIUS,L. (1976) Immunohistochemical studies using antibodies to leucine-enkephalin: initial observations on the nervous system of the rat. Neuroscience 1, 349-351. GtSPEN, W. H., BLrrr~LAAR,J., WmGANT,V., TERENrUS,L. & DEWIND,D. (1976) Interaction between ACTH fragments, brain opiate receptors and morphine induced analgesia. Eur. J. Pharmacol. (in press). HUGHES,J. (1975) Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine. Brain Res. 88, 295-308. HUGHES,J., SMITH,T. W., KOSTERLITZ,H. W., FOTHERGILL,L. A., MORGAN,B. A. & Monms, H. R. (1975) Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature, Lond. 258, 577-579. PERT, C. B. & SNYDER,S. H. (1973) Opiate receptors: demonstration in nervous tissue. Science 179, 10111014.
PERT, C. B. & SNYDER,S. H. (1974) Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol. Pharmacol. 10, 868-879. SCATCHARD,G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660--672. SIMON,E. J., HILLER,J. M. & EDELMAN,I. (1973) Stereospecific binding of the potent narcotic analgesic 3H--etorphine to rat-brain homogenate. Proc. natn. Acad. Sci. 70, 1947-1949. SNYDER,S. H. (1975) Opiate receptor in normal and drug altered brain function. Nature, Lond. 257, 185-189. TEREN~S, L. (1973) Characteristics of the "receptor" for narcotic analgesics in synaptic plasma membrane fraction from rat brain. Actapharm. toxieoL 33, 377-384. TERENmS,L. (1974) A rapid assay for the narcotic receptor in rat brain: application to methadone analogues. Acta pharm, toxicol. 34, 88-91.
58
LAPS TERENIUS
TE~N~.% L. & WAHLSTR6M,A. (1975) Search for an endogenous ligand for the opiate receptor. Acre physiol, scand. 94, 74-81.
TEREr,a-OS,L. & WAmSTR6M, A. (1976) A method for site selectivity analysis applied to opiate receptors. Eur. J. Pharmacol. (in press). TERENIUS,L., WAm.STR6~, A., LII,a)EeERG, G., KAa~SON, S. & RAGNARSSON,U. (1976) Opiate receptor affinity of peptides related to leu-enkephalin. Biochem. Biophys. Res. Communs 71, 175-179.
OPIOID PEPTIDES A N D OPIATES DIFFER IN RECEPTOR SELECTIVITY LARS TERENIUS Department of Medical Pharmacology, University of Uppsala, Box 573, S-751 23 Uppsala, Sweden SUMMARY (1) The binding properties of opioid receptors in whole rat cerebrum have been studied with tritium-labelled dihydromorphine, naltrexone and Leu-enkephalin as radioindicators and various opioid peptides and opiates as competitors. (2) Dihydromorphine shows high affinity binding to sites which are competitively blocked by various opiate agonists and antagonists (DHM sites). (3) Naltrexone binds to additional sites which also strongly bind other narcotic antagonists (NAL sites). (4) The morphinomimetic enkephalins show high selectivity for DHM sites while ACTH fragments and somatostatin show less selectivity. (5) Leu-enkephalin appears to bind to separate sites (EKN sites) with similar affinity to that for DHM sites. (6) Both dihydromorphine and naltrexone show higher affinity for DHM sites than for EKN sites. (7) In conclusion, at least three kinds of opioid binding sites are observable. These differences in receptor populations may relate to functional differences.
Key Words---Opioid; opioid receptor; morphine; naltrexone; enkephalin.
INTRODUCTION FOLLOWING the original observation that opiates bind to specific receptors in the C N S (Pert & Snyder, 1973; Simon, Hiller & Edelman, 1973; Terenius, 1973) a considerable a m o u n t o f w o r k has been directed to the question o f whether natural ligands for these receptors exist. Such ligands, previously u n k n o w n with regard to interaction with the opiate receptors, have n o w been characterized, and surprisingly found to be low-molecular weight peptides (Hughes, 1975; Terenius & Wahlstr6m, 1975). Hughes, Smith, Kosterlitz, Fothergill, M o r g a n & Morris (1975) recently succeeded in isolating, identifying and synthesizing two related pentapeptides, the enkephalins, with opiate receptor affinity. This p r o m p t e d us to prepare labelled Leu-enkephalin and to c o m p a r e its receptor binding properties with those o f the narcotic agonist, dihydromorphine and the narcotic antagonist, naltrexone. MATERIALS A N D METHODS
Drugs Tritium-labelled dihydromorphine (DHM) at 75 Ci/mmole was obtained from the Radiochemical Centre, Amersham, England. Tritium-labelled naltrexone at 16 Ci/mmole was a gift from Dr. R. Willette, NIMH, Rockville, Md., U.S.A. Leu-enkephalin-3H was prepared in this laboratory from [3,5-diiodo-Tyr(1), leucine(5)]-enkephalin which was obtained from Dr. U. Ragnarsson, Dept. of Biochemistry, University of Uppsala. The iodine atoms were replaced by tritium in a catalysed exchange procedure. Labile tritium was removed and the reaction mixture was chromatographed on a silica gel column (Partisil 10; 20 × 0.45 cm) in a high-pressure liquid chromatography apparatus. The eluant was butanol-acetic acid-water (4:1:1 v/v). Analytical thin-layer chromatography in several systems gave a radiochemical purity of > 98 ~o. The labelled compound also behaved chromatographically as Leu-enkephalin on gel filtration and it was specifically absorbed by antibodies raised against Leu-enkephalin (Elde, HOkfelt, Johansson & Terenius, 1976). Its specific activity was 44 Ci/mmole. 53
54
LARS TERENIUS
Unlabelled dihydromorphine was prepared by the author by catalytic reduction of morphine, unlabelled naloxone was a gift from Endo Labs, and Leu-enkephalin was synthesized here (Terenius, Wahlstr6m, Lindeberg, Karlsson & Ragnarsson, 1976).
Chromatographic identification of radioactivity derived from Leu-enkephalin during incubation Leu-enkephalin was found to be metabolically unstable when incubated with synaptic plasma membrane preparations. Incubation medium (3-5 ml) with labelled peptide which had been exposed to these membrane preparations was heated to 100°C for 15 rain. The medium was cooled to room temperature and run through a Sephadex G10 column (Terenius & Wahlstr6m, 1975) eluted in 0.2 M HAc. The fractions of radioactivity eluted later than the majority of inorganic salts. Thus freeze-drying yielded an essentially salt-free residue to which cold Leu-enkephalin, all possible tyrosine-containing fragments and tyrosine were added in excess before the mixture was spotted onto analytical silica gel thin-layer plates (Fertigplatten, Merck, Darmstadt). The plates were developed in butanol-acetic acid-water (4:1 : 1 v/v) which separates Leu-enkephalin and all possible tyrosine containing peptide fragments completely. After development, the plates were weakly stained with ninhydrin, then the silica layer was scraped down into scintillation vials, eluted and counted for tritium activity.
Tests for receptor affinity The experimental procedure for testing receptor affinity has been described elsewhere (Terenius, 1974). A synaptic plasma membrane fraction of rat cerebrum is incubated with a tritium-labelled opioid. Incubation is terminated by centrifugation in the cold, which separates bound opioid (pellet) from free (supernatant). All incubations are run in a near-physiologic buffer, pH 7.4, at 25°C. The incubation time varied and is given in legends to each separate experiment. Several experiments were aimed at comparing the inhibiting activity of an unlabelled compound against two different labelled drugs (the radioindicators). To maximize the resolving capacity of such analysis, incubations were run in chemically identical solutions, but with different radioindicators. This will result in an identical indicator error with each solution. An example: the competitive affinities of various peptides were tested against dihydromorpbine-aH and naltrexone-3H. Then, two indicator solutions were prepared, (a) dihydromorphine-3H, 0.36 nM + naltrexone, 0.21 riM, (b) dihydromorphine, 0.36 nM + naltrexone-3H, 0.21 riM. Each experimental run included samples without competitor (controls) and those with a large excess (10 -6 M) of nonlabelled counterpart of the radioindicator. Binding in the presence of this excess was not considered and was subtracted from all experimental values. The effect of various competitors was expressed in percent of the corrected control value and the concentrations giving 50 ~o binding inhibition (ICjo) were estimated graphically. Scatchard analysis (Scatchard, 1949) was done using increasing concentrations of labelled Leu-enkephalin. In the calculations of dissociation constants, corrections were made for nonspecific binding remaining in the presence of 10 -6 M carrier (cf. Chamness & McGuire, 1975).
RESULTS When different opioids were tested for competitive affinity against dihydromorphine-3H or naltrexone-3H it became apparent that differences in relative affinities are present. Table TABLE I. SELECTIVITY OF VARIOUS AGENTS FOR OPIOID RECEPTORS
Agent
IC~o (riM) against DHM
Met-enkephalin* Somatostatin ACTHI_2, Dihydromorphine* Naltrexone*
NAL
17 460 10,000 50,000 30,000 60,000 4.0 150 0.20 0.22
ICso ] ICso NAL [ D H M 27 5 2 38 1.1
The incubation solutions were: (DHM), dihydromorphine-3H, 0.36 nM + naltrexone 0.21 riM; (NAL), dihydromorphine, 0.36 n M + naltrexone3H 0.21 riM. Incubations were run at 25°C for 40 rain. * Data from Terenius & Wahlstr6m (1976).
OPIOID RECEPTORSELECTIVITY
55
I summarizes the results with some peptides as competitors and compares them with those obtained with morphine or naltrexone. Apparently, Met-enkephalin is very similar to dihydromorphine in its receptor selectivity, while somatostatin shows lower selectivity, and the ACTH fragment shows very low selectivity approaching that of the almost unselective naloxone. A similar study of relative affinities was attempted comparing the results on incubation with Leu-enkephalin-3H and dihydromorphine-3H. Studies with the labelled enkephalin were obscured by the rapid metabolism. After I0 min of incubation of 0.3 nM Leu-enkephalin with synaptic plasma membrane fractions at 25°C, 45 ~o was intact substance, while after 40 min only 25 ~ remained in the enkephalin fraction. However, the specific fraction of total bound radioactivity remained essentially the same during an incubation period of 5-20 min. A 10-min incubation period was chosen for all subsequent experiments. A Scatchard plot of the binding of Leu-enkephalin to synaptic plasma membranes revealed two components (Fig. 1). Addition of an excess of non-labelled Leu-enkephalin, 10-6M final concentration, saturated the high affinity sites. After correction for the nonspecific binding occurring with this excess of carrier, only the high-affinity site was discernible. The corrected dissociation constants in two experiments were 0.21 × 10-9M and 0.30 × 10-9M, respectively. The following experiments were designed to test whether various opiates could inhibit binding of the labelled Leu-enkephalin (Table II). All active narcotics inhibited binding, albeit at rather high concentrations. The inhibition was stereospecific, since the levo-isomer of each antipodal pair showed the highest inhibitory potency. The high activity of levorphanol and the inactivity of dextrorphan is particularly striking. Finally, an investigation was performed to determine whether various agents would preferentially inhibit the binding of labelled dihydromorphine or labelled Leu-enkephalin. It was found that dihydromorphine or naloxone showed comparatively poor inhibiting activity against Leu-enkephalin-3H, but were much more efficient against dihydromorphine-3H. Leu-enkephalin, on the other hand, was about equally effective against both radioindicators (Fig. 2). Similar results were obtained with Met-enkephalin (not shown). 0.04 0.03 LJ.
o.oz
=_, . . . . . . . . . . . . . .
e....~..~.
0.01
0 0
1000
2OO0
3000
4OOO
I 5O00
! 6000
B (DPM)
FIG. 1. Scatchard analysis of the binding of tritium-labelled Leu-cnkcphalin to rat brain synaptic pl~qma membranes. B = bound, F = free substance. Closed circles were obtained in the presence of 10 - e M nonlabelled substance.
56
LAPS TEREN1US TABLE II. EFFECT OF
OPIATES O N L E U - E N K E P H A L I N - 3 H BINDING TO OPIOID RECEPTORS
Opiate
Concentration (m)
levo-Methadone
~oremaining binding
10 -a 10 -7 10- " 10 -7 10- s 10-7 10- s 10-7
dextro-Methadone Levorphanol Dextrorphan
71 64 99 75 67 37 102 100
Incubation was carried out for 10 min at 25°C. Values are per cent of a control (corrected for nonspecific binding, see Methods) without competitors. There were 3-4 samples in each group. DISCUSSION A current trend in neurobiology is to find biochemical correlates to functional parameters. Such analysis is also being attempted in the field o f natural and synthetic opioids. The first step in the action o f these molecules is to combine with a receptor. I n biochemical terms such a receptor is characterized by its ability to specifically bind the proper agents and no others. Based on their structural selectivity in competition analysis, specific opioid receptors have been f o u n d to exist in the C N S (Pert & Snyder, 1973; Simon et aL, 1973; Terenius, 1973). It soon became apparent, however, that somewhat different results were obtained, dependent u p o n whether a narcotic agonist, such as d i h y d r o m o r p h i n e or etorphine, or a narcotic antagonist, such as naloxone or naltrexone, were used as radioactive indicator drugs (Pert & Snyder, 1974). This is also illustrated in Table I. Studies o f a large n u m b e r o f narcotic agents in the competition assays showed a correlation between binding
I00
Z u.I i
o
80
~ 6o "~ 40 i
8 '~
2O
10
I
40
I
60
% of c o n t r o l
I
80
I00
OHM
FIG. 2. Selectivity of various substances for opioid receptors using dihydromorphine (DHM) or Leu-enkephalin (Leu-EKN) as radioindicators. The incubation solutions were: (DHM) dihydromorphine-3H, 0.24 n M + Leu-enkephalin, 0.30 riM; (Leu-EKN), dihydromorphine, 0.24 n M + Leu-enkephalin-3H, 0.30 riM. The following competitors were tested: © © Dihydromorphine 10-9, 10-s M; /k A Naloxone 3 × 10 -1°, 3 × 10-9 M; • • Leu-enkephalin 3 × 10-9, 3 × 10 -s i.
OPIOID RECEPTORSELECTIVITY
57
properties and pharmacological profile, that is, namotic agonism versus narcotic antagonism. One would, for instance from data in Table I, expect A C T H I _ 2 , to be a narcotic antagonist. In fact, A C T H fragments do show this property (Gispen, Buitelaar, Wiegant, Terenius & de Wied, 1976). It has been postulated that opioid receptors can exist in two forms, one specifically binding narcotic agonists, the other narcotic antagonists (Snyder, 1975). Since the natural opioids occurring in the mammalian nervous system are peptides and consequently chemically very different from natural or synthetic opiates, it became of interest to evaluate their interaction with opioid receptors and to compare them with those of the opioids. Direct incubation with Leu-enkephalin-3H revealed a high-affinity site (Fig. 1). Interestingly, when this binding is analysed by competition techniques and compared with the binding of dihydromorphine-3H, evidence for heterogeneity is present (Fig. 2). Thus, dihydromorphine and naloxone are poor inhibitors of Leu-enkephalin-3H binding. This can only be explained if the receptor population is heterogenous and if Leuenkephalin differs in selectivity between these sites. The functions of these additional sites, revealed by using Leu-enkephalin-aH as the radioindicator, are not known at the present time. In analogy with the results described above on agonist-antagonist discrimination, it is tempting to assume that the difference may play a functional role. One interesting property of the Leu-enkephalin sites will be their relative insensitivity to naloxone. I am grateful to Mr. L. Johansson and Mrs. I. Eriksson for experimental assistance. The work was supported by the Swedish Medical Research Council (B76-25X-03766-05). REFERENCES CnA~,W.SS, G. C. & McG~Rr, W. L. (1975) Scatchard plots: common errors in correction and interpretation. Steroids 26, 538-542. ELDE, R., H6KVELT,T., JOnANSSON,O. & TEmENIUS,L. (1976) Immunohistochemical studies using antibodies to leucine-enkephalin: initial observations on the nervous system of the rat. Neuroscience 1, 349-351. GtSPEN, W. H., BLrrr~LAAR,J., WmGANT,V., TERENrUS,L. & DEWIND,D. (1976) Interaction between ACTH fragments, brain opiate receptors and morphine induced analgesia. Eur. J. Pharmacol. (in press). HUGHES,J. (1975) Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine. Brain Res. 88, 295-308. HUGHES,J., SMITH,T. W., KOSTERLITZ,H. W., FOTHERGILL,L. A., MORGAN,B. A. & Monms, H. R. (1975) Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature, Lond. 258, 577-579. PERT, C. B. & SNYDER,S. H. (1973) Opiate receptors: demonstration in nervous tissue. Science 179, 10111014.
PERT, C. B. & SNYDER,S. H. (1974) Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol. Pharmacol. 10, 868-879. SCATCHARD,G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660--672. SIMON,E. J., HILLER,J. M. & EDELMAN,I. (1973) Stereospecific binding of the potent narcotic analgesic 3H--etorphine to rat-brain homogenate. Proc. natn. Acad. Sci. 70, 1947-1949. SNYDER,S. H. (1975) Opiate receptor in normal and drug altered brain function. Nature, Lond. 257, 185-189. TEREN~S, L. (1973) Characteristics of the "receptor" for narcotic analgesics in synaptic plasma membrane fraction from rat brain. Actapharm. toxieoL 33, 377-384. TERENmS,L. (1974) A rapid assay for the narcotic receptor in rat brain: application to methadone analogues. Acta pharm, toxicol. 34, 88-91.
58
LAPS TERENIUS
TE~N~.% L. & WAHLSTR6M,A. (1975) Search for an endogenous ligand for the opiate receptor. Acre physiol, scand. 94, 74-81.
TEREr,a-OS,L. & WAmSTR6M, A. (1976) A method for site selectivity analysis applied to opiate receptors. Eur. J. Pharmacol. (in press). TERENIUS,L., WAm.STR6~, A., LII,a)EeERG, G., KAa~SON, S. & RAGNARSSON,U. (1976) Opiate receptor affinity of peptides related to leu-enkephalin. Biochem. Biophys. Res. Communs 71, 175-179.