Brain Opiate Receptors Research Articles

Agonist—antagonist properties of phenoxybenzamine in antinociception and opiate dependence tests

The doe—response curve for phenoxybenzamine inhibition of stereospecific 3H-naloxone binding in brain homogenates was found to shift to the right in the presence of 100 mM NaCl. From the ratios of the IC 50 in the presence of Na + to the IC 50 in the presence of Na + calculated in this study for phenoxybenzine and some opiates it was predicted that phenoxybenzamine would have mixed agonist—antagonist properties in antinociceoption and that its antagonist activity would be in the rank order: butorphanol (1.8) ⪢ phenoxybenzamine (4.3) > M2230 (5.7) > pentazocine (6.9) ⪢ levorphanol (13.6). Phenoxybenzamine antagonized the oxymorphone-induced Straub tail reaction in mice and induced the abstinence syndrome in non-withdrawn morpine-dependent mice as predicted by the Na + response ratio. In the mouse tail flick assay phenoxybenzamine acted like the mized agonist—antagonist benzomorphinas as predicted. In “physical dependence capabilty” phenoxybenzamine was intermediate between butorphanol and pentazocine as predicted by the Na + response ratios. The success of the Na + response ratio derived from phenoxybenzamine's properties in the vitro opiate binding assay in predicting the behavior and rank order of phenoxybenzamine in in vivo tests, plus the ability of naloxone to antagonize phenoxybenzamine's antinociceptive action suggest that phenoxybenzamine's antinociceptive action may be mediated by the brain opiate receptor rather than by central α-adrenergic receptor blockade.

Pharmacological and immunological characterization of the LEU 5 analogue of human β-endorphin

The potencies of β h-endorphin, Met (O) 5- β h-endorphin, and syntheticLeu 5- β h-endorphin have been compared in three bioassays of opioid activity, and in two radioimmunoassays. In all assays, a peptide isolated from hemodialysates from a psychotic patient behaved likeLeu 5- β h-endorphin; it has been distinguished unambiguously from β h-endorphin and Met(O) 5- β h-endorphin.Leu 5- β h-endorphin was one-fifth as potent as β h-endorphin in guinea pig ileum myenteric plexus, but was only slightly less active in mouse vas deferens and in guinea pig brain opiate receptor binding assay. The low cross-reactivity ofLeu 5- β h-endorphin relative to β h-endorphin with an antiserum raised to β-endorphin suggests that the preferred solution conformations of these peptides are different. In all bioassays β h-endorphin was 2- to 3-fold less potent than β c-endorphin.

Regional distribution of a high-affinity enkephalin-degrading peptidase (‘enkephalinase’) and effects of lesions suggest localization in the vicinity of opiate receptors in brain

The distribution of a high affinity enkephalin-dipeptidylcarboxypeptidase between regions of mouse brain is markedly heterogenous and parallels that of opiate receptors. Furthermore intrastriatal administration of kainic acid as well as interruption of the nigrostriatal dopaminergic pathway by 6-hydroxydopamine (6-OHDA) lead to similar decreases in this peptidase activity and in the number of opiate receptors. On the contrary, no correlation was found betwen low affinity enkephalin degrading enzymes and opiate receptors.

Opiate receptors on catecholaminergic neurones in brain

The identification of neuronal systems bearing opiate receptors in brain is helping to elucidate the acute as well as the chronic behavioural actions of morphine-like drugs. Lesion studies indicate that opiate receptors are to be found on dopaminergic and noradrenergic cells, not only on their nerve-endings, but probably also on their cell bodies. Can all opiate receptors therefore be regarded as postsynaptic receptors for opioid peptide-containing neurones? What are the actions they mediate on catecholaminergic transmission? Do these features contribute to our understanding of the mechanisms of addiction? As shown in this article, recent data are bringing some light on these various questions.

RADIOIMMUNOASSAY OF METHIONINE ENKEPHALIN AND INTERFERENCE BY BRAIN FACTOR OF IM MUNOREACTI VIT Y AND OPIATE RECEPTOR BINDING ACTIVITY

We developed a radioimmunoassay of methionine-enkephalin (met-enk) which was found to be highly specific to the peptide and 1 pmole was the detection limit. Interference by the following peptides was practically negligible; leucine- enkephalin, LAP- or CNBr-treated met-enk, α- and β-endorphins. Morphine and its congeners were totally inert. Measurement of met-enk in rat brain P2 fraction revealed besides met-enk, the presence of the factor which cross-reacted with the antiserum and also appeared to bind with the brain opiate receptor. Though the nature of the factor has not yet been characterized, it is suggested that for qualitative assessment of the tissue level of met-enk, a method to selectively inactivate either the peptide or the factor should be developed first

Brain Opiate Receptor Concentrations are Increased in Adult Spontaneously Hypertensive Rats

The saturable binding of 3H-naltrexone in the brains of eight week old spontaneously hypertensive rats (SHR) is about twice that measured in corresponding normotensive WKY rats. This increase is dependent on age since in three and four week old SHR and WKY rats no difference in binding is observed. Scatchard analysis of the saturation curves for the adult animals revealed that the change in binding is due to an increase in the number of binding sites and does not reflect a difference in binding affinity. The increase in opiate receptor content of SHR rats coincides with the appearance of elevated blood pressure in these animals, and supports a concept in which an interaction between the endorphins and the endocrine system may be involved in the mechanisms controlling hypertension.

A stereochemically-constrained enkephalin analog: α-Aminoisobutyryl 2 methionine 5 enkephalinamide

FEBS LettersVolume 96, Issue 2 p. 273-276 Full-length articleFree Access A stereochemically-constrained enkephalin analog α-Aminoisobutyryl2 methionine5 enkephalinamide R. Nagaraj, R. Nagaraj Molecular Biophysics Unit and Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, IndiaSearch for more papers by this authorP. Balaram, Corresponding Author P. Balaram Molecular Biophysics Unit and Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, IndiaAddress correspondence to: P. BalaramSearch for more papers by this author R. Nagaraj, R. Nagaraj Molecular Biophysics Unit and Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, IndiaSearch for more papers by this authorP. Balaram, Corresponding Author P. Balaram Molecular Biophysics Unit and Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, IndiaAddress correspondence to: P. BalaramSearch for more papers by this author First published: December 15, 1978 https://doi.org/10.1016/0014-5793(78)80416-5Citations: 16AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 J. Hughes, T.W. Smith, J.W. Kosterlitz, L.A. Fothergill, B.A. Morgan, H.R. Morris, Nature, 258, (1975), 577– 579. 2 R. Simantov, S.H. Snyder, Life Sci., 18, (1976), 781– 788. 3 M. Anteunis, A.K. Lala, C. Garbay-Jaureguiberry, B.P. Roques, Biochemistry, 16, (1977), 1462– 1466. 4 B.P. Roques, C. Garbay-Jaureguiberry, R. Oberlin, M. Anteunis, A.K. Lala, Nature, 262, (1976), 778– 779. 5 C.R. Jones, V. Garsky, W.A. Gibbons, Biochem. Biophys. Res. Commun., 76, (1977), 619– 625. 6 H.E. Bleich, A.R. Day, R.J. Freer, J.A. Glaser, Biochem. Biophys. Res. Commun., 74, (1977), 592– 598. 7 M.A. Khaled, M.M. Long, W.D. Thompson, R.J. Bradley, G.B. Brown, D.W Urry, Biochem. Biophys. Res. Commun., 76, (1977), 224– 231. 8 A. Goldstein, J. Goldstein, B.M. Cox, Life Sci., 17, (1975), 1643– 1654. 9 A.F. Bradbury, D.G. Smythe, C.R. Snell, Nature, 260, (1976), 165– 166. 10 P.W. Schiller, C.F. Yam, M. Lis, Biochemistry, 16, (1977), 1831– 1838. 11 F.A. Gorin, G.R. Marshall, Proc. Natl. Acad. Sci. USA, 74, (1977), 5179– 5183. 12 J.L. De Coen, C. Humblet, M.H.J. Koch, FEBS Lett., 73, (1977), 38– 42. 13 Y. Isogai, G. Nemethy, H.A. Scheraga, Proc. Natl. Acad. Sci. USA, 74, (1977), 414– 418. 14 F.A. Momany, Bicohem. Biophys. Res. Commun., 75, (1977), 1098– 1103. 15 G.H. Loew, S.K. Burt, Proc. Natl. Acad. Sci. USA, 75, (1978), 7– 11. 16 C.B. Pert, S.H. Snyder, Proc. Natl. Acad. Sci. USA, 70, (1973), 2243– 2247. 17 G.D. Smith, J.F. Griffin, Science, 199, (1978), 1214– 1216. 18 N. Shamala, R. Nagara, P. Balaram, Biochem. Biophys. Res. Commun., 79, (1977), 292– 298. 19 N. Shamala, R. Nagaraj, B.V. Venkataram Prasad, D. Prashanth, P. Balaram, Int. Symp. Biomol. Struct. Conformation and Evolution, Madras, abst. 130/H17, (1978), 20 Nagara, R., Shamal, N. and Balaram, P. (1978) submitted. 21 G.R. Marshall, H.E. Bosshard, Circ. Res., 30/31, (1972), 143– 150. supp. II 22 A.W. Burgess, S.J. Leach, Biopolymers, 12, (1973), 2599– 2605. 23 V.Z. Pletnev, E.P. Gromov, E.M. Popov, Khim. Prir. Soedin., 9, (1973), 224– 229. 24 G.N. Ramachandran, V. Sasisekharan, Adv. Prot. Chem., 23, (1968), 283– 437. 25 M. Bremer, W. Huber, Helv. Chim. Acta, 36, (1953), 1109– 1115. 26 E. Schnabel, Liebigs Ann. Chem., 702, (1967), 188– 196. 27 Md. Abu Khaled, V. Renugopalakrishnan, D.W. Urry, J. Am. Chem. Soc., 98, (1976), 7547– 7553. 28 C.B. Pert, A. Pert, J.K. Chang, B.T.W. Fong, Science, 194, (1976), 330– 332. 29 D.H. Coy, A.J. Kastin, A.V. Schally, O. Morin, N.C. Caron, F. Labrie, J.M. Walker, P. Fertel, G.G. Bernstson, C.A. Sandman, Biochem. Biophys. Res. Commun., 73, (1976), 632– 637. 30 R. Chandrasekaran, A.V. Lakshminarayanan, U.V. Pandya, G.N. Ramachandran, Biochim. Biophys. Acta, 303, (1973), 14– 27. 31 C.M. Venkatachalam, Biopolymers, 6, (1968), 1425– 1436. 32 G.R. Marshall, F.A. Gorin, J. Meienhofer, M. Goodman Peptides — Proc. 5th Am. Peptide Symp. (1977), John Wiley and Sons 84– 87. Citing Literature Volume96, Issue2December 15, 1978Pages 273-276 ReferencesRelatedInformation

Lack of opiate receptor involvement in centrally induced calcitonin analgesia

Calcitonin (CT) injected into the brain ventricles (ICV) of conscious rabbits induced an analgesia not reversable by naloxone which could be repeatedly elicited (for 5 days), while tolerance to morphine developed. CT and morphine synergized in vivo when administered ICV in combination. CT did not alter electrically-induced contractions of guinea-pig myenteric plexus-longitudinal muscle and no displace of 3H-dihydromorphine by CT was observed in brain opiate receptor preparations. We have concluded that the mechanism of centrally induced CT analgesia may be opiate-independent.

Endorphin in rat pituitary glands: Its distribution within the gland, and age related changes in gland content in male and female rats

Endorphins were extracted with glacial acetic acid:acetone from freshly microdissected single lobes of rat pituitary and assayed for inhibition of 3H-etorphine stereospecific binding to rat brain opiate receptors. Highest tissue concentrations of endorphin were found in pars intermedia and pars distalis, with minimal activity observed in pars nervosa. In addition, mature female rats exhibited a significantly higher anterior lobe endorphin content than males, reflecting the larger gland weight, although no significant differences were found between neurointermediate lobe endorphin content of either sex. Pituitary endorphin content increased substantially with age, rising rapidly between the fifth and tenth week after birth. This elevation was still apparent at twenty-five weeks. These studies emphasize the importance of employing rats of closely similar age and weight in any experiments designed to examine pituitary endorphin function, and suggest that ontological development of pituitary endorphin may proceed in a manner similar to that of other pituitary hormones (e.g., MSH) derived from the same precursor peptide.

Phospholipase A inhibition of opiate receptor binding can be reversed by albumin

OPIATE receptors in animal and human brain have been shown to be tightly associated with cell membranes1–3. Our laboratory has reported the solubilisation of an etorphine–receptor complex4, but to date it has not been possible to obtain an active receptor in soluble form. Nevertheless, it has been possible to study many biochemical characteristics of the membrane-bound receptor. Its sensitivity to sulphhydryl reagents and a number of other protein reagents5,6 as well as to proteolytic enzymes1,7 suggests the participation of protein(s) in opiate binding. The evidence is less clear with respect to a function for phospholipids. Opiate binding activity of cell membrane preparations from rat brain has been shown to be exquisitely sensitive to phospholipase A of Vipera russelli venom (in the ng ml−1 range)8, but very insensitive to phospholipase A present in the venom of Crotalus adamenteus1 or that derived from pig pancreas (Lin & Simon, unpublished). Phospholipase C is inhibitory only in very high concentrations1,8. The great sensitivity of opiate receptors to the Russell's viper enzyme and to ionic and non-ionic detergents4,7 points to a possible role for phospholipids in the binding process. We report here that inhibition of opiate binding by phospholipase A can be reversed almost completely by incubation of the enzyme-treated membranes with bovine serum albumin (BSA).

Receptor affinity and pharmacological potency of a series of narcotic analgesic, anti-diarrheal and neuroleptic drugs

A series of 26 drugs was tested for in vitro binding to opiate receptors in the presence and absence of 0.1 M NaCl. The results were correlated with assays for in vivo pharmacological potency. Highly significant correlation was found between binding in the presence and absence of sodium ions and analgesic potency. For 10 drugs tested for anti-diarrheal potency significant correlation was observed with binding to brain opiate receptors when binding was carried out in sodium-containing medium. These data add support to the hypothesis that stereospecific opiate binding sites are pharmacological receptors which mediate analgesia and anti-diarrheal action. We found that neuroleptics can bind to opiate receptors with affinities in the micromolar range, in agreement with reports by others. The anti-diarrheal compound loperamide exhibits no significant central opiate effects but binds to opiate receptors from brain in vitro with high affinity. Evidence is presented suggesting that the lack of specific analgesic effect is the result of poor penetration through the blood-brain barrier. Our results lend further support to the similarity of opiate receptors in the brain and in the intestinal tract.

Hormone—receptor interactions: [4‐Carboranylalanine, 5‐leucine] ‐ enkephalin as a structural probe for the opiate receptor

The new phenylalanine analogue, L-carboranylalanine (Car) [l] was recently found to be a good probe for investigating some special properties of the aromatic recognition site of chymotrypsin [2] . The icosahedral side chain of Car is somewhat larger than the phenyl ring of Phe, rotating about its 1,4 axis. Yet it is easily accomodated by chymotrypsin; the concomitant deformation of the recognition site propagated to the mechanistic site (especially Ser 195) is probably the reason that Car esters are not hydrolysed by the enzyme. One of the aims of our laboratory is to study hormone-receptor interactions. After the promising results with chymotrypsin as a model, we are investigating the use of Car replacements of Phe, Tyr, and Trp in polypeptide hormones. Phenylalanine in position 4 seems to be quite important for enkephalin action, because a number of analogues with replacements of this amino acid by others all show reduced or even missing opiate receptor affinity and biological activity (review [3] ; see also [4-61). An introduction of Car into this position seemed ideally suitable to see whether or not carboranylalanine analogues of peptide hormones can be expected to have exceptional features. We have therefore prepared [4carboranylalanine, S-leucine] enkephalin, H.Tyr-Gly-Gly-Car-Leu.OH and investigated its ability to displace [3H]naloxone from rat brain opiate receptor preparations according to the technique developed in the laboratory of Snyder [7] . [S-Methionine] -enkephalin, [S-leucine] enke-

Properties of the peripheral opiate receptors in the cat nicitating membrane

The noradrenaline overflow and the contractile response elicited by nerve stimulation of the muscle were inhibited by 1 μM morphine in the cat nictitating membrane. This concentration of morphine did not modify the response of the muscle to exogenous noradrenaline. The inhibitory effect of morphine was increased by low Na + (50 mM), whereas the capacity of naloxone as antagonist to morphine was higher with 150 mM than with 50 mM Na +. These results suggest that the peripheral opiate receptors which interact with noradrenergic neurotransmission could show a sodium allosteric transformation similar to that described for the brain opiate receptor. The effect of morphine was enhanced by manganese ion in the presence of normal Na +. The responses of the cat nictitating membrane to nerve stimulation were not altered in the presence of the protein modifying reagent DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] but the effect of morphine on the adrenergic neurotransmission was diminished by DTNB with 150 mM Na +. It is postulated that the affinity of the ligands for presynaptic receptors which regulate adrenergic neurotransmission might be modified during the physiological changes in ion concentration which accompany nerve depolarization.

Gamma endorphin, alpha endorphin and Met-enkephalin are formed extracellularly from lipotropin C fragment.

A SERIES of peptides derived from lipotropin has been reported to possess morphinomimetic properties. The C fragment (or β endorphin, residues 61–91) was isolated from pituitary1,2 and shown to be present in brain3, the C′ fragment (61–87) occurs in pituitary1, γ endorphin (61–77) and α endorphin (61–76) were obtained after acid extraction of tissue of hypothalamic and pituitary origin4, and Met-enkephalin (61–65) was found in extracts of brain5,6. Examination of the ability of these peptides to displace specifically bound 3H-naloxone from brain opiate receptors showed that C fragment was much more potent than the shorter peptides7 and investigation of the antinociceptive properties showed that C fragment alone produces profound and long-lasting analgesia8–11. In addition, various other central activities are exhibited by C fragment12–14. We report here that C fragment can be degraded by membrane bound enzymes from rat brain to form the smaller opiate-like peptides.

Structural requirements for opioid activity of analogues of the enkephalins.

Structure-activity relations of a series of analogues of the two endo­genous morphine-like peptides, leucine-enkephalin and methionine-enkephalin are examined on the basis of ( a ) effects on the mouse vas deferens and the guinea pig ileum and ( b ) affinities for the rat brain opiate receptor. In the mouse vas deferens, metabolism of the peptides by proteolysis is not a major influence on activity. In contrast, however, brain opiate re­ceptor preparations contain an abundance of proteolytic enzymes, the effects of which can be minimized by conducting opiate receptor binding assays at 0 °C and in the presence of bacitracin. The potentiation of biological activity and opiate receptor binding affinity by replacing the Gly 2 residue in the natural enkephalins by d-Ala, is discussed both in terms of increased stability of the Tyr-d-Ala bond to aminopeptidases and of the stabilization of the peptide conform­ation as present in the receptor-peptide complex. The substitution of the Leu 5 - or Met 5 -residue by the corresponding d-amino acid contributes little to proteolytic stability, which emphasizes that the predominating site at which metabolism occurs is the Tyr 1 -Gly 2 bond. Of the analogues described, [d-Ala 2 , d-Leu 5 ]-enkephalin is the most active peptide in the three assay systems, the mouse vas deferens, the guinea pig ileum and the rat brain opiate receptor preparations. Substitutions by the respective d-amino acids d-Tyr and d-Phe at positions 1 and 4 reduce both the potency and binding affinity and emphasize the importance of stereochemical acceptability at these positions. The promotion of receptor binding by d-amino acids is examined, particularly with respect to implied peptide conformations. The experi­mental data have been analysed for the relative influence of metabolic and conformational factors.

Analgesic activity of lipotropin C fragment depends on carboxyl terminal tetrapeptide

A SERIES of peptides with morphine-like activity has been identified in extracts of pituitary1–3 and brain4–7. With the exception of Leu-enkephalin, all those characterised represent some fraction of the 31-residue C fragment—a specific activation product of lipotropin1,8. Several attempts have been made to define the receptor requirements of opiate peptides9,10 and to make potent and stable agonists5,11,12, but essentially all the studies have been based on the easily synthesised NH2-terminal pentapeptide of C fragment, Met-enkephalin. Unlike the enkephalins, C fragment has a high potency in its central actions13–16, in vivo and in vitro, and it is relatively stable to brain peptidases17–19. C fragment is the only naturally occurring peptide to possess strong analgesic properties and have a long lasting action20. It is clear that specific regions of the molecular structure, not present in γ and α endrophin or Met-enkephalin, have a role in the expression of its central activity. We present here evidence that the COOH-terminal tetrapeptide of C fragment, Lys-Lys-Gly-Gln, is responsible for the unique affinity of C fragment for brain opiate receptors and that the presence of all four residues is essential for the high analgesic potency.

The metabolic stability of the enkephalins

[Met 5] and [Leu 5]-enkephalins inhibit 3H-naloxone binding to brain opiate receptors much more effectively at 4°C than at 25°C. At 25°C several protease inhibitors potentiate the action of the enkephalins. Bacitracin is the most effective of these. In the presence of 100 μg/ml of bacitracin, the potencies of [Met 5]- and [Leu 5]-enkephalins are similar to those at 4°C. Some protease inhibitors, such as TLCK and TAME, are effective by themselves in inhibiting the binding of 3H-naloxone. Enkephalins with D-amino acids in the 2-position are equally effective at 0°C and at 25°C and their action is not potentiated by bacitracin. In particular, [D-Ala 2]-enkephalins do not seem to be significantly degraded by the membrane enzymes which destroy the natural enkephalins.

β-Endorphin: Structure-activity relationships in the guinea pig ileum and opiate receptor binding assays

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.

Induction of tolerance to the analgesic action of lipotropin in C-fragment.

THE C-Fragment of β-lipotropin (lipotropin 61-91), was first discovered as an intact peptide in the pituitary gland1,2 and it has recently been shown to be present in brain3. The peptide binds strongly to brain opiate receptors in vitro4,5 and has potent opiate-like actions on the guinea pig ileum and mouse vas deferens15. Reports from our laboratory described the potent analgesic properties of C-Fragment when injected into the cerebral ventricles of the cat4–6, and in subsequent studies its analgesic effects have been demonstrated in rodents7–9. The results reported here confirm the analgesic potency of C-Fragment administered intracerebrally in the rat, and show that chronic administration of the peptide leads to the development of tolerance to its analgesic actions.

Interaction between acth fragments, brain opiate receptors and morphine-induced analgesia

The present study confirms that N-terminal fragments of ACTH have an affinity for rat brain opiate receptors in vitro. Such peptides, devoid of corticotrophic activity, were found to inhibit morphine-induced analgesia if they also possessed affinity for opiate receptors in vitro. The structure—activity relationship for these two parameters is comparable to that observed for the same peptides on the induction of excessive grooming.