Department of Medicine, New York University Medical Center, New York, New York.

 

INTRODUCTION

 

Research on the mode of action of narcotic analgesic drugs and the mechanism of the development of tolerance and physical dependence is one of the oldest of scientific pursuits. For many years the first step in the action of these drugs was postulated to be their binding to highly specific "receptor" sites. This specific drug-receptor interaction was thought to trigger certain chemical or physical changes leading to the observed pharmacological responses. The reason for such a receptor postulate was the striking structural and steric specificity of many of the actions of narcotic Analgesics. Rather minor structural changes have major effects on the pharmacology of the molecule, leading in some instances to the formation of antagonists, drugs whose main action consists of counteracting the effects of other opiates. While the receptor postulate dates back two to three decades, the discovery of the existence of specific opiate receptors occurred only about three years ago.

 

DISCOVERY OF STEREOSPECIFIC BINDING SITES IN ANIMAL BRAIN

 

Binding of opiates to tissue homogenates was demonstrated some years ago in our laboratory using equilibrium dialysis (1). However, attempts to measure specific binding, defined as binding of labelled dihydromorphine, sensitive to displacement by the specific antagonist, nalorphine, were unsuccessful.

 

More recently Goldstein et al. (2) were the first to utilize the property of stereospecificity to search for opiate receptors. A series of modifications of the Goldstein procedure led to the discovery of stereospecific binding sites in homogenates of rat brain independently and simultaneously in three laboratories (3-5). In our laboratory we used 3H-etorphine, a narcotic analgesic of enormous potency (about 10,000 times as potent as morphine in rats), as our labelled ligand. The hope, which was realized, was that the great potency of this drug might reflect, at least in part, high affinity for the receptor. 3H-etorphine of high specific activity was incubated with rat brain homogenate at very low concentrations (10-19-10-9M) in the presence of a large excess of either unlabelled levorphanol (L) or its inactive enantiomer, dextrorphan (D). The homogenate was centrifuged and the pellet washed twice with cold buffer by recentrifugation. Radioactivity in the washed pellet was determined by liquid scintillation counting. Stereospecific binding was defined as that portion of the binding that is prevented by excess L but not by D. Using this procedure, most of the binding (7080%) was found to be stereospecific. More recently a more rapid filtration technique was used by Pert and Snyder (5) and this has been adopted in our laboratory. Typical recent results from our laboratory are shown in Table 1.

 

PROPERTIES OF STEREOSPECIFIC OPIATE BINDING SITES

 

The binding sites have to date been found only in the central nervous systems of vertebrates and in the innervation of certain other tissues known to be sensitive to opiates, such as the guinea pig ileum and the mouse vas deferens. The sites are tightly bound to cell membranes. Enrichment of stereospecific binding in the synaptosome fraction (4,6,7) suggests that the binding sites are primarily present near synapses. Whether they are located post or pre-synaptically has not yet been determined.

 

Binding of opiates is saturable and half saturation (a measure of affinity) occurs at concentrations that are comparable to the brain concentrations at which these drugs are thought to exert their pharmacological effects. In a number of studies (3,8,9) excellent correlation has been found between the in vivo potency of a large number of opiates and their affinity for stereospecific binding sites.

 

The opiate binding sites are very sensitive to proteolytic enzymes, such as trypsin, chymotrypsin and pronase (3,10) as well as to a variety of reagents known to react with functional groups of proteins, the most thoroughly studied of which are reagents that react with sulfhydryl (SH) groups (11). A protein (or proteins) is therefore essential for stereospecific binding of opiates. Sensitivity to phospholipase A (10) suggests a role for phospholipids. All properties of the stereo-specific binding sites so far examined are consistent with their being the recognition and binding components of pharmacologically important opiate receptors. These sites will henceforth be referred to as receptors.

 

DISTRIBUTION OF OPIATE RECEPTORS IN THE BRAIN

 

The availability of human brain tissue obtained during autopsies at the Office of the Chief Medical Examiner of the City of New York, permitted us to establish the existence of opiate receptors in human brain. A study of the distribution of receptor sites in over forty anatomical regions of human brain was undertaken in collaboration with Dr. John Pearson (12). Levels of binding were found to vary greatly from region to region. High binding (0.3-0.4 pmoles/mg protein) was found in alL regions of the limbic system except the hippocampus which has a rather low level of opiate binding. All regions consisting primarily of white matter, the cerebellum and regions of the brain stem were very low or virtually devoid of binding. High binding was also found in certain non-limbic areas such as the locus coeruleus and the pulvinar. Very similar results were reported by Kuhar et al. (13) for monkey brain.

 

THE POSSIBLE PHYSIOLOGICAL ROLE OF OPIATE RECEPTORS

 

Many investigators have suggested that, in order to survive the eons of evolution, opiate receptors must possess a physiological function that conveys a selective advantage on the organism that carries them. This led to a search for an endogenous ligand for the receptor. Exploration of all known neurotransmitters or modulators met with uniformly negative results. This then prompted the search for the existence of a previously unknown opiate-like molecule in the brain. Two laboratories were successful about the same time in demonstrating opiate-like activity in aqueous extracts of pig brain. Hughes et al. (14) showed that such extracts inhibited electrically stimulated contractions of isolated guinea pig ileum and mouse vas deferens. This inhibition was reversed by naloxone. Terenius and Wahistrom (15) showed that something in aqueous brain extracts was able to compete with labelled opiates for binding to opiate receptors. More recently Hughes et al. (16) reported that the active principle of their extract consists of two pentapeptides with the structures H-Tyr-Gly-Gly-Phe-Met-OH and H-Tyr-Gly-Gly-Phe-Leu-OH. A larger peptide with opiate-like properties was found in bovine pituitary by Goldstein and his collaborators (17,18). These opiate-like peptides, or endorphins, are discussed in detail by Dr. Goldstein. The question of their physiological role has not yet been answered. However, it is attractive to speculate that the endorphins and their receptors represent components of an endogenous pain-suppression system. At any rate, if a function is found for these peptides, the physiological role of opiate receptors will be clarified. For further detail see Loh and Law (chapter 19 in this volume).

 

CONFORMATIONAL FORMS OF THE OPIATE RECEPTOR

 

The findings in our laboratory (3) that increasing salt concentrations resulted in the reduction of opiate binding while no such effect was seen by Pert and Snyder (5) for naloxone binding, led us to suggest that this might reflect a difference in the manner in which agonists and antagonists bind to receptors. Evidence to support such a difference was obtained by Pert et al. (19) who also discovered that the effect was highly specific for sodium salts which reduced the binding of agonists but increased the binding of antagonists. Other alkali metal ions such as Kt, Rb+ and Cs+ do not exhibit this discriminatory effect, while Lit is partially effective. Detailed studies of the effect of sodium (20) led to the finding that the results are most readily explained by the interconversion of two conformational forms of the receptor. The conformation prevalent in a media containing Nat has a higher affinity for antagonists and a lower affinity for agonists than the conformation that exists in Nat-free media. Independent evidence for this interconversion was obtained by a study of the kinetics of receptor inactivation by the SH-reagent, Nethylmaleimide (NEM) (11). As shown in Figure 1, in the presence of sodium, SH-groups of the receptors are markedly less accessible to inactivation (t12 of 30 min instead of 8 min). This protection shows the same ion specificity (Table 2) and response to sodium (Figure 2) as the changes in ligand affinity. The significance of the ability of the receptor to change its conformation is not yet known, but the great specificity of sodium in producing this phenomenon leads us to suspect that it may be important.

Fig. 1. Kinetics of inactivation of stereospecific 3H -nal - trexone binding by NEM and protection by pretreatment with naltrexone. Inactivation was carried out at 37 degrees. P2 fraction was preincubated for five minutes with or without unlabelled naltrexone (3 nM) before addition of NEM. For the binding assay 3H-naltrexone was added to final concentration of 2 nM (total concentration of naltrexone 5 nM). From reference 11.

 

To study the detailed chemical composition and functioning of opiate receptors it will be necessary to solubilize them off cell membranes and purify them. Some progress in this direction has been achieved in our laboratory (21). Cell membranes from rat brain are allowed to bind 3H-etorphine. The bound membranes are freed of unbound drug and concentrated by centrifugation and re-suspension in a smaller volume 'of buffer. The membranes are then treated with a 1% solution of the non-ionic detergent BRIJ 36T. Ultracentrifugation of this suspension at 100,000 x g yields a clear supernatant that contains most of the radioactivity. By use of chromatography on XAD-4 resin it was determined that 25-30% of the radioactivity in the supernatant is still bound to a large macromolecule (Figure 3). The molecular weight of this complex, as determined by chromatography on Sepharose 6B, was 350,000 (Figure 4). Evidence was obtained that the solubilized macromolecular moiety attached to etorphine has properties identical to those of the opiate receptor. To date, the free macromolecule obtained by allowing the etorphine to dissociate is not able to rebind opiates stereospecifically. Efforts to modify the conditions to allow us to obtain a soluble receptor capable of binding opiates in solutions are in progress.

 

Fig. 3. Elution profile on XAD-4 of Brij extract of P2 membranes,-bound with 3H-etorphine. P2 membranes (2 mg of protein per milliliter) were incubated with 3H-etorphine (1 x 10-9M, 20.7 Ci/mmole) and subsequently extracted with 1% Brij 36T. A 1-ml sample of the supernatant, after ultracentrifugation, was placed on a column (2 by 10 cm) of XAD-4 (Rohm and Haas) and eluted with cold 0.05M tris buffer. (A) 3H-etorphine bound in the presence of 10-6M dextrorphan. (o) 3H-etorphine bound in the presence of 10-6M levorphanol. (A) 3H -etorphine added to Brij extract of P2 membranes subsequent to extraction and ultracentrifugation. (o) Protein concentration is given in micrograms per milliliter. Data from reference 21, copyright 1975 by the American Association for the Advancement of Science.

 

Fig. 4. Estimation of the molecular weight of solubilized 3H-etorphine-bound complex on Sepharose 6B. Gel filtration was carried out on a column, 1 by 52 cm; the eluting solution was 0.05M tris buffer, pH 7.4. Data are expressed as Key; by definition Kav=(Ve-Vo/Vt-V0), where Ve is the elution volume corresponding to the peak concentration of solute (marker proteins monitored by absorbance at 280 nm, 3H-, etorphine-bound complex monitored by radioactivity determination), V6 is the void volume as determined by the appearance of dextran blue, and Vt is the total liquid volume as determined with free 3H-etorphine. Vo and Vt values were 20 and 65 ml, respectively. The relation between the logarithm of the molecular weight and Kai, was used to obtain the molecular weight of the 3H-etorphine-bound complex. Data from reference 21, copyright 1975 by the American Association for the Advancement of Science.

 

SUMMARY

 

Considerable evidence has been accumulated demonstrating that stereospecific binding sites for opiates and their antagonists are the long-sought opiate receptors that mediate the pharmacological effects of these drugs. To date evidence suggests that one type of receptor can exist in several conformational states, but the question of the existence of multiple receptors for the many responses evoked by opiates is still unsettled.

 

The discovery of opiate receptors has recently given rise to another very exciting finding, namely, the existence of polypeptides in animal and human brain that can bind to the receptors and exhibit opiate-like activities. The study of the interaction of receptors with exogenous and endogenous ligands. The reactions triggered by these interactions, and the isolation and purification of receptor molecules should within the foreseeable future give us considerable insight into the mode of action of narcotic analgesics. An understanding of the physiological role of the receptor and its endogenous ligands may also lead to greater comprehension of aspects of brain function.

 

REFERENCES

 

1. Van Praag, D. and Simon, E.J.: Studies on the intracellular distribution and tissue binding of dihydromorphine7,8-H3 in the rat. Proc. Soc. Exp. Biol. Med. 122:6-11 (1966).

2. Goldstein, A., Lowney, K.I. and Pal, B.K.: Stereospecific and nonspecific interactions of the morphine congener levorphanol in subcellular fractions of mouse brain. Proc. Natl. Acad. Sci. U.S. 68:1742-1747 (1971).

3. Simon, E.J., Hiller, J.M. and Edelman, I.: Stereospecific binding of the potent narcotic analgesic (3H) etorphine to rat-brain homogenate. Proc. Natl. Acad. Sci. U.S. 70:1947-1949 (1973).

4. Terenius, L.: Stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex. Acta Pharmacol. Toxicol. 32:317-320 (1973).

5. Pert, C.B. and Snyder, S.H.: Opiate receptor: Demonstration in nervous tissue. Science 179:1011-1014 (1973).

6. Pert, C.B., Snowman, A.M. and Snyder, S.H.: Localization of opiate receptor binding in synaptic membranes of rat brain. Brain Res. 70:184-188 (1974).

7. Hitzeman, R.J., Hitzeman, B.A. and Loh, H.H.: Binding of 3H-naloxone in the mouse brain: Effect of ions and tolerance development. Life Sci. 14:2393-2404 (1974).

8. Wilson, R.S., Rogers, M.E., Pert, C.B. and Snyder, S.H.: Homologous N-alkylnorketobemidones. Correlation of receptor binding with analgesic potency. J. Med. Chem. 18:240-242 (1975).

9. Creese, I. and Snyder, S.H.: Receptor binding and pharmacological activity of opiates in the guinea-pig intestine. J. Pharm. exp. Ther. 194:205-219 (1975).

10. Pasternak, G.W. and Snyder, S.H.: Opiate receptor binding: Effects of enzymatic treatments. Mot. Pharmacol. 10:183-193 (1973).

11. Simon, E.J. and Groth, J.: Kinetics of opiate receptor inactivation by sulfhydryl reagents: Evidence for conformational change in presence of sodium ions. Proc. Natl. Acad. Sci. U.S. 72:2404-2407 (1975).

12. Hiller, J.M., Pearson, J. and Simon, E.J.: Distribution of stereospecific binding of the potent narcotic analgesic etorphine in the human brain: Predominance in the limbic system. Res. Commun. Chem. Pathol. Pharmacol. 6:1052-1062 (1973).

13. Kuhar, M.J., Pert, C.B. and Snyder, S.H.: Regional distribution of opiate receptor binding in monkey and human brain. Nature 245:447-450 (1973).

14. Hughes, J.: Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine. Brain Res. 88:295-308 (1975).

15. Terenius, L. and Wahlstrom, A.: Inhibitor(s) of narcotic receptor binding in brain extracts and cerebrospinal fluid. Acta Pharmacol. Toxicol. 35:55 (1974).

16. Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A. and Morris, H.R.: Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258:577-579 (1975).

17. Teschemacher, H., Opheim, K.E., Cox, B.M. and Goldstein, A.: A peptide-like substance from pituitary that acts like morphine. 1. Isolation. Life Sci. 16:1771-1776 (1975).

18. Cox, B.M., Opheim, K.E., Teschemacher, H. and Goldstein, A.: A peptide-like substance from pituitary that acts like morphine. 2. Purification and properties. Life Sci. 16:1777-1782 (1975).

19. Pert, C.B. and Snyder, S.H.: Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol. Pharmacol. 10:868-879 (1974).

20. Simon, E.J., Hiller, J.M., Groth, J. and Edelman, I.: Further properties of stereospecific opiate binding sites in rat brain: On the nature of the sodium effect. J. Pharm. exp. Ther. 192:531-537 (1975).

21. Simon, E.J., Hiller, J.M. and Edelman, I.: Solubilization of a stereospecific opiate-macromolecular complex from rat brain. Science 190:389-390 (1975).