INTRODUCTION

 

The recent discoveries of the existence of opiate receptors and of endogenous opioid peptides in the central nervous system of animals and man have raised hopes that an understanding of the molecular mechanism of drug addiction may soon be achieved. It is clear from the title of this chapter that that day is not yet here. I will give a brief chronological review of the significant discoveries and developments and will then summarize recent research that bears on the possible involvement of the endogenous opioid system in drug addiction.

 

THE DISCOVERY OF OPIATE RECEPTORS

 

The hypothesis that narcotic analgesics must bind to highly specific sites or receptors in the central nervous system (CNS) in order to produce their many well-known responses has been held by some investigators for several decades. The evidence for the existence of such receptor sites was compelling. It consisted primarily of the remarkable stereospecificity and, for certain parts of the molecules, structural specificity displayed by many of the pharmacological actions of narcotic analgesic drugs.

 

As thousands of analogues of morphine were synthesized in search of the still mythical nonaddictive analgesic it became clear that one enantiomer of a racemic mixture (usually the levo-rotatory one) was generally much more active than the other. Moreover, some parts of the molecule could be drastically altered with relatively little change in potency, whereas tampering with certain regions had dramatic effects. The most interesting and best studied such region is the substituent on the tertiary nitrogen, one of the functional groups essential for narcotic analgesic activity.

 

When the methyl group is substituted by a larger alkyl group, eg, an allyl or cyclopropylmethyl group, analgesic potency is reduced and the drug takes on the new pharmacological role of a potent, specific antagonist against many of the actions of morphine and related narcotics. Some drugs have both agonist and antagonist properties, while others, such as naloxone and naltrexone (the N-allyl and N-cyclopropylmethyl analogues, resp. of oxymorphone), are "pure" antagonists. The synthesis of mixed agonist-antagonist drugs as candidates for analgesics with low addiction liability has been a major enterprise in pharmaceutical company laboratories in recent years. Moreover, the pure antagonist naltrexone, longer acting than naloxone, has shown some promise for the treatment of heroin addicts.

 

The kinds of specificities described above are most easily explained by interaction with binding sites that exhibit complementary specificity. The search for such specific binding sites or opiate receptors began in the 1950s and bore fruit in the early 1970s. It was easy to show binding of opiates to cell constituents (Van Praag, 1966) but to distinguish specific from nonspecific binding proved difficult.

 

It was the measurement of stereospecific binding that led to success. Ingoglia and Dole (1970) were the first to apply stereospecificity to the search for receptors by injecting 1- and d-methadone into the lateral ventricle of rats, but found no difference in the rate of diffusion of the enantiomers. Goldstein et al (1971) devised a method for measuring stereospecific binding of 3H-levorphanol in mouse brain homogenates. They reported that only 2 percent of the total binding was stereospecific and the properties and distribution of this binding turned out to be quite different from those of the subsequently discovered "receptors." Upon purification this binding material proved to be cerebroside sulfate.

 

In 1973 our laboratory (Simon et al, 1973) and those of Snyder (1973) and Terenius (1973), using modifications of the Goldstein procedure, independently and simultaneously, reported the observation in animal brain homogenates of stereospecific binding of opiates that represented the major portion of the total binding. Since that time stereospecific binding studies have been done in many laboratories and much evidence has been accumulated suggesting that these stereospecific sites are indeed receptors that are responsible for many of the pharmacological actions of the opiates. They have been found in man (Hiller et al, 1973) and in all vertebrates so far studied. Very recently it has been reported that they also exist in some invertebrates (Stefano, 1980).

 

PROPERTIES AND DISTRIBUTION OF OPIATE RECEPTORS

 

The properties of the opiate binding sites have been studied extensively and their distribution in the brain and spinal cord has been mapped in considerable detail by dissection and in vitro binding measurement (Hiller, 1973; Kuhar, 1973) as well as by autoradiography (Pert, 1975; Atweh, 1977; Atweh, 1977; Atweh, 1977).

 

Stereospecific binding is saturable and total binding amounts to 15-20 pmol of opiate per gram of rat brain. Affinities range from KD of 0.25 nM for a potent fentanyl analogue (Stahl et al, 1977) to little or no affinity for drugs devoid of opiate activity. The average dissociation constants for effective narcotic analgesics are in the 1-10 nM range. The pH optimum for binding is in the physiological range with a fairly broad optimum between 6.5 and 8.0. The addition of salts to the incubation mixture tends to reduce binding. Sodium represents an interesting exception. Its presence causes inhibition of agonist binding, whereas the binding of most antagonists is significantly increased. This highly specific effect of sodium (exhibited to some extent by Li+ but not by any of the other alkali metals, K+, Rb+, or Cs+) has been shown in our laboratory to be the result of a conformational change in the opiate receptor (Simon et al, 1975).

 

The inhibition of stereospecific binding by proteolytic enzymes (Simon et al, 1973; Pasternak, 1973) and a variety of protein reagents, including sulfhydryl reagents, suggests the involvement of protein moieties in opiate binding. The role of phospholipids is yet to be established. Binding is inhibited by some, but not all, phospholipase A preparations (Simon et al, 1973; Pasternak, 1973) but not by phospholipases C or D. Moreover, we have shown (Lin, 1978) that inhibition by phospholipase A can be reversed by washing the membrane preparation with a solution of bovine serum albumin, suggesting that the nature of the phospholipid environment may be very important for the active conformation of the opiate receptor.

 

The extensive mapping studies can be summarized here only briefly. The highest levels of opiate receptors are found in the areas of the limbic system and in the regions that have been implicated in the pathways involved in pain perception and modulation, such as the periventricular and periaqueductal gray areas, the medial thalamus, the nucleus raphe magnus and the substantia gelatinosa of the spinal cord. It has been suggested that the limbic system receptors may be involved in opiate-induced euphoria (or dysphoria) and in the affective aspects of pain perception.

 

Perhaps the most convincing evidence suggesting that stereospecific binding has pharmacological relevance comes from a number of studies that show excellent correlation between pharmacological potencies and in vitro binding affinities for a large number of drugs, varying in analgesic potencies over 5-6 orders of magnitude (Stahl et al, 1977; Wilson et al, 1975).

 

DISCOVERY AND MAPPING OF ENDOGENOUS OPIOID PEPTIDES

 

The evidence that the brain of all vertebrates investigated, from the hagfish to man, contains opiate receptors led investigators to raise the question why such receptors for plant-derived substances exist in the CNS and have survived eons of evolution. A physiological role for opiate receptors that confers a selective advantage on the organisms seemed probable, suggesting the presence of an endogenous opiatelike ligand for the receptor. This notion was reinforced by the finding in the early 1970s that electrical stimulation of certain brain areas was able to mobilize an endogenous pain-relieving system, resulting in long-lasting analgesia (Reynolds, 1969; Mayer et al, 1971).

 

None of the many known neurotransmitters or neurohormones was found to exhibit high affinity for opiate receptors. A number of laboratories therefore initiated a search for new opiatelike substances in extracts of animal brain. This search was successful first in the laboratories of Hughes and Kosterlitz (1975) and of Terenius and Wahlstrom (1974). Goldstein and his collaborators (1975) at about the same time, reported opioid activity in extracts of pituitary glands. Hughes utilized the in vitro bioassays for opiates, namely naloxone-reversible inhibition of electrically evoked contraction of the mouse vas deferens or the guinea pig ileum, while Terenius assayed endygenous opioid activity by measuring ability of brain extracts and fractions to compete with labeled opiates for receptor binding.

 

These studies culminated in the identification of the opioid substances in extracts of pig brain by Hughes et al (1975). They reported that the activity resided in two pentapeptides, Tyr-Gly-Gly-Phe-Met and Tyr-Gly-GlyPhe-Leu, which they named methionine (Met) and leucine (Leu) enkephalin. Hughes et al also reported the interesting observation that the sequence of Met-enkephalin was present as amino acid residues 61-65 in the pituitary hormone 13-lipotropin ((3LPH). This hormone had been isolated in 1965 from pituitary glands by C.H. Li (1964). It possessed weak lipolytic activity which was never seriously thought to be its real function. The report of Hughes et al, along with that of the Goldstein group of the existence of opioid activity in the pituitary gland, led Guillemin to examine the extracts of pig hypo-thalami and pituitary glands (remaining in his freezer from his Nobel prize winning identification of hypothalamic releasing factors). Two polypeptides with opioid activity were found and sequenced (Ling et al, 1976). They proved to have structures identical with amino acid sequences 61-76 and 61-77 of PLPH. Meanwhile, potent opioid activity was found in the C-terminal fragment of OLPH (LPH 61-91) in two laboratories (Bradbury et al, 1976; Cox et al, 1976) while the intact PLPH molecule was inactive. The proliferation of endogenous peptides with opioid activity caused the author of this paper to suggest the term "endorphin" (a contraction of "endogenous" and "morphine") which has been widely accepted. The C-terminal fragment was renamed 13-endorphin by Li, while LPH 61-76 and 61-77 were named a and 7-endorphin, respectively, by Guillemin.

 

Recently, a number of additional peptides with opioid activity has been reported. One of the most important of these is a peptide from the pituitary characterized by A. Goldstein and collaborators (1979). The peptide was named dynorphin by the authors because of its potent opioid activity in bioassay systems. It has been found in certain areas of the CNS in addition to the pituitary.

 

All the opioid peptides exhibit opiatelike activity when injected intraventricularly. This activity includes analgesia, respiratory depression and a variety of behavioral changes including the production of a rigid catatonia. The pharmacological effects of the enkephalins are very fleeting, presumably due to their rapid destruction by peptidases. The longer-chain endorphins are more stable and produce long-lived effects. Thus, analgesia from intraventricular administration of 0-endorphin can last 3-4 hours. All of the responses to endorphins are reversible by opiate antagonists, such as naloxone. There have been reports that certain analogues of enkephalin can produce analgesia after systemic injection or even oral ingestion (Roemer et al, 1977).

 

Distribution of enkephalins has been studied by biochemical (Simantov et al, 1976) as well as by bioassay (Hughes et al, 1977) and immunohistochemical techniques (Elde et al, 1976; Simantov et al, 1976). The distribution of enkephalins in the CNS shows considerable, though not complete, correlation with the distribution of opiate receptors. Thus, the globus pallidus has a very high density of enkephalin (or at least enkephalinlike immunoreactive material) while it is low in opiate receptors. Certain cortical areas dense in opiate receptors have low levels of enkephalin.

 

In the earlier studies of HafeIt and colleagues (1976) the immunofluorescence was all found in nerve fibers and terminals but not in cell bodies. In a more recent paper, this group (Wilda et al, 1977) utilized colchicine which is known to arrest axonal transport. After such treatment it was possible to find immunofluorescence in cell bodies after treatment with antiserum to Met-enkephalin. More than 20 cell groups containing enkephalin have so far been observed in the brain and spinal cord, a number somewhat larger than the 15 catecholamine cell groups known to exist in rat brain. The authors felt that their results indicate that these perikarya possess the machinery for enkephalin biosynthesis. This was the first indication that enkephalin is probably not derived from large endorphin precursors, since levels of 0-lipotropin and 0-endorphin are very low in some of the areas that are found to have high enkephalin levels.

 

Studies on the distribution of 3-endorphin in the laboratories of Guillemin (1977) and Watson (1977) have provided convincing evidence for a distribution that is very different from that of the enkephalins. This has led to the suggestion that the CNS has separate enkephalinergic and endorphiergic neuronal systems. 0-endorphin is present in the pituitary, where there is little or no enkephalin, as well as in certain regions of the brain. Brain (3-endorphin seems to originate in a single set of neurons located in the periarcuate region of the hypothalamus, with axons projecting throughout the brain stem and into areas of the forebrain.

 

NARCOTIC ADDICTION

 

There are a number of ways in which the endogenous opioid system might be involved in drug addiction. There could be changes in the number or properties of opiate receptors, altered levels of enkephalins and/or endorphins and finally changes in the metabolism of the opioid peptides. Experiments probing these possible changes will be summarized.

 

All opioid peptides will produce tolerance and physical dependence when injected repeatedly. Cross-tolerance with plant alkaloid opiates has also been shown. This does not prove that tolerance/dependence develop to endogenously produced and released endorphins nor that these peptides and their receptors are involved in the formation of tolerance and dependence to narcotics.

 

A theory that predated the biochemical demonsiration of opiate receptors is one that suggests changes in either the number or binding characteristics of opiate receptors. A change in binding affinities similar to that seen when sodium concentration is increased during in vitro binding is especially attractive, since sensitivity to agonists decreases during tolerance formation while sensitivity to antagonists increases dramatically. Klee and Streaty (1974) examined this question in whole rat brain and found no changes in the number or affinities of opiate binding sites. We felt that this might be explicable by a "drowning out" of changes occurring in only a few brain regions. However, an examination of receptor number and binding affinities in the medial thalamus, periventricular gray region and caudate nucleus in collaboration with K. Bonnet (1976) gave equally negative results. Whereas these three areas have high levels of receptors and/or have been implicated in various aspects of opiate action, the possibility still remains that these were not the appropirate areas to examine. However, it is at least equally possible that detectable changes in receptors do not occur during chronic morphinization.

 

The absence of changes in opiate binding during chronic morphinization of animals has given rise to the notion that the alterations occur in steps subsequent to the binding of opiates to their receptor. Several years ago Collier and Roy (1974) reported that opiates inhibit prostaglandin El-stimulated adenylate cyclase in rat brain homogenate. This interesting observation has proved difficult to reproduce. However, a similar observation from studies in a cell culture system has lent credence and support to this finding. Neuroblastoma x glioma hybrid cells in culture were shown to contain opiate receptors (Sharma et al, 1975). The receptor binding of opiates and endogenous opioids results in inhibition of basal as well as prostaglandin Erstimulated adenylate cyclase. When these cultures are grown in the presence of morphine inhibition of adenylate cyclase requires increasing concentrations of opiates, a finding that has been suggested as the cellular equivalent of tolerance. It is due to an increase in enzyme activity that seems to be induced by the presence of opiate in the culture medium. Moreover, a putative cellular equivalent of withdrawal is also observed. When cells grown in morphine are placed in drug-free medium or treated with naloxone there is a dramatic overproduction of cyclic AMP (cAMP). The relevance of these results to the CNS of intact animals has yet to be established.

 

Observations of Collier and his collaborators provide further evidence that cAMP may play a role in chronic effects of opiates. Treatment of naive animals with inhibitors of phosphodiesterase, the enzyme that destroys cAMP, results in symptoms that closely resemble the withdrawal syndrome from opiates. This has been termed quasi morphine withdrawal syndrome (QMWS) by Collier who suggests that it results from increased brain levels of cAMP (1974).

 

The possibility that a change in endorphin level might be observed during tolerance/dependence development has also received attention. A report by Simantov and Snyder (1976) that enkephalin levels are elevated in brains of tolerant rats has been refuted by experiments from the same laboratory (1977). The earlier work which had been done using a radioreceptor assay was not supported when the much more specific radioimmunoassay was used.

 

Herz's group (1979) found little change in the level of 0-endorphin immunoreactivity 10 days after morphine pellet implantation in rats. However, when the period of exposure to morphine was extended to one month or longer a 60 percent decrease of [3-endorphin-like immunoreactivity from the intermediate/posterior lobe of the pituitary was observed. There was also a decrease in some brain areas such as septum and midbrain, but the level in the hypothalamus was unaltered. Some decrease in enkephalin levels in the striatum and the pituitary was also reported. The authors admit that interpretation of these data is difficult, especially since attempts to repeat these experiments with the potent narcotic analgesic etorphine were unsuccessful and since the period of pellet implantation far exceeded the period required for the development of tolerance and physical dependence.

 

Recently there has been a report (Su et al, 1978) that the intravenous administration of 4 mg of human 0-endorphin to human addicts led to dramatic improvement in severe abstinence syndromes. There was no euphoria and little adverse effect. In a double-blind study it was found that subjects were able to distinguish morphine and 13-endorphin. After endorphin treatment they felt thirsty, dizzy, sleepy, warm, and had "a strange feeling throughout the body." However, all these symptoms disappeared in 20 minutes whereas the beneficial effects of endorphin on the withdrawal syndrome lasted for several days. The long-lasting suppression of especially the most severe symptoms of abstinence (vomiting, diarrhea, tremor, and restlessness) by a single dose of /3-endorphin suggested to the authors the possibility that this endogenous peptide may indeed have a role in the mechanism of tolerance/dependence development to opiates.

 

An exciting recent discovery that could have a bearing on our understanding of the role of the endogenous opioid system in addiction was made simultaneously in Paris and at Stanford University. Malfroy et al (1978) and Sullivan et al (1978) reported the existence of a membrane-bound peptidase that appears to be relatively specific for the breakdown of enkephalins. This "enkephalinase" is a carboxydipeptidase, ie, it splits enkephalin between the glycine in position 3 and the phenylalanine. The Schwartz group reported that the level of this enzyme increases significantly during chronic morpilinization of rats. Other groups have found less dramatic changes, but this finding deserves watching. An enzyme, called by the authors enkephalinase A, has recently been purified by Gorenstein et al (1980).

 

A recent study of plasma (3-endorphin levels in narcotic addicts and non-addict control subjects showed a large decrease of plasma [3-endorphin in the addicts (Ho et al, 1980). However, the antiserum used for the radioimmunoassays must have been rather non-specific, since the control levels of 13-endorphin were about 1000 pg/ml plasma. Many other laboratories have reported anywhere from borderline detectable levels to 30 pg/ml.

For completeness I should like to mention two recent developments of considerable interest for which the relationship to the opiate receptor is still unknown.

 

Walter et al (1978) reported that it was possible to suppress the abstinence syndrome when rats were withdrawn from chronic morphine by administration of the dipeptide Z-Pro-D-Leu. There was no effect on the analgesic response to morphine. The mechanism of this phenomenon is not understood.

 

Based on the abundant literature which seems to implicate catecholamines in the actions of opiates, Gold et al (1978) treated human heroin addicts with clonidine. In a double-blind, placebo-controlled study clonidine eliminated objective signs and subjective symptoms of opiate withdrawal for 4-6 hours in all addicts. In an open pilot study the same patients did well while taking clonidine for one week. All of the patients had been addicted to opiates for 6-10 years and had been on methadone for 6-60 months at the time of the study. The authors suggest that their success with clonidine indicates that abstinence may be produced by an interaction between opiate receptors and alpha-2 adrenergic receptors in the mediation of effects by endogenous opiates in noradrenergic areas such as the locus coeruleus.

 

CONCLUDING COMMENTS

 

In spite of the enormous activity in research on opiate receptors and endorphins, the physiological function of this system has not yet been established. Involvement of the endogenous opioid system has been suggested for pain suppression, narcotic addiction, mental diseases, in particular, schizophrenia and depression, sexual activity, and overeating. Proof is not yet available for any of these roles. The best evidence exists for an involvement of endogenous opioids and their receptors in the modulation of pain. It is based primarily on the observation that several types of nondrug induced analgesiA, such as electrical stimulation analgesia, acupuncture and placebo analgesia are reversible by naloxone. The release of enkephalins and 0- endorphin into CSF during analgesia has also been reported.

 

As can be seen from the above summary, evidence for the participation of the endogenous opioid system in narcotic addiction is still very sparse and largely indirect. Moreover, the changes that have been reported deal with a possible involvement in the development of tolerance and physical dependence. Evidence for a role of the endorphin system in such important aspects of drug abuse as psychic dependence and drug hunger is even more difficult to obtain.

 

Nevertheless, there is considerable optimism among researchers in this field. This optimistic outlook is based in part on the conviction that it is highly improbable that endogenous opioid peptides would not be involved in the actions of exogenous opiates, which they resemble so closely in their pharmacology. If opiate receptors are involved in the acute effects of opiates (for which the evidence is much better) then why should they not have a role in the chronic effects of the same drugs?

Moreover, the evidence cited, while admittedly unconvincing, is sufficiently "teasing" to encourage further research along these lines. One of the reasons for the slow progress in this area to date is the lack of appropriate research tools, many of which are just now becoming available. Thus, we are just beginning to understand the biosynthesis and metabolic fate of opioid peptides. This knowledge is essential to a recognition of alterations in the metabolism or turnover of the peptides during chronic morphinization.

 

When it was found that there are no changes in opiate receptors there was no awareness that several different subclasses of opiate receptors exist (see chapter by Dr. R.S. Zukin for a discussion of multiple opiate receptors). A similar statement applies to the measurement of opioid peptides. Only the enkephalins and 13-endorphin have been measured, yet several other peptides have since been found to exist.

 

Thus, I wish to terminate this discussion on an upbeat note by making the prediction that an involvement of the opiate receptor—opioid peptide system in the biochemical mechanism of opiate addiction will be delineated within the next few years. It is not beyond the realm of possibility that a role for this system may also be found in certain other types of substance abuse or compulsive behavior.

 

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