Addiction Research Foundation, Palo Alto, California, 94304.

 

In 1856, Claude Bernard presented his memorable lectures on the South American arrow poison curare. Ushering in the era of modern experimental pharmacology, he localized the unique muscle-paralyzing action of this drug to receptor sites in the neuromuscular junction. It took three-quarters of a century, through the discoveries of Langley, Hunt, Loewi, Dale and others, before it became clear that the curare receptors had an endogenous ligand, and that this ligand was acetylcholine.

 

The subsequent history of pharmacology teaches that, although the universal proposition is unprovable, it has been frequently the case that drug receptors are really receptors for endogenous ligands, that the efficacy of drugs is an accident of molecular architecture.

 

Our discovery of opiate receptors limited to membranes of neural tissue and with a very high degree of stereospecificity (1) was followed within two years by the development of a greatly improved methodology (2,3) which permitted detailed characterization of the receptors. A very uneven regional distribution in the central nervous system suggested specific neuropharmacologic functions, such as pain modulation. The appearance of these receptors at the vertebrate stage of evolution further suggested some specific functional role. Thus, it became obvious that there should be an endogenous ligand. Our own search for such a ligand began in 1972, but not until 1974, did we turn our attention to hydrophilic compounds like peptides. Meanwhile, the painstaking and persistent efforts of Hughes led to identification of methionine enkephalin and leucine enkephalin, two pentapeptides obtained from brain (4,5). By 1975, we had discovered a pituitary peptide with typical opioid effect, much larger than the enkephalins. These developments are summarized in another chapter of this book (6).

 

When a large and a small peptide are known to produce essentially the same biologic effect, it is parsimonious to assume that the large is precursor to the small, and we argued that interpretation at the Airlie House meeting of the International Narcotic Research Club in 1975 (7). Indeed, glutathione (a tripeptide) is the largest peptide known to be synthesized in the vertebrate organism by a direct enzymic peptide synthetase mechanism, so it was also likely, a priori, that the pentapeptide enkephalin was synthesized as part of a larger sequence by the usual ribosomal translation mechanism. When the structure of Met-enkephalin became known, and it was evident that its sequence appeared uniquely as residues 61-65 of the pituitary peptide a-lipotropin (0-LPH), the probability increased greatly that this pituitary peptide of 91 residues was the precursor (prohormone).

 

Was it possible that pituitary a-LPH was cleaved, to yield an active opioid peptide (Endorphin)?* In collaboration with C.H. Li we showed that the whole a-LPH molecule was inactive, but that all fragments cleaved at the 60-61 bond had endorphin activity (8). Thus, in the enkephalin sequence (Tyr-Gly-Gly-Phe-Met), Tyr must have a free amino terminus, so that the basic free a-nitrogen of Tyr can furnish the cationic group required in all known ligands that interact with the opiate receptor. We also showed directly, with a synthetic opioid heptapeptide, that blocking this a-nitrogen abolished opioid activity (9).

 

What is the relationship between pituitary and brain endorphin? It seemed possible that the pentapeptide enkephalins of brain were actually derived from pituitary endorphin [e.g., a-LPH-(61-91)] by successive cleavages, followed b¢ passage from the blood into the brain. Hypophysectomized rats, however, showed no decline in their content of brain opioid peptide activity up to a month after operation in comparison with sham operated controls (10). It follows that brain endorphins, including the enkephalins, are synthesized within the brain. We have now shown that opioid activity extracted from brain is associated with peptides in the 3000-dalton range [the size of a-LPH-(61-91)] or larger, as well as with smaller peptides of the size of enkephalin. It remains uncertain at this time whether the native form that interacts with the opiate receptors is large, or whether the pentapeptide is cleaved in vivo and released into synaptic clefts (11).

 

Future research on opiates will certainly be concerned with the physiologic role of the endorphins. We have now shown that there are several distinct types of endorphin, both in pituitary and in brain. One type is derived from a-lipotropin (a-LPH) and comprises a-LPH-(61-91) and various smaller fragments of the class a-LPH-(61-n), the smallest of which is S-LPH-(61-65) (Met-enkephalin). In brain, we find endorphin activity associated with a peptide in the 3000- dalton range, and like methionine-enkephalin, its activity is reduced by cyanogen bromide. It is possible, therefore, that in the native state enkephalins exist as part of a longer sequence, and it is even possible that they arise as degradation artifacts during the isolation procedure. A smaller endorphin we find in beef brain has an apparent molecular weight of about 1200-1400. Its biologic activity is unaffected by cyanogen bromide treatment (thus, it contains no critically placed Met residue), and unlike the endorphins derived from a-LPH, its activity is greatly reduced by trypsin treatment. It could possibly contain Leu-enkephalin, but its structure must be different in some way from that of a-LPH. Finally, in pituitary, we find that most of the endorphin activity is due to peptides that are not identical to the B-LPH endorphins. These endorphins appear to be more potent than a-LPH-(61-91) or Met-enkephalin, while their sensitivity to cyanogen bromide suggests that they may contain Met-enkephalin.

 

The existence of different endorphins suggests the possibility that there are different endorphinergic systems in brain, probably interacting with non-identical opiate receptors. Pharmacologic evidence had already suggested the presence of different classes of opiate receptor. Martin (12) proposed three types of receptor, designated p, K, and a, based upon differential effects of various classes of opiate agonists and mixed agonist-antagonists. Hutchinson et al. (13) showed that the receptors in guinea pig myenteric plexus and in mouse vas deferens behaved differently with respect to relative affinities for agonists and antagonists.

 

The most significant research problem concerning the endorphins is to elucidate their physiologic roles. Surprisingly, naloxone, which blocks the opiate receptors, has little pharmacologic effect of its own. It is possible, though unlikely, that even when this antagonist completely blocks the effects of high doses of exogenous opiates, it is incapable of blocking the actions of endorphin released in very close proximity. More likely, the endorphinergic systems are not tonically active, but function on a standby basis, being called into play only by circumstances we have not yet fully learned to duplicate. Electrical stimulation of the periaqueductal grey produces analgesia, which is partially blocked by naloxone (14). Acupuncture analgesia is also blocked to some degree by naloxone (15). And there is some evidence that naloxone increases the sensitivity of mice to the noxious effect of a hot plate (16). On the other hand, this antagonist failed to alter the threshold of aversive stimulation at which rats escape from foot shock (17). It seems improbable that a neural system should exist to antagonize acute pain (which, after all, has important survival value), but possibly one function of endorphin is to obtund chronic pain. The definitive experiments have not yet been carried out.

 

It is noteworthy that opiate analgesia is at least in part a consequence of the profound alteration of effective state produced by opiates, causing indifference to all aversive stimuli, including pain. Respiratory depression, release of growth hormone, and inhibition of release of luteinizing hormone are among the many specific effects of opiates. It may well be that the several endorphins serve as neurohumoral agents in a variety of systems, much as do the well known neurotransmitters like acetylcholine and norepinephrine. It is interesting that the opiate receptors are found in high density, not only in pain pathways, but also in areas of brain (e.g., amygdala) which are not thought to be involved in pain.

 

It has long been apparent the opiate effects are not uniquely related to any of the known neurotransmitters, although changes in content and turnover of several transmitters are associated with opiate actions and with tolerance and dependence. Rather, the opiate receptors seem to subserve a general inhibitory function in various neuronal pathways. I proposed (18) that administration of an exogenous opiate might result in suppression of synthesis of endogenous opioids, by analogy to the effects of administering other hormones. Negative feedback loops control the output of hormones and neurotransmitters to maintain appropriate stimulation of target cells. Administration of thyroid hormone, for example, shuts down endogenous synthesis and release by the thyroid gland. Thus, prolonged administration of heroin, saturating the opiate (endorphin) receptors, could cause a shutdown of endorphin production. Some aspects of the withdrawal syndrome could, therefore, be due to endorphin deficiency. Since the neurotransmitter changes in opiate tolerance and dependence are reversed rather quickly upon opiate withdrawal, it is possible that the primary abstinence syndrome is caused by neurotransmitter imbalance, while the secondary, prolonged abstinence syndrome (with its disturbance of affective state) could be caused by endorphin deficiency. The possibility should also be entertained that a pre-existing, genetically determined endorphin deficiency could predispose to opiate addiction.

 

Finally, the fact that opiates are primary reinforcers in operant self-administration paradigms suggest that endorphins may play a central role in the "reward system".

 

It is provocative that one of the few clear effects of naloxone at low dosage is to disrupt appetitive behavior in rats (19,20). One may also speculate that a common pathway shared by all drugs of abuse could involve endorphin and the opiate (endorphin) receptors. The recent reports that ethanol induced calcium depletion from brain membranes in vivo is blocked by naloxone (21) open a fruitful new line of research on the relationship between ethanol and the opiates--research that could lead to better understanding of multiple drug abuse.

 

It is on theoretical grounds improbable that endorphins (or congeners of endorphins) will prove to be non-addicting analgesics. Nor is it likely that useful new medications will result from molecular modification of the endorphin structures. On the contrary, if the endorphins had been discovered before opiates were known, the challenge would certainly have been to develop stable and lipophilic non-peptide analogues to pass the blood-brain barrier and fit the analgesic endorphin receptors. The outcome of such efforts would undoubtedly have been the development of agents like levorphanol, etorphine, meperidine, and methadone. Future research, however, is likely to lead us to more fundamental understanding of the processes of primary reinforcement, tolerance, and dependence, which underlie opiate addiction and other addictive diseases.

 

ACKNOWLEDGEMENTS

 

It is difficult, in describing the work of a whole laboratory group, to ascribe particular experimental advances accurately. The work upon which this chapter is based was carried out by Drs. B.M. Cox, K.E. Opheim, H. Teschemacher, S. Gentlemen, T.-P. Su, and by L.I. Lowney, A.L. Cheung, and M. Ross. These investigations were supported by grants DA-972 and DA-1199 from the National Institute on Drug Abuse.

 

* Endorphin is a generic term to describe opioid peptides without designating any particular chemical structure.

 

REFERENCES

 

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

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

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

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

5. 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).

6. Loh, H.H. and Law, P.-Y.: Pharmacology of endogenous opiate-like peptides, Alcohol and Opiates: Neurochemical and Behavioral Mechanisms. Edited by Blum, K., Basic Books, Inc., New York, New York (1976) (chapter in this volume).

7. Goldstein, A. (editor): The Opiate Narcotics: Neurochemical Mechanisms in Analgesia and Dependence. Proceedings of The International Narcotic Research Club Conference, May 21-24, 1975, Airlie House, Virginia. Pergamon Press, New York (1975).

8. Cox, B.M., Goldstein, A. and Li, C.H.: Opioid activity of a peptide [0-LPH-(61-91)], derived from 13-lipotropin. Proc. Nat. Acad. Sci. U.S.A. 73:1821-1823 (1976).

9. Goldstein, A., Goldstein, J.S. and Cox, B.M.: A synthetic peptide with morphine-like pharmacologic action. Life Sci. 17:1643-1654 (1975).

10. Cheung, A.L. and Goldstein, A.: Failure of hypophysectomy to alter brain content of opioid peptides (endor phins). Life Sci., in press (1976).

11. Goldstein, A.: Opioid peptides (endorphins) in pituitary and brain. Science, in press (1976).

12. Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E., and Gilbert, P.E.: The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmacol. Exp. Ther., in press (1976).

13. Hutchinson, M., Kosterlitz, H.W., Leslie, F.M., Water-field, A.A. and Terenius, L.: Assessment in the guinea-pig ileum and mouse vas deferens of benzomorphans which have strong antinociceptive activity but do not substitute for morphine in the dependent monkey. Brit. J. Pharmacol. 55:541-546 (1975).

14. Akil, H., Mayer, D.J. and Liebeskind, J.C.: Antagonism of stimulation-produced analgesia by naloxone, a narcotic antagonist. Science 191:961-962 (1976).

15. Mayer, D.J., Price, D.D., Raffi, A. and Barber J.: Proc. 1st World Congress on Pain, in press (1976).

16. Jacob, J.J., Tremblay, E.C. and Colombel, M.C.: Facilitation de reactions nociceptives par la naloxone chez la souris et chez le rat. Psychopharmacologia 37:217-223 (1974).

17 Goldstein, A., Pryor, G.T., Otis, L.S. and Larsen, F.: On the role of endogenous opioid peptides: Failure of naloxone to influence shock escape threshold in the rat. Life Sci. 18:599-604 (1976).

18. Goldstein, A.: Are opiate tolerance and dependence reversible: Implications for the treatment of heroin addiction, Biological and Behavioral Approaches to Drug Dependence. Edited by Cappell, H. and LeBlanc, A.E., Proceedings of the International Symposia on Alcohol and Drug Research, Addiction Research Foundation, Toronto, Canada (1975).

19. Holtzman, S.G.: Behavioral effects of separate and combined administration of naloxone and d-amphetamine. J. Pharmacol. Exp. Ther. 189:51-62 (1974).

20. Holtzman, S.G.: Effects of narcotic antagonists on fluid intake in the rat. Life Sci. 16:1465-1470 (1975).

21. Ross, D.H., Medina, M.A. and Cardenas, H.L.: Morphine and ethanol: Selective depletion of regional brain calcium. Science 186:63-65 (1974).