New York State Office of Drug Abuse Services, Testing and Research Laboratory, Brooklyn, New York, 11217.

 

INTRODUCTION

 

Narcotic analgesic drugs, or the opiates (more properly the opioids), have varied chemical and pharmacological characteristics, ranging chemically from the simple meperidine molecule to the structurally complex etorphine molecule (Figure 1), and ranging behaviorally from the euphoriant heroin to the antidiarrheal agent, diphenoxylate. All opiates possess, however, the characteristics that define the drug class: the ability to induce tolerance and physical and psYchic dependence with chronic drug use (1).

The neurochemical mechanisms underlying the phenomena of tolerance, abstinence and drug-seeking behavior have been explored in depth, especially during the past ten years. In this overview, I shall describe briefly the results of some of the studies and the conclusions that may be reached.

 

SITES OF OPIATE ACTION IN THE NERVOUS SYSTEM

 

Anatomical Sites 

 

Opiates produce responses in most brain areas, in the pituitary gland, ganglion cells and spinal cord, as well as at some neuronal junctions outside the central nervous system (2). Opiates depress impulse transmission only at a few neuronal junctions in the autonomic nervous system; these sites are not Characteristic of species, organ or tissue (3). Impulse flow in cholinergic junctions in the guinea-pig ileum and rat vagus, in adrenergic neuronal junctions in the cat nictitating membrane and mouse vas deferens, is inhibited by morphine and other opiates (3). In most mammalian species opiates increase the contractile activity of the intestine, possibly via a serotonergic neuron (4). In the spinal cord, a direct action of opiates is demonstrated in experiments in which the drugs are shown to alter spinal reflexes equally well in intact and in spinally-transsected animals (5). There are, on the other hand, other effects of morphine on spinal neurons involv,ing afferent transmission that require an intact cord (6). In the pons-medulla, where vegetative functions such as respiration, regulation of blood pressure and emesis are sited, opiates act to disturb these functions (2). Temperature regulation is affected by the intrahypothalamic application of opiates, and the hypophyseal-hypothalamic hormonal system is also affected by opiate administration (7). The limbic system including the neocortex has also been implicated in opiate actions, particularly behavioral responses (8). Thus, the sites of opiate action are located throughout the central nervous system and at some peripheral neuronal junctions.

 

Neurochemical Sites 

 

When radiolabelled opiates are administered to laboratory animals, the drugs are found in all brain regions with a temporal and spatial distribution related to the lipid solubilities of the drugs rather than to their pharmacological potencies (9). However, the potency of one opiate, etorphine, is so high that an effective dose via the intracerebral route is about 1/100th that of morphine; a dose that diminishes nonspecific binding so that specific changes may be measured. The binding of 3H-etorphine in synaptic membranes of rat brain is reduced to one-half when naloxone is also injected in doses sufficient to block the pharmacological responses of etorphine (10).

 

Opiates bind to brain tissue in vitro also, in an interaction that is stereospecific, with high affinity of drug to receptor and a saturable binding (11-13). Isolated synaptic membranes from many brain areas contain opiate receptors, with a heterogeneous distribution (14,15). Thus, synaptic membranes are identified as the site of the initial drug:receptor interaction.

 

Molecular Sites 

 

The stereospecific binding of opiates to their receptors is competitive, with EC50 related to the potencies of the drugs (16). The binding of agonists and antagonists can be distinguished by sodium ions, which decrease agonist binding and enhance antagonist binding (17). The ratios of EC50 values determined in the presence and absence of 100 mM NaC1 range from 1.0 for naloxone and naltrexone (the purest antagonists) to 12-60 for agonists, with mixed agonist-antagonists in the intermediate range (16).

 

The opiate receptor has not been isolated as yet. However, an opiate:receptor complex has been solubilized from a rat brain synaptosomal preparation with the non-ionic detergent, Brij 36T (18). The bound opiate is dissociated by treatment with proteolytic enzymes, sulfhydral reagents and heat, suggesting that the receptor is a protein (18).

 

After the initial drug:receptor complex is formed, it must produce a transduction of the information to secondary effector processes. A tentative identification of the cyclic nucleotide systems as the mediators of this transduction is supported by two general lines of evidence: (1) the nature of the cAMP and cGMP systems as amplifiers in the regulation of neurotransmission by ions, neurotransmitters and neurohormones (19), and (2) the changes found experimentally in the levels of cyclic nucleotides and the sensitivity of cyclases in brain (20-22) and in neuronal cells in culture (23,24). The nature of the further reactions in the neuron, and elsewhere, is related to the specific functions of the cells involved.

 

The development of tolerance may be considered as a series of adaptive responses to the continual presence of a potent drug. Since tolerance had been demonstrated in many neurochemical parameters, I shall discuss those for which a biochemical mechanism is known in appropriate sections of this chapter.

 

 

Catecholamines 

 

The administration of morphine or other opiates to laboratory animals induces alterations in the content and the rate of turnover of catecholamines in brain. Both dopamine and norepinephrine levels fall after opiates are administered acutely (25). The effect is dose-dependent and multiphasic (26,27). The most prominent effect of opiates on the turnover of catecholamines is a sharp rise in the rate of dopamine biosynthesis in striatum after a single dose of morphine (29,30). Tolerance develops in this biochemical parameter as opiates are used chronically (31).

 

The administration of non-opiate drugs to animals often affects the response to opiates. Drugs that tend to increase the levels of dopamine at the receptor (such as iproniazid, apomorphine or cocaine) generally enhance the pharmacological responses to opiates, while drugs that decrease dopamine levels (such as haloperidol, alpha-methylDOPA) inhibit opiate action (32).

 

Acetylcholine 

 

Opiates have an antirelease action at cholinergic synapses, and effect antagonized by naloxone and absent in tolerant animals (33). The antirelease activity has been demonstrated in cortical slices, in perfused brain and on the cortical surface, as well as in vivo (34). In morphine-tolerant animals, the dose-effect curve for inhibition of neocortical acetylcholine release is shifted to the right, i.e. tolerance develops in this response (35). During withdrawal from morphine use, brain acetylcholine is utilized at an enhanced rate (36), and an increase in the level of free acetylcholine is found, suggesting that release is enhanced after drug removal (37)

 

In low concentrations, opiates prevent contraction of stimulated guinea-pig ileum preparations by inhibiting acetylcholine release (38). Short term tolerance to this effect has been attributed to a disuse supersensitivity phenomenon: after exposure to an opiate for a period of time, the ileum increases its response to a fixed dose of acetylcholine three to fivefold (39).

 

Serotonin

 

The acute administration of morphine to rodents accelerates the rate of biosynthesis of serotonin in brain (40). This effect is located in the forebrain (41). The biochemical correlate for this effect may be changes in tryptophan hydroxylase activity in nerve-ending fractions of forebrain areas; the synaptosomal enzyme activity decreases two or three hours after initial dose of morphine, and is increased in tolerant animals (42). An inhibitor of serotonin biosynthesis, pdhlorophenylalanine, antagonizes the pharmacological responses to morphine (43-45). The ratio of serotonin to catecholamines in brain seems to be more important than actual levels of the biogenic amines for morphine-induced analgesia (46), temperature alterations (47,48) or effects on oxytremorine analgesia (49). In these studies, norepinephrine seems to be a morphine antagonist, while serotonin potentiates morphine responses.

 

The stimulation of contraction' of the dog intestine by morphine occurs at the same time that endogenous serotonin is released (4), an effect antagonized by naloxone (50). Tolerance develops in this biochemical parameter in tolerant animals (50).

 

Other Neurotransmitters 

 

GABA levels seem to be important for the expression of some pharmacological responses to morphine administration. When GABA levels in brain are increased either by administering GABA, or an inhibitor of GABA catabolism, aminooxyacetic acid, the responses to acute morphine are reduced (51). The development of tolerance and dependence are accelerated by these same agents (51). In subcortical areas of brain of morphine-tolerant rats, the levels of GABA are increased. Thus, GABA acts as an opiate antagonist (in the pharmacological sense of the word).

 

Histamine release in peripheral tissues is stimulated by morphine (52). In the CNS, however, no changes in histamine levels have been seen in animals treated acutely with morphine. In morphine-tolerant animals, however, hypothalamic levels of histamine are reduced, and are even lower in the hypothalamus of animals undergoing withdrawal (53).

 

Unique Role for One Neurotransmitter 

 

The studies just described suggest that all known neurotransmitters are involved in the actions of opiates (and in the expression of these actions), and that no one neurotransmitter plays a unique role in either opiate action or dependence.

 

ROLE OF NEUROREGULATORS IN OPIATE ACTIONS AND DEPENDENCE

 

Ions

 

Any disturbance of the ionic milieu of the neuronal cell can be expected to alter neuronal function. However, calcium ions seem to have a specific role in the action of opiates. The analgesic effect of morphine in mice is depressed by the intracisternal administration of calcium ions, and potentiated by the administration of a calcium ion-chelator, EDTA (54). Brain levels of this ion are lower in mice treated with morphine, an effect to which tolerance develops (55). In rats, a depletion of calcium ions is produced by morphine administration (56,57). Not only calcium ions, but also magnesium ions and manganese ions increase the AD50 for morphine (58). These latter investigators have concluded the pharmacological responses to morphine in mice are directly related to the intracellular levels of calcium ions in brain (58). Lanthanum ions, which are antagonists of calcium ions in several biological systems, produce analgesia when introduced into the CSF, an effect antagonized by calcium ions or naloxone (59).

 

The monovalent ion, lithium has a sedative effect in man and animals. In mice, morphine is able to reverse the inhibition of spontaneous locomotor activity induced by lithium ions (60). Conversely, lithium ions reverse some morphine responses; namely, analgesia (61) and body temperature regulation (62).

 

Prostaglandins 

 

In the guinea-pig ileum, morphine inhibits the contractions evoked by prostaglandins (PG) as well as by electrical stimulation (63). In rat brain homogenates, opiates inhibit the stimulation of cAMP formation by PGs (64). Similar effects have been observed in cultured hybrid cells (neuroblastoma X glioma) (23,24). At low concentrations of opiate, the responsiveness of adenylate cyclase to PGs is enhanced, while at higher concentrations, the enzyme is inhibited (23). Since PGs hyperpolarized neuronal cells (65), thus inhibiting the transmission of the nerve impulse, a reversal of PG-induced hyperpolarization by opiates would lead to the activation of inhibitory pathways, and a selective depression of neuronal activity.

 

Cyclic Nucleotides 

 

When cAMP is injected systemically into rodents together with morphine, the analgesic response is antagonized (66). In morphine-tolerant animals, the administration of cAMP systemically (67) or into the CSF (68) increases withdrawal signs. Adenine, adenosine and ATP also decrease the acute effects of opiates (69), suggesting that the adenosine moiety may be responsible for the effects.

 

Opiate administration to animals has varying effects on the levels of cyclic nucleotides and the activity of adenylate and guanylate cyclases (70). Both the nucleotide levels and the cyclase activities may be expected to fluctuate widely over a short time period in whole tissue, making it difficult to discern relevant alterations in either parameter. In hybrid cells in culture, however, opiates can be shown to have effects on the cyclic nucleotide system. The stimulation of adenylate cyclase activity by PGEs is inhibited by morphine (71). An effect that is stereospecific is the increase in cGMP levels induced by morphine in these cells (65). In rat brain, synaptosomal adenylate cyclase activity is not affected by opiates added in vitro (21). Dopamine-sensitive adenylate cyclase in synaptosomal preparations from rat striatum is not inhibited by opiates (20). Thus, opiates do not act directly on postsynaptic dopamine receptors in this brain region, as neuroleptics do.

 

Peptides 

 

Responses to morphine are altered by the simultaneous administration of neuroactive peptides. For example: an analog of vasopressin increases the analgesic effect of morphine in mice (72) and somatostatin antagonizes the stimulation of growth hormone (GH) release induced by morphine (73). B-melanotropin and adrenocorticotropin (ACTH) 1_24 antagonize the pharmacological responses to morphine (74) and substance P abolishes the abstinence syndrome (75). The injection of thyrotropin-releasing hormone (TRH) into certain brain areas produces shaking behavior in rats very like the abstinent shaking produced by the micro-injection of naloxone into the same areas in morphine-tolerant animals (76).

It may be necessary to re-examine some of these effects in light of the possibility that the peptide preparations may have been contaminated with the endogenous morphine-like peptides recently discovered.

Pituitary Peptide Hormones 

 

The administration of narcotic analgesic drugs to man or animalsiroduces changes in the rates of release of peptide hormones from the anterior and posterior gland. Acute administration of morphine increases the secretion of ACTH from the pituitary: this is a relatively specific effect since it is blocked by nalorphine, and induced only with the L-isomer of methadone (77). Chronic morphine treatment reduces ACTH release to a low normal level in man (78) and in animals (79). Low doses of morphine increase the secretion of GH, while higher doses increase the secretion of both GH and gonadotropins (80). In contrast, morphine inhibits the release of TSH (81). The relationship of the hypothalamus and other brain areas to the pituitary gland is complex (Figure 2). Neuronal cells in discrete hypothalamic nuclei contain the oligopeptide releasing factors that are transported to the pituitary where they influence the rate of tropic hormone release. For some of the effects of opiates on the release of peptide hormones from the anterior pituitary, the hypothalamic dopaminergic neurons are the mediators. The neurons controlling LH-RF, GH-IF, prolactin-inhibitory factor and MIF are all dopaminergic and inhibited by morphine and methadone (82), thus the release of LH is inhibited while the release of the other peptide hormones is accelerated by the inhibition of inhibitory factors.

 

Morphine produces an antidiurectic response that is effected in part by the release of antidiuretic hormone from the posterior pituitary gland (83). With the development of tolerance, the antidiurectic effect disappears and a diuretic effect may sometimes be found (84). The peptide hormones of the posterior pituitary gland, vasopressin and oxytocin, are synthesized in the supraoptic and paraventricular nuclei of the hypothalamus, presumably from a larger peptide (85). It is possible that the effects of opiates on these hormones is also mediated by neurotransmitters in the hypothalamus.

 

The regulation of synthesis and release of hypophyseal peptide hormones by neural pathways and by feedback control by pituitary hormones, hormones in the target tissue and hypothalamic releasing factors is suggested in Figure 2. An additional pathway for the regulation is the short feedback loop whereby pituitary hormones enter the CSF in the basilar cisterns and are transported to the periventricular organ system of the hypothalamus, and to other brain areas, and hypothalamic releasing hormones are released into the CSF to return directly to the site of their origin (86,87). These routes may be important for the dissemination of the morphine-like peptides.

 

Unique Role for a Neuroregulatory System 

 

The studies described above suggest that all neuroregulatory systems play a role in the actions of opiates, and that neuronal responses to the presence of opiates are mediated through normal cell mechanisms.

 

ENDORPHINS

 

The presence in nervous tissue of endogenous ligands, substances that bind stereospecifically to opiate receptors, was suggested by the unique characteristics of the receptors. The discovery of possible ligand activity was first reported by Terenius and Wahlstrom (88) and by Hughes and his colleagues (89). Other groups have described the partial purification of 'morphine-like' factors from brain (90) and from the pituitary gland (91). The chemical identification of brain 'morphine-like' factor as two pentapeptides with the following structure: tyr-gly-gly-phe-X, with X=methionine or leucine, and the recognition that the met-peptide is a sequence of the pituitary peptide hormone, 13-lipotropin, was made by Hughes, Kosterlitz and their colleagues (92). These pentapeptides are called enkephalins. However, the generic name for the enkephalins and other substances with morphine-like activity is endorphin. Segments of f3-lipotropin containing the met-enkephalin sequence of amino acids bind stereospecifically to opiate receptors, and act like opiates in isolated tissue preparations, guinea-pig ileum and mouse vas deferens (93,94). Some of the endorphins are shown in Figure 3. Morphine-like pharmacological activity in vivo has also been demonstrated for the endorphins when the destruction of the peptides by tissue esterases is prevented (95). Opiate-like effects, antagonism by naloxone, tolerance and cross-tolerance have all been demonstrated for the endorphins (96-98). Thus, these peptides are endogenous substances with all of the specific biological activity of the narcotic analgesic drugs.

 

Endorphins are distributed throughout the central nervous system (99). The regulation of their biosynthesis and catabolism, and the sites of their biosynthesis and catabolism, and the sites of their biosynthesis and action, await further study. It is possible that the small peptides may be synthesized in many parts of brain by the usual ribosomal type of protein biosynthesis, or that they are synthesized without ribosomal participation (like glutathione), or that they are formed by hydrolysis of larger peptides synthesized in the anterior pituitary gland. In our laboratory, we have been able to radiolabel enkephalins in vivo by injecting 3H-amino acid precursors into the rat CSF and isolating labelled enkephalins from the rat brain after thirty minutes (100). These experiments do not shed light on the site of enkephalin formation, but do suggest that endorphins are readily formed in brain. An understanding of the role that endorphins play in the actions of opiates is in its infancy. This knowledge will be most useful because the synthesis, metabolism and function of the endorphin system must be involved in the mechanisms of action of narcotic analgesic drugs in the central nervous system.

 

SUMMARY

 

1. Narcotic analgesic drugs act throughout the central nervous system and at some peripheral neuramuscular junctions.

2. Opiate receptors at these sites may be identified by characteristic stereospecific binding of narcotic agonists and antagonists.

3. The initial drug:receptor interaction occurs in synaptic membranes, and may be followed by transduction through the cyclic nucleotide system.

4. Dopamine, norepinephrine, serotonin, acetylcholine and other neurotransmitters play a role in the action of opiates, but none play a unique role.

5. The neuroregulatory systems in brain also are involved in the mechanisms of action of the opiates.

6. The naturally occurring endorphins can be expected to play an important part in the mechanisms of opiate action, tolerance and dependence.

 

REFERENCES

 

1. WHO Expert Committee on Drug Dependence: Drug Dependence: Its Significance and Characteristics. Bull. World Health Org. 32:721-734 (1965).

2. Borison, H.L.: Sites of action of narcotics in the nervous system, Narcotic Drugs: Biochemical Pharmacology. Edited by Clouet, D.H., Plenum Press, New York, p. 342364 (1971).

3. Kosterlitz, H.W. and Waterfield, A.A.: In vitro models in the study of structure-activity relationships of narcotic analgesics. Ann. Rev. Pharmacol. 15:29-47 (1975).

4. Burks, T.F.: Gastrointestinal pharmacology. Ann. Rev. Pharmacol. 16:15-32 (1976).

5. Martin, W.R., Eades, C.G., Thompson, W.O., Thompson, J.A. and Flanary, H.G.: Morphine physical dependence in the dog. J. Pharmacol. exp. Therap. 189:759-771 (1974).

6. Takagi, H., Satoh, M., Doi, T., Kawasaki, K. and Akaike, A.: Indirect and direct depressive effects of morphine on activation of lamina V cells of the spinal dorsal horn induced by intra-arterial injection of bradykinin. Arch. Internat. Pharmacol. Ther. 221:96-104 (1976).

7. George, R.: Drug effects on endocrine function. Prog. Brain Res. 39:339-346 (1973).

8. Ervin, F.R.: Effects of opioids on electrical activity of deep structures in the human brain, Addictive States. Edited by Wikler, A., Williams and Wilkins, Baltimore, p. 150-156 (1968).

9. Clouet, D.H. and Williams, N.: Localization in brain particulate fractions of narcotic analgesic drugs administered intracisternally to rats. Biochem. Pharmacol. 22:1283-1293 (1973).

10. Mule, S.J., Casella, G. and Clouet, D.H.: The specificity of binding of the narcotic agonist etorphine in synaptic membranes of rat brain in vivo. Psychopharmacol. 44:125-129 (1975).

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

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

13. Simon, E.J., Hiller, J.M. and Edelman, I.: Stereospecific binding of the potent narcotic analgesic 38-etorphine to rat brain homogenate. Proc. Nat. Acad. Sci. 70:19471949 (1973).

14. 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).

15. Hiller, J.M., Pearson, J. and Simon E.J.: Distribution of stereospecific binding of the potent narcotic analgesic etorphine in the human brain. Res. Comm. Chem. Path. Pharmacol. 6:1052-1062 (1973).

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

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

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

19. Greengard, P.: Cyclic nucleotides, protein phosphorylation and neuronal function. Adv. Cyclic Nucleo. Res. 5:585-601 (1975).

20. Iwatsubo, K. and Clouet, D.H.: Dopamine-sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine or haloperidol. Biochem. Pharmacol. 24: 1499-1503 (1975).

21. Clouet, D.H., Gold, G.J. and Iwatsubo, K.: Effects of narcotic analgesic drugs on the cyclic adenosine 3',5'- monophosphate-adenylate cyclase system in rat brain. Brit. J. Pharmacol. 54:541-548 (1975).

22. Racagni, G., Zsilla, G., Guidotti, A. and Costa, E.: Accumulation of cGMP in striatum of rats injected with narcotic analgesics: Antagonism by naltrexone. J. Pharm. Pharmacol. 28:258-260 (1976).

23. Traber, J., Gullis, R. and Hamprecht, B.: Influence of opiates on the levels of adenosine 3',5'-cyclic monophosphate in neuroblastoma X glioma hybrid cells, The Opiate Narcotics. Edited by Goldstein, A., Pergamon Press, New York, p. 111-116 (1975).

24. Klee, W.A., Sharma, S.K. and Nirenberg, M.: Opiate receptors as regulators of adenylate cyclase, The Opiate Narcotics. Edited by Goldstein, A., Pergamon Press, New York, p. 117-122 (1975).

25. Takagi, H. and Nakama, M.: Studies on the mechanism of action of tetrabenazine as a morphine antagonist. Japan. J. Pharmacol. 18:54-58 (1968).

26. Rethy, C.R., Smith, C.B. and Villarreal, J.: Effects of narcotic analgesics upon locomotor activity and brain catecholamine content of the mouse. J. Pharmacol. exp. Ther. 176:472-279 (1971).

27. Ahtee, L.: Catelepsy and stereotyped behavior in rats treated with methadone chronically: Relation to brain homovanillic acid levels. J. Pharm. Pharmacol. 25:649651 (1973).

28. Fukui, K. and Takagi, H.: Effect of morphine on cerebral content of metabolites of dopamine in normal and tolerant mice: Its possible relation to analgesic action. Brit. J. Pharmacol. 44:45-51 (1972).

29. Clouet, D.H. and Ratner, M.: Catecholamine biosynthesis in the brains of rats treated with morphine. Science 168:854-856 (1970).

30. Costa, E., Carenzi, A., Guidotti, A. and Revuelta, A.: Narcotic analgesics and the regulation of catecholamine stores, Frontiers in Catecholamine Research. Edited by Costa, E. and Usdin, E., Pergamon Press, New York, p. 1003-1010 (1973).

31. Smith, C.B., Sheldon, M.I., Bednarczyk, H.J. and Villarreal, J.: Morphine-induced increases in the incorporation of 14C-tyrosine into14 C-dopamine and I4Cnorepinephrine in the mouse brain. J. Pharmacol. exp. Ther. 180:547-557 (1972).

32. Clouet, D.H.: Possible roles of catecholamines in the action of narcotic drugs, Catecholamines and Behavior, Vol. II. Edited by Friedhof, A.J., Plenum Publishing Co., New York, p. 167-189 (1975).

33. Labreque, G. and Domino, E.F.: Tolerance to and physical dependence on morphine. J. Pharmacol. exp. Ther. 191:189200 (1974).

34. Domino, E.F., Vasko, M.R. and Wilson, A.E.: Mixed depressant and stimulant actions of morphine and their relationship to brain acetylcholine. Life Sci. 18:361376 (1976).

35. Large, W.A. and Milton, A.S.: The effects of acute and chronic administration on brain acetylcholine levels. Brit. J. Pharmacol. 38:451P-452P (1970).

36. Crossland, J.: Acetylcholine and morphine abstinence syndrome, Drugs and Cholinergic Mechanisms in the CNS. Edited by Heilbronn, E. and Winter, A., Forsvarets Forsk., Stockholm, p. 634-645 (1970).

37. Domino, E.F. and Wilson, A.: Enhanced brain acetylcholine utilization during morphine withdrawal in the rat. Nature 243:285-286 (1973).

38. Cox, B.M. and Weinstock, M.: The effects of analgesic drugs on the release of acetylcholine from electrically-stimulated guinea-pig ileum. Brit. J. Pharmacol. 27:8192 (1966).

39. Shoham, S. and Weinstock, M.: The role of supersensitivity to acetylcholine in the production of tolerance to morphine in stimulated guinea-pig ileum. Brit. J. Pharmacol. 52:597-603 (1974).

40. Yarborough, G.G., Buxbaum, D.M. and Sanders-Bush, E.: Biogenic amines and narcotic effects. J. Pharmacol. exp. Ther. 185:328-334 (1973).

41. Azmitia, E.C., Hess, P. and Reis, D.: Tryptophan hydroxylase changes in midbrain of the rat after chronic morphine administration. Life Sci. 9:633-637 (1970).

42. Knapp, S. and Mandell, A.J.: Narcotic drugs: Effects on the serotonin biosynthetic systems of the brain. Science 177:1209-1211 (1972).

43. Tenen, S.S.: Antagonism of the analgesic effect of morphine and other drugs by p-chlorophenylalanine, a serotonin depletor. Psychopharmacol. 12:278-285 (1968).

44. Berney, S.A. and Buxbaum, D.M.: The effect of morphine on catecholamine turnover and its relationship to morphine-induced motor activity. Pharmacologist 15:202 (1973).

45. Way, E.L., Loh, H. and Schen, F.H.: Morphine tolerance, physical dependence and the synthesis of brain serotonin. Science 162:1290-1292 (1968).

46. Sparkes, C.G. and Spencer, P.S.J.: Antinociceptive activity of morphine after injection of biogenic amines in cerebral ventricles of the conscious cat. Brit. J. Pharmacol. 42:230-241 (1971).

47. Feldberg, W. and Sherwood, S.L.: Injection of drugs into the lateral ventricles of the cat. J. Physiol. 107:372-381 (1954).

48. Oka, T., Nozaki, M. and Hosoya, E.: The effect of cholinergic antagonists on the increases of spontaneous motor activity and body weight induced by the administration of morphine to tolerant rats. Psychopharmacol. 23:231-238 (1971).

49. Calcutt, C.R., Doggett, N.S. and Spencer, P.S.J.: Modification of the antinociceptive effect of morphine by centrally administered ouabain and dopamine.

50. 518-526 (1974).

51. Ho., I.K., Loh, H.H. and Way, E.L.: Influence of GABA on morphine analgesia, tolerance and physical dependence. Proc. West. Pharmacol. Soc. 16:4-7 (1973).

52. Schmidt, C.F. and Livingston, A.E.: The action of morphine on mammalian circulation. J. Pharmacol. exp. Ther. 47:411-420 (1933).

53. Henwood, R.W. and Mazurkiewicz-Kilwecki, I.M.: Possible role of brain histamine in morphine addiction, The Opiate Narcotics. Edited by Goldstein, A., Pergamon Press, New York, p. 209-210 (1975).

54. Kakunaga, T., Kaneto, H. and Hano, K.: Significance of the calcium ion in morphine analgesia. J. Pharmacol. exp. Ther. 153:134-141 (1966).

55. Shikimi, T., Kaneto, H. and Hano, K.: Effect of morphine on the liberation of acetylcholine from the mouse cerebral cortex slices in relation to Ca++ concentration in the medium. Jap. J. Pharmacol. 17:136-137 (1967).

56. Ross, D.H.: Tolerance to morphine-induced Ca++ depletion in regional brain areas: Characterization with reserpine and protein synthesis inhibitors. Brit. J. Pharmacol. 55:431-437 (1975).

57. Sanghvi, I.S. and Gershon, S.: Morphine dependent rats: Blockade of precipitated abstinence by calcium. Life Sci. 18:649-654 (1976).

58. Harris, R.A., Loh, H.H. and Way, E.L.: Effects of divalent cations, cation chelators and an ionophore on morphine analgesia and tolerance. J. Pharmacol. exp. Ther. 195:488-498 (1975).

59. Harris, R.A., Twamoto, E.T., Loh, H.H. and Way, E.L.: Analgetic effects of lanthanum: Cross-tolerance with morphine. Brain Res. 100:221-225 (1975).

60. Lal, S. and Sourkes, T.L.: Potentiation and inhibition of the amphetamine stereotypy in rats by neuroleptics and other agents. Arch. Inter. Pharmacodyn. 199:289301 (1972).

61. Jensen, J.: The effect of prolonged lithium ingestion on morphine actions in the rat. Acta Pharmacol. Toxicol. 35:395-402 (1974).

62. Tulunay, F.C., Kiran, B.K. and Kaymakcalan, S.: Interaction between morphine and lithium. Acta Med. Turc. 8: Psychopharmacol. 21:111 -114 (1971).  vBurks, T.F. and Grubb, tolerance in intestine. M.N.: Sites of acute morphine Pharmacol. exp. Ther. 191:  51-60 (1971).

63. Jaques. R.: Morphine as an inhibitor of prostaglandin E1 in the isolated guinea-pig intestine. Experientia 25: 1059-1060 (1969).

64. Collier, H.O.J. and Roy, A.C.: Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenates. Nature 248:24-27 (1974).

65. Traber, J., Reiser, G., Fisher, K. and Hamprecht, D.: Measurements of cAMP and membrane potential in neuroblastoma-glioma hydrid cells. Opiates and adrenergic agonists cause effects opposite to prostaglandin El. FEBS Lett 52:327-333 (1975).

66. Ho, I.K., Loh, H.H. and Way, E.L.: Effect of cAMP on morphine analgesia, tolerance and dependence. Nature 238:397-398 (1972).

67. Ho, I.K., Loh, H.H. and Way, E.L.: Cyclic adenosine monophosphate antagonism of morphine analgesia. J. Pharmacol. exp. Ther. 185:336-346 (1973).

68. Collier, H.O.J. and Francis, D.L.: Morphine abstinence is associated with increased brain cAMP. Nature 255-: 159-162 (1975).

69. Gourley, D.R.J. and Beckner, S.K.: Antagonism of morphine analgesia by adenine, adenosine and adenine nucleotides. Proc. Soc. Exp. Biol. Med. 144:774-778 (1973).

70. Clouet, D.H.: Opiate and the brain adenylate cyclase system, Tissue Responses to Addictive Drugs. Edited by Ford, D.H. and Clouet, D.H., Spectrum Publications, Hollishead, New York, p. 99-105 (1976).

71. Sharma, S.K., Nirenberg, M. and Klee, W.A.: Morphine receptors as regulators of adenylate cyclase activity. Proc. Nat. Acad. Sci. 72:590-594 (1975).

72. Krivoy, W.A., Zimmermann, E. and Lande, S.: Facilitation of the development of resistance to morphine analgesia by des-glycinamide lys-vasopressin. Proc. Nat. Acad. Sci. 71:1852-1856 (1974).

73. Ferland, L., Labrie, F., Coy, D.H., Arimura, A. and Schally, A.V.: Inhibition by somatostatin analogs of plasma GH levels stimulated by morphine in the rat. Mol. Cell. Endocrinol. 4:79-88 (1976).

74. Zimmermann, E. and Krivoy, W.A.: Antagonism between morphine and the polypeptides ACTH, ACTHi_ 24 and MSH in the nervous system. Prog. Brain Res. 39:383-392 (1973).

75. Stern, P. and Hadzovic, S.: Pharmacological analysis of central actions of substance P. Arch. Inter. Pharmacodyn 202:259-262 (1973).

76. Wei, E., Sigal, S., Loh, H.H. and Way, E.L.: TRH and shaking behavior in the rat. Nature 253:739-740 (1975).

77. George, R. and Way, E.L.: Studies on the mechanisms of pituitary-adrenal activation by morphine. Brit. J. Pharmacol. 10:260-264 (1955).

78. Eisenmann, A.J., Fraser, H.T. and Brooks, J.W.: Urinary excretion and plasma levels of 17-hydroxycorticosteroids during a cycle of addiction to morphine. J. Pharmacol. exp. Therap. 132:226-231 (1961).

79. Slusher, M.G. and Browning, B.: Morphine inhibition of plasma corticosteroid levels in chronic catheterized rats. J. Pharmacol. exp. Ther. 200:1032-1034 (1961).

80. Kokka, N., Garcia, J.F. and Elliott, H.W.: Effects of acute and chronic administration of narcotic drugs on growth hormone and corticotropin secretion in rats. Progr. Brain Res. 39:347-360 (1973).

81. George, R. and Lomax, P.: The effects of morphine, chlorpromazine and reserpine on pituitary-thyroid activity in rats. J. Pharmacol. exp. Ther. 150:129-136 (1965).

82. Clemens, J.A. and Sawyer, B.D.: Evidence that methadone stimulates prolactin release by dopamine receptor blockade. Endocrinol. Res. Comm. 1:373-378 (1971).

83. George, R. and Way, E.L.: The role of the hypothalamus in pituitary-adrenal activity and antidiuresis by morphine. J. Pharmacol. exp. Ther. 125:111-117 (1959).

84. Inturissi, C.E. and Fujimoto, J.M.: Development of tolerance to the antidiuretic action of morphine in the rat. Eur. J. Pharmacol. 2:301-307 (1968).

85. Livett, B.G.: Immunochemical studies on the storage and axonal transport of neurophysins in the hypothalamoneurohypophyseal system. Ann. N.Y. Acad. Sci. 248:112133 (1975).

86. Witter, A.: The in vivo fate of brain oligopeptides. Biochem. Pharmacol. 24:2025-2030 (1975).

87. Kawakami, M. and Sakuma, Y.: Electrophysiological evidence for possible participation of periventricular neurons in anterior pituitary regulation. Brain Res. 101:79-94 (1976).

88. Terenius, L. and Wahlstrom, A.: Search for the endogenous ligand for the opiate receptor. Acta Physiol. Scand. 94:74-81 (1974).

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

90. Simantov, R. and Snyder, S.H.: Morphine-like peptides in mamalian brain: Isolation, structure elucidation, and interactions with the opiate receptor. Proc. Nat. Acad. Sci. 73:2515-2519 (1976).

91. Cox, B.M., Goldstein, A. and Li, C.H.: Opioid activity of a peptide, (3-lipotropin (61-91), derived from alipotropin. Proc. Nat. Acad. Sci. 73:1821-1823 (1976).

92. 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).

93. Kosterlitz, H.W. and Hughes, J.: Some thoughts on the significance of enkephalin, the endogenous ligand, The Opiate Narcotics. Edited by Goldstein, A., Pergamon Press, p. 245-250 (1975).

94. Li, C.H. and Chung, D.: Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary glands. Proc. Nat. Acad. Sci. 73:1145-1148 (1976).

95. Wei, E.: Chronic, intracerebral infusion of opiates and peptides with osmotic minipumps, and the development of physical dependence, Cellular Effects of Opiates. Edited by Kosterlitz, H.W., North-Holland, Amsterdam (1976).

96. Lazarus, L.H., Ling, N. and Guillemin, R.: 0-lipotropin as a prohormone for the morphinomimetic peptides endorphins and enkephalins. Proc. Nat. Acad. Sci. 73:21562159 (1976).

97. Buscher, H.H., Hill, R.C. Romer, D., Cardinaux, F., Closse, A., Hauser, D. and Pless, J.: Evidence for the analgesic activity of enkephalin in the mouse. Nature 26/:423-425 (1976).

98. Minneman, K.P. and Iversen, L.L.: Enkephalin and opiate narcotics increase cyclic GMP accumulation in slices of rat neostriatum. Nature 262:313-314 (1976).

99. Simantov, R., Kuhar, M.J., Pasternak, G.W. and Snyder, S.H.: The regional distribution of a morphine-like factor enkephalin in monkey brain. Brain Res. 106:189-197 (1976).

100. Clouet, D.H. and Ratner, M.: The incorporation of 3Hglycine into enkephalin in the brains of morphine-treated rats, Cellular Effects of Opiates. Edited by Kosterlitz, H.W., North-Holland, Amsterdam (1976).