Horace H. Loh and Ping-Yee Law

Langley Porter Neuropsychiatric Institute and Department of Pharmacology, University of California, San Francisco, California, 94122.

* This work was supported in part by DA-4RG-012 and by DA-00564. H.H.L. is a recipient of a NIMH Research Scientist Career Development Award, K2-DA-70554. This manuscript reviews the literature to May 30, 1976.

 

INTRODUCTION

 

The presence of endogenous substances which interact with existing cellular receptors has been observed in numerous systems. Steroids express their action by interacting with cytoplasmic receptors in the target tissue. Peptide hormones, e.g., insulin, express their action by interacting with their respective membrane receptors and thus altering the intracellular level of c-AMP. Since it was demonstrated by Terenius (1), Pert and Snyder (2), Simon et a/. (3), and Wong and Horng (4) that analgesic action of the opiates probably was the consequence of a stereospecific interaction between the opiates and receptors in the CNS, isolation of an endogenous substance which has high affinity for such receptors is probable. The interaction between the opiates and the receptors is highly stereospecific, the receptors have functional specificity and only compounds with opiate agonistic or antagonistic properties have appreciable affinity for the receptor [neurotransmitters have no affinity (4,5)]. Intuitively, it is unlikely that there be a highly specific receptor with functional activity and no endogenous ligands to interact with. Such an endogenous substance was proposed initially to exist in the neural tissues by Collier (6) in 1972, and later by Goldstein (7) in 1973. Their postulation was not without experimental basis. Earlier reports by Murray and Miller (8) and Huidobro and Miranda (9) suggested the presence of an endogenous agonist-like substance. Murray and Miller reported the lowering of narcotic AD50 with a pituitary extract of oxytocin which was absent when synthetic oxytocin was used instead. Huidobro observed the modification of morphine response in mice by extracts from mice chronically treated with morphine. More important, the intrinsic activity of naloxone in various systems further supports the postulation of the existence of an endogenous compound. It has been observed that naloxone (1) increases acetylcholine release in the in vitro preparation of guinea pig ileum (10); (2) enhances the nociceptive response in rats and mice (11); and (3) reverses electroanalgesia in rats (12). Such observations could be attributed to naloxone antagonizing the action of an endogenous morphine-like substance although Goldstein et al. (13-15) have found otherwise. It is the purpose of this chapter to review the results of the search for endogenous morphine-like substances, namely enkephalins, a- and 8-endorphin and other related peptides. We will also review the present evidence for the existence of an antagonist-like substance and the numerous peptides which modulate morphine effect, namely adrenocorticotropic hormone (ACTH), melanocytestimulating hormone (MSH), thyrotropin-releasing hormone (TRH) and Substance P.

 

ENDOGENOUS SUBSTANCES WITH MORPHINE-LIKE ACTIVITY

 

Before the isolation of any substance, specific assay methods to distinguish such substances from others have to be available. Stereospecific binding of radioactive opiates to the synaptic plasma membrane (SPM) fractions as suggested by Goldstein et a/. (16), and the bioassay of mouse vas deferens and the longitudinal muscle preparation of the guinea pig myenteric plexus as described by Kosterlitz et al. (17,18) have been the methods of choice for the in vitro assay of any morphine-like substance. In both cases, steps have to be taken to ensure that any observed inhibition in stereospecific binding, or in electrically stimulated contraction of mouse vas deferens and guinea pig ileum by any substance, is an opiate specific event. For the receptor binding assay, since the interaction between agonist and receptors is greatly affected by cations (19,20), inhibition in 3H-opiate binding to the membrane fractions by any morphine-like substance must be sensitive to Nal' concentration. In the case of bioassay, inhibition of the electrically stimulated contraction in the vas deferens or ileum, by any substance, has to be reversible, or blocked by, the opiate antagonist, naloxone, and not by other receptor blockers such as propranolol or hexamethonium. Only after any endogenous substance exhibits such properties in the binding assay and the bioassay can it be considered as an opiate-like substance.

 

Enkephalin 

 

Enkephalin* is the first endogenous substance isolated from mammalian brains which inhibits morphine-like activity in vitro. Hughes (21), Terenius and Wahlstrom (22,23), and later Pasternak et al. (24), independently reported the existence of a factor, which inhibited the electrically stimulated contraction of mouse vas deferens and guinea pig ileum (21), and also inhibited 3H-dihydromorphine binding to the SPM (22-24). Although the methods of isolation were different in all three cases, and the molecular weight estimation varied among the three groups (800-1200), the properties of their factors were so similar that we consider that all three groups probably have the same compound: enkephalin. This proved to be the case, for recently Simantov and Snyder reported the isolation and purification of their factor from the bovine brain which turned out to have the same sequence as enkephalin (25).

 

Localization

 

A relatively high level of enkephalin is found in the brain (0.42-0.55 pg of normorphine equivalence per gm. rat brain) (21). It is unevenly distributed in the brain and the pattern of distribution closely resembles the distribution of opiate receptors in the brain (26,27). The release or destruction of enkephalin could partially explain the increase in opiate binding in the membrane preparation after preincubation (28). Enkephalin is further suggested to be contained in the synaptosomal fraction by Pasternak et al. (24). They obtained their major activity in the P2 (crude mitochondria and nerve endings) fraction, and the activity was released into the supernatant when the material was lysed. These authors suggested that enkephalin may be a putative neurotransmitter in certain nerves. A morphine-like substance with enkephalin properties was found in human cerebrospinal fluid (CSF) by Terenius (29) and in a peripheral system which has morphine receptors, namely guinea pig ileum (Hughes, personal communication).

 

Structure and Properties

 

Enkephalin was purified from porcine brain by Hughes et al. (30,31) and from bovine brain by Simantov and Snyder (25). In both cases, using mass spectrometry and Edman degradation, enkephalin was determined to be a mixture of two pentapeptides with the amino acid sequence of H-Tyr-Gly-Gly-Phe-Met-OH (Metenkephalin) and H-Tyr-Gly-Gly-Phe-Leu-OH (Leu-enkephalin). In porcine brain, the ratio of Met-enkephalin to Leu-enkephalin is 4:1. The ratio is reversed in the bovine brain. The sequence provides the important tyramine moiety and the 13-bend to fit the opiate receptors as suggested by Goldstein et al. (32), Bradbury et al. (33) and Horn and Rodgers (34). As a matter of fact, the primary sequence of enkephalin bears a remarkable resemblance to the heptapeptide synthesized by Goldstein et al. (32) which has slight opiate activity in the in vitro assays (H-Tyr-Gly-Gly-Gly-Lys-Met-Gly-OH). Molecular weight derived from this structure is significantly lower than the ones reported (640 vs. 800-1200). Such a discrepancy arose because of the interaction of enkephalin with the column materials as demonstrated by Simantov and Snyder with a Bio-gel column (25).

 

Enkephalin was found to be a very stable peptide. In aqueous solution at pH 5-6, it retained its activity for several months at -20°C (21). It could be heated to 100°C for sixty minutes at neutral pH without loss of activity (25). But enkephalin is very sensitive to proteolytic enzymes (21, 24). The ability to inhibit ileum contractions and opiate binding could be destroyed by carboxypeptidase A and leuaneaminopeptidase (21,24). It is not sensitive to trypsin or neuraminidase. Whether the peptide is sensitive to chymotrypsin is not well resolved. Hughes reported enkephalin was not sensitive to chymotrypsin treatment (21), but Pasternak et al. reported the binding activity of enkephalin was affected by the enxyme (24). It is possible that the difference could result from the amount of enzyme used in the two studies. Since Hughes used 10 ug/ml vs. 1.0 mg/ml for Pasternak et al., a small contamination of exopeptidase in the chymotrypsin preparation would be magnified at the high concentration of enzyme and not at the lower.

 

Biological and Pharmacological Properties

 

Both Met-enkephalin and Leu-enkephalin possess potent agonist-like activity in vitro. They produced a dose-related inhibition of electrocally stimulated contractions of mouse vas deferens and guinea pig ileum (21). This inhibition could only be stereospecifically reversed or blocked by opiate antagonists (30). Enkephalin is selectively active in the tissues known to possess opiate receptors. Metenkephalin was reported to be twenty times more potent than normorphine in mouse vas deferens and equipotent with normorphine in guinea pig ileum (31). Cox et al. (35) reported that synthetic Met-enkephalin was only one-third as potent as normorphine in the ileum preparations. Data obtained in our laboratory agree with Cox et al. Nevertheless, the difference in the potency in the two bioassays reported by Hughes et al. (31) is striking. The authors attributed the variation to the different amount of proteolytic enzyme activity present in the preparations. But, with the reported difference in pA2 value of naloxone to reverse enkephalin and normorphine (7.89 + 0.06 and 8.60 + 0.06, respectively) (30), and the stability of the preparation in the incubation mixture for up to five minutes (21), a difference in the potency between mouse vas deferens and guinea pig ileum could result from the dissimilar effect of the peptides on the adrenergic and cholinergic neurons. Though no tachyphylaxis could be produced by repeated administration of the peptides to mouse vas deferens or the guinea pig ileum, it is possible to demonstrate a cross tolerance between morphine and Met-enkephalin (36). The ID50 of Met-enkephalin increased 3- to 400-fold in the guinea pig ileum preparation from animals implanted with two or four morphine pellets for three days (36). This argues strongly that the site of action of enkephalin is similar to that of morphine, as suggested by theoretical calculations reported by Goldstein et al. (32) and Horn and Rodgers (34).

 

Enkephalin also shows activity in competing for opiate binding in SPM (22,23) or membrane preparations of rat (21, 22-25), or guinea pig brain (21,23,35). Double reciprocal analysis of the inhibition indicated that it is competitive (23). Binding of the peptide is further demonstrated to be at or near the opiate binding sites by the inhibition of the peptide activity with membranes pretreated with trypsin, chymotrypsin and sulfhydryl reagents (24). Stereospecific binding of opiates was shown to be affected by such reagents (28). Furthermore, the inhibition of 3H-naloxone binding by enkephalin is influenced by the concentration of cations (23-25, 37). The IC50 for Met-enkephalin increased from 8 nM to 30 nM in the presence of 100 mM Na+ (25). In the presence of 1.0 mM Mntt, the IC50 decreased to 2.7 nM (25). Since enkephalin competed for 3H-morphine binding 40-fold better than 3H-naloxone binding (37), and the fact that Nat decreased agonist binding and Mntt enhanced agonist binding, enkephalin has definite agonistic properties in the in vitro binding system. With the exception of Hughes et al. (31), who reported that Met-enkephalin was three times more potent than normorphine in competing for opiate binding, all other reports (23,25,35,37) indicate that enkephalin is relatively less potent than normorphine. The value varied from 30% to 60% as potent.

 

Attempts to demonstrate any antinociceptive properties of enkephalin have been unsuccessful. Until recently, enkephalin administered centrally produced no effect in mice or rats. Only with the injection of 100 to 200 jig of Met-enkephalin intraventricularly, did enkephalin produce a slight increase in the latency of tail-flick response (38). The effect was observed by Belluzzi et al. to be transient (dissipated after ten to twelve minutes) and had a latency of two to six minutes after injection. The potency of enkephalin was much lower than that of morphine. Two hundred pg of Met-enkephalin produced and effect similar to 10 pg of morphine sulfate (38). Similar results were obtained by Loh et al. (39) in determining the effect of Met-enkephalin in tail-flick, hotplate, and writhing tests in mice. Fifty pg of Met-enkephalin administered intracerebrally produced a weak analgesic response in all three tests, and the activity of enkephalin only lasted for five to ten minutes. Both groups were able to block the response to Met-enkephalin by pretreatment with naloxone. The transient effect of Metenkephalin, and its low potency when compared with morphine sulfate in vivo, were generally believed to be due to the peptide sensitivity towards exopeptidases. Such an explanation might not be sufficient, since Wei and Loh (40) recently demonstrated development of opiate-like dependent behavior when rats were constantly infused in the periaqueductal grey region with a total of 15 pg Met-enkephalin, or less, for seventy hours. All three signs of dependent behavior, wet shakes, teeth chattering and escaping phenomenon, were observed with the rats infused with Met-enkephalin. Rapid degradation of the peptides would be expected to eliminate any change in enkephalin level in the brain, and hence block the development of deperdence on the peptide.

 

Endorphins 

 

The term endorphin, a combination of endogenous and morphine, was proposed by Eric Simon to signify any substance with opiate activity, such as the previously described substance, enkephalin. Indications of the existence of endorphins, other than enkephalin, are strong. Murray and Miller's report on the lowering of ADso by a pituitary preparation of oxytocin was not observed in the hypophysectomized rats (8), thus suggesting the involvement of the pituitary glands. Moreover, there were numerous reports on pituitary peptides modulating the response to morphine [see review by George and Lomax (41)]. So the search for endorphin(s) present in the pituitary is on. The search is further prompted by the finding of Cox et al. (42) that a crude preparation of ACTH contained morphine-like activity which is absent in synthetic ACTH. [Goldstein's group named such a compound "pituitary opioid peptide-1 (POP-1)".] More recently, following structural studies of Met-enkephalin showing that its sequence is identical to residues 61-65 of a-lipotropin hormone (a-LPH), several peptides derived from the parent molecule, B-LPH, have been found to have opiate-like activity. Since two such peptides, "a- and a-endorphin" and POP-1 were all isolated from the pituitary, the probability that they were related to each other is high.

 

POP-1

 

Cox et a/. (42) were the first to report the presence of any endogenous morphine activity in the extract of pituitary. They were able to demonstrate the activity with the guinea pig ileum assay in a crude preparation of ACTH (42), and in bovine pituitary extract (43). Their POP-1 activity in the ileum was reversible and blocked by naloxone. POP-1 has some properties similar to those of enkephalin. The substance is heat stable, shows activity in both the mouse vas deferens and guinea pig ileum preparation, and shows a positive Na+ effect in the competition binding assay with H-naloxone. But POP-1 differs from enkephalin in that its apparent molecular weight is larger, 1750, and its sensitivity toward various proteolytic enzymes is different from that of enkephalin. It is not sensitive to carboxypeptidase A and B, leucineaminopeptidase, ribonuclease, phospholipase A and B and phosphodiesterase. But POP-1 is sensitive to trypsin and chymotrypsin. It is possible that POP-1 is a molecule containing the pentapeptide sequence of enkephalin within its structure. Since the activity of the POP-1 was not completely destroyed by trypsin, there should be POP's other than those which have been isolated so far.

 

a- and (3-Endorphin

 

It was initially noted by Hughes et al. that the amino acid sequence of Met-enkephalin is present as residues 61-65 of a-LPH isolated from the pituitary glands of sheep (44), pig (45), camel (46), and man (47) (Figure 1). Hughes et a/. thus suggested the possibility of a-LPH being the "pro-endorphin" in the pituitary. Since peptides with the pentapeptide sequence at the N-terminal had been isolated from the pituitary (48), and the same peptides could be obtained from 0-LPH by mild digestion with trypsin in vitro (49), it was not unlikely that these peptides from a-LPH would have opiate activity.

The first such peptide was isolated by Guillemin et al. (50) from the porcine pituitary. Using the technique of high pressure liquid chromatography, Guillemin et al. were able to demonstrate opiate activity in a hexadecapeptide, termed a-endorphin, with the bioassays and the binding assay. The peptide is 0.6 the potency of the normorphine in guinea pig ileum preparation. After sequencing the peptide with mass spectrometry, the structure of a-endorphin was observed to be identical to that of residues 61-76 of a-LPH. This further supported the original hypothesis that a-LPH may be the "pro-endorphin" molecule.

 

Concurrently, Li and Chung (46) reported the isolation of and untriakontapeptide named 6-endorphin, with opiate activity from camel pituitary glands. The peptide sequence of a-endorphin was determined to be identical to those of residues 61-91 of a-LPH. In the binding assay, a-endorphin is three to five times more potent than normorphine in competing for 3H-opiate binding (46). It elicited a positive Na+ effect, although the effect was comparatively smaller in magnitude than that of Met-enkephalin (37). This might explain the lack of in vivo activity of Met-enkephalin and the potent antinociceptive response to 8-endorphin (38,39). It is of interest that the parent molecule, 8-LPH and peptides with N-terminal sequence other than the pentapeptide, e.g., 0-MSH (residues 41-58 of a-LPH), have no activity in the binding assay (35,37). When the activity of 0-endorphin was determined with the guinea pig ileum preparation, it was found to be 0.4 times as potent as normorphine (35). 8-MSH and a-LPH at concentration up to 10-6M were found to be inactive. Only at a very high concentration of a-LPH did Cox et al. (35) find some delayed activity.

 

By fractioning the ileum bath fluid with a Bio-gel P6 column, Cox et al. were able to demonstrate the appearance of binding activity at fractions with molecular weight less than 3000. Seemingly, a-LPH was converted to smaller active fragments. Indirectly, this further supported the importance of the pentapeptide sequence of Met-enkephalin at the N-terminal.

 

In a detailed study, Bradbury et al. (37) determined the sequences in a-LPH which have opiate-like activity in the binding assay. They found that the intermediate peptides of 5-endorphin, peptides with the sequence corresponding to residues 61-68, 61-69, 61-87, 61-89 of $-LPH, all have agonist-like activity, i.e., they all compete for 3H-morphine binding better than 4I-naloxone binding and have a positive Ma+ effect. No activity was observed with a-LPH, y-LPH (158), N-fragment (1-38), a-MSH (41-58) or the 70-79 fragment. So all active fragments contain the pentapeptide sequence of Met-enkephalin at their N-terminal. There is a 30-fold difference in potency between a-endorphin and Met-enkephalin, 61-68, 61-69 and 61-87. Moreover, 61-89 is 20-fold more potent than 61-87, indicating the importance of the two lysine residues on 88 and 89 of B-endorphin. It is further demonstrated in the guinea pig ileum by Seidah et al. (51) that fragment 61-82 is equipotent to Met-enkephalin while 66-91 is completely devoid of activity. a-endorphin in their preparation is 10-fold more potent than Met-enkephalin. So with a small variation in the primary structure, there is a large difference in potency. The secondary structure and perhaps the tertiary structure probably have important roles in the in vitro and in vivo activity of a-endorphin.

 

The in vivo activity of a-endorphin has been demonstrated by Loh et al. (39) recently. It was found to be a potent antinociceptive agent when injected intracerebrally (39) (i.c.) and intravenously (52) (i.v.). When 0-endorphin was administered intracerebrally into the mice, a naloxone reversible inhibition in the tail-flick, hot plate and writhing response was observed. This was found to be a dose dependent phenomenon and lasted for 60-90 minutes. On molar basis, a-endorphin is 18-33 times more potent than morphine sulfate (Table 1). Similar results were obtained with i.v. injections with the exception that a-endorphin is 3- to 4-fold more potent than morphine sulfate (52). It is not the conversion of a-endorphin to an active molecule which yielded the observed antinociceptive activity, since a-LPH was at least eighty times less potent than a-endorphin. Also, a-endorphin, fragment 61-69 and the tryptic digest of a-endorphin were found to be inactive when 40 pg, 20 pg and 10 pg per 25 g mouse was administered i.c., respectively. The fact that $-endorphin is acting like an opioid is substantiated by the cross tolerance and cross dependence studies. It was demonstrated that the AD50 of 0-endorphin increased with chronic treatment of mice with morphine (53). Furthermore, the withdrawal syndromes observed in morphine-dependent mice could be blocked by 8-endorphin (53). Recently, Wei et al. have also found that constant infusion of 8-endorphin produced dependent behavior in rats (40). Thus, all in vivo data indicate that a-endorphin mediates its effect via pathways similar to those of opiate analgesics.

 

Others

 

One possible candidate for being a morphine-like substance is bradykinin, a nonapeptide liberated upon treatment of plasma with trypsin or snake venom. When bradykinin was administered intraventricularly, increase in the threshold of the electrical stimuli applied to the tooth pulp of rabbit was observed (54,55). This antinociceptive activity of bradykinin was fast in onset (maximal ten minutes after injection) and is dissipated within sixty minutes. There was some functional specificity. Addition of amino acids to the N-terminal arginine of bradykinin decreased the antinociceptive potency even though the effects on vascular permeability and blood pressure increase (55). Although the activity of bradykinin was demonstrated not to be due to desensitization of the surrounding area of the tooth pulp, without the control experiments with the opiate antagonist, naloxone, bradykinin could not be considered to be an endorphin. Moreover, the affinity of bradykinin in the in vitro receptor binding assay was relatively low (IC50 > 10-5M) (56). Probably, the antinociceptive effect of bradykinin is caused by some physiological effect which bradykinin and morphine have in common, e.g., depletion of norepinephrine (57,58). Pretreatment with reserpine abolished morphine (59) and bradykinin (54) effects on the threshold of electric stimuli applied to the tooth pulp of rabbit.

 

Another candidate to be an endorphin is the morphine-like compound isolated by Levy et al. (60). Using an antibody specific for morphine, they were able to extract a morphine-like compound from the brain of rat, rabbit, cat and calf. This compound has a regional distribution similar to that of enkephalin, with the exception that this compound is present in the cerebellum. Chemical, chromatographic and immunological tests showed great similarities of the compound to morphine. But the compound is neither sensitive to any proteolytic enzymes nor sensitive to heating in 6 N HC1. More important, although this compound showed activity in the guinea pig ileum, the inhibition of the contraction was not reversed or blocked by naloxone. Thus, based on the criteria previously discussed, this compound is not an endorphin.

 

ENDOGENOUS SUBSTANCE WITH ANTAGONIST-LIKE ACTIVITY

 

Direct demonstration of the existence of an endogenous antagonist-like factor has not been successful. There are numerous reports supporting the hypothesis of the existence of such a factor. Reports such as the blockade of tolerance development to morphine by administration of protein synthesis inhibitors actinomycin D (71), puromycin (62) or cycloheximide (63) support the hypothesis. The numerous observations in the literature that ACTH and 13-MSH antagonize morphine effects also indicate the existence of an endogenous antagonist-like substance (see review by Zimmerman and Krivoy on ACTH and a-msm effect on morphine) (64). But so far, the identity of such a substance has not been clearly resolved.

 

One possible candidate for endogenous antagonist-like substance is the "tolerance factor" of Ungar. Ungar and Cohen (65) and later Ungar and Galvan (66) were able to demonstrate the transfer of tolerance with a chymotrypsin sensitive factor from brain extract of rats chronically treated with morphine to the naive animals. Intraperitoneal injection of such factor to the naive animal produced tolerance to morphine from three hours to ninety-six hours. Recently, Ungar (67) reported the purification of a factor and determined its structure to be H-Arg-Tyr-Gly-Gly-Phe-Met-OH. This structure has a molecular weight such that it might be the same compound which Terenius (68) reported to have antagonistic properties in the binding assay and could only be found in tolerant animals. But Ungar's in vivo observation could not be reproduced by Smith and Takemori (69). Furthermore, the method of administration (intraperitoneal) and the long duration in the action of this factor make the significance of this observation unclear. Whether the hexapeptide, H-Arg-Tyr-Gly-Gly-PheMet-OH, is the endogenous antagonist-like substance could not be answered. For this hexapeptide has no antagonistic property in the mouse vas deferens (67) or the guinea pig ileum (70).

 

Other peptides which might be endogenous antagonist-like substances are ACTH and 13-MSH. It was observed that (3-MSH blocked the morphine inhibitory effect on the polysynaptic reflexes in cat spinal cord (71), that ACTH 1_24 antagonized the morphine effect on the isolate spinal cord (71,72) and the electric foot shock in rats (73), and that (3-MSH release was stimulated by morphine (74). But the antagonistic activity of these two peptides might not be at the opiate receptor sites, since both peptides have low affinity for the opiate binding site (56,75). IC50 for ACTHi_ 24 was 3 uM, comparatively higher than that of naloxone (2.4 nM) (37) and that of morphine (4.4 nM) (37). Moreover, the action of ACTHi_ 24 most likely is mediated via glucocorticoids, for the antagonistic activity of ACTHi_ 24 toward morphine was not observed in adrenalectomized animals (73,76). Only after supplementing such animals with dexamethasone could the effect of ACTH1.. 24 be observed.

 

Another two peptides which modulate morphine's effect in vivo are TRH and Substance P. The distribution of TRH (77,78) and Substance P (79) in the brain suggested that these two peptides have important roles in synaptic function. When TRH was injected at the periaqueductal grey area (the site of morphine action) of rat brain, shaking behavior similar to that observed when naloxone was administered to dependent animals was observed (80). A crude preparation of Substance P antagonized the excitatory action of morphine on mice (81, 82). But this effect of Substance P was not observed with the purified preparation. Conversely, when synthetic Substance P was administered to mice, it abolished the abstinence syndrome (jumping) in morphinized mice (83). Without any activity in competing for opiate binding (55), the role of TRH and Substance P on morphine action, once again, might not be at the receptor level.

 

DISCUSSION

 

In this chapter, we have reviewed the present evidence for the existence of any peptides which have opiate activity. As discussed earlier in the chapter, any substance which is considered to be endorphin must interact with the opiate receptors in the binding assay and should have activity in the bioassays which is reversible by or blocked by naloxone. The only peptides which satisfy both these requirements are enkephalin, a- and 0-endorphin and a few $-LPH-related peptides. But the relative inactivity of enkephalin in vivo raises a question whether the in vitro assay methods yield a quantitative measurement for predicting analgesic potency for any endorphins. A previous explanation of the in vivo activity of enkephalin has been the degradation of the peptide by exopeptidases. Since the demonstration of the potent activity of a-endorphin in vivo, such an explanation might not be valid. Since the potency of c3-endorphin to the binding assay is very sensitive to peptide length, any degradation by exopeptidases in vivo would diminish its activity. On the contrary, 0-endorphin was found to be more active in vivo than in vitro (35,37,39,46). Moreover, the ability of a constant infusion of enkephalin to produce opiate-like dependent behavior in rats indicated that the level of enkephalin could be maintained at a higher than normal level in the brain. Since both in vitro methods are basically a measurement of receptor binding potential of the peptides, interactions between the "binding sites" (or regulation sites) and the "analgesic sites" (or catalytic sites) could not be measured. Probably, the secondary and tertiary structures of the peptide also play a vital role in the in vivo activity.

 

The demonstration that 0-endorphin has in vivo antinociceptive activity further emphasized the role of 0-LPH as the "prohormone" or the "pro-endorphin" in the pituitary. With the tWo existing peptides, a-msli and 0-endorphin having functional activity, it is not unlikely that more peptides derived from 0-LPH would have functional activity. Since it has been well established that introduction of an allyl group will convert the opiate molecule from an agonist to an antagonist, it is attractive to postulate that with the introduction of other amino acids to the 0-endorphin molecule, preferably at the N-terminal, the peptide would have antagonistic properties. Even though the hexapeptide proposed by Ungar has no antagonistic property (70,84), any addition of amino acid to that molecule might yield the long-sought endogenous antagonist-like substance. With a-mmi having some antagonistic properties, it might be of interest to investigate the peptides with the sequence of residues 41-91.

 

If such an antagonist-like factor exists, it might yield some insight into the problem of tolerance and dependence development. It has been suggested by Kosterlitz and Hughes (85) and more recently by Simantov and Snyder (86) that enkephalin is a neuromodulator or a putative neurotransmitter, controlling certain inhibitory mechanisms determining the rate of transmitter release. With a negative feedback mechanism on the synthesis of enkephalin, from the constantly stimulated receptor when opiates are introduced, tolerance will develop when more and more opiates are required to maintain such an inhibitory mechanism or enkephalin biosynthesis. Such a postulation could not explain the recent observation by Simantov and Snyder (86) that the enkephalin level in the brain "increases" during tolerance development. We would like to postulate that the brain contains not only endorphin, it also contains an antagonist-like factor (ALF) which might have its origin from the a-LPH molecule. The endorphin(s) might regulate the inhibitory neurons and the ALF regulate the excitatory neurons. In the normal non-analgesic state, these two compounds would counteract each other. With the introduction of morphine, the equilibrium would shift to favor the inhibitory neurons and thus the analgesic state. In order to counteract the morphine effect the synthesis of ALF would increase. Together with the negative feedback of morphine on the synthesis of endorphin(s), the two phenomena would act synergistically to produce a high level of tolerance with a small change in the level of the two endogenous compounds. With the removal of morphine, the equilibrium would favor the excitatory neurons because of the rise of the ALF and the fall of the endorphin(s) level, thus resulting in the observed abstinence syndrome, jumping, teeth chattering and wet shakes.

 

In conclusion, the demonstration of the presence of endorphin is one of the most exciting findings in the field of narcotic research in recent years. Although the role of the peptides isolated so far in tolerance development is not known, we feel that a major step has been taken in the direction of solving the problem.

 

We will use the name enkephalin throughout this chapter only for the peptides with amino acid sequence H-Tyr-GlyGly-Phe-Met-OH and H-Tyr-Gly-Gly-Phe-Leu-OH as suggested by J. Hughes.

 

REFERENCES

 

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4. Wong, D.T. and Horng, J.S.: Stereospecific interaction of opiate narcotics in binding of 3H-Dihydromorphine to membranes of rat brain. Life Sciences 13:1543-1556 (1973).

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