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Some thoughts about opiopeptins, peptides with opiate-like activity
Dependence, 11(1983) Elsevier Scientific Publishers Ireland Ltd.
Drug and Alcohol
23
23-31
SOME THOUGHTS ABOUT OPIOPEPTINS, PEPTIDES WITH OPIATELIKE ACTIVITY
E. LEONG WAY Department
ofPharmacology,
University
of California,
San Francisco,
CA 94143
(U.S.A.)
When the announcement was made late in 1975, that two closely related pentapeptides (enkephalins) with opiate-like activity had been isolated from porcine brain, there was much excitement. For the biologists not particularly interested in opiates, the finding meant that there were new fields to plow because the novel substances in all probability must be associated with unknown neurons with important physiologic functions. To the pharmacologists, however, especially those who had been engaged in opiate research, this was tremendously uplifting. I recall saying to myself: “After being engaged in opiate research for three decades perhaps, I shall now have the opportunity to learn in my lifetime how morphine produces analgesia, tolerance and physical dependence.”
Even without any facts, it was not too difficult for me to rationalize and formulate a plausible hypothesis involving the newly discovered peptides to explain heroin addiction. Persons finding it difficult to cope with the stresses of today’s competitive society must have a deficiency of enkephalin. Instinctively, they crave heroin to remedy this aberration, and taking the replacement drug, gain instantaneous relief. However, this alleviation can only be temporary because repeated administration of the narcotic turns off the synthesis of the native opioid ligand and this results in further depletion of an already reduced supply. A compensatory mechanism must then develop to certain biochemical processes ordinarily inhibited by enkephalin. As they become increasingly suppressed by repeated high doses of the exogenous substitute a counter-adaptive response results in the inhibited sites developing suprasensitivity, and when the heroin is discontinued, the suprasensitive processes became manifest by an overshoot rebound or the withdrawal syndrome. Thus, now that the endogenous ligand is known it should not be too difficult to determine its physiologic role and sort out the processes it regulates. This knowledge should pave the way to explain acute opiate effects as well as tolerance and physical dependence. Seven years later I am somewhat less ebullient but still full of hope and optimism. Since the isolation of the enkephalins several more peptides with greater potency have been identified and the literature on peptides with 0376-8716/83/$03.00 D 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
24
opiate-like activity, which I shall henceforth call opiopeptins rather than endorphins to reduce confusion, has been accumulating at a dizzy accelerating pace. However, the pieces in the jig-saw puzzle have not fallen in place sufficiently to give a clear picture of what their roles are. When it clears, almost certainly we will have a better understanding of how opiates act. In the meantime, we do not have to stop our conjectures which started right after the first publication on the enkephahns but the information is now far more complex than I had ever realized. In presenting my thoughts on these matters, I leaned heavily on original papers cited in two of our very recent reviews [ 1,221 and three monographs [3--51. Two enkephalins were isolated first, both pentapeptides, and these were named methionine- or met-enkephalin and leucine- or leu-enkephalin by Hughes, Kosterlitz and associates in 1975; methionine and leucine being the respective terminal groups of the common tetra peptide residue tyrosyl, glycyl, glycyl, phenylalanyl [ 1,3,5]. The Britishers pointed out that the five amino acid sequence of met-enkephalin was contained in the 91-amino acid peptide, fi-lipotropin (p-LPH) isolated from sheep pituitaries in 1964 by Li [3,4] but at the time this fact did not seem to have too much bearing on opiate action. The more immediate thoughts were that morphine and its surrogates must be mimicking the effects of the enkephalins either directly as exogenous substitutes or indirectly by preserving the effects of the enkephalins through altering their disposition (stimulating release, inhibiting breakdown or preventing reuptake). The simplest view was that the alkaloids were substituting for the enkephalins and this generally has been borne out by the laboratory data. The findings provide little support for the likelihood that the alkaloidal opiates might mediate their effects by preventing the destruction or reuptake of the enkephalins. While it was puzzling that there should be two enkephalins, since it could not be explained, not much thought was devoted to the matter during these early stages. The enkephalins were believed to be the prime peptides involved in opioid action for a number of reasons. Firstly, both met- and leu-enkephalin were the first to be identified as opiate-like substances and this was accomplished by using two classic preparations for assessing opiate activity, namely the guinea pig ileum and the mouse vas deferens. Secondly, they were about equal or more active than morphine in these preparations and since their isolation was so arduous, it seemed rather unlikely that peptides with even greater potency might exist. Thirdly, the distribution of the enkephalins in the brain followed surprisingly close to that of the opiate receptors or at least to the binding sites for morphine and its surrogates [1,3--5-j. The first inkling that the story was not so simple occurred in 1976 when p-endorphin was isolated independently in the laboratories of Li and Smyth and found by Loh et al. [3] to be far more potent analgetic than the enkephdns or morphine. However, at the time @-endorphin was found only in
25
the pituitary and not in the brain. Furthermore, since met-enkephalin represented a fragment of fl-endorphin and since it was found in several-fold higher concentrations than leu-enkephalin [l], it appears reasonable to assume that it might be the prime peptide species acting at opioid receptor sites in the CNS [ 3-51. Some puzzling questions concerning the enkephalins began to surface when workers were unable to demonstrate convincingly analgetic properties for the two compounds. Even when the compounds were injected intracerebrally, they evinced at best only a slight transient antinociceptive effect. The enkephalin proponents argued that the failure to produce analgesia was due to their rapid destruction rather than a lack in intrinsic activity. These arguments did not hold water because at the time it had already been demonstrated that the half-life of the enkephalin, although short, should be still long enough to produce measurable analgesia for a few minutes. Moreover, it had already been demonstrated by Wei and Loh (1976) that slow infusion of a small dose of met-enkephalin could produce tolerance [ 1,3]. A more plausible explanation was provided in 1977 by Kosterlitz, one of the discoverers of the enkephalins, who proposed that the enkephalins were acting on opiate receptors different from those of morphine [ 11. In presenting his revisedviews, Kosterlitz paid tribute to Martin who had developed the concept of receptor dualism in opiate action but these works had been largely ignored. Martin probably began thinking that opiates might act on more than one receptor after Lasagna and Beecher discovered serendipitously in 1954 that nalorphine alleviated pain [4]. Even though it did not make sense that a competitive antagonist for morphine should display analgetic properties, facts prevailed over reason and an intense research for a superior surrogate for nalorphine was launched. Martin compared the effect of some of these compounds in the spinal dog preparation and in humans, and as a consequence of his studies, proposed that there were at least three distinct opiate receptors which he named cc, K and u. His hypothesis is summarized in one of his more recent papers [6]. Martin’s hypothesis provided a plausible answer for the analgetic effects of nalorphine and certain other agonist-antagonists (e.g. pentazocine, cyclazotine, ketocyclazocine and later butorphanol). Although such compounds are p antagonists, they also possess K agonist activity. To accommodate the psychotomimetic effects of these compounds, Martin proposed that they were activating still another receptor, the u receptor. Although these studies stirred little interest in the beginning, after the discovery of the enkephalins and the study by Lord et al. in 1977 demonstrating different potency ratios for morphine and the enkephalin on the guinea pig ileum and mouse vas deferens [ 11, the concept of multiple opiate receptors has gained increasing acceptance although no receptor has been isolated. Even though these receptors remain figments of the mind of pharmacologists, they have considerable heuristic value. However, we are beginning
26
to be overburdened with too many receptors. In addition to ~1,K and u receptors, in vitro evidence indicate that there are also 6,~ and E receptors and more may be forthcoming, including subsets of the p receptor. Some of the receptor types may be peculiar to a particular species or one receptor may have different conformations [ 11. An unfortunate matter is that the receptor characterized by in vitro studies can not always be related to in vivo results. The most consistent data have been obtained on the ~1receptor. The rank order of activity of p-agonists (morphine and its alkaloidal congeners) in the guinea pig ileum has been found to correspond reasonably well not only to analgetic activity in experimental animals and humans but also to their binding affinities in brain homogenates [ 11. The K -receptor is less well established. It is also present in the guinea pig ileum and the order of activity of K -agonists on this preparation yields good correlation with analgetic potency. However, binding studies in brain with K -agonists have not provided as clear-cut data as desired. With respect to the u-receptor, there is no reliable in vitro method for assessing pharmacologic activity although binding studies suggest a relationship with phencyclidine. The 6 receptor has been identified in the mouse vas deferens and a relationship between activity in this preparation and binding data with met- and leu-enkephalin may exist but, as yet, there is no simple behavioral test on an intact animal that can be correlated with these data. The other receptors are even more nebulous, each having been identified only in vitro on a preparation peculiar to a particular species [1,51. A major part of the problem in identifying the various receptors lies in the fact that none of the peptides with opiate-like activity can solely be identified with one receptor. The enkephalins are predominantly 6 agonists, at least in the mouse vas deferens, but in the guinea pig ileum they appear to act as p-agonists and they have little or no K action. Binding studies reveal that the enkephalins displace their peptide analogs better than morphine or other alkaloidal I_C agonists in specific brain areas but such studies need to be related to behavioral effects in an intact animal [ 11. fl-Endorphin has wide ranging effects exhibiting I-(, K and 6 activity by the in vitro tests [1,3--51 but this versatility complicates the thinking. Lee and her associates propose that in order for fl-endorphin to achieve its effect, it must attach simultaneously to both the I-( and 6 receptors [ 71. Dynorphin has been suggested to be the native ligand for the K -receptor and there is good evidence to support this notion at least in the guinea pig ileum. We first made this suggestion in 1981 [9] and subsequently Herz’s and Goldstein’s laboratories independently supported our findings and notions. More recent studies indicate, however that the story is not so simple and that dynorphin has more than just K agonist activity. Lee and associates reported in 1981 that dynorphin can suppress p agonist (morphine) abstinence and restore sensitivity to morphine in animals that have been rendered tolerant to morphine [8]. Another interesting finding is that when
27
the seventeen amino acid chain length of dynorphin is reduced to seven or five, K activity disappears and 1-1and 6 activity become apparent [9]. Not only is it difficult to assign a particular opiopeptin to one receptor type, it should be noted also that none of the peptides which have been isolated, has been established unequivocably to be the real species activating the receptor. Such an answer will not be easy to obtain, the problem being, whether the native ligand’is the most potent peptide present in small amounts or a less potent one present in much higher quantities. Thus, we might argue for example, that met- and leu-enkephalin because of their relatively low potency, may not be true native ligands but rather are stable active end-products of some larger residues found in the respective laboratories of Matsuo, Udenfriend and Costa and which have been shown to possess greater potency than the pentapeptides [ 1,5]. To display a little more optimism, the muddied picture is beginning to clear. All native peptides with opioid-like activity appear to belong to one of three major classes which possess separate neuronal pathways that can be categorized as enkephalinergic, endorphinergic and dynorphinergic. The precursor protein associated with each type is also distinct and can be hydrolyzed into several residues that exhibit opiate-like activity [ 71. The enkephalinergic system appears to have the most diversified functions in the brain. Its neuronal pathways are most widespread and their distribution correlates well with opiate binding site density. The ability of the enkephalins to inhibit neuronal firing and to inhibit substance P release point to the possibility that, if they are not neurotransmitters, they can modulate release of transmitters throughout the central and peripheral nervous systems [ 1,7]. The precursor protein for the system, proenkephalin (or proenkephalin A) contains six copies of met-enkephalin and one of leu-enkephalin
[lOI.
The endorphinergic system seems to be closely related to pituitary function. It may well play the intermediary in modulating the release of trophic factors from the hypothalamus that regulate the release of hormones from the pituitary gland. In particular, prolactin, growth hormone and antidiuretic hormone release has been shown to be inhibited by fl-endorphin whereas release of luteinizing hormone is stimulated. The endorphinergic system has a highly selective distribution, having a few projections from the hypothalamus extending mainly to the diencephalon and dorsal medial thalamus. The precursor for the endorphinergic system, pro-opiomelanocortin, contains ACTH, melanocyte stimulating hormone, and P-endorphin with met-enkephalin at its amino terminal residue [4,5]. The dynorphinergic system has a distribution that often closely overlaps with the enkephalinergic system but in some instances there are distinct differences; certain dynorphin pathways are closely associated with those of the posterior pituitary. The precursor protein of pre-dynorphin (or preenkephalin B) contains three sequences of leu-enkephalin that are part of a-neo-endorphin, (dynorphin A) and dynorphin B (rimorphine) [ 101.
28
The recentness of the discovery of the dynorphinergic system makes it difficult to make conjectures about its functions. A relevant question to ask concerns its connection with the enkephalinergic and endorphinergic systems. Why should there be three opiopeptin systems? Recent evidence from Lee’s and our laboratory support the possibility that the dynorphinergic system might have direct influence over the other two systems [8,9]. As endogenous ligands whose effects are mimicked to varying degrees by morphine, the opiopeptins must somehow be involved not only with acute morphine effects but chronic ones as well. As might be expected therefore, tolerance to morphine conveys cross-tolerance to /3-endorphin and the enkephalins. Cross-dependence is also exhibited by the opiopeptins as evidenced by the fact that fl-endorphin has been shown by Wei and Loh in 1976 to suppress morphine abstinence in animals and in 1980 by Su et al. in humans [ 3,4]. Indeed, primary dependence to some opiopeptins can be produced by sustained administration of low doses as demonstrated for P-endorphin and met-enkephalin in 1976 by Wei and Loh [ 3,4]. Dynorphin is highly effective in suppressing morphine abstinence and although its analgetic activity is controversial, Lee’s group have shown that analgetic sensitivity to morphine in the morphine tolerant animal is restored after dynorphin administration [8]. These findings are surprising in that as mentioned earlier, in the guinea pig ileum dynorphin behaves essentially as a K agonist [ 91. Clearly then, dynorphin is not just a K agonist. In any event, assuming that morphine effects are associated with the opiopeptins, then morphine must alter certain functions that are regulated by these endogenous ligands. Processes that are likely to be affected are those that might alter neurotransmitter function. In order to elucidate the mechanisms involved in opiate-induced analgesia, tolerance, and physical dependence, many investigators have attempted to implicate several neurotransmitters. After altering the synthesis, storage, release, or degradation of acetylcholine, dopamine, norepinephrine, and serotonin, the consequences of such maneuvers on analgesia, and the tolerant-dependent state were investigated. The conclusions, if not the findings, were controversial because a participatory role of any given neurotransmitter could be associated to some degree with the morphine response selected for evaluation. These findings led us to conclude [2] that opiate effects may be mediated by some basic mechanism common to all the aforementioned neurotransmitters. One mutual thread relates to considerable evidence indicating that opioid agonists can inhibit the release of many neurotransmitters, including acetylcholine, dopamine and norepinephrine. Since Ca ‘+ has been implicated in neurotransmitter release, we decided to examine the interrelationships between calcium and morphine. Such a study appeared worthwhile also because electrophysiologic evidence support a role for Ca” in opiate effects. There is mounting evidence that opiates act primarily as inhibitors of specific neurons. Iontophoretic application of opiates in the brain typically inhibits the firing of
29
sensitive neurons. In instances where the effects of opiates are found to be excitatory, they have been found to be due to disinhibition. The inhibitory effect of opiates appears to be mediated presynaptically by inhibiting neurotransmitter release. Thus, Mudge et al. in 1979 found that an enkephalin analog-D-ala’ enkephalin amide, not only inhibits depolarized Ca”-dependent release of substance P from sensory neurons grown in dispersed cell culture but also decreases the duration of the Ca” action potential recorded from the cell body, They suggested that a similar effect on CaZ’ channels located at nerve endings could explain the action of opiates on neurotransmitter release [2]. There is accumulating evidence that Ca ‘+ is involved in opiate action. Studies from our laboratory and others reveal that after acute administration of an opiate, a lowering of brain Ca” occurs at nerve endings, especially in synaptic vesicles. In contrast, after chronic morphine administration there is an increase of Ca2’ at the same sites that is directly related to the degree of tolerance development. We have also found that manipulations, which increased brain Ca”, antagonized opiate effects while lowering the Ca” enhanced opiate effects. Indeed, it was possible to demonstrate analgesia with such Ca2+ antagonists as lanthanum and EGTA, a calcium chelator. Furthermore, La3+ can attenuate the signs and symptoms of morphine withdrawal, presumably by lowering neuronal Ca”. Thus, there appears to be two opposing effects of opiates on neuronal Ca2’, an immediate one to reduce Ca2+ and a delayed counteradaptive response to reverse the acute lowering effect on Ca 2+. It is our thesis that these two opposing actions of morphine can be utilized as a framework to explain such properties as analgesia, tolerance and physical dependence. Our operational hypothesis [ 21 is that the nociceptive state is regulated by the Ca” level within the neuron, a lowering effects analgesia while an elevation results in hyperalgesia. Adaptation to the lowering process occurs and it is cumulative so that with continuous opiate administration there is a gradual build up of Ca 2+. The consequence would be tolerance development since, to produce analgesia, more opiate would be required to lower the elevated neuronal Ca2’. Under such conditions a new elevated steady state for Ca2+ becomes established whereby lowering of Ca” becomes more difficult and maintenance of the state requires the presence of the opiate (physical dependence). The abstinence syndrome would then reflect a supersensitive state to opiate lack or a hyperirritable response to Ca” excess that is ordinarily inhibited by the presence of opiates. A case for CAMP in opiate action can also be made [12] but the results are not as convincing as with Ca”. After we demonstrated that CAMP antagonizes opiate action, three laboratories (Collier, Klee and Hamprecht) independently obtained data to support that opiate effects are mediated through inhibition of adenylate cyclase. If this be the case, then opiates should not only consistently inhibit adenylate cyclase in model systems in which opiates are known to elicit pharmacologic actions but they should also
30
elevate cellular CAMP. Furthermore, opiates should produce effects that are antagonized by CAMP administration or by manipulatidns that tend to increase CAMP such as by phosphodiesterase inhibition. All four criteria can be met by selecting a different model system for each parameter but even in such instances the data are controversial. Although inhibitors of adenylate cyclase can be readily demonstrated in tissue culture of the neuroblastoma X glioma (NG 108-15) cell line, the effect is not so readily shown in the brain of animals injected with morphine. As an example of this point, although most investigators report CAMP antagonism of opiate action, others claim it produces analgesia. Undoubtedly, CAMP must be involved in opiate action but whether it is a crucial initiating event or merely involved secondarily in the cascade of a triggered happening is quite another matter. Likely, the Ca” and CAMP effects are related. It has been proposed, for example, that CAMP may facilitate Ca2’ entry and morphine, by inhibiting adenylate cyclase, should decrease intracellular CAMP and thereby decrease Ca2’ entry and neurotransmitter release. However, since the effects of CAMP are also dependent on Ca”, it is difficult to say whether CAMP is the chicken or the egg. In conclusion, a clear understanding of the mechanisms involved in morphine effects in analgesia, euphoria, tolerance, physical dependence and compulsive usage must await the elucidation of the physiologic role of the opiopeptins. There is now considerable evidence implicating Ca2’ in opiate action and likely these effects are related to the function of opiopeptins. While pharmacologic data can often be misleading, there is evidence indicating that neuropeptides act to inhibit intracellular mediators of neurohormones and neurotransmitters. The mediators within the cell serve to effect the change triggered by these substance, that is, they act as ‘second messengers’. Agents serving as second messengers include CAMP and Ca’” which may alter neurotransmitter release. Perhaps by the twentieth volume of Drug and Alcohol Dependence, we might have the answers and I might still be around. REFERENCES 1 J.P. Huidobro-Toro and E. Leong Way, Opiates, in: D.G. Grahame-Smith (Ed.), Psychopharmacology, Part I, Preclinical Psychopharmacology, Excerpta Medica, Amsterdam, Chap. 8. 2 D. Chapman and E. Leong Way, Pharmacologic consequences of calcium interactions with opioid alkaloids and peptides, in: R.G. Rahwan and D. Witiak (Eds.), Calcium Regulation and Drug Design, ACS Symposium Series, 201, American Chemical Society, Washington, 1982,202 pp. 3 H.H. Loh and D.H. Ross, NeurochemicaJ mechanisms of opiates and endorphins, Raven Press, New York, 1979,563 pp. 4 C.H. Li (Ed.), Hormonal proteins and peptides, Academic Press, New York, 1981, 359 pp. 5 J.B. Malick and R.M.S. Bell, Endorphins, chemistry, physiology, pharmacology and clinical relevance, Marcel Dekker, New York, 1982,296 pp.
31 6 7 8 9 10 11
W.R. Martin et al., J. Pharmacol. Exp. Ther., 197 (1976) 517. V. Hijllt, Trends Neurosci. (1983) in press. C. Tulunay et al., J. Pharmacol. Exp. Ther., 219 (1981) 296. K. Yoshimura et al., J. Pharmacol. Exp. Ther., 222 (1982) 71. H. Kakidani et al., Nature, 298 (1982) 245. H.O.J. Collier, Nature 293 (1982) 625.
Drug and Alcohol
23
23-31
SOME THOUGHTS ABOUT OPIOPEPTINS, PEPTIDES WITH OPIATELIKE ACTIVITY
E. LEONG WAY Department
ofPharmacology,
University
of California,
San Francisco,
CA 94143
(U.S.A.)
When the announcement was made late in 1975, that two closely related pentapeptides (enkephalins) with opiate-like activity had been isolated from porcine brain, there was much excitement. For the biologists not particularly interested in opiates, the finding meant that there were new fields to plow because the novel substances in all probability must be associated with unknown neurons with important physiologic functions. To the pharmacologists, however, especially those who had been engaged in opiate research, this was tremendously uplifting. I recall saying to myself: “After being engaged in opiate research for three decades perhaps, I shall now have the opportunity to learn in my lifetime how morphine produces analgesia, tolerance and physical dependence.”
Even without any facts, it was not too difficult for me to rationalize and formulate a plausible hypothesis involving the newly discovered peptides to explain heroin addiction. Persons finding it difficult to cope with the stresses of today’s competitive society must have a deficiency of enkephalin. Instinctively, they crave heroin to remedy this aberration, and taking the replacement drug, gain instantaneous relief. However, this alleviation can only be temporary because repeated administration of the narcotic turns off the synthesis of the native opioid ligand and this results in further depletion of an already reduced supply. A compensatory mechanism must then develop to certain biochemical processes ordinarily inhibited by enkephalin. As they become increasingly suppressed by repeated high doses of the exogenous substitute a counter-adaptive response results in the inhibited sites developing suprasensitivity, and when the heroin is discontinued, the suprasensitive processes became manifest by an overshoot rebound or the withdrawal syndrome. Thus, now that the endogenous ligand is known it should not be too difficult to determine its physiologic role and sort out the processes it regulates. This knowledge should pave the way to explain acute opiate effects as well as tolerance and physical dependence. Seven years later I am somewhat less ebullient but still full of hope and optimism. Since the isolation of the enkephalins several more peptides with greater potency have been identified and the literature on peptides with 0376-8716/83/$03.00 D 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
24
opiate-like activity, which I shall henceforth call opiopeptins rather than endorphins to reduce confusion, has been accumulating at a dizzy accelerating pace. However, the pieces in the jig-saw puzzle have not fallen in place sufficiently to give a clear picture of what their roles are. When it clears, almost certainly we will have a better understanding of how opiates act. In the meantime, we do not have to stop our conjectures which started right after the first publication on the enkephahns but the information is now far more complex than I had ever realized. In presenting my thoughts on these matters, I leaned heavily on original papers cited in two of our very recent reviews [ 1,221 and three monographs [3--51. Two enkephalins were isolated first, both pentapeptides, and these were named methionine- or met-enkephalin and leucine- or leu-enkephalin by Hughes, Kosterlitz and associates in 1975; methionine and leucine being the respective terminal groups of the common tetra peptide residue tyrosyl, glycyl, glycyl, phenylalanyl [ 1,3,5]. The Britishers pointed out that the five amino acid sequence of met-enkephalin was contained in the 91-amino acid peptide, fi-lipotropin (p-LPH) isolated from sheep pituitaries in 1964 by Li [3,4] but at the time this fact did not seem to have too much bearing on opiate action. The more immediate thoughts were that morphine and its surrogates must be mimicking the effects of the enkephalins either directly as exogenous substitutes or indirectly by preserving the effects of the enkephalins through altering their disposition (stimulating release, inhibiting breakdown or preventing reuptake). The simplest view was that the alkaloids were substituting for the enkephalins and this generally has been borne out by the laboratory data. The findings provide little support for the likelihood that the alkaloidal opiates might mediate their effects by preventing the destruction or reuptake of the enkephalins. While it was puzzling that there should be two enkephalins, since it could not be explained, not much thought was devoted to the matter during these early stages. The enkephalins were believed to be the prime peptides involved in opioid action for a number of reasons. Firstly, both met- and leu-enkephalin were the first to be identified as opiate-like substances and this was accomplished by using two classic preparations for assessing opiate activity, namely the guinea pig ileum and the mouse vas deferens. Secondly, they were about equal or more active than morphine in these preparations and since their isolation was so arduous, it seemed rather unlikely that peptides with even greater potency might exist. Thirdly, the distribution of the enkephalins in the brain followed surprisingly close to that of the opiate receptors or at least to the binding sites for morphine and its surrogates [1,3--5-j. The first inkling that the story was not so simple occurred in 1976 when p-endorphin was isolated independently in the laboratories of Li and Smyth and found by Loh et al. [3] to be far more potent analgetic than the enkephdns or morphine. However, at the time @-endorphin was found only in
25
the pituitary and not in the brain. Furthermore, since met-enkephalin represented a fragment of fl-endorphin and since it was found in several-fold higher concentrations than leu-enkephalin [l], it appears reasonable to assume that it might be the prime peptide species acting at opioid receptor sites in the CNS [ 3-51. Some puzzling questions concerning the enkephalins began to surface when workers were unable to demonstrate convincingly analgetic properties for the two compounds. Even when the compounds were injected intracerebrally, they evinced at best only a slight transient antinociceptive effect. The enkephalin proponents argued that the failure to produce analgesia was due to their rapid destruction rather than a lack in intrinsic activity. These arguments did not hold water because at the time it had already been demonstrated that the half-life of the enkephalin, although short, should be still long enough to produce measurable analgesia for a few minutes. Moreover, it had already been demonstrated by Wei and Loh (1976) that slow infusion of a small dose of met-enkephalin could produce tolerance [ 1,3]. A more plausible explanation was provided in 1977 by Kosterlitz, one of the discoverers of the enkephalins, who proposed that the enkephalins were acting on opiate receptors different from those of morphine [ 11. In presenting his revisedviews, Kosterlitz paid tribute to Martin who had developed the concept of receptor dualism in opiate action but these works had been largely ignored. Martin probably began thinking that opiates might act on more than one receptor after Lasagna and Beecher discovered serendipitously in 1954 that nalorphine alleviated pain [4]. Even though it did not make sense that a competitive antagonist for morphine should display analgetic properties, facts prevailed over reason and an intense research for a superior surrogate for nalorphine was launched. Martin compared the effect of some of these compounds in the spinal dog preparation and in humans, and as a consequence of his studies, proposed that there were at least three distinct opiate receptors which he named cc, K and u. His hypothesis is summarized in one of his more recent papers [6]. Martin’s hypothesis provided a plausible answer for the analgetic effects of nalorphine and certain other agonist-antagonists (e.g. pentazocine, cyclazotine, ketocyclazocine and later butorphanol). Although such compounds are p antagonists, they also possess K agonist activity. To accommodate the psychotomimetic effects of these compounds, Martin proposed that they were activating still another receptor, the u receptor. Although these studies stirred little interest in the beginning, after the discovery of the enkephalins and the study by Lord et al. in 1977 demonstrating different potency ratios for morphine and the enkephalin on the guinea pig ileum and mouse vas deferens [ 11, the concept of multiple opiate receptors has gained increasing acceptance although no receptor has been isolated. Even though these receptors remain figments of the mind of pharmacologists, they have considerable heuristic value. However, we are beginning
26
to be overburdened with too many receptors. In addition to ~1,K and u receptors, in vitro evidence indicate that there are also 6,~ and E receptors and more may be forthcoming, including subsets of the p receptor. Some of the receptor types may be peculiar to a particular species or one receptor may have different conformations [ 11. An unfortunate matter is that the receptor characterized by in vitro studies can not always be related to in vivo results. The most consistent data have been obtained on the ~1receptor. The rank order of activity of p-agonists (morphine and its alkaloidal congeners) in the guinea pig ileum has been found to correspond reasonably well not only to analgetic activity in experimental animals and humans but also to their binding affinities in brain homogenates [ 11. The K -receptor is less well established. It is also present in the guinea pig ileum and the order of activity of K -agonists on this preparation yields good correlation with analgetic potency. However, binding studies in brain with K -agonists have not provided as clear-cut data as desired. With respect to the u-receptor, there is no reliable in vitro method for assessing pharmacologic activity although binding studies suggest a relationship with phencyclidine. The 6 receptor has been identified in the mouse vas deferens and a relationship between activity in this preparation and binding data with met- and leu-enkephalin may exist but, as yet, there is no simple behavioral test on an intact animal that can be correlated with these data. The other receptors are even more nebulous, each having been identified only in vitro on a preparation peculiar to a particular species [1,51. A major part of the problem in identifying the various receptors lies in the fact that none of the peptides with opiate-like activity can solely be identified with one receptor. The enkephalins are predominantly 6 agonists, at least in the mouse vas deferens, but in the guinea pig ileum they appear to act as p-agonists and they have little or no K action. Binding studies reveal that the enkephalins displace their peptide analogs better than morphine or other alkaloidal I_C agonists in specific brain areas but such studies need to be related to behavioral effects in an intact animal [ 11. fl-Endorphin has wide ranging effects exhibiting I-(, K and 6 activity by the in vitro tests [1,3--51 but this versatility complicates the thinking. Lee and her associates propose that in order for fl-endorphin to achieve its effect, it must attach simultaneously to both the I-( and 6 receptors [ 71. Dynorphin has been suggested to be the native ligand for the K -receptor and there is good evidence to support this notion at least in the guinea pig ileum. We first made this suggestion in 1981 [9] and subsequently Herz’s and Goldstein’s laboratories independently supported our findings and notions. More recent studies indicate, however that the story is not so simple and that dynorphin has more than just K agonist activity. Lee and associates reported in 1981 that dynorphin can suppress p agonist (morphine) abstinence and restore sensitivity to morphine in animals that have been rendered tolerant to morphine [8]. Another interesting finding is that when
27
the seventeen amino acid chain length of dynorphin is reduced to seven or five, K activity disappears and 1-1and 6 activity become apparent [9]. Not only is it difficult to assign a particular opiopeptin to one receptor type, it should be noted also that none of the peptides which have been isolated, has been established unequivocably to be the real species activating the receptor. Such an answer will not be easy to obtain, the problem being, whether the native ligand’is the most potent peptide present in small amounts or a less potent one present in much higher quantities. Thus, we might argue for example, that met- and leu-enkephalin because of their relatively low potency, may not be true native ligands but rather are stable active end-products of some larger residues found in the respective laboratories of Matsuo, Udenfriend and Costa and which have been shown to possess greater potency than the pentapeptides [ 1,5]. To display a little more optimism, the muddied picture is beginning to clear. All native peptides with opioid-like activity appear to belong to one of three major classes which possess separate neuronal pathways that can be categorized as enkephalinergic, endorphinergic and dynorphinergic. The precursor protein associated with each type is also distinct and can be hydrolyzed into several residues that exhibit opiate-like activity [ 71. The enkephalinergic system appears to have the most diversified functions in the brain. Its neuronal pathways are most widespread and their distribution correlates well with opiate binding site density. The ability of the enkephalins to inhibit neuronal firing and to inhibit substance P release point to the possibility that, if they are not neurotransmitters, they can modulate release of transmitters throughout the central and peripheral nervous systems [ 1,7]. The precursor protein for the system, proenkephalin (or proenkephalin A) contains six copies of met-enkephalin and one of leu-enkephalin
[lOI.
The endorphinergic system seems to be closely related to pituitary function. It may well play the intermediary in modulating the release of trophic factors from the hypothalamus that regulate the release of hormones from the pituitary gland. In particular, prolactin, growth hormone and antidiuretic hormone release has been shown to be inhibited by fl-endorphin whereas release of luteinizing hormone is stimulated. The endorphinergic system has a highly selective distribution, having a few projections from the hypothalamus extending mainly to the diencephalon and dorsal medial thalamus. The precursor for the endorphinergic system, pro-opiomelanocortin, contains ACTH, melanocyte stimulating hormone, and P-endorphin with met-enkephalin at its amino terminal residue [4,5]. The dynorphinergic system has a distribution that often closely overlaps with the enkephalinergic system but in some instances there are distinct differences; certain dynorphin pathways are closely associated with those of the posterior pituitary. The precursor protein of pre-dynorphin (or preenkephalin B) contains three sequences of leu-enkephalin that are part of a-neo-endorphin, (dynorphin A) and dynorphin B (rimorphine) [ 101.
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The recentness of the discovery of the dynorphinergic system makes it difficult to make conjectures about its functions. A relevant question to ask concerns its connection with the enkephalinergic and endorphinergic systems. Why should there be three opiopeptin systems? Recent evidence from Lee’s and our laboratory support the possibility that the dynorphinergic system might have direct influence over the other two systems [8,9]. As endogenous ligands whose effects are mimicked to varying degrees by morphine, the opiopeptins must somehow be involved not only with acute morphine effects but chronic ones as well. As might be expected therefore, tolerance to morphine conveys cross-tolerance to /3-endorphin and the enkephalins. Cross-dependence is also exhibited by the opiopeptins as evidenced by the fact that fl-endorphin has been shown by Wei and Loh in 1976 to suppress morphine abstinence in animals and in 1980 by Su et al. in humans [ 3,4]. Indeed, primary dependence to some opiopeptins can be produced by sustained administration of low doses as demonstrated for P-endorphin and met-enkephalin in 1976 by Wei and Loh [ 3,4]. Dynorphin is highly effective in suppressing morphine abstinence and although its analgetic activity is controversial, Lee’s group have shown that analgetic sensitivity to morphine in the morphine tolerant animal is restored after dynorphin administration [8]. These findings are surprising in that as mentioned earlier, in the guinea pig ileum dynorphin behaves essentially as a K agonist [ 91. Clearly then, dynorphin is not just a K agonist. In any event, assuming that morphine effects are associated with the opiopeptins, then morphine must alter certain functions that are regulated by these endogenous ligands. Processes that are likely to be affected are those that might alter neurotransmitter function. In order to elucidate the mechanisms involved in opiate-induced analgesia, tolerance, and physical dependence, many investigators have attempted to implicate several neurotransmitters. After altering the synthesis, storage, release, or degradation of acetylcholine, dopamine, norepinephrine, and serotonin, the consequences of such maneuvers on analgesia, and the tolerant-dependent state were investigated. The conclusions, if not the findings, were controversial because a participatory role of any given neurotransmitter could be associated to some degree with the morphine response selected for evaluation. These findings led us to conclude [2] that opiate effects may be mediated by some basic mechanism common to all the aforementioned neurotransmitters. One mutual thread relates to considerable evidence indicating that opioid agonists can inhibit the release of many neurotransmitters, including acetylcholine, dopamine and norepinephrine. Since Ca ‘+ has been implicated in neurotransmitter release, we decided to examine the interrelationships between calcium and morphine. Such a study appeared worthwhile also because electrophysiologic evidence support a role for Ca” in opiate effects. There is mounting evidence that opiates act primarily as inhibitors of specific neurons. Iontophoretic application of opiates in the brain typically inhibits the firing of
29
sensitive neurons. In instances where the effects of opiates are found to be excitatory, they have been found to be due to disinhibition. The inhibitory effect of opiates appears to be mediated presynaptically by inhibiting neurotransmitter release. Thus, Mudge et al. in 1979 found that an enkephalin analog-D-ala’ enkephalin amide, not only inhibits depolarized Ca”-dependent release of substance P from sensory neurons grown in dispersed cell culture but also decreases the duration of the Ca” action potential recorded from the cell body, They suggested that a similar effect on CaZ’ channels located at nerve endings could explain the action of opiates on neurotransmitter release [2]. There is accumulating evidence that Ca ‘+ is involved in opiate action. Studies from our laboratory and others reveal that after acute administration of an opiate, a lowering of brain Ca” occurs at nerve endings, especially in synaptic vesicles. In contrast, after chronic morphine administration there is an increase of Ca2’ at the same sites that is directly related to the degree of tolerance development. We have also found that manipulations, which increased brain Ca”, antagonized opiate effects while lowering the Ca” enhanced opiate effects. Indeed, it was possible to demonstrate analgesia with such Ca2+ antagonists as lanthanum and EGTA, a calcium chelator. Furthermore, La3+ can attenuate the signs and symptoms of morphine withdrawal, presumably by lowering neuronal Ca”. Thus, there appears to be two opposing effects of opiates on neuronal Ca2’, an immediate one to reduce Ca2+ and a delayed counteradaptive response to reverse the acute lowering effect on Ca 2+. It is our thesis that these two opposing actions of morphine can be utilized as a framework to explain such properties as analgesia, tolerance and physical dependence. Our operational hypothesis [ 21 is that the nociceptive state is regulated by the Ca” level within the neuron, a lowering effects analgesia while an elevation results in hyperalgesia. Adaptation to the lowering process occurs and it is cumulative so that with continuous opiate administration there is a gradual build up of Ca 2+. The consequence would be tolerance development since, to produce analgesia, more opiate would be required to lower the elevated neuronal Ca2’. Under such conditions a new elevated steady state for Ca2+ becomes established whereby lowering of Ca” becomes more difficult and maintenance of the state requires the presence of the opiate (physical dependence). The abstinence syndrome would then reflect a supersensitive state to opiate lack or a hyperirritable response to Ca” excess that is ordinarily inhibited by the presence of opiates. A case for CAMP in opiate action can also be made [12] but the results are not as convincing as with Ca”. After we demonstrated that CAMP antagonizes opiate action, three laboratories (Collier, Klee and Hamprecht) independently obtained data to support that opiate effects are mediated through inhibition of adenylate cyclase. If this be the case, then opiates should not only consistently inhibit adenylate cyclase in model systems in which opiates are known to elicit pharmacologic actions but they should also
30
elevate cellular CAMP. Furthermore, opiates should produce effects that are antagonized by CAMP administration or by manipulatidns that tend to increase CAMP such as by phosphodiesterase inhibition. All four criteria can be met by selecting a different model system for each parameter but even in such instances the data are controversial. Although inhibitors of adenylate cyclase can be readily demonstrated in tissue culture of the neuroblastoma X glioma (NG 108-15) cell line, the effect is not so readily shown in the brain of animals injected with morphine. As an example of this point, although most investigators report CAMP antagonism of opiate action, others claim it produces analgesia. Undoubtedly, CAMP must be involved in opiate action but whether it is a crucial initiating event or merely involved secondarily in the cascade of a triggered happening is quite another matter. Likely, the Ca” and CAMP effects are related. It has been proposed, for example, that CAMP may facilitate Ca2’ entry and morphine, by inhibiting adenylate cyclase, should decrease intracellular CAMP and thereby decrease Ca2’ entry and neurotransmitter release. However, since the effects of CAMP are also dependent on Ca”, it is difficult to say whether CAMP is the chicken or the egg. In conclusion, a clear understanding of the mechanisms involved in morphine effects in analgesia, euphoria, tolerance, physical dependence and compulsive usage must await the elucidation of the physiologic role of the opiopeptins. There is now considerable evidence implicating Ca2’ in opiate action and likely these effects are related to the function of opiopeptins. While pharmacologic data can often be misleading, there is evidence indicating that neuropeptides act to inhibit intracellular mediators of neurohormones and neurotransmitters. The mediators within the cell serve to effect the change triggered by these substance, that is, they act as ‘second messengers’. Agents serving as second messengers include CAMP and Ca’” which may alter neurotransmitter release. Perhaps by the twentieth volume of Drug and Alcohol Dependence, we might have the answers and I might still be around. REFERENCES 1 J.P. Huidobro-Toro and E. Leong Way, Opiates, in: D.G. Grahame-Smith (Ed.), Psychopharmacology, Part I, Preclinical Psychopharmacology, Excerpta Medica, Amsterdam, Chap. 8. 2 D. Chapman and E. Leong Way, Pharmacologic consequences of calcium interactions with opioid alkaloids and peptides, in: R.G. Rahwan and D. Witiak (Eds.), Calcium Regulation and Drug Design, ACS Symposium Series, 201, American Chemical Society, Washington, 1982,202 pp. 3 H.H. Loh and D.H. Ross, NeurochemicaJ mechanisms of opiates and endorphins, Raven Press, New York, 1979,563 pp. 4 C.H. Li (Ed.), Hormonal proteins and peptides, Academic Press, New York, 1981, 359 pp. 5 J.B. Malick and R.M.S. Bell, Endorphins, chemistry, physiology, pharmacology and clinical relevance, Marcel Dekker, New York, 1982,296 pp.
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W.R. Martin et al., J. Pharmacol. Exp. Ther., 197 (1976) 517. V. Hijllt, Trends Neurosci. (1983) in press. C. Tulunay et al., J. Pharmacol. Exp. Ther., 219 (1981) 296. K. Yoshimura et al., J. Pharmacol. Exp. Ther., 222 (1982) 71. H. Kakidani et al., Nature, 298 (1982) 245. H.O.J. Collier, Nature 293 (1982) 625.