Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, Bethesda, Maryland, 20014.

 

Cyclic nucleotides, and particularly cyclic AMP, are believed to play important roles in the nervous system as second messengers of synaptic communication (1-4). Thus, catecholamines functioning through 8-adrenergic receptors, dopamine, histamine, adenosine, E prostaglandins and some other putative neurotransmitters and neuromodulators are believed to function as substances which stimulate adenylate cyclase and thus raise cyclic AMP levels in cells with the appropriate receptors. Morphine and the other opiate-like substances have been shown to interact, directly or indirectly, with Many of these transmitter and modulator substances (5-7), and it was therefore natural to ask whether the opiates might not also function via the adenylate cyclase system.

 

Studies by Collier and Roy (8,9) with brain homogenates provided the first biochemical evidence that morphine may function as a specific inhibitor of adenylate cyclase. These workers found that morphine, levorphanol, methadone, heroin, etorphine and pethidine, but not dextrorphan, inhibit the PGE1 stimulated conversion of (3H)-ATP to (3H)-cyclic AMP in whole homogenates of rat brain, and that this inhibition is prevented by naloxone. These experiments demonstrated the approiate specificity and pharmacological relevance of the inhibition of opiates of cyclic AMP formation in brain homogenates. Although a number of workers have not been able to demonstrate such inhibitory effects of opiates on cyclic AMP production in brain homogenates (10-12), recent work by Wilkenin and Makman (13) in homogenates of monkey amygdala confirms at least in large part, the results of Collier and Roy, as do some recent experiments with brain slices by Minneman and Iversen (14). Nevertheless, the situation in brain seems excessively complicated due, in part, to the large number of cell types and functional regions represented both in homogenate and in slice preparations. Regional heterogeneity of response in the brain is well demonstrated in experiments in which cyclic nucleotide levels are measured after in vivo manipulations (15,16).

 

In an effort to find a more homogeneous cell population with which to study the mechanism of morphine action, Klee and Nirenberg (17) screened a number of cell lines, which are maintained in continuous culture, for the presence of opiate receptors. One of the cell lines examined, neuroblastoma x glioma hybrid NG108-15, was found to possess a large number of morphine receptors (2-3 x 105/cell) and this cell was chosen for further study. The hybrid cells had been prepared (18) by virus-induced fusion of a mouse neuroblastoma cell line (clone N18-TG2) (19), and a rat glioma cell line (clone C6-BU1) (20), neither of which, interestingly, contains large numbers of morphine receptors. Clone N18-TG2 has subsequently been shown to contain a measurable, but still relatively small, number of receptors when a modified assay is used (21). The properties of the opiate receptor of NG108-15 hybrid cells are, in all ways tested (17) similar to those found in rat brain homogenates (22-26).

a. Measured in reaction mixtures identical in composition with those used in the assay of adenylate cyclase, by displacement of (3H)-naloxone. Data from Sharma, Nirenberg and Klee (21).

 

Sharma, Nirenberg and Klee (21) found that the adenylate cyclase activity of homogenates of NG108-15 is sensitive to inhibition by opioid agonists such as morphine, etorphine and levorphanol at concentrations which are consistent with the pharmacological potency of these drugs and which are also similar to the concentrations at which they bind to the opiate receptor (Table 1). Furthermore, dextrorphan, the inactive stereoisomer of levorphanol does not inhibit the adenylate cyclase of these cells nor does the opiate antagonist, naloxone. The latter substance will, however, effectively antagonize the action of morphine and other opiates on adenylate cyclase, at concentrations which are consistent with the known affinity of naloxone for the opiate receptor. Furthermore, the adenylate cyclase activity of homogenates of the parental cell lines neuroblastoma N18-TG2 (containing few opiate receptors) and glioma C6-BUl (containing no opiate receptors) is inhibited only slightly and not at all, respectively (21). Thus, these studies, which have been confirmed in other laboratories (10, 27), establish the functional linkage of morphine receptors as inhibitors to adenylate cyclase.

 

Study of cyclic AMP levels in intact NG108-15 cells by Traber, Hamprecht and their colleagues (28-31) as well as Sharma et al. (21,22) has led to the same conclusion. Cyclic AMP levels of these cells are raised many fold by prostaglandin E1 (32) or adenosine (33) and these increases are blocked by opiates in a naloxone reversible, and stereospecific way (29, 33). Basal cyclic AMP levels are also found to be lowered by opiates (21,33).

 

Endogenous opiate peptides, the enkephalins (34,35) and others (36-39), have been recently characterized and are the objects of extraordinarily intense investigations. These facinating substances are undoubtedly of physiological importance (40,41) and apparently supply the answer to the question of the purpose for which opiate receptors exist in the vertebrate nervous system. It was natural, therefore, to ask whether the enkephalins and other endogenous opiate peptides function as receptor-mediated inhibitors of adenylate cyclase as do the standard opiates. These pentapeptides have been shown by Klee and Nirenberg (42) to be among the most potent receptor-mediated inhibitors of adenylate cyclase known and by Brandt et al. (43) to inhibit cAMP accumulation in NG108-15 hybrid cells. Their properties are much like those of morphine except that they are short acting, presumably due to rapid proteolytic degradation (44,45). Longer fragments of 0-lipotropin as well as other peptides with opiate-like properties have also been shown to inhibit the adenylate cyclase of NG108-15 cells in a naloxone reversible manner (46), and sometimes with a somewhat longer duration of action than that of the enkephalin. We may exect studies in the future of the interaction of a series of related peptides with the opiate receptor and with adenylate cyclase to lead to new insights into the mechanism of action of these receptors. Certainly, the information which has emerged thus far needs to be supplemented with more detailed studies.

 

The problems of opiate tolerance and dependence are characteristic of all examples of this class of drug which have been studied in sufficient depth. Studies of adenylate cyclase and cyclic AMP levels in neuroblastoma x glioma NG108-15 cells have also led to interesting new insights into these phenomena. Hybrid cells, when cultured for a number of hours in the continued presence of opiates (31,47) or of enkephalin (48,49) develop a compensatory mechanism which results in tolerance to the effects of these substances simultaneously rendering the cells dependent upon the presence of opiates for normal cellular function. The biochemical basis for this tolerance and dependence appears to be the development of an increased amount of adenylate cyclase activity (47). An experiment demonstrating the development of increased adenylate cyclase activity in response to cell culture in the presence of morphine is shown in Table 2, experiment 1. Enzyme activity is seen to increase to such an extent that, in the morphine treated cells, activity measured in the presence of added morphine is equal to that of control cells measured in the absence of inhibitor. In the assay, morphine, which had been present during culture of the cells has been removed by washing prior to homogenization. Cells which have responded to opiates or opiate peptides by a compensatory increase in adenylate cyclase activity are dependent upon opiates since the rapid withdrawal of these substances by means of the antagonist naloxone will result in dramatic increases in cyclic AMP levels to values well aove those normally found (Table 2, experiment 2).

As we have seen, opiates have two types of effects upon adenylate cyclase in NG108-15 cells: an immediate inhibition followed by a slowly developing increase in total activity. This dual regulation (47) can account for all of the known effects of narcotics, although other contributory mechanisms cannot be ruled out at present. A diagrammatic summary of the dual regulation of adenylate cyclase by opiates is presented in figure 1. The figure shows that opiates produce an immediate fall in cellular cyclic AMP levels (panel A) due to inhibition of adenylate cyclase without change in its total activity (panel B). Upon continued exposure to opiates the cells respond by an increase in the total amount of adenylate cyclase activity which (even though the enzyme is still inhibited by morphine) brings cyclic AMP levels back to normal and the cells are now tolerant to opiates. An important feature of the opiate receptor mediated inhibition of adenylate cyclase, is that incubation of enzyme activity is never complete, even at saturating amounts of inhibitor (21). If inhibition of enzyme activity were complete, tolerance could not be produced by the mecahnism described here except at low, subsaturating concentrations of drugs. The model also demonstrated that tolerant cells are dependent upon opiates since rapid withdrawal of morphine results in a release of the inhibition of an abnormally high amount of adenylate cyclase activity with the consequent formation of abnormally large amounts of cyclic AMP. The dual regulation model is consistent with the compensating hypertrophy proposed by Collier and Roy (8), and is a specific example of the enzyme inhibition and expansion models proposed by the Goldsteins (50) and Shuster (51) and by the homeostatic model proposed by Himmelsbach (52). It has many features which are testable in animals and some evidence that this type of mechanism operates in whole animals has been produced (16,53-56). On the other hand, many features of the model are, as yet, only poorly understood. Thus, neither the mechanism of the receptor mediated inhibition of adenylate cyclase activity nor that of the receptor mediated delayed positive response is understood.

 

Morphine receptors can now confidently be considered to be, in reality, polypeptide hormone (or neurotransmitter) receptors, and they are most probably located on the cell surface (17). Their coupling with adenylate cyclase may be via the same, as yet unknown, types of mechanisms which couple glucagon, ACTH or other peptide hormone receptors with the enzyme. Although there is some evidence which implicates protein synthesis in the mechanism of acquisition of opiate tolerance and dependence, it is not at all clear that the increased adenylate cyclase activity is due to the synthesis of an increased number of molecules of enzyme. Conceivably, the increase in activity observed may reflect altered control mechanisms and not more enzyme. These questions are currently being explored in a number of laboratories and their resolution should do much to increase our understanding of the mechanism of opiate action.

 

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