Does naloxone always act as an opiate antagonist?

Life Sciences, Vol. 33, Sup. I, 1983, pp. 739-742 Printed in the U.S.A.

Pergamon Press

DOES NALOXONE ALWAYS ACT AS AN OPIATE ANTAGONIST? A. A.-B. Badawy, M. Evans, N. F. Punjani and C. J. Morgan South Glamorgan Health Authority, Addiction Unit Research Laboratory, Whitchurch Hospital, Cardiff CF4 7XB~ Wales, U.K. (Received in final form June 26, 1983) Summary Evidence for the ability of the opiate antagonist naloxone to block a variety of metabolic effects exerted by morphine and non-opiate drugs is reviewed. Naloxone prevents or reverses the following effects in the rat: (a) the chronic morphine-induced increase in liver [NADPH]; (b) the consequent chronic morphine-induced inhibition of liver tryptophan pyrrolase activity; (c) the resultant chronic morphine-induced enhancement of brain 5-hydroxytryptamine synthesis; (d) the similar effects on liver and brain tryptophan metabolism exerted chronically by other drugs of dependence (ethanol, nicotine and phenobarbitone)~ (e) the acute ethanol-induced increase in the hepatic [NADH]/[NAD ] ratio. Naloxone also (f) inhibits basal and stimulated lipolysis in fed and 24hr-starved rats. This leads to prevention of (g) the consequent increase in the availability of circulating free tryptophan, and (h) the resultant tryptophan-mediated decrease in liver 5-aminolaevulinate synthase activity. The question of how many of these effects involve changes in endogenous opiates or at opiate receptors is not clearly understood at present and thus merits investigation. However, because most of the above effects are explained on biochemical grounds, and in view of evidence from behavioural and pharmacological studies [see (1)], the possibility must be considered that many of the actions of naloxone may be unrelated to its opiate-receptor-antagonistic properties. The opiate-receptor antagonist naloxone (17-allyl-4,5~-epoxy-3,14-dihydroxynormorphinan-6-one) has been used extensively in studies in which antagonism of morphine or other agents was considered a criterion for implicating endogenous opiates and/or their receptors in the actions of such agents. A recent review (1) has, however, pointed out that, although many effects of naloxone are due to blockade of opiate receptors, there are many findings strongly suggesting that this agent may act in a variety of instances by mechanisms unrelated to such a blockade. Such findings include the reported [for references, see (1)] antagonism by naloxone of: (a) the antinociceptive actions of certain non-opiate drugs; (b) the central nervous system depressant actions of non-opiate depressants, including ethanol; (c) the behavioural actions of drugs that interact with dopaminergic systems; (d) the biological actions of drugs and agents that interact with GABAergic systems~ (e) the behaviou~cal, pharmacological and electrophysiological actions of a group of miscellaneous agents, including ACTHl_24 , adenosine, N,N-dimethyltryptamine and lysergic acid diethylamide. In addition to antagonism of these biological actions, naloxone has been shown in this Laboratory to antagonize or prevent a variety of biochemical changes in the rat caused by morphine and non-opiate drugs in a number of metabolic 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

740

Biochemical Effects of Naloxone

Vol. 33, Sup. I, 1983

systems. The purpose of this article is therefore to review these novel biochemical findings with naloxone and the biochemical rationale behind the investigations which led to their demonstration. It is hoped that such an account will serve to illustrate the versatility of the biological role of naloxone and, thereby, stimulate additional interest in, and further work on, the mechanisms of the various actions of this opiate antagonist. Chronic effects of morphine on tryptophan disposition and metabolism in liver and brain and on liver NADPH concentration: We have demonstrated (2) the ability of chronic morphine administration to inhibit the activity of rat liver tryptophan pyrrolase (tryptophan 2,3-dioxygenase, EC 1.13.11.11) by increasing the hepatic concentration of the allosteric inhibitor NADPH, and presented evidence (3) strongly suggesting that, by decreasing hepatic tryptophan degradation, this inhibition increases the availability of the circulating amino acid to the brain, thus leading to an enhancement of cerebral 5-hydroxytryptamine (5-HT) synthesis. Because naloxone has been known for sometime (4) to antagonize the morphine effect on 5-HT metabolism, it was considered of interest to find out if such an antagonism is also associated with a blockade of the morphine effects on tryptophan disposition and on liver tryptophan pyrrolase activity and [NADPH]. Naloxone was indeed found (3) to reverse the chronic morphine-induced increase in liver [NADPH] and the associated inhibition of liver pyrrolase activity. Moreover, time-course studies (3,5) showed that the naloxone reversal of the morphine effects on liver and brain tryptopnan metabolism was a rapid phenomenon~ being achieved as early as 1Omin after antagonist administration (Img/kg, intraperitoneally). These findings therefore not only strongly suggest that chronic morplnine administration enhances brain 5-HT synthesis by inhibiting liver tzlrptophan pyrrolase activity, but also demonstrate the ability of naloxone to reverse or antagonize the effects of morphine on tryptophan disposition and on the hepatic metabolism of tryptophan and pyridine dinucleotides. Chronic effects of ethanol~ nicotine and phenobarbitone on tryptophan disposition and metabolism in liver and brain and on liver pyridine dinucleotide (phosphate) concentrations: Chronic administration of ethanol, nicotine and phenob~rbitone has been shown (2,6) to inhibit rat liver tryptophan pyrrolase activity by increasing hepatic [NADPH]; additionally, the ethanol treatment increased [NAEH]. As is the case with morphine, this pyrrolase inhibition by the above three non-opiate drugs of dependence was shown (3,7) to increase tryptophan availability to the brain and, hence, to enhance cerebral 5-HT synthesis. In view of these similarities between all four drugs of dependence, it was considered of interest to find out if naloxone is also capable of reversing the chronic effects of the above three non-opiate drugs of dependence on tryptophan disposition and metabolism in liver and brain. Naloxone (~mg/kg) was indeed found (8) to reverse all these effects. An additional finding of interest was that naloxone caused (3,8) an increase in serum corticosterone concentration in rats chronically treated with ethanol, morphine, nicotine or phenobarbitone; the mechanism(s) of this effect requires investigation. Acute ethanol intoxication and the redox states of the hepatic NAD(P) couples: If, as was previously shown (8), naloxone is capable of reversing the chronic ethanol-induced disturbance in the redox states of the above pyridine dinucleotide
Vol. 33, Sup. I, 1983

Biochemical Effects of Naloxone

741

to 92mg/dl. In experiments so far unreported, we found that naloxone is an even-faster accelerater of ethanol removal from the circulation, being capable of producing the same effect quantitatively as above within 15min of administration. Thus in addition to causing rapid arousal in intoxicated individuals, naloxone appears to possess two of the most important properties needed in an ideal 'amethystic agent' (10), namely those of rapid onset of action and fast acceleration of ethanol removal. As well as causing a rapid arousal, naloxone is capable of antagonizing many behavioural and other effects of acute ethanol intoxication, and we suggested (9,11) that pharmacokinetic antagonism may be one mechanism by which naloxone may act in this condition. Acute effects of morphine and other lipolytic agents on liver 5-aminolaevulinate s~nthase activity and the haem saturation of tryptophan pyrrolase: Before describing any findings with naloxone in this section, it may be useful to state that hepatic haem biosynthesis is regulated by feedback mechanisms exerted at the first and rate-limiting step of the pathway, that catalyzed by 5-aminolaevulinate synthase (EC 2.3.1.37; 5-ALA-S) most likely by a small~ rapidly-turningover and readily-exchangeable pool of haem commonly referred to as the regulatory-haem pool. There is also considerable evidence (12,~3) strongly suggesting that this regulatory-haem pool is very closely associated with the hepatic haemoprotein tryptophan pyrrolase, and demonstrating the existence of an inverse relationship between the haem saturation of this latter enzyme and 5-ALA-S activity. That acute morphine administration to rats increases the haem saturation of tryptophan pyrrolase in liver has been demonstrated by us (2) and confirmed in a subsequent study (14) in which it was also shown that morphine enhances the activity of the major haem-catabolizing enzyme, haem oxygenase (EC 1.14.99.3), and that this enhancement can be prevented by naloxone. Although it was suggested (14) that morphine increases haem availability for saturation of tryptophan pyrrolase and for enhancement of haem oxygenase activity by releasing the haem of the major hepatic haemoprotein, cytochrome P-450, we presented evidence (13) strongly suggesting that morphine influences tryptophan pyrrolase and haem oxygenase (and also decreases 5-ALA-S activity) by acting via tryptophan [for the effects of the latter on pyrrolase and 5-ALA-S, see (%5)3 secondarily to the increase in tryptophan availability to the liver following its displacement from serum-protein binding sites by non-esterified fatty acids, whose circulating levels are increased as a consequence of stimulation of lipolysis by the morphine treatment. In support of this latter mechanism, we found (13) that other lipolytic agents (such as endotoxin and theophylline), as well as morphine itself, exerted similar effects on serum non-esterified fatty acid concentration, serum tryptophan binding and availability, liver tryptophan concentration, liver 5-ALA-S activity and the haem saturation of liver tryptophan pyrrolase, and that all these effects are preventable by pretreatment of rats with the ~-adrenoceptor-blocking agent propranolol. Because naloxone was shown (14) previously to prevent the morphine-induced enhancement of haem oxygenase activity, it was considered of interest to find out if it can also block the associated effects on 5-ALA-S activity and the haem saturation of trlaptophan pyrrolase. Naloxone did indeed preven~ all the effects of morphine on haem and trlaptophan metabolism and disposition, and was also capable of preventing the similar effects exerted by the other two lipolytic agents endotoxin and theophylline (13). Inhibition of lipolysis and prevention of the lipolysis-induced chanqes in tryptophan disposition by naloxone: The above findings with naloxone led us to consider the possibility that this opiate antagonist may prevent the increase in tr!aptophan availability to the liver caused by administration of the above lipolytic agents. This was found to be so (%3) and it was also shown that naloxone exerted this effect by preventing the increase in serum non-esterified fatty acid concentration and the consequent rise in that of free serum

742

Biochemical Effects of Naloxone

Vol. 33, Sup. I, 1983

tryptophan in starved rats. These effects of naloxone were observed at i.5hr after intraperitoneal administration of a 5mg/kg dose to starved rats given the lipolytic agents at 0.5hr after naloxone. Additionally, in control starved rats (i.e. those treated with 0.9~ NaCI instead of lipolytic agents), naloxone caused significant decreases in concentrations of serum non-esterified fatty acids, free serum tmyptophan, total serum tryptophan and liver tr!rptophan (13). Naloxone also lowered serum non-esterified fatty acid concentration in fed rats both under basal conditions and after theophylline administration. These results therefore demonstrate the ability of naloxone to influence lipolysis and tryptoph~n disposition, the precise mechanism(s) of which clearly requires detailed investigation. General conclusions and conrnents: The above account has shown that haloxone antagonizes or prevents a variety of biochemical effects exerted by morphine and various non-opiate drugs on a number of metabolic systems, such as liver and brain tryptophan metabolism, tryptophan disposition, liver haem metabolism, liver pyridine dinucleotide metabolism and lipolysis. Many of these observations may be of importance in relation to a number of clinical practices. To the biochemist, many of the above effects of naloxone need not be explained by implicating endogenous opiates and/or receptors. However, the question of how many of these effects really involve such receptors can only be answered by performing further experiments using a number of criteria (I) including comparison of antagonism by stereospecific isomers of opiate antagonists. Acknowledgements: Reviewed work from this Laboratory was supported by grants from the Welsh Office and the Wellcome Trust. We are also grateful to Endo Laboratories and the DuPont Organization for a generous gift of naloxone and to Mr. ~. Dacey for skilful animal maintenance. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15.

J. SAWYNOK, C. PINSKY and F. S. L A B ~ L ~ , Life Sci. 25, 1621-1631 (%979). A. A.-B. BADAWY and M. EVANS, Adv. Exp. Med. Biol. 59, 229-251 (1975). A. A.-B. BADAWY, N. F. PUNJANI and M. EVANS, Biochem. J. 196, 161-170 (1981). F. H. SHEN, H. H. LOH and E. L. WAY, J. Pharmacol. Exp. Ther. 175, 427-434 (1970). A. A.-B. BADAWY and M. EVANS, Br. J. Pharmacol. 74, 511-513 (1981). A. A.-B. BADAWY and M. EVANS, Biochem. J. 148, 425-432 (1975). A. A.-B. BADAWY, N. F. PUNJANI and M. EVANS, Biochem. J. 178, 575-580 (1979). A. A.-B. BADAWY, M. EVANS and N. F. PUNJANI, Br. J. Pharmacol. 74, 489494 (1981). A. A.-B. BADAWY and M. EVANS, Br. J. Pharmacol. 74, 514-516 (1981). R. L. ALKANA and E. P. NORLE, Biochemistry and Pharmacology of Ethanol (eds E. MAJCHROWICZ and E. P. NOBLE), vol. 2, pp. 349-374, Plenum Press, New York (1979). A. A.-B. BADAWY and M. EVANS, ~nn. Intern. Med. 98, 672 (1983). A. A.-B. BADAWY, A. N. WELCH and C. J. MORGAN, Biochem. J. 206, 441-449 (1982). A. A.-B. BADAWY and C. J. MORGAN, Biochem. J. 206, 451-460 (1982). D. GURANTZ and M. A. CORREIA, Biochem. Pharmacol. 30, 1529-1536 (1981). A. A.-B. BADAWY, A. N. WELCH and C. J. MORGAN, Biochem. J. 198, 309-314 (1981).