Effects of naltrexone on plasma corticosterone in opiate-naive rats: A central action

Life Sciences, Vol. 34, pp. I185-i191 Printed in the U.S.A.

Pergamon Press

EFFECTS OF NALTREXONE ON PLASMA CORTICOSTERONE IN OPIATE-NAIVE RATS: A CENTRAL ACTION Richard M. Eisenberg University of Minnesota, Duluth School of Medicine Department of Pharmacology Duluth, Minnesota 55812 (Received in final form January 17, 1984)

Summary We have previously reported the elevation of plasma corticosterone by i.v. naloxone HC1 (NX). This work has been extended, with the current study, showing a similar effect with naltrexone HC1 (NTX) and that this effect is due to a central action of the drug. Using opiate-naive male rats with chronic i°v. catheters, stereotaxically placed intraeerebroventrieular (ICV) cannula guides where necessary, and sound-attenuated one-way vision boxes, serial blood samples were obtained from conscious unrestrained animals. NTX (5.0, 10.0, or 20.0 mg/kg i.v.) resulted in a significant increase in plasma eorticosterone 15 rain following injection. I°V. administration of the methylbromide salts of each drug, which do not cross the blood-brain barrier, did not produce this same elevation in hormone level at several doses (0.4, 1.0, 2.0, 10.0 or 20.0 mg/kg). In contrast, ICV injection of either of the quaternary salts (50 pg/10 ul/animal) resulted in an immediate and sustained rise in plasma cortieosterone. Results obtained demonstrate that NTX has a similar effect on plasma corticosterone as NX at the appropriate doses and that the effect is a central rather than a peripheral one. Naloxone and naltrexone have been considered as pure narcotic antagonists, showing an effect subsequent to opiate administration. Low doses of naloxone have been e f f e c t i v e l y used to antagonize the plasma cortieosterone elevation following morphine or levorphanol (1-3) as well as other opiate effects. The traditional concept of narcotic antagonist activity, however, is being reconsidered since the discovery of the endogenous opiate-like peptides. It would be expected that naloxone and similar drugs should have an action by altering neuronal pathways where endogenous opiates are involved. The semantic dilemma of whether this is an a~onis~ or {KlJ~gg.lli~action may be irresolvable. P r e v i o u s r e s u l t s from our l a b o r a t o r y have shown that sufficient doses of naloxone will produce a dose-related elevation in corticosterone in o p i a t e - n a i v e a n i m a l s (4). N u m e r o u s r e p o r t s h a v e suggested that narcotic antagonists can evoke a v a r i e t y of r e s p o n s e s in t h e a b s e n c e of n a r c o t i c p r e t r e a t m e n t . T h e s e e f f e c t s a r e on n e u r o e n d o c r i n e systems, stress-induced analgesia, and actions of non-opiate drugs of abuse. With respect to the hypothalamo-pituitary-adrenal axis, in addition to our previous results, dose-related increases in basal plasma corticosterone following naloxone have been reported in the mouse (5), the rat (6) and in humans (7,8), as well as enhanced corticosterone stress responses (9). Additionally, low doses of 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.

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naloxone have a direct effect on adrenoeortieal secretion and may potentiate the action of ACTH on the gland (17). The purpose of the present study is to examine the effeets of another "purer opiate antagonist, naltrexone, on the hypothalamo-pituitary-axis and to show that previously described effects are not unique to naloxone. Further, the relative significance of these effects as the result of central rather than systemic drug a c t i o n is determined. Methods Male rats (Harlan Sprague/Dawley, Madison, WI) weighing 200-350 grams were used. They were maintained in individual cages following s u r g e r y at an ambient temperature of (21o) and light cycle (on 06:00-20.'00 hrs). Standard rat chow and water (containing tetracycline, 0.8 mg/ml) were provided ad libitum. Each animal was allowed to acclimate to the surroundings and routine for at least 5 days prior to surgery. Experimental proeedure: The four-day procedure began with the surgical placement of the catheter via the external jugular vein to the entrance of the right atrium as previously described (2). At the same time, in the required experiments, a eannula guide (20 gauge stainless steel) was stereotaxically placed in the appropriate position for intracerebroventrieular (ICV) injections. This implantation and the utilization of individual sound-proofed, one-way vision boxes allows for the serial sampling of blood from conscious, unrestrained animals. In order to maintain catheter pateney during the three day interval between surgery and blood sampling, catheters were flushed periodically with 0.1-0.2 ml heparin/saline, 500U/ml. In addition, animals were placed in the experimental chambers during this interval in order that they could accommodate to the surroundings. On day 4, rats were placed and eonneeted in the chambers: an external sampling tube was attached to the i.v. port and a 25 gauge injection eannula was inserted through the guide into the lateral ventricle. Two hours were allowed to elapse before blood sampling began at 09:30 hrs. This allowed for stabilization of the plasma eortieosterone levels after handling. Each 0.6 ml blood sample was withdrawn following the removal of the void volume of the sampling tube. To reduce the effects of blood loss involved with sequential sampling, the fluid volume was replaced by saline after the first sample. After each subsequent sampling of blood, the cellular fraction from the previous sample, resuspended in saline, was injected. The samples were then transferred to test tubes and centrifuged, and the plasma separated and stored at 0o. Plasma corticosterone was determined by a modification of the fluorometric method of Glick et al. (10). In our procedure, methylene chloride was substituted for chloroform in the extraction of corticosterone and the dilute sodium hydroxide wash was omitted. Experimental drugs and s t a t i s t i c a l analysis: All drug solutions for i.v. injection were made so that the fluid volume injected was 1.0 ml/kg b.w. Equivalent amounts of the saline vehicle were administered to controls. Drugs for ICV administration were dissolved in 10 vl of saline and slowly injected over 20 - 30 sec. Dye was injected at the time of sacrifice and the brain was examined to verify the injection site. Doses were calculated as the salt. Statistical analysis was done using two-way analysis of variance for repeated measures to determine dose and time effects of treatment and one-way analysis of variance with Seheffe's test to determine significant differences between group means using 95% confidence limits. Results

The data in Figure 1 show a dose-related effect on plasma eortieosterone after

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Naltrexone on Plasma Cortlcosterone

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FIG. 1 Effects of naltrexone HCI, injected intravenously a f t e r 0 m i n u t e s , on p l a s m a e o r t i e o s t e r o n e (mean + S.E. in ~g/dl). * indicates a significant d i f f e r e n c e c o m p a r e d t o s a l i n e t r e a t m e n t at t h e r e s p e c t i v e time interval, numbers in parentheses are animals per group. 40-

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FIG. 2 E f f e c t s of n a l o x o n e m e t h y l b r o m i d e , i n j e c t e d i n t r a v e n o u s l y a f t e r 0 minutes, on plasma eorticosterone (mean + / - S.E. in ~ g / d l ) , numbers in parentheses are animals per group. n a l t r e x o n e and s u g g e s t t h a t t h e e f f e c t i v e d o s e must be in e x c e s s of 2.0 mg/kg. Both the magnitude of the hormone elevation and the duration of e f f e c t appear to be related to the dose of drug according to a two-wa~-~nalysis of variance. The question o f w h e t h e r t h e e f f e c t is the r e s u l t o f drug a c t i o n on c e n t r a l or p e r i p h e r a l r e c e p t o r s w a s a p p r o a c h e d using t h e q u a t e r n a r y salts of the narcotic antagonists. The r e s u l t s in F i g u r e s 2 and 3 s h o w t h e e f f e c t s of naloxone methlybromide and naltrexone methylbromide, respectively, on plasma eortieosterone

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40

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FIG. 4 Effects of naloxone m e t h y l b r o m i d e (NXMBr) o r n a l t r e x o n e methylbromide (NTXMBr), i n j e e t e d i n t r a e e r e b r o v e n t r i e u l a r l y a f t e r 0 minutes, on plasma corticosterone (mean + S.E. in v g/dl). * indicates a significant difference compared to saline t r e a t m e n t at t h e r e s p e c t i v e time interval, numbers in parentheses are animals per group. when the drugs were administered i.v. The mean plasma hormone levels, at each test point, were compared to the saline control group at the corresponding time interval. In all instances, there did not appear to be the significant elevation in plasma corticosterone that was originally observed with the tertiary salts. There seems to be a pattern of elevated eorUcesterone over time with the three lower doses of naloxone m e t h y l b r o m i d e and with all of the doses of naltrexone

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methylbromide. Statistical analysis has shown that the hormone changes is related to time only and not drug treatment. Additional statistical comparisons of the actions of naltrexone, 10 and 20 mg/kg (Figure i), with the methylbromide salt at the same doses (Figure 3) were made. These show that the hormone elevations at 15 and 30 rain. following the tertiary compound were significantly higher than after the quaternary compound. At 60 rain., the corticosterone level after naloxone (I0 mg/kg) was lower than after the quaternary salt. The higher mean value 60 min. following saline (Figure 3) is not statistically different from the zero-time value. The data in Figure 4 show quite different responses. There were immediate and sustained hormone increases when the quaternary compounds were injected directly into the cerebroventricular system (50 u g/10 u I/animal).

Discussion The present findings that the effects of naloxone are duplicated by another "pure" antagonist, naltrexone, support the notion that these drugs have effects of their own and that previous observations were not due to a unique property of naloxone. Elevations of plasma corticosterone in response to naloxone administrationhave been reported by several investigators. Originally, Kokka and George (II) observed an increase following 46 mg/kg in the rat. T h i s was confirmed by studies in our laboratory and others using doses in excess of what is traditionally used to antagonize morphine's action (4,5). A more recent report suggests that doses of 0.25 - 2.0 mg/kg are also effective stimulants of this system (6). The question of the significance of a direct action of narcotic antagonists on the adrenal gland to elevate hormone levels was raised by Lymangrover et al. (17). They showed that low doses of naloxone stimulated steroidogenesis in isolated adrenocortical tissue and potentiated the action of ACTH. The current results suggest that the central actions may be more predominant following systemic administration. There are several possible explanations for these data that are worth considering: i) with sufficient dose, naloxone and naltrexone have direct agonist actions on central or peripheral opiate receptors similar to that of morphine-like compounds; 2) opiate receptors have a role in the negative feedback system controlling the hypothalamo-pituitary-adrenal axis - - opiate antagonists disinhibit the system resulting in a stimulation; and 3) naloxone or naltrexone precipitate a withdrawal stress response in normal animals "dependent" on endogenous opiates. On examining the first alternative, there appears to be mountingevidence from the numerous reports over the last several years that opiate antagonists produce an effect in the absence of narcotic pretreatment. Several neuroendocrineaxes appear to be affected by naloxone. Both LH and FSH secretion are elevated (12,13), with t o l e r a n c e occurring to this effect (14), while prolactin levels are decreased following naloxone (13,15). The response of TSH to TRH ( 1 6 ) and the adrenoeortical response to ACTH (17) are both enhanced. The results presented in this report and our previous data (4) argue against such a simple agonist action since tolerance to the naloxone-inducedcortieosterone effect has not been shown as one might anticipate if the action of naloxone was similar to that of morphine.

The s e c o n d h y p o t h e s i s i n v o l v i n g o p i a t e r e c e p t o r s in the n e g a t i v e f e e d b a c k of the hypothalamo-pituitary-adrenal a x i s m a y h a v e s o m e basis. Both ACTH and b e t a -endorphin a r e d e r i v e d from a c o m m o n p r e c u r s o r , p r o - o p i o e o r t i n (18), a n d b o t h s u b s t a n c e s are s e c r e t e d concomitantly (19). That the s e c r e t i o n of both substances are under the influence of CRF and under negative f e e d b a c k control a p p e a r s t o b e s u p p o r t e d by t h e f i n d i n g t h a t s t r e s s - i n d u c e d a n a l g e s i a is a n t a g o n i z e d by dexamethasone pretreatment and attenuated by naloxone (20). Thus, t h e f e e d b a c k s y s t e m may be s e n s i t i v e to both e o r t i e o s t e r o i d s and endogenous o p i a t e s . Naloxone or naltrexone blockade of these r e c e p t o r s would r e s u l t in a f a c i l i t a t i o n o f C R F r e l e a s e and an i n e r e a s e in p l a s m a c o r t i e o s t e r o n e . The d a t a a l s o s u p p o r t the

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concept that a central mechanism is involved and that there is some proximity to the cerebroventricular system. The third explanation of the results assumes that an "addiction" or "dependence" normally exists to endogenous opiates similar to that which can be produced with exogenous opiates. When naloxone or naltrexone is administered at sufficient doses, a withdrawal response is precipitated as in animals acutely or chronically treated with morphine resulting in a plasma corticosterone increase (1,3). It has been demonstrated that exogenously administered endorphins can prevent withdrawal in the morphine-dependent animal (21). Additionally, endogenous opiates, injected directly into the cerebroventricular system can produce dependence such that naloxone will produce the characteristic withdrawal signs (22). These findings support the notion that the peptides do produce dependence, at l e a s t pharmacologically. Whether the opiate antagonists in the present studies produce the plasma corticosterone elevation by precipitating a "withdrawal" response can not be ruled out at the present time. The current data show that our original finding that naloxone elevates plasma corticosterone is not unique to this antagonist. Further, mechanisms involved appear to be predominantly central in origin, have some proximity to the cerebro-ventricular system, and presumably involve a disruption of endogenous opiate function. Ackn~)wledgments The author wishes to express his appreciation for technical assistance provided by Miss Mary Wilkinson, Mr. Allen Odean, and Mrs. Judy Ostern. The following drugs were generously supplied: naloxone and naltrexone (Endo Laboratories, Wilmington, DE) and naloxone and naltrexone methylbromide (Boehringer Ingelheim, Ridgefield, CT). This study was supported by USPHS grant DA 02015. References 1.

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