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Effects of naloxone on plasma corticosterone in the opiate-naive rat
Life Sciences, Vol. 26, pp. 935-943 Printed in the U.S.A.
Pergamon Press
EFFECTS OF NALOXONE ON PLASMA CORTICOSTERONE IN THE OPIATE-NAIVE RAT Richard M. Eisenberg Department of Pharmacology University of Minnesota, Duluth School of Medicine Duluth, Minnesota 55812 (Received in final form January 22, 1980) Sumnary Naloxone HCl (NX) has long been considered to be a pure narcotic antagonist, having an effect only subsequent to pretreatment with a narcotic. Characteristically, low doses of NX have been used to antagonize the effects of analgesic doses of narcotics and to precipitate withdrawal in chronically treated animals. In this study, the effects of high doses of NX (2.0-20.0 mg/kg) on changes in plasma corticosterone were examined in the opiate-naive animal. Using male rats with chronic intravenous catheters and one-way vision boxes, injections were made and serial blood samples were obtained in the conscious, unrestrained animal. The acute administration of NX to the opiate-naive animal produced a dose-related increase in plasma corticosterone with respect to both amplitude and duration. NX (10.0 mg/kg i.v.) produced a significant elevation in hormone level at 15 and 30 minutes. With NX (20.0 mg/kg i.v.) the duration of the response was extended to 60 minutes. To examine whether short-term tolerance to this effect could be produced, animals were given a single pretreatment with either NX (10.0 mg/kg) or saline i.v. Two hours later NX produced a similar elevation in hormone level in both groups. The effect of chronic injection of NX was also studied. Animals pretreated with either NX (10.0 mg/kg) or saline S.C. once daily for 7 days did not show a significant difference following the subsequent administration of NX. In both cases, a significant elevation of plasma corticosterone resulted. The results suggest that NX may have a direct effect on opiate receptors resulting in an elevation of plasma hormone levels or NX may be disrupting an endogenous opiate-receptor interaction producing a stress response. Naloxone has long been considered as a pure narcotic antagonist, having a demonstrable effect subsequent only to treatment with a narcotic. In our laboratory, as in others, low doses of naloxone have been used effectively to antagonize the plasma corticosterone elevation induced by a narcotic (1,2). This
A preliminary report of these results was presented at the annual meeting of the Endocrine Society, Anaheim, CA, 1978. The author wishes to acknowledge the expert technical assistance provided by Mrs. Patricia Melis. This project was supported by USPHSgrant DAO2015. 0024-3205/80/120935-09$02.00/0 Copyright (c) 1980 Pergamon Press Ltd
936
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Vol. 26, No. 12, 1980
classical concept of naloxone activity is now.under thorough reinvestigation since the discovery of the endogenous morphine-like substances--the endorphins (3,495). It would now appear that there should be a presumptive action by naloxone alone simply by disrupting neural.pathways where endorphins or enkephalins are involved. Numerous reports suggest that naloxone is capable of evoking a pharmacological response in the absence of a narcotic. Intraperitoneal administration of 0.25.0 mg/kg of naloxone in the rat produced an elevation of serum LH and FSH and lowered serum prolactin and growth hormone (6). Higher doses in the mouse result in a dose-related elevation of plasma corticosterone (7). Hyperalgesia in rats has been reported after pretreatment with naloxone prior to hot plate test (8), exposure to footshock (9), subjection to cold water stress (lo), acetic acid injection (11) or bradykinin injection (12). Further, naloxone similarly altered pain perception in human subjects (13,14). Interestingly, naloxone has been reported to interact with a variety of non-opiate drugs. The behavioral effects of chlorpromazine in the pigeon (15), d-amphetamine in the rat (16,17), d-amphetamine, ethanol, and LSD in the mouse (T8,19,20) have been altered by moderate doses of naloxone. Analgesia produced a nitrous oxide (21), phenowbenzamine (22,23), and ketamine (24) has been antagonized by naloxone. The current study examines the effects of naloxone on plasma corticosterone. This parameter is characteristically elevated by acute treatment with analgesic doses of morphine (1) and levorphanol (2) and during spontaneous and naloxoneinduced narcotic withdrawal (25,2). Additionally, the effects of acute and chronic naloxone pretreatment on the plasma corticosterone response was also investigated. Materials and Methods Male rats (ARS SpraguelOawley, Madison, Wise.) weighing 200-350 grams were utilized in this study. All animals were placed in individual cages maintained at constant temperature (21OC) and light cycle (lights on at 06:OO to 20:00 hrs). Normal laboratory chow and water containing 0.8 mg/ml tetracycline were provided ad libitum. Each animal was allowed to acclimate to the surroundings and routine xrmst five days prior to surgery and experimentation. The surgical implantation of catheters and the utilization of individual sound-proofed, one-way vision boxes during serial sampling of blood from conscious, unrestrained animals have been described previously (26). Surgical/experimental procedure: 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 already described (2). In order to maintain catheter patency during the three day interval between surgery and blood sampling, catheters were flushed with 0.1-0.2 ml heparinized-saline, 500 U/ml, on days 2 and 3. In addition, animals were placed in the experimental chambers daily during this interval in order that they could accomnodate to the surroundings. On day 4. the animal was placed into the experimental chamber and connected to the injection/sampling tube at least two hours prior to any blood sampling to allow for stabilization of the plasma corticosterone levels after handling. Blood sampling in each experiment began either at 09:30 or lo:15 hrs. 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 the 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, suspended in saline, was injected. The samples were then transferred to test tubes and centrifuged, and
Vol. 26, No. 12, 1980
Naloxone and Corticosterone
937
the plasma separated and stored at O°C. Plasma corticosterone was determined by a modification of the fluorometric method of Glick et al. (27). In our procedure, methylene chloride is substituted for chlorofozs the extraction of corticosterone and the dilute sodium hydroxide was omitted. Experimental drugs and statistical analysis: Naloxone solutions were made in 0.9% saline so that the fluid volume injected was 1.0 ml/kg b.w. Equivalent amounts of saline were administered to controls. The doses of naloxone HCl were calculated at the salt. The naloxone was a gift from Endo Laboratories, Garden City, New York. In all of the data presented in this study, the posttreatment plasma corticosterone levels are shown as the mean percent of the pretreatment (or time zero) plasma corticosterone levels. For the data reduction, individual plasma corticosterone levels for each post-treatment time interval were calculated as a percent of the pretreatment level for each animal and this value was then transformed to the log. Statistical analysis of the transform data for each time interval was done using analysis of variance where t-values were calculated using the pooled variance estimates. Levels of significance are expressed in each case. Results The first experiment shown in figure 1 was designed to show the effects of several doses of naloxone on plasma corticosterone over a four-hour period.
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FIG. 1 Effects of naloxone HCl on plasma corticosterone. Values are shown as the mean percentage of zero time 2 S.E. The pretreatment hormone level for all animals is 7.9 + 0.8 pg% (mean f S.E.) and is shown as 100% at zero time. Numbers at graph symbols indicate animals per group. In some cases standard errors were omitted for clarity. Significant responses are indicated by *, please refer to text.
Naloxone and Corticosterone
938
Vol. 26, No. 12, 1980
Saline or one of three doses of naloxone (2.0, 10.0, or 20.0 mg/kg) were injected i.v. inznediatelyfollowing the withdrawal of the first blood sample at time zero. The two higher doses of the drug produced a significantly elevated (pcO.002) dose-related corticosterone response at 15 and 30 minutes following treatment. With the highest dose, 20 mg/kg, the response was still significantly greater (~~0.02) than the control at 60 minutes but at 180 minutes the value was significantly lower than the control (p
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FIG. 2 Effects of naloxone HCl (10.0 mg/kg) on plasma corticosterone after a single i.v. pretreatment with either saline or naloxone. Values are shown as the mean percentage of zero time f S.E. The pretreatment hormone level for the naloxone/saline group is 7.6 + 1.6 pg%, for the saline/naloxone group it is 6.9 + 1.6 pg%, and for the naloxone/naloxone group it is 7.4 + 2.2 !Jg%. Each is shown as 100% at zero time. Numbers at graph symbols indicate animals per group. In some cases standard errors were omitted for clarity. Significant responses: *p
240
939
Naloxone and Corticosterone
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Three groups of naive animals were treated with a combination of saline and/or naloxone. The first intravenous injection was made 120 minutes prior to time zero. Both groups treated pith naloxone, 10 mg/kg i.v., after tima zero showed an almost identical plasma corticosterone response over the four-hour sampling period without regard to whether the pretreatment had been saline or naloxone (10 mg/kg). Hormone levels after the saline/naloxone combination were significantly elevated at the 15, 30. 60 and 120 minute intervals (p
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FIG. 3 Effects of naloxone HCl (10.0 mg/kg) on plasma corticosterone after once-daily injections of naloxone or saline for 7 days. Values are shown as the mean percentage of zero time f S.E. The zero time hormone level for the saline pretreated group is 6.8 * 0.8 Pg% and for the naloxone pretreated group it is 3.7 + 0.8 ug%. Each is shown as 100% at zero time. Numbers at graph symbols indicate animals per group. In some cases standard errors were omitted for clarity.
940
Naloxone and Corticosterone
Vol. 26, No. 12, 1980
with either saline or naloxone, 10 mg/kg, for 7 days. On the day of the serial blood sampling (day 8), animals were given their respective subcutaneous injections just prior to being placed in the experimental chambers. Naloxone, 10 mg/kg, was administered intravenously via the injection/sampling tube after time zero. Both the saline and drug-pretreated groups showed an elevation in hormone levels at 15 and 30 minutes. There was no statistical difference between the two groups. The pattern of response was similar to that observed in the acute response shown in figure 1. At the later time intervals there was no difference between the groups. Discussion The observation that a high dose of naloxone is capable of elevating plasma corticosterone in rats was previously made by Kokka and George (1). In their extensive investigation of various factors influencing growth hormone and corticotropin secretion, they found two small but significant increases at intervals following naloxone. More recently, naloxone, 50 mg/kg, in mice, was shown to produce an abrupt increase in plasma corticosterone which peaked at 30 minutes (7). In both studies the drug was administered by the intraperitoneal route and blood samples were obtained at suitable intervals by decapitation. These findings are in agreement with the observations presented herein where injections were made and serial blood samples were obtained in conscious, undisturbed animals. It would appear that there are at least three possible explanations for the finding that this apparently pure narcotic antagonist is having an effect on plasma corticosterone which is similar to the agonist: 1) at high doses naloxone has a direct agonist action on opiate receptors; 2) endorphins have inhibitory activity on the hypothalamo-pituitary-adrenal.axis--naloxonedisinhibits the axis resulting in CRF release; and 3) naloxone precipitates a withdrawal stress response to the "dependence" on endogenous opiates. A theoretical discussion is presented below with the understanding that the present data are not yet sufficient to permit conclusions to be drawn. Examining the first alternative, there appears to be some evidence supporting agonist activity for naloxone. Very high doses of the drug have been shown to elicit clonic-tonic convulsions in several species (29). In behavioral studies in the pigeon, non-toxic doses of naloxone appeared to possess agonist activity (30). More recently, Frank & Marwaha (31) examined opiate receptors on frog muscle fibers. Their data suggest that high concentrations of naloxone will depress tissue excitability in a similar fashion to opiate agonists. Furthermore, low doses of naltrexone will antagonize that excitability depression. The results of the pretreatment experiments (figures 2 & 3) speak against this explanation, since chronic treatment with narcotic agonists normally produce tolerance and this was not observed. The second hypothesis explaining the action of naloxone on plasma corticosterone involves the negative feedback control of the hypothalamo-pituitary-adrenal axis. This explanation postulates that one of the endogenous opiates participates as part of an inhibitory pathway in the control of CRF release--the initial step in the release of corticosterone--possibly by presynaptic inhibition (32). Administration of naloxone would disinhibit the system resulting in an elevation of plasma corticosterone. As part of this feedback mechanism, changes in endorphin levels would have to parallel the changes in the activity of the system. Such a parallel has been demonstrated by Guilemin -et al. (33)
Vol. 26, No. 12, 1980
Naloxone and Corticosterone
941
who showed that stress-induced corticotropin secretion results in an equimolar release of beta-endorphin, probably originating from the same precursor molecule (34). Thus, as corticotropin secretion increases, the initial step--CRF release--is progressively depressed by increased endogenous opiate activity. Consistent with the explanation is the finding that leu-enkephalin has an inhibitory effect on CRF-induced release of corticotropin from cultured anterior pituitary cells (35). One assumption is that the opiate receptor inhibiting CRF release, is distinct from the opiate receptors on which morphine and other agonists act to stimulate the ultimate release of corticosterone. With this model, it would be anticipated that a stress stimulus in a naloxone-pretreated animal would result in a greater corticosterone response than in the absence of the drug. This experiment was attempted by Gibson -et al. (7) with inconclusive results. The third explanation of the present results is that an "addiction" or "dependency" exists to endogenous opiates. Naloxone, at sufficient dose, precipitates a "withdrawal" stress response similar to what was observed in the chronically morphinized animal (25). Functionally, this may be more complex than simply the disinhibition of CRF release. It has been shown that exogenously administered endorphins can substitute for morphine in the habituated animal (36) and that the endogenous opiates, when administered by the intraventricular route, can produce the dependent condition to narcotics in which naloxone will precipitate the characteristic withdrawal signs (37). This supports the concept that, at least pharmacologically, the peptides produce narcotic dependence. Physiologically, however, endogenous opiates may serve as part of a pain response system, which when altered by naloxone, results in the corticosterone stress response. Some explanation should be made, at this point, about the variability of resting plasma corticosterone and the fluctuation in hormone levels following drug injections. This is particularly relevant to the data presented in figure 1. A more detailed discussion has been made in previous reports (2,28). Briefly, this variability is like that which was found in previous studies when the technique of sequential blood sampling was employed. We have attempted to carefully examine variables in the procedure to explain what appears to be a consistent plasma corticosterone rise between 11:30 a.m. and 1:x) p.m. The elevation does not appear to be attributable to the intervening sampling with the concomnitant saline or red cell replacement. The same rise was observed in unpublished experiments where the 15 and 30 minute samples were omitted. Additionally, it does not appear to be correlated with the initial i.v. injection since time zero levels are low in figure 2, which is 120 minutes following i.v. naloxone. We have also examined the use of heparin in the saline that is used to fill the dead space in the sampling tube by substituting bacteriostatic/ovroqen-free saline and found that this had no effect. Hematocrits did .._ _ not significantly change during the sampling protocol. Our only conclusion at this time is that this elevation is due to an as vet unknown factor or that it is a component of the normal rhythmical pattern which may be somehow phased through experimental manipulation. A pattern which can be altered by drug treatment or previous stimulation of corticosterone secretion. This may be either a primary or secondary effect; thus, observations made after drug intervention may include the drug effect, as well as the system attempting to reestablish itself. The finding that naloxone's action is not altered by itmnediateor prolonged Pretreatment is difficult to interpret with the data currently available. The
942
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half-life of the drug in rat brain and plasma has been estimated at 0.4 hours after the administration of l-10 mg/kg s.c., suggesting that its biological activity may be short-lived (38). Previous work from this laboratory, however, indicates that a dose of 1.0 mg/kg i.v. offered sufficient protection from the actions of a low dose of levorphanol for at least 3 hours (2). The site of action at high doses, though, may be quite different and unrelated. Further findings elucidating the mechanism by which naloxone elevates corticosterone will hopefully clarify the reasons for the lack of effect of pretreatment. References N. KOKKA and R. GEORGE, Narcotics and the Hypothalamus, E. Zimmerman and R. George, Raven Press, New York (1974). 2. R.M. EISENBERG and S.B. SPARBER, J. Pharm. Exp. Ther. 211364-369 (1979). J. HUGHES, Brain. Res. 88 295-308 (1975). :: J. HUGHES, T.W. SMITH, m. KOSTERLITZ, L.A. FOTHERSILL, B.A. MORGAN and H.R. MORRIS, Nature 258 577-579 (1975). L. TERNEIUS and A. WSTROM, Acta. Physiol. Stand. 94 74-81 (1975). z: J.F. BRUNI, D. VAN VUST, S. MARSHALL and J. MEITES, Efe Sci. 21 461-466 (1977) 7. A. GIBSON, M. GINSBURG, M. HALL, and S.L. HART, Br. J. PharmacX!j 139-146 f1979)_ ,.-._,_ P. GREVERT, E.R. BAIZMAN and A. GOLDSTEIN, Life Sci. 23 723-728 (1978). 9": R. KAPLAN and S.D. GLICK, Life Sci. 24 2309-2312 (197v. 10. R.J. BODNAR, D.D. KELLY, A. SPIASSIAFC. EHRENBERG, and M. GLUSMAN, Pharmac. Biochem. & Behav. 8 667-672. 11. N. KOKKA and A.S. FAIRHURST, Life Sci. 21 975-980 (1977). 12. M. SATOH, S. KAWAKIRI, M. YAMAMOTO, H. fi&INO and H. TAKASI, Life Sci. -24 685-690 (1979). M.S. BUCHSBAUM, G.C. DAVIS and W.E. BUNNEY, JR., Nature 270 620-622 (1977). ;:: G.C. DAVIS, M.S. BUCHSBAUM, W.E. BUNNEY, JR., Life Sci. 23449-1460 (1978). D.E. McMILLAN, Psychopharmacologia (Berl.) 19 128-133 (lv13). ;:: S.G. HOLTZMAN and R.E. JEWETT, J. Pharmac. Ep. Therap. 187 380-390 (1973). S.G. HOLTMAN, J. Phannac. Exp. Therap. 189 51-60 (1974).;:: P.W. DETTMAR, A. COWAN and D.S. WALTER,-&%ropharm. 171041-1044 (1978). 19. l_i_i8yIDDAUGH,E. READ and W. BOSSAN, Pharmac. Biochem., Behav. 2157-160 1.
20. 21.
26. 22:: 29. 30. 31.
A.P. FiRTZIGER and R. FISCHER, Psychopharm. 54 313-314 (1977). B.A. BERKOWITZ, A.D. FINCK and S.H. NGAI, J.-Pharmac. Exp. Therap. 203 539-547 (1977). V.R. SPIEHLER and L. PAALZOW, Life Sci. 24 2125-2132 (1979). V.R. SPIEHLER and L. RANDALL, Eur. J. Pharmac. 55 389-395 (1979). M.L. LAKIN and W.D. WINTERS, Proc. West Pharmac. Sot. 21 27-30 (1978). S.B. SPARBER, L. GELLERT, L. LICHTBLAU and R.M. EISENBRG, Factors Affectinq the Action of Narcotics, M.W. Adler, L. Manara and R. Samanin, Raven Press, New York (1978) E.T. KNYCH and R.M. EISENBERG, Neuroendocr. 29 110-118 (1979). D. GLICK, D.V. REDLICH and S. LEVINE, Endocriii.74 653-655 (1964). S.B. SPARBER, V.F. GELLERT and L. FOSSOM, NeurocGmical Mechanisms of Opiates and Endorphins, H.H. Loh and D.H. Ross, Raven Press, New York (1979). H. BLUMBERG and H.B. DAYTON, Agonist and Antaqonist Actions of Narcotic Analgesic Drugs, H,W. Kosterlitz, H.O.J. Collier and J.E. Villarreal, Univ. Park Press. Baltimore (1973). D.E. McMILLAN, P.S. WOLF and R.A. CARCHMAN, J. Pharmac. Exp. Therap. 175 443-458 (1970). G.B. FRANK and J. MARWAHA, J. Pharmac. Exp. Therap. 209 382-388 (1979).
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32. 33. 34. 35. 36. 37. 38.
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S.H. SfiYDER,Sci. Am. 236(3) 44-56 (1977). R. GUILLEMIN, T.M. VARXJ. ROSSIER, S. MINI&N. LING, C. RIVIER, W. VALE, and F. BLOOM, Sci. 197 1367-1369 (1977). R.E. MAINS, B.A. EIPPER, and M. LING, Proc. Nat'l. Acad. Sci., USA 3 3014-4018 (1977). K. HASHIMOTO, S. YUNOKI, J. TAKAHARA and T. OFUJI, Endocrin. Japon.26_103-109 (1979). J.I. SZEKELY, A.Z. RONAI, Z. DUNAI-KDVACS, E. MISLECZ, S. BAJUSZ, and L. GRAF, Life Sci. 20 1259-1264 (1977). E. WE1 andT. LOH, Sci. 193 1262-1263 (1976). A.L. MISRA, R.B. PONTANI7.L. VADLAMINI and S.J. MULE, J. Pharmac. Exp. Therap. x 257-268 (7976).
Pergamon Press
EFFECTS OF NALOXONE ON PLASMA CORTICOSTERONE IN THE OPIATE-NAIVE RAT Richard M. Eisenberg Department of Pharmacology University of Minnesota, Duluth School of Medicine Duluth, Minnesota 55812 (Received in final form January 22, 1980) Sumnary Naloxone HCl (NX) has long been considered to be a pure narcotic antagonist, having an effect only subsequent to pretreatment with a narcotic. Characteristically, low doses of NX have been used to antagonize the effects of analgesic doses of narcotics and to precipitate withdrawal in chronically treated animals. In this study, the effects of high doses of NX (2.0-20.0 mg/kg) on changes in plasma corticosterone were examined in the opiate-naive animal. Using male rats with chronic intravenous catheters and one-way vision boxes, injections were made and serial blood samples were obtained in the conscious, unrestrained animal. The acute administration of NX to the opiate-naive animal produced a dose-related increase in plasma corticosterone with respect to both amplitude and duration. NX (10.0 mg/kg i.v.) produced a significant elevation in hormone level at 15 and 30 minutes. With NX (20.0 mg/kg i.v.) the duration of the response was extended to 60 minutes. To examine whether short-term tolerance to this effect could be produced, animals were given a single pretreatment with either NX (10.0 mg/kg) or saline i.v. Two hours later NX produced a similar elevation in hormone level in both groups. The effect of chronic injection of NX was also studied. Animals pretreated with either NX (10.0 mg/kg) or saline S.C. once daily for 7 days did not show a significant difference following the subsequent administration of NX. In both cases, a significant elevation of plasma corticosterone resulted. The results suggest that NX may have a direct effect on opiate receptors resulting in an elevation of plasma hormone levels or NX may be disrupting an endogenous opiate-receptor interaction producing a stress response. Naloxone has long been considered as a pure narcotic antagonist, having a demonstrable effect subsequent only to treatment with a narcotic. In our laboratory, as in others, low doses of naloxone have been used effectively to antagonize the plasma corticosterone elevation induced by a narcotic (1,2). This
A preliminary report of these results was presented at the annual meeting of the Endocrine Society, Anaheim, CA, 1978. The author wishes to acknowledge the expert technical assistance provided by Mrs. Patricia Melis. This project was supported by USPHSgrant DAO2015. 0024-3205/80/120935-09$02.00/0 Copyright (c) 1980 Pergamon Press Ltd
936
Naloxone and Corticosterone
Vol. 26, No. 12, 1980
classical concept of naloxone activity is now.under thorough reinvestigation since the discovery of the endogenous morphine-like substances--the endorphins (3,495). It would now appear that there should be a presumptive action by naloxone alone simply by disrupting neural.pathways where endorphins or enkephalins are involved. Numerous reports suggest that naloxone is capable of evoking a pharmacological response in the absence of a narcotic. Intraperitoneal administration of 0.25.0 mg/kg of naloxone in the rat produced an elevation of serum LH and FSH and lowered serum prolactin and growth hormone (6). Higher doses in the mouse result in a dose-related elevation of plasma corticosterone (7). Hyperalgesia in rats has been reported after pretreatment with naloxone prior to hot plate test (8), exposure to footshock (9), subjection to cold water stress (lo), acetic acid injection (11) or bradykinin injection (12). Further, naloxone similarly altered pain perception in human subjects (13,14). Interestingly, naloxone has been reported to interact with a variety of non-opiate drugs. The behavioral effects of chlorpromazine in the pigeon (15), d-amphetamine in the rat (16,17), d-amphetamine, ethanol, and LSD in the mouse (T8,19,20) have been altered by moderate doses of naloxone. Analgesia produced a nitrous oxide (21), phenowbenzamine (22,23), and ketamine (24) has been antagonized by naloxone. The current study examines the effects of naloxone on plasma corticosterone. This parameter is characteristically elevated by acute treatment with analgesic doses of morphine (1) and levorphanol (2) and during spontaneous and naloxoneinduced narcotic withdrawal (25,2). Additionally, the effects of acute and chronic naloxone pretreatment on the plasma corticosterone response was also investigated. Materials and Methods Male rats (ARS SpraguelOawley, Madison, Wise.) weighing 200-350 grams were utilized in this study. All animals were placed in individual cages maintained at constant temperature (21OC) and light cycle (lights on at 06:OO to 20:00 hrs). Normal laboratory chow and water containing 0.8 mg/ml tetracycline were provided ad libitum. Each animal was allowed to acclimate to the surroundings and routine xrmst five days prior to surgery and experimentation. The surgical implantation of catheters and the utilization of individual sound-proofed, one-way vision boxes during serial sampling of blood from conscious, unrestrained animals have been described previously (26). Surgical/experimental procedure: 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 already described (2). In order to maintain catheter patency during the three day interval between surgery and blood sampling, catheters were flushed with 0.1-0.2 ml heparinized-saline, 500 U/ml, on days 2 and 3. In addition, animals were placed in the experimental chambers daily during this interval in order that they could accomnodate to the surroundings. On day 4. the animal was placed into the experimental chamber and connected to the injection/sampling tube at least two hours prior to any blood sampling to allow for stabilization of the plasma corticosterone levels after handling. Blood sampling in each experiment began either at 09:30 or lo:15 hrs. 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 the 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, suspended in saline, was injected. The samples were then transferred to test tubes and centrifuged, and
Vol. 26, No. 12, 1980
Naloxone and Corticosterone
937
the plasma separated and stored at O°C. Plasma corticosterone was determined by a modification of the fluorometric method of Glick et al. (27). In our procedure, methylene chloride is substituted for chlorofozs the extraction of corticosterone and the dilute sodium hydroxide was omitted. Experimental drugs and statistical analysis: Naloxone solutions were made in 0.9% saline so that the fluid volume injected was 1.0 ml/kg b.w. Equivalent amounts of saline were administered to controls. The doses of naloxone HCl were calculated at the salt. The naloxone was a gift from Endo Laboratories, Garden City, New York. In all of the data presented in this study, the posttreatment plasma corticosterone levels are shown as the mean percent of the pretreatment (or time zero) plasma corticosterone levels. For the data reduction, individual plasma corticosterone levels for each post-treatment time interval were calculated as a percent of the pretreatment level for each animal and this value was then transformed to the log. Statistical analysis of the transform data for each time interval was done using analysis of variance where t-values were calculated using the pooled variance estimates. Levels of significance are expressed in each case. Results The first experiment shown in figure 1 was designed to show the effects of several doses of naloxone on plasma corticosterone over a four-hour period.
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FIG. 1 Effects of naloxone HCl on plasma corticosterone. Values are shown as the mean percentage of zero time 2 S.E. The pretreatment hormone level for all animals is 7.9 + 0.8 pg% (mean f S.E.) and is shown as 100% at zero time. Numbers at graph symbols indicate animals per group. In some cases standard errors were omitted for clarity. Significant responses are indicated by *, please refer to text.
Naloxone and Corticosterone
938
Vol. 26, No. 12, 1980
Saline or one of three doses of naloxone (2.0, 10.0, or 20.0 mg/kg) were injected i.v. inznediatelyfollowing the withdrawal of the first blood sample at time zero. The two higher doses of the drug produced a significantly elevated (pcO.002) dose-related corticosterone response at 15 and 30 minutes following treatment. With the highest dose, 20 mg/kg, the response was still significantly greater (~~0.02) than the control at 60 minutes but at 180 minutes the value was significantly lower than the control (p
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FIG. 2 Effects of naloxone HCl (10.0 mg/kg) on plasma corticosterone after a single i.v. pretreatment with either saline or naloxone. Values are shown as the mean percentage of zero time f S.E. The pretreatment hormone level for the naloxone/saline group is 7.6 + 1.6 pg%, for the saline/naloxone group it is 6.9 + 1.6 pg%, and for the naloxone/naloxone group it is 7.4 + 2.2 !Jg%. Each is shown as 100% at zero time. Numbers at graph symbols indicate animals per group. In some cases standard errors were omitted for clarity. Significant responses: *p
240
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Three groups of naive animals were treated with a combination of saline and/or naloxone. The first intravenous injection was made 120 minutes prior to time zero. Both groups treated pith naloxone, 10 mg/kg i.v., after tima zero showed an almost identical plasma corticosterone response over the four-hour sampling period without regard to whether the pretreatment had been saline or naloxone (10 mg/kg). Hormone levels after the saline/naloxone combination were significantly elevated at the 15, 30. 60 and 120 minute intervals (p
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FIG. 3 Effects of naloxone HCl (10.0 mg/kg) on plasma corticosterone after once-daily injections of naloxone or saline for 7 days. Values are shown as the mean percentage of zero time f S.E. The zero time hormone level for the saline pretreated group is 6.8 * 0.8 Pg% and for the naloxone pretreated group it is 3.7 + 0.8 ug%. Each is shown as 100% at zero time. Numbers at graph symbols indicate animals per group. In some cases standard errors were omitted for clarity.
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Vol. 26, No. 12, 1980
with either saline or naloxone, 10 mg/kg, for 7 days. On the day of the serial blood sampling (day 8), animals were given their respective subcutaneous injections just prior to being placed in the experimental chambers. Naloxone, 10 mg/kg, was administered intravenously via the injection/sampling tube after time zero. Both the saline and drug-pretreated groups showed an elevation in hormone levels at 15 and 30 minutes. There was no statistical difference between the two groups. The pattern of response was similar to that observed in the acute response shown in figure 1. At the later time intervals there was no difference between the groups. Discussion The observation that a high dose of naloxone is capable of elevating plasma corticosterone in rats was previously made by Kokka and George (1). In their extensive investigation of various factors influencing growth hormone and corticotropin secretion, they found two small but significant increases at intervals following naloxone. More recently, naloxone, 50 mg/kg, in mice, was shown to produce an abrupt increase in plasma corticosterone which peaked at 30 minutes (7). In both studies the drug was administered by the intraperitoneal route and blood samples were obtained at suitable intervals by decapitation. These findings are in agreement with the observations presented herein where injections were made and serial blood samples were obtained in conscious, undisturbed animals. It would appear that there are at least three possible explanations for the finding that this apparently pure narcotic antagonist is having an effect on plasma corticosterone which is similar to the agonist: 1) at high doses naloxone has a direct agonist action on opiate receptors; 2) endorphins have inhibitory activity on the hypothalamo-pituitary-adrenal.axis--naloxonedisinhibits the axis resulting in CRF release; and 3) naloxone precipitates a withdrawal stress response to the "dependence" on endogenous opiates. A theoretical discussion is presented below with the understanding that the present data are not yet sufficient to permit conclusions to be drawn. Examining the first alternative, there appears to be some evidence supporting agonist activity for naloxone. Very high doses of the drug have been shown to elicit clonic-tonic convulsions in several species (29). In behavioral studies in the pigeon, non-toxic doses of naloxone appeared to possess agonist activity (30). More recently, Frank & Marwaha (31) examined opiate receptors on frog muscle fibers. Their data suggest that high concentrations of naloxone will depress tissue excitability in a similar fashion to opiate agonists. Furthermore, low doses of naltrexone will antagonize that excitability depression. The results of the pretreatment experiments (figures 2 & 3) speak against this explanation, since chronic treatment with narcotic agonists normally produce tolerance and this was not observed. The second hypothesis explaining the action of naloxone on plasma corticosterone involves the negative feedback control of the hypothalamo-pituitary-adrenal axis. This explanation postulates that one of the endogenous opiates participates as part of an inhibitory pathway in the control of CRF release--the initial step in the release of corticosterone--possibly by presynaptic inhibition (32). Administration of naloxone would disinhibit the system resulting in an elevation of plasma corticosterone. As part of this feedback mechanism, changes in endorphin levels would have to parallel the changes in the activity of the system. Such a parallel has been demonstrated by Guilemin -et al. (33)
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Naloxone and Corticosterone
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who showed that stress-induced corticotropin secretion results in an equimolar release of beta-endorphin, probably originating from the same precursor molecule (34). Thus, as corticotropin secretion increases, the initial step--CRF release--is progressively depressed by increased endogenous opiate activity. Consistent with the explanation is the finding that leu-enkephalin has an inhibitory effect on CRF-induced release of corticotropin from cultured anterior pituitary cells (35). One assumption is that the opiate receptor inhibiting CRF release, is distinct from the opiate receptors on which morphine and other agonists act to stimulate the ultimate release of corticosterone. With this model, it would be anticipated that a stress stimulus in a naloxone-pretreated animal would result in a greater corticosterone response than in the absence of the drug. This experiment was attempted by Gibson -et al. (7) with inconclusive results. The third explanation of the present results is that an "addiction" or "dependency" exists to endogenous opiates. Naloxone, at sufficient dose, precipitates a "withdrawal" stress response similar to what was observed in the chronically morphinized animal (25). Functionally, this may be more complex than simply the disinhibition of CRF release. It has been shown that exogenously administered endorphins can substitute for morphine in the habituated animal (36) and that the endogenous opiates, when administered by the intraventricular route, can produce the dependent condition to narcotics in which naloxone will precipitate the characteristic withdrawal signs (37). This supports the concept that, at least pharmacologically, the peptides produce narcotic dependence. Physiologically, however, endogenous opiates may serve as part of a pain response system, which when altered by naloxone, results in the corticosterone stress response. Some explanation should be made, at this point, about the variability of resting plasma corticosterone and the fluctuation in hormone levels following drug injections. This is particularly relevant to the data presented in figure 1. A more detailed discussion has been made in previous reports (2,28). Briefly, this variability is like that which was found in previous studies when the technique of sequential blood sampling was employed. We have attempted to carefully examine variables in the procedure to explain what appears to be a consistent plasma corticosterone rise between 11:30 a.m. and 1:x) p.m. The elevation does not appear to be attributable to the intervening sampling with the concomnitant saline or red cell replacement. The same rise was observed in unpublished experiments where the 15 and 30 minute samples were omitted. Additionally, it does not appear to be correlated with the initial i.v. injection since time zero levels are low in figure 2, which is 120 minutes following i.v. naloxone. We have also examined the use of heparin in the saline that is used to fill the dead space in the sampling tube by substituting bacteriostatic/ovroqen-free saline and found that this had no effect. Hematocrits did .._ _ not significantly change during the sampling protocol. Our only conclusion at this time is that this elevation is due to an as vet unknown factor or that it is a component of the normal rhythmical pattern which may be somehow phased through experimental manipulation. A pattern which can be altered by drug treatment or previous stimulation of corticosterone secretion. This may be either a primary or secondary effect; thus, observations made after drug intervention may include the drug effect, as well as the system attempting to reestablish itself. The finding that naloxone's action is not altered by itmnediateor prolonged Pretreatment is difficult to interpret with the data currently available. The
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half-life of the drug in rat brain and plasma has been estimated at 0.4 hours after the administration of l-10 mg/kg s.c., suggesting that its biological activity may be short-lived (38). Previous work from this laboratory, however, indicates that a dose of 1.0 mg/kg i.v. offered sufficient protection from the actions of a low dose of levorphanol for at least 3 hours (2). The site of action at high doses, though, may be quite different and unrelated. Further findings elucidating the mechanism by which naloxone elevates corticosterone will hopefully clarify the reasons for the lack of effect of pretreatment. References N. KOKKA and R. GEORGE, Narcotics and the Hypothalamus, E. Zimmerman and R. George, Raven Press, New York (1974). 2. R.M. EISENBERG and S.B. SPARBER, J. Pharm. Exp. Ther. 211364-369 (1979). J. HUGHES, Brain. Res. 88 295-308 (1975). :: J. HUGHES, T.W. SMITH, m. KOSTERLITZ, L.A. FOTHERSILL, B.A. MORGAN and H.R. MORRIS, Nature 258 577-579 (1975). L. TERNEIUS and A. WSTROM, Acta. Physiol. Stand. 94 74-81 (1975). z: J.F. BRUNI, D. VAN VUST, S. MARSHALL and J. MEITES, Efe Sci. 21 461-466 (1977) 7. A. GIBSON, M. GINSBURG, M. HALL, and S.L. HART, Br. J. PharmacX!j 139-146 f1979)_ ,.-._,_ P. GREVERT, E.R. BAIZMAN and A. GOLDSTEIN, Life Sci. 23 723-728 (1978). 9": R. KAPLAN and S.D. GLICK, Life Sci. 24 2309-2312 (197v. 10. R.J. BODNAR, D.D. KELLY, A. SPIASSIAFC. EHRENBERG, and M. GLUSMAN, Pharmac. Biochem. & Behav. 8 667-672. 11. N. KOKKA and A.S. FAIRHURST, Life Sci. 21 975-980 (1977). 12. M. SATOH, S. KAWAKIRI, M. YAMAMOTO, H. fi&INO and H. TAKASI, Life Sci. -24 685-690 (1979). M.S. BUCHSBAUM, G.C. DAVIS and W.E. BUNNEY, JR., Nature 270 620-622 (1977). ;:: G.C. DAVIS, M.S. BUCHSBAUM, W.E. BUNNEY, JR., Life Sci. 23449-1460 (1978). D.E. McMILLAN, Psychopharmacologia (Berl.) 19 128-133 (lv13). ;:: S.G. HOLTZMAN and R.E. JEWETT, J. Pharmac. Ep. Therap. 187 380-390 (1973). S.G. HOLTMAN, J. Phannac. Exp. Therap. 189 51-60 (1974).;:: P.W. DETTMAR, A. COWAN and D.S. WALTER,-&%ropharm. 171041-1044 (1978). 19. l_i_i8yIDDAUGH,E. READ and W. BOSSAN, Pharmac. Biochem., Behav. 2157-160 1.
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A.P. FiRTZIGER and R. FISCHER, Psychopharm. 54 313-314 (1977). B.A. BERKOWITZ, A.D. FINCK and S.H. NGAI, J.-Pharmac. Exp. Therap. 203 539-547 (1977). V.R. SPIEHLER and L. PAALZOW, Life Sci. 24 2125-2132 (1979). V.R. SPIEHLER and L. RANDALL, Eur. J. Pharmac. 55 389-395 (1979). M.L. LAKIN and W.D. WINTERS, Proc. West Pharmac. Sot. 21 27-30 (1978). S.B. SPARBER, L. GELLERT, L. LICHTBLAU and R.M. EISENBRG, Factors Affectinq the Action of Narcotics, M.W. Adler, L. Manara and R. Samanin, Raven Press, New York (1978) E.T. KNYCH and R.M. EISENBERG, Neuroendocr. 29 110-118 (1979). D. GLICK, D.V. REDLICH and S. LEVINE, Endocriii.74 653-655 (1964). S.B. SPARBER, V.F. GELLERT and L. FOSSOM, NeurocGmical Mechanisms of Opiates and Endorphins, H.H. Loh and D.H. Ross, Raven Press, New York (1979). H. BLUMBERG and H.B. DAYTON, Agonist and Antaqonist Actions of Narcotic Analgesic Drugs, H,W. Kosterlitz, H.O.J. Collier and J.E. Villarreal, Univ. Park Press. Baltimore (1973). D.E. McMILLAN, P.S. WOLF and R.A. CARCHMAN, J. Pharmac. Exp. Therap. 175 443-458 (1970). G.B. FRANK and J. MARWAHA, J. Pharmac. Exp. Therap. 209 382-388 (1979).
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S.H. SfiYDER,Sci. Am. 236(3) 44-56 (1977). R. GUILLEMIN, T.M. VARXJ. ROSSIER, S. MINI&N. LING, C. RIVIER, W. VALE, and F. BLOOM, Sci. 197 1367-1369 (1977). R.E. MAINS, B.A. EIPPER, and M. LING, Proc. Nat'l. Acad. Sci., USA 3 3014-4018 (1977). K. HASHIMOTO, S. YUNOKI, J. TAKAHARA and T. OFUJI, Endocrin. Japon.26_103-109 (1979). J.I. SZEKELY, A.Z. RONAI, Z. DUNAI-KDVACS, E. MISLECZ, S. BAJUSZ, and L. GRAF, Life Sci. 20 1259-1264 (1977). E. WE1 andT. LOH, Sci. 193 1262-1263 (1976). A.L. MISRA, R.B. PONTANI7.L. VADLAMINI and S.J. MULE, J. Pharmac. Exp. Therap. x 257-268 (7976).