The effects of chronic morphine treatment on ... - Springer Link

Psychopharmacology

Psychopharmacology (1983) 81 : 1O- 13

9

Springer-Verlag 1983

The Effects of Chronic Morphine Treatment on Neurotensin-Induced Antinociception Daniel Luttinger 1, 2,., Susan K. Burgess 1, Charles B. Nemeroff 1' a, and Arthur J. Prange Jr. z' 2, 3 i BiologicalSciences Research Center, University of North Carolina, Chapel Hill, NC 27514, USA 2 The Neurobiology Program, University of North Carolina, Chapet Hill, NC 27514, USA 3 .Department of Psychiatry, University of North Carolina, School of Medicine, Chapel Hill, NC 27514, USA

Abstract. Previous studies have shown that the opioid antagonist naloxone does not alter neurotensin (NT)-induced antinociception. In the present studies, tolerance to morphine in mice significantly attenuated NT-induced antinociception, but not NT-induced hypothermia. In addition, centrally administered NT inhibited naloxone-precipitated jumping in morphine-dependent mice. These results indicate complex interactions between NT-induced antinociception and opioid systems. Key words: Morphine - Neurotensin Hypothermia - Naloxone - Tolerance

Analgesia -

Neurotensin (NT) is an endogenous peptide that is localized in the gut and brain (Carraway and Leeman 1976). Injection of NT into the CNS produces a variety of effects (Nemeroff et al. 1983 for review). One of the well-described effects of NT, originally reported by Clineschmidt and McGuffin (1977), is dose-dependent antinociception (i.e., a reduced responsiveness to painful stimuli). The antinociception induced by NT is extremely potent both in magnitude and duration. In a study in which the antinociceptive potency of 12 peptides was tested on a molar basis, only fl-endorphin was more potent than NT (Nemeroff et al. 1979). Antinociception following central NT administration has been observed in both rats and mice and in a variety of analgesia-screening tests (Clineschmidt et al. 1979; Osbahr et al. 1981). Pharmacological characterization of this response has shown that it is not altered by the opiate antagonist naloxone (Clineschmidt et al. 1979; Osbahr et al. 1981). In addition, NT does not inhibit 12sI-(D-ala2, D-leuS)-enkephalin binding to the 4-thioguanine-resistant mouse neuroblastoma opiate receptors (R. J. Miller, personal communication). Several different receptor blockers do not antagonize NT-induced antinociception, including serotonin (5-HT) antagonists, muscarinic and nicotinic cholinergic antagonists, H1- and H2-histaminergic antagonists, a- and fl-noradrenergic antagonists, and a dopamine (DA) antagonist (Clineschmidt et al. 1979). NT-induced antinociception, however, is antagonized by central or peripheral administration of thyrotropin-releasing hormone (TRH) whereas fi-endorphineinduced antinociception is not (Holaday et al. 1978; Osbahr et al. 1981). *

Present address:

Sterling Winthrop Research Institute, Rensselaer,

NY 12144, USA Offprint requests to :

D. Luttinger

In rats, intracerebral injections of NT have been found to produce antinociception in a few discrete regions, and these regions are distinct from those where NT is active in producing hypothermia (Kalivas et al. 1981). NT consistently produced an antinociceptive response when administered into the central amygdaloid nucleus, area perilemniscus, pars medialis, mesencephalic periaqueductal grey, and medial reticular area in the rostral medulla. In many of these NTsensitive sites, morphine- or opiate-like peptides have also been found to produce an antinociceptive response. For instance, morphine has been found to produce antinociception when injected into the central amygdaloid nucleus (Rodgers 1977), and the mesencephalic periaqueductal grey (Bennett and Mayer 1979; Yaksh and Rudy 1978). The fact that there are several brain regions where both NT and morphine produce an antinociceptive response suggests possible interactions. To further characterize NT-induced antinociception, the effect of morphine tolerance on NT-induced antinociception was assessed.

Materials and Methods Male Swiss-Webster mice (Flow Laboratories, Dublin, VA, USA), weighing 2 5 - 35 g, were used in all experiments. The mice were housed six per cage in a controlled environment (12-h light-dark cycle) animal facility with food and water continuously available. The mice were housed in this manner for at least 1 week prior to use in an experiment. Mice were made tolerant and physically dependent on morphine by SC implantation of a 75 mg morphine pellet for 3 days. The morphine pellet was implanted while the mouse was anesthetized with ether. An incision was made in the back, near the base of the neck, the pellet inserted, and the incision closed with a wound clip. After 3 days, the pellet was removed under ether anesthesia. In the tolerance studies, sham-operated mice (no pellet implanted) served as controls. Antinociception was assessed 24 h after pellet removal with a copper hot-plate (IITC, Landing, N J, USA) set at a temperature of 51 ~ 52 ~C. The mice were placed with all four paws on the hot plate and the time (to the nearest 0.1 s) for the mice either to lick their paws or to jump was recorded by a trained observer who was blind to the treatment regimen. An arbitrary cutoff of 30s was used to score animals not responding to the noxious stimulus. Ten mice were used per treatment. Three measures of response latency were taken 20min apart before intracisternal (IC) injection of NT, fl-endorphin, or vehicle (0.9 % saline) in a volume of 10 gl.

II

Under light ether anesthesia the mice were injected IC and tested every 20 min over a l-h period postinjection. The effects of morphine tolerance on the rate of decay of NT immunoreactivity following injection of NT (10 pg IC) was studied. NT concentrations in whole mouse brain at 10, 20, and 40 min after NT injection were assessed by radioimmunoassay, as previously described (Manberg et al. 1983). The effects of NT injection on the immediate change in whole brain concentration of NT was assessed only in nondependent animals. Six mice were used at each time point, The effects of morphine tolerance on NT-induced hypothermia were also investigated. The mice were rendered tolerant as described. At 24 h after pellet removal, NT (1 gg IC) was injected and rectal temperature measured every 30 min for 2 h in an environment maintained at 25 ~C. Ten mice were used in each treatment group, The effects o f I C NT on the naloxone-induced withdrawal response of morphine-dependent mice was studied. Mice Were rendered dependent on morphine by 72 h implantation of a morphine pellet, as described. At 6 h after pellet removal, the ability of naloxone ( 2 5 - 1 , 0 0 0 gg/kg SC) to precipitate jumping was assessed. NT (t0 gg) or vehicle (0.9 ~ saline) were injected IC 30min prior to naloxone. Stereotyped jumping has previously been shown to be a characteristic sign of the opiate-withdrawal response in mice (Way et al. 1969). A 30cm square Plexiglas platform 64cm above the floor was used. Four to six mice were placed on the platform immediately following naloxone injection and the number of mice that jumped off the platform was recorded. Mice remaining on the platform after 15 min were removed, and they were considered to have not manifested withdrawal, Morphine pellets were provided by the National Institute of Drug Abuse (Bethesda, MD, USA). Human fi-endorphin was a gift from the National Institute of Mental Health (Bethesda, MD, USA). Morphine sulfate was purchased from Merck (St. Louis, MO, USA) and NT, from Bachem (Torrance, CA, USA). Statistical significance ( P < 0.05) for data derived from response latencies on the hot plate and NT-induced hypothermia were assessed with Dunnett's test for multiple comparisons after A N O V A (Dunnett 1964). The significance of decay in NT concentrations was assessed with Student's t-test. The data pertaining to the effects on NT on naloxoneprecipitated withdrawal was assessed by probit analysis according to the methods of Litchfield and Wilcoxon (1949). Results

The SC implantation of a 75 mg morphine pellet for 72h produced consistent opioid tolerance. #-Endorphin (5 pg IC), when administered to normal mice, produced significant antinociception that persisted for at least 60 rain. In contrast, in mice previously implanted with morphine pellets, the duration of antinociception induced by #-endorphin was markedly attenuated (Fig. 1). Antinociception induced by NT (10gg IC) was also attenuated by previous morphine pellet treatment (Fig. 2). In fact, morphine tolerance completely blocked NT-induced antinociception. Furthermore, NT doses of 5 - 3 0 gg produced comparable results. Thus, with all doses of NT tested, the increase in response latency from baseline was significantly attenuated in morphine-tolerant mice. The reduction in the antinociceptive response after IC NT in morphine-tolerant mice was apparently not due to an

30 ,,, 25 03 +1 03

,;~

\

T

20

0 o 03 15

m U-I0 03 03 z oa_ 5 co 03 oc

O SHAM OPERATED/SALINE 9 SHAM OPERATED/B-ENDORPHIN D MORPHINE PELLETED/SALINE 9 MORPHINE PELLETED/~-ENDORPHIN

I

I

PREINJECTION

20

I

I

40

60

TIME (MINUTES)

Fig. 1. The effect of #-endorphin (5 gg IC) on response latency on the hot plate (51 ~ 52~ in morphine-tolerant and n ontolerant mice. Ten mice were utilized in each treatment group. * P< 0.05 when compared to saline-injected controls (Dunnett's test for multiple comparisons)

3o

/////// _~ tll/I _ ~

,,i 03 25 +l 03 c3 o7 20 ~ 15 w ~_ ~- ~0 03 co zo a_ 03 5 03 fr

0 9 El 9

SHAM-OPERATED/SALINE SHAM-OPERATED/NEUROTENSIN MORPHINE PELLETED/SALINE MORPHINE PELLETED/NEUROTENSIN

I

r

PREINJECTION

20

I

40

I

60

TIME (MINUTES) Fig. 2. The effect ofneurotensin (10 gg IC) on response latency on the hot plate (51 ~176 in morphine-tolerant and nontolerant mice. Ten mice were used in each treatment group. * P < 0.05 when compared to salineinjected controls (Dunnett's test for multiple comparisons)

alteration in the metabolism of administered NT. The half-life of 10 gg NT after IC injection was approximately 11 min, and morphine tolerance did not alter this (Fig. 3). In a similar way, morphine tolerance did not alter endogenous NT concentration in whole mouse brain. NT (10 gg IC) inhibited naloxone-precipitated jumping in morphine-dependent mice. There was approximately a tenfold shift in the naloxone dose-response curve after IC NT treatment. The EDs0 for naloxone in morphine-dependent mice injected IC with saline was 97 gg/kg and, in mice injected IC with NT, the EDso was 943 gg/kg. In addition, the slope of the dose-response curve for naloxone-precipitated jumping after NT was not as steep. Naloxone-induced diarrhea and

t2 Table 1. The effect of Morphine tolerance on neurotensin-induced hypothermia in mice. Mice were sham-operated or made tolerant to morphine by SC implantation of a 75 mg morphine pellet for 72 h. At 24 h after pellet removal the mice were lightly anesthetized with ether and injected intracisternalty with either vehicle (0.9 % saline) or neurotensin (1 ~g). There were ten mice per group Pretreatment

IC treatment

No pellet No pellet Morphine pellet Morphine p e l l e t

Colonic temperature (~ +_SEM)

saline neurotensin (1 gg) saline neurotensin (1 ~g)

0 min

30 min

60 min

90 min

120 min

38.0 _+0.2 37.7 _+0.1 37.2 • 0.2 37.5 +_0.2

37.1 _+0.4 33.8 • 0.5* 36.7 +_0.3 34.4 +_0.3*

36.4 • 0.5 34.8 _+0.4* 36.7 +_0.4 34.4 • 0.2*

36.8 • 0.6 35.5 • 0.4 36.2 • 0.5 35.3 -2_0.9

36.4 • 0.6 35.6 + 0.6 36.2 • 0.3 34.6 + 0.9

* P < 0.05 compared to the respective saline-treated mice (Dunnett's test for multiple comparisons)

1800 w o9 +1 1500 Z W I-0cY 1200

0 SHAMOPERATED 9 MORPHINEPELLETED

CL

E 900 c:n o..

z 600 or) Z w I-" 500 0 II1 W Z

~

NON-INJECTEDCONTROLS

i

I

I

I

0

I0

20

40

TIME

(MINUTES)

Fig. 3. The rate of decay of immunoreactive neurotensin in whole mouse brain after exogenously administered neurotensin (10pg IC) in morphine-tolerant and nontolerant mice. Six mice were used in each treatment group

piloerection in morphine-dependent mice did not appear to be affected by IC NT treatment. The effect of morphine tolerance on the CNS effects of NT exhibited some specificity. Although morphine tolerance attenuated NT-induced antinociception, NT-induced hypothermia was unchanged (Table 1). Discussion

The present studies demonstrate that morphine tolerance reduces NT-induced antinociception in mice. The results indicate that morphine interacts as some level with NT neuronal systems. Because the analgesia induced by morphine is completely antagonized by the opiate receptor antagonist naloxone, while NT-induced analgesia is unaffected, the interaction is probably not at a common receptor. Further evidence of an interaction between N T and opioid systems is provided by the findings in the mouse physically dependent on morphine undergoing naloxone-precipitated withdrawal. NT decreased naloxone-precipitated jumping, but not naloxone-precipitated piloerection or diarrhea. This effect on naloxone-precipitated jumping in the morphine-

dependent mouse has also been observed with T R H (Bhargava 1980). The fact that, in this respect, N T and T R H exhibit similar actions is intriguing given that for many behaviors these peptides exert opposite actions (Burgess et al. 1983). In fact, T R H has been shown to antagonize NT-, but not /~-endorphin-induced antinociception (Osbahr et al. 1981 ; Holaday et al. 1978). Interestingly, NT does not affect ethanol withdrawal in ethanol-dependent rats (Frye et al. 1981), suggesting that the action of N T on withdrawal from opiates is not a non-specific effect on all forms of withdrawal. The 'cross-tolerance' between morphine and NT was shown to exhibit some specificity. This was demonstrated by the lack of effect on NT-induced hypothermia by tolerance to morphine. Previously, our group found that NTinduced hypothermia is not affected by tolerance to ethanol (Prange et al. 1983). Furthermore, morphine tolerance did not alter the rate of decay of radioimmunoassayed NT after exogenous administration. This suggests that morphine tolerance does not alter NT-induced antinociception simply by enhancing the rate of NT degradation. We also observed no difference in whole brain concentration of endogenous NT in morphine-dependent mice when compared with morphine-naive mice. Morley et al. (1980) observed that morphine addiction increased NT concentration in the thalamus of rats. Regional differences in endogenous or exogenously applied NT may have been masked while examining whole mouse brain. This, or species differences may explain the discrepancy between our findings and those of Morley et al. (1980). An IC injection of NT (10gg) increased whole brain concentrations of this peptide approximately 17-fold. This does not imply, however, that a 17-fold increase in NT is necessary for inducing antinociception. The critical issue, which is not assessed by this study, is the change in NT concentration at the active site. At present this cannot be determined. Indeed, given that NT-induced antinociception lasts for approximately 2 h (Clineschmidt and McGuffin 1977) and injected NT is virtually gone within l h suggests that the 17-fold increase in concentration may not be necessary. In conclusion, we have demonstrated that tolerance to morphine selectively attenuates NT-induced antinociception without affecting NT-induced hypothermia. This suggests that tolerance to morphine alters the mechanisms involved in NT-induced antinociception. The specific mechanisms responsible for this effect are unknown at present.

Acknowledgements. This work was supported by brants from NIMH (MH 22356, MH 32316, MH 34121, MH 33127, MH 14277) and

13 NICHHD (HD 03110). The authors appreciate the technical assistance of Barbara Gau and Donald A. Stanley. We gratefully acknowledge the assistance of Faygele ben Miriam in the preparation of this manuscript. We would also like to thank the National Institute of Mental Health for the human fl-endorphin.

References Bennett GJ, Mayer DJ (1979) Inhibition of spinal cord intemeurons by narcotic microinjection and focal electrical stimulation in the periaqueductal central grey matter. Brain Res 172: 243 - 257 Bhargava HN (1980) The effects of thyrotropin-releasing hormone on the central nervous system responses to chronic morphine administration. Psychopharmacology 68:185 - 189 Burgess SK, Luttinger D, Hemandez DE, Kalivas PW, Nemeroff CB, Prange AJ Jr (1983) Antagonism of the central nervous system effects of neurotensin by thyrotropin-releasing hormone. In: Griffiths EC, Bennett GW (eds) Thyrotropin-releasing hormone (TRH) Satellite Symposium of the Eight ISN Meeting. Raven Press, New York, pp 291 - 302 Carraway R, Leeman SE (1976) Characterization of radioimmunoassayable neurotensin in the rat: Its differential distribution in the central nervous system, small intestine and stomach. J Biol Chem 251:7045-7052 Clineschmidt BV, McGuffin JC (1977) Neurotensin administered intracisternally inhibits responsiveness of mice to noxious stimuli. Eur J Pharmacol 46:395 - 396 Clineschmidt BV, McGuffin JC, Bunting PB (1979) Neurotensin: Antinocisponsive action in rodents. Eur J Pharmacol 54:129-139 Dunnett CW (1964) New tables for comparisons with a control. Biometrics 20:483--49t Frye GD, Luttinger D, Nemeroff DB, Vogel RA, Prange AJ Jr, Breese GR (1981) Modification of the actions of' ethanol by centrally active peptides. Peptides 2: 99 - 106 Holaday JW, Tseng LF, L6h HH, Li CH (1978) Thyrotropin-releasing hormone antagonizes fi-endorphin hypothermia and catalepsy. Life Sci 22:1537- 1544

Kalivas PW, Nemeroff CB, Bau BA, Prange AJ Jr (1981) Brain regions mediating neurotensin-induced antinociception. Pain (Suppl) 1:5105 Litchfield JT Jr, Wilcoxon F (1949) A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 96:99-113 Manberg PJ, Youngblood WW, Nemeroff CB, Rosser MN, Iversen LL, Prange AJ Jr, Kizer JS (1983) Regional distribution ofneurotensin in human brain. J Neurochem (in press) Morley JE, Yamada T, Walsh JH, Lamers CB, Wong H, Shulkes A, Damassa DA, Gordon J, Carolson HE, Hershman JM (1980) Morphine addiction and withdrawal alters brain peptide concentrations. Life Sci 26:2239-2244 Nemeroff CB, Osbahr AJ, Manberg PJ Erwin GN, Prange AJ Jr (1979) Alterations in nociception and body temperature after intracis ternal administration of neurotensin, /~-endorphin, other endogenous peptides and morphine. Proc Natl Acad Sci USA 76:5368-5371 Nemeroff CB, Luttinger D, Prange AJ Jr (1983) Neurotensin and bombesin. In: Iversen LL, Iversen SD, Snyder SH (eds) The handbook of psychopharmacology. Plenum, New York (in press) Osbahr III AJ, Nemeroff CB, Luttinger D, Mason GA, Prange AJ Jr (1981) Neurotensin-induced antiuociception in mice: Antagonism by thyrotropin-releasing hormone. J Pharmacol Exp Ther 217:645-651 Prange AJ Jr, Nemeroff CB, Chappell PB, Kalivas PW, Luttinger D, Stanley DA, Frye GD (1983) Lack of tolerance to neurotensininduced hypothermia in the ethanol tolerant mouse. Alcohol Clin Exp Res (in press) Rodgers RJ (1977) Elevation of aversive threshold in rats by intraamygdaloid injection of morphine sulfate. Pharmacol Biochem Behav 6:385- 390 Way EL, L6h HH, Shen FH (1969) Simultaneous quantitative assessment of morphine tolerance and physical dependence. J Pharmacol Exp Ther 167:1-8 Yaksh TL, Rudy TA (1978) Narcotic analgetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques. Pain 4:299-359 Received September 23, 1982; Final version January 25, 1983