- Email: [email protected]
Cholecystokinin receptor binding in morphine analgesia: Tolerance, withdrawal and abstinence
Nruropptides 1:. Longman
( 1994)
26, 379-383
Group Ltd 1994
Cholecystokinin Receptor Binding in Morphine Analgesia: Tolerance, Withdrawal and Abstinence M. WELIN”,
J. HARRO*S,
R. YUKHANANOVS,
F. NYBERGt
and L. ORELAND”
*Departments of Medical Pharmacology and tPharmacology, Uppsala University, Biomedical Centre, Uppsala, Sweden, #Laboratory of Psychopharmacology, Institute of General and Molecular Pathology, Tartu University, Tartu, Estonia and Slnstitute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia (Reprint requests to LO)
Abstract-The effect of morphine treatment on cholecystokinin (CCK) receptor binding in rat cerebral cortex was investigated. Subcutaneous implantation and removal of Alzet miniosmotic pumps, releasing morphine, permitted us to establish the phases of initial analgesia, tolerance to the analgesic action of morphine, morphine withdrawal and abstinence. CCK receptor binding in rat cerebral cortex never differed from the values obtained from animals implanted with saline-releasing minipumps. The results of the present study suggest that the putative changes in the interaction between opioidergic and CCKergic neurotransmission at different stages of morphine treatment and withdrawal are not caused by changes of CCK receptor binding properties.
Introduction Cholecystokinin (CCK) is a neuropeptide involved in opioid analgesia. Large doses of CCK induce naloxone-reversible analgesia,’ but small and probably physiologically more relevant amounts of this peptide counteract the antinociceptive effect of exogenous and endogenous opioids.2.3 Recent findings have led to the hypothesis that CCK acts as an endogenous antagonist of opioids in the central nervous system (see ref. 4 for review). Thus, the Date received Date accepted
I September 10 January
1993 1994
Correspondence to: Professor Lars Oreland, Uppsala University Department of Medical Pharmacology, Box 593, Biomedicum, S-75124 Uppsala. Sweden
administration of CCK receptor antagonists such as proglumide, lorglumide, devazepide, L365,260 and C1988 results in a marked enhancement of opioid-induced analgesia in rats, monkeys and humans.5m” Furthermore, tolerance to the analgesic effect of morphine is prevented when CCK receptors are blocked.5,6.“.“.‘3.‘4The analysis of pharmacological data suggests that blockade of CCKB receptors is involved in the enhancement of morphine analgesia and in preventing tolerance to the antinociceptive action of morphine. On the other hand, CCK receptor antagonists do not attenuate naloxone-precipitated morphine withdrawaLh,13.‘” However, the blockade of CCK, receptors enhanced the ability of morphine to induce conditioned place preference,” suggesting that while
379
380 the dependence to morphine may not be linked to the CCKergic neurotransmission, the euphoric action of morphine is. There are studies examining the acute CCKopioid interactions directly, but so far the data concerning the action of long-term opioid receptor stimulation on CCKergic neurotransmission are very scarce. Recently, however, Pohl and colleaguesI examined the effect of both acute and repeated morphine treatment on brain CCK and CCK mRNA levels. No effect of morphine treatment on these measures was found and thus, the working hypothesis that chronic stimulation of opioid receptors triggers a progressive compensatory increase in the activity of CCK-containing neurons was not supported. However, in this study, the antinociceptive effect of morphine was not tested. Furthermore, the increase in CCKergic activity could occur at the postsynaptic level, with changes of the biochemical characteristics of CCK, receptors. We have, therefore, studied the binding characteristics of CCK receptors in rat cerebral cortex at different behaviourally validated stages of morphine treatment and withdrawal.
Materials and methods
Adult male Sprague-Dawley rats (Alab, Sollentuna, Sweden), weighing 220-240 g at the beginning of the experiment, were housed in an environment with controlled humidity (60%) temperature (24°C) and lighting (lights on 9.00-21.00). Animals were provided with food and water ad libitum and adapted to these conditions for at least 7 days before experimentation. From the day before the implantation of minipumps until sacrifice the rats were housed in individual cages. All rats were randomly divided into 4 groups, each consisting of 6 morphine-treated and 5 control animals. Miniosmotic pumps (Alzet, 2MLl) were filled with morphine acetate (70 mg/ml) or saline and were implanted subcutaneously under diethyl ether anaesthesia on the back of the animals. Animals of the analgesia group were decapitated 24 h and those of the tolerance group 120 h after the pump implantation. Rats belonging to the withdrawal group were killed 24 h and those forming the abstinence group 48 h after pump removal. In
NEUROPEPTIDES
both of these groups the pumps had been removed 5 days (120-124 h) after implantation. Behaviouval testing
Animals were weighed every day and their water consumption monitored. The pain sensitivity was measured by the paw pressure test using a commercially available apparatus (Ugo Basile, Comerio, Italy). Briefly, the pressure was applicated through a bullet-shaped plastic tip to the plantar surface of the hind paw. The pressure was continuously increased at a rate of 480 or 320 mN/s and the pressure at which a rapid removal of the paw was observed was assigned as the response threshold. Radioligand binding
After decapitation, brain was removed from the skull and cortical tissue rapidly dissected on ice. CCK receptor binding was performed as described earlier.17 In brief, brain tissue was homogenized in 20 volumes of ice-cold Tris-HCl (pH 7.4) using a Potter-S glass-teflon homogenizer (1000 rpm, 10 passes) The membranes were washed twice in the same buffer by centrifugation and resuspension. The final pellets were homogenized in HEPES buffer (10 mM HEPES; 130 mM NaCl; 5 mM MgCl?; 5 mM KCl; 1 mM EDTA; pH 6.5 adjusted with 5 N NaOH) containing bovine serum albumin (0.5 mg/ml). CCK-8 receptor labelling was carried out in the presence of 0.0553 nM tritiated ligand, propionyl-(3H)CCK-8-sulphated (spec. act. 78.0 Ci/mmol, Amersham Radiochemicals) at room temperature in a total incubation volume of 0.5 ml. Caerulein (100 nM) was added to determine nonspecific binding. Before adding the radioligand, membrane suspension was preincubated with or without displacer for 30 min at room temperature. Incubation was initiated by adding the radioligand and terminated after 90 min by rapid filtration over Whatman GF/B filters using a Brandel Cell Harvester (M-24s). The filters were washed with 10 ml cold incubation buffer, dried and assayed for radioactivity by liquid scintillation spectrometry. Data analysis
Saturation curves obtained in radioligand binding experiments were analyzed using non-linear least
CCK RECEPTOR BINDING
IN MORPHINE
0
q
”
0
12
24
48
381
ANALGESIA
12
I I
control morphine
96
120
Time (hours) following pump implantation
Cl control H
i;
morphine
I
I
”
6
Time
12 (hours)
48 21 32 18 following pump removal
Fig. 1 Pain sensitivity of rats treated with morphine in the paw pressure test. Spotted and striped columns correspond to salineand morphine-treated groups respectively. (A) Pain sensitivity after implantation of the minipumps. (B) Hyperalgesia after removal of the morphine-releasing osmotic minipumps. Data are expressed as means f SEM and were analyzed by ANOVA followed by Kruskal-Wallis test.
squares regression.” Parametric or non-parametric one-way analysis of variance (ANOVA) was used initially for the statistical evaluation of the data, followed by Scheffe’s test, as appropriate.
sensitivity had disappeared 96 h after the pump implantation, indicative of the development of tolerance to the analgesic action of morphine. Tolerance remained persistent until sacrifice. In contrast to the control animals, there was a considerable drop in the body weight (data not shown) and water consumption of the morphine-treated rats during the following 24 h after removal of implanted minipumps. Subsequently, the animals started to gain weight and the amount of water consumed became normal in the next 24 h (Fig. 2A,B). Approximately 18 h after the pumps were removed, apparent hyperalgesia was observed in the morphine-treated group (Fig. IA). This hyperalgesia had disappeared at 48 h following pump removal. Thus. certain symptoms of morphine withdrawal were present over a definite time period. In the present study, there was no difference between saline- and morphine-treated rats in cortical CCK receptor binding at any stage (Table). Recently it was demonstrated that neither after acute morphine treatment nor during morphine tolerance there was any change in CCK-like immunoreactivity and preproCCK mRNA levels in a variety of regions of the brain and spinal cord.16 With regard to acute morphine treatment, similar results have been reported by others.” Thus, increased sensitivity of CCKergic neurotransmission appears not to be the primary reason for morphine tolerance and dependence, since neither presynaptic CCK-containing neurons nor postsynaptic CCK receptors seem to undergo adaptational changes. Two limitations of our study, however, should be considered. First, our investigation was restricted to the cerebral cortex and
mh
u “al-
2
Results and discussion
‘=, 120-
Since the development of opioid dependence is strongly influenced by the drug treatment schedule, it seems essential to shape out the periods of analgesia, tolerance, withdrawal and abstinence based on behavioural measurement. The first phase of morphine action after the implantation of minipumps, morphine analgesia, was evident already 12 h after implantation (Fig. 1A). This difference between morphine- and saline-treated rats in pain
.g
E
loo-
s
E a %
8 5 r
80. 60-
‘$4oJ.,.,.,.,.,.,.,., 168 l&e
(hik)%hw~g
p121p
i~~lar&oion
Fig. 2 The water consumption of rats treated with either saline (empty circles) or morphine (filled circles).
382
NEUROPEPTIDES
CCK receptor binding in the cerebral cortex of morphine-treated rats at the stage of analgesia, tolerance, withdrawal and abstinence
Table
Morphine 3 ma,r Analgesia Tolerance Withdrawal Abstinence
3.75kO.25 3.02kO.21 3.56 + 0.28 1.89iO.21
and
control
Control fG
B muI
Kt
0.64+0.07 0.58+0.11 0.52+0.07 0.21 5 0.04
4.02 f 0.23 3.04+0.18 3.28 +0.23 2.02TO.09
0.74kO.08 0.6OkO.13 0.36 f 0.05 0.27 * 0.03
Rats were killed either 24 h after the pump implantation (analgesia group), 120 h after the pump implantation (tolerance group), 24 h after the pump removal (withdrawal group) or 48 h after the pump removal (abstinence group). Data are expressed as mean k SEM of 3d separate analyses. The apparent maximal number of binding sites, B,,,, is expressed as pmol/g w/w tissue; the affinity constant Kd as nmol/l. Analysis of variance indicated that morphine-treated rats did not differ from controls at any stage of drug influence.
the possibility of changes at CCK receptors in other brain regions cannot be ruled out. Nevertheless, it is obvious that CCK receptor regulation is not generally changed after morphine treatment, at least with regard to the CCKB receptor type. Second, since stress has been reported to influence brain CCK levels and CCK receptor characteristics,20.‘1 and repeated handling may reduce behavioral sensitivity to CCK receptor stimulation,” it should be taken into account that manipulations with the rats and single-housing could interfere with the binding results. However, it is evident from the present study that tolerance to the antinociceptive effect of morphine as well as morphine dependence can develop without changes at CCKB receptors. There is ample behavioural evidence that pharmacological manipulations of CCKergic activity do influence morphine opiate analgesia and tolerance (see Introduction), but the data available at the present time suggest that the adaptation to the long-term morphine treatment does not affect CCKergic neurotransmission directly. References Jiirna, I. and Zetler, G. Antinociceptive effect of centrally administered caerulein and cholecystokinin octapeptlde (CCK-8). Eur. J. Pharmacol. 1981; 73: 323-331. Faris, P. L., Komisaruk. B. R., Watkins L. R. and Mayer, D. J. Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 1983; 219: 316312. Itoh, S., Katsuura, G. and Maeda, Y. Caerulein and cholecystokinin suppress j%endorphin-induced analgesia in the rat. Eur. J. Pharmacol. 1982; 80: 421425. Baber, N. S., Dourish. C. T. and Hill, D. R. The role of
5.
6.
I.
8.
9.
IO.
11.
12.
13.
14.
CCK, caerulein, and CCK antagonists in nociception. Pain 1989; 39: 307-328. Dourish, C. T., Hawley, D. and Iversen, S. D. Enhancement of morphine analgesia and prevention of morphine tolerance in the rat by the cholecystokinin antagonist L-364,718. Eur. J. Pharmacol. 1988; 147: 469472. Dourish, C. T., O’Neill, M. F., Coughlan, J., Kitchener, S. J.. Hawley, D. and Iversen, S. D. The selective CCK-B receptor antagonist L-365,260 enhances morphine analgesia and prevents morphine tolerance in the rat. Eur. J. Pharmacol. 1990; 176: 3544. Dourish, C. T., O’Neill, M. F., Schaffer, L. W., Siegl, P. K. S. and Iversen, S. D. The cholecystokinin receptor antagonist devazepide enhances morphine-induced analgesia but not morphine-induced respiratory depression in the squirrel monkey. J. Pharmacol. Exp. Ther. 1990; 255: 11581165. Kellstein, D. E. and Mayer, D. J. Spinal co-administration of cholecystokinin antagonists with morphine prevents the development of opioid tolerance. Pain 1991; 47: 221-229. Watkins, L. R., Kinscheck. I. B. and Mayer, D. J. Potentiation of morphine analgesia by the cholecystokinin antagonist proglumide. Brain Res. 1985; 327: 169-180. Wiesenfeld-Hallin. Z., Xu, X -J., Hughes, J.. Horwell, D. C. and Hlikfelt, T. PD134308. a selective antagonist of cholecystokinin type B receptor, enhances the analgesic effect of morphine and synergistically interacts with intrathecal galanin to depress spinal nociceptive reflexes. Proc. Natl. Acad. Sci. USA 1990; 87: 7105-7109. Tang, J., Chou. J.. Iadarola, M.. Yang, H.-Y. T. and Costa, E. Proglumide prevents and curtails acute tolerance to morphme in rats. Neuropharmacology 1984; 23: 715-718. Lavigne, G. J., Hargreaves. K. M.. Schmidt, E. A. and Dionne, R. A. Proglumide potentiates morphine analgesia for acute postsurgical pain. Clin. Pharmacol. Ther. 1989; 45: 666673. Panerai, A. E., Rovati, L. C., Cocco, E., Sacerdote, P. and Mantegazza, P. Dissociation of tolerance and dependence to morphine: a possible role for cholecystokinin. Brain Res. 1987; 410: 52-60. Xu, X.-J., Wiesenfeld-Hallin, Z., Hughes, J., Horwell, D. C. and Hiikfelt. T. CI988, a selective antagonist of cholecystokinin, receptors, prevents morphine tolerance in the rat. Br. J. Pharmacol. 1992; 105: 591-596.
CCK RECEPTOR BINDING IN MORPHINE ANALGESIA
15. Higgins, G. A.. Nguyen: P. and Sellers, E. M. Morphine place conditioning is differentially affected by CCKA and CCK, receptor antagonists. Brain Res. 1992: 572: 208-215. 16. Pohl, M.. Collin. E.. Benoliel. J. J.. Bourgoin. S., Cesselin. F. and Hamon, M. Cholecystokinin (CCK)-like material and CCK mRNA levels in the rat brain and spinal cord after acute or repeated morphine treatment. Neuropeptides 1992: 21: 1933200. J. and Oreland. L. Age-related differences of 17. Harro, cholecystokinin receptor binding in the rat brain. Prog. Nemo-Psychopharmacol. Biol. Psychiat. 1992; 16: 3699 375. R. J. Emfitter. a non-linear regression data 18. Leatherbarrow. analysis program for the IBM PC. Amsterdam: Elsevier Science Publishers, 1987.
383 19. Rosen. A. and Brodin, E. Effect of acute morphine treatment on peptide levels in the peri-aqueductal grey. Acta Physiol. &and. 1989: 136: 493494. 20. Siegel, R. A., Dtiker, E.-M., Fuchs. E.. Pahnke, L. and Wuttke, W. Responsiveness of mesolimbic, mesocortical, septal and hippocampal cholecystokinin and substance P neuronal systems to stress. in the male rat. Neurochem. Int. 1985: 6: 7833789. 21. Harro. J.. Kiivet. R.-A.. Lang. A. and Vasar. E. Rats with anxious or non-anxious type of exploratory behaviour differ in their brain CCK-8 and benzodiazepine receptor characteristics. Behav. Brain Res. 1990: 39: 63371. 22. Harro. J.. Pold, M. and Vasar, E. Anxiogenic-like action of caerulein, a CCK-8 receptor agonist. in the mouse: influence of acute and subchronic diazepam treatment. Naunyn-Schmiedebergs Arch. Pharmacol. 1990; 341: 62267.
( 1994)
26, 379-383
Group Ltd 1994
Cholecystokinin Receptor Binding in Morphine Analgesia: Tolerance, Withdrawal and Abstinence M. WELIN”,
J. HARRO*S,
R. YUKHANANOVS,
F. NYBERGt
and L. ORELAND”
*Departments of Medical Pharmacology and tPharmacology, Uppsala University, Biomedical Centre, Uppsala, Sweden, #Laboratory of Psychopharmacology, Institute of General and Molecular Pathology, Tartu University, Tartu, Estonia and Slnstitute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia (Reprint requests to LO)
Abstract-The effect of morphine treatment on cholecystokinin (CCK) receptor binding in rat cerebral cortex was investigated. Subcutaneous implantation and removal of Alzet miniosmotic pumps, releasing morphine, permitted us to establish the phases of initial analgesia, tolerance to the analgesic action of morphine, morphine withdrawal and abstinence. CCK receptor binding in rat cerebral cortex never differed from the values obtained from animals implanted with saline-releasing minipumps. The results of the present study suggest that the putative changes in the interaction between opioidergic and CCKergic neurotransmission at different stages of morphine treatment and withdrawal are not caused by changes of CCK receptor binding properties.
Introduction Cholecystokinin (CCK) is a neuropeptide involved in opioid analgesia. Large doses of CCK induce naloxone-reversible analgesia,’ but small and probably physiologically more relevant amounts of this peptide counteract the antinociceptive effect of exogenous and endogenous opioids.2.3 Recent findings have led to the hypothesis that CCK acts as an endogenous antagonist of opioids in the central nervous system (see ref. 4 for review). Thus, the Date received Date accepted
I September 10 January
1993 1994
Correspondence to: Professor Lars Oreland, Uppsala University Department of Medical Pharmacology, Box 593, Biomedicum, S-75124 Uppsala. Sweden
administration of CCK receptor antagonists such as proglumide, lorglumide, devazepide, L365,260 and C1988 results in a marked enhancement of opioid-induced analgesia in rats, monkeys and humans.5m” Furthermore, tolerance to the analgesic effect of morphine is prevented when CCK receptors are blocked.5,6.“.“.‘3.‘4The analysis of pharmacological data suggests that blockade of CCKB receptors is involved in the enhancement of morphine analgesia and in preventing tolerance to the antinociceptive action of morphine. On the other hand, CCK receptor antagonists do not attenuate naloxone-precipitated morphine withdrawaLh,13.‘” However, the blockade of CCK, receptors enhanced the ability of morphine to induce conditioned place preference,” suggesting that while
379
380 the dependence to morphine may not be linked to the CCKergic neurotransmission, the euphoric action of morphine is. There are studies examining the acute CCKopioid interactions directly, but so far the data concerning the action of long-term opioid receptor stimulation on CCKergic neurotransmission are very scarce. Recently, however, Pohl and colleaguesI examined the effect of both acute and repeated morphine treatment on brain CCK and CCK mRNA levels. No effect of morphine treatment on these measures was found and thus, the working hypothesis that chronic stimulation of opioid receptors triggers a progressive compensatory increase in the activity of CCK-containing neurons was not supported. However, in this study, the antinociceptive effect of morphine was not tested. Furthermore, the increase in CCKergic activity could occur at the postsynaptic level, with changes of the biochemical characteristics of CCK, receptors. We have, therefore, studied the binding characteristics of CCK receptors in rat cerebral cortex at different behaviourally validated stages of morphine treatment and withdrawal.
Materials and methods
Adult male Sprague-Dawley rats (Alab, Sollentuna, Sweden), weighing 220-240 g at the beginning of the experiment, were housed in an environment with controlled humidity (60%) temperature (24°C) and lighting (lights on 9.00-21.00). Animals were provided with food and water ad libitum and adapted to these conditions for at least 7 days before experimentation. From the day before the implantation of minipumps until sacrifice the rats were housed in individual cages. All rats were randomly divided into 4 groups, each consisting of 6 morphine-treated and 5 control animals. Miniosmotic pumps (Alzet, 2MLl) were filled with morphine acetate (70 mg/ml) or saline and were implanted subcutaneously under diethyl ether anaesthesia on the back of the animals. Animals of the analgesia group were decapitated 24 h and those of the tolerance group 120 h after the pump implantation. Rats belonging to the withdrawal group were killed 24 h and those forming the abstinence group 48 h after pump removal. In
NEUROPEPTIDES
both of these groups the pumps had been removed 5 days (120-124 h) after implantation. Behaviouval testing
Animals were weighed every day and their water consumption monitored. The pain sensitivity was measured by the paw pressure test using a commercially available apparatus (Ugo Basile, Comerio, Italy). Briefly, the pressure was applicated through a bullet-shaped plastic tip to the plantar surface of the hind paw. The pressure was continuously increased at a rate of 480 or 320 mN/s and the pressure at which a rapid removal of the paw was observed was assigned as the response threshold. Radioligand binding
After decapitation, brain was removed from the skull and cortical tissue rapidly dissected on ice. CCK receptor binding was performed as described earlier.17 In brief, brain tissue was homogenized in 20 volumes of ice-cold Tris-HCl (pH 7.4) using a Potter-S glass-teflon homogenizer (1000 rpm, 10 passes) The membranes were washed twice in the same buffer by centrifugation and resuspension. The final pellets were homogenized in HEPES buffer (10 mM HEPES; 130 mM NaCl; 5 mM MgCl?; 5 mM KCl; 1 mM EDTA; pH 6.5 adjusted with 5 N NaOH) containing bovine serum albumin (0.5 mg/ml). CCK-8 receptor labelling was carried out in the presence of 0.0553 nM tritiated ligand, propionyl-(3H)CCK-8-sulphated (spec. act. 78.0 Ci/mmol, Amersham Radiochemicals) at room temperature in a total incubation volume of 0.5 ml. Caerulein (100 nM) was added to determine nonspecific binding. Before adding the radioligand, membrane suspension was preincubated with or without displacer for 30 min at room temperature. Incubation was initiated by adding the radioligand and terminated after 90 min by rapid filtration over Whatman GF/B filters using a Brandel Cell Harvester (M-24s). The filters were washed with 10 ml cold incubation buffer, dried and assayed for radioactivity by liquid scintillation spectrometry. Data analysis
Saturation curves obtained in radioligand binding experiments were analyzed using non-linear least
CCK RECEPTOR BINDING
IN MORPHINE
0
q
”
0
12
24
48
381
ANALGESIA
12
I I
control morphine
96
120
Time (hours) following pump implantation
Cl control H
i;
morphine
I
I
”
6
Time
12 (hours)
48 21 32 18 following pump removal
Fig. 1 Pain sensitivity of rats treated with morphine in the paw pressure test. Spotted and striped columns correspond to salineand morphine-treated groups respectively. (A) Pain sensitivity after implantation of the minipumps. (B) Hyperalgesia after removal of the morphine-releasing osmotic minipumps. Data are expressed as means f SEM and were analyzed by ANOVA followed by Kruskal-Wallis test.
squares regression.” Parametric or non-parametric one-way analysis of variance (ANOVA) was used initially for the statistical evaluation of the data, followed by Scheffe’s test, as appropriate.
sensitivity had disappeared 96 h after the pump implantation, indicative of the development of tolerance to the analgesic action of morphine. Tolerance remained persistent until sacrifice. In contrast to the control animals, there was a considerable drop in the body weight (data not shown) and water consumption of the morphine-treated rats during the following 24 h after removal of implanted minipumps. Subsequently, the animals started to gain weight and the amount of water consumed became normal in the next 24 h (Fig. 2A,B). Approximately 18 h after the pumps were removed, apparent hyperalgesia was observed in the morphine-treated group (Fig. IA). This hyperalgesia had disappeared at 48 h following pump removal. Thus. certain symptoms of morphine withdrawal were present over a definite time period. In the present study, there was no difference between saline- and morphine-treated rats in cortical CCK receptor binding at any stage (Table). Recently it was demonstrated that neither after acute morphine treatment nor during morphine tolerance there was any change in CCK-like immunoreactivity and preproCCK mRNA levels in a variety of regions of the brain and spinal cord.16 With regard to acute morphine treatment, similar results have been reported by others.” Thus, increased sensitivity of CCKergic neurotransmission appears not to be the primary reason for morphine tolerance and dependence, since neither presynaptic CCK-containing neurons nor postsynaptic CCK receptors seem to undergo adaptational changes. Two limitations of our study, however, should be considered. First, our investigation was restricted to the cerebral cortex and
mh
u “al-
2
Results and discussion
‘=, 120-
Since the development of opioid dependence is strongly influenced by the drug treatment schedule, it seems essential to shape out the periods of analgesia, tolerance, withdrawal and abstinence based on behavioural measurement. The first phase of morphine action after the implantation of minipumps, morphine analgesia, was evident already 12 h after implantation (Fig. 1A). This difference between morphine- and saline-treated rats in pain
.g
E
loo-
s
E a %
8 5 r
80. 60-
‘$4oJ.,.,.,.,.,.,.,., 168 l&e
(hik)%hw~g
p121p
i~~lar&oion
Fig. 2 The water consumption of rats treated with either saline (empty circles) or morphine (filled circles).
382
NEUROPEPTIDES
CCK receptor binding in the cerebral cortex of morphine-treated rats at the stage of analgesia, tolerance, withdrawal and abstinence
Table
Morphine 3 ma,r Analgesia Tolerance Withdrawal Abstinence
3.75kO.25 3.02kO.21 3.56 + 0.28 1.89iO.21
and
control
Control fG
B muI
Kt
0.64+0.07 0.58+0.11 0.52+0.07 0.21 5 0.04
4.02 f 0.23 3.04+0.18 3.28 +0.23 2.02TO.09
0.74kO.08 0.6OkO.13 0.36 f 0.05 0.27 * 0.03
Rats were killed either 24 h after the pump implantation (analgesia group), 120 h after the pump implantation (tolerance group), 24 h after the pump removal (withdrawal group) or 48 h after the pump removal (abstinence group). Data are expressed as mean k SEM of 3d separate analyses. The apparent maximal number of binding sites, B,,,, is expressed as pmol/g w/w tissue; the affinity constant Kd as nmol/l. Analysis of variance indicated that morphine-treated rats did not differ from controls at any stage of drug influence.
the possibility of changes at CCK receptors in other brain regions cannot be ruled out. Nevertheless, it is obvious that CCK receptor regulation is not generally changed after morphine treatment, at least with regard to the CCKB receptor type. Second, since stress has been reported to influence brain CCK levels and CCK receptor characteristics,20.‘1 and repeated handling may reduce behavioral sensitivity to CCK receptor stimulation,” it should be taken into account that manipulations with the rats and single-housing could interfere with the binding results. However, it is evident from the present study that tolerance to the antinociceptive effect of morphine as well as morphine dependence can develop without changes at CCKB receptors. There is ample behavioural evidence that pharmacological manipulations of CCKergic activity do influence morphine opiate analgesia and tolerance (see Introduction), but the data available at the present time suggest that the adaptation to the long-term morphine treatment does not affect CCKergic neurotransmission directly. References Jiirna, I. and Zetler, G. Antinociceptive effect of centrally administered caerulein and cholecystokinin octapeptlde (CCK-8). Eur. J. Pharmacol. 1981; 73: 323-331. Faris, P. L., Komisaruk. B. R., Watkins L. R. and Mayer, D. J. Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 1983; 219: 316312. Itoh, S., Katsuura, G. and Maeda, Y. Caerulein and cholecystokinin suppress j%endorphin-induced analgesia in the rat. Eur. J. Pharmacol. 1982; 80: 421425. Baber, N. S., Dourish. C. T. and Hill, D. R. The role of
5.
6.
I.
8.
9.
IO.
11.
12.
13.
14.
CCK, caerulein, and CCK antagonists in nociception. Pain 1989; 39: 307-328. Dourish, C. T., Hawley, D. and Iversen, S. D. Enhancement of morphine analgesia and prevention of morphine tolerance in the rat by the cholecystokinin antagonist L-364,718. Eur. J. Pharmacol. 1988; 147: 469472. Dourish, C. T., O’Neill, M. F., Coughlan, J., Kitchener, S. J.. Hawley, D. and Iversen, S. D. The selective CCK-B receptor antagonist L-365,260 enhances morphine analgesia and prevents morphine tolerance in the rat. Eur. J. Pharmacol. 1990; 176: 3544. Dourish, C. T., O’Neill, M. F., Schaffer, L. W., Siegl, P. K. S. and Iversen, S. D. The cholecystokinin receptor antagonist devazepide enhances morphine-induced analgesia but not morphine-induced respiratory depression in the squirrel monkey. J. Pharmacol. Exp. Ther. 1990; 255: 11581165. Kellstein, D. E. and Mayer, D. J. Spinal co-administration of cholecystokinin antagonists with morphine prevents the development of opioid tolerance. Pain 1991; 47: 221-229. Watkins, L. R., Kinscheck. I. B. and Mayer, D. J. Potentiation of morphine analgesia by the cholecystokinin antagonist proglumide. Brain Res. 1985; 327: 169-180. Wiesenfeld-Hallin. Z., Xu, X -J., Hughes, J.. Horwell, D. C. and Hlikfelt, T. PD134308. a selective antagonist of cholecystokinin type B receptor, enhances the analgesic effect of morphine and synergistically interacts with intrathecal galanin to depress spinal nociceptive reflexes. Proc. Natl. Acad. Sci. USA 1990; 87: 7105-7109. Tang, J., Chou. J.. Iadarola, M.. Yang, H.-Y. T. and Costa, E. Proglumide prevents and curtails acute tolerance to morphme in rats. Neuropharmacology 1984; 23: 715-718. Lavigne, G. J., Hargreaves. K. M.. Schmidt, E. A. and Dionne, R. A. Proglumide potentiates morphine analgesia for acute postsurgical pain. Clin. Pharmacol. Ther. 1989; 45: 666673. Panerai, A. E., Rovati, L. C., Cocco, E., Sacerdote, P. and Mantegazza, P. Dissociation of tolerance and dependence to morphine: a possible role for cholecystokinin. Brain Res. 1987; 410: 52-60. Xu, X.-J., Wiesenfeld-Hallin, Z., Hughes, J., Horwell, D. C. and Hiikfelt. T. CI988, a selective antagonist of cholecystokinin, receptors, prevents morphine tolerance in the rat. Br. J. Pharmacol. 1992; 105: 591-596.
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