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Supersensitivity of brain opiate receptor subtypes after chronic naltrexone treatment
Life Sciences, Vol. 31, pp. 1401-1404 Printed in the U.S.A.
Pergamon Prese
SUPERSENSITIVITY OF BRAIN OPIATE RECEPTOR SUBTYPES AFTER CHRONIC NALTREXONE TREATMENT Ann Tempel, R. Suzanne Zukin and Eliot L. Gardner Departments of Biochemistry, Neuroscience and Psychiatry Albert Einstein College of Medicine Bronx, New York i0461 (Received in final form June 14, 1982) Summary Chronic administration of the narcotic antagonist naltrexone resulted in a marked increase in brain opiate receptors. Similar changes were observed for putative Mu, Delta, and Kappa opiate receptor subtypes. In contrast, only a modest increase was observed for the putative Sigma receptor. Withdrawal from chronic naltrexone treatment resulted in a decrease from elevated receptor levels to nearly control receptor levels in a period of about 6 days, as revealed by [3H] etorphine binding. These results may shed light on the mechanisms of opiate dependence and withdrawal. The phenomenon of supersensitivity has been well documented in many receptor systems, including the dopaminergic (1,2) and, more recently, enkephalinergic systems (3,4,5,6). The phenomena of tolerance and addiction make the opiate system an interesting one in which to study neuronal plasticity. Zukin and coworkers (6), have shown that chronic blockade of opiate receptors with the antagonist naltrexone led to a nearly two-fold increase in both Mu and Delta opiate receptors in the central nervous system. The extent of opiate receptor change varied throughout the brain; largest increases were observed in mesolimbic and frontal cortex areas. In addition, the newly synthesized or unmasked opiate receptors were more sensitive to guanyl nucleotide modulation. The present studies explored the effects of chronic naltrexone treatment on Kappa and Sigma opiate receptors as well as the possibility of downregulation following naltrexone withdrawal. Methods For experiments exploring drug-induced supersensitivity of opiate receptor subtypes, rats were implanted subcutaneously with 2 pellets containing 30 mg naltrexone each (7). Eight days after pellet implantation animals were sacrificed, brains removed, and tissue prepared as described (6) with the inclusion of a 30 minute incubation at 37°C following the second resuspension. For binding assays, i ml aliquots were incubated in triplicate with [3H] etorphine (in the presence or absence of 10-5M levorphanol) or [3H] Nallylnorcyclazocine (SKF) or [3H] ethylketocyclazocine (EKC) (in the presence or absence of IO-5M cyclazocine) for 45 minutes at 4°C. For the study exploring the effects of withdrawal from chronic naltrexone treatment, animals were implanted with naltrexone pellets for 8 days. Pellets were removed on the eighth day and animals were sacrificed at various intervals after pellet withdrawal. Brain tissue was prepared and homogenates assayed as described above using [3HI 0024-3205/82/121401-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1402
Opiate Receptor Subtype Supersensitivity
Vol. 31, No.s 12 & 13, 1982
etorphine. Data were analyzed by linear regression analysis. Biphasic binding curves were analyzed by an independent linear regression analysis of each linear component. Each experiment was repeated at least twice. Results Eight days after naltrexone pellet implantation, the density of [3H] etorphine binding sites was 95% greater in brains from naltrexone-treated than in those from control animals (Fig. 1). [3H] EKC binding to brain (Fig. 2) revealed a 74% increase in the number of high affinity (putative Mu (6)) sites and a i05% increase in the low affinity (putative Kappa (6)) sites. These increases in binding reflect a change in density with no apparent change in affinity. Scatchard analysis of [3H] SKF binding to brain (Fig. 3) revealed a i08% increase in the number of high affinity (putative Mu (6)) sites, but only a 27% increase in the low affinity (putative Sigma (6)) sites. Table i summarizes the effects of long-term naltrexone treatment on the various opiate receptor subtypes.
800
,oo.
a°°i
,001
.o:,,
,ooi,,,o. ,oo. 0
"\
.....
i ,o1
.......
100 200 : ~ 0 400 500 600 7 0 0 8 0 0 9 0 0
~OUNO]#mo#/mg~o~) FIG. 1 Scatcha~d plots of [3H] etorphine binding to whole brain homogenates of naltrexone-treated and control rats.
o
4o 8o ~zo ~ o z o o z ~ 2 s o ~ o
4oo
(BOUN~(fmol/mgpm~) FIG. 2 Scatehard plots of [3H] EKC binding to whole brain homogenates of naltrexonetreated and control rats.
In the naltrexone withdrawal study (Fig. 4) [3H] etorphine binding to rat brain revealed a steady decrease in receptor number with no change in affinity for the first 6 days following pellet removal. At six days, brain tissue from animals originally treated with naltrexone showed an opiate receptor density that did not differ, within experimental error, from that of control brain. At ten days following pellet withdrawal, brain tissue from the animals treated originally with naltrexone showed an opiate receptor density 35% ± 3% higher than that of control brain (in 3 out of 3 sets of animals). By 14 days, naltrexone-brain values were essentially the same as control levels, and remained so, for at least 4 weeks following withdrawal from naltrexone (by Scatchard analysis at 7 day intervals).
soo
Vol. 31, No.s 12 & 13, 1982
5tA 50 -
Opiate Receptor Subtype Supersensitivity
1403
~Control
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DAYSAFTERNALTREXONEPELLETWITHDRAWAL
[BOUND] (fmol/mg I~oteie)
FIG. 4
FIG. 3 Scatchard plots of [3H] SKF binding to whole brain homogenates of naltrexone-treated and control rats.
Time-course of the decrease in brain opiate receptor number following withdrawal from chronic naltrexone treatment. Pellets were removed on day "zero".
Discussion In rats treated chronically with naltrexone there were approximately twofold increases in the high and low affinity binding sites of [3~H] DHM (putative Mu ligand), [3HI DADLE (putative Delta ligand), [3H] etorphine and [3HI EKC (putative Mu and Kappa ligand). A similar increase was observed for the [3H] SKF high affinity site (putative Mu receptor), but only a modest change was found in the low affinity site (putative Sigma receptor). Thus, the sites identified as Mu, Delta, and Kappa appear to be regulated in a coordinated manner during chronic administration of the classical opiate antagonist naltrexone. It is interesting to note that the putative Sigma site is not so regulated. Numerous studies have shown that Sigma behavioral effects are not reversible by classical opiate antagonists such as naloxone (8). Thus, as previously suggested, the sigma receptor is not really an opiate receptor, but a site of psychotomimetic action for drugs of several classes. It is also interesting that both up- and down regulation can be demonstrated in the opiate system in analogy to the dopaminergic system. These studies may shed light on the phenomenaof tolerance and dependence.
1404
Opiate Receptor Subtype Supersensitivity
Vol. 31, No.s 12 & 13, 1982
TABLE i Radiolabelled Ligand Binding To Opiate Receptor Subtypes After Chronic Naltrexone Treatment
Control (fmol/mg Protein)
[3H] dihydromorphine (Mu ligand Bmax(1) Bmax(2)
Naltrexone-treated (fmol/mg Protein)
% Change
(6) 80 120
130 220
63 ~ 83 ÷
3.2 4.2
[3H] DADLE *(6) (Delta ligand)
350
640
83 ~
4.2
[3H] etorphine
490
957
95 ~
4.8
Bmax(1) Bmax(2)
23 229
40 470
74 ± 7.4 I05 ~ i0.6
Bmax(1) Bmax(2)
38 121
79 154
i08 ~ 10.8 27 ~ 2.8
[3H] EKC
[3H] SKF
* D-Ala 2, D-Leu5-enkephalin.
References i. 2. 3. 4. 5. 6.
7. 8.
I. CREESE, D. BURT and S. SNYDER, Science, 197 596-598 (1977). E. GARDNER, R. ZUKIN AND M. MAKMAN, Brain Res., 194 232-239 (1980). R. SCHULZ, M. WUSTER AND A. HERZ, Naun, Schmied, Arch. Pharmacol., 306 93-96 (1979). A. TANG and R. COLLINS, Eur. J. Pharmacol., 47 473-474 (1978). R. LAHTI and R. COLLINS, Eur. J. Pharmacol., 51 185-186 (1978). R. ZUKIN, J. SUGARMAN, M. FITZ-SYAGE, E. GARDNER, S. ZUKIN AND A. GINTZLER, Brain Res., in press, (1982). A. MISRA and R. PONTANI, J. Pharm. Pharmacol., 30 325-326 (1978). R. ZUKIN and S. ZUKIN, Life Sciences, 29 2681-2690 (1981).
Pergamon Prese
SUPERSENSITIVITY OF BRAIN OPIATE RECEPTOR SUBTYPES AFTER CHRONIC NALTREXONE TREATMENT Ann Tempel, R. Suzanne Zukin and Eliot L. Gardner Departments of Biochemistry, Neuroscience and Psychiatry Albert Einstein College of Medicine Bronx, New York i0461 (Received in final form June 14, 1982) Summary Chronic administration of the narcotic antagonist naltrexone resulted in a marked increase in brain opiate receptors. Similar changes were observed for putative Mu, Delta, and Kappa opiate receptor subtypes. In contrast, only a modest increase was observed for the putative Sigma receptor. Withdrawal from chronic naltrexone treatment resulted in a decrease from elevated receptor levels to nearly control receptor levels in a period of about 6 days, as revealed by [3H] etorphine binding. These results may shed light on the mechanisms of opiate dependence and withdrawal. The phenomenon of supersensitivity has been well documented in many receptor systems, including the dopaminergic (1,2) and, more recently, enkephalinergic systems (3,4,5,6). The phenomena of tolerance and addiction make the opiate system an interesting one in which to study neuronal plasticity. Zukin and coworkers (6), have shown that chronic blockade of opiate receptors with the antagonist naltrexone led to a nearly two-fold increase in both Mu and Delta opiate receptors in the central nervous system. The extent of opiate receptor change varied throughout the brain; largest increases were observed in mesolimbic and frontal cortex areas. In addition, the newly synthesized or unmasked opiate receptors were more sensitive to guanyl nucleotide modulation. The present studies explored the effects of chronic naltrexone treatment on Kappa and Sigma opiate receptors as well as the possibility of downregulation following naltrexone withdrawal. Methods For experiments exploring drug-induced supersensitivity of opiate receptor subtypes, rats were implanted subcutaneously with 2 pellets containing 30 mg naltrexone each (7). Eight days after pellet implantation animals were sacrificed, brains removed, and tissue prepared as described (6) with the inclusion of a 30 minute incubation at 37°C following the second resuspension. For binding assays, i ml aliquots were incubated in triplicate with [3H] etorphine (in the presence or absence of 10-5M levorphanol) or [3H] Nallylnorcyclazocine (SKF) or [3H] ethylketocyclazocine (EKC) (in the presence or absence of IO-5M cyclazocine) for 45 minutes at 4°C. For the study exploring the effects of withdrawal from chronic naltrexone treatment, animals were implanted with naltrexone pellets for 8 days. Pellets were removed on the eighth day and animals were sacrificed at various intervals after pellet withdrawal. Brain tissue was prepared and homogenates assayed as described above using [3HI 0024-3205/82/121401-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1402
Opiate Receptor Subtype Supersensitivity
Vol. 31, No.s 12 & 13, 1982
etorphine. Data were analyzed by linear regression analysis. Biphasic binding curves were analyzed by an independent linear regression analysis of each linear component. Each experiment was repeated at least twice. Results Eight days after naltrexone pellet implantation, the density of [3H] etorphine binding sites was 95% greater in brains from naltrexone-treated than in those from control animals (Fig. 1). [3H] EKC binding to brain (Fig. 2) revealed a 74% increase in the number of high affinity (putative Mu (6)) sites and a i05% increase in the low affinity (putative Kappa (6)) sites. These increases in binding reflect a change in density with no apparent change in affinity. Scatchard analysis of [3H] SKF binding to brain (Fig. 3) revealed a i08% increase in the number of high affinity (putative Mu (6)) sites, but only a 27% increase in the low affinity (putative Sigma (6)) sites. Table i summarizes the effects of long-term naltrexone treatment on the various opiate receptor subtypes.
800
,oo.
a°°i
,001
.o:,,
,ooi,,,o. ,oo. 0
"\
.....
i ,o1
.......
100 200 : ~ 0 400 500 600 7 0 0 8 0 0 9 0 0
~OUNO]#mo#/mg~o~) FIG. 1 Scatcha~d plots of [3H] etorphine binding to whole brain homogenates of naltrexone-treated and control rats.
o
4o 8o ~zo ~ o z o o z ~ 2 s o ~ o
4oo
(BOUN~(fmol/mgpm~) FIG. 2 Scatehard plots of [3H] EKC binding to whole brain homogenates of naltrexonetreated and control rats.
In the naltrexone withdrawal study (Fig. 4) [3H] etorphine binding to rat brain revealed a steady decrease in receptor number with no change in affinity for the first 6 days following pellet removal. At six days, brain tissue from animals originally treated with naltrexone showed an opiate receptor density that did not differ, within experimental error, from that of control brain. At ten days following pellet withdrawal, brain tissue from the animals treated originally with naltrexone showed an opiate receptor density 35% ± 3% higher than that of control brain (in 3 out of 3 sets of animals). By 14 days, naltrexone-brain values were essentially the same as control levels, and remained so, for at least 4 weeks following withdrawal from naltrexone (by Scatchard analysis at 7 day intervals).
soo
Vol. 31, No.s 12 & 13, 1982
5tA 50 -
Opiate Receptor Subtype Supersensitivity
1403
~Control
_z ,qe,
6o 0
2
_z
Na/trexone Trea~e~
,,~ ao u
_z
ae 20
iOl
|p i!
!
,
0
,
,
,
,
'%. ~. ,
40 80
,
.
.
.
.
.
,
.
0
120 160 200 240
I
2
,
I
4
,
I
6
,
I
8
,
I
I0
,
I
,
I
12 "14
DAYSAFTERNALTREXONEPELLETWITHDRAWAL
[BOUND] (fmol/mg I~oteie)
FIG. 4
FIG. 3 Scatchard plots of [3H] SKF binding to whole brain homogenates of naltrexone-treated and control rats.
Time-course of the decrease in brain opiate receptor number following withdrawal from chronic naltrexone treatment. Pellets were removed on day "zero".
Discussion In rats treated chronically with naltrexone there were approximately twofold increases in the high and low affinity binding sites of [3~H] DHM (putative Mu ligand), [3HI DADLE (putative Delta ligand), [3H] etorphine and [3HI EKC (putative Mu and Kappa ligand). A similar increase was observed for the [3H] SKF high affinity site (putative Mu receptor), but only a modest change was found in the low affinity site (putative Sigma receptor). Thus, the sites identified as Mu, Delta, and Kappa appear to be regulated in a coordinated manner during chronic administration of the classical opiate antagonist naltrexone. It is interesting to note that the putative Sigma site is not so regulated. Numerous studies have shown that Sigma behavioral effects are not reversible by classical opiate antagonists such as naloxone (8). Thus, as previously suggested, the sigma receptor is not really an opiate receptor, but a site of psychotomimetic action for drugs of several classes. It is also interesting that both up- and down regulation can be demonstrated in the opiate system in analogy to the dopaminergic system. These studies may shed light on the phenomenaof tolerance and dependence.
1404
Opiate Receptor Subtype Supersensitivity
Vol. 31, No.s 12 & 13, 1982
TABLE i Radiolabelled Ligand Binding To Opiate Receptor Subtypes After Chronic Naltrexone Treatment
Control (fmol/mg Protein)
[3H] dihydromorphine (Mu ligand Bmax(1) Bmax(2)
Naltrexone-treated (fmol/mg Protein)
% Change
(6) 80 120
130 220
63 ~ 83 ÷
3.2 4.2
[3H] DADLE *(6) (Delta ligand)
350
640
83 ~
4.2
[3H] etorphine
490
957
95 ~
4.8
Bmax(1) Bmax(2)
23 229
40 470
74 ± 7.4 I05 ~ i0.6
Bmax(1) Bmax(2)
38 121
79 154
i08 ~ 10.8 27 ~ 2.8
[3H] EKC
[3H] SKF
* D-Ala 2, D-Leu5-enkephalin.
References i. 2. 3. 4. 5. 6.
7. 8.
I. CREESE, D. BURT and S. SNYDER, Science, 197 596-598 (1977). E. GARDNER, R. ZUKIN AND M. MAKMAN, Brain Res., 194 232-239 (1980). R. SCHULZ, M. WUSTER AND A. HERZ, Naun, Schmied, Arch. Pharmacol., 306 93-96 (1979). A. TANG and R. COLLINS, Eur. J. Pharmacol., 47 473-474 (1978). R. LAHTI and R. COLLINS, Eur. J. Pharmacol., 51 185-186 (1978). R. ZUKIN, J. SUGARMAN, M. FITZ-SYAGE, E. GARDNER, S. ZUKIN AND A. GINTZLER, Brain Res., in press, (1982). A. MISRA and R. PONTANI, J. Pharm. Pharmacol., 30 325-326 (1978). R. ZUKIN and S. ZUKIN, Life Sciences, 29 2681-2690 (1981).