Brain Research, 526 (1990) 270-275 Elsevier
270 BRES 15828
Decrease in
-opioid receptor binding capacity in rat brain after chronic PL017 treatment
Pao-Luh Tao t, Haw-Yen Lee 1, Li-Ren Chang I and Horace H. Loh 2 t Department of Pharmacology, National Defense Medical Center, Taipei (Taiwan) and 2Department of Pharmacology, University of Minnesota, Minneapolis, MN 55414 (U.S.A.) (Accepted 6 March 1990)
Key words: Brain opioid receptor; PL017; Receptor, p-opioid; Down regulation
In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or D-Ala2,o-LeuS-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases,/t-opioid receptor binding capacity was attenuated together with the d-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both/~ and d receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the/~-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5-20/~g/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the ADs0 of the peptide to elicit the antinociceptive response and about 14-fold increase in the EDso of the peptide to elicit the catatonic effect. When g- and d-binding was determined using [3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20-30%) in the/~-opioid receptor binding but not in d-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [3H]DAMGO saturation binding data revealed a decrease in Bmax values and no change in the Kd values. Hence,/~-opioid receptors can be specifically regulated by ligand in the brain as d-receptors are in neuroblastoma x glioma NG 108-15 cells. Chronic activation of the g-opioid receptor in the brain would result in the down-regulation of the binding sites.
INTRODUCTION Alteration in ligand-receptor interaction during chronic drug treatment has been suggested as a possible mechanism underlying opioid tolerance. In neonatal rats, Tempel et al. 26 found that morphine could produce a down regulation of brain ~t-opioid receptors during the first week of life. However, most studies investigating this issue have found that chronic morphine treatment of adult animals results in no change in brain opioid receptor levels 13'1523, or even an increase lz'21. In the clonal cell line, neuroblastoma x glioma NG108-15 hybrid cells, down-regulation of 6-0pioid receptors is observed following chronic treatment with opioid agonists 16'~7. However, chronic treatment with partial agonists, such as morphine (in this system), does not have this effect. Accordingly, we reasoned that the failure to observe down-regulation of opioid receptors in adult rat brain might have resulted from not using the appropriate agonist. Some support for this notion is also provided by studies by Dingledine et al. 6. They found that chronic treatment of hippocampal slices in vitro with D-Aia z, D-Leu 5-
enkephalin ( D A D L E ) resulted in a decrease of 6receptors, but not of/~-receptors. Incubation with the /~-agonists morphine or morphiceptin had no effect on either/~- or &receptors, although physiologic tolerance to morphiceptin was observed. Dingledine et al. concluded that down-regulation could account for tolerance to 6-agonists in this system, but not tolerance to/~-agonists. Recently, we reported decreases in both /~- and 6-0pioid receptor binding in rats chronically treated with etorphine 24. A n d in a subsequent study 25, we obtained somewhat similar results following chronic administration of D A D L E , except that chronic D A D L E treatment preferentially reduced 6-0pioid receptor binding activity. Because etorphine and D A D L E can each interact with both ~- and d-receptors 8,14,28, one could argue that the observed decrease in/t-opioid receptor number reflects activation of 6 receptors. In order to further demonstrate that ~-opioid receptors are under similar control as d-opioid receptors, we treated rats chronically with CH3Phe3,D-Pro4-morphiceptin (PL017) in the present study. This opioid peptide was chosen because of its high selectivity toward the ~-opioid receptor, and because it is more stable than morphiceptin in vivo 1'3'18.
Correspondence: E-L. Tao, Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan, R.O.C. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
271 TABLE I
TABLE II
Alteration in antinociception potency of PLO17 during chronic PLO17 treatment
Alteration in catatonia potency of PLOI7 during chronic PLOI7 treatment
The ability of PL017 to produce antinociceptive responses in rats treated with PL017 according to the paradigm outlined in text was determined by the up-down method of Dixon 7. In each group, n > 6 rats were used in the determination of the ADso values. The degree of tolerance was calculated as: ADs0 after treatment/ADso before treatment.
The ability of PL017 to produce catatonia responses in rats treated with PL017 according to the paradigm outlined in text was determined by the up-down method of Dixon 7. In each group n > 6 rats were used in the determination of the EDso values. The degree of tolerance was calculated as: EDso after treatment/EDso before treatment (0 day).
Duration
Duration
P L017 E D so (#g/kg b. wt. )
Degree of tolerance
0 days I day 3 days 5 days
1.79 + 2.40 + 7.25 + 24.2 +
1.0 1.3 4.0 13.5
PLO17 ADjo (#g/kg b. wt.)
Degree of tolerance
Before treatment After treatment 1 day 3days 5 days
1.28 + 0.17 1.53+0.12 1.41 + 0.17
2.97 + 0.4 18.5+2.4 39.1 + 2.7
2.3 12.1 27.7
0.25 0.19 0.98 1.0
g for 20 min. Excess PL017 was removed by washing the P2 membranes first with 1:100 w/v of 50 mM Tris buffer (pH 7.4) containing 100 mM NaCI, 5 p M GTP and 1 mM phenylmethyl sulfonyl fluoride; and second with the same volume of this buffer without GTP. These two washes were carried out by incubating the membrane mixtures at 37 *C for 15 rain, and then membranes were pelleted by centrifuging at 22,000 g for 20 min. The membranes were washed finally with 50 mM Tris buffer (pH 7.4) at 4 *C to remove NaC1 before binding assays. The washed membranes were stored in a freezer at -70 ° C .
MATERIALS AND METHODS
Treatment of animals with PLO17 and pharmacological tests Male Sprague-Dawley rats weighing 160-250 g were treated with PL017 via a stainless-steel cannula implanted into the left lateral cerebroventricle (i.c.v.) according to the coordinates: P 1.0 mm, L 1.25 mm, V 4-5 mm using bregma as zero. After the rats had recovered from the operation (3 days), PL017 was administered i.c.v, to determine the ADso dose by the tail-flick antinociceptive response 5 and EDso dose for catatonia effect 27 according to the up-down method as described by Dixon 7. Afterwards, the animals were chronically treated with PL017 by daily administration of the peptide at 09.00 and 17.00 h starting with a 2.5/~g/kg i.c.v, dose on day 1, followed by 5/~g/kg on day 2, 10/tg/kg on day 3, 15 gg/kg on day 4 and 20 #g/kg on day 5. After chronic PL017 treatment of 1, 3 or 5 days, the antinociceptic ADso values or catatonic EDso values of PL017 were re-determined by the up-down method. For acute PL017 treatment, rats were injected i.c.v, with 2.5 jug/kg PL017 20 min prior to sacrifice.
Opioid receptor binding assays Opioid receptor binding assays in triplicates were carded out with [3H]diprenorphine or [3H]DAMGO in the presence of 5 mM Mg 2+ and a final volume of 1.0 ml of Tris buffer at pH 7.4 for 90 rain at 24 °C, using 5 # M diprenorphine or 1/zM D A M G O for non-specific binding determinations. In the studies in which the relative/~- and 6-opioid receptor binding was determined, [3H]diprenorphine was used as radioactive ligand, 1/zM morphiceptin 4 was added to a set of triplicates, and 1 # M DPDPE 9 was added to another set of triplicates. Thus, /z-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 g M morphiceptin; and the 6-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 / t M of DPDPE. After the opioid receptor binding had reached equilibrium, incubations were terminated by collecting the membranes on Whatman GF/B filters, and the filters were washed three times with 5 ml of 10 mM "Iris buffer (pH 7.4) at 4 °C. The amount of membrane protein used in each assay was determined by the method of Lowry et al. 19.
Membrane preparations After either chronic or acute treatment with PL017, the rats were sacrificed by decapitation and the brains excised. Membranes were prepared from the brain regions of cortex, midbrain (including thalamus and hypothalamus) and striatum by homogenizing the tissues in 0.32 M sucrose buffered with 50 mM "Iris buffer (pH 7.4) in a tissue/buffer ratio of 1:10. After the removal of nuclei by the 1000 x g x 10 min centrifugation procedure, P2 membranes were pelleted by centrifuging the 1000 g for 10 min supernatants at 22,000
TABLE III
Cross-tolerance to D A D L E after chronic PLO17 treatment The ADso of PL017 or D A D L E in rats with PL017 according to the paradigm outlined in text was determined by the up-down method of Dixon 7. In each group, n > 6 rats were used in the determination of the ADs0 values. The % of cross-tolerance was determined by: (degree of tolerance to DADLE/degree of tolerance to PL017) x 100%.
Duration
0days 1 day 3 days 5 days
PL017
DA DLE
% of cross-tolerance
A D ao (Izg/kg b. wt. )
Degree of tolerance
A 1)5o (gglkg b. wt. )
Degree of tolerance
1.17 + 3.35 + 22.3 + 44.5 +
2.9 19.1 38.0
13.6 + 20.2 + 27.2 + 55.1 +
1.5 2.0 4.0
*P < 0.01; two-tailed t-test.
0.18 0.16" 2.0* 3.1"
1.1 1.8" 1.6" 6.1"
1.5/2.9 2.0/19.1 4.0/38.0
(51.7%) (10.5%) (10.5%)
272
Materials [3H]diprenorphine (41 Ci/mmol) was supplied by Amersham, [3H]DAMGO (35 Ci/mmol) was supplied by NEN, diprenorphineHC1 was obtained from the National Institute on Drug Abuse (Bethesda, MD), the opioid peptides morphiceptinTM and PL0173, DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol)1° and DPDPE (D-pen2, D-penS-enkephalin) 2° were purchased from Peninsula Labs. (Belmont, CA). All other chemicals used were reagent grade and were supplied by Sigma (St. Louis, MO). RESULTS
Development o f tolerance to chronic PL017 treatment PL017 has been shown to have antinociceptic, catatonic and physical d e p e n d e n t effects 3. Prior to investigating the possible alteration in opioid r e c e p t o r binding activity during chronic PL017 t r e a t m e n t , the ability of the present p a r a d i g m to p r o d u c e tolerance response was d e t e r m i n e d , using the antinociceptive response to the tail-flick test and c a t a t o n i a response. The ADs0 and EDs0 values of PL017 to elicit antinociception and catatonia respectively, animals which had not been t r e a t e d with PL017 were c o m p a r e d with those of the same animals which were previously t r e a t e d with increasing doses of PL017 for 1, 3 and 5 days. A s s u m m a r i z e d in Table I, the average ADs0 value of i.c.v, administered PL017 to p r o d u c e the antinociceptive response in the tail-flick test in the 3 groups of animals was 1.41 + 0.10/~g/kg. A f t e r 1 day of administration of PL017, i.e. 2 i.c.v, injections of 2 . 5 / t g / k g of PL017, the ADso increased from 1.28 + 0.17 ktg/kg to 2.97 + 0.40/~g/kg, i.e. 2.3-fold tolerance h a d been p r o d u c e d . F u r t h e r m o r e , further chronic injection of PL017 resulted in a t i m e - d e p e n d e n t increase of
TABLE IV
[~H]Diprenorphine binding to membranes from various brain regions after chronic PL017 treatment Specific binding of 2 nM [3H]diphrenorphine with washed membranes from various brain regions were carried out as described in Methods. The values represent the averages + S.E.M. of the binding data from 6 separate animals. The values in parentheses are the percentages of specific binding remained after chronic PL017 treatment as compared to that in membranes isolated from rats treated acutely with PL017.
[~H] Diprenorphine bound (fmol/mg protein)
Acute PL017 Chronic PL017 1 day 3 days 5 days
Cortex
Midbrain
Striatum
221.3 + 7.4
240.9 + 10.1
370.2+ 11.3
236.3 + 17.6 (106.8%) 218.5 + 6.7 (98.7%) 159.8 + 5.6* (72.2%)
250.4+ 10.3 (103.9%) 218.7 + 11.2 (90.8%) 181.9 + 9.5* (75.5%)
361.1+ 11.4 (97.5%) 360.3+ 14.9 (97.3%) 302.0 + 17.4" (81.6%)
*P < 0.05; Duncan multi-range test.
the ADs0 values of the o p i o i d p e p t i d e . A s shown in Table I, after 5 days of chronic t r e a t m e n t , the ADso increased from 1.41 + 0.17/~g/kg to 39.1 + 2.7/~g/kg, i.e. 27.7-fold tolerance to antinociceptive effects had b e e n p r o d u c e d . As to the catatonia response, there was also a timed e p e n d e n t increase of the EDs0 values of PL017 after chronic t r e a t m e n t , although the d e g r e e of tolerance was less than that for antinociceptive response as shown in Table II. T h e r e f o r e , under the current p a r a d i g m of multiple i.c.v. PL017 injections, animals d e v e l o p e d a high degree of tolerance to the o p i o i d peptide.
Cross-tolerance to D A D L E after chronic P L 0 1 7 treatment The selectivity of PL017 t o w a r d the p - o p i o i d r e c e p t o r was d e m o n s t r a t e d by the relative lack of cross-tolerance toward D A D L E after chronic PL017 t r e a t m e n t . A s r e p o r t e d in previous studies 25, the antinociceptive effect of D A D L E after i.c.v, administration is possibly due to the activation of the 6-opioid receptor. W h e n animals were t r e a t e d with PL017 for 3 o r 5 days and the ability
TABLE V
Relative alteration in p- and t~-opioid receptor binding after chronic PL017 treatment Specific binding of 2 nM [3H]diprenorphine to washed membranes from various brain regions of rats chronically or acutely treated with PL017 were carried out in the presence of 5 mM Mg2+ as described in Methods. In order to determine the relative contribution of/~- and 6-opioid receptor to the specific binding, [3H]diprenorphine binding was carried out in the presence of morphiceptin, a p-opioid receptor selective ligand or DPDPE, a 6-1igand. Thus, /~-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 pM morphiceptin; and the 6-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 /~M DPDPE. The values represent the averages of determinations obtained with membrane preparations from 6 different animals. The values in parentheses are the percentage of binding remained after chronic PL017 treatment as compared to that of acute PL017 treatment. Duncan multirange test was used to analyze these data.
[3H]Diprenorphine binding (fmol/mg protein)
A. p-Opioid receptor Acute PL017 Chronic PL017 1 day 3days 5days B. 6-Opioid receptor Acute PL017 Chronic PL017 1 day 3 days 5days
Cortex
Midbrain
Striatum
47.0 + 4.1
87.1 + 9.9
49.8 + 7.4
38.7 + 3.8 41.3+3.6 37.5+1.9" (79.8%)
65.6 + 6.7 86.5+5.8 59.5+8.0* (68.3%)
53.6 + 6.9 54.1+7.9 27.1+4.4" (54.4%)
61.2 + 7.1
66.3 + 10.7
69.3 + 7.1
57.5 + 7.6 58.0 + 5.3 54.2+9.3
60.3 + 10.7 51.4 + 9.6 67.1+8.5
69.5+6.9
273 Mldbrain 0.14 "
a
, ~ 0.12 -
control
0.10 g.
m
0.08 0.06 0.04 0.02 0.00 0.00 0.01
0.02 0.03 0.04 0.05 0.06 0.07 Bound (nM)
Fig. 1. Scatchard analysis of [3H]DAMGO saturation binding to midbrain membranes of control rats (i.e. acute PL017 treated rats) ([~) or rats treated with PL017 for 5 days (m). Saturation binding was measured with 10 different concentrations of [3H]DAMGO, 0.05-5.0 nM. The resulting specific binding data were analysed by a non-linear curve fitting program (Ligand 1). The Bmax values were 129.5 fmol/mg (0.067 nM) (D) and 94.3 fmol/mg (0.056 nM) (m); and the K d values were 0.48 nM (17) and 0.45 nM (m), respectively. Data shown are from one representative experiment. Comparable results were obtained in two independent sets of experiments.
of D A D L E to elicit antinociceptiion was determined, a 2to 4-fold increase in the ADs0 values of DADLE was observed, corresponding to only 10% cross-tolerance between D A D L E and PL017 (Table III).
Effect of chronic PLO17 treatment on opioid receptor binding activities Total opioid receptor binding before and after chronic PL017 treatment was determined with 2 nM of [3H]-
DISCUSSION
0.201
L ~
u •
-PL017 +PL017 (10nM)
0.05~ 0.00 0.00
,
,
,
,
0.02
0.04
0.06
0.0a
Bound (nM) Fig. 2. Scatchard analysis of [3H]DAMGO saturation binding to midbrain membranes of control rats in the absence ([2) or presence ( I ) of 10 nM PL017, saturation binding was measured with 12 different concentrations of [3H]DAMGO, 0.05-5.0 nM. The resulting specific binding data were analysed by non-linear curve fitting program (Ligand 1). The B~,ax values were 0.066 nM (I7) and 0.064 nM ( I ) ; and the K d values were 0.37 nM (El) and 2.24 nM ( I ) , respectively. Data shown are from one representative experiment. Comparable results were obtained in two independent sets of
experiments.
diprenorphine in 3 different rat brain regions: cortex, midbrain and striatum. As shown in Table IV, a significant decrease (P ~< 0.05, Duncan multi-range test) in the amount of [3H]diprenorphine bound was found in all 3 brain regions after 5 days of PL017 administration. There was a decrease of about 25-30% binding in the midbrain and cortex, and a 20% decrease in the striatum. The question of which opioid receptor type activity was being altered by chronic PL017 treatment was determined by measuring binding of [3H]diprenorphine in the presence of morphiceptin or DPDPE as described in Methods. It was observed that only p-opioid receptor activity was attenuated by the chronic opioid peptide treatment as summarized in Table V. There was no significant change of 6-opioid receptor activity after chronic PL017 treatment. The observed decrease in p-opioid receptor binding could be due to the presence of non-washed PL017. However, we previously showed that our washing paradigm was able to remove the lipophilic alkaloid ligand etorphine from P2 membrane24- Furthermore, when Scatchard analysis was carried out with [3H]DAMGO, a 27% decrease in the Bm~ value was observed in membrane isolated from rats chronically treated with PL017, while no significant difference in K d was found (Fig. 1). Thus, the observed decrease in the opioid receptor binding activity after chronic PL017 treatment was due to a decrease of number of binding sites and not due to a decrease in the affinity of the ligand for the receptor. The latter would be expected if residual PL017 were the source of the decrease in binding (Fig. 2).
In previous studies 24"25, we demonstrated that chronic treatment of rats with either etorphine 24 or D A D L E 25 resulted in the reduction of opioid receptor binding activities during the course of tolerance development. Although p-opioid receptor binding was attenuated more obviously than 6-binding after chronic etorphine treatment, and the reverse was found for chronic DADLE treatment, the lack of selectivity of these ligandss'14'28 leaves open the possibility that down-regulation of one receptor type is mediated by interaction with another type. To rule this out, we used the highly p-receptor selective ligand PL0171,3,1s to chronically treat the animal. When rats were chronically treated with PL017, a high degree of tolerance to the antinociceptic and catatonic effects of PL017 (30- to 40-fold and about 14-fold, respectively) were observed (Tables I-III). One might argue that if the animal was catatonic due to the drug effect of PL017, the tail-flick latency is a reflection of the
274 catatonic effect of the peptide. It is true that prior to drug treatment, the ADs0 (antinociceptic effect) and EDs0 (catatonic effect) in Tables I and II are similar (1.41 + 0.10/~g/kg vs. 1.79 + 0.25/~g/kg). However, after PL017 treatment for 3 days or 5 days, the ADs0 was much higher than the EDs0 (18.5 + 2.4/~g/kg vs. 7.25 + 0.98/~g/kg and 39.1 + 2.7 ~g/kg vs. 24.2 + 1.0 /~g/kg). Thus, after chronic PL017 treatment, even if the animal was catatonic, it could still show a tail-flick response. Hence the antinociceptive measurement is not a reflection of the catatonic response to the peptide. When the animal was highly tolerant to the antinociceptic effect of PL017 after 3 days or 5 days treatment with this opioid peptide, it only showed about 10% cross-tolerance to D A D L E (Table III). This minimal effect on the putative 6-receptor effect clearly suggests that chronic effect of PL017 was mainly at the g-opioid receptor. In fact, even the small degree of D A D L E tolerance that was observed could reflect mediation of D A D L E antinociception through g-opioid receptors. The observed decrease in opioid receptor binding after 5 days of PL017 treatment could be due to the presence of unwashed PL017. If this were the case, then Scatchard analysis of the saturation binding data should in theory reveal a decrease in the affinity of the ligand for the receptor, or an increase in the K d value. This was confirmed by the experiment shown in Fig. 2, when 10 nM PL017 was added to the incubation buffer, the K d of [3H]DAMGO binding increased from 0.37 nM to 2.24 nM, but the B.~ax was not significantly altered. However, when Scatchard analysis was carried out on the saturation binding data obtained with midbrain membranes isolated from rats treated with PL017 for 5 days, a decrease only in the Bmax value was observed, the K d value was still the same (Fig. 1). Thus, the observed decrease in the opioid receptor binding activity after chronic PL017 treatment
should be due to a decrease of number of binding sites. While our data clearly showed selective down-regulation of/~-opioid receptors, as in other chronic opioid treatment studies 24,25, there was a lack of correlation between the degree and time course of receptor downregulation and the loss of the pharmacological effects of the drug. Although there was a 19-fold increase in the ADs0 value after 3 days of chronic PL017 treatment, there was no detectable down-regulation of the/~-opioid receptor in this period. Thus, while down-regulation of the receptor is one cellular adaptation process induced by chronic presence of opioid agonists as in N G cells, it is not the cause of tolerance to opioid antinociception. In conclusion, down-regulation of the /~-opioid receptor could be shown in the rats after 5 days chronic treatment of PL017 by i.c.v, route. Because of the relative selectivity of the peptide used, and the relatively small change in 6-opioid receptor activities during chronic PL017 treatment, it can be concluded that the observed decrease in the/~-opioid receptor activities was not due to the activation of the 6-opioid receptor. Conversely, we have also shown that b-opioid receptor density can be reduced by a 6-opioid receptor selective ligand (DPDPE) without any alteration in the/~-opioid receptor activities (unpublished observations). Thus, opioid receptors, at least the /t- and b-receptor types, can be selectively down-regulated by chronic treatment with sufficiently selective agonists. These data support the notion of distinct ,u- and 6-opioid receptors, and are not easily consistent with models based on interconversion or interaction of the receptor types 2'H'19.
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Acknowledgements. The present studies were supported in part by US PHS Grant DA-00564 and Grant NSC 77-0412-B016-33 from the National Science Council, Republic of China. H.H.L. is the recipient of a National Institute of Drug Abuse Career Award K-02-DA-70554.
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delta receptor selectivities, Life Sci., 33 Suppl. I (1983) 447-450. 21 Pert, C.B. and Snyder, S.H., Opiate receptor binding enhancement by opiate administration in vivo, Biochem. Pharmacol., 25 (1976) 847-853. 22 Russell, R.D., Leslie, J.B., Su, Y.-F., Watkins, W.D. and Chang, K.-J., Continuous intrathecal opioid analgesia: tolerance and cross-tolerance of mu and delta spinal opioid receptors, J. Pharmacol. Exp. Ther., 240 (1987) 150-158. 23 Simon, E.J. and Hiller, J.B., In vitro studies on opiate receptors and their ligands, Fed. Proc., 37 (1978) 141-146. 24 Tao, P.L., Law, P.Y. and Loh, H.H., Decrease in 6- and /~-opioid receptor binding capacity in rat brain after chronic etorphine treatment, J. Pharmacol. Exp. Ther., 240 (1987) 809-816. 25 Tao, P.L., Chang, L.-R., Law, P.Y. and Loh, H.H., Decrease in 6-opioid receptor density in rat brain after chronic D-AIa2, o-LeuS-enkephalin treatment, Brain Research, 462 (1988) 313320. 26 Tempel, A., Habas, J.E., Paredes, W. and Barr, G.A., Morphine-induced downregulation of /~-opioid receptors in neonatal rat brain, Brain Research, 469 (1988) 129-133. 27 Tseng, L.F., Loh, H.H. and Li, C.H., Human fl-endorphin: development of tolerance and behavioral activity in rats, Biochem. Biophys. Res. Commun., 74 (1977) 390-396. 28 Wolozin, B.L., and Pasternak, G.W., Classification of multiple morphine and enkephalin binding sites in the central nervous system, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 6181-6185.
270 BRES 15828
Decrease in
-opioid receptor binding capacity in rat brain after chronic PL017 treatment
Pao-Luh Tao t, Haw-Yen Lee 1, Li-Ren Chang I and Horace H. Loh 2 t Department of Pharmacology, National Defense Medical Center, Taipei (Taiwan) and 2Department of Pharmacology, University of Minnesota, Minneapolis, MN 55414 (U.S.A.) (Accepted 6 March 1990)
Key words: Brain opioid receptor; PL017; Receptor, p-opioid; Down regulation
In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or D-Ala2,o-LeuS-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases,/t-opioid receptor binding capacity was attenuated together with the d-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both/~ and d receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the/~-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5-20/~g/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the ADs0 of the peptide to elicit the antinociceptive response and about 14-fold increase in the EDso of the peptide to elicit the catatonic effect. When g- and d-binding was determined using [3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20-30%) in the/~-opioid receptor binding but not in d-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [3H]DAMGO saturation binding data revealed a decrease in Bmax values and no change in the Kd values. Hence,/~-opioid receptors can be specifically regulated by ligand in the brain as d-receptors are in neuroblastoma x glioma NG 108-15 cells. Chronic activation of the g-opioid receptor in the brain would result in the down-regulation of the binding sites.
INTRODUCTION Alteration in ligand-receptor interaction during chronic drug treatment has been suggested as a possible mechanism underlying opioid tolerance. In neonatal rats, Tempel et al. 26 found that morphine could produce a down regulation of brain ~t-opioid receptors during the first week of life. However, most studies investigating this issue have found that chronic morphine treatment of adult animals results in no change in brain opioid receptor levels 13'1523, or even an increase lz'21. In the clonal cell line, neuroblastoma x glioma NG108-15 hybrid cells, down-regulation of 6-0pioid receptors is observed following chronic treatment with opioid agonists 16'~7. However, chronic treatment with partial agonists, such as morphine (in this system), does not have this effect. Accordingly, we reasoned that the failure to observe down-regulation of opioid receptors in adult rat brain might have resulted from not using the appropriate agonist. Some support for this notion is also provided by studies by Dingledine et al. 6. They found that chronic treatment of hippocampal slices in vitro with D-Aia z, D-Leu 5-
enkephalin ( D A D L E ) resulted in a decrease of 6receptors, but not of/~-receptors. Incubation with the /~-agonists morphine or morphiceptin had no effect on either/~- or &receptors, although physiologic tolerance to morphiceptin was observed. Dingledine et al. concluded that down-regulation could account for tolerance to 6-agonists in this system, but not tolerance to/~-agonists. Recently, we reported decreases in both /~- and 6-0pioid receptor binding in rats chronically treated with etorphine 24. A n d in a subsequent study 25, we obtained somewhat similar results following chronic administration of D A D L E , except that chronic D A D L E treatment preferentially reduced 6-0pioid receptor binding activity. Because etorphine and D A D L E can each interact with both ~- and d-receptors 8,14,28, one could argue that the observed decrease in/t-opioid receptor number reflects activation of 6 receptors. In order to further demonstrate that ~-opioid receptors are under similar control as d-opioid receptors, we treated rats chronically with CH3Phe3,D-Pro4-morphiceptin (PL017) in the present study. This opioid peptide was chosen because of its high selectivity toward the ~-opioid receptor, and because it is more stable than morphiceptin in vivo 1'3'18.
Correspondence: E-L. Tao, Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan, R.O.C. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
271 TABLE I
TABLE II
Alteration in antinociception potency of PLO17 during chronic PLO17 treatment
Alteration in catatonia potency of PLOI7 during chronic PLOI7 treatment
The ability of PL017 to produce antinociceptive responses in rats treated with PL017 according to the paradigm outlined in text was determined by the up-down method of Dixon 7. In each group, n > 6 rats were used in the determination of the ADso values. The degree of tolerance was calculated as: ADs0 after treatment/ADso before treatment.
The ability of PL017 to produce catatonia responses in rats treated with PL017 according to the paradigm outlined in text was determined by the up-down method of Dixon 7. In each group n > 6 rats were used in the determination of the EDso values. The degree of tolerance was calculated as: EDso after treatment/EDso before treatment (0 day).
Duration
Duration
P L017 E D so (#g/kg b. wt. )
Degree of tolerance
0 days I day 3 days 5 days
1.79 + 2.40 + 7.25 + 24.2 +
1.0 1.3 4.0 13.5
PLO17 ADjo (#g/kg b. wt.)
Degree of tolerance
Before treatment After treatment 1 day 3days 5 days
1.28 + 0.17 1.53+0.12 1.41 + 0.17
2.97 + 0.4 18.5+2.4 39.1 + 2.7
2.3 12.1 27.7
0.25 0.19 0.98 1.0
g for 20 min. Excess PL017 was removed by washing the P2 membranes first with 1:100 w/v of 50 mM Tris buffer (pH 7.4) containing 100 mM NaCI, 5 p M GTP and 1 mM phenylmethyl sulfonyl fluoride; and second with the same volume of this buffer without GTP. These two washes were carried out by incubating the membrane mixtures at 37 *C for 15 rain, and then membranes were pelleted by centrifuging at 22,000 g for 20 min. The membranes were washed finally with 50 mM Tris buffer (pH 7.4) at 4 *C to remove NaC1 before binding assays. The washed membranes were stored in a freezer at -70 ° C .
MATERIALS AND METHODS
Treatment of animals with PLO17 and pharmacological tests Male Sprague-Dawley rats weighing 160-250 g were treated with PL017 via a stainless-steel cannula implanted into the left lateral cerebroventricle (i.c.v.) according to the coordinates: P 1.0 mm, L 1.25 mm, V 4-5 mm using bregma as zero. After the rats had recovered from the operation (3 days), PL017 was administered i.c.v, to determine the ADso dose by the tail-flick antinociceptive response 5 and EDso dose for catatonia effect 27 according to the up-down method as described by Dixon 7. Afterwards, the animals were chronically treated with PL017 by daily administration of the peptide at 09.00 and 17.00 h starting with a 2.5/~g/kg i.c.v, dose on day 1, followed by 5/~g/kg on day 2, 10/tg/kg on day 3, 15 gg/kg on day 4 and 20 #g/kg on day 5. After chronic PL017 treatment of 1, 3 or 5 days, the antinociceptic ADso values or catatonic EDso values of PL017 were re-determined by the up-down method. For acute PL017 treatment, rats were injected i.c.v, with 2.5 jug/kg PL017 20 min prior to sacrifice.
Opioid receptor binding assays Opioid receptor binding assays in triplicates were carded out with [3H]diprenorphine or [3H]DAMGO in the presence of 5 mM Mg 2+ and a final volume of 1.0 ml of Tris buffer at pH 7.4 for 90 rain at 24 °C, using 5 # M diprenorphine or 1/zM D A M G O for non-specific binding determinations. In the studies in which the relative/~- and 6-opioid receptor binding was determined, [3H]diprenorphine was used as radioactive ligand, 1/zM morphiceptin 4 was added to a set of triplicates, and 1 # M DPDPE 9 was added to another set of triplicates. Thus, /z-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 g M morphiceptin; and the 6-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 / t M of DPDPE. After the opioid receptor binding had reached equilibrium, incubations were terminated by collecting the membranes on Whatman GF/B filters, and the filters were washed three times with 5 ml of 10 mM "Iris buffer (pH 7.4) at 4 °C. The amount of membrane protein used in each assay was determined by the method of Lowry et al. 19.
Membrane preparations After either chronic or acute treatment with PL017, the rats were sacrificed by decapitation and the brains excised. Membranes were prepared from the brain regions of cortex, midbrain (including thalamus and hypothalamus) and striatum by homogenizing the tissues in 0.32 M sucrose buffered with 50 mM "Iris buffer (pH 7.4) in a tissue/buffer ratio of 1:10. After the removal of nuclei by the 1000 x g x 10 min centrifugation procedure, P2 membranes were pelleted by centrifuging the 1000 g for 10 min supernatants at 22,000
TABLE III
Cross-tolerance to D A D L E after chronic PLO17 treatment The ADso of PL017 or D A D L E in rats with PL017 according to the paradigm outlined in text was determined by the up-down method of Dixon 7. In each group, n > 6 rats were used in the determination of the ADs0 values. The % of cross-tolerance was determined by: (degree of tolerance to DADLE/degree of tolerance to PL017) x 100%.
Duration
0days 1 day 3 days 5 days
PL017
DA DLE
% of cross-tolerance
A D ao (Izg/kg b. wt. )
Degree of tolerance
A 1)5o (gglkg b. wt. )
Degree of tolerance
1.17 + 3.35 + 22.3 + 44.5 +
2.9 19.1 38.0
13.6 + 20.2 + 27.2 + 55.1 +
1.5 2.0 4.0
*P < 0.01; two-tailed t-test.
0.18 0.16" 2.0* 3.1"
1.1 1.8" 1.6" 6.1"
1.5/2.9 2.0/19.1 4.0/38.0
(51.7%) (10.5%) (10.5%)
272
Materials [3H]diprenorphine (41 Ci/mmol) was supplied by Amersham, [3H]DAMGO (35 Ci/mmol) was supplied by NEN, diprenorphineHC1 was obtained from the National Institute on Drug Abuse (Bethesda, MD), the opioid peptides morphiceptinTM and PL0173, DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol)1° and DPDPE (D-pen2, D-penS-enkephalin) 2° were purchased from Peninsula Labs. (Belmont, CA). All other chemicals used were reagent grade and were supplied by Sigma (St. Louis, MO). RESULTS
Development o f tolerance to chronic PL017 treatment PL017 has been shown to have antinociceptic, catatonic and physical d e p e n d e n t effects 3. Prior to investigating the possible alteration in opioid r e c e p t o r binding activity during chronic PL017 t r e a t m e n t , the ability of the present p a r a d i g m to p r o d u c e tolerance response was d e t e r m i n e d , using the antinociceptive response to the tail-flick test and c a t a t o n i a response. The ADs0 and EDs0 values of PL017 to elicit antinociception and catatonia respectively, animals which had not been t r e a t e d with PL017 were c o m p a r e d with those of the same animals which were previously t r e a t e d with increasing doses of PL017 for 1, 3 and 5 days. A s s u m m a r i z e d in Table I, the average ADs0 value of i.c.v, administered PL017 to p r o d u c e the antinociceptive response in the tail-flick test in the 3 groups of animals was 1.41 + 0.10/~g/kg. A f t e r 1 day of administration of PL017, i.e. 2 i.c.v, injections of 2 . 5 / t g / k g of PL017, the ADso increased from 1.28 + 0.17 ktg/kg to 2.97 + 0.40/~g/kg, i.e. 2.3-fold tolerance h a d been p r o d u c e d . F u r t h e r m o r e , further chronic injection of PL017 resulted in a t i m e - d e p e n d e n t increase of
TABLE IV
[~H]Diprenorphine binding to membranes from various brain regions after chronic PL017 treatment Specific binding of 2 nM [3H]diphrenorphine with washed membranes from various brain regions were carried out as described in Methods. The values represent the averages + S.E.M. of the binding data from 6 separate animals. The values in parentheses are the percentages of specific binding remained after chronic PL017 treatment as compared to that in membranes isolated from rats treated acutely with PL017.
[~H] Diprenorphine bound (fmol/mg protein)
Acute PL017 Chronic PL017 1 day 3 days 5 days
Cortex
Midbrain
Striatum
221.3 + 7.4
240.9 + 10.1
370.2+ 11.3
236.3 + 17.6 (106.8%) 218.5 + 6.7 (98.7%) 159.8 + 5.6* (72.2%)
250.4+ 10.3 (103.9%) 218.7 + 11.2 (90.8%) 181.9 + 9.5* (75.5%)
361.1+ 11.4 (97.5%) 360.3+ 14.9 (97.3%) 302.0 + 17.4" (81.6%)
*P < 0.05; Duncan multi-range test.
the ADs0 values of the o p i o i d p e p t i d e . A s shown in Table I, after 5 days of chronic t r e a t m e n t , the ADso increased from 1.41 + 0.17/~g/kg to 39.1 + 2.7/~g/kg, i.e. 27.7-fold tolerance to antinociceptive effects had b e e n p r o d u c e d . As to the catatonia response, there was also a timed e p e n d e n t increase of the EDs0 values of PL017 after chronic t r e a t m e n t , although the d e g r e e of tolerance was less than that for antinociceptive response as shown in Table II. T h e r e f o r e , under the current p a r a d i g m of multiple i.c.v. PL017 injections, animals d e v e l o p e d a high degree of tolerance to the o p i o i d peptide.
Cross-tolerance to D A D L E after chronic P L 0 1 7 treatment The selectivity of PL017 t o w a r d the p - o p i o i d r e c e p t o r was d e m o n s t r a t e d by the relative lack of cross-tolerance toward D A D L E after chronic PL017 t r e a t m e n t . A s r e p o r t e d in previous studies 25, the antinociceptive effect of D A D L E after i.c.v, administration is possibly due to the activation of the 6-opioid receptor. W h e n animals were t r e a t e d with PL017 for 3 o r 5 days and the ability
TABLE V
Relative alteration in p- and t~-opioid receptor binding after chronic PL017 treatment Specific binding of 2 nM [3H]diprenorphine to washed membranes from various brain regions of rats chronically or acutely treated with PL017 were carried out in the presence of 5 mM Mg2+ as described in Methods. In order to determine the relative contribution of/~- and 6-opioid receptor to the specific binding, [3H]diprenorphine binding was carried out in the presence of morphiceptin, a p-opioid receptor selective ligand or DPDPE, a 6-1igand. Thus, /~-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 pM morphiceptin; and the 6-opioid receptor binding was defined as the amount of [3H]diprenorphine specific binding displaceable with 1 /~M DPDPE. The values represent the averages of determinations obtained with membrane preparations from 6 different animals. The values in parentheses are the percentage of binding remained after chronic PL017 treatment as compared to that of acute PL017 treatment. Duncan multirange test was used to analyze these data.
[3H]Diprenorphine binding (fmol/mg protein)
A. p-Opioid receptor Acute PL017 Chronic PL017 1 day 3days 5days B. 6-Opioid receptor Acute PL017 Chronic PL017 1 day 3 days 5days
Cortex
Midbrain
Striatum
47.0 + 4.1
87.1 + 9.9
49.8 + 7.4
38.7 + 3.8 41.3+3.6 37.5+1.9" (79.8%)
65.6 + 6.7 86.5+5.8 59.5+8.0* (68.3%)
53.6 + 6.9 54.1+7.9 27.1+4.4" (54.4%)
61.2 + 7.1
66.3 + 10.7
69.3 + 7.1
57.5 + 7.6 58.0 + 5.3 54.2+9.3
60.3 + 10.7 51.4 + 9.6 67.1+8.5
69.5+6.9
273 Mldbrain 0.14 "
a
, ~ 0.12 -
control
0.10 g.
m
0.08 0.06 0.04 0.02 0.00 0.00 0.01
0.02 0.03 0.04 0.05 0.06 0.07 Bound (nM)
Fig. 1. Scatchard analysis of [3H]DAMGO saturation binding to midbrain membranes of control rats (i.e. acute PL017 treated rats) ([~) or rats treated with PL017 for 5 days (m). Saturation binding was measured with 10 different concentrations of [3H]DAMGO, 0.05-5.0 nM. The resulting specific binding data were analysed by a non-linear curve fitting program (Ligand 1). The Bmax values were 129.5 fmol/mg (0.067 nM) (D) and 94.3 fmol/mg (0.056 nM) (m); and the K d values were 0.48 nM (17) and 0.45 nM (m), respectively. Data shown are from one representative experiment. Comparable results were obtained in two independent sets of experiments.
of D A D L E to elicit antinociceptiion was determined, a 2to 4-fold increase in the ADs0 values of DADLE was observed, corresponding to only 10% cross-tolerance between D A D L E and PL017 (Table III).
Effect of chronic PLO17 treatment on opioid receptor binding activities Total opioid receptor binding before and after chronic PL017 treatment was determined with 2 nM of [3H]-
DISCUSSION
0.201
L ~
u •
-PL017 +PL017 (10nM)
0.05~ 0.00 0.00
,
,
,
,
0.02
0.04
0.06
0.0a
Bound (nM) Fig. 2. Scatchard analysis of [3H]DAMGO saturation binding to midbrain membranes of control rats in the absence ([2) or presence ( I ) of 10 nM PL017, saturation binding was measured with 12 different concentrations of [3H]DAMGO, 0.05-5.0 nM. The resulting specific binding data were analysed by non-linear curve fitting program (Ligand 1). The B~,ax values were 0.066 nM (I7) and 0.064 nM ( I ) ; and the K d values were 0.37 nM (El) and 2.24 nM ( I ) , respectively. Data shown are from one representative experiment. Comparable results were obtained in two independent sets of
experiments.
diprenorphine in 3 different rat brain regions: cortex, midbrain and striatum. As shown in Table IV, a significant decrease (P ~< 0.05, Duncan multi-range test) in the amount of [3H]diprenorphine bound was found in all 3 brain regions after 5 days of PL017 administration. There was a decrease of about 25-30% binding in the midbrain and cortex, and a 20% decrease in the striatum. The question of which opioid receptor type activity was being altered by chronic PL017 treatment was determined by measuring binding of [3H]diprenorphine in the presence of morphiceptin or DPDPE as described in Methods. It was observed that only p-opioid receptor activity was attenuated by the chronic opioid peptide treatment as summarized in Table V. There was no significant change of 6-opioid receptor activity after chronic PL017 treatment. The observed decrease in p-opioid receptor binding could be due to the presence of non-washed PL017. However, we previously showed that our washing paradigm was able to remove the lipophilic alkaloid ligand etorphine from P2 membrane24- Furthermore, when Scatchard analysis was carried out with [3H]DAMGO, a 27% decrease in the Bm~ value was observed in membrane isolated from rats chronically treated with PL017, while no significant difference in K d was found (Fig. 1). Thus, the observed decrease in the opioid receptor binding activity after chronic PL017 treatment was due to a decrease of number of binding sites and not due to a decrease in the affinity of the ligand for the receptor. The latter would be expected if residual PL017 were the source of the decrease in binding (Fig. 2).
In previous studies 24"25, we demonstrated that chronic treatment of rats with either etorphine 24 or D A D L E 25 resulted in the reduction of opioid receptor binding activities during the course of tolerance development. Although p-opioid receptor binding was attenuated more obviously than 6-binding after chronic etorphine treatment, and the reverse was found for chronic DADLE treatment, the lack of selectivity of these ligandss'14'28 leaves open the possibility that down-regulation of one receptor type is mediated by interaction with another type. To rule this out, we used the highly p-receptor selective ligand PL0171,3,1s to chronically treat the animal. When rats were chronically treated with PL017, a high degree of tolerance to the antinociceptic and catatonic effects of PL017 (30- to 40-fold and about 14-fold, respectively) were observed (Tables I-III). One might argue that if the animal was catatonic due to the drug effect of PL017, the tail-flick latency is a reflection of the
274 catatonic effect of the peptide. It is true that prior to drug treatment, the ADs0 (antinociceptic effect) and EDs0 (catatonic effect) in Tables I and II are similar (1.41 + 0.10/~g/kg vs. 1.79 + 0.25/~g/kg). However, after PL017 treatment for 3 days or 5 days, the ADs0 was much higher than the EDs0 (18.5 + 2.4/~g/kg vs. 7.25 + 0.98/~g/kg and 39.1 + 2.7 ~g/kg vs. 24.2 + 1.0 /~g/kg). Thus, after chronic PL017 treatment, even if the animal was catatonic, it could still show a tail-flick response. Hence the antinociceptive measurement is not a reflection of the catatonic response to the peptide. When the animal was highly tolerant to the antinociceptic effect of PL017 after 3 days or 5 days treatment with this opioid peptide, it only showed about 10% cross-tolerance to D A D L E (Table III). This minimal effect on the putative 6-receptor effect clearly suggests that chronic effect of PL017 was mainly at the g-opioid receptor. In fact, even the small degree of D A D L E tolerance that was observed could reflect mediation of D A D L E antinociception through g-opioid receptors. The observed decrease in opioid receptor binding after 5 days of PL017 treatment could be due to the presence of unwashed PL017. If this were the case, then Scatchard analysis of the saturation binding data should in theory reveal a decrease in the affinity of the ligand for the receptor, or an increase in the K d value. This was confirmed by the experiment shown in Fig. 2, when 10 nM PL017 was added to the incubation buffer, the K d of [3H]DAMGO binding increased from 0.37 nM to 2.24 nM, but the B.~ax was not significantly altered. However, when Scatchard analysis was carried out on the saturation binding data obtained with midbrain membranes isolated from rats treated with PL017 for 5 days, a decrease only in the Bmax value was observed, the K d value was still the same (Fig. 1). Thus, the observed decrease in the opioid receptor binding activity after chronic PL017 treatment
should be due to a decrease of number of binding sites. While our data clearly showed selective down-regulation of/~-opioid receptors, as in other chronic opioid treatment studies 24,25, there was a lack of correlation between the degree and time course of receptor downregulation and the loss of the pharmacological effects of the drug. Although there was a 19-fold increase in the ADs0 value after 3 days of chronic PL017 treatment, there was no detectable down-regulation of the/~-opioid receptor in this period. Thus, while down-regulation of the receptor is one cellular adaptation process induced by chronic presence of opioid agonists as in N G cells, it is not the cause of tolerance to opioid antinociception. In conclusion, down-regulation of the /~-opioid receptor could be shown in the rats after 5 days chronic treatment of PL017 by i.c.v, route. Because of the relative selectivity of the peptide used, and the relatively small change in 6-opioid receptor activities during chronic PL017 treatment, it can be concluded that the observed decrease in the/~-opioid receptor activities was not due to the activation of the 6-opioid receptor. Conversely, we have also shown that b-opioid receptor density can be reduced by a 6-opioid receptor selective ligand (DPDPE) without any alteration in the/~-opioid receptor activities (unpublished observations). Thus, opioid receptors, at least the /t- and b-receptor types, can be selectively down-regulated by chronic treatment with sufficiently selective agonists. These data support the notion of distinct ,u- and 6-opioid receptors, and are not easily consistent with models based on interconversion or interaction of the receptor types 2'H'19.
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Acknowledgements. The present studies were supported in part by US PHS Grant DA-00564 and Grant NSC 77-0412-B016-33 from the National Science Council, Republic of China. H.H.L. is the recipient of a National Institute of Drug Abuse Career Award K-02-DA-70554.
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