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The Effect of μ-Opioid Receptor Antisense on Morphine Potency and Antagonist-Induced Supersensitivity and Receptor Upregulation
Brain Research Bulletin, Vol. 42, No. 6, pp. 479–484, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(96)00375-9
The Effect of m-Opioid Receptor Antisense on Morphine Potency and Antagonist-Induced Supersensitivity and Receptor Upregulation SUKRUT SHAH, ALOKESH DUTTAROY, BILLY T. CHEN, JOANNE CARROLL AND BYRON C. YOBURN 1 Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, 8000 Utopia Parkway, St. John’s University, Queens, NY 11439 [Received 20 June 1996; Revised 11 September 1996; Accepted 23 September 1996] ABSTRACT: The present study examined the effect of in vivo antisense oligodeoxynucleotide treatment on naltrexone (NTX)induced functional supersensitivity and m-opioid receptor upregulation in mice. On day 1 mice were implanted SC with a NTX or placebo pellet and injected IT and ICV with dH2O or oligodeoxynucleotides. The oligodeoxynucleotides were designed so that they were either perfectly complementary to the first 18 bases of the coding region of mouse m-opioid receptor mRNA, or had one (Mismatch-1) or four (Mismatch-4) mismatches. On days 3, 5, 7, and 9, mice were again injected IT and ICV with dH2O or one of the oligodeoxynucleotides. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia or sacrificed for saturation binding studies ([ 3H]DAMGO). Naltrexone increased the analgesic potency of morphine in dH2O treated mice by É 70%. In binding studies, NTX significantly increased density of brain (É 60%) and spinal cord (É 140%) m-opioid receptors without affecting affinity. The m-opioid antisense and the oligodeoxynucleotide with one mismatch (Mismatch-1) significantly reduced the potency of morphine by É twofold in placebotreated mice. The oligodeoxynucleotide with four mismatches (Mismatch-4) did not significantly alter morphine potency. When placebo-treated mice were treated with either the antisense to the mouse m-opioid receptor, Mismatch-4 or Mismatch-1 there were no significant changes in the density of m-opioid receptors. Thus, m-opioid antisense significantly reduced morphine potency without changing m-opioid receptor density. When NTX and oligodeoxynucleotide treatments were combined, there was no change in NTX-induced supersensitivity and m-opioid receptor upregulation. These data suggest that opioid antagonist-induced supersensitivity and upregulation of m-opioid receptors does not involve changes in gene expression. Q 1997 Elsevier Science Inc.
in adaptive responses to drug treatment. Several investigators have examined the effect of antisense treatment on a variety of opioid receptor mediated effects both in vivo and in vitro. Treatment with antisense oligodeoxynucleotides to d [ 2,3,6,7,16,17,23,25,26 ], m [ 9,21,22 ] , and k opioid receptors [1,10 ] has been reported to reduce the potency of agonists acting at each receptor site. It is presumed that antisense treatment reduces the translation of mRNA to protein and that there is subsequent decrease in the protein coded for by the message. This decrease in receptors might explain the loss of agonist potency following antisense treatment. Consistent with this possibility, modest decreases in d opioid receptor density following antisense treatment have been reported [ 3,7,23 ] . Similar decreases in receptor density have been observed in other neurotransmitter systems following antisense [ 28,29 ] . Antisense treatment can be used to evaluate if changes in gene expression are involved in regulation of opioid receptor density. A standard model of opioid receptor regulation is antagonist-induced receptor upregulation and functional supersensitivity ( e.g., [18,24,31,33 – 35 ) . If antagonist ( e.g., naltrexone — NTX ) treatment acts by increasing gene expression and, hence, mRNA, then it might be expected that antisense treatment would interfere with supersensitivity and receptor upregulation. The present study examined the effect of mopioid receptor antisense treatment on naltrexone-induced supersensitivity to the analgesic potency of morphine and mopioid receptor upregulation. Furthermore, the effect of varying the number of nucleotide mismatches on morphine potency was also determined.
KEY WORDS: Antisense, Oligodeoxynucleotide, Upregulation, Supersensitivity, Opioid analgesia, m-Opioid receptor, Naltrexone, Morphine, Mismatch, Tail flick.
METHOD Subjects Male, Swiss–Webster mice (22–24 g) obtained from Taconic Farms (Germantown, NY) were used throughout. The animals were maintained 5–10 per cage with free access to food and water and housed for at least 24 h prior to experimentation. Mice were used only once.
INTRODUCTION Cloning of the opioid receptors has allowed the development of unique approaches to examining the role of gene expression 1
To whom requests for reprints should be addressed.
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SHAH ET AL. TABLE 1 THE EFFECT OF m-OPIOID ANTISENSE OLIGODEOXYNUCLEOTIDE ADMINISTRATION ON NTXINDUCED SUPERSENSITIVITY TO MORPHINE
Treatment
Placebo-dH2O Placebo-Mismatch-4 NTX-dH2O Placebo-Antisense NTX-Antisense
Morphine ED50 (95% CL)
3.19 2.50 1.87 5.91 2.01
Potency (Relative to Placebo-dH2O)
Potency (Relative to Placebo-Antisense)
1.00 1.28 1.71* 0.54* 1.59*
— — — 1.00 2.94†
(2.64–3.84) (2.03–3.04) (1.50–2.32) (4.97–7.03) (1.62–2.48)
On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV with dH2O, Antisense or Mismatch-4 (IT Å 10 mg; ICV Å 20 mg). On days 3, 5, 7, and 9 mice were again injected ICV and IT with the same drugs. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia using the tailflick and a cumulative dose– response protocol. The results presented are the combined data from three independent experiments (n Å 6– 8/group for each experiment). * p õ 0.05 compared to placebo-dH2O; † p õ 0.01 compared to placebo-antisense.
Oligodeoxynucleotide Synthesis and Administration An antisense oligodeoxynucleotide ( 5 *-GCC GGC GCT GCT GTC CAT-3 * ) complimentary to the first 18 nucleotides of the coding region of the mouse m-opioid receptor [19 ] was synthesized. A mismatch ( Mismatch-4 ) oligodeoxynucleotide was also constructed ( 5 *-GCC GGC CGT GCT GCT CAT3 * ) in which the nucleotides at positions 7 and 8 and 14 and 15 were reversed. We also tested an antisense oligodeoxynucleotide ( 5 *-GCC GGT GCT GCT GTC CAT-3 * ) complimentary to the first 18 nucleotides of the coding region of the rat m-opioid receptor [ 9 ] . This rat oligodeoxynucleotide ( Mismatch-1 ) has one mismatch for the mouse m-opioid receptor
mRNA nucleotide at the 6th position. Oligodeoxynucleotides were reconstituted in sterile dH2O at a final concentration of 5 mg / ml. Procedure Placebo and NTX pellets were implanted and removed from the nape of the neck while the mice were lightly anesthesized with halothane:oxygen ( 4:96 ) . Intrathecal ( IT; 2 ml ) and intracerebroventricular ( ICV; 4 ml ) injections were made as described previously [14,32 ] . On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV with dH2O or oligodeoxynucleotides ( IT
FIG. 1. The effect of m-opioid antisense and mismatch administration on morphine analgesia and NTX-induced supersensitivity. On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV (IT Å 10 mg; ICV Å 20 mg) with dH2O, Antisense (left panel), Mismatch-4 (left panel) or Mismatch-1 (right panel). On days 3, 5, 7, and 9 mice were again injected ICV and IT. After the final injections on day 9, placebo and NTX pellets were removed. Twenty-four hours later mice were tested for morphine analgesia using the tailflick assay. Each panel is the combined results from two to three independent experiments (n Å 6–8/ group for each experiment), each with its own control group (PlacebodH2O).
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TABLE 2 THE EFFECT OF m-OPIOID ANTISENSE (MISMATCH-1) OLIGODEOXYNUCLEOTIDE ADMINISTRATION ON NTX-INDUCED SUPERSENSITIVITY TO MORPHINE
Treatment
Placebo-dH2O NTX-dH2O Placebo-Mismatch-1 NTX-Mismatch-1
Morphine ED50 (95% CL)
2.66 1.69 5.24 1.92
Potency (Relative to Placebo-dH2O)
Potency (Relative to Placebo-Antisense)
1.00 1.57* 0.51† 1.39
— — 1.00 2.73‡
(2.09–3.36) (1.29–2.20) (4.23–6.40) (1.45–2.45)
On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV with dH2O or Mismatch-1 (IT Å 10 mg; ICV Å 20 mg). On days 3, 5, 7, and 9 mice were again injected ICV and IT with the same drugs. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia using the tailflick and a cumulative dose–response protocol. The results presented are the combined data from 2 independent experiments (n Å 6–8/group for each experiment). * p õ 0.05 and † p õ 0.01 compared to Placebo-dH2O; ‡ p õ 0.01 compared to Placebo-Antisense.
Å 10 mg, ICV Å 20 mg ) ( n Å 6 – 8 / group ) . On days 3, 5, 7, and 9 mice were again injected ICV and IT with dH2O or oligodeoxynucleotides. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were sacrificed, and brain and spinal cord removed for binding studies. Other mice were weighed and then tested in morphine analgesia dose – response studies ( see below ) .
Analgesia Assay Analgesia (antinociception) was determined using the tailflick assay in which a beam of light was focused on the dorsal tail surface approximately 2 cm from the tip of the tail. The intensity of the light was adjusted so that baseline flick latencies determined prior to morphine administration were 2–4 s. If a mouse failed to flick by 10 s following morphine administration, the test was terminated and a latency of 10 s was recorded. Mice that had a latency of 10 s were defined as analgesic. Mice were tested for analgesia 30 min following morphine. All testing was conducted in a blind manner.
FIG. 2. The effect NTX and oligodeoxynucleotide treatment on m-opioid receptor density in mouse brain. On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV (IT Å 10 mg; ICV Å 20 mg) with dH2O, Antisense (A sense), Mismatch-1 (Mis 1) or Mismatch-4 (Mis 4). On days 3, 5, 7, and 9 mice were again injected ICV and IT. After the final injections on day 9, placebo and NTX pellets were removed. Twenty-four hours later mice were sacrificed and whole brain was removed and saturation binding experiments were conducted using [ 3H] DAMGO. Data are means ( { SEM) from two to four experiments for each treatment condition. *Indicates significantly different from Placebo-dH2O ( p õ 0.05).
Cumulative Dose–Response Protocol A cumulative dose–response protocol was used for all studies. All mice in a treatment group (6–8/group) were injected SC with a starting dose of morphine (1 mg/kg) and tested for analgesia 30 min later. Mice that were not analgesic were given a second dose (increment dose) of morphine within 5 min of testing and then tested for analgesia again 30 min later. This cumulative dose–response protocol was continued until at least 75% of the mice were analgesic. The starting and increment doses used were the same for control and treated mice in each study. The actual morphine doses used in the cumulative dose– response protocol were determined previously [12]. Data from cumulative dosing is presented such that the percent of mice that are analgesic is plotted against the total (cumulative) dose administered. Opioid Receptor Binding Mice were sacrificed (n Å 2/group) and whole brain and spinal cord rapidly removed, weighed, and then homogenized in 80 vol of ice-cold 50 mM Tris buffer (pH 7.4). Homogenates were then centrifuged at 15,000 rpm for 15 min, the supernatant discarded and the pellet resuspended in buffer and centrifuged again, and then frozen at 0807C until analysis. The pellets were thawed, resuspended in Tris buffer, incubated (30 min at 257C), centrifuged, and finally resuspended in 20–80 vol of 50 mM phosphate buffer (pH 7.2). An aliquot (200 ml) of the homogenate was then assayed in triplicate in tubes containing 0.04–5.0 nM [ 3H] [D-Ala 2-MePhe 4-Gly(ol 5 )] enkephalin (DAMGO). Nonspecific binding was determined in the presence of 1000 nM levorphanol. Brain and cord homogenates were incubated for 90 min at 257C. Incubation was terminated by the addition of icecold phosphate buffer and the samples were filtered over GF/B glass fiber filters. Filters were washed three times with cold buffer, transferred to vials, scintillation cocktail added, and then counted using liquid scintillation spectroscopy. Counts per minute (CPMs) were converted to disintegrations per minute (DPMs) using the external standard method. Protein was determined using a microassay technique based on the method of Bradford [4] using reagent purchased from BIO-RAD (Richmond, CA). Binding experiments were conducted two to four times for each treatment condition. Drugs All oligodeoxynucleotides were purchased form Midland Certified Reagents (Midland, TX). Morphine sulfate was ob-
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FIG. 3. The effect NTX and oligodeoxynucleotide treatment on m-opioid receptor density in mouse spinal cord. On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV (IT Å 10 mg; ICV Å 20 mg) with dH2O, Antisense (A sense) or Mismatch-4 (Mis 4). On days 3, 5, 7, and 9 mice were again injected ICV and IT. After the final injections on day 9, placebo and NTX pellets were removed. Twenty-four hours later mice were sacrificed and spinal cord was removed and saturation binding experiments were conducted using [ 3H] DAMGO. Data are means ( { SEM) from three experiments for each treatment condition. *Indicates significantly different from PlacebodH2O (p õ 0.05).
tained from Penick Laboratories (Newark, NJ). Placebo and NTX pellets were obtained from Research Triangle Institute (Research Triangle Park, NC) through the Research Technology Branch of the National Institute on Drug Abuse. NTX and placebo pellets were wrapped in nylon mesh before SC implantation. Data Analysis Quantal dose–response data were analyzed by Probit Analysis [13] using a computerized program (BLISS 21, Department of Statistics, University of Edinburgh) that estimates ED50s, 95% confidence limits, relative potency and determines significant differences between ED50s. Bmaxs and Kd s were determined from saturation studies using nonlinear regression (Prism ver 1.03, Graphpad Software, San Diego, CA). In all cases binding data were fit best by a one-site model. Significant differences for binding studies were based on ANOVA and post hoc tests. RESULTS NTX increased the analgesic potency of morphine in dH2Otreated mice by É70% ( Table 1, Fig. 1, left panel ) compared to the placebo-dH2O group. The potency of morphine was significantly reduced by É twofold in the placebo-antisense group relative to the placebo-dH2O group. The ED50 for the placebo-mismatch-4 group did not differ from control ( placebo-dH2O ) . Thus, NTX increased and antisense decreased the analgesic potency of morphine. When NTX and antisense treatments were combined, the supersensitivity effect was still present relative to the placebo-antisense and the placebodH2O groups. There was no significant difference between the NTX-dH2O and NTX-antisense groups. The effect of an oligodeoxynucleotide with one mismatch on morphine potency and NTX-induced supersensitivity was also determined (Table 2, Fig 1, right panel). An oligodeoxynucleotide with one mismatch effectively reduced the potency of morphine by É twofold. As previously (Table 1), NTX significantly increased the analgesic potency of morphine. When NTX and mismatch-1 treatments were combined, the supersensitivity effect was present relative to the appropriate control group (pla-
cebo-mismatch-1). However, supersensitivity to morphine for the NTX-mismatch-1 group approached (p õ 0.10), but did not reach, statistical significance compared to the placebo-dH2O group. There was no significant difference between the NTXdH2O and NTX-mismatch-1 groups. Binding experiments (Figs. 2 and 3) indicated that NTX significantly increased the density of brain, F (6, 11) Å 11.3, p õ 0.01, and spinal cord, F (4, 10) Å 4.9, p õ 0.02, m-opioid receptors without affecting affinity (Kd range in brain and cord, respectively Å 0.56–0.67 nM, 0.75–0.96 nM; Fs ° 1.00, p ú 0.05). NTX alone increased receptor density by É60% in brain and É140% in cord. When mice were treated with either the antisense to the mouse m-opioid receptor, mismatch-4 or mismatch-1 (binding studies in brain only) there were no significant changes in the density of receptors (p ú 0.05). Similarly, when NTX treatment was combined with antisense administration (brain and cord) or mismatch-1 (brain) there was no significant change in NTX-induced upregulation. We did not examine the effect of mismatch-4 on NTX-induced upregulation in brain or spinal cord since this oligodeoxynucleotide had no effect on morphine’s potency in analgesia studies (see Table 1). DISCUSSION The present results show that in vivo treatment with an oligodeoxynucleotide complementary to a portion of the mRNA for the mouse m-opioid receptor can reduce the analgesic potency of morphine. Similarly, an antisense complementary to the first 18 nucleotides of the rat m-opioid receptor (mismatch-1), with a single nucleotide mismatch for the murine m receptor, also significantly reduced morphine potency. However, a oligodeoxynucleotide with four noncomplementary nucleotides (mismatch-4) did not alter the analgesic potency of morphine. While one mismatch in an oligodeoxynucleotide can alter affinity for an mRNA, the location of the mismatch and the actual nucleotide substituted are also important considerations [11]. Our results that an oligodeoxynucleotide with one mismatch (mismatch-1) is equally effective to an oligodeoxynucleotide with no mismatches indicate that the single mismatch is benign; a finding that may reflect the similarities between mouse and rat m-opioid receptor mRNAs [9,19]. On the other hand, an oligodeooxynucleotide with four mismatches was ineffective in reducing morphine’s potency. Consistent with prior studies, NTX produced significant functional supersensitivity to morphine and significant increases in m-opioid receptor density as determined in whole brain and spinal cord homogenates [18,31,33,34,35). Thus, the binding assay, as well as the functional assay, are clearly responsive to treatments that regulate opioid receptors. However, we found no effect of antisense treatment on brain or spinal cord m receptors, although antisense treatment did reduce the analgesic potency of morphine by approximately twofold. Although other investigators have reported changes in d-opioid receptors following in vivo antisense using autoradiography [6] and homogenate binding assays [3,7,23], to our knowledge, decreases in m binding have not been reported following in vivo treatment. The failure to observe an effect of antisense on binding cannot be due to the ineffectiveness of this treatment, because morphine potency was reduced by antisense treatment. Furthermore, the lack of effect on binding is not due to the insensitivity of the binding assay, because increases in receptors were observed following NTX treatment. It is not readily apparent why there is a discrepancy between the binding and functional assays, but in other neurotransmitter systems there is precedent for changes in function with very modest changes in protein following antisense. For example, Weiss and
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colleagues found that antisense oligodeoxynucleotides directed at the D2 dopamine receptor almost completely blocked the behavioral effects of the D2 agonist quinpirole while D2 receptor binding was decreased by only É15–25% [30]. Weiss and colleagues have attributed this discrepancy to the possible selective action of antisense on a very small pool of functional receptors that are responsible for the majority of behavioral, but not binding, changes [20]. Perhaps a similar state of affairs exists for the murine m-opioid receptor in that binding studies detect both functional and nonfunctional receptors and antisense only inhibits production of a small pool of functional receptors. Similarly, better correlation between binding and function following antisense treatment may be observed if select brain regions were examined, although the increases in density in whole brain and cord following NTX suggests that the current assay techniques can readily detect changes in binding. When antisense and NTX treatment were combined, antisense did not block NTX-induced supersensitivity or receptor upregulation. This finding agrees with previous reports that reversible (which reliably increases receptor density) and irreversible in vivo antagonist treatment (which occasions an increase in receptors following depletion), do not act to increase m-opioid receptor gene expression as determined by quantitation of mRNA [5,8,15,27]. Thus, antagonist-induced increases in m-opioid receptor density may not depend upon de novo synthesis of receptors and may reflect changes in the rate of posttranslational processing such as activation of an existing pool of ‘‘cryptic’’ receptors. It is possible, however, that with different dosing protocols for antisense (e.g., more frequent, higher dose) an effect on NTX-induced supersensitivity and upregulation may have be observed. In summary, an antisense oligodeoxynucleotide to the mouse m-opioid receptor reduced morphine potency without changing the density of m-opioid receptors. Furthermore, antisense treatment did not block opioid antagonist-induced supersensitivity or upregulation. These data suggest that increases in opioid receptors following antagonist treatment do not depend upon increases in gene expression. ACKNOWLEDGEMENTS
This research was supported in part by National Institute on Drug Abuse (NIDA) Grants DA 04185. Dr. M. T. Turnock did not provide much technical help, but nothing happens without her.
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The Effect of m-Opioid Receptor Antisense on Morphine Potency and Antagonist-Induced Supersensitivity and Receptor Upregulation SUKRUT SHAH, ALOKESH DUTTAROY, BILLY T. CHEN, JOANNE CARROLL AND BYRON C. YOBURN 1 Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, 8000 Utopia Parkway, St. John’s University, Queens, NY 11439 [Received 20 June 1996; Revised 11 September 1996; Accepted 23 September 1996] ABSTRACT: The present study examined the effect of in vivo antisense oligodeoxynucleotide treatment on naltrexone (NTX)induced functional supersensitivity and m-opioid receptor upregulation in mice. On day 1 mice were implanted SC with a NTX or placebo pellet and injected IT and ICV with dH2O or oligodeoxynucleotides. The oligodeoxynucleotides were designed so that they were either perfectly complementary to the first 18 bases of the coding region of mouse m-opioid receptor mRNA, or had one (Mismatch-1) or four (Mismatch-4) mismatches. On days 3, 5, 7, and 9, mice were again injected IT and ICV with dH2O or one of the oligodeoxynucleotides. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia or sacrificed for saturation binding studies ([ 3H]DAMGO). Naltrexone increased the analgesic potency of morphine in dH2O treated mice by É 70%. In binding studies, NTX significantly increased density of brain (É 60%) and spinal cord (É 140%) m-opioid receptors without affecting affinity. The m-opioid antisense and the oligodeoxynucleotide with one mismatch (Mismatch-1) significantly reduced the potency of morphine by É twofold in placebotreated mice. The oligodeoxynucleotide with four mismatches (Mismatch-4) did not significantly alter morphine potency. When placebo-treated mice were treated with either the antisense to the mouse m-opioid receptor, Mismatch-4 or Mismatch-1 there were no significant changes in the density of m-opioid receptors. Thus, m-opioid antisense significantly reduced morphine potency without changing m-opioid receptor density. When NTX and oligodeoxynucleotide treatments were combined, there was no change in NTX-induced supersensitivity and m-opioid receptor upregulation. These data suggest that opioid antagonist-induced supersensitivity and upregulation of m-opioid receptors does not involve changes in gene expression. Q 1997 Elsevier Science Inc.
in adaptive responses to drug treatment. Several investigators have examined the effect of antisense treatment on a variety of opioid receptor mediated effects both in vivo and in vitro. Treatment with antisense oligodeoxynucleotides to d [ 2,3,6,7,16,17,23,25,26 ], m [ 9,21,22 ] , and k opioid receptors [1,10 ] has been reported to reduce the potency of agonists acting at each receptor site. It is presumed that antisense treatment reduces the translation of mRNA to protein and that there is subsequent decrease in the protein coded for by the message. This decrease in receptors might explain the loss of agonist potency following antisense treatment. Consistent with this possibility, modest decreases in d opioid receptor density following antisense treatment have been reported [ 3,7,23 ] . Similar decreases in receptor density have been observed in other neurotransmitter systems following antisense [ 28,29 ] . Antisense treatment can be used to evaluate if changes in gene expression are involved in regulation of opioid receptor density. A standard model of opioid receptor regulation is antagonist-induced receptor upregulation and functional supersensitivity ( e.g., [18,24,31,33 – 35 ) . If antagonist ( e.g., naltrexone — NTX ) treatment acts by increasing gene expression and, hence, mRNA, then it might be expected that antisense treatment would interfere with supersensitivity and receptor upregulation. The present study examined the effect of mopioid receptor antisense treatment on naltrexone-induced supersensitivity to the analgesic potency of morphine and mopioid receptor upregulation. Furthermore, the effect of varying the number of nucleotide mismatches on morphine potency was also determined.
KEY WORDS: Antisense, Oligodeoxynucleotide, Upregulation, Supersensitivity, Opioid analgesia, m-Opioid receptor, Naltrexone, Morphine, Mismatch, Tail flick.
METHOD Subjects Male, Swiss–Webster mice (22–24 g) obtained from Taconic Farms (Germantown, NY) were used throughout. The animals were maintained 5–10 per cage with free access to food and water and housed for at least 24 h prior to experimentation. Mice were used only once.
INTRODUCTION Cloning of the opioid receptors has allowed the development of unique approaches to examining the role of gene expression 1
To whom requests for reprints should be addressed.
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SHAH ET AL. TABLE 1 THE EFFECT OF m-OPIOID ANTISENSE OLIGODEOXYNUCLEOTIDE ADMINISTRATION ON NTXINDUCED SUPERSENSITIVITY TO MORPHINE
Treatment
Placebo-dH2O Placebo-Mismatch-4 NTX-dH2O Placebo-Antisense NTX-Antisense
Morphine ED50 (95% CL)
3.19 2.50 1.87 5.91 2.01
Potency (Relative to Placebo-dH2O)
Potency (Relative to Placebo-Antisense)
1.00 1.28 1.71* 0.54* 1.59*
— — — 1.00 2.94†
(2.64–3.84) (2.03–3.04) (1.50–2.32) (4.97–7.03) (1.62–2.48)
On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV with dH2O, Antisense or Mismatch-4 (IT Å 10 mg; ICV Å 20 mg). On days 3, 5, 7, and 9 mice were again injected ICV and IT with the same drugs. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia using the tailflick and a cumulative dose– response protocol. The results presented are the combined data from three independent experiments (n Å 6– 8/group for each experiment). * p õ 0.05 compared to placebo-dH2O; † p õ 0.01 compared to placebo-antisense.
Oligodeoxynucleotide Synthesis and Administration An antisense oligodeoxynucleotide ( 5 *-GCC GGC GCT GCT GTC CAT-3 * ) complimentary to the first 18 nucleotides of the coding region of the mouse m-opioid receptor [19 ] was synthesized. A mismatch ( Mismatch-4 ) oligodeoxynucleotide was also constructed ( 5 *-GCC GGC CGT GCT GCT CAT3 * ) in which the nucleotides at positions 7 and 8 and 14 and 15 were reversed. We also tested an antisense oligodeoxynucleotide ( 5 *-GCC GGT GCT GCT GTC CAT-3 * ) complimentary to the first 18 nucleotides of the coding region of the rat m-opioid receptor [ 9 ] . This rat oligodeoxynucleotide ( Mismatch-1 ) has one mismatch for the mouse m-opioid receptor
mRNA nucleotide at the 6th position. Oligodeoxynucleotides were reconstituted in sterile dH2O at a final concentration of 5 mg / ml. Procedure Placebo and NTX pellets were implanted and removed from the nape of the neck while the mice were lightly anesthesized with halothane:oxygen ( 4:96 ) . Intrathecal ( IT; 2 ml ) and intracerebroventricular ( ICV; 4 ml ) injections were made as described previously [14,32 ] . On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV with dH2O or oligodeoxynucleotides ( IT
FIG. 1. The effect of m-opioid antisense and mismatch administration on morphine analgesia and NTX-induced supersensitivity. On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV (IT Å 10 mg; ICV Å 20 mg) with dH2O, Antisense (left panel), Mismatch-4 (left panel) or Mismatch-1 (right panel). On days 3, 5, 7, and 9 mice were again injected ICV and IT. After the final injections on day 9, placebo and NTX pellets were removed. Twenty-four hours later mice were tested for morphine analgesia using the tailflick assay. Each panel is the combined results from two to three independent experiments (n Å 6–8/ group for each experiment), each with its own control group (PlacebodH2O).
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TABLE 2 THE EFFECT OF m-OPIOID ANTISENSE (MISMATCH-1) OLIGODEOXYNUCLEOTIDE ADMINISTRATION ON NTX-INDUCED SUPERSENSITIVITY TO MORPHINE
Treatment
Placebo-dH2O NTX-dH2O Placebo-Mismatch-1 NTX-Mismatch-1
Morphine ED50 (95% CL)
2.66 1.69 5.24 1.92
Potency (Relative to Placebo-dH2O)
Potency (Relative to Placebo-Antisense)
1.00 1.57* 0.51† 1.39
— — 1.00 2.73‡
(2.09–3.36) (1.29–2.20) (4.23–6.40) (1.45–2.45)
On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV with dH2O or Mismatch-1 (IT Å 10 mg; ICV Å 20 mg). On days 3, 5, 7, and 9 mice were again injected ICV and IT with the same drugs. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia using the tailflick and a cumulative dose–response protocol. The results presented are the combined data from 2 independent experiments (n Å 6–8/group for each experiment). * p õ 0.05 and † p õ 0.01 compared to Placebo-dH2O; ‡ p õ 0.01 compared to Placebo-Antisense.
Å 10 mg, ICV Å 20 mg ) ( n Å 6 – 8 / group ) . On days 3, 5, 7, and 9 mice were again injected ICV and IT with dH2O or oligodeoxynucleotides. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were sacrificed, and brain and spinal cord removed for binding studies. Other mice were weighed and then tested in morphine analgesia dose – response studies ( see below ) .
Analgesia Assay Analgesia (antinociception) was determined using the tailflick assay in which a beam of light was focused on the dorsal tail surface approximately 2 cm from the tip of the tail. The intensity of the light was adjusted so that baseline flick latencies determined prior to morphine administration were 2–4 s. If a mouse failed to flick by 10 s following morphine administration, the test was terminated and a latency of 10 s was recorded. Mice that had a latency of 10 s were defined as analgesic. Mice were tested for analgesia 30 min following morphine. All testing was conducted in a blind manner.
FIG. 2. The effect NTX and oligodeoxynucleotide treatment on m-opioid receptor density in mouse brain. On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV (IT Å 10 mg; ICV Å 20 mg) with dH2O, Antisense (A sense), Mismatch-1 (Mis 1) or Mismatch-4 (Mis 4). On days 3, 5, 7, and 9 mice were again injected ICV and IT. After the final injections on day 9, placebo and NTX pellets were removed. Twenty-four hours later mice were sacrificed and whole brain was removed and saturation binding experiments were conducted using [ 3H] DAMGO. Data are means ( { SEM) from two to four experiments for each treatment condition. *Indicates significantly different from Placebo-dH2O ( p õ 0.05).
Cumulative Dose–Response Protocol A cumulative dose–response protocol was used for all studies. All mice in a treatment group (6–8/group) were injected SC with a starting dose of morphine (1 mg/kg) and tested for analgesia 30 min later. Mice that were not analgesic were given a second dose (increment dose) of morphine within 5 min of testing and then tested for analgesia again 30 min later. This cumulative dose–response protocol was continued until at least 75% of the mice were analgesic. The starting and increment doses used were the same for control and treated mice in each study. The actual morphine doses used in the cumulative dose– response protocol were determined previously [12]. Data from cumulative dosing is presented such that the percent of mice that are analgesic is plotted against the total (cumulative) dose administered. Opioid Receptor Binding Mice were sacrificed (n Å 2/group) and whole brain and spinal cord rapidly removed, weighed, and then homogenized in 80 vol of ice-cold 50 mM Tris buffer (pH 7.4). Homogenates were then centrifuged at 15,000 rpm for 15 min, the supernatant discarded and the pellet resuspended in buffer and centrifuged again, and then frozen at 0807C until analysis. The pellets were thawed, resuspended in Tris buffer, incubated (30 min at 257C), centrifuged, and finally resuspended in 20–80 vol of 50 mM phosphate buffer (pH 7.2). An aliquot (200 ml) of the homogenate was then assayed in triplicate in tubes containing 0.04–5.0 nM [ 3H] [D-Ala 2-MePhe 4-Gly(ol 5 )] enkephalin (DAMGO). Nonspecific binding was determined in the presence of 1000 nM levorphanol. Brain and cord homogenates were incubated for 90 min at 257C. Incubation was terminated by the addition of icecold phosphate buffer and the samples were filtered over GF/B glass fiber filters. Filters were washed three times with cold buffer, transferred to vials, scintillation cocktail added, and then counted using liquid scintillation spectroscopy. Counts per minute (CPMs) were converted to disintegrations per minute (DPMs) using the external standard method. Protein was determined using a microassay technique based on the method of Bradford [4] using reagent purchased from BIO-RAD (Richmond, CA). Binding experiments were conducted two to four times for each treatment condition. Drugs All oligodeoxynucleotides were purchased form Midland Certified Reagents (Midland, TX). Morphine sulfate was ob-
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FIG. 3. The effect NTX and oligodeoxynucleotide treatment on m-opioid receptor density in mouse spinal cord. On day 1 mice were implanted SC with a 15 mg NTX pellet or placebo pellet and injected IT and ICV (IT Å 10 mg; ICV Å 20 mg) with dH2O, Antisense (A sense) or Mismatch-4 (Mis 4). On days 3, 5, 7, and 9 mice were again injected ICV and IT. After the final injections on day 9, placebo and NTX pellets were removed. Twenty-four hours later mice were sacrificed and spinal cord was removed and saturation binding experiments were conducted using [ 3H] DAMGO. Data are means ( { SEM) from three experiments for each treatment condition. *Indicates significantly different from PlacebodH2O (p õ 0.05).
tained from Penick Laboratories (Newark, NJ). Placebo and NTX pellets were obtained from Research Triangle Institute (Research Triangle Park, NC) through the Research Technology Branch of the National Institute on Drug Abuse. NTX and placebo pellets were wrapped in nylon mesh before SC implantation. Data Analysis Quantal dose–response data were analyzed by Probit Analysis [13] using a computerized program (BLISS 21, Department of Statistics, University of Edinburgh) that estimates ED50s, 95% confidence limits, relative potency and determines significant differences between ED50s. Bmaxs and Kd s were determined from saturation studies using nonlinear regression (Prism ver 1.03, Graphpad Software, San Diego, CA). In all cases binding data were fit best by a one-site model. Significant differences for binding studies were based on ANOVA and post hoc tests. RESULTS NTX increased the analgesic potency of morphine in dH2Otreated mice by É70% ( Table 1, Fig. 1, left panel ) compared to the placebo-dH2O group. The potency of morphine was significantly reduced by É twofold in the placebo-antisense group relative to the placebo-dH2O group. The ED50 for the placebo-mismatch-4 group did not differ from control ( placebo-dH2O ) . Thus, NTX increased and antisense decreased the analgesic potency of morphine. When NTX and antisense treatments were combined, the supersensitivity effect was still present relative to the placebo-antisense and the placebodH2O groups. There was no significant difference between the NTX-dH2O and NTX-antisense groups. The effect of an oligodeoxynucleotide with one mismatch on morphine potency and NTX-induced supersensitivity was also determined (Table 2, Fig 1, right panel). An oligodeoxynucleotide with one mismatch effectively reduced the potency of morphine by É twofold. As previously (Table 1), NTX significantly increased the analgesic potency of morphine. When NTX and mismatch-1 treatments were combined, the supersensitivity effect was present relative to the appropriate control group (pla-
cebo-mismatch-1). However, supersensitivity to morphine for the NTX-mismatch-1 group approached (p õ 0.10), but did not reach, statistical significance compared to the placebo-dH2O group. There was no significant difference between the NTXdH2O and NTX-mismatch-1 groups. Binding experiments (Figs. 2 and 3) indicated that NTX significantly increased the density of brain, F (6, 11) Å 11.3, p õ 0.01, and spinal cord, F (4, 10) Å 4.9, p õ 0.02, m-opioid receptors without affecting affinity (Kd range in brain and cord, respectively Å 0.56–0.67 nM, 0.75–0.96 nM; Fs ° 1.00, p ú 0.05). NTX alone increased receptor density by É60% in brain and É140% in cord. When mice were treated with either the antisense to the mouse m-opioid receptor, mismatch-4 or mismatch-1 (binding studies in brain only) there were no significant changes in the density of receptors (p ú 0.05). Similarly, when NTX treatment was combined with antisense administration (brain and cord) or mismatch-1 (brain) there was no significant change in NTX-induced upregulation. We did not examine the effect of mismatch-4 on NTX-induced upregulation in brain or spinal cord since this oligodeoxynucleotide had no effect on morphine’s potency in analgesia studies (see Table 1). DISCUSSION The present results show that in vivo treatment with an oligodeoxynucleotide complementary to a portion of the mRNA for the mouse m-opioid receptor can reduce the analgesic potency of morphine. Similarly, an antisense complementary to the first 18 nucleotides of the rat m-opioid receptor (mismatch-1), with a single nucleotide mismatch for the murine m receptor, also significantly reduced morphine potency. However, a oligodeoxynucleotide with four noncomplementary nucleotides (mismatch-4) did not alter the analgesic potency of morphine. While one mismatch in an oligodeoxynucleotide can alter affinity for an mRNA, the location of the mismatch and the actual nucleotide substituted are also important considerations [11]. Our results that an oligodeoxynucleotide with one mismatch (mismatch-1) is equally effective to an oligodeoxynucleotide with no mismatches indicate that the single mismatch is benign; a finding that may reflect the similarities between mouse and rat m-opioid receptor mRNAs [9,19]. On the other hand, an oligodeooxynucleotide with four mismatches was ineffective in reducing morphine’s potency. Consistent with prior studies, NTX produced significant functional supersensitivity to morphine and significant increases in m-opioid receptor density as determined in whole brain and spinal cord homogenates [18,31,33,34,35). Thus, the binding assay, as well as the functional assay, are clearly responsive to treatments that regulate opioid receptors. However, we found no effect of antisense treatment on brain or spinal cord m receptors, although antisense treatment did reduce the analgesic potency of morphine by approximately twofold. Although other investigators have reported changes in d-opioid receptors following in vivo antisense using autoradiography [6] and homogenate binding assays [3,7,23], to our knowledge, decreases in m binding have not been reported following in vivo treatment. The failure to observe an effect of antisense on binding cannot be due to the ineffectiveness of this treatment, because morphine potency was reduced by antisense treatment. Furthermore, the lack of effect on binding is not due to the insensitivity of the binding assay, because increases in receptors were observed following NTX treatment. It is not readily apparent why there is a discrepancy between the binding and functional assays, but in other neurotransmitter systems there is precedent for changes in function with very modest changes in protein following antisense. For example, Weiss and
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colleagues found that antisense oligodeoxynucleotides directed at the D2 dopamine receptor almost completely blocked the behavioral effects of the D2 agonist quinpirole while D2 receptor binding was decreased by only É15–25% [30]. Weiss and colleagues have attributed this discrepancy to the possible selective action of antisense on a very small pool of functional receptors that are responsible for the majority of behavioral, but not binding, changes [20]. Perhaps a similar state of affairs exists for the murine m-opioid receptor in that binding studies detect both functional and nonfunctional receptors and antisense only inhibits production of a small pool of functional receptors. Similarly, better correlation between binding and function following antisense treatment may be observed if select brain regions were examined, although the increases in density in whole brain and cord following NTX suggests that the current assay techniques can readily detect changes in binding. When antisense and NTX treatment were combined, antisense did not block NTX-induced supersensitivity or receptor upregulation. This finding agrees with previous reports that reversible (which reliably increases receptor density) and irreversible in vivo antagonist treatment (which occasions an increase in receptors following depletion), do not act to increase m-opioid receptor gene expression as determined by quantitation of mRNA [5,8,15,27]. Thus, antagonist-induced increases in m-opioid receptor density may not depend upon de novo synthesis of receptors and may reflect changes in the rate of posttranslational processing such as activation of an existing pool of ‘‘cryptic’’ receptors. It is possible, however, that with different dosing protocols for antisense (e.g., more frequent, higher dose) an effect on NTX-induced supersensitivity and upregulation may have be observed. In summary, an antisense oligodeoxynucleotide to the mouse m-opioid receptor reduced morphine potency without changing the density of m-opioid receptors. Furthermore, antisense treatment did not block opioid antagonist-induced supersensitivity or upregulation. These data suggest that increases in opioid receptors following antagonist treatment do not depend upon increases in gene expression. ACKNOWLEDGEMENTS
This research was supported in part by National Institute on Drug Abuse (NIDA) Grants DA 04185. Dr. M. T. Turnock did not provide much technical help, but nothing happens without her.
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