Anesth Analg 2003;97:1458-1463
© 2003 International Anesthesia Research Society
PAIN MEDICINE
Mu-Opioid Receptor mRNA Regulation During Morphine Tolerance in the Rat Peripheral Nervous System
Thomas Meuser, MD*,
Thorsten Giesecke, MD*,
Anja Gabriel, MD*,
Maria Horsch, MD*,
Rainer Sabatowski, MD*,
Jürgen Hescheler, MD
,
Stefan Grond, MD
, and
Pamela Pierce Palmer, MD PhD
Departments of *Anesthesiology and
Neurophysiology, University of Cologne, Cologne, Germany,
Department of Anesthesiology, University of Halle Wittenberg, Halle, Germany, and the
Department of Anesthesia, University of California, San Francisco, CA
Address correspondence and reprint requests to Dr. med. Tom Meuser, MD, Department of Anesthesiology and Intensive Care Medicine, University of Cologne, 50924 Cologne, Germany. Address email to Tom.Meuser{at}uni-koeln.de
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Abstract
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In vivo data on opioid receptor mRNA regulation after agonist exposure in the peripheral nervous system are lacking. Therefore, we studied the impact of morphine treatment on the regulation of mu-opioid receptor mRNA during behavioral signs of tolerance in rat peripheral sensory ganglia. Nineteen rats were treated in 2 groups with either morphine (10 mg/kg subcutaneously) or saline over 4 days, and a subset of rats received naloxone on the fifth day followed by either morphine injection on the sixth day or death to obtain dorsal root ganglia for mRNA analysis. Animals were tested on the hot plate during treatment days. To assess the levels of mu-opioid receptor mRNA, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used with the co-amplification of the "housekeeping" gene cyclophilin as internal control. Morphine treatment over 4 days induced tolerance as reflected on the hot-plate test by a significant reduction of paw-withdrawal latency from 242% to 99% above baseline. Using RT-PCR we demonstrated a down-regulation of mu-opioid receptor mRNA by 62% after morphine exposure (P < 0.05). After acute withdrawal of morphine from the mu-receptor by naloxone, the mu-opioid receptor mRNA levels in the dorsal root ganglia were restored to control levels within 24 h and the paw-withdrawal latency also returned to 280% above control. These data suggest that the peripheral nervous system may be an important site of opioid tolerance development.
IMPLICATIONS: The peripheral nervous system is a possible site of opioid receptor tolerance. We show the development of behavioral tolerance and mu-opioid receptor mRNA down-regulation in the dorsal root ganglia in rats after chronic morphine treatment. Both this mRNA down-regulation and behavioral tolerance reverse after 24 h of naloxone treatment.
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Introduction
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Tolerance is a major limitation of the long-term clinical use of opioids. Studies in rats have shown tolerance to be pharmacodynamic, receptor-specific, and reversible if the agonist is removed (1,2). However, the cellular and molecular mechanisms of opioid tolerance are poorly understood. In addition to receptor internalization and alterations in cyclic adenosine monophosphate and G-protein signaling, receptor down-regulation has attracted attention as a mechanism of tolerance. In the central nervous system (CNS) of guinea pigs, chronic morphine exposure produced a 15% decrease of mu-opioid receptor mRNA in the mediobasal hypothalamus but no changes of mRNA levels in other regions of the brain (3). Comparable studies in rat brains failed to detect any modification of mu-opioid receptor mRNA after continuous morphine treatment (4,5).
In addition to CNS effects of opioids, it is widely accepted that the mu-opioid receptor participates in the modulation of nociception at primary afferent terminals (6–8). We reported in an earlier reverse transcriptase-polymerase chain reaction (RT-PCR) study that the mRNA encoding the cloned mu-opioid receptor is expressed in rat dorsal root ganglia (DRG) (7). Many other studies also have documented mu-opioid receptor mRNA and protein expression in sensory ganglia (9,10).
With the recent emphasis in the literature on the peripheral actions of opioids, there has been speculation that the peripheral nervous system (PNS) is a possible site for opioid tolerance. Specifically, tolerance after daily systemic, intrathecal, intracerebroventricular, or local injection was investigated in mice in vivo (8). The 50% effective dose (ED50) value for systemic morphine shifted to the right more than twofold after 5 days, whereas no change occurred after supraspinal or spinal administration alone. However, morphine’s ED50 value in the tail-flick test after local administration shifted more than 19-fold. The authors propose that, after chronic morphine exposure, opioid tolerance is predominantly mediated at the peripheral level, and that the PNS should be further investigated regarding the molecular mechanisms of opioid tolerance (8). Similarly, the incubation of cultured rat DRG neurons with a mu receptor-selective agonist resulted in a dose-dependent reduction (
70%) of the expressed mu receptor proteins. Furthermore, an up-regulation (40%) of mu-receptor protein occurred after preincubation with naloxone (11). Thus, regulatory processes on the level of transcription and/or receptor protein expression may contribute to opioid tolerance in the PNS. However, data on opioid receptor mRNA levels and their regulation after chronic agonist exposure in the PNS in vivo are lacking. Therefore, we studied the impact of chronic morphine treatment on mu-opioid receptor mRNA regulation in the PNS during behavioral signs of tolerance.
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Methods
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The experiments were conducted in accordance with the guidelines of the International Association for the Study of Pain and approved by the German Government Animal Investigation Committee. Nineteen male Sprague-Dawley rats (Harlan Winkelmann, Borchen, Germany) weighing 300–400 g were housed with lights on 6 AM to 6 PM and with food and water available ad libitum. Animals were randomly assigned to two treatment groups. Eight control rats received 0.5 mL sodium chloride 0.9% (saline), whereas 11 other rats received 0.5 mL morphine (10 mg/kg; MSI®, Mundipharma, Limburg, Germany). Drugs were administered subcutaneously twice daily with a consistent schedule in the morning and the evening for 4 days. In the morning of the fifth day, four control rats and four morphine rats were killed. The remaining four rats in the control group received no further morphine injections, but received 2 injections of naloxone (0.5 mg/kg) on the fifth day in the morning and evening and were killed on the sixth day. Four rats in the morphine-treatment group also received 2 injections of naloxone (0.5 mg/kg) on the fifth day in the morning and evening and were killed on the sixth day. The 3 remaining rats from the morphine-treatment group received 2 injections of naloxone (0.5 mg/kg) on the fifth day in the morning and evening but then also received morphine (10 mg/kg) injection in the morning on the sixth day.
Hot-plate tests were performed at 48°C by a blinded observer. Latency to hind paw licking, stamping, or jumping was recorded. The cutoff time was 45 s. Each rat was trained on the hot plate three times before testing. Rats were tested 30 min before (baseline) and 30 min after the first injection on the first day. On days 2–4, rats were tested 30 min before and 30 min after the last injection of each day and, in the groups treated additionally with naloxone, 30 min before and after the last injection of naloxone on the fifth day. The group of rats that received morphine injection in the morning of the sixth day was tested both 30 min before and after this injection.
Under pentobarbital anesthesia (60 mg/kg, intraperitoneal), bilateral lumbar DRG (L3-5) from 16 rats were carefully excised to avoid contamination from surrounding tissue. They were frozen immediately in liquid nitrogen and stored at -80°C until analyzed using the RT-PCR technique. Total RNA from six DRGs of each rat was extracted using the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany). Co-purified DNA was ultimately digested with DNase I. The amount and purity of the total RNA was determined by spectrophotometry (BioPhotometer®, Eppendorf Germany) at 260 nm and 280 nm. A nucleic acid preparation with an A260/A280 ratio of 
2.0 was considered as pure.
To assess the levels of mu-opioid receptor mRNA, a quantitative, noncompetitive method for RT-PCR was used. This well established method uses the coamplification of the constitutively expressed "housekeeping" gene cyclophilin-A, an isomerase that has been shown to be constant in quantitative mRNA assays and is used as an internal control by normalizing the expression levels of the target gene of interest (12). The method of PCR needs to be standardized for PCR conditions (primer kinetics, cDNA template concentrations, cycle numbers) because the amplification rate remains constant during the exponential phase of the PCR reaction if there is sufficient substrate (primers, enzymes, nucleotides, magnesium) and no inhibitory PCR reaction by-products (12). Custom primers were ordered (Life Technologies, Karlsruhe, Germany) and standard curves for each primer pair were produced to ensure linear reproducibility. It is crucial that the PCR results are taken from the exponential phase of the amplification because, under these conditions, the ratio of the amplified products between any two samples represents the initial ratio of the RNA messages in the starting material.
After the exponential phase, the amplification decreases significantly because the PCR reaction reaches the saturation phase. We consistently coamplified cyclophilin with the RT-PCR and calculated ratios between the unregulated cyclophilin and the potentially regulated target gene. As the amount of original total RNA was known, as was the amount of amplified cDNA compared to a standardized DNA ladder, we were able to calculate the abundance of the transcript of the target gene within the origin total RNA. The sequence-specific synthetic oligodeoxyribonucleotide primers coding for the opioid receptor and cyclophilin were designed according to the sequences from the GenBank database using the BLAST tool from the National Center for Biotechnology Information (NCBI, Bethesda, MD; http://www.ncbi.nlm.nih.gov), all with same lengths (20 bp), G/C-contents (55%) and, thus, melting points (Table 1). They were derived from regions flanking introns so that amplification products of cDNA could be distinguished from residual genomic DNA. The primers were checked for not overlapping and not matching with any other known sequence. RT-PCR was performed using the Titan® "One Tube" RT-PCR System (Roche Diagnostics, Mannheim, Germany). Enzymes used for the reverse transcription were avian myeloblastosis virus RT and for the PCR a mix of Taq DNA polymerase and Pwo DNA polymerase. RT-PCR reactions for cyclophilin and mu-opioid receptor of each animal were performed in separate, thin-walled reaction tubes, but together in the same working steps to ensure consistent conditions. Each PCR amplification with a volume of 25 µL contained 100 ng total RNA, 0.4 µM of each primer, 0.2 mM of each deoxynucleotide (dNTP), 5 mM dithiothreitol, 5 U RNase-Inhibitor, 1.5 mM MgCl2, and 0.5 µL enzyme mix. The reactions were done in a Perkin-Elmer 2400 thermocycler using the following program: reverse transcription at 55°C for 30 min, denaturation at 94°C for 2 min, then for each cycle denaturation at 94°C (30 s), annealing at 62°C (30 s), and extension at 68°C (1 min). The number of cycles was 35; the program ended with 7 min at 68°C and storage at 4°C. Every PCR was accompanied by one negative control reaction without template RNA. PCR products were analyzed by gel electrophoresis on an ethidium bromide stained 2% Seakem LE agarose gel (FMC Bioproducts) in tris-borate-EDTA buffer. Gels were visualized on an ultraviolet light table, photographed and analyzed with the Kodak Digital Science 1D Image Analysis Software (Eastman Kodak, Rochester, NY). The software analyzed size and intensity of the bands from the digital image and calculated the cDNA content of each band by comparing with the DNA marker XIV standard (Roche Diagnostics, Mannheim, Germany). For each sample, the cDNA content of the opioid receptor band was divided by the value for the corresponding cyclophilin band. The resulting ratios reflect the relative amount of the opioid receptor mRNA in the extracted total RNA. The RT-PCR bands were purified and subcloned into the pCR® 2.1-TOPO® vector (Invitrogen, Groningen, NL). The inserts were sequenced with an ABI PrismTM 377 DNS Sequencer (Perkin Elmer/Applied Biosystems) with the TaqFS dye-terminator cycle-sequencing kit.
For statistical analysis, the mean values of the hot-plate tests of each rat before the first injection (baseline values) were compared among all 19 rats with one-way ANOVA. Changes within and between the different treatment groups were tested with the Student-Newman-Keuls test. Calculated relative opioid receptor mRNA levels in different treatment groups were compared using a one-way ANOVA and the Student-Newman-Keuls test in case of statistically significant differences. All values are presented as mean ± SEM. A value of P < 0.05 was considered significant.
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Results
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On day one, the hot-plate mean baseline values given in seconds delay in reaction between the groups before the first injection of either saline or morphine (saline, 8 ± 0.5 s; morphine, 7 ± 0.4 s) were not statistically different. Morphine or saline was injected twice daily for 4 days. Thirty minutes after the first injection of morphine, the mean hot-plate latency time increased 242% (24 ± 3 s; P < 0.001) above baseline. The mean hot-plate latency of the control rats did not change compared to baseline levels over the 4 days of saline treatment (data not shown). On day 4, before the last injection of morphine, baseline hot-plate latency values of the morphine group were not different from baseline values on day 1, indicating that hyperalgesia in the hind paw had not developed during the 4-day hot-plate testing. Thirty minutes after the last morphine injection on day 4, the mean hot-plate latency time of 14 ± 1 s was significantly shorter (reduced to 99% above baseline values) than on day 1 after the first injection (P = 0.002). After administration of naloxone on the fifth day, the mean hot-plate latency of the morphine-naloxone group decreased to its smallest value, but was not statistically different from baseline or saline control (Fig. 1). The rats that received morphine on day 6 after naloxone treatment on day 5 had a complete return of their behavioral response to morphine exhibited by a hot-plate latency of 27 ± 2 s (280% above baseline).
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Figure 1. Hot-plate latency times to paw withdrawal are given in seconds and are presented as mean ± SEM. Morphine treatment in rats days 1–4 (n = 11) was followed by naloxone-treatment during day 5 (n = 7) and morphine treatment again on day 6 (n = 3). Injections were performed twice per day. Day 1 baseline latencies were measured 30 min before the first injection of morphine and postdose latencies were measured 30 min after this first injection. Latencies on days 2–5 were measured after the second injection of morphine or naloxone. On day 6 animals only received a morning morphine injection (**P = 0.002).
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After RNA isolation and RT-PCR, amplification products were run electrophoretically on agarose gels. For each individual animal, gel analysis was performed from which the individual ratios to cyclophilin were derived. The gel electrophoresis bands of cyclophilin and mu-opioid receptor were located at their expected lengths; the negative controls showed no gel band (Fig. 2). The results of the gel electrophoresis bands were digitally measured and statistically analyzed. We calculated the ratio by dividing the mu-opioid receptor band by the value of the corresponding cyclophilin band for each rat. The calculated mean ratios were 0.95 ± 0.14 (mean ± SEM) in the control group, 0.36 ± 0.19 in the morphine group, and 0.92 ± 0.17 in the morphine + naloxone group. The control ratio was made to correspond to 100% control; therefore, relative to the control animals, the mu-opioid receptor mRNA levels of the morphine-tolerant rats were down-regulated by 62% after 4 days (P < 0.05). After naloxone treatment on day 5, the mu-opioid receptor mRNA levels returned to control levels (Fig. 3). After 4 days of saline, mRNA levels after naloxone treatment were not different from control (ratio data not graphed). The gel electrophoresis bands of one rat out of each group were purified and subcloned. All sequenced RT-PCR bands were identical with the cloned and published cyclophilin and mu-opioid receptor sequences.
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Figure 2. Gel electrophoresis (2% agarose gels stained with ethidium bromide) pictures. A, control rat after 4 days administration of saline; B, morphine-treated rat after 4 days administration of morphine; C, morphine-treated rat after 4 days administration of morphine plus naloxone on day 5. Within each gel picture from left to right: DNA reference ladder (column 1), negative control w/o template RNA (column 2), cyclophilin amplification product at 235 bp length (column 3), and mu-opioid receptor amplification product at 510 bp length (column 4).
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Figure 3. Mean ratio control was designated as 100% control. In the morphine-tolerant rats, the mu-opioid receptor mRNA levels were down-regulated by 62% after 4 days compared with control (*P < 0.05). After naloxone, the mu-opioid receptor mRNA levels were restored to 97% of control. Naloxone treatment alone was not significantly different from control (P = 0.132, data not shown). Data shown as mean ± SEM.
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Discussion
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The major finding of the present study is that chronic morphine exposure leads to a down-regulation of mu-opioid receptor mRNA in peripheral sensory ganglia of rats showing behavioral signs of morphine tolerance.
Morphine treatment over 4 days induced tolerance, as reflected on the hot-plate test by a significant reduction of withdrawal latency from 242% to 99% above baseline. With RT-PCR we demonstrated a down-regulation of mu-opioid receptor mRNA by 62% after morphine exposure. After acute withdrawal of morphine from the mu-receptor, the mu-opioid receptor mRNA levels were restored to control level within 24 hours. Similarly, the behavioral signs of tolerance were also reversed, with an increase in paw-withdrawal latency to 280% above baseline.
For quantification of mRNA, we excised lumbar DRG because peripheral sensory ganglia have been proven as a valid site for morphine tolerance in the PNS of rodents (8,11). Furthermore, it has been shown that among DRG levels, opioid receptor mRNA density is highest in the lumbar region (13). Along with glia cells, the DRG consists of both small and large diameter primary afferent neurons. Small-diameter, slowly conducting primary afferent neurons of the A
- and C-fiber type are involved in nociception and pain transmission. It has been shown in a quantitative in situ hybridization study that both small and large diameter neurons of the rat DRG express mu- and -opioid receptor mRNA, but most DRG cells expressing these receptor mRNAs are small and likely to be involved in nociceptive pathways (6). As whole DRG were used in this study, it is possible that observed changes in mu-opioid receptor mRNA reflected changes in non-neuronal as well as neuronal cell populations.
One of the major limitations of the clinical use of opioids is their tendency to induce tolerance. The underlying molecular mechanisms are poorly understood. Various mechanisms of morphine tolerance, such as receptor internalization or desensitization of G-protein-coupling, have been discussed in the literature. Although receptor internalization is common with G-protein-coupled receptors, morphine has not been shown to stimulate the internalization of mu-opioid receptors (14,15). It is this lack of receptor internalization that is thought to be one of the reasons for the more rapid tolerance development with morphine. The mu-opioid receptor is chronically exposed to morphine and not allowed to internalize and become re-expressed and re-coupled to the G-protein. Receptor internalization and re-expression occur constantly and do not result in an overall decrease in the number of expressed mu-opioid receptors.
Mu-opioid receptor down-regulation, however, is reflected as an overall decrease in the level of receptor protein, or mRNA on the transcriptional level, and studies on CNS regions have yielded inconsistent results. Three studies demonstrate no change in mu-opioid receptor mRNA levels in brains of morphine-tolerant rats (4,5,16), although a down-regulation of mu-opioid receptor mRNA has been found in the mediobasal hypothalamus of morphine-tolerant guinea pigs with no changes in other brain regions (3).
In the PNS, only one study on isolated rat DRG verified that in vitro exposure to opioid agonist, DAMGO, a selective mu-opioid receptor ligand, resulted in a decrease of opioid receptor binding sites (11). It has been shown that the induction of opioid tolerance after chronic morphine exposure is more pronounced in the PNS than in the CNS (8). There is no question that opioid tolerance occurs at the level of the CNS, even though receptor down-regulation may not play a significant role in that process. Many animal studies have demonstrated that intrathecal administration of opioid agonist results in decreased analgesia over time (17,18). Yet it is intriguing to consider the results of the present study and those of Kolesnikov et al. (8), which suggest that, with systemic administration of opioids, the periphery may be an important site of opioid tolerance development.
We found that one day of treatment with naloxone in the absence of morphine reversed both the down-regulation of mu-opioid receptor mRNA expression in the DRG and also reversed the behavioral signs of tolerance. An important study by Ibuki et al. (19) found that daily naloxone treatment during continuous intrathecal administration of morphine over an 8-day period resulted in more rapid development of tolerance to morphine. This effect was thought to be attributable to an increase in spinal excitatory amino acid release during daily opioid withdrawal, thereby activating spinal N-methyl-D-aspartate receptors. Our one-day reversal with naloxone probably did not result in significant chronic spinal excitatory amino acid release compared with the previous study and, therefore, the morphine analgesia returned to day 1 levels after this day of mu-receptor "off-time."
This is the first study to show in vivo that chronic exposure to morphine down-regulates mu-opioid receptor mRNA levels in rat peripheral sensory ganglia. The down-regulation of mu-opioid receptor mRNA coincides with behavioral signs of tolerance. Future studies are needed to investigate whether down-regulation of mu-opioid receptor mRNA leads to a consecutive down-regulation of mu-opioid receptor protein density.
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Acknowledgments
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Supported by grants 134/1998 and 6/2000 of the Köln Fortune Program/Faculty of Medicine, University of Cologne, Germany. Dr. Pamela Pierce Palmer was supported by NIH grant AR45570.
The authors thank the following individuals at the University of Cologne for their outstanding help: W. Buzello, MD, professor and chairman of anesthesia; A. Hagemann, doctoral candidate; and C. Rabb, animal care facilities staff.
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Accepted for publication May 29, 2003.
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Abstract
Introduction
