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NEUROPHARMACOLOGY
Departments of Instrumental Analysis (M.Y., T.F.) and Biochemistry (S.K.), Faculty of Pharmaceutical Science, Fukuoka University, Fukuoka, Japan; and Clinical Pharmacokinetics (A.M., H.T., S.H.) and Pharmaceutics (S.O.), Division of Clinical Pharmacy Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
Received for publication
June 22, 2005
Accepted
August 17, 2005.
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Abstract |
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; Frederickson et al., 1977; Ohdo et al., 1988, 2001; Koyanagi and Ohdo, 2002; Koyanagi et al., 2003). Daily variations in biological functions such as gene expression and protein synthesis are thought to be important factors affecting the efficacy of drugs. Recent molecular studies of the circadian biological clock system have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of 24-h rhythms (Antoch et al., 1997; Gekakis et al., 1998; Sangoram et al., 1998; Jin et al., 1999; Kume et al., 1999; Ripperger et al., 2000; Shearman et al., 2000). In mammals, the 24-h rhythms in different tissues are coordinated by a master clock located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The master clock follows a daily light/dark cycle, and, in turn, synchronizes subsidiary oscillators in other brain regions and many peripheral tissues through neural and/or hormonal signals (Kalsbeek et al., 1996; Terazono et al., 2003). These subsidiary oscillators coordinate a variety of biological processes, producing 24-h rhythms in physiology and behavior. The daily rhythm of glucocorticoid secretion from the adrenal cortex is controlled by the hypothalamus-pituitary-adrenal (HPA) axis, which in turn is regulated by the SCN (Moore and Eichler, 1972). Glucocorticoids are involved in the regulation of a variety of physiological functions, and circadian changes in the circulating glucocorticoid levels are thought to affect the efficacy and/or toxicity of many drugs.
The analgesic effect of morphine varies according to the time of day when it is administered (Frederickson et al., 1977; Naber et al., 1981; Yoshida et al., 2003). The dosing time-dependent difference in the analgesic effect is associated with the 24-h variation in µ-opioid receptor function in the brainstem (Yoshida et al., 2003). Although an interaction has been suggested between the glucocorticoid and µ-opioid receptor system (Min et al., 1994; Wendel and Hoehe, 1998), the relevance of 24-h variation in glucocorticoid secretion in the analgesic effect of morphine remains unclear. In this study, we found that the µ-opioid receptor in the mouse brainstem was expressed in response to glucocorticoid stimuli. CRH-deficient mice, disrupting the 24-h rhythm of glucocorticoid secretion, failed to show a time-dependent variation in µ-opioid receptor function. We therefore used these animals to investigate the relevance of 24-h variation in glucocorticoid secretion in the analgesic effect of morphine.
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Materials and Methods |
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Animals and Treatment. A breeding colony of CRH-deficient mice (C57BL/6 129Sv genetic background) was purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed three to six per cage in a standardized light/dark cycle (lights on from 7:00 AM to 7:00 PM) at a room temperature of 24 ± 1°C and a humidity of 60 ± 10% with food and water ad libitum. Genotypes were determined by PCR method as described previously (Muglia et al., 1995). During periods referred to as darkness, dim red light was used to aid the treatment of mice. To investigate the temporal profiles of plasma corticosterone levels, blood samples were drawn by orbital sinus collection at 9:00 AM, 1:00 PM, 5:00 PM, 9:00 PM, 1:00 AM, and 5:00 AM. In the brainstem of wild-type mice, µ-opioid receptor expression peaked at 1:00 AM and declined at 9:00 AM, whereas the mRNA levels of µ-opioid receptor in the CRH-deficient mice remained at low throughout the day (data not shown). We therefore compared the function of µ-opioid receptor between the two genotypes at 9:00 AM and 1:00 AM. To examine the influence of glucocorticoid on µ-opioid receptor expression, CRH-deficient mice were administered subcutaneously (s.c.) with corticosterone (0.25, 0.5, and 1.0 mg/kg) or vehicle (propylene glycol; 0.1 ml/10 g body weight), at 7:00 AM, and total RNA of the brainstem was isolated at 4 h after drug injection. To explore whether glucocorticoid influences the analgesic effect of morphine, CRH-deficient mice were administered morphine (15 mg/kg i.p.) or saline at 4 h after injection of corticosterone (0.25, 0.5, and 1.0 mg/kg s.c.) or vehicle (propylene glycol; 0.1 ml/10 g body weight s.c.).
Determination of Plasma Corticosterone Concentration. Plasma samples were obtained after centrifugation at 3000 rpm for 3 min. These samples were heated at 56°C for 30 min to displace the corticosterone-binding protein. The plasma corticosterone concentration was determined by radioimmunosorbent assay (corticosterone [125I] assay system; GE Healthcare).
Determination of the Analgesic Effect. A thermal technique (hot-plate analgesia meter MK-350; Muromati Kikai Co., Ltd., Tokyo, Japan) was used to evaluate analgesic latency after morphine or saline injection (Kavaliers and Hirst, 1983). The surface of the plate was maintained at a temperature of 55 ± 0.5°C. The analgesic latency was determined at 30 min after morphine or saline injection. Time (in seconds) to either hind paw licking or jumping was recorded as pain response latency. To avoid heat injury, mice not responding after 120 s were removed from the hot-plate. The latency of those mice was 120 s. The time of 120 s was set in a previous paper (Bansinath et al., 1990). To avoid the likelihood of habituation or tolerance of the mice to hot-plate, animals were not used repeatedly.
Quantitative RT-PCR Analysis. Total RNA from the mouse brainstems was extracted as follows. The brain was excised quickly, the cerebral cortex and cerebellum were removed, and the brainstem was isolated on an ice-cold Petri dish using the brain atlas of Franklin and Paxinos (1997). Total RNA from the brainstem of individual mice was extracted separately using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA of the mouse µ-opioid receptor (GenBank accession number U19380
[GenBank]
) and GAPDH (GenBank accession number M88354
[GenBank]
) were synthesized and amplified using Superscript One-Step RT-PCR (Invitrogen). To quantify mRNAs, kinetic analysis of the amplified products, ensuring that signals were derived only from the exponential phase of amplifications, was performed in each sample as follows. After the first 24 cycles of amplification, an aliquot of 6 µg/ml was drawn for electrophoresis and the tubes were submitted to one more cycle of PCR. This procedure was repeated until 28 cycles had been performed. The PCR products were run on 3% agarose gel. After the gel was stained with ethidium bromide, the density of each band was analyzed using Kodak 1D image analysis software (Eastman Kodak Co.). The exponential phase of GAPDH amplification in all experimental conditions was located between the 24th and the 27th cycles, and the exponential phases of µ-opioid receptor gene were located between the 26th and the 28th cycles. Plotting the amounts of PCR products versus cycle numbers allowed us to recognize that the amplified efficiency of GAPDH and µ-opioid receptor gene is comparable. Therefore, the amplified products were quantified at the 27th cycle. The ratio of the amplified target to the amplified internal control (calculated by dividing the value of the µ-opioid receptor by that of GAPDH) was compared among the groups.
Specific µ-Opioid Receptor Binding Assay. The brainstem was homogenized in 1 ml of ice-cold 50 mM Tris-HCl buffer, pH 7.4. The homogenate was then centrifuged at 15,000 rpm for 15 min at 4°C. The obtained pellet was resuspended in 1 ml of Tris-HCl buffer, pH 7.4, and incubated at 37°C for 15 min, and then the homogenate was centrifuged again. The pellet was resuspended in 3 ml of Tris-HCl buffer. The protein concentration was approximately 2 mg/ml using Lowry's method (DC protein assay; Bio-Rad, Hercules, CA). The binding assay was performed with a reaction mixture (total volume, 200 µl) containing 100 µl of aliquot of brainstem homogenate, 0.156 to 5 nM [3H]DAMGO. Nonspecific binding was determined by using 10 nM naloxone. After incubation at 37°C for 30 min, the reaction mixture, together with 100 µl of Tris-HCl buffer to wash the tube, was laid over the 300-µl ice-cold fetal bovine serum and centrifuged at 10,000 rpm for 1 min at 4°C. The supernatant was removed, and the pellet was transferred to scintillation vials with 10 ml of scintillation cocktail and counted using a liquid scintillation counter (LSC-1000; Aloka Co., Mitaka, Tokyo, Japan) after standing for 6 h. Specific binding is the difference between binding determined in the absence of ligand and in the presence of ligand and is calculated as follows: specific binding (femtomoles per milligram of protein) = [total binding (femtomoles per milligram of protein)] – [nonspecific binding (femtomoles per milligram of protein)].
Brainstem Slice Culture. Brains were quickly removed from the skulls of CRH-deficient mice. Brainstem slices (500 µm in thickness) were prepared using mouse brain matrix (RBM-2000C; ASI Instruments, Inc., Warren, MI). The slices were preincubated for 30 min with 1 ml of Krebs-Ringer buffer (128.9 mM NaCl, 4.2 mM KCl, 1.5 mM CaCl2, 22.4 mM NaHCO3, 1.2 mM KH2PO4, 1.3 mM MgSO4, and 10 mM d-glucose) in the presence or absence of RU486 (10 µM). After the preincubation, corticosterone was added to the incubation buffer to give final concentration of 60 ng/ml. Four hours after treatment with corticosterone, total RNA from the brainstem slices was extracted as described above. The mRNA levels of µ-opioid receptor were determined by RT-PCR method.
Statistical Analysis. The significance of 24-h variation in each parameter was tested by analysis of variance. The statistical significance of differences among groups was evaluated by analysis of variance and the Bonferroni multiple comparison test. A 5% level of probability was considered significant.
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; Naber et al., 1981; Yoshida et al., 2003). On the other hand, there was no significant time-dependent variation in an amount of [3H]DAMGO binding in the brainstem of CRH-deficient mice; the amount of [3H]DAMGO binding levels remained at the daytime (9:00 AM) levels of wild-type mice at both time points (Fig. 2B). These findings suggest that time-dependent variation in µ-opioid receptor expression in the mouse brainstem is controlled by the HPA axis. Loss of Dosing Time-Dependent Difference in the Analgesic Effect of Morphine in CRH-Deficient Mice. We next investigated the influence of dosing time on the ability of morphine to increase pain endurance. The wild-type mice given saline showed significant time-dependent variation in the latency of their response to thermal stimulus, with significantly longer latency time during the dark phase (1:00 AM) than during the light phase (9:00 AM) (P < 0.01; Fig. 3). The latency time after a single injection of morphine (15 mg/kg i.p.) at 9:00 AM or 1:00 AM was significantly longer compared with that after saline injection at the corresponding dosing time (P < 0.01, respectively). Furthermore, the analgesic effect was significantly enhanced in mice injected with morphine at 1:00 AM than at 9:00 AM (P < 0.01).
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By contrast, CRH-deficient mice given saline showed no significant time-dependent variation in the latency of their response to the thermal stimulus (Fig. 3). Although the latency time was significantly prolonged by a single injection of morphine (15 mg/kg i.p.) at 9:00 AM and at 1:00 AM (P < 0.01, respectively), there was no significant dosing time-dependent difference in the analgesic effect. These findings suggest that rhythmic changes in the function of the HPA axis influence the analgesic effect of morphine by regulating the expression of the µ-opioid receptor.
Influence of Glucocorticoid on mRNA Levels of the µ-Opioid Receptor in the Mouse Brainstem. In the final set of experiments, we tested whether the µ-opioid receptor in the mouse brainstem is expressed in response to glucocorticoid stimuli. A single injection of corticosterone to CRH-deficient mice resulted in the induction of the µ-opioid receptor mRNA in a dose-dependent manner (Fig. 4A). Significant induction of µ-opioid receptor mRNA was found in mice with an injection of 1.0 mg/kg corticosterone (P < 0.05; Fig. 4A). The mRNA levels of µ-opioid receptor observed in CRH-deficient mice after the corticosterone injection was similar to daily peak levels of µ-opioid receptor expression in wild-type mice (Fig. 2B). Furthermore, a significant accumulation of µ-opioid receptor mRNA was also observed when cultured brainstem slices were exposed to 60 ng/ml corticosterone for 4 h (P < 0.01; Fig. 4B). However, the corticosterone-induced accumulation of µ-opioid receptor mRNA in the brainstem slices was significantly inhibited by pretreating the slices with glucocorticoid receptor (GR) antagonist RU486 (P < 0.01).
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Treatment of CRH-deficient mice with corticosterone dose dependently enhanced the analgesic effect of morphine (Fig. 4C). Significant potentiation of the analgesic effect of morphine was observed when the mice were pretreated with 1.0 mg/kg corticosterone (P < 0.05; Fig. 4C). These findings suggest that glucocorticoid hormone can enhance the analgesic effect of morphine through the induction of µ-opioid receptor expression.
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Discussion |
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The mRNA levels of the µ-opioid receptor and its binding capacity in the brainstem of wild-type mice fluctuated in a 24-h phase-dependent manner. The analgesic effect of morphine in wild-type mice was significantly enhanced by administering the drug when the µ-opioid receptor function was increased. These results are consistent with previous findings that the µ-opioid receptor function in nocturnally active rodents increased during their active period (Naber et al., 1981; Yoshida et al., 2003). It has been suggested that the time-dependent variation in [3H]DAMGO binding in mouse brainstem is attributable to the changes in the number of µ-opioid receptor rather than that in their binding affinities (Yoshida et al., 2003). Therefore, the time-dependent variation in the number of µ-opioid receptor seems to cause a dosing time-dependent difference in the analgesic effect of morphine.
In the brainstem of CRH-deficient mice, no significant time-dependent variation in µ-opioid receptor function was observed. Furthermore, the analgesic effect of morphine in these animals did not significantly vary according to their administration time. A single administration of 1.0 mg/kg corticosterone could induce the expression of µ-opioid receptor mRNA in a CRH-deficient mouse brainstem. The mRNA levels of µ-opioid receptor observed in CRH-deficient mice after the corticosterone injection was similar to daily peak of µ-opioid receptor mRNA levels in wild-type mice. Furthermore, at 1 h after the injection of 1.0 mg/kg corticosterone to CRH-deficient mice, the plasma concentration reached similar to daily peak of endogenous corticosterone levels in wild-type mice (Supplemental Fig. S1). Therefore, the time-dependent increase and decrease in the circulating glucocorticoid levels may be involved in the regulation of circadian change in the µ-opioid receptor function in mouse brainstem.
Daily peak of corticosterone concentration in plasma of wild-type mice was observed around the late light phase, whereas the levels of µ-opioid receptor mRNA and its binding capacity were increased during the mid dark phase. Although the plasma corticosterone concentration reached a peak level at 1 h after injection (Supplemental Fig. S1), the levels of µ-opioid receptor mRNA in the brainstem significantly increased from 4 h to 8 h after the glucocorticoid stimuli (Supplemental Fig. S2). Therefore, the function of µ-opioid receptor in the mouse brainstem seems to increase at several hours after the maximal glucocorticoid concentration. This may account for difference in daily peak times between plasma corticosterone levels and µ-opioid receptor function.
Glucocorticoids exert their action on gene expression through the activation of cytoplasmic GRs that bind to glucocorticoid response elements (GREs). The GRE is located in the 5'-flanking region of the µ-opioid receptor gene of mice and humans (Min et al., 1994; Wendel and Hoehe, 1998). The presence of a GRE in the µ-opioid receptor gene is consistent with the interaction between glucocorticoid and the µ-opioid receptor function. In fact, the corticosterone-induced accumulation of µ-opioid receptor mRNA in the brainstem slices was significantly inhibited by pretreating the slices with GRs antagonist RU486. These findings suggest that glucocorticoid can directly act on brainstem cells, thereby inducing the expression of µ-opioid receptor through GRs. This may explain how glucocorticoid exerts a long-lasting effect on morphine antinociception. A previous study by Pieretti et al. (1994), however, showed that systemic administration of dexamethasone reduced the antinociception induced by DAMGO. They evaluated the analgesic effect of DAMGO at 0.5 h after dexamethasone injection. Whether dexamethasone acutely inhibits the µ-opioid receptor function is unclear. The inhibitory effect might be due to the difference in the biological potencies between dexamethasone and corticosterone.
In the current model, the mammalian circadian clock system is hierarchically organized: the master pacemaker in the SCN governs subsidiary oscillators in other brain regions and many peripheral tissues through neural and/or humoral signals (Balsalobre et al., 1998; Terazono et al., 2003). These subsidiary oscillators coordinate a variety of biological processes, producing overt rhythms in physiology and behavior. Circadian secretion of glucocorticoids acts to synchronize peripheral clocks, thereby coordinating the physiological functions according to the circadian time (Balsalobre et al., 2000). Our present findings also suggest that glucocorticoid hormones participate in the circadian control of the pain threshold by coordinating µ-opioid receptor expression.
The findings in this animal model suggest a mechanism underlying the time-dependent variation in µ-opioid receptor function. The rhythmic secretion of glucocorticoids seems to induce a circadian coordination of µ-opioid receptor expression in the mouse brainstem. A potent analgesic effect of morphine could be expected by administering the drug when µ-opioid receptor function was increased. Therefore, our results may also provide clues to select the most appropriate time for morphine administration
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Footnotes |
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ABBREVIATIONS: SCN, suprachiasmatic nuclei; HPA, hypothalamus-pituitary-adrenal; CRH, corticotrophin-releasing hormone; [3H]DAMGO, [D-ala2,N-methyl-phe4,glyol5][tyrosyl-3,5-3H]-enkephalin; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RU486, 17
-hydroxy-11-[4-dimethylamino phenyl]-17
-[1-propynyl]estra-4,9-dien-3-one; GR, glucocorticoid receptor; GRE, glucocorticoid-response element.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Shigehiro Ohdo, Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582 Japan. E-mail: ohdo{at}phar.kyushu-u.ac.jp
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