Neurogastroenterology & Motility Neurogastroenterol Motil (2014) 26, 1396–1407
doi: 10.1111/nmo.12387
Circular muscle contraction in the mice rectum plays a key role in morphine-induced constipation H. ONO ,* A. NAKAMURA ,* K. MATSUMOTO ,†,‡ S. HORIE ,† G. SAKAGUCHI *
& T. KANEMASA *
*Pain & Neurology, Medicinal Research Laboratories, Shionogi and Co., Ltd., Toyonaka, Osaka, Japan †Laboratory of Pharmacology, Josai International University, Togane, Chiba, Japan ‡Division of Pathological Sciences, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, Japan
Key Messages
• Opioid-induced constipation is considered as a major and difficult problem in the clinical field. • Focusing on morphine response of the small and large intestinal regions, we aimed to identify the key region involved in opioid-induced constipation.
• Using in vitro isometric recording of the circular muscle contraction and in vivo bead expulsion time assay in mice, we found that the rectum is likely the key region for opioid-induced constipation.
Abstract Background Although opioids induce intestinal muscle contraction and provoke constipation, the intestinal region(s) that contribute to the constipation have remained unclear. We report here a region-specific response of intestinal muscle contraction to morphine and its correlation with in vivo constipation. Methods Regions of mice small and large intestines were dissected histologically and circular muscle contractile responses were measured using isometric transducers. Bead expulsion assays were performed to assess in vivo constipation. Key Results The strongest contraction in response to morphine was detected in the rectum. The distal and transverse colon also showed strong contractions, whereas weak responses were detected in the proximal colon, jejunum, and ileum. Regarding the sustainability of muscle contractions during morphine exposure, prolonged waves were detected only in the rectum, while the waves diminished gradually in other regions. To identify the mechanism(s) underlying this
difference, we focused on nitric oxide synthase (NOS). In the distal colon, decreased contraction during morphine exposure was recovered by application of a NOS inhibitor (L-NAME), while a NOS substrate (L-arginine) enhanced contractile degradation. In contrast L-NAME and L-arginine modestly affected the sustained contraction in the rectum. To confirm the correlation with constipation, beads were inserted into the transverse colon, distal colon, or rectum after morphine administration and expulsion times were examined. Beads tended to stop at the rectum even when inserted in the deeper colonic regions. Conclusions & Inferences The rectum showed the greatest response to morphine in both in vitro and in vivo analyses, therefore it may play a key role for opioidinduced constipation. Keywords constipation, morphine, nitric oxide (NO), rectum, l-opioid receptor (MOR). Abbreviations: AUC, area-under-the-curve; MOR, l-opioid receptor; NO, nitric oxide; NOS, nitric oxide synthase; PCR, polymerase chain reaction.
Address for Correspondence Toshiyuki Kanemasa, PhD, Pain & Neurology, Medicinal Research Laboratories, Shionogi & Co., Ltd, 1-1, 3-chome, Futaba-cho, Toyonaka, Osaka 561-0825, Japan. Tel: +81-6-6331-6654; fax: +81-748-88-6508; e-mail: [email protected] Received: 28 November 2013 Accepted for publication: 6 June 2014
INTRODUCTION Morphine is a l-opioid receptor (MOR) agonist that is used clinically for patients suffering from moderate-to-
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induced contractile response in the individual regions of the intestines. In addition, the locus that makes the greatest contribution to opioid-induced constipation is not clear. Thus, to assess the contribution of the various intestinal regions in opioid-induced constipation, in the present study, we compared morphine-induced circular muscle contractile potency and sustainability in histologically known regions (jejunum, ileum, proximal, transverse, distal colon, and rectum) of the mouse intestine. We found that contractile responses to morphine differed among the regions. Furthermore, this contractile efficacy was correlated with in vivo bead propulsion data. Thus, we identified the intestinal region responsible for morphine-induced constipation.
severe pain. Although morphine and other MOR agonists show potent analgesic effects, their side effects, such as constipation, nausea, and drowsiness, often make their continuous use difficult.1 Among these side effects, opioid-induced constipation is known to occur in more than 90% of patients given opioids for pain management.1 In addition, although tolerance to other side effects often occurs, constipation seems resistant to tolerance and continues during opioid use.2,3 Thus, constipation is considered a major and difficult to avoid clinical issue. To date, numerous pharmacological studies of the mechanisms of MOR agonist-induced constipation have been reported. This is considered to be a direct effect of MORs because gastrointestinal transit inhibition is abolished in MOR-knockout animals.4 Expression of the MOR has been detected in many central and peripheral tissues. Although opioids act on MOR in the central nerves system and indirectly induce constipation,5,6 it is also widely accepted that opioids act directly on MORs in intestinal neurons, changing gastrointestinal motility, consequently inducing constipation.7,8 Indeed, a peripheral MOR antagonist has been shown to relieve morphine-induced constipation in animal studies and clinical trials.9,10 In the intestine, autoradiographic binding of MOR agonists has been detected.11 In addition, high MOR expression has been identified in myenteric plexus, which regulates neuronal activity in the intestines.12,13 Because MORs are also expressed in the internal border of the circular muscles, the inner muscle layers of myenteric plexus, agonists are thought to act on the nerve terminals of these neurons and affect muscle activity.13–16 In contrast, in the longitudinal smooth muscle layers located at the outer layers of the myenteric plexus, MOR expression has been reported to be lower than that in the circular muscles.17 In addition, because the circular muscle layers are typically much thicker than the longitudinal muscles and play the dominant role in peristalsis,18 we assumed that the circular muscles play a more important role in opioidinduced constipation. In studies examining tissue preparations in vitro, treatment with several MOR agonists has been shown to reduce circular muscle relaxation by inhibiting inhibitory neurotransmitter release and increasing phasic contractile activity of muscles, reducing peristaltic movement from the proximal (oral) to the distal (anal) side of the intestine.15,19–21 Many of these reports use primarily the ileum and colon to compare the effects of each agonist. Despite these, to our knowledge, there is little information regarding the magnitude of the opioid-
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MATERIALS AND METHODS Animals Male ddY mice (5–7-week old; Japan SLC, Hamamatsu, Japan) were used. Animals were housed in a room maintained at 23 1 °C under a 12/12-h light/dark cycle and allowed access to water and food ad libitum. All procedures for animal experiments were approved by the Animal Care and Use Committee of Shionogi Research Laboratories, Osaka, Japan, and were consistent with the internal guidelines for animal experiments and in adherence to the ethics policy of Shionogi & Co., Ltd (Osaka, Japan).
Drugs Morphine hydrochloride was from Shionogi & Co., Ltd. Naloxone was from Tocris Bioscience (Bristol, UK). N’-nitro-L-argininemethyl ester hydrochloride (‘L-NAME’) and S-nitroso-N-acetyl-D, L-penicillamine (‘SNAP’) were from Cayman Chemical Company (Ann Arbor, MI, USA). L-arginine was from Sigma-Aldrich (Tokyo, Japan). Agents were dissolved in 0.9% physiological saline (Otsuka Pharmaceutical, Tokyo, Japan) for in vivo experiments and in purified water for in vitro experiments.
Contractile analysis of mouse circular muscles Prior to the experiment, mice were individually habituated in a cage with mesh wire inserted in the bottom; food was restricted for 12 h and water was available ad libitum. Mice were sacrificed by cervical spine fracture/dislocation and the small and large intestines were removed and placed in Krebs–Henseleit solution (NaCl 112.08 mM, KCl 5.90 mM, CaCl2 1.97 mM, MgCl2 1.18 mM, NaH2PO4 1.22 mM, NaHCO3 25.00 mM, and glucose 11.49 mM) bubbled with 95% O2 and 5% CO2 at 37 °C until use. Segments of the jejunum (10-cm distal to the stomach) and ileum (5-cm proximal to the cecum) were dissected from the small intestine, and the proximal colon (0.5-cm distal to the cecum), transverse colon (3-cm proximal to the anus), distal colon (1.5-cm proximal to the anus), and rectum (0.3-cm proximal to the anus) were dissected from the large intestine (8 mm in length). Tissues were placed under 0.5-g tension with a thin triangle hook (side length, 8 mm) suspended along the axis of the circular muscle in a 10-mL organ bath (Krebs–Henseleit solution bubbled with 95% O2 and 5% CO2 at 37 °C) and equilibrated for 5 min until drug treatment. Drugs were
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administered by 10 lL in a total volume of 10 mL. Contractions were recorded before and after exposure to each drug (morphine, naloxone, L-NAME, SNAP, and L-arginine) with an isometric transducer (TB-611T; Nihon Kohden, Tokyo, Japan) connected to an amplifier (AD-632J; Nihon Kohden) and PowerLab/4SP (AD Instrument, Castle Hill, NSW, Australia) and analyzed using the LabChart 6.1.3 software (AD Instrument). The waves of the contractions were analyzed in terms of area-under-the-curve (AUC) using the LabChart software. Each tissue was used only one time in each experiment.
recording methods. The ring preparations of the small (jejunum and ileum) and large (proximal, transverse, transverse to distal, distal colon, and rectum) intestines were equilibrated in organ baths for 5 min, and then contractions were measured during morphine exposure. Fig. 1A illustrates the regions sampled for each segment, and Fig. 1B shows representative recorded changes in contractile waves in the mouse intestine before and after morphine exposure (10 lM). In the jejunum, ileum, and proximal colon, rhythmical contractions were detected both before and after morphine treatment, and the amplitude of the waves exhibited slight changes after morphine treatment. In the transverse colon to the rectum, the amplitude of waves before morphine was similar, and was increased soon after morphine treatment. Dose–response changes were measured for 3 min during morphine (0.1–100 lM) exposure and were assessed in terms of AUC (Fig. 1C). The relative AUC values, compared with those assessed prior to the experiment, increased dose-dependently in the large intestine, with the highest value in the rectum. In addition, AUC values in the large intestine were higher in the terminal side of the large intestine. Weak responses were detected in the small intestine (Fig. 1B). Because the doses used here were similar to previous studies in mice,16 we assume that the contractile responses detected were not due to use of excessive doses of morphine. The results suggested differences in the contractile ability of each locus of the intestinal circular muscles, and that the rectum showed the greatest response to morphine. Using naloxone, we determined whether the contractile changes in the colonic regions and rectum were due to MOR stimulation by morphine. The dose of naloxone used was 1 lM, because this dose has been reported to antagonize MOR selectively.19 In all segments examined, morphine (10 lM) was added as described in Fig. 1, followed 5 min later by naloxone; changes in the waves were then assessed. Naloxone inhibited morphineinduced contractions, toward baseline levels, in all samples examined (Fig. 2A). The relative AUC values, compared with those assessed prior to the experiment, showed significant increase after morphine treatment, but those were decreased after 3 min of naloxone treatment in the rectum and distal colon (Fig. 2B). Because of the low-level contractile responses to morphine in the other colonic regions, statistically significant differences were not detected in these regions (data not shown). According to these results, the contractions induced by morphine treatment were predominantly due to MOR activation in the intestinal circular muscle fragments.
Quantitative RT-PCR Total RNA was prepared from each fragment of the small and large intestine (8-mm-long ring fragments sampled as for the contractile analysis, above) using the RNeasy Mini kit (Qiagen, Tokyo, Japan). Reverse transcription was performed on total RNA (0.5 lg) using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Tokyo, Japan). Samples contained 19 SYBR Premix Ex Taq II, 19 ROX II (Takara, Shiga, Japan), 10-lM gene-specific primers, and 500-ng synthesized cDNA in a volume of 20 lL. Quantitative polymerase chain reaction (PCR) was performed in duplicate using an Applied Biosystems 7500/7500 Fast Real-time PCR system (Life Technologies) as follows: 1 cycle at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as an internal control. The gene-specific primers for MOR (MA086354 F and R) and GAPDH (MA050371 F and R) were purchased from Takara.
Large intestinal transit Large intestinal transit (from transverse colon to rectum) was assessed using a bead expulsion assay. Although the insertion depth of each bead is commonly 2 cm from the anus in mouse experiments,22,23 we used depths of insertion of 0.5, 1, 1.5, 2, 2.5, or 3 cm from the anus, to determine the locus associated with constipation among the transverse colon (3 cm from the anus), distal colon (2.5, 2, 1.5 cm from the anus), and rectum (1, 0.5 cm from the anus). Prior to the experiment, mice were habituated individually as described above, with food restricted for 12 h. For the experiment, a glass bead (2-mm diameter; Hamada Rika, Osaka, Japan) was inserted through the anus using a thin plastic tube to determine the control value of the bead expulsion time. Saline or morphine (s.c.) was administered to each mouse; 10 min later, a glass bead was inserted as in the pre-experiment and the expulsion time for each mouse was measured.
Statistical analysis Data are shown as means SEM. The SAS software (ver. 9; SAS Institute, Cary, NC, USA) was used for statistical analyses. The significance of differences among groups was assessed by one-way or two-way ANOVA followed by Dunnett’s comparison test. In all analyses, p < 0.05 was taken to indicate statistical significance.
RESULTS Effects of morphine on mouse intestinal circular muscle fragments We first examined the contractile response of the mouse muscle fragments to morphine using isometric
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Figure 1 Morphine-induced contractions of circular muscle fragments in mouse small and large intestines. (A) A model of the mouse intestine. Each intestinal region was sampled at the locus shown. (B) Effects of morphine (10 lM) on the small (jejunum and ileum) and large (proximal, transverse, transverse to distal, distal colon, and rectum) intestines of a single mouse. The arrow indicates morphine treatment. (C) Dose–response of contractile changes during the initial 3 min of morphine (0.1–100 lM) exposure was determined by calculating the area-under-the-curve (AUC). Left and right graphs show the small and large intestines, respectively. *p < 0.05, **p < 0.01, vs the pretreatment value, n = 6–16. Each value represents the mean SEM.
tial MOR expression, we next investigated the expression levels of MOR in each region. Although verifying MOR protein expression levels by immunohistochemistry or Western blotting may be the ideal method, there are at present no commercially available
Measurement of MOR mRNA expression levels in the small and large intestines To reveal whether the differences in contractile responses among the segments were due to differen-
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Figure 2 Effect of l-opioid receptor antagonist (naloxone) on morphine-induced circular muscle contraction. (A) Naloxone (1 lM) was administered after 5-min exposure to morphine (10 lM). Black and gray arrows indicate the timing of morphine and naloxone treatments, respectively. (B) Contractile responses to 3-min exposure to morphine and naloxone determined by area-under-the-curve (AUC). **p < 0.01, #p < 0.05, and n.s. (non-significant), vs the pretreatment value (distal colon; p = 0.81, rectum; p = 0.81 for morphine with naloxone), n = 8. Each value represents the mean SEM.
antibodies specific for MOR.24 We thus compared mRNA expression levels in the fragments of the intestine by quantitative RT-PCR. Primers were selected from the sequences of exons 2 and 3 of the mouse MOR, because these regions are conserved among the known MOR splicing variants.25 Expression was 10-fold higher in the large intestine than the small intestine segments. There were no significant differences in expression among the segments of the small intestine and large intestine (Fig. 3). Thus, circular muscle contraction was correlated with MOR expression in the small and large intestines, but not among the various regions of the large intestine.
Continuous contractions of large intestinal circular muscles during morphine exposure Although marked responses to morphine were detected in the transverse colon to the rectum (Fig. 1B), the continuity of the contractions during morphine exposure seemed to differ between the rectum and colonic regions. We next compared the sustainability of the morphine-induced contractile responses of the circular muscles of the transverse colon to the rectum by exposure to morphine for longer durations. During 30 min of morphine (10 lM) treatment, contractions were measured at 5-min intervals from 0 min (time of morphine administration) to 30 min. In the rectum,
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Figure 3 l-opioid receptor (MOR) expression in the small and large intestines. Total mRNA was extracted from each segment of the intestine, dissected as in Fig. 1, and MOR expression levels were determined by quantitative RT-PCR. **p < 0.01, vs the rectum, n = 4. Each value represents the mean SEM.
layers.26 To ascertain the role of NOS in the degradation of the contractions in the colon, the effect of a NOS inhibitor (L-NAME) was examined. Using the same methods as in Fig. 1, tissues were prepared and habituated in an organ bath for 5 min. Morphine (10 lM) was then added and after 20 min of exposure, L-NAME (50 lM) was added and the contractile responses before and after L-NAME treatment were measured. Because the transverse and transverse to distal colon exhibited less marked responses to morphine (Fig. 4), we compared the effects of L-NAME in the distal colon and rectum. In the distal colon, morphine-evoked contractions decreased during exposure. At this time, additional treatment with L-NAME resulted in gradual recovery of the contractile responses to a similar extent to the contractions at 0–3 min of morphine treatment (Fig. 5A). In the rectum, no response to L-NAME was detected. As shown in Fig. 5B, the decreased AUC value during 15–18 min of morphine exposure compared with 0–3 min of initial morphine exposure was recovered significantly with L-NAME treatment in the distal colon, while the AUC was unchanged in the rectum. These data suggest that contractile degradation during morphine exposure in the colonic circular muscles is a result of NOS production. We further focused on NOS–NO pathway to understand the mechanism underlying difference in the morphine-induced continuous contraction between the colon and rectum. To see the contribution of NO on contractile degradation, effect of NO donor SNAP was examined. Using the same method as in Fig. 1, the distal colon and rectum were equilibrated to the organ bath and morphine (10 lM) was first administered. After 3 min of morphine exposure, which is the timing before contractile degradation occurs in the distal colon as shown in Fig. 4, SNAP (50 lM) was added to the bath and contractile responses were measured for 3 min, respectively. As shown in Fig. 6A and B, SNAP mark-
strong and continued contractile responses were detected until the end of the measurements (Fig. 4). In contrast, although contractile changes were detected at first, those waves were gradually attenuated in the transverse, transverse to distal, and distal colon. Thus, continuous contraction of the circular muscles was detected only in the rectum, suggesting that the mechanism of contraction may differ between the rectum and colon.
Contribution of nitric oxide (NO) to the contractile degradation in the colon In the mouse intestine, more than 50% of enteric neurons express nNOS,26 and expression and production levels differ according to region, causing region-specific reductions in muscle contraction. In this regard, the colon has the highest density of nitric oxide synthase (NOS)-immunoreactive neurons, mainly in the fibers of the myenteric plexus, projecting to the circular muscle
Figure 4 Sustainability of the contractile response during morphine exposure in the large intestine. Morphine (10 lM)-induced contractile responses in the transverse colon to the rectum were measured during 30-min exposure to morphine at 5-min intervals from 0 min (time of morphine administration) for 30 min determined by area-under-thecurve (AUC). *p < 0.05, vs the pretreatment value, n = 10–14. Each value represents the mean SEM.
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Figure 5 Effect of NOS inhibitor (L-NAME) on morphine-induced circular muscle contraction in the distal colon and rectum. (A) L-NAME (50 lM) was added after 20-min exposure to morphine (10 lM). Black and gray arrows indicate the timing of morphine and L-NAME treatment, respectively. (B) Contractile responses to 3-min exposure to morphine and L-NAME determined by area-under-the-curve (AUC). **, ##p < 0.01, and n.s. (nonsignificant), vs 15–18 min of morphine exposure (rectum; p = 0.82 for 0–3 min morphine treatment, and p = 0.44 for morphine with L-NAME), n = 8–10. Each value represents the mean SEM.
Effects of morphine on bead transit latency in the regions of the large intestine
edly and similarly decreased the contraction of the distal colon and rectum, suggesting that these regions have ability to respond to NO and that NO has an important role for inhibiting circular muscle contraction. We next examined the contribution of NOS to the degradation of contraction during morphine exposure in the colon by using the NO substrate (L-arginine). Using the similar method as in Fig. 4, morphine (10 lM) was added with or without L-arginine (200 lM) to each organ bath and contractions of the distal colon and rectum were measured at 5-min intervals from 0 min (time of morphine or morphine+L-arginine administration) to 30 min. As shown in Fig. 6C, coadministration of Larginine significantly inhibited the AUC value of morphine-evoked contraction in the distal colon, while it had modest effect in the rectum. As NOS converts Larginine to NO and citrulline in accordance with its activity, the effect of L-arginine reflects the NOS activity level. Therefore, these results also suggested the different activation of NOS between the colon and rectum during morphine exposure, which may cause different contractile sustainability in these regions.
Morphine-evoked contraction in the large intestine increased with proximity to the anus (Fig. 1A). We thus examined whether those in vitro results were correlated with opioid-induced constipation in vivo using a bead expulsion assay. To confirm the effective dose of morphine, the morphine dose–response was examined by inserting a bead at a 2-cm depth from the anus 10 min after morphine administration (Fig. 7A). Morphine treatment resulted in a dosedependent increase in bead expulsion time, and 3 and 10 mg/kg morphine sufficiently delayed bead expulsion compared with pre-experiment values. We thus used 3 mg/kg morphine and measured bead expulsion times after insertion at various depths from the anus. In terms of the depth of bead insertion, 0.5 and 1 cm were intended to show the constipation effect in the rectum, 1.5, 2 and 2.5 cm for the distal colon, and 3 cm for the transverse colon. Following systemic morphine administration, beads were
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Figure 6 Effect of nitric oxide (NO) donor (SNAP) and NO substrate (L-arginine) on morphine-induced circular muscle contraction in the distal colon and rectum. (A) SNAP (50 lM) was added after 3-min exposure to morphine (10 lM). Black and gray arrows indicate the timing of morphine and SNAP treatment, respectively. (B) Contractile responses to 3-min exposure to morphine and SNAP determined by area-under-the-curve (AUC). **p < 0.01, #p < 0.05, and n.s. (non-significant), vs the pretreatment value (distal colon; p = 0.23, rectum; p = 0.61 for morphine with SNAP), n = 7. (C) Effect of L-arginine (200 lM) on contraction during 30-min morphine exposure (10 lM). *p < 0.05 between morphine and morphine+L-arginine group, n = 10–11. Each value represents the mean SEM.
parts of the intestines, makes a major contribution to morphine-induced constipation.
inserted and the bead expulsion time was measured. At all depths examined, morphine significantly increased the bead expulsion time compared with the prevalue (Fig. 7B). The bead expulsion time did not differ according to depth of insertion, indicating that the rectum plays an important role in morphineinduced constipation. These in vivo results are consistent with the in vitro data (Fig. 1), suggesting that the greater and longer contractile response of the circular muscles in the rectum, compared to the other
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DISCUSSION In the present study, we focused on the effects of morphine on mouse intestinal circular muscle contraction and compared the contractile waves among the histologically known regions. The strongest contraction after morphine treatment was detected in the rectum.
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rectum. Therefore, the rectum is the region primarily involved in opioid-induced constipation. Morphine-induced circular muscle contraction was greater in the large intestine than the small intestine. Although the quantitative RT-PCR experiments were performed using whole-tissue RNA extracted from the respective segments of the intestine, and we did not examine MOR expression at the myenteric plexus in detail, levels of contraction—in terms of AUC values— between the small and large intestines seemed to be correlated with MOR mRNA levels. Previous study supports our finding of weak MOR expression in the mouse ileum, that electrical stimulation-evoked contraction was not inhibited by morphine.27 In this regard, it was assumed that low MOR expression caused the weak effect of morphine in the small intestine. Another possible mechanism for the difference between the small and large intestines, different contributions of intracellular signaling between the small and large intestines has been reported. Raehal et al. found that G protein-coupled receptor kinases 6 (GRK6)-knockout mice expelled beads inserted from the anus more rapidly than did wild-type controls after morphine administration.23 In contrast, lack of GRK6 had no effect on morphine-induced delay in the small intestine, as determined by charcoal meal transit, between these animals. A similar difference between the small and large intestines has also been reported in b-arrestin2-knockout animals. Lack of b-arrestin2 reversed both the acute morphine-evoked bead expulsion delay and reduced defecation in the large intestine, whereas it had no effect in the small intestine.28 In addition, lack of b-arrestin2 enhanced morphineinduced contractile tolerance of the large intestine, but not of the small intestine.21,29 Although b-arrestin2 is well known as a key adaptor protein in receptor endocytosis, it also functions as a positive regulator of the intracellular signaling that up-regulates mitogen-activated protein kinase pathways,19,30 which is assumed to mediate constipation.21 In these respects, activation of the intracellular pathways mediating MOR might be fundamentally different between the small and large intestines, and this might cause lesser contribution of small intestine on opioid-induced constipation. In the large intestine, MOR mRNA levels were similar in all segments examined by RT-PCR, whereas the morphine contractile response was stronger in the distal colon and rectum. We considered two possibilities to explain this. Firstly, this may be caused by the different thickness of the muscle along the large intestine. Our preliminary data using carbachol, a
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Figure 7 Large intestinal transit measured by bead expulsion assay. (A) A glass bead (2-mm diameter) was inserted 2 cm from the anus 10 min after morphine (s.c.) administration. Bead expulsion times for each mouse were measured. **p < 0.01, vs the pretreatment value, n = 6–9. (B) Glass bead was inserted 0.5–3 cm in 0.5-cm intervals from the anus before and after morphine (3 mg/kg, s.c.) administration. No significant difference according to insertion depth was detected pre- or posttreatment. p > 0.05, vs pre- (p = 0.68–1.0) or post- (p = 0.38–0.97) 0.5-cm insertion values, respectively, n = 5–7. Each value represents the mean SEM.
Although the colonic regions examined also showed marked contraction, the responses were greater on the distal side than the proximal side. The response in the jejunum and ileum was markedly lower than in the large intestine, with only slight changes in contraction upon morphine treatment. Furthermore, we compared the sustainability of the contractions during morphine exposure among the regions of the large intestine. While there were contractile degradations in the transverse colon to distal colon, a continuous response was detected in the rectum. These results are consistent with the in vivo bead expulsion assay results; after morphine administration, inserted beads seemed to be propelled from the oral to anal side, stopping at the
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to respond to NO if there is enough levels of NO. It is reported that intrinsic density of NOS-immunoreactive cells is higher in the colon, vs other parts of the intestines under the normal state.34 In addition, NOS shows higher activity in the proximal side than the distal side of the large intestine,34 which may also account for the prolonged contraction in the rectum compared to the colon. Morphine is known to affect NOS activity through MOR located on nitrergic nerves.19,26,35 According to the human saphenous vein, NO release starts to increase in 5 min after morphine administration.36 Thus, in addition to difference in intrinsic NOS activity, different contractile degradation between colon and rectum may also result from difference in morphine-induced NOS activation. Although the time course and region specificity of NOS activation by morphine in the intestine is not known, it is possible that additional NOS activation by morphine enhanced contractile degradation during 30-min exposure in the colon. Although little is known about the mechanism of the difference in contractile sustainability between the colon and rectum, these two possibilities, i.e., regional- and morphine-dependent differences, abundant NOSs are produced and may account for contractile degradation of the circular muscles in the colonic regions but not in the rectum. The in vivo bead expulsion assay showed consistent results, suggesting the importance of the rectum in opioid-induced constipation. Raehal et al. (2005) showed that a single injection of morphine could reduce defecation even after 6 h, indicating that our results were not due to elimination of morphine itself.29 In our study, the expulsion time after morphine administration did not differ according to bead insertion depth. Thus, it seems that fecal pellets move through the colon to the rectum whether they are inserted into the transverse or distal regions of the colon, and then stop at the rectum. According to our in vitro data, because of the non-sustained contraction of the circular muscles in the colon, the bead may transit these areas and then stop at the rectum due to the high amplitude and prolonged contractions. Although few data suggest a relationship between the rectum and opioid-induced constipation, opioidaddicted patients who undergo colectomies are reported to remain constipated, possibly due to rectal dysfunction.37 In addition, opioid-used patients are considered to associate with stool-filled rectum with an impacted fecal mass in the anorectal region.38 Thus, our results suggest that the mechanism of opioidinduced constipation involves abnormal contractions in the rectum that can induce fecal incontinence.
muscarinic receptor agonist, showed that circular muscles undergo larger contractions in the distal regions (distal colon and rectum) vs the proximal regions (proximal and transverse colon; data not shown). Although no report is available in mice, human anatomical studies of the distal colon to the rectum show that the thickness of the circular muscle layer increases toward the anus,31 consistent with this hypothesis. A second possibility is that while we assume that MOR mRNA levels in the ring samples of the intestines are similar, MOR expression in the myenteric plexus may be higher on the anal than the oral side, which may induce higher contractions toward the rectum. Although we could not use immunohistochemical approach,24 reports using affinity-purified antibody to the C-terminus of rat MORs showed positive staining of the neurons of the myenteric plexus in all regions in the intestine with different expression levels according to regions.13 Because we have little evidence for these two hypotheses, we should further examine for their participation to explain the different contraction of the large intestinal regions in the future. We found the continuous contraction in the rectum, but not in the colonic regions. To identify the mechanism underlying these differences, it was considered that MOR desensitization levels are different among regions. Although no report is available for the rectum, it has been reported that repeated exposure to DAMGO or fentanyl lead to b-arrestin2-associated MOR desensitization and following contractile degradation in the colon but not in the ileum, suggesting the different mechanism leading to MOR desensitization among the intestinal regions.29 In addition to this, it was also considered that different intracellular mechanism that leads to contractile degradation may be different. On account of the second possibility, we focused on NOS–NO pathway because this was thought to be most likely to influence circular muscle contraction. In the circular muscle layers, NO is an important inhibitory neurotransmitter to regulate muscle contraction.16,32,33 We found the opposite effect of NOS inhibitor (L-NAME) and NO substrate (L-arginine) on the distal colon contraction that decreased contraction was recovered by L-NAME to the levels during the initial morphine treatment, while contractile degradation was enhanced by L-arginine. It was noteworthy that less effect of these reagents were observed in the rectum, suggested that lower activation of NOS–NO causes lower degradation of contraction. This is also supported by SNAP results that the rectum has ability
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ingly, the rectum is likely the key region involved in opioid-induced constipation.
Although we did not examine the effect of NO in the in vivo assay, earlier study has shown that L-NAME can dose-dependently and completely block the fecal pellet propulsion of the large intestine.33 However, alternative neurons such as cholinergic and serotonergic neurons and the neuronal network of these neurons are also important for peristaltic movement. Therefore, future studies are needed to clarify the mechanisms controlling opioid-induced constipation. Because previous reports have addressed morphineinduced contraction of circular muscle segments in specific regions of the intestine, our study represents the first comprehensive analysis to distinguish contractile strength among the known regions of the small and large intestine. To our knowledge, this is the first report of the likely significance of the rectum, which showed a higher contractile response to morphine with sustained contractions compared with the other regions of the intestine. These data suggest the mechanism of delayed bead transit in the rectum. Accord-
REFERENCES 1 Twycross RG, Lack SA. Control of Alimentary Symptoms in Far Advanced Cancer. London: Churchill Livingstone, 1986: 166–207. 2 Schug SA, Zech D, Grond S. Adverse effects of systemic opioid analgesics. Drug Saf 1992; 7: 200–13. 3 Holzer P, Ahmedzai SH, Niederle N, Leyendecker P, Hopp M, Bosse B, Spohr I, Reimer K. Opioid-induced bowel dysfunction in cancer-related pain: causes, consequences, and a novel approach for its management. Opioid Manag 2009; 5: 145–51. 4 Roy S, Liu HC, Loh HH. Mu-Opioid receptor-knockout mice: the role of mu-opioid receptor in gastrointestinal transit. Brain Res Mol Brain Res 1998; 56: 281–3. 5 Stewart JJ, Weisbrodt NW, Burks TF. Central and peripheral actions of morphine on intestinal transit. J Pharmacol Exp Ther 1978; 205: 547–55. 6 Mori T, Shibasaki Y, Matsumoto K, Shibasaki M, Hasegawa M, Wang E, Masukawa D, Yoshizawa K et al. Mechanisms that underlie l-opioid receptor agonist-induced constipation; differential involvement of l-opioid receptor sites and responsible regions. J Pharmacol Exp Ther 2013; 347: 91–9. 7 Daniel EE, Sutherland WH, Bogoch A. Effects of morphine and other
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FUNDING No funding is associated with the article.
DISCLOSURE None of the authors have any competing interest to disclose relating to this submission. H. Ono, A. Nakamura, G. Sakaguchi, and T. Kanemasa are employees of Shionogi and Co., Ltd, the manufacture of morphine.
AUTHOR CONTRIBUTION HO performed all the research; HO and AN analyzed the data and wrote the article; KM and SH instructed the methods of in vivo and in vitro methods; HO, AN, KM, SH, GS, and TK conceived and directed research. All authors discussed the results and commented on the manuscript.
drugs on motility of the terminal ileum. Gastroenterology 1959; 36: 510–23. Tavani A, Bianchi G, Ferretti P, Manara L. Morphine is most effective on gastrointestinal propulsion in rats by intraperitoneal route: evidence for local action. Life Sci 1980; 27: 2211–7. Foss JF, Bass AS, Goldberg LI. Doserelated antagonism of the emetic effect of morphine by methylnaltrexone in dogs. J Clin Pharmacol 1993; 33: 747–51. Gatti A, Sabato AF. Management of opioid-induced constipation in cancer patients: focus on methylnaltrexone. Clin Drug Investig 2012; 32: 293–301. DeHaven-Hudkins DL, DeHaven RN, Little PJ, Techner LM. The involvement of the mu-opioid receptor in gastrointestinal pathophysiology: therapeutic opportunities for antagonism at this receptor. Pharmacol Ther 2008; 117: 162–87. Sternini C, Spann M, Anton B, Keith DE Jr, Bunnett NW, von Zastrow M, Evans C, Brecha NC. Agonist-selective endocytosis of mu opioid receptor by neurons in vivo. Proc Natl Acad Sci U S A 1996; 93: 9241–6. Bagnol D, Mansour A, Akil H, Watson SJ. Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract. Neuroscience 1997; 81: 579– 91.
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14 Kromer W. Endogenous and exogenous opioids in the control of gastrointestinal motility and secretion. Pharmacol Rev 1988; 40: 121–62. 15 Wood JD, Galligan JJ. Function of opioids in the enteric nervous system. Neurogastroenterol Motil 2004; 16: 17–28. 16 Iwata H, Tsuchiya S, Nakamura T, Yano S. Morphine leads to contraction of the ileal circular muscle via inhibition of the nitrergic pathway in mice. Eur J Pharmacol 2007; 574: 66– 70. 17 Bitar KN, Makhlouf GM. Selective presence of opiate receptors on intestinal circular muscle cells. Life Sci 1985; 37: 1545–50. 18 Kosterlitz HW, Lees GM. Pharmacological analysis of intrinsic intestinal reflexes. Pharmacol Rev 1964; 16: 301–39. 19 Lenard L Jr, Halmai V, Barth o L. Morphine contracts the guinea pig ileal circular muscle by interfering with a nitric oxide mediated tonic inhibition. Digestion 1999; 60: 562–6. 20 Ross GR, Gabra BH, Dewey WL, Akbarali HI. Morphine tolerance in the mouse ileum and colon. J Pharmacol Exp Ther 2008; 327: 561–72. 21 Kang M, Maguma HT, Smith TH, Ross GR, Dewey WL, Akbarali HI. The role of b-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal
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tract. J Pharmacol Exp Ther 2012; 340: 567–76. Coates MD, Johnson AC, GreenwoodVan Meerveld B, Mawe GM. Effects of serotonin transporter inhibition on gastrointestinal motility and colonic sensitivity in the mouse. Neurogastroenterol Motil 2006; 18: 464–71. Raehal KM, Schmid CL, Medvedev IO, Gainetdinov RR, Premont RT, Bohn LM. Morphine-induced physiological and behavioral responses in mice lacking G protein-coupled receptor kinase 6. Drug Alcohol Depend 2009; 104: 187–96. Smith TH, Grider JR, Dewey WL, Akbarali HI. Morphine decreases enteric neuron excitability via inhibition of sodium channels. PLoS ONE 2012; 7: e45251. Majumdar S, Grinnell S, Le Rouzic V, Burgman M, Polikar L, Ansonoff M, Pintar J, Pan YX et al. Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects. Proc Natl Acad Sci USA 2011; 108: 19778–83. Baccari MC, Traini C, Garella R, Cipriani G, Vannucchi MG. Relaxin exerts two opposite effects on mechanical activity and nitric oxide synthase expression in the mouse
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colon. Am J Physiol Endocrinol Metab 2012; 303: E1142–50. Smith CF, Waldron C, Brook NA. Opioid receptors in the mouse ileum. Arch Int Pharmacodyn Ther 1988; 291: 122–31. Raehal KM, Walker JK, Bohn LM. Morphine side effects in beta-arrestin 2 knockout mice. J Pharmacol Exp Ther 2005; 314: 1195–201. Maguma HT, Dewey WL, Akbarali HI. Differences in the characteristics of tolerance to l-opioid receptor agonists in the colon from wild type and b-arrestin2 knockout mice. Eur J Pharmacol 2012; 685: 133–40. Whalen EJ, Rajagopal S, Lefkowitz RJ. Therapeutic potential of b-arrestinand G protein-biased agonists. Trends Mol Med 2011; 17: 126–39. Shafik A, Doss S, Asaad S, Ali YA. Rectosigmoid junction: anatomical, histological, and radiological studies with special reference to a sphincteric function. Int J Colorectal Dis 1999; 14: 237–44. Manaka H, Manaka Y, Kostolanska F, Fox JE, Daniel EE. Release of VIP and substance P from isolated perfused canine ileum. Am J Physiol 1989; 257: G182–90. Foxx-Orenstein AE, Grider JR. Regulation of colonic propulsion by
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enteric excitatory and inhibitory neurotransmitters. Am J Physiol 1996; 271: G433–7. Takahashi T, Owyang C. Regional differences in the nitrergic innervation between the proximal and the distal colon in rats. Gastroenterology 1998; 115: 1504–12. Stefano GB, Zhu W, Cadet P, Bilfinger TV, Mantione K. Morphine enhances nitric oxide release in the mammalian gastrointestinal tract via the l3 opiate receptor subtype: a hormonal role for endogenous morphine. J Physiol Pharmacol 2004; 55: 279–88. Stefano GB. Autoimmunovascular regulation: morphine and anandamide and ancondamide stimulated nitric oxide release. J Neuroimmunol 1998; 83: 70–6. Christiansen J, Rasmussen OO. Colectomy for severe slow-transit constipation in strictly selected patients. Scand J Gastroenterol 1996; 31: 770–3. McMillan SC. Assessing and managing opiate-induced constipation in adults with cancer. Cancer Control 2004; 11: 3–9.
doi: 10.1111/nmo.12387
Circular muscle contraction in the mice rectum plays a key role in morphine-induced constipation H. ONO ,* A. NAKAMURA ,* K. MATSUMOTO ,†,‡ S. HORIE ,† G. SAKAGUCHI *
& T. KANEMASA *
*Pain & Neurology, Medicinal Research Laboratories, Shionogi and Co., Ltd., Toyonaka, Osaka, Japan †Laboratory of Pharmacology, Josai International University, Togane, Chiba, Japan ‡Division of Pathological Sciences, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, Japan
Key Messages
• Opioid-induced constipation is considered as a major and difficult problem in the clinical field. • Focusing on morphine response of the small and large intestinal regions, we aimed to identify the key region involved in opioid-induced constipation.
• Using in vitro isometric recording of the circular muscle contraction and in vivo bead expulsion time assay in mice, we found that the rectum is likely the key region for opioid-induced constipation.
Abstract Background Although opioids induce intestinal muscle contraction and provoke constipation, the intestinal region(s) that contribute to the constipation have remained unclear. We report here a region-specific response of intestinal muscle contraction to morphine and its correlation with in vivo constipation. Methods Regions of mice small and large intestines were dissected histologically and circular muscle contractile responses were measured using isometric transducers. Bead expulsion assays were performed to assess in vivo constipation. Key Results The strongest contraction in response to morphine was detected in the rectum. The distal and transverse colon also showed strong contractions, whereas weak responses were detected in the proximal colon, jejunum, and ileum. Regarding the sustainability of muscle contractions during morphine exposure, prolonged waves were detected only in the rectum, while the waves diminished gradually in other regions. To identify the mechanism(s) underlying this
difference, we focused on nitric oxide synthase (NOS). In the distal colon, decreased contraction during morphine exposure was recovered by application of a NOS inhibitor (L-NAME), while a NOS substrate (L-arginine) enhanced contractile degradation. In contrast L-NAME and L-arginine modestly affected the sustained contraction in the rectum. To confirm the correlation with constipation, beads were inserted into the transverse colon, distal colon, or rectum after morphine administration and expulsion times were examined. Beads tended to stop at the rectum even when inserted in the deeper colonic regions. Conclusions & Inferences The rectum showed the greatest response to morphine in both in vitro and in vivo analyses, therefore it may play a key role for opioidinduced constipation. Keywords constipation, morphine, nitric oxide (NO), rectum, l-opioid receptor (MOR). Abbreviations: AUC, area-under-the-curve; MOR, l-opioid receptor; NO, nitric oxide; NOS, nitric oxide synthase; PCR, polymerase chain reaction.
Address for Correspondence Toshiyuki Kanemasa, PhD, Pain & Neurology, Medicinal Research Laboratories, Shionogi & Co., Ltd, 1-1, 3-chome, Futaba-cho, Toyonaka, Osaka 561-0825, Japan. Tel: +81-6-6331-6654; fax: +81-748-88-6508; e-mail: [email protected] Received: 28 November 2013 Accepted for publication: 6 June 2014
INTRODUCTION Morphine is a l-opioid receptor (MOR) agonist that is used clinically for patients suffering from moderate-to-
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induced contractile response in the individual regions of the intestines. In addition, the locus that makes the greatest contribution to opioid-induced constipation is not clear. Thus, to assess the contribution of the various intestinal regions in opioid-induced constipation, in the present study, we compared morphine-induced circular muscle contractile potency and sustainability in histologically known regions (jejunum, ileum, proximal, transverse, distal colon, and rectum) of the mouse intestine. We found that contractile responses to morphine differed among the regions. Furthermore, this contractile efficacy was correlated with in vivo bead propulsion data. Thus, we identified the intestinal region responsible for morphine-induced constipation.
severe pain. Although morphine and other MOR agonists show potent analgesic effects, their side effects, such as constipation, nausea, and drowsiness, often make their continuous use difficult.1 Among these side effects, opioid-induced constipation is known to occur in more than 90% of patients given opioids for pain management.1 In addition, although tolerance to other side effects often occurs, constipation seems resistant to tolerance and continues during opioid use.2,3 Thus, constipation is considered a major and difficult to avoid clinical issue. To date, numerous pharmacological studies of the mechanisms of MOR agonist-induced constipation have been reported. This is considered to be a direct effect of MORs because gastrointestinal transit inhibition is abolished in MOR-knockout animals.4 Expression of the MOR has been detected in many central and peripheral tissues. Although opioids act on MOR in the central nerves system and indirectly induce constipation,5,6 it is also widely accepted that opioids act directly on MORs in intestinal neurons, changing gastrointestinal motility, consequently inducing constipation.7,8 Indeed, a peripheral MOR antagonist has been shown to relieve morphine-induced constipation in animal studies and clinical trials.9,10 In the intestine, autoradiographic binding of MOR agonists has been detected.11 In addition, high MOR expression has been identified in myenteric plexus, which regulates neuronal activity in the intestines.12,13 Because MORs are also expressed in the internal border of the circular muscles, the inner muscle layers of myenteric plexus, agonists are thought to act on the nerve terminals of these neurons and affect muscle activity.13–16 In contrast, in the longitudinal smooth muscle layers located at the outer layers of the myenteric plexus, MOR expression has been reported to be lower than that in the circular muscles.17 In addition, because the circular muscle layers are typically much thicker than the longitudinal muscles and play the dominant role in peristalsis,18 we assumed that the circular muscles play a more important role in opioidinduced constipation. In studies examining tissue preparations in vitro, treatment with several MOR agonists has been shown to reduce circular muscle relaxation by inhibiting inhibitory neurotransmitter release and increasing phasic contractile activity of muscles, reducing peristaltic movement from the proximal (oral) to the distal (anal) side of the intestine.15,19–21 Many of these reports use primarily the ileum and colon to compare the effects of each agonist. Despite these, to our knowledge, there is little information regarding the magnitude of the opioid-
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MATERIALS AND METHODS Animals Male ddY mice (5–7-week old; Japan SLC, Hamamatsu, Japan) were used. Animals were housed in a room maintained at 23 1 °C under a 12/12-h light/dark cycle and allowed access to water and food ad libitum. All procedures for animal experiments were approved by the Animal Care and Use Committee of Shionogi Research Laboratories, Osaka, Japan, and were consistent with the internal guidelines for animal experiments and in adherence to the ethics policy of Shionogi & Co., Ltd (Osaka, Japan).
Drugs Morphine hydrochloride was from Shionogi & Co., Ltd. Naloxone was from Tocris Bioscience (Bristol, UK). N’-nitro-L-argininemethyl ester hydrochloride (‘L-NAME’) and S-nitroso-N-acetyl-D, L-penicillamine (‘SNAP’) were from Cayman Chemical Company (Ann Arbor, MI, USA). L-arginine was from Sigma-Aldrich (Tokyo, Japan). Agents were dissolved in 0.9% physiological saline (Otsuka Pharmaceutical, Tokyo, Japan) for in vivo experiments and in purified water for in vitro experiments.
Contractile analysis of mouse circular muscles Prior to the experiment, mice were individually habituated in a cage with mesh wire inserted in the bottom; food was restricted for 12 h and water was available ad libitum. Mice were sacrificed by cervical spine fracture/dislocation and the small and large intestines were removed and placed in Krebs–Henseleit solution (NaCl 112.08 mM, KCl 5.90 mM, CaCl2 1.97 mM, MgCl2 1.18 mM, NaH2PO4 1.22 mM, NaHCO3 25.00 mM, and glucose 11.49 mM) bubbled with 95% O2 and 5% CO2 at 37 °C until use. Segments of the jejunum (10-cm distal to the stomach) and ileum (5-cm proximal to the cecum) were dissected from the small intestine, and the proximal colon (0.5-cm distal to the cecum), transverse colon (3-cm proximal to the anus), distal colon (1.5-cm proximal to the anus), and rectum (0.3-cm proximal to the anus) were dissected from the large intestine (8 mm in length). Tissues were placed under 0.5-g tension with a thin triangle hook (side length, 8 mm) suspended along the axis of the circular muscle in a 10-mL organ bath (Krebs–Henseleit solution bubbled with 95% O2 and 5% CO2 at 37 °C) and equilibrated for 5 min until drug treatment. Drugs were
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administered by 10 lL in a total volume of 10 mL. Contractions were recorded before and after exposure to each drug (morphine, naloxone, L-NAME, SNAP, and L-arginine) with an isometric transducer (TB-611T; Nihon Kohden, Tokyo, Japan) connected to an amplifier (AD-632J; Nihon Kohden) and PowerLab/4SP (AD Instrument, Castle Hill, NSW, Australia) and analyzed using the LabChart 6.1.3 software (AD Instrument). The waves of the contractions were analyzed in terms of area-under-the-curve (AUC) using the LabChart software. Each tissue was used only one time in each experiment.
recording methods. The ring preparations of the small (jejunum and ileum) and large (proximal, transverse, transverse to distal, distal colon, and rectum) intestines were equilibrated in organ baths for 5 min, and then contractions were measured during morphine exposure. Fig. 1A illustrates the regions sampled for each segment, and Fig. 1B shows representative recorded changes in contractile waves in the mouse intestine before and after morphine exposure (10 lM). In the jejunum, ileum, and proximal colon, rhythmical contractions were detected both before and after morphine treatment, and the amplitude of the waves exhibited slight changes after morphine treatment. In the transverse colon to the rectum, the amplitude of waves before morphine was similar, and was increased soon after morphine treatment. Dose–response changes were measured for 3 min during morphine (0.1–100 lM) exposure and were assessed in terms of AUC (Fig. 1C). The relative AUC values, compared with those assessed prior to the experiment, increased dose-dependently in the large intestine, with the highest value in the rectum. In addition, AUC values in the large intestine were higher in the terminal side of the large intestine. Weak responses were detected in the small intestine (Fig. 1B). Because the doses used here were similar to previous studies in mice,16 we assume that the contractile responses detected were not due to use of excessive doses of morphine. The results suggested differences in the contractile ability of each locus of the intestinal circular muscles, and that the rectum showed the greatest response to morphine. Using naloxone, we determined whether the contractile changes in the colonic regions and rectum were due to MOR stimulation by morphine. The dose of naloxone used was 1 lM, because this dose has been reported to antagonize MOR selectively.19 In all segments examined, morphine (10 lM) was added as described in Fig. 1, followed 5 min later by naloxone; changes in the waves were then assessed. Naloxone inhibited morphineinduced contractions, toward baseline levels, in all samples examined (Fig. 2A). The relative AUC values, compared with those assessed prior to the experiment, showed significant increase after morphine treatment, but those were decreased after 3 min of naloxone treatment in the rectum and distal colon (Fig. 2B). Because of the low-level contractile responses to morphine in the other colonic regions, statistically significant differences were not detected in these regions (data not shown). According to these results, the contractions induced by morphine treatment were predominantly due to MOR activation in the intestinal circular muscle fragments.
Quantitative RT-PCR Total RNA was prepared from each fragment of the small and large intestine (8-mm-long ring fragments sampled as for the contractile analysis, above) using the RNeasy Mini kit (Qiagen, Tokyo, Japan). Reverse transcription was performed on total RNA (0.5 lg) using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Tokyo, Japan). Samples contained 19 SYBR Premix Ex Taq II, 19 ROX II (Takara, Shiga, Japan), 10-lM gene-specific primers, and 500-ng synthesized cDNA in a volume of 20 lL. Quantitative polymerase chain reaction (PCR) was performed in duplicate using an Applied Biosystems 7500/7500 Fast Real-time PCR system (Life Technologies) as follows: 1 cycle at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as an internal control. The gene-specific primers for MOR (MA086354 F and R) and GAPDH (MA050371 F and R) were purchased from Takara.
Large intestinal transit Large intestinal transit (from transverse colon to rectum) was assessed using a bead expulsion assay. Although the insertion depth of each bead is commonly 2 cm from the anus in mouse experiments,22,23 we used depths of insertion of 0.5, 1, 1.5, 2, 2.5, or 3 cm from the anus, to determine the locus associated with constipation among the transverse colon (3 cm from the anus), distal colon (2.5, 2, 1.5 cm from the anus), and rectum (1, 0.5 cm from the anus). Prior to the experiment, mice were habituated individually as described above, with food restricted for 12 h. For the experiment, a glass bead (2-mm diameter; Hamada Rika, Osaka, Japan) was inserted through the anus using a thin plastic tube to determine the control value of the bead expulsion time. Saline or morphine (s.c.) was administered to each mouse; 10 min later, a glass bead was inserted as in the pre-experiment and the expulsion time for each mouse was measured.
Statistical analysis Data are shown as means SEM. The SAS software (ver. 9; SAS Institute, Cary, NC, USA) was used for statistical analyses. The significance of differences among groups was assessed by one-way or two-way ANOVA followed by Dunnett’s comparison test. In all analyses, p < 0.05 was taken to indicate statistical significance.
RESULTS Effects of morphine on mouse intestinal circular muscle fragments We first examined the contractile response of the mouse muscle fragments to morphine using isometric
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Figure 1 Morphine-induced contractions of circular muscle fragments in mouse small and large intestines. (A) A model of the mouse intestine. Each intestinal region was sampled at the locus shown. (B) Effects of morphine (10 lM) on the small (jejunum and ileum) and large (proximal, transverse, transverse to distal, distal colon, and rectum) intestines of a single mouse. The arrow indicates morphine treatment. (C) Dose–response of contractile changes during the initial 3 min of morphine (0.1–100 lM) exposure was determined by calculating the area-under-the-curve (AUC). Left and right graphs show the small and large intestines, respectively. *p < 0.05, **p < 0.01, vs the pretreatment value, n = 6–16. Each value represents the mean SEM.
tial MOR expression, we next investigated the expression levels of MOR in each region. Although verifying MOR protein expression levels by immunohistochemistry or Western blotting may be the ideal method, there are at present no commercially available
Measurement of MOR mRNA expression levels in the small and large intestines To reveal whether the differences in contractile responses among the segments were due to differen-
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Figure 2 Effect of l-opioid receptor antagonist (naloxone) on morphine-induced circular muscle contraction. (A) Naloxone (1 lM) was administered after 5-min exposure to morphine (10 lM). Black and gray arrows indicate the timing of morphine and naloxone treatments, respectively. (B) Contractile responses to 3-min exposure to morphine and naloxone determined by area-under-the-curve (AUC). **p < 0.01, #p < 0.05, and n.s. (non-significant), vs the pretreatment value (distal colon; p = 0.81, rectum; p = 0.81 for morphine with naloxone), n = 8. Each value represents the mean SEM.
antibodies specific for MOR.24 We thus compared mRNA expression levels in the fragments of the intestine by quantitative RT-PCR. Primers were selected from the sequences of exons 2 and 3 of the mouse MOR, because these regions are conserved among the known MOR splicing variants.25 Expression was 10-fold higher in the large intestine than the small intestine segments. There were no significant differences in expression among the segments of the small intestine and large intestine (Fig. 3). Thus, circular muscle contraction was correlated with MOR expression in the small and large intestines, but not among the various regions of the large intestine.
Continuous contractions of large intestinal circular muscles during morphine exposure Although marked responses to morphine were detected in the transverse colon to the rectum (Fig. 1B), the continuity of the contractions during morphine exposure seemed to differ between the rectum and colonic regions. We next compared the sustainability of the morphine-induced contractile responses of the circular muscles of the transverse colon to the rectum by exposure to morphine for longer durations. During 30 min of morphine (10 lM) treatment, contractions were measured at 5-min intervals from 0 min (time of morphine administration) to 30 min. In the rectum,
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Figure 3 l-opioid receptor (MOR) expression in the small and large intestines. Total mRNA was extracted from each segment of the intestine, dissected as in Fig. 1, and MOR expression levels were determined by quantitative RT-PCR. **p < 0.01, vs the rectum, n = 4. Each value represents the mean SEM.
layers.26 To ascertain the role of NOS in the degradation of the contractions in the colon, the effect of a NOS inhibitor (L-NAME) was examined. Using the same methods as in Fig. 1, tissues were prepared and habituated in an organ bath for 5 min. Morphine (10 lM) was then added and after 20 min of exposure, L-NAME (50 lM) was added and the contractile responses before and after L-NAME treatment were measured. Because the transverse and transverse to distal colon exhibited less marked responses to morphine (Fig. 4), we compared the effects of L-NAME in the distal colon and rectum. In the distal colon, morphine-evoked contractions decreased during exposure. At this time, additional treatment with L-NAME resulted in gradual recovery of the contractile responses to a similar extent to the contractions at 0–3 min of morphine treatment (Fig. 5A). In the rectum, no response to L-NAME was detected. As shown in Fig. 5B, the decreased AUC value during 15–18 min of morphine exposure compared with 0–3 min of initial morphine exposure was recovered significantly with L-NAME treatment in the distal colon, while the AUC was unchanged in the rectum. These data suggest that contractile degradation during morphine exposure in the colonic circular muscles is a result of NOS production. We further focused on NOS–NO pathway to understand the mechanism underlying difference in the morphine-induced continuous contraction between the colon and rectum. To see the contribution of NO on contractile degradation, effect of NO donor SNAP was examined. Using the same method as in Fig. 1, the distal colon and rectum were equilibrated to the organ bath and morphine (10 lM) was first administered. After 3 min of morphine exposure, which is the timing before contractile degradation occurs in the distal colon as shown in Fig. 4, SNAP (50 lM) was added to the bath and contractile responses were measured for 3 min, respectively. As shown in Fig. 6A and B, SNAP mark-
strong and continued contractile responses were detected until the end of the measurements (Fig. 4). In contrast, although contractile changes were detected at first, those waves were gradually attenuated in the transverse, transverse to distal, and distal colon. Thus, continuous contraction of the circular muscles was detected only in the rectum, suggesting that the mechanism of contraction may differ between the rectum and colon.
Contribution of nitric oxide (NO) to the contractile degradation in the colon In the mouse intestine, more than 50% of enteric neurons express nNOS,26 and expression and production levels differ according to region, causing region-specific reductions in muscle contraction. In this regard, the colon has the highest density of nitric oxide synthase (NOS)-immunoreactive neurons, mainly in the fibers of the myenteric plexus, projecting to the circular muscle
Figure 4 Sustainability of the contractile response during morphine exposure in the large intestine. Morphine (10 lM)-induced contractile responses in the transverse colon to the rectum were measured during 30-min exposure to morphine at 5-min intervals from 0 min (time of morphine administration) for 30 min determined by area-under-thecurve (AUC). *p < 0.05, vs the pretreatment value, n = 10–14. Each value represents the mean SEM.
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Figure 5 Effect of NOS inhibitor (L-NAME) on morphine-induced circular muscle contraction in the distal colon and rectum. (A) L-NAME (50 lM) was added after 20-min exposure to morphine (10 lM). Black and gray arrows indicate the timing of morphine and L-NAME treatment, respectively. (B) Contractile responses to 3-min exposure to morphine and L-NAME determined by area-under-the-curve (AUC). **, ##p < 0.01, and n.s. (nonsignificant), vs 15–18 min of morphine exposure (rectum; p = 0.82 for 0–3 min morphine treatment, and p = 0.44 for morphine with L-NAME), n = 8–10. Each value represents the mean SEM.
Effects of morphine on bead transit latency in the regions of the large intestine
edly and similarly decreased the contraction of the distal colon and rectum, suggesting that these regions have ability to respond to NO and that NO has an important role for inhibiting circular muscle contraction. We next examined the contribution of NOS to the degradation of contraction during morphine exposure in the colon by using the NO substrate (L-arginine). Using the similar method as in Fig. 4, morphine (10 lM) was added with or without L-arginine (200 lM) to each organ bath and contractions of the distal colon and rectum were measured at 5-min intervals from 0 min (time of morphine or morphine+L-arginine administration) to 30 min. As shown in Fig. 6C, coadministration of Larginine significantly inhibited the AUC value of morphine-evoked contraction in the distal colon, while it had modest effect in the rectum. As NOS converts Larginine to NO and citrulline in accordance with its activity, the effect of L-arginine reflects the NOS activity level. Therefore, these results also suggested the different activation of NOS between the colon and rectum during morphine exposure, which may cause different contractile sustainability in these regions.
Morphine-evoked contraction in the large intestine increased with proximity to the anus (Fig. 1A). We thus examined whether those in vitro results were correlated with opioid-induced constipation in vivo using a bead expulsion assay. To confirm the effective dose of morphine, the morphine dose–response was examined by inserting a bead at a 2-cm depth from the anus 10 min after morphine administration (Fig. 7A). Morphine treatment resulted in a dosedependent increase in bead expulsion time, and 3 and 10 mg/kg morphine sufficiently delayed bead expulsion compared with pre-experiment values. We thus used 3 mg/kg morphine and measured bead expulsion times after insertion at various depths from the anus. In terms of the depth of bead insertion, 0.5 and 1 cm were intended to show the constipation effect in the rectum, 1.5, 2 and 2.5 cm for the distal colon, and 3 cm for the transverse colon. Following systemic morphine administration, beads were
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Figure 6 Effect of nitric oxide (NO) donor (SNAP) and NO substrate (L-arginine) on morphine-induced circular muscle contraction in the distal colon and rectum. (A) SNAP (50 lM) was added after 3-min exposure to morphine (10 lM). Black and gray arrows indicate the timing of morphine and SNAP treatment, respectively. (B) Contractile responses to 3-min exposure to morphine and SNAP determined by area-under-the-curve (AUC). **p < 0.01, #p < 0.05, and n.s. (non-significant), vs the pretreatment value (distal colon; p = 0.23, rectum; p = 0.61 for morphine with SNAP), n = 7. (C) Effect of L-arginine (200 lM) on contraction during 30-min morphine exposure (10 lM). *p < 0.05 between morphine and morphine+L-arginine group, n = 10–11. Each value represents the mean SEM.
parts of the intestines, makes a major contribution to morphine-induced constipation.
inserted and the bead expulsion time was measured. At all depths examined, morphine significantly increased the bead expulsion time compared with the prevalue (Fig. 7B). The bead expulsion time did not differ according to depth of insertion, indicating that the rectum plays an important role in morphineinduced constipation. These in vivo results are consistent with the in vitro data (Fig. 1), suggesting that the greater and longer contractile response of the circular muscles in the rectum, compared to the other
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DISCUSSION In the present study, we focused on the effects of morphine on mouse intestinal circular muscle contraction and compared the contractile waves among the histologically known regions. The strongest contraction after morphine treatment was detected in the rectum.
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rectum. Therefore, the rectum is the region primarily involved in opioid-induced constipation. Morphine-induced circular muscle contraction was greater in the large intestine than the small intestine. Although the quantitative RT-PCR experiments were performed using whole-tissue RNA extracted from the respective segments of the intestine, and we did not examine MOR expression at the myenteric plexus in detail, levels of contraction—in terms of AUC values— between the small and large intestines seemed to be correlated with MOR mRNA levels. Previous study supports our finding of weak MOR expression in the mouse ileum, that electrical stimulation-evoked contraction was not inhibited by morphine.27 In this regard, it was assumed that low MOR expression caused the weak effect of morphine in the small intestine. Another possible mechanism for the difference between the small and large intestines, different contributions of intracellular signaling between the small and large intestines has been reported. Raehal et al. found that G protein-coupled receptor kinases 6 (GRK6)-knockout mice expelled beads inserted from the anus more rapidly than did wild-type controls after morphine administration.23 In contrast, lack of GRK6 had no effect on morphine-induced delay in the small intestine, as determined by charcoal meal transit, between these animals. A similar difference between the small and large intestines has also been reported in b-arrestin2-knockout animals. Lack of b-arrestin2 reversed both the acute morphine-evoked bead expulsion delay and reduced defecation in the large intestine, whereas it had no effect in the small intestine.28 In addition, lack of b-arrestin2 enhanced morphineinduced contractile tolerance of the large intestine, but not of the small intestine.21,29 Although b-arrestin2 is well known as a key adaptor protein in receptor endocytosis, it also functions as a positive regulator of the intracellular signaling that up-regulates mitogen-activated protein kinase pathways,19,30 which is assumed to mediate constipation.21 In these respects, activation of the intracellular pathways mediating MOR might be fundamentally different between the small and large intestines, and this might cause lesser contribution of small intestine on opioid-induced constipation. In the large intestine, MOR mRNA levels were similar in all segments examined by RT-PCR, whereas the morphine contractile response was stronger in the distal colon and rectum. We considered two possibilities to explain this. Firstly, this may be caused by the different thickness of the muscle along the large intestine. Our preliminary data using carbachol, a
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Figure 7 Large intestinal transit measured by bead expulsion assay. (A) A glass bead (2-mm diameter) was inserted 2 cm from the anus 10 min after morphine (s.c.) administration. Bead expulsion times for each mouse were measured. **p < 0.01, vs the pretreatment value, n = 6–9. (B) Glass bead was inserted 0.5–3 cm in 0.5-cm intervals from the anus before and after morphine (3 mg/kg, s.c.) administration. No significant difference according to insertion depth was detected pre- or posttreatment. p > 0.05, vs pre- (p = 0.68–1.0) or post- (p = 0.38–0.97) 0.5-cm insertion values, respectively, n = 5–7. Each value represents the mean SEM.
Although the colonic regions examined also showed marked contraction, the responses were greater on the distal side than the proximal side. The response in the jejunum and ileum was markedly lower than in the large intestine, with only slight changes in contraction upon morphine treatment. Furthermore, we compared the sustainability of the contractions during morphine exposure among the regions of the large intestine. While there were contractile degradations in the transverse colon to distal colon, a continuous response was detected in the rectum. These results are consistent with the in vivo bead expulsion assay results; after morphine administration, inserted beads seemed to be propelled from the oral to anal side, stopping at the
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to respond to NO if there is enough levels of NO. It is reported that intrinsic density of NOS-immunoreactive cells is higher in the colon, vs other parts of the intestines under the normal state.34 In addition, NOS shows higher activity in the proximal side than the distal side of the large intestine,34 which may also account for the prolonged contraction in the rectum compared to the colon. Morphine is known to affect NOS activity through MOR located on nitrergic nerves.19,26,35 According to the human saphenous vein, NO release starts to increase in 5 min after morphine administration.36 Thus, in addition to difference in intrinsic NOS activity, different contractile degradation between colon and rectum may also result from difference in morphine-induced NOS activation. Although the time course and region specificity of NOS activation by morphine in the intestine is not known, it is possible that additional NOS activation by morphine enhanced contractile degradation during 30-min exposure in the colon. Although little is known about the mechanism of the difference in contractile sustainability between the colon and rectum, these two possibilities, i.e., regional- and morphine-dependent differences, abundant NOSs are produced and may account for contractile degradation of the circular muscles in the colonic regions but not in the rectum. The in vivo bead expulsion assay showed consistent results, suggesting the importance of the rectum in opioid-induced constipation. Raehal et al. (2005) showed that a single injection of morphine could reduce defecation even after 6 h, indicating that our results were not due to elimination of morphine itself.29 In our study, the expulsion time after morphine administration did not differ according to bead insertion depth. Thus, it seems that fecal pellets move through the colon to the rectum whether they are inserted into the transverse or distal regions of the colon, and then stop at the rectum. According to our in vitro data, because of the non-sustained contraction of the circular muscles in the colon, the bead may transit these areas and then stop at the rectum due to the high amplitude and prolonged contractions. Although few data suggest a relationship between the rectum and opioid-induced constipation, opioidaddicted patients who undergo colectomies are reported to remain constipated, possibly due to rectal dysfunction.37 In addition, opioid-used patients are considered to associate with stool-filled rectum with an impacted fecal mass in the anorectal region.38 Thus, our results suggest that the mechanism of opioidinduced constipation involves abnormal contractions in the rectum that can induce fecal incontinence.
muscarinic receptor agonist, showed that circular muscles undergo larger contractions in the distal regions (distal colon and rectum) vs the proximal regions (proximal and transverse colon; data not shown). Although no report is available in mice, human anatomical studies of the distal colon to the rectum show that the thickness of the circular muscle layer increases toward the anus,31 consistent with this hypothesis. A second possibility is that while we assume that MOR mRNA levels in the ring samples of the intestines are similar, MOR expression in the myenteric plexus may be higher on the anal than the oral side, which may induce higher contractions toward the rectum. Although we could not use immunohistochemical approach,24 reports using affinity-purified antibody to the C-terminus of rat MORs showed positive staining of the neurons of the myenteric plexus in all regions in the intestine with different expression levels according to regions.13 Because we have little evidence for these two hypotheses, we should further examine for their participation to explain the different contraction of the large intestinal regions in the future. We found the continuous contraction in the rectum, but not in the colonic regions. To identify the mechanism underlying these differences, it was considered that MOR desensitization levels are different among regions. Although no report is available for the rectum, it has been reported that repeated exposure to DAMGO or fentanyl lead to b-arrestin2-associated MOR desensitization and following contractile degradation in the colon but not in the ileum, suggesting the different mechanism leading to MOR desensitization among the intestinal regions.29 In addition to this, it was also considered that different intracellular mechanism that leads to contractile degradation may be different. On account of the second possibility, we focused on NOS–NO pathway because this was thought to be most likely to influence circular muscle contraction. In the circular muscle layers, NO is an important inhibitory neurotransmitter to regulate muscle contraction.16,32,33 We found the opposite effect of NOS inhibitor (L-NAME) and NO substrate (L-arginine) on the distal colon contraction that decreased contraction was recovered by L-NAME to the levels during the initial morphine treatment, while contractile degradation was enhanced by L-arginine. It was noteworthy that less effect of these reagents were observed in the rectum, suggested that lower activation of NOS–NO causes lower degradation of contraction. This is also supported by SNAP results that the rectum has ability
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ingly, the rectum is likely the key region involved in opioid-induced constipation.
Although we did not examine the effect of NO in the in vivo assay, earlier study has shown that L-NAME can dose-dependently and completely block the fecal pellet propulsion of the large intestine.33 However, alternative neurons such as cholinergic and serotonergic neurons and the neuronal network of these neurons are also important for peristaltic movement. Therefore, future studies are needed to clarify the mechanisms controlling opioid-induced constipation. Because previous reports have addressed morphineinduced contraction of circular muscle segments in specific regions of the intestine, our study represents the first comprehensive analysis to distinguish contractile strength among the known regions of the small and large intestine. To our knowledge, this is the first report of the likely significance of the rectum, which showed a higher contractile response to morphine with sustained contractions compared with the other regions of the intestine. These data suggest the mechanism of delayed bead transit in the rectum. Accord-
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FUNDING No funding is associated with the article.
DISCLOSURE None of the authors have any competing interest to disclose relating to this submission. H. Ono, A. Nakamura, G. Sakaguchi, and T. Kanemasa are employees of Shionogi and Co., Ltd, the manufacture of morphine.
AUTHOR CONTRIBUTION HO performed all the research; HO and AN analyzed the data and wrote the article; KM and SH instructed the methods of in vivo and in vitro methods; HO, AN, KM, SH, GS, and TK conceived and directed research. All authors discussed the results and commented on the manuscript.
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