1Division of Surgery, Danderyd Hospital, SE-182 88 Stockholm; 2Department of Gastroenterology and Hepatology, Karolinska Hospital, Karolinska Institutet, SE-171 Stockholm, Sweden; 3Department of Physiology and Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203; and 4Neurology/Gastrointestinal CEDD, GlaxoSmithKline, Harlow, Essex, CM19 5 AW United Kingdom
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ABSTRACT |
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migrating motor complex; SB-334867-A; enteric ganglia; myenteric plexus
Few studies have examined the peripheral effects of orexins. Plasma concentrations of OXA are increased during fasting in humans (12) and are lower in obese subjects compared with normal-weight subjects in the fasted state (1). We have recently demonstrated (16) that intravenous infusion of OXA and OXB in rats replaces the migrating myoelectric complex (MMC) by a pattern of irregular spiking; however, the mechanism behind this effect has not been clarified.
Recently, a selective orexin receptor 1 (OX1R; SB-334867-A) antagonist has been developed (23). Intraperitoneal administration of SB-334867-A results in decreased food intake and body weight gain as well as increased metabolic rate (7). There have been no studies on OX1R antagonists and gastrointestinal motility, although SB-334867-A has recently been shown to block OXA-induced contractions in the guinea pig small intestine (15).
The aim of this study was to investigate the mechanism of OXA action on small bowel motility and to determine whether OXA has a physiological role in the small bowel motor control of fasted conscious rats. We also determined whether OXA-containing neurons display nitric oxide (NO) synthase (NOS)-like immunoreactivity [neuronal NOS (nNOS)] in the rat duodenum.
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MATERIAL AND METHODS |
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Fasting small bowel motility studies. All experiments started with a control recording of basal myoelectric activity, during which four activity fronts of the MMC propagated over all three recording sites during a period of 60 min. Infusion of OXA (Bachem AG, Bubendorf, Switzerland) was started immediately after the fifth activity front had passed the first electrode site, using a microinjection pump (model CMA 100; Carnegie Medicine, Stockholm, Sweden).
In a first set of dose-response experiments, OXA at doses of 50 (n = 8), 250 (n = 8), 500 (n = 13), 1,000 (n = 9), and 5,000 (n = 7) pmol·kg1·min1 was administered intravenously for 60 min, and the effect on small bowel motility was recorded.
In a second set of experiments, the effect of OXA on small bowel motility was studied in rats subjected to a bilateral surgical subdiaphragmatic vagotomy (n = 7). In addition to vagotomy, a pyloroplasty was performed and the animals were fed standard rat chow. After a 60-min control period, intravenous infusion with OXA (500 pmol·kg1·min1) was given for 60 min. The dose 500 pmol·kg1·min1 was chosen because this dose was the closet to the ED50 in dose-response experiments.
In a third set of experiments, the effect of pretreatment of guanethidine (Ismelin, CIBA-Geigy, Basel, Switzerland) was studied on the response to OXA on fasting motility (n = 7). An intravenous bolus of guanethidine (3 mg/kg1) was given 24 h before experiments. After a 1-h control period, OXA (500 pmol·kg1·min1) was given as intravenous infusion for 60 min. The dose 500 pmol·kg1·min1 was chosen for the same reasons as above.
In a fourth set of experiments, the effect of the NOS inhibitor N-nitro-L-arginine (L-NNA) (Sigma, St. Louis, MO) was studied on the response to OXA at doses of 500 and 5,000 pmol·kg1·min1 (each n = 7). After the control period, OXA was infused intravenously for 60 min. After propagated activity fronts were resumed, L-NNA at a dose of 1 mg/kg was given intravenously 10 min before infusion with OXA was repeated for another 60 min. In a separate experiment, L-NNA alone (1 mg/kg) was administered and myoelectric activity was recorded for 60 min. The dose of 1 mg/kg of L-NNA has in previous studies been shown to exert a reliable pharmacological blockade without disturbing unspecific stimulation of the MMC pattern (8, 13). The doses of 500 and 5,000 pmol·kg1·min1 were used to evaluate the dependence of half-maximal and maximal responses of OXA on NO.
Studies with the OX1R antagonist. In a first set of experiments, rats (n = 6) were prepared as above. After a 60-min control period, an intravenous bolus of the OX1R antagonist SB-334867-A [10 mg/kg dissolved in DMSO (Sigma) to 0.33 ml] was administered immediately after termination of an activity front at the duodenal level, and motor activity was studied for another 60 min. In a separate set of experiments, DMSO was given alone (n = 4).
In a second set of experiments, rats (n = 6) were prepared as above. After a 60-min control period, OXA was administered intravenously for 60 min at a dose of 500 pmol·kg1·min1. After a 30-min washout period and normal fasting motility resumed, an intravenous bolus of SB-334867-A (10 mg/kg) was given and intravenous infusion of OXA (500 pmol·kg1·min1) and SB-334867-A (0.1 mg·kg1·min1) was given for 60 min.
Tissue preparation for immunohistochemisty. Male Sprague-Dawley rats (300350 g, n = 6) were killed with an intravenous overdose of pentobarbital sodium (Apoteksbolaget, Umeå, Sweden). Segments (1 cm) of duodenum (immediately distal to the pylorus) were removed, washed through the lumen, and placed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h. After fixation, the preparations were washed and stored in PBS containing sodium azide (1%). Material to be sectioned was cryoprotected overnight (at 4°C) in PBS containing 30% (wt/vol) sucrose, embedded in optimal cutting temperature (Miles Scientific, Naperville, IL), frozen with liquid N2, and sectioned (10 µm) using a Leica cryostat-microtome (Leica Microsystems, Deerfield, IL).
For whole mount preparations, segments of duodenum were opened along the mesenteric border, and the resulting rectangular sheet of intestine was stretched, pinned flat on balsa wood, and fixed as above. After fixation, the preparations were washed in PBS and then dissected into layers. Two whole mounts, one containing the submucosal and the other the myenteric plexus, were prepared as previously described (10).
Immunocytochemistry. To locate OXA protein in the tissue by immunocytochemistry, preparations were exposed to PBS containing 0.5% Triton X-100 and 4% horse serum for 30 min and then incubated with primary antibodies against OXA (affinity-purified rabbit polyclonal, diluted 1:2,000, Alpha Diagnostic International, San Antonio, TX). After preparations were washed with PBS, they were incubated (for 3 h) with affinity-purified donkey anti-rabbit secondary antibodies conjugated to indocarbocyanine (Cy3; diluted 1:2,000; Jackson ImmunoResearch Laboratories, West Grove, PA) or FITC (1:500; Jackson ImmunoResearch Laboratories). Parallel control sections were included that were incubated with normal goat serum instead of primary antibodies. No immunostaining was observed when a control IgG was substituted for the primary antibody.
Double-label immunocytochemistry was used to identify the cells that displayed OXA immunoreactivity and OXR1-immunoreactivity (rabbit polyclonal, diluted 1:500, Chemicon International, Temecula, CA) using primary antibodies raised in different species in conjunction with species-specific secondary antibodies [donkey anti-sheep (Jackson ImmunoResearch Laboratories), diluted 1:200] coupled to contrasting fluorophores (FITC or Cy3, as above). Primary antibodies against nNOS (sheep polyclonal, diluted 1:5,000, Chemicon International) were also used. To enhance visualization of peptide immunoreactivity in nerve cell bodies, rats were injected with colchicine (5 mg/kg) intraperitoneally. Twenty-four hours later, the rats were killed.
The tissues were placed in coverslips in Vectashield (Vector Laboratories, Burlingame CA). Preparations were examined by using a Radiance 2000 laser scanning confocal microscope (BioRad, San Francisco, CA) attached to an Axioskop 2 microscope (Carl Zeiss, Thornwood, NY). Images of 512 x 512 pixels were obtained and processed by using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA) and printed with a Kodak (XLS-8600) printer.
Ethics, data, and statistical analysis. The local Ethics committee for animal experimentation in Stockholm, Sweden approved the experimental protocol.
The main characteristic feature of myoelectric activity of the small intestine in the fasted state, the activity front (phase III) of the MMC, was defined as a period of clearly distinguishable intense spiking activity with an amplitude at least twice that of the preceding baseline, propagating aborally through the whole recording segment and followed by a period of quiescence. The MMC cycle length, duration, and propagation velocity of the activity fronts were calculated (2) as a mean of the 60-min study period. All statistical evaluations are against the control period for experiment; however, control (saline) results are shown as a mean for all control data for that set of experiments. When no activity front was observed during the 60-min infusion period, a value of 61 min was assigned as the measured value. When only one or two activity fronts were observed, followed by a long period of quiescence, the MMC cycle length was calculated from the activity front preceding the start of the infusion to the activity front observed and then from the observed activity front during the infusion to the end of the infusion period plus 1 min.
Data are expressed as median (range), unless otherwise stated. The data were assessed for statistical significance using the two-sided Mann-Whitney U-test or Wilcoxon signed rank test for matched pairs at P < 0.05, as appropriate.
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RESULTS |
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After bilateral subdiaphragmatic vagotomy or pretreatment with guanethidine, the response of the small intestine to OXA was not different from that seen in untreated rats. The duration or propagation velocity of activity fronts in the gut segment was not affected (Table 2).
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Pretreatment with L-NNA (1 mg/kg) resulted in a significant reduction of the cycle length compared with control. Pretreatment with L-NNA abolished the response of the small intestine to OXA at both 500 and 5,000 pmol·kg1·min1, so that the addition of L-NNA to the intravenous infusion of OXA was not different from control but significantly different from OXA alone. Again, no effect was seen on the duration or propagation velocity of activity fronts in the gut segment (Table 3).
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OX1R antagonist induces activity fronts and decreases MMC cycle length. Thirty-six (1848) seconds after a bolus of SB-334867-A, an activity front was induced in all animals. During the 60-min study period, the MMC cycle length decreased after administration of the OX1R antagonist compared with control. The OX1R antagonist did not affect the duration of the activity front or the propagating velocity. Administration of DMSO alone did not affect the MMC pattern (Fig. 1, Table 4).
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The effect of OXA on fasting motility is OX1R specific. The concomitant administration of OXA and SB-334867-A resulted in a significant reduction of the MMC cycle length compared with OXA alone and not different from control. The duration of the activity front and propagating velocity were unchanged (Fig. 2, Table 5).
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Localization of OXA and nNOS in the duodenum. As previously reported (16), OXA immunoreactivity was found in nerve cell bodies and fibers in the rat duodenum (Fig. 3). OXA-immunoreactive neurons were found in submucosal (not illustrated) and myenteric (Fig. 3A) ganglia. The immunoreactivity in the cytoplasm of such cells was relatively low but higher in colchicine-treated animals. No immunoreactivity was found in control sections processed without the primary antisera (not illustrated).
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Further studies were done to determine whether the neurons with OXA immunoreactivity also contained nNOS. OXA-immunoreactive submucosal neurons did not contain nNOS (not illustrated); however, in the myenteric plexus, a subset of OXA-immunoreactive neurons (Fig. 3A) displayed nNOS immunoreactivity (Fig. 3B). Some nNOS-immunoreactive neurons in the myenteric plexus were encircled by OXA-immunoreactive nerve fibers (Fig. 3, C and D). A subset of OXR1-immunoreactive neurons (Fig. 3E) in the myenteric plexus also contains nNOS (Fig. 3F). OXA-immunoreactive nerve fibers also contained nNOS (data not shown).
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DISCUSSION |
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Intravenous infusions of OXA at doses ranging between 50 and 5,000 pmol·kg1·min1 increased the MMC cycle length without affecting the duration or propagation velocity of the activity fronts in a dose-dependent manner. Yet, the effect of OXA at higher doses seemed to be slightly variable. In our previous study (16), quiescence was achieved at 500 pmol·kg1·min1. Similar results were seen with OXA 500 pmol·kg1·min1 in experiments with L-NNA, whereas in the dose-response experiments, quiescence was not seen in some animals even at 500 and 1,000 pmol·kg1·min1. The reason for this is not clear. The half-life and degradation of peripheral OXA is not known. An individual response variability to OXA might explain our findings, but no such variations have been described elsewhere.
The administration of the selective OX1R antagonist SB-334867-A induced an activity front in 30 s in all animals and shortened the MMC cycle length by 25% compared with control situation. This suggests that OXA and OX1R are involved in the regulation of fasting motility. Also, because the combined administration of OXA and SB-334867-A resulted in a significant reduction in MMC cycle length compared with OXA alone, this suggests that the effect of OXA on fasting motility is OX1R specific. Motilin has been suggested to be involved in the regulation of the MMC and plasma concentrations of motilin fluctuate with the different phases of the MMC in humans (24). Plasma concentrations of OXA have been shown to increase during fasting in rats (18) and humans (12); however, it is unknown whether plasma OXA fluctuates during the different phases of the MMC or postprandially. Previous work with SB-334867-A (7) suggests that the compound has anorectic and thermogenic properties and may be used as a potential treatment for non-insulin-dependent diabetes mellitus and obesity. Our data indicate that there may also be a role for the SB-334867-A and OXA in the treatment of gastrointestinal motor disorders.
Intravenous infusion of OXA produced a comparable inhibitory effect as did VIP on the MMC (13). Because OXA nerve fibers in the circular muscle of the duodenum also contain VIP (16), the inhibitory effect of orexins on the MMC may be mediated by VIP or, at least, involve similar mechanisms as VIP (16). We have previously shown (16) that in the rat duodenum, OXA immunoreactivity occurs in endocrine cells, nerve cell bodies, and nerve fibers that innervate myenteric and submucosal ganglia and circular muscle. We have also found that VIP and OXA are colocalized, implying that the OXA-immunoreactive neurons are secretomotor neurons, interneurons, and/or inhibitory motoneurons. The mucosa contained OXA-immunoreactive nerve fibers; however, the innervation of the mucosa was sparse compared with the dense OXA innervation of ganglia and muscle. Thus OXA neurons are more likely to be interneurons and/or motoneurons (16). About 45% of submucosal nerve cells in the rat ileum contain VIP (14). These cells include secretomotor neurons and interneurons projecting within the submucosal plexus and to neurons in the myenteric plexus (4, 19). Myenteric VIP-containing cells are thought to be inhibitory motoneurons that mediate relaxation of the circular muscle (5).
In this study, we extended our studies of OXA distribution in the rat duodenum to include nNOS. We demonstrated that a subset of OXA neurons in the myenteric plexus contains nNOS as well as numerous nerve fibers in the circular muscle. NO has been demonstrated to be a transmitter of inhibitory neurons of the enteric nervous system (21) and to mediate relaxations of the gut (3). In this paper, we demonstrate that the NOS inhibitor L-NNA diminished the response of OXA on fasting small bowel motility. Although L-NNA alone reduced the cycle length of the MMC by a median of 8 min, pretreatment with L-NNA before OXA infusion decreased the cycle length by 42 min. The present results suggest that OXA modulates duodenal and jejunal motility through peripheral actions of the peptide, at least partly through an L-arginine/NO pathway.
In support of our results, in vitro studies of OXA on mouse small intestine demonstrate that OXA partially mediates nonadrenergic, noncholinergic relaxation through the activation of NOS-containing myenteric neurons (22). The results of Satoh et al. (22) suggest that OXA activates NOS neurons directly or indirectly though activation of interneurons within the myenteric plexus. Furthermore, spontaneous relaxations of the rat duodenum in between activity fronts of the MMC are NO dependent, whereas relaxation of the duodenum during activity fronts seems to be modulated, but not directly mediated, by NO. Rather, ATP seems to be the mediator of relaxation during the activity front of the MMC (6). Thus, although somewhat speculative, it is possible that the inhibition of activity fronts of the MMC seen by OXA in this study may be the result of NO modulation of ATP-mediated relaxation of the rat duodenum during activity front of the MMC.
In summary, this study demonstrates that the inhibitory effect of OXA on fasting small bowel motility is at least partly dependent on an L-arginine/NO pathway and seems to be mediated by OX1R. OXA seems to have a physiological role regulating the MMC, because a selective OX1R antagonist shortened the MMC cycle length. On the basis of our findings, it seems likely that OXA plays a significant role in modulating gastrointestinal function and may be involved in the processing of nutrients and energy control in addition to the central stimulation of food intake.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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