Antiexudative Effects of Opioids and Expression of - and - Opioid Receptors during Intestinal Inflammation in Mice: Involvement of Nitric Oxide

The study evaluates the effects of (KOR), (DOR), and -opioid receptor (MOR) agonists on the inhibition of plasma extravasation during acute and chronic intestinal inflammation in mice. The antiexudative effects of KOR and DOR agonists in animals treated with nitric oxide synthase (NOS) inhibitors and their protein levels in the gut (whole jejunum and mucosa) and spinal cord of mice with chronic intestinal inflammation were also measured. Inflammation was induced by the intragastric administration of one (acute) or two (chronic) doses of croton oil. Plasma extravasation was measured using Evans blue and protein levels by Western blot and immunoprecipitation. Plasma extravasation was significantly increased 2.7 times during chronic inflammation. The potency of the KOR agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolydinyl)cyclohexyl]benzeneazetamine (U50,488H) inhibiting plasma extravasation was enhanced 26.3 times during chronic compared with acute inflammation. [D-Pen,D-Pen]-Enkephalin (DPDPE) (a DOR agonist) was also 11.8 times more potent during chronic inflammation, whereas the antiexudative effects of fentanyl (a MOR agonist) were not significantly altered. Receptor-specific antagonists reversed the effects. Protein levels of KOR and DOR in the whole jejunum and mucosa were significantly increased after chronic inflammation. Treatment with NOS inhibitors N nitro-L-arginine methyl ester or L-N-(1-iminoethyl)-lysine hydrochloride diminished plasma extravasation and inhibited the increased antiexudative effects of U50,488H and DPDPE during chronic intestinal inflammation. The data show that the enhanced antiexudative effects of KOR and DOR agonists could be related to an increased expression of KOR and DOR in the gut and that the release of nitric oxide may play a role augmenting the effects of opioids during chronic inflammation. Peripheral inflammation induces the local release of numerous chemical mediators that, among other effects, sensitize the peripheral terminals of primary afferents inducing pain and hyperalgesia; these terminals also release neuropeptides (Richardson and Vasko, 2002) that participate in the local inflammatory response by inducing vasodilatation, plasma extravasation, and edema (Karimian and Ferrell, 1994; Amann et al., 1995; Siney and Brain, 1996). Opioids reduce plasma extravasation induced by peripheral inflammation by binding to specific opioid receptors located in the central and peripheral nervous systems (Taylor et al., 2000; Romero et al., 2005). Opioid receptors are found in the central and peripheral nervous system as well as in non-neuronal sites such as the vascular endothelium and immune cells (Mansour et al., 1994; Cadet et al., 2000; Saeed et al., 2000; Tomassini et al., 2003). In the gut, opioid receptors are present in the myenteric and submucosal plexuses as well as in epithelial cells (Lang et al., 1996; Bagnol et al., 1997; Pol et al., 2001) and modulate several intestinal functions such as motility and This work was supported by grants from Comisión Interministerial de Ciencia y Tecnologica (SAF2003-02578), Fondo de Investigaciones Sanitarias (C03/06 and PI030245) (Madrid, Spain), and Generalitat de Catalunya (2001SRG00409) (Barcelona, Spain). Part of these results was presented as a communication to the 34th International Narcotics Research Conference, Perpignan, France (July 2003). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.091991. ABBREVIATIONS: MOR, -opioid receptor(s); DOR, -opioid receptor(s); KOR, -opioid receptor(s); NOS, nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; EB, Evans blue; EU, extravasation unit(s); PBS, phosphate-buffered saline; U50,488H, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolydinyl)cyclohexyl]benzeneazetamine; DPDPE, [D-Pen2,5]-enkephalin; nor-BNI, nor-binaltorphimine dihydrochloride; L-NAME, N -nitro-L-arginine methyl ester; L-NIL, L-N-(1-iminoethyl)-lysine; D-NAME, N -nitro-D-arginine methyl ester; ANOVA, analysis of variance. 0022-3565/06/3161-261–270$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 91991/3067006 JPET 316:261–270, 2006 Printed in U.S.A. 261 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from secretion under certain pathological conditions (Valle et al., 2000; Pol and Puig, 2004). Intestinal inflammation increased the transcription and expression of MOR located in the myenteric plexus and is responsible for the enhanced antitransit effects of MOR agonists in these experimental conditions (Pol et al., 2001). Inflammation of the gut also increased transcription and protein levels of DOR in the myenteric and submucosal plexuses, a fact that could explain the increased effects of DOR agonists on the inhibition of gastrointestinal transit and intestinal permeability (Pol et al., 2003). Although the expression of KOR in the submucosal plexusmucosa of mice was also enhanced after chronic inflammation (Pol et al., 2003), a definite role for this receptor and its precise location in the mucosal layer have not been established. Nitric oxide produced by the three nitric-oxide synthase (NOS) isoforms—neuronal NOS, inducible (iNOS), and endothelial (eNOS)—is as a neurotransmitter in the central and peripheral nervous systems. Evidence indicates that nitric oxide is involved in the behavioral and antinociceptive effects of opioids (Nozaki-Taguchi and Yamamoto, 1998; Manzanedo et al., 2004; Tasatargil and Sadan, 2004). Nitric oxide is also implicated in several pathophysiological processes such as inflammation and regulation of gene expression. Thus, we have recently demonstrated that nitric oxide derived from iNOS was implicated in the enhanced antitransit effects of morphine as well as in the enhanced transcription of MOR gene observed during intestinal inflammation (Pol et al., 2005). In contrast, nitric oxide suppressed the transcription of KOR gene in stem cells cultures (Park et al., 2002). No studies have been carried out to evaluate the effects of nitric oxide in the antiexudative effects of KOR and DOR agonists during intestinal inflammation. In a model of intestinal inflammation induced by croton oil, the aims of the present investigation were to evaluate 1) the effects of specific opioid receptor agonists on plasma extravasation during acute and chronic intestinal inflammation; 2) the contribution of endogenous opioid peptides released during inflammation in plasma extravasation; 3) the role of nitric oxide in the enhanced effects of KOR and DOR agonists during chronic inflammation; and 4) the expression of KOR and DOR in the intestine (whole jejunum and dissected mucosal) and spinal cord of animals with and without chronic intestinal inflammation. Materials and Methods Animals. Male Swiss CD-1 mice, weighing 25 to 30 g, were used in all experiments. Mice were housed under 12-h/12-h light/dark conditions in a room with controlled temperature (22°C) and humidity (66%). Animals had free access to food and water and were used after a minimum of 4 days acclimatization to the housing conditions. All experiments were conducted between 9:00 AM and 6:00 PM. The study protocol was approved by the local committee of animal use and care of our Institution, in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Intestinal Inflammation. Two types of intestinal inflammation (acute and chronic) were used in our study. Acute inflammation was induced by the intragastric administration of a single dose (0.05 ml) of croton oil (Sigma-Aldrich, St. Louis, MO) diluted in olive oil (1:1); control animals received the same volume of intragastric saline. Mice in the chronic treatment group were gavaged with a second dose of croton oil or saline (0.05 ml) 24 h after the first dose (Fig. 1A). In both instances, mice were fasted for 18 h before croton oil or saline administration, except for free access to water, which was available for the duration of the study. In the acute treatment group, plasma extravasation was evaluated 3 h 10 min after croton oil or saline, whereas in the chronic treatment experiments plasma extravasation was measured 96 h 10 min after the first dose of croton oil. These time points were selected based on previous studies from our laboratory, demonstrating that they were the times of maximal epithelial injury in both models of intestinal inflammation (Puig and Pol, 1998). Plasma Extravasation. The extravasation of plasma proteins to the small intestine was assessed with Evans Blue (EB) dye using a technique adapted from Udaka et al. (1970). At 3 or 96 h after the first dose of croton oil (for the acute and chronic inflammation, respectively), animals were briefly anesthetized with halothane and injected intravenously with 50 mg/kg EB (85 l) to allow subsequent quantification of plasma extravasation. For each animal, a sterile insulin syringe coupled to a 0.33 12.7-mm needle was used. Animals were sacrificed by cervical dislocation 10 min times after EB administration, and the small intestine was carefully removed, washed with saline, and the first 24 cm from the pyloric valve was reserved. EB was extracted by incubation of this fragment in 6 ml of Fig. 1. A, experimental design showing the sequence of croton oil (CO) or saline (SS) administration and the time (h) of evaluation of Evans blue (EB) in acute and chronic treatment groups. B, plasma EU values in animals with acute (AC-CO) or chronic (CR-CO) intestinal inflammation. Each column represented the mean values S.E.M from 10 animals. , significant differences compared with animals with acute inflammation (P 0.01; Student’s t test). 262 Jiménez et al. at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from formamide at 60°C for 24 h. The EB extracted was quantified by spectrophotometry (SmartSpec3000; Bio-Rad, Hercules, CA) at 620 nm and expressed in extravasation units (EU). One EU was defined as 0.001 units of absorbance (Ohishi and Odagiri, 1984). Plasma extravasation, defined as the increase in plasma protein due to inflammation, was obtained by subtracting from the EB values obtained in the gut of animals with acute or chronic inflammation the corresponding EB values of control animals (without inflammation) using the following equation: plasma extravasation (EU) EB inflamed intestine EB control intestine. Tissue Isolation. Small intestine (jejunum) and the thoracic section of the spinal cord from animals with and without chronic intestinal inflammation were excised, placed in sterile Microfuge tubes, snap-frozen in liquid nitrogen, and stored at 80°C until assay. The dissection of the mucosa of the gut was performed by placing segments of jejunum in ice-chilled phosphate-buffered saline (PBS). Then, the gut was opened longitudinally to expose the mucosal side, which was subsequently pinned to a silicone elastomercoated Petri dish. The mucosal layer was separated from the remaining layers by performing a careful superficial scraping with a glass slide, and samples from four animals were pooled into one experimental sample. Then, samples were frozen in liquid nitrogen and stored at 80°C. All dissections were performed under a stereomi-