Protective role of M3 muscarinic acetylcholine receptor in indomethacin-induced small intestinal injury

Protective role of M3 muscarinic acetylcholine receptor in indomethacin-induced small intestinal injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective role of M3 muscarinic acetylcholine receptor in indomethacin-induced small intestinal injury Yoko Igarashi-Hisayoshi, Eikichi Ihara, Xiaopeng Bai, Yoshimasa Tanaka, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3217047/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Aug, 2024 Read the published version in Journal of Molecular Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Background EP 4 prostanoid receptor (EP 4 R) contributes to the intestinal epithelial barrier function, and inhibition of prostaglandin E (PGE) production by non-steroidal anti-inflammatory drugs (NSAIDs) plays a central role in NSAID-induced enteropathy. However, given that M 3 muscarinic acetylcholine receptor (M 3 R)-selective agents are unavailable, how M 3 R regulates the intestinal epithelial barrier function remains unclear. The present study explored how M 3 R is involved in the regulation of the intestinal epithelial barrier function and its pathophysiological role in NSAID-induced enteropathy. Methods Using the novel highly-selective M 3 positive allosteric modulator PAM-369 that we recently developed, we evaluated the role of M 3 R in the intestinal epithelial barrier function ex vivo by measuring the short circuit current (Isc) of intestinal epithelium with a Ussing chamber system and examined whether or not M 3 R protects against small intestinal injury in indomethacin-treated mice. Results Both the PGE 1 derivative misoprostol and carbachol similarly increased the Isc in a concentration-dependent manner. The Isc increases were abolished either by receptor antagonists (an EP 4 R antagonist and a M 3 R antagonist, respectively) or by removal of extracellular Cl − . PAM-369 increased the Isc by potentiating M 3 R, which could contribute to enhanced intestinal epithelial barrier function. Treatment with PAM-369 ameliorated small intestinal injury in indomethacin-treated mice. Importantly, the M 3 R expression was significantly up-regulated, and PAM-369 potentiation of M 3 R was augmented in indomethacin-treated mice compared to untreated mice. Conclusions These findings show that M 3 R plays a role in maintaining the intestinal epithelial barrier function. M 3 R is a promising target for treating or preventing NSAID-induced enteropathy. intestinal epithelial barrier function positive allosteric modulator M3 muscarinic acetylcholine receptor NSAID-induced enteropathy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to relieve pain, reduce inflammation, and bring down a fever. However, despite their medical benefits, NSAIDs also induce side effects. One such side effect is drug-induced gastrointestinal injury. The recently developed modality of video capsule and balloon-assisted small intestinal endoscopy has shown that NSAIDs affect the small intestine as well as the stomach [ 1 , 2 , 3 ]. Several studies have reported that NSAID treatment increases intestinal permeability [ 4 ]. Indeed, as many as 70% of long-term users of NSAIDs show small intestinal injury, including 30% with erosions or ulcers [ 5 ]. Although appropriate treatment for NSAID-induced enteropathy is an urgent medical and social need, there is currently none available. While gastric acid suppression by proton pump inhibitors (PPIs) is effective for preventing NSAID-induced gastropathy, these agents are not effective or even worsen NSAID-induced enteropathy [ 6 , 7 ]. The intestinal epithelial barrier function is determined by the balance between mucosal offense and defense factors, such as the formation of a mucin layer and intestinal epithelial water secretion [ 8 ]. The Cl − transport across the intestinal epithelium is crucial for maintaining intestinal epithelial water secretion, thereby helping not only flush out noxious substances and harmful organisms but also form mucin layers in combination with secreted mucin [ 9 , 10 ]. EP 4 prostanoid receptor (EP 4 R) plays a crucial role in the maintenance of the intestinal epithelial barrier function by inducing intestinal epithelial Cl − secretion [ 11 ], and impairment of the intestinal barrier function by inhibiting PGE production is a primary cause of NSAID-induced enteropathy [ 1 ]. Although EP 4 R agonists are reasonably effective against NSAID-induced enteropathy [ 12 , 13 ], misoprostol, a prostaglandin E 1 (PGE 1 ) derivative, is currently clinically used for NSAID-induced gastropathy but not for NSAID-induced enteropathy due to side effects of severe diarrhea [ 14 , 15 ]. The M 3 muscarinic acetylcholine receptor (M 3 R) is a member of the G-protein-coupled, muscarinic acetylcholine receptor family, which mediate cholinergic neurotransmission at effector cells. Five types of muscarinic receptors (M 1 R, M 2 R, M 3 R, M 4 R, and M 5 R) have been identified, among which M 1 R and M 3 R are mainly expressed in intestinal epithelial cells. Due to a lack of receptor subtype-selective compounds, whether or not M 3 R plays a role in the maintenance of the intestinal epithelial barrier function remains unclear, and whether or not activation of M 3 R is protective against NSAID-induced enteropathy has not been addressed. Muscarinic receptors were recently shown to have an orthosteric site for native ligands of acetylcholine and an allosteric site for regulation of receptor activity [ 16 ]. Since M 3 R shares low homology of its allosteric site with other muscarinic receptors, this site can be a target for the synthesis of selective modulators for M 3 R. We recently developed a novel M 3 positive allosteric modulator (PAM), PAM-369, and confirmed that it actually acts as a PAM for M 3 R [ 17 ]. Since PAMs for M 3 R do not activate M 3 R without orthosteric ligands, M 3 PAM can retain spatiotemporal fidelity of physiological regulation or native signaling patterns [ 18 , 19 ]. Given the above, the present study explored using PAM-369 whether or not M 3 R protects against NSAID-induced enteropathy in a mouse model of indomethacin-induced small intestinal injury. Materials and Methods Animals Adult male C57BL/6J mice (Charles River Laboratories Inc., Kanagawa, Japan) between 7 and 9 weeks old were used for experiments. All mice were maintained in specific-pathogen-free facilities at Kyushu University (Fukuoka, Japan). All experiments were approved by the Committee on Animal Research of Kyushu University (A19-263). Chemicals PAM-369 was synthesized at Mochida Pharmaceuticals, Inc. (Tokyo, Japan) [ 17 ]. Carbachol, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), dimethyl sulfoxide (DMSO), forskolin, indomethacin, misoprostol, and neostigmine bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). ONO-AE3-208 was obtained from Cayman Chemical (Ann Arbor, MI, USA). For the ex vivo experiment, PAM-369, 4-DAMP, and forskolin were dissolved in DMSO as stock. Carbachol and neostigmine were dissolved in distilled water. ONO-AE3-208 was dissolved in a 1:1 solution of DMSO and phosphate-buffered saline (PBS). Misoprostol was dissolved in 100% ethanol as stock. For animal administration, PAM-369 was dissolved in 0.5% methylcellulose. Indomethacin was dissolved in 2% Na 2 CO 3 . The stock of misoprostol in 100% ethanol was diluted with PBS. Solutions Tissue preparation was conducted in normal extracellular solution (NES, pH 7.4) containing the following (in mmol/L): 137.4 NaCl, 5.9 KCl, 1.2 CaCl 2 , 1.2 MgCl 2 , 11.6 HEPES, and 11.5 glucose. All Ussing chamber experiments, except for that involving the Cl − free condition, were performed in a Krebs bicarbonate ringer (KBR) solution containing the following (in mmol/L): 119 NaCl, 21 NaHCO 3 , 2.4 K 2 HPO 4 , KH 2 PO 4 , 1.2 CaCl 2 , 1.2 MgCl 2 , and 10 glucose. The solution was gassed with 95% O 2 and 5% CO 2 , with a resulting pH of 7.4. For the Cl − free experiment, NaCl, CaCl 2 , and MgCl 2 were equimolarly substituted by Na-gluconate, Ca-(gluconate) 2 , and Mg-(gluconate) 2 , respectively. Tissue preparation The segments of mouse ileum were cleaned gently with cold NES, and then opened along the mesenteric border. The ileum segments were pinned to a silicon rubber plate with the serosal side up. The seromuscular layer was peeled off using microforceps by blunt dissection under a binocular microscope, and ileal muco-submucosal tissues for the Ussing chamber experiments were prepared. Ussing chamber experiments Ileal muco-submucosal tissues were mounted onto 0.25-cm 2 sliders dedicated to a Ussing chamber. Both mucosal and serosal sides of tissues were bathed in 3 mL oxygenated KBR solution separately. The solution was bubbled with 95% O 2 and 5% CO 2 and kept at 37°C during experiments by a circulating water bath. Ileal muco-submucosal tissues were short circuited by a voltage clamp (VCC MC6; Physiologic Instruments, San Diego, CA, USA), and the short-circuit current (Isc; µA/cm 2 ), which reflects transepithelial ion transport, was continuously monitored and recorded by Acquire & Analyze (Physiologic Instruments). After a 30-min equilibrium period, the compounds, including PAM-369, carbachol, misoprostol, and several inhibitors designed to meet the objectives, were applied. At the end of each experiment, the ileal muco-submucosal tissues were all stimulated by addition of forskolin (10 − 5 M) at the serosal side to obtain a reference response. In all experiments other than the Cl-free one, the ileal muco-submucosal tissues that had a good response to forskolin with a ΔIsc > 20 µA/cm 2 were only used for analyses. Induction of indomethacin-induced small intestinal injury After an overnight 14-h fast, mice were treated with indomethacin (15 mg/10 mL/kg) subcutaneously just before resuming feed. At 24 h after the administration of indomethacin, the mice were sacrificed under isoflurane anesthesia, and their small intestines were excised. To examine the lesion area, 1% Evans blue (5 mL/kg) was intravenously injected into mice 30 min before sacrifice. The areas of lesions in small intestines were calculated with the Image J software program. Drug administration to the mice with indomethacin-induced small intestinal injury PAM-369 (3, 10 and 30 mg/10 mL/kg) was orally administered once a day for 1 week, and 1 h after the last administration of PAM-369 (day 7), indomethacin was subcutaneously administered to induce small intestinal injury. Misoprostol (0.1 mg/10 mL/kg) was orally administered three times a day (1 h before and 3 and 6 h after treatment with indomethacin), since misoprostol has a short half-life in plasma [ 20 ]. Indomethacin-treated control mice were administered vehicle of 0.5% methylcellulose or 0.1% ethanol for PAM-369 or misoprostol, respectively, at the same time. Experimental protocols are shown in Supplemental Fig. 1a and b. The evaluation of ion secretion of ileal sub-mucosal tissues after indomethacin administration After an overnight 14-h fast, mice were treated with indomethacin (15 mg/10 mL/kg) subcutaneously just before resuming feed. Just before (0 h) and 3 and 6 h after the administration of indomethacin, the mice were sacrificed under isoflurane anesthesia. Some of the excised small intestines were immersed in RNAlater (Ambion, Austin, TX, USA) immediately after collection, transferred to − 30°C freezers, and stored until further analyses. Ileal muco-submucosal tissue was prepared for measurement of Isc as described above. Determination of mRNA expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from tissue samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Real-time quantitative RT-PCR analyes were performed as previously described [ 17 ]. The expression of mRNAs associated with cholinergic signaling, including the M 1 (Mm01231010_m1), M 2 (Mm01167087_m1), and M 3 (Mm01338410_m1) receptors, and choline acetyltransferase (ChAT) (Mm01221880_m1) was determined by quantitative PCR using FAM-labeled TaqMan Gene Expression Assay Reagents (Applied Biosystems, Foster City, CA, USA). Each target gene was normalized by glyceraldehyde-3-phosphate dehydrogenase (Mm99999915_g1) with the comparative ΔΔCt method. Statistical analyses All data are presented as the mean ± standard error. A statistical software package JMP software program (SAS Institute, Cary, NC, USA) was used to analyze the data. Statistical analyses were performed using Student’s unpaired t -test between two groups, a one-way analysis of variance (ANOVA) followed by Dunnett’s test for three or more groups, and a two-way ANOVA for three or more groups split on two variables. P < 0.05 was considered to be significant. Results Ion secretion activity of misoprostol in mouse ileum We first investigated the ion secretion activity of misoprostol in mouse ileum with a Ussing chamber. The prostaglandin E 1 (PGE 1 ) derivative misoprostol (0.1–10 µM) increased the Isc in a concentration-dependent manner (Fig. 1 a and b). The increase in the Isc obtained with 1 µM misoprostol was 12.7 ± 1.20 µA/cm 2 (n = 3) (Fig. 1 c). Pretreatment with ONO-AE3-208 (3 µM), an EP 4 R antagonist, almost abolished the misoprostol (1 µM)-induced increase in the Isc (Fig. 1 d). The extent of the misoprostol-induced increase in the Isc in the presence of ONO-AE3-208 (1.03 ± 0.50 µA/cm 2 ; n = 4) was significantly lower than that in the absence (8.25 ± 1.35 µA/cm 2 ; n = 4) ( p < 0.01) (Fig. 1 d and f), indicating that misoprostol-induced ion secretion activity was mediated by EP 4 R in mouse ileum. Removal of extracellular Cl − abolished the increase in the Isc induced not only by misoprostol (0.22 ± 0.20 µA/cm 2 ; n = 4) ( p < 0.01) but also by forskolin (Fig. 1 e and f), indicating that Cl − played a central role in misoprostol-induced ion secretion activity. Ion secretion activity of carbachol in mouse ileum. First, we examined effects of carbachol (0.1–10 µM) on Isc (Fig. 2 a and b). While 0.1 µM carbachol did not increase the Isc (0.64 ± 0.26 µA/cm 2 ; n = 3), carbachol did increase the Isc in a concentration-dependent manner between 0.3 and 10 µM. Since the increase in the Isc induced by 1 µM carbachol (13.83 ± 2.06 µA/cm 2 ; n = 3) (Fig. 2 c) was comparable to that induced by 1 µM misoprostol (12.7 ± 1.20 µA/cm 2 ) (Fig. 1 c), 1 µM carbachol was used in subsequent experiments. The carbachol-induced increase in the Isc was almost abolished either by pretreatment with the M 3 R antagonist 4-DAMP (0.32 ± 0.51 µA/cm 2 , n = 3, p < 0.01) or removal of extracellular Cl − (0.44 ± 0.16 µA/cm 2 , n = 4, p < 0.01) versus no pretreatment (9.97 ± 1.42 µA/cm 2 ; n = 8), indicating that stimulation of M 3 R by carbachol also caused Cl − secretion in mouse ileum (Fig. 2 d and e). Effects of PAM-369 on exogenously added carbachol-induced and endogenously produced acetylcholine-induced ion secretion activities in mouse ileum PAM-369 itself has no effect on any receptors, but it can generate an action on the target receptor under conditions where the orthosteric site is bound by either endogenously produced acetylcholine or exogenously added ligands, such as carbachol. Therefore, we investigated the effects of PAM-369 on the carbachol-induced increase in the Isc. While 1 µM PAM-369 did not induce a rapid increase in the Isc, it did enhance the carbachol (1 µM)-induced increase in the Isc in a dose-dependent manner (Fig. 3 a and b). The carbachol (1 µM)-induced increase in the Isc was significantly enhanced by pretreatment with PAM-369 at 1 µM (21.3 ± 1.70 µA/cm 2 ; n = 5) ( p < 0.05) and at 10 µM (20.9 ± 2.69 µA/cm 2 ; n = 8) ( p < 0.01), compared with the increase in the Isc without PAM-369 (9.51 ± 1.87 µA/cm 2 ; n = 7). However, the application of 10 µM PAM-369 itself gradually increased the basal Isc (Fig. 3 c). Pretreatment with 0.1 µM 4-DAMP almost abolished both the PAM-369-induced basal increase in the Isc (from 8.53 ± 1.82 to 0.91 ± 0.44 µA/cm 2 , p < 0.05) and the PAM-369-potentiated carbachol-induced increase in the Isc (from 21.8 ± 2.97 to 4.69 ± 1.24 µA/cm 2 , p < 0.01) (Fig. 3 d and e), indicating that the increase in the Isc associated with 10 µM PAM-369 was mediated by M 3 R. Next, we examined the effects of neostigmine, an acetylcholine esterase inhibitor, on the PAM-369-associated response. As shown in Fig. 3 f, g, and h, pretreatment with neostigmine significantly augmented the PAM-369-induced basal increase in the Isc (from 8.53 ± 1.82 to 15.8 ± 2.47 µA/cm 2 ) ( p < 0.05) but had no effect on the carbachol-induced increase in the Isc (from 21.8 ± 2.97 to 24.7 ± 2.90 µA/cm 2 ). Effects of misoprostol and potentiation of M 3 R by PAM-369 on indomethacin-induced small intestinal injury We examined whether and to what extent misoprostol and potentiation of M 3 R by PAM-369 can protect against indomethacin-induced small intestinal injury. Single subcutaneous treatment of indomethacin at 15 mg/kg caused acute ulcers and erosions in the small intestine, especially the ileum (Fig. 4 c, left). Oral administration of 0.1 mg/kg misoprostol 3 times a day (0.3 mg/kg/day) significantly ameliorated the indomethacin-induced injury. The area of indomethacin-induced ulcers treated with misoprostol (10.4 ± 1.03 mm 2 ; n = 6) was significantly smaller than that treated with vehicle (16.8 ± 2.35 mm 2 ; n = 6) ( p < 0.05) (Fig. 4 a). Furthermore, oral administration of PAM-369 at 10 and 30 mg/kg/day for 1 week also ameliorated indomethacin-induced intestinal injury (Fig. 4 b). The area of indomethacin-induced ulcers pretreated with PAM-369 at 30 mg/kg/day (8.58 ± 2.84 mm 2 ; n = 9) was significantly lower than that treated with vehicle (22.7 ± 2.99 mm 2 ; n = 8) ( p < 0.01). Notably, indomethacin-induced intestinal injury was almost completely prevented in four out of eight mice (Fig. 4 c). The mRNA expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α was upregulated by indomethacin treatment in the ileum segments both with and without visible ulcers. Treatment with PAM-369 at 30 mg/kg/day tended to suppress the expression of those inflammatory cytokine genes, especially in non-ulcer areas (Supplemental Fig. 3). Altered ion secretion responses of mouse ileum after indomethacin treatment We next investigated the ion secretion activities in the ileum harvested just before (0 h) and 3 and 6 h after administration of indomethacin. The carbachol-induced increase in the Isc was slightly augmented at 3 h (22.9 ± 2.22 µA/cm 2 ; n = 6) and 6 h (24.9 ± 3.36 µA/cm 2 ; n = 6) compared to at 0 h (17.1 ± 2.22 µA/cm 2 ; n = 5) (Fig. 5 a and b). As noted, the basal response of the Isc to PAM-369 was significantly enhanced as the treatment time of indomethacin increased. The basal increase in the Isc induced by 1 µM PAM-369 in the ileum harvested at 3 h (6.43 ± 0.55 µA/cm 2 , n = 6) and 6 h (7.63 ± 0.81 µA/cm 2 , n = 6) after indomethacin administration was significantly higher than that just before (0 h) indomethacin administration (2.89 ± 0.49 µA/cm 2 , n = 5) ( p < 0.01) (Fig. 5 c and d). Furthermore, the increase in the Isc induced by 10 µM PAM-369 in the ileum harvested at 3 h (17.1 ± 1.24 µA/cm 2 , n = 4) and 6 h (16.2 ± 1.33 µA/cm 2 , n = 4) was significantly higher than that just before (0 h) indomethacin administration (10.1 ± 1.67 µA/cm 2 , n = 4) ( p < 0.05) (Fig. 5 e and f). Time course of the expression of cholinergic - associated genes in mouse ileum after indomethacin treatment To investigate the mechanisms by which the responses of the Isc to carbachol and PAM-369 were potentiated in the ileum harvested at 3 and 6 h after indomethacin administration, we examined whether or not the expression of cholinergic genes was changed under those conditions. The mRNA expression of M 1 R, M 2 R, and ChAT at 3 and 6 h after indomethacin administration did not markedly differ from just before (0 h) indomethacin administration (Fig. 6 a, b, and d). However, the mRNA expression of M 3 R was significantly increased at 3 and 6 h after indomethacin administration compared to that just before (0 h) indomethacin administration (Fig. 6 c). Discussion Whether or not M 3 R was involved in the intestinal epithelial barrier function has been unclear, as receptor subtype-selective compounds are not currently available and mice lacking the M 3 R seemed to retain cholinergic intestinal ion transport, probably due to compensatory mechanisms [ 21 ]. In the present study using a novel highly selective M 3 PAM (PAM-369), we showed for the first time that M 3 R does help maintain the intestinal epithelial barrier function via induction of intestinal epithelial Cl − secretion in a similar manner to EP 4 R, and potentiation of M 3 R by M 3 PAM ameliorated indomethacin-induced small intestinal injury. This study demonstrated that both EP 4 R and M 3 R contribute to the intestinal epithelial Cl − secretion in a similar manner, using the application of receptor antagonists and an extracellular Cl − -free condition. Intestinal epithelial Cl − secretion through apical Cl − channels, mainly cystic fibrosis transmembrane conductance regulator (CFTR), is known to be indispensable for the formation of water secretion and the mucin layer, which plays a crucial role in the intestinal mucosal barrier function [ 8 , 10 , 22 , 23 ]. Cl − secretion is accompanied by secretion of Na + in a paracellular pathway, which is followed by water secretion [ 24 ]. One important point of the present study is that the involvement of M 3 R in intestinal epithelial Cl − secretion has been definitely determined using PAM-369 in combination with complete blockade of the Isc increase via 4-DAMP, an M 3 R antagonist. It is considered that activation of EP 4 R enhances apical Cl − conductance via CFTR through an increase in cyclic adenosine monophosphate (cAMP). In contrast, it is reported that activation of M 3 R reinforces basolateral K + efflux in a Ca 2+ -dependent manner, leading to an increased uptake of Cl − into epithelial cells via Na/K/2Cl cotransporter (NKCC1), which results in an increased driving force for intestinal epithelial Cl − secretion [ 24 ]. Use of PAM-369 also led us to discover the important fact that a small amount of endogenous acetylcholine continues to be produced basally in intestinal tissue. Application of PAM-369 at a high concentration slowly but significantly increased the basal Isc in the absence of carbachol, even though PAM-369 exerts no effects on the gastrointestinal tract in the absence of an orthosteric ligand for M 3 R. This was further confirmed by the finding that the PAM-369-induced increase in basal Isc was significantly potentiated by neostigmine. Given that 4-DAMP had no effects on the basal Isc, the amount of basally produced acetylcholine is not sufficient to cause a basal increase in the Isc itself; however, the basal response to acetylcholine was unmasked by allosteric changes of M 3 R by 10 µM PAM-369. We were unable to clarify in our study why a small amount of endogenous acetylcholine continued to be produced basally. It has been reported that a basal level of acetylcholine release was also observed in unstimulated bladder tissue [ 25 ]. One speculation is that basal stimulation of M 3 R with acetylcholine at a very low level that cannot induce any response itself might contribute to the conduct of an appropriate reaction or avoid an unexpectedly strong reaction by adjusting the M 3 R sensitivity to orthosteric ligands. Further studies will be required to clarify this point in the future. The present study successfully demonstrated for the first time that M 3 R was a viable therapeutic target for NSAID-induced enteropathy. Impairment of the intestinal epithelial barrier function by inhibition of the PGE-EP 4 signal transduction pathway is a primary cause of NSAID-induced enteropathy. An EP 4 R agonist reportedly alleviated indomethacin-induced small intestinal injury in rodents [ 12 ]. Consistent with previous studies, we confirmed that activation of EP 4 R by misoprostol increased intestinal epithelial Cl − secretion ex vivo , and misoprostol was indeed effective in preventing indomethacin-induced small intestinal injury in our in vivo mice model. Although misoprostol is currently clinically available, common side effects, such as abdominal pain and diarrhea, are not usually tolerated by patients with NSAID-induced enteropathy in clinical practice [ 14 , 15 ]. Since M 3 R activation can induce intestinal epithelial Cl − secretion in a similar manner to EP 4 R activation, we hypothesized that M 3 R would be a viable substitute for EP 4 R in preventing NSAID-induced enteropathy. As expected, potentiation of M 3 R ameliorated indomethacin-induced small intestinal injury in a similar manner to misoprostol. This study provided further evidence that M 3 R is a viable therapeutic target for NSAID-induced enteropathy. It should be noted in the present study that the PAM-369-induced increase in the basal Isc was significantly enhanced after indomethacin treatment, indicating that M 3 R can be enhanced by induction of NSAID-induced enteropathy. Indeed, the cholinergic system, including M 3 R and ChAT, can reportedly be enhanced in acute small intestinal inflammation to prevent further progression of organ damage [ 26 ]. Consistent with the results concerning the Isc in the Ussing chamber, we confirmed that the expression of M 3 R (but not M 1 R or M 2 R) was significantly up-regulated by indomethacin treatment, while the ChAT expression was unchanged. Given that 4-DAMP had no effect on the basal Isc but the PAM-369-induced increase in basal Isc was potentiated by indomethacin treatment, not only the increased expression of M 3 R itself but also the increased sensitivity of the orthostatic site and/or allosteric site to M 3 R may be present during indomethacin treatment. It is unlikely that functional M 3 R is newly synthesized in a short time [ 27 ], but its up-regulation by indomethacin treatment is possibly brought by endocytic trafficking [ 28 ]. Further investigations will be needed to clarify the mechanism underlying how M 3 R signaling was enhanced under conditions where the production of prostaglandin is suppressed. More importantly, combined treatment of misoprostol and PAM-369 had no additional effects (Supplemental Fig. 2), suggesting that both agents may exert their effects through the same final pathway, such as CFTR. In NSAID-induced enteropathy, up-regulation of M 3 R signaling might be induced in order to compensate for the down-regulated PGE-EP 4 pathway in terms of the intestinal epithelial barrier function (Fig. 7 ). In this regard, M 3 R may indeed be a therapeutic target for NSAID-induced enteropathy. In conclusion, M 3 R plays a role in intestinal epithelial Cl − secretion in a similar manner to EP 4 R in terms of the intestinal mucosal barrier function. Suppression of prostaglandin production by indomethacin induced an increased expression of M 3 R and enhanced M 3 R signaling, possibly via a compensatory mechanism. The further potentiation of M 3 receptor by PAM-369 ameliorated indomethacin-induced small intestinal injury. M 3 R is a promising target for the treatment or prevention of NSAID-induced enteropathy. Abbreviations ChAT, choline acetyltransferase; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; DMSO, dimethyl sulfoxide; EP 4 R, EP 4 prostanoid receptor; IK Ca , intermediate conductance calcium-sensitive potassium channel; IL, interleukin; Isc, short circuit current; KBR, Krebs bicarbonate ringer; M 3 R, M 3 muscarinic acetylcholine receptor; NES, normal extracellular solution; NKCC, Na/K/2Cl cotransporter; NSAID, nonsteroidal anti-inflammatory drug; PAM, positive allosteric modulator; PGE, prostaglandin E; PPI, proton pump inhibitor. Declarations Acknowledgments: The authors thank the laboratory members for their hard work and thorough discussion and Japan Medical Communication (http://www.japan-mc.co.jp) for editing a draft of this manuscript. Funding: This project has been executed using the institutions’ (Kyushu University and Mochida Pharmaceutical Co., Ltd.) budgets including a grant from Mochida Pharmaceutical Co., Ltd. to EI and YO for this collaborative research. The funders provided support in the form of salaries for authors YI, CH, and YT (Mochida Pharmaceutical Co., Ltd.). This study was also supported in part by the Japan Society for the Promotion of Science KAKENHI (23K07440 and 22K19530). Disclosures: YI and YT are the employees of Mochida Pharmaceutical Co., Ltd. whose company partly funded this study. EI belongs to an endowed course supported by companies including Ono Pharmaceutical Co., Ltd., Miyarisan Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Otsuka Pharmaceutical Factory, Inc., Fujifilm Medical Co., Ltd., Terumo Corporation, FANCL Corporation, Ohga Pharmacy, and Abbott Japan, LLC. EI receives a lecture honorarium from Takeda Pharmaceutical Company. YO conducts collaborative research with Fujifilm Medical Co., Ltd. and FANCL Corporation. Author contributions: Yoko Igarashi-Hisasyoshi, Eikichi Ihara, Yoshimasa Tanaka, Haruei Ogino, and Takatoshi Chinen proposed the research design. Yoko Igarashi-Hisayoshi and Xiaopeng Bai conducted experiments. Yoko Igarashi-Hisasyoshi performed data analysis. Eikichi Ihara and Yasushi Taguchi contributed new reagents or analytic tools. Yoko Igarashi-Hisasyoshi and Eikichi Ihara mainly wrote the manuscript. Xiaopeng Bai, Yoshimasa Tanaka, Haruei Ogino, and Takatoshi Chinen reviewed the manuscript. Yoshihiro Ogawa supervised this study. All authors contributed to the interpretation of the study. Data Availability: The data presented in this work are available upon request from the corresponding author. Ethical approval: The ethics governing the use and conduct of experiments on animals were strictly observed, and the experimental protocol was approved by the Committee on Animal Research of Kyushu University (A19-263). References Higuchi K, Umegaki E, Watanabe T et al (2009) Present status and strategy of NSAIDs-induced small bowel injury. J Gastroenterol 44:879-888. https://doi.org/10.1007/s00535-009-0102-2 Endo H, Sakai E, Kato T et al (2015) Small bowel injury in low-dose aspirin users. J Gastroenterol 50:378-386. https://doi.org/10.1007/s00535-014-1028-x Matsumoto T, Kudo T, Esaki M et al (2008) Prevalence of non-steroidal anti-inflammatory drug-induced enteropathy determined by double-balloon endoscopy: a Japanese multicenter study. Scand J Gastroenterol 43:490-496. https://doi.org/10.1080/00365520701794121 Edogawa S, Peters SA, Jenkins GD et al (2018) Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota. FASEB J, fj201800560R. https://doi.org/10.1096/fj.201800560R Bjarnason I, Scarpignato C, Holmgren E, Olszewski M, Rainsford KD, Lanas A (2018) Mechanisms of Damage to the Gastrointestinal Tract From Nonsteroidal Anti-Inflammatory Drugs. Gastroenterology 154:500-514. https://doi.org/10.1053/j.gastro.2017.10.049 Washio E, Esaki M, Maehata Y et al (2016) Proton Pump Inhibitors Increase Incidence of Nonsteroidal Anti-Inflammatory Drug-Induced Small Bowel Injury: A Randomized, Placebo-Controlled Trial. Clin Gastroenterol Hepatol 14:809-815.e801. https://doi.org/10.1016/j.cgh.2015.10.022 Wallace JL, Syer S, Denou E et al (2011) Proton pump inhibitors exacerbate NSAID-induced small intestinal injury by inducing dysbiosis. Gastroenterology 141:1314-1322, 1322.e1311-1315. https://doi.org/10.1053/j.gastro.2011.06.075 McGuckin MA, Lindén SK, Sutton P, Florin TH (2011) Mucin dynamics and enteric pathogens. 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J Pharmacol Sci 114:248-261. https://doi.org/10.1254/jphs.10r06cr Kunikata T, Araki H, Takeeda M, Kato S, Takeuchi K (2001) Prostaglandin E prevents indomethacin-induced gastric and intestinal damage through different EP receptor subtypes. J Physiol Paris 95:157-163. https://doi.org/10.1016/s0928-4257(01)00021-3 Taha AS, McCloskey C, McSkimming P, McConnachie A (2018) Misoprostol for small bowel ulcers in patients with obscure bleeding taking aspirin and non-steroidal anti-inflammatory drugs (MASTERS): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Gastroenterol Hepatol 3:469-476. https://doi.org/10.1016/S2468-1253(18)30119-5 Watanabe T, Sugimori S, Kameda N et al (2008) Small bowel injury by low-dose enteric-coated aspirin and treatment with misoprostol: a pilot study. Clin Gastroenterol Hepatol 6:1279-1282. https://doi.org/10.1016/j.cgh.2008.06.021 Kruse AC, Hu J, Pan AC et al (2012) Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482:552-556. https://doi.org/10.1038/nature10867 Igarashi-Hisayoshi Y, Ihara E, Bai X et al (2023) Determination of Region-Specific Roles of the M. Dig Dis Sci 68:439-450. https://doi.org/10.1007/s10620-022-07637-y Burford NT, Traynor JR, Alt A (2015) Positive allosteric modulators of the μ-opioid receptor: a novel approach for future pain medications. Br J Pharmacol 172:277-286. https://doi.org/10.1111/bph.12599 Jakubík J, El-Fakahany EE (2010) Allosteric Modulation of Muscarinic Acetylcholine Receptors. Pharmaceuticals (Basel) 3:2838-2860. https://doi.org/ 10.3390/ph3092838. Tang OS, Gemzell-Danielsson K, Ho PC (2007) Misoprostol: pharmacokinetic profiles, effects on the uterus and side-effects. Int J Gynaecol Obstet 99:S160-167. https://doi.org/10.1016/j.ijgo.2007.09.004 Hirota CL, McKay DM (2006b) M3 muscarinic receptor-deficient mice retain bethanechol-mediated intestinal ion transport and are more sensitive to colitis. Can J Physiol Pharmacol 84:1153-1161. https://doi.org/10.1139/y06-068 Gustafsson JK, Lindén SK, Alwan AH, Scholte BJ, Hansson GC, Sjövall H (2015) Carbachol-induced colonic mucus formation requires transport via NKCC1, K⁺ channels and CFTR. Pflugers Arch 467:1403-1415. https://doi.org/10.1007/s00424-014-1595-y Garcia MA, Yang N, Quinton PM (2009) Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion. J Clin Invest 119:2613-2622. https://doi.org/10.1172/JCI38662 Barrett KE, Keely SJ (2000) Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol 62:535-572. https://doi.org/10.1146/annurev.physiol.62.1.535 Yoshida M, Inadome A, Maeda Y et al (2006) Non-neuronal cholinergic system in human bladder urothelium. Urology, 67:425-430. https://doi.org/10.1016/j.urology.2005.08.014 Pohl CS, Lennon EM, Li Y, DeWilde MP, Moeser AJ (2018) S. Typhimurium challenge in juvenile pigs modulates the expression and localization of enteric cholinergic proteins and correlates with mucosal injury and inflammation. Auton Neurosci, 213:51-59. https://doi.org/10.1016/j.autneu.2018.05.009 Nathanson NM (2008) Synthesis, trafficking, and localization of muscarinic acetylcholine receptors. Pharmacol Ther, 119:33-43. https://doi.org/10.1016/j.pharmthera.2008.04.006 Zenko D, Hislop JN (2018) Regulation and trafficking of muscarinic acetylcholine receptors. Neuropharmacology, 136:374-382. https://doi.org/10.1016/j.neuropharm.2017.11.017 Supplementary Files M3PAMNSAIDSupplementalMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 22 Aug, 2024 Read the published version in Journal of Molecular Medicine → Version 1 posted Editorial decision: Major Revisions Needed 13 Feb, 2024 Reviewers agreed at journal 23 Oct, 2023 Reviewers invited by journal 07 Aug, 2023 Editor assigned by journal 01 Aug, 2023 First submitted to journal 29 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3217047","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":224726725,"identity":"ec6bf34a-b024-47a5-a3b0-3779ec3be629","order_by":0,"name":"Yoko 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06:58:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3217047/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3217047/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00109-024-02474-0","type":"published","date":"2024-08-22T15:57:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":41444243,"identity":"ef0b3432-0603-4579-bf42-b9503a4d1019","added_by":"auto","created_at":"2023-08-11 14:23:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241159,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of misoprostol on the Isc in mouse ileum. a: Representative recordings of 0.1, 0.3, 1, 3, and 10 μM misoprostol (Miso, serosal side)-induced Isc increase. Fifteen minutes after the last test substance application, forskolin (FSK, 10 μM, serosal side) was applied to confirm the viability of the tissue in all experiments. b: The cumulative results obtained from three independent experiments for the concentration-dependent Isc response to misoprostol are shown. c, d: Representative recordings of the misoprostol (1 μM, serosal side)-induced Isc increase in the absence (c) or presence (d) of ONO-AE3-208 (3 μM, serosal side), which was applied 15 min before misoprostol application. e: A representative recording of the misoprostol (1 μM, serosal side)-induced Isc increase under Cl\u003csup\u003e-\u003c/sup\u003e-free conditions. f: The cumulative results obtained from 3-4 independent experiments for c, d, and e. Data are shown as the means ± SE. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, significantly different from the misoprostol (1 μM) response (Dunnett’s test).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/a0d093f4a562d4ad93247442.png"},{"id":41441908,"identity":"b80a513c-a6d8-4ad6-8a2e-952ec39998ae","added_by":"auto","created_at":"2023-08-11 14:15:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":240072,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of carbachol on the Isc in mouse ileum. a: Representative recordings of 0.1, 0.3, 1, 3, and 10 μM carbachol-induced Isc increase. b: The cumulative results obtained from three independent experiments for concentration-dependent Isc response to carbachol are shown. c, d: Representative recording of the carbachol (1 μM, serosal side)-induced Isc increase in the absence (c) or presence (d) of 4-DAMP (0.1 μM, serosal side), which was applied 15 min before carbachol application. e: A representative recording of the carbachol (1 μM, serosal side)-induced Isc increase under Cl\u003csup\u003e-\u003c/sup\u003e-free conditions. f: The cumulative results obtained from 3-4 independent experiments for c, d, and e. Data are shown as the means ± SE. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, significantly different from the carbachol (1 μM) response (Dunnett’s test).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/db847bc70fef7b0fb76ef525.png"},{"id":41444244,"identity":"a08faafb-8b04-41c9-aefd-f56ac69e96d3","added_by":"auto","created_at":"2023-08-11 14:23:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":416448,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of PAM-369 on the carbachol-induced and basal Isc in mouse ileum. a: A representative recording of the carbachol (1 μM, serosal side)-induced Isc increase in the absence or presence of PAM-369 (0.1 μM, serosal side), which was applied 15 min before carbachol application. b: The cumulative results obtained from 4-8 independent experiments for the concentration-dependent effects of PAM-369 on the carbachol-induced Isc increase are shown. c: A representative recording of changes in the basal and carbachol (1 μM, serosal side)-induced Isc potentiated by PAM-369 (10 μM, serosal side) in the absence or presence of 4-DAMP (0.1 μM, serosal side). d, e: The cumulative results from 4-7 independent experiments for the effects of 4-DAMP on the increase in the basal (d) and carbachol-induced (e) Isc potentiated by PAM-369 are shown. f: A representative recording of changes in the basal and carbachol (1 μM, serosal side)-induced Isc potentiated by PAM-369 (10 μM, serosal side) in the presence of neostigmine (1 μM, serosal side). g, h: The cumulative results from 4-7 independent experiments for the effects of neostigmine increase on the basal (g) and carbachol-induced (h) Isc potentiated by PAM-369 are shown. Data are shown as the means ± SE. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, significantly different from the control response in the absence of PAM-369 (Dunnett’s test). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, significantly different between the two indicated groups (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/7e817e6e6d9df77db3d8243f.png"},{"id":41441911,"identity":"94ebc3ca-3b3a-4135-a589-d736536f66a2","added_by":"auto","created_at":"2023-08-11 14:15:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727187,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of misoprostol and PAM-369 on indomethacin-induced small intestinal injury in mouse. a, b: The cumulative results of effects of misoprostol (a, n=6) or PAM-369 (b, n=3-9) on the ulcer area of small intestine in indomethacin-treated mice. Misoprostol (0.1 mg/10 mL/kg) or vehicle was orally applied 3 times a day, 1 h before and 3 and 6 h after indomethacin administration. PAM-369 (3, 10, and 30 mg/10 mL/kg) or vehicle was orally applied once a day for 1 week. Mice were fasted for 14 h, and 1 h after the first administration of misoprostol or last administration of PAM-369 (day\u0026nbsp;7), indomethacin (15 mg/10 mL/kg) was subcutaneously administered. Mice were fed soon after indomethacin administration. After 24 h, mice were sacrificed, and their small intestines were excised. Evans blue (5 mL/kg) was intravenously injected into mice 30 min before sacrifice. The areas of lesions in small intestines were calculated with the Image J software program. c: Macroscopic views of small intestinal ulcerative lesions in indomethacin-treated mice in the absence (left) or presence (right) of 30 mg/kg PAM-369. The lesions were stained dark blue with 1% Evans blue. Data are shown as the means ± SE. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, significantly different between the two indicated groups (Student’s \u003cem\u003et\u003c/em\u003e-test). \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, significantly different from the vehicle administration (Dunnett’s test).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/efbc993ecfbc957b70eb79c7.png"},{"id":41441909,"identity":"81a61e61-ae65-4884-adc0-e969dac2ab98","added_by":"auto","created_at":"2023-08-11 14:15:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":460793,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of indomethacin treatment on activity of M\u003csub\u003e3\u003c/sub\u003e receptor signaling in mouse ileum. a, c, e: Representative recordings of changes in the Isc induced by carbachol (1 μM, serosal side) without PAM-369 (a) and treated with 1 μM (c) or 10 μM (e) PAM-369 in mouse ileum before (0 h) and after 3 and 6 h after indomethacin administration. b, d, f: The cumulative results obtained from 5-6 independent experiments for a, c, e, respectively, are shown. Mice were fasted for 14 h, and indomethacin (15 mg/10 mL/kg) was subcutaneously administered. Before (0 h) and 3 and 6 h after administration, mice were sacrificed, and their small intestines were excised. Data are shown as the means ± SE. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, significantly different from the response of the ileum just before (0 h) indomethacin administration (Dunnett’s test).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/7b2288e6d9aface7cc67dd88.png"},{"id":41441913,"identity":"43d78503-a601-4468-a8c6-df292de59fcf","added_by":"auto","created_at":"2023-08-11 14:15:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":136503,"visible":true,"origin":"","legend":"\u003cp\u003eThe mRNA expression of cholinergic-associated genes in mouse ileum treated with indomethacin. a-d: The mRNA expression of M\u003csub\u003e1\u003c/sub\u003e (a), M\u003csub\u003e2\u003c/sub\u003e (b), M\u003csub\u003e3\u003c/sub\u003e (c), and ChAT (d) in mouse ileum just before (0 h) and 3 and 6 h after indomethacin administration (n=6). Mice were fasted for 14 h, and indomethacin (15 mg/10 mL/kg) was subcutaneously administered. Before (0 h) and 3 and 6 h after indomethacin administration, mice were sacrificed, and their small intestines were excised. Data are shown as the means ± SE. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, significantly different from the expression in the ileum just before indomethacin administration (Dunnett’s test).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/1a0e860628cbe9a88c6dedc1.png"},{"id":41441914,"identity":"1b379b46-8c5c-47df-9a15-4d94e20caf60","added_by":"auto","created_at":"2023-08-11 14:15:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":398654,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced M\u003csub\u003e3\u003c/sub\u003e receptor signaling in NSAID-induced small intestinal injury. a: In normal small intestine, both EP\u003csub\u003e4\u003c/sub\u003e and M\u003csub\u003e3\u003c/sub\u003e receptor signaling pathways contribute to Cl\u003csup\u003e-\u003c/sup\u003e secretion to maintain the mucosal barrier function. b: In indomethacin-treated small intestine, the M\u003csub\u003e3\u003c/sub\u003e receptor signaling pathway is enhanced, possibly to compensate for the down-regulated EP\u003csub\u003e4\u003c/sub\u003e receptor signaling pathway, to maintain the mucosal barrier function. ACh, acetylcholine; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; IKCa, intermediate conductance calcium-sensitive potassium channel; NKCC, Na/K/2Cl cotransporter; PGE, prostaglandin E.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/e7f228a8e61ccd1735ca4d3e.png"},{"id":63300340,"identity":"fbeca1c2-575e-491c-9446-d5747d5f8433","added_by":"auto","created_at":"2024-08-26 16:13:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3228219,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/eb494c1e-0fb0-4130-8052-b6d6ce823321.pdf"},{"id":41441915,"identity":"776ba5cf-41e8-470f-822e-19ae86dbfc65","added_by":"auto","created_at":"2023-08-11 14:15:15","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":227493,"visible":true,"origin":"","legend":"","description":"","filename":"M3PAMNSAIDSupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3217047/v1/393a43cd6f401776773acc29.docx"}],"financialInterests":"","formattedTitle":"Protective role of M3 muscarinic acetylcholine receptor in indomethacin-induced small intestinal injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to relieve pain, reduce inflammation, and bring down a fever. However, despite their medical benefits, NSAIDs also induce side effects.\u003c/p\u003e \u003cp\u003eOne such side effect is drug-induced gastrointestinal injury. The recently developed modality of video capsule and balloon-assisted small intestinal endoscopy has shown that NSAIDs affect the small intestine as well as the stomach [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Several studies have reported that NSAID treatment increases intestinal permeability [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Indeed, as many as 70% of long-term users of NSAIDs show small intestinal injury, including 30% with erosions or ulcers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although appropriate treatment for NSAID-induced enteropathy is an urgent medical and social need, there is currently none available. While gastric acid suppression by proton pump inhibitors (PPIs) is effective for preventing NSAID-induced gastropathy, these agents are not effective or even worsen NSAID-induced enteropathy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe intestinal epithelial barrier function is determined by the balance between mucosal offense and defense factors, such as the formation of a mucin layer and intestinal epithelial water secretion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e transport across the intestinal epithelium is crucial for maintaining intestinal epithelial water secretion, thereby helping not only flush out noxious substances and harmful organisms but also form mucin layers in combination with secreted mucin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. EP\u003csub\u003e4\u003c/sub\u003e prostanoid receptor (EP\u003csub\u003e4\u003c/sub\u003eR) plays a crucial role in the maintenance of the intestinal epithelial barrier function by inducing intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and impairment of the intestinal barrier function by inhibiting PGE production is a primary cause of NSAID-induced enteropathy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although EP\u003csub\u003e4\u003c/sub\u003eR agonists are reasonably effective against NSAID-induced enteropathy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], misoprostol, a prostaglandin E\u003csub\u003e1\u003c/sub\u003e (PGE\u003csub\u003e1\u003c/sub\u003e) derivative, is currently clinically used for NSAID-induced gastropathy but not for NSAID-induced enteropathy due to side effects of severe diarrhea [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe M\u003csub\u003e3\u003c/sub\u003e muscarinic acetylcholine receptor (M\u003csub\u003e3\u003c/sub\u003eR) is a member of the G-protein-coupled, muscarinic acetylcholine receptor family, which mediate cholinergic neurotransmission at effector cells. Five types of muscarinic receptors (M\u003csub\u003e1\u003c/sub\u003eR, M\u003csub\u003e2\u003c/sub\u003eR, M\u003csub\u003e3\u003c/sub\u003eR, M\u003csub\u003e4\u003c/sub\u003eR, and M\u003csub\u003e5\u003c/sub\u003eR) have been identified, among which M\u003csub\u003e1\u003c/sub\u003eR and M\u003csub\u003e3\u003c/sub\u003eR are mainly expressed in intestinal epithelial cells. Due to a lack of receptor subtype-selective compounds, whether or not M\u003csub\u003e3\u003c/sub\u003eR plays a role in the maintenance of the intestinal epithelial barrier function remains unclear, and whether or not activation of M\u003csub\u003e3\u003c/sub\u003eR is protective against NSAID-induced enteropathy has not been addressed.\u003c/p\u003e \u003cp\u003eMuscarinic receptors were recently shown to have an orthosteric site for native ligands of acetylcholine and an allosteric site for regulation of receptor activity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Since M\u003csub\u003e3\u003c/sub\u003eR shares low homology of its allosteric site with other muscarinic receptors, this site can be a target for the synthesis of selective modulators for M\u003csub\u003e3\u003c/sub\u003eR. We recently developed a novel M\u003csub\u003e3\u003c/sub\u003e positive allosteric modulator (PAM), PAM-369, and confirmed that it actually acts as a PAM for M\u003csub\u003e3\u003c/sub\u003eR [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Since PAMs for M\u003csub\u003e3\u003c/sub\u003eR do not activate M\u003csub\u003e3\u003c/sub\u003eR without orthosteric ligands, M\u003csub\u003e3\u003c/sub\u003e PAM can retain spatiotemporal fidelity of physiological regulation or native signaling patterns [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the above, the present study explored using PAM-369 whether or not M\u003csub\u003e3\u003c/sub\u003eR protects against NSAID-induced enteropathy in a mouse model of indomethacin-induced small intestinal injury.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult male C57BL/6J mice (Charles River Laboratories Inc., Kanagawa, Japan) between 7 and 9 weeks old were used for experiments. All mice were maintained in specific-pathogen-free facilities at Kyushu University (Fukuoka, Japan). All experiments were approved by the Committee on Animal Research of Kyushu University (A19-263).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003ePAM-369 was synthesized at Mochida Pharmaceuticals, Inc. (Tokyo, Japan) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Carbachol, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), dimethyl sulfoxide (DMSO), forskolin, indomethacin, misoprostol, and neostigmine bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). ONO-AE3-208 was obtained from Cayman Chemical (Ann Arbor, MI, USA). For the \u003cem\u003eex vivo\u003c/em\u003e experiment, PAM-369, 4-DAMP, and forskolin were dissolved in DMSO as stock. Carbachol and neostigmine were dissolved in distilled water. ONO-AE3-208 was dissolved in a 1:1 solution of DMSO and phosphate-buffered saline (PBS). Misoprostol was dissolved in 100% ethanol as stock. For animal administration, PAM-369 was dissolved in 0.5% methylcellulose. Indomethacin was dissolved in 2% Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The stock of misoprostol in 100% ethanol was diluted with PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSolutions\u003c/h2\u003e \u003cp\u003eTissue preparation was conducted in normal extracellular solution (NES, pH 7.4) containing the following (in mmol/L): 137.4 NaCl, 5.9 KCl, 1.2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1.2 MgCl\u003csub\u003e2\u003c/sub\u003e, 11.6 HEPES, and 11.5 glucose. All Ussing chamber experiments, except for that involving the Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e free condition, were performed in a Krebs bicarbonate ringer (KBR) solution containing the following (in mmol/L): 119 NaCl, 21 NaHCO\u003csub\u003e3\u003c/sub\u003e, 2.4 K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1.2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1.2 MgCl\u003csub\u003e2\u003c/sub\u003e, and 10 glucose. The solution was gassed with 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e, with a resulting pH of 7.4. For the Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e free experiment, NaCl, CaCl\u003csub\u003e2\u003c/sub\u003e, and MgCl\u003csub\u003e2\u003c/sub\u003e were equimolarly substituted by Na-gluconate, Ca-(gluconate)\u003csub\u003e2\u003c/sub\u003e, and Mg-(gluconate)\u003csub\u003e2\u003c/sub\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTissue preparation\u003c/h2\u003e \u003cp\u003eThe segments of mouse ileum were cleaned gently with cold NES, and then opened along the mesenteric border. The ileum segments were pinned to a silicon rubber plate with the serosal side up. The seromuscular layer was peeled off using microforceps by blunt dissection under a binocular microscope, and ileal muco-submucosal tissues for the Ussing chamber experiments were prepared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eUssing chamber experiments\u003c/h2\u003e \u003cp\u003eIleal muco-submucosal tissues were mounted onto 0.25-cm\u003csup\u003e2\u003c/sup\u003e sliders dedicated to a Ussing chamber. Both mucosal and serosal sides of tissues were bathed in 3 mL oxygenated KBR solution separately. The solution was bubbled with 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e and kept at 37\u0026deg;C during experiments by a circulating water bath. Ileal muco-submucosal tissues were short circuited by a voltage clamp (VCC MC6; Physiologic Instruments, San Diego, CA, USA), and the short-circuit current (Isc; \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e), which reflects transepithelial ion transport, was continuously monitored and recorded by Acquire \u0026amp; Analyze (Physiologic Instruments). After a 30-min equilibrium period, the compounds, including PAM-369, carbachol, misoprostol, and several inhibitors designed to meet the objectives, were applied. At the end of each experiment, the ileal muco-submucosal tissues were all stimulated by addition of forskolin (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M) at the serosal side to obtain a reference response. In all experiments other than the Cl-free one, the ileal muco-submucosal tissues that had a good response to forskolin with a ΔIsc\u0026thinsp;\u0026gt;\u0026thinsp;20 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e were only used for analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInduction of indomethacin-induced small intestinal injury\u003c/h2\u003e \u003cp\u003eAfter an overnight 14-h fast, mice were treated with indomethacin (15 mg/10 mL/kg) subcutaneously just before resuming feed. At 24 h after the administration of indomethacin, the mice were sacrificed under isoflurane anesthesia, and their small intestines were excised. To examine the lesion area, 1% Evans blue (5 mL/kg) was intravenously injected into mice 30 min before sacrifice. The areas of lesions in small intestines were calculated with the Image J software program.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eDrug administration to the mice with indomethacin-induced small intestinal injury\u003c/h2\u003e \u003cp\u003ePAM-369 (3, 10 and 30 mg/10 mL/kg) was orally administered once a day for 1 week, and 1 h after the last administration of PAM-369 (day 7), indomethacin was subcutaneously administered to induce small intestinal injury. Misoprostol (0.1 mg/10 mL/kg) was orally administered three times a day (1 h before and 3 and 6 h after treatment with indomethacin), since misoprostol has a short half-life in plasma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Indomethacin-treated control mice were administered vehicle of 0.5% methylcellulose or 0.1% ethanol for PAM-369 or misoprostol, respectively, at the same time. Experimental protocols are shown in Supplemental Fig.\u0026nbsp;1a and b.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe evaluation of ion secretion of ileal sub-mucosal tissues after indomethacin administration\u003c/h2\u003e \u003cp\u003eAfter an overnight 14-h fast, mice were treated with indomethacin (15 mg/10 mL/kg) subcutaneously just before resuming feed. Just before (0 h) and 3 and 6 h after the administration of indomethacin, the mice were sacrificed under isoflurane anesthesia. Some of the excised small intestines were immersed in RNAlater (Ambion, Austin, TX, USA) immediately after collection, transferred to \u0026minus;\u0026thinsp;30\u0026deg;C freezers, and stored until further analyses. Ileal muco-submucosal tissue was prepared for measurement of Isc as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of mRNA expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from tissue samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Real-time quantitative RT-PCR analyes were performed as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The expression of mRNAs associated with cholinergic signaling, including the M\u003csub\u003e1\u003c/sub\u003e (Mm01231010_m1), M\u003csub\u003e2\u003c/sub\u003e (Mm01167087_m1), and M\u003csub\u003e3\u003c/sub\u003e (Mm01338410_m1) receptors, and choline acetyltransferase (ChAT) (Mm01221880_m1) was determined by quantitative PCR using FAM-labeled TaqMan Gene Expression Assay Reagents (Applied Biosystems, Foster City, CA, USA). Each target gene was normalized by glyceraldehyde-3-phosphate dehydrogenase (Mm99999915_g1) with the comparative ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. A statistical software package JMP software program (SAS Institute, Cary, NC, USA) was used to analyze the data. Statistical analyses were performed using Student\u0026rsquo;s unpaired \u003cem\u003et\u003c/em\u003e-test between two groups, a one-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s test for three or more groups, and a two-way ANOVA for three or more groups split on two variables. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIon secretion activity of misoprostol in mouse ileum\u003c/h2\u003e \u003cp\u003eWe first investigated the ion secretion activity of misoprostol in mouse ileum with a Ussing chamber. The prostaglandin E\u003csub\u003e1\u003c/sub\u003e (PGE\u003csub\u003e1\u003c/sub\u003e) derivative misoprostol (0.1\u0026ndash;10 \u0026micro;M) increased the Isc in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). The increase in the Isc obtained with 1 \u0026micro;M misoprostol was 12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Pretreatment with ONO-AE3-208 (3 \u0026micro;M), an EP\u003csub\u003e4\u003c/sub\u003eR antagonist, almost abolished the misoprostol (1 \u0026micro;M)-induced increase in the Isc (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The extent of the misoprostol-induced increase in the Isc in the presence of ONO-AE3-208 (1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;4) was significantly lower than that in the absence (8.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;4) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and f), indicating that misoprostol-induced ion secretion activity was mediated by EP\u003csub\u003e4\u003c/sub\u003eR in mouse ileum. Removal of extracellular Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e abolished the increase in the Isc induced not only by misoprostol (0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;4) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but also by forskolin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and f), indicating that Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e played a central role in misoprostol-induced ion secretion activity.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIon secretion activity of carbachol in mouse ileum.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFirst, we examined effects of carbachol (0.1\u0026ndash;10 \u0026micro;M) on Isc (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). While 0.1 \u0026micro;M carbachol did not increase the Isc (0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;3), carbachol did increase the Isc in a concentration-dependent manner between 0.3 and 10 \u0026micro;M. Since the increase in the Isc induced by 1 \u0026micro;M carbachol (13.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) was comparable to that induced by 1 \u0026micro;M misoprostol (12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), 1 \u0026micro;M carbachol was used in subsequent experiments. The carbachol-induced increase in the Isc was almost abolished either by pretreatment with the M\u003csub\u003e3\u003c/sub\u003eR antagonist 4-DAMP (0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) or removal of extracellular Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e (0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) versus no pretreatment (9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;8), indicating that stimulation of M\u003csub\u003e3\u003c/sub\u003eR by carbachol also caused Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion in mouse ileum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEffects of PAM-369 on exogenously added carbachol-induced and endogenously produced acetylcholine-induced ion secretion activities in mouse ileum\u003c/em\u003e \u003c/p\u003e \u003cp\u003ePAM-369 itself has no effect on any receptors, but it can generate an action on the target receptor under conditions where the orthosteric site is bound by either endogenously produced acetylcholine or exogenously added ligands, such as carbachol. Therefore, we investigated the effects of PAM-369 on the carbachol-induced increase in the Isc. While 1 \u0026micro;M PAM-369 did not induce a rapid increase in the Isc, it did enhance the carbachol (1 \u0026micro;M)-induced increase in the Isc in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). The carbachol (1 \u0026micro;M)-induced increase in the Isc was significantly enhanced by pretreatment with PAM-369 at 1 \u0026micro;M (21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;5) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and at 10 \u0026micro;M (20.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;8) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), compared with the increase in the Isc without PAM-369 (9.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;7). However, the application of 10 \u0026micro;M PAM-369 itself gradually increased the basal Isc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Pretreatment with 0.1 \u0026micro;M 4-DAMP almost abolished both the PAM-369-induced basal increase in the Isc (from 8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82 to 0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the PAM-369-potentiated carbachol-induced increase in the Isc (from 21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97 to 4.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and e), indicating that the increase in the Isc associated with 10 \u0026micro;M PAM-369 was mediated by M\u003csub\u003e3\u003c/sub\u003eR.\u003c/p\u003e \u003cp\u003eNext, we examined the effects of neostigmine, an acetylcholine esterase inhibitor, on the PAM-369-associated response. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g, and h, pretreatment with neostigmine significantly augmented the PAM-369-induced basal increase in the Isc (from 8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82 to 15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but had no effect on the carbachol-induced increase in the Isc (from 21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97 to 24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of misoprostol and potentiation of M\u003csub\u003e3\u003c/sub\u003eR by PAM-369 on indomethacin-induced small intestinal injury\u003c/h2\u003e \u003cp\u003eWe examined whether and to what extent misoprostol and potentiation of M\u003csub\u003e3\u003c/sub\u003eR by PAM-369 can protect against indomethacin-induced small intestinal injury. Single subcutaneous treatment of indomethacin at 15 mg/kg caused acute ulcers and erosions in the small intestine, especially the ileum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, left). Oral administration of 0.1 mg/kg misoprostol 3 times a day (0.3 mg/kg/day) significantly ameliorated the indomethacin-induced injury. The area of indomethacin-induced ulcers treated with misoprostol (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 mm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;6) was significantly smaller than that treated with vehicle (16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35 mm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;6) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Furthermore, oral administration of PAM-369 at 10 and 30 mg/kg/day for 1 week also ameliorated indomethacin-induced intestinal injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The area of indomethacin-induced ulcers pretreated with PAM-369 at 30 mg/kg/day (8.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84 mm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;9) was significantly lower than that treated with vehicle (22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99 mm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;8) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, indomethacin-induced intestinal injury was almost completely prevented in four out of eight mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The mRNA expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α was upregulated by indomethacin treatment in the ileum segments both with and without visible ulcers. Treatment with PAM-369 at 30 mg/kg/day tended to suppress the expression of those inflammatory cytokine genes, especially in non-ulcer areas (Supplemental Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAltered ion secretion responses of mouse ileum after indomethacin treatment\u003c/h2\u003e \u003cp\u003eWe next investigated the ion secretion activities in the ileum harvested just before (0 h) and 3 and 6 h after administration of indomethacin. The carbachol-induced increase in the Isc was slightly augmented at 3 h (22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;6) and 6 h (24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.36 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;6) compared to at 0 h (17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e; n\u0026thinsp;=\u0026thinsp;5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b). As noted, the basal response of the Isc to PAM-369 was significantly enhanced as the treatment time of indomethacin increased. The basal increase in the Isc induced by 1 \u0026micro;M PAM-369 in the ileum harvested at 3 h (6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;6) and 6 h (7.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;6) after indomethacin administration was significantly higher than that just before (0 h) indomethacin administration (2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;5) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and d). Furthermore, the increase in the Isc induced by 10 \u0026micro;M PAM-369 in the ileum harvested at 3 h (17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;4) and 6 h (16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;4) was significantly higher than that just before (0 h) indomethacin administration (10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e, n\u0026thinsp;=\u0026thinsp;4) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eTime course of the expression of cholinergic\u003c/em\u003e-\u003cem\u003eassociated genes in mouse ileum after indomethacin treatment\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTo investigate the mechanisms by which the responses of the Isc to carbachol and PAM-369 were potentiated in the ileum harvested at 3 and 6 h after indomethacin administration, we examined whether or not the expression of cholinergic genes was changed under those conditions. The mRNA expression of M\u003csub\u003e1\u003c/sub\u003eR, M\u003csub\u003e2\u003c/sub\u003eR, and ChAT at 3 and 6 h after indomethacin administration did not markedly differ from just before (0 h) indomethacin administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b, and d). However, the mRNA expression of M\u003csub\u003e3\u003c/sub\u003eR was significantly increased at 3 and 6 h after indomethacin administration compared to that just before (0 h) indomethacin administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhether or not M\u003csub\u003e3\u003c/sub\u003eR was involved in the intestinal epithelial barrier function has been unclear, as receptor subtype-selective compounds are not currently available and mice lacking the M\u003csub\u003e3\u003c/sub\u003eR seemed to retain cholinergic intestinal ion transport, probably due to compensatory mechanisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study using a novel highly selective M\u003csub\u003e3\u003c/sub\u003e PAM (PAM-369), we showed for the first time that M\u003csub\u003e3\u003c/sub\u003eR does help maintain the intestinal epithelial barrier function via induction of intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion in a similar manner to EP\u003csub\u003e4\u003c/sub\u003eR, and potentiation of M\u003csub\u003e3\u003c/sub\u003eR by M\u003csub\u003e3\u003c/sub\u003e PAM ameliorated indomethacin-induced small intestinal injury.\u003c/p\u003e \u003cp\u003eThis study demonstrated that both EP\u003csub\u003e4\u003c/sub\u003eR and M\u003csub\u003e3\u003c/sub\u003eR contribute to the intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion in a similar manner, using the application of receptor antagonists and an extracellular Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e-free condition. Intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion through apical Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e channels, mainly cystic fibrosis transmembrane conductance regulator (CFTR), is known to be indispensable for the formation of water secretion and the mucin layer, which plays a crucial role in the intestinal mucosal barrier function [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion is accompanied by secretion of Na\u003csup\u003e+\u003c/sup\u003e in a paracellular pathway, which is followed by water secretion [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. One important point of the present study is that the involvement of M\u003csub\u003e3\u003c/sub\u003eR in intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion has been definitely determined using PAM-369 in combination with complete blockade of the Isc increase via 4-DAMP, an M\u003csub\u003e3\u003c/sub\u003eR antagonist. It is considered that activation of EP\u003csub\u003e4\u003c/sub\u003eR enhances apical Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e conductance via CFTR through an increase in cyclic adenosine monophosphate (cAMP). In contrast, it is reported that activation of M\u003csub\u003e3\u003c/sub\u003eR reinforces basolateral K\u003csup\u003e+\u003c/sup\u003e efflux in a Ca\u003csup\u003e2+\u003c/sup\u003e-dependent manner, leading to an increased uptake of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e into epithelial cells via Na/K/2Cl cotransporter (NKCC1), which results in an increased driving force for intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUse of PAM-369 also led us to discover the important fact that a small amount of endogenous acetylcholine continues to be produced basally in intestinal tissue. Application of PAM-369 at a high concentration slowly but significantly increased the basal Isc in the absence of carbachol, even though PAM-369 exerts no effects on the gastrointestinal tract in the absence of an orthosteric ligand for M\u003csub\u003e3\u003c/sub\u003eR. This was further confirmed by the finding that the PAM-369-induced increase in basal Isc was significantly potentiated by neostigmine. Given that 4-DAMP had no effects on the basal Isc, the amount of basally produced acetylcholine is not sufficient to cause a basal increase in the Isc itself; however, the basal response to acetylcholine was unmasked by allosteric changes of M\u003csub\u003e3\u003c/sub\u003eR by 10 \u0026micro;M PAM-369.\u003c/p\u003e \u003cp\u003eWe were unable to clarify in our study why a small amount of endogenous acetylcholine continued to be produced basally. It has been reported that a basal level of acetylcholine release was also observed in unstimulated bladder tissue [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. One speculation is that basal stimulation of M\u003csub\u003e3\u003c/sub\u003eR with acetylcholine at a very low level that cannot induce any response itself might contribute to the conduct of an appropriate reaction or avoid an unexpectedly strong reaction by adjusting the M\u003csub\u003e3\u003c/sub\u003eR sensitivity to orthosteric ligands. Further studies will be required to clarify this point in the future.\u003c/p\u003e \u003cp\u003eThe present study successfully demonstrated for the first time that M\u003csub\u003e3\u003c/sub\u003eR was a viable therapeutic target for NSAID-induced enteropathy. Impairment of the intestinal epithelial barrier function by inhibition of the PGE-EP\u003csub\u003e4\u003c/sub\u003e signal transduction pathway is a primary cause of NSAID-induced enteropathy. An EP\u003csub\u003e4\u003c/sub\u003eR agonist reportedly alleviated indomethacin-induced small intestinal injury in rodents [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consistent with previous studies, we confirmed that activation of EP\u003csub\u003e4\u003c/sub\u003eR by misoprostol increased intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion \u003cem\u003eex vivo\u003c/em\u003e, and misoprostol was indeed effective in preventing indomethacin-induced small intestinal injury in our \u003cem\u003ein vivo\u003c/em\u003e mice model. Although misoprostol is currently clinically available, common side effects, such as abdominal pain and diarrhea, are not usually tolerated by patients with NSAID-induced enteropathy in clinical practice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Since M\u003csub\u003e3\u003c/sub\u003eR activation can induce intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion in a similar manner to EP\u003csub\u003e4\u003c/sub\u003eR activation, we hypothesized that M\u003csub\u003e3\u003c/sub\u003eR would be a viable substitute for EP\u003csub\u003e4\u003c/sub\u003eR in preventing NSAID-induced enteropathy. As expected, potentiation of M\u003csub\u003e3\u003c/sub\u003eR ameliorated indomethacin-induced small intestinal injury in a similar manner to misoprostol.\u003c/p\u003e \u003cp\u003eThis study provided further evidence that M\u003csub\u003e3\u003c/sub\u003eR is a viable therapeutic target for NSAID-induced enteropathy. It should be noted in the present study that the PAM-369-induced increase in the basal Isc was significantly enhanced after indomethacin treatment, indicating that M\u003csub\u003e3\u003c/sub\u003eR can be enhanced by induction of NSAID-induced enteropathy. Indeed, the cholinergic system, including M\u003csub\u003e3\u003c/sub\u003eR and ChAT, can reportedly be enhanced in acute small intestinal inflammation to prevent further progression of organ damage [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consistent with the results concerning the Isc in the Ussing chamber, we confirmed that the expression of M\u003csub\u003e3\u003c/sub\u003eR (but not M\u003csub\u003e1\u003c/sub\u003eR or M\u003csub\u003e2\u003c/sub\u003eR) was significantly up-regulated by indomethacin treatment, while the ChAT expression was unchanged. Given that 4-DAMP had no effect on the basal Isc but the PAM-369-induced increase in basal Isc was potentiated by indomethacin treatment, not only the increased expression of M\u003csub\u003e3\u003c/sub\u003eR itself but also the increased sensitivity of the orthostatic site and/or allosteric site to M\u003csub\u003e3\u003c/sub\u003eR may be present during indomethacin treatment. It is unlikely that functional M\u003csub\u003e3\u003c/sub\u003eR is newly synthesized in a short time [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], but its up-regulation by indomethacin treatment is possibly brought by endocytic trafficking [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Further investigations will be needed to clarify the mechanism underlying how M\u003csub\u003e3\u003c/sub\u003eR signaling was enhanced under conditions where the production of prostaglandin is suppressed. More importantly, combined treatment of misoprostol and PAM-369 had no additional effects (Supplemental Fig.\u0026nbsp;2), suggesting that both agents may exert their effects through the same final pathway, such as CFTR. In NSAID-induced enteropathy, up-regulation of M\u003csub\u003e3\u003c/sub\u003eR signaling might be induced in order to compensate for the down-regulated PGE-EP\u003csub\u003e4\u003c/sub\u003e pathway in terms of the intestinal epithelial barrier function (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In this regard, M\u003csub\u003e3\u003c/sub\u003eR may indeed be a therapeutic target for NSAID-induced enteropathy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, M\u003csub\u003e3\u003c/sub\u003eR plays a role in intestinal epithelial Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e secretion in a similar manner to EP\u003csub\u003e4\u003c/sub\u003eR in terms of the intestinal mucosal barrier function. Suppression of prostaglandin production by indomethacin induced an increased expression of M\u003csub\u003e3\u003c/sub\u003eR and enhanced M\u003csub\u003e3\u003c/sub\u003eR signaling, possibly via a compensatory mechanism. The further potentiation of M\u003csub\u003e3\u003c/sub\u003e receptor by PAM-369 ameliorated indomethacin-induced small intestinal injury. M\u003csub\u003e3\u003c/sub\u003eR is a promising target for the treatment or prevention of NSAID-induced enteropathy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eChAT, choline acetyltransferase; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; DMSO, dimethyl sulfoxide; EP\u003csub\u003e4\u003c/sub\u003eR, EP\u003csub\u003e4\u003c/sub\u003e prostanoid receptor; IK\u003csub\u003eCa\u003c/sub\u003e, intermediate conductance calcium-sensitive potassium channel; IL, interleukin; Isc, short circuit current; KBR, Krebs bicarbonate ringer; M\u003csub\u003e3\u003c/sub\u003eR, M\u003csub\u003e3\u003c/sub\u003e muscarinic acetylcholine receptor; NES, normal extracellular solution; NKCC, Na/K/2Cl cotransporter; NSAID, nonsteroidal anti-inflammatory drug; PAM, positive allosteric modulator; PGE, prostaglandin E; PPI, proton pump inhibitor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors thank the laboratory members for their hard work and thorough discussion and Japan Medical Communication (http://www.japan-mc.co.jp) for editing a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis project has been executed using the institutions’ (Kyushu University and Mochida Pharmaceutical Co., Ltd.) budgets including a grant from Mochida Pharmaceutical Co., Ltd. to EI and YO for this collaborative research. The funders provided support in the form of salaries for authors YI, CH, and YT (Mochida Pharmaceutical Co., Ltd.). This study was also supported in part by the Japan Society for the Promotion of Science KAKENHI (23K07440 and 22K19530).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures:\u0026nbsp;\u003c/strong\u003eYI and YT are the employees of Mochida Pharmaceutical Co., Ltd. whose company partly funded this study. EI belongs to an endowed course supported by companies including Ono Pharmaceutical Co., Ltd., Miyarisan Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Otsuka Pharmaceutical Factory, Inc., Fujifilm Medical Co., Ltd., Terumo Corporation, FANCL Corporation, Ohga Pharmacy, and Abbott Japan, LLC. EI receives a lecture honorarium from Takeda Pharmaceutical Company. YO conducts collaborative research with Fujifilm Medical Co., Ltd. and FANCL Corporation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Yoko Igarashi-Hisasyoshi, Eikichi Ihara, Yoshimasa Tanaka, Haruei Ogino, and Takatoshi Chinen proposed the research design. Yoko Igarashi-Hisayoshi and Xiaopeng Bai conducted experiments. Yoko Igarashi-Hisasyoshi performed data analysis. Eikichi Ihara and Yasushi Taguchi contributed new reagents or analytic tools. Yoko Igarashi-Hisasyoshi and Eikichi Ihara mainly wrote the manuscript. Xiaopeng Bai, Yoshimasa Tanaka, Haruei Ogino, and Takatoshi Chinen reviewed the manuscript. Yoshihiro Ogawa supervised this study. All authors contributed to the interpretation of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe data presented in this work are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003eThe ethics governing the use and conduct of experiments on animals were strictly observed, and the experimental protocol was approved by the Committee on Animal Research of Kyushu University (A19-263).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHiguchi K, Umegaki E, Watanabe T et al (2009) Present status and strategy of NSAIDs-induced small bowel injury. J Gastroenterol 44:879-888. https://doi.org/10.1007/s00535-009-0102-2\u003c/li\u003e\n\u003cli\u003eEndo H, Sakai E, Kato T et al (2015) Small bowel injury in low-dose aspirin users. J Gastroenterol 50:378-386. https://doi.org/10.1007/s00535-014-1028-x\u003c/li\u003e\n\u003cli\u003eMatsumoto T, Kudo T, Esaki M et al (2008) Prevalence of non-steroidal anti-inflammatory drug-induced enteropathy determined by double-balloon endoscopy: a Japanese multicenter study. Scand J Gastroenterol 43:490-496. https://doi.org/10.1080/00365520701794121\u003c/li\u003e\n\u003cli\u003eEdogawa S, Peters SA, Jenkins GD et al (2018) Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota. FASEB J, fj201800560R. https://doi.org/10.1096/fj.201800560R\u003c/li\u003e\n\u003cli\u003eBjarnason I, Scarpignato C, Holmgren E, Olszewski M, Rainsford KD, Lanas A (2018) Mechanisms of Damage to the Gastrointestinal Tract From Nonsteroidal Anti-Inflammatory Drugs. Gastroenterology 154:500-514. https://doi.org/10.1053/j.gastro.2017.10.049\u003c/li\u003e\n\u003cli\u003eWashio E, Esaki M, Maehata Y et al (2016) Proton Pump Inhibitors Increase Incidence of Nonsteroidal Anti-Inflammatory Drug-Induced Small Bowel Injury: A Randomized, Placebo-Controlled Trial. Clin Gastroenterol Hepatol 14:809-815.e801. https://doi.org/10.1016/j.cgh.2015.10.022 \u003c/li\u003e\n\u003cli\u003eWallace JL, Syer S, Denou E et al (2011) Proton pump inhibitors exacerbate NSAID-induced small intestinal injury by inducing dysbiosis. Gastroenterology 141:1314-1322, 1322.e1311-1315. https://doi.org/10.1053/j.gastro.2011.06.075 \u003c/li\u003e\n\u003cli\u003eMcGuckin MA, Lind\u0026eacute;n SK, Sutton P, Florin TH (2011) Mucin dynamics and enteric pathogens. Nat Rev Microbiol 9:265-278. https://doi.org/10.1038/nrmicro2538 \u003c/li\u003e\n\u003cli\u003eHirota CL, McKay DM (2006a) Cholinergic regulation of epithelial ion transport in the mammalian intestine. Br J Pharmacol 149:463-479. https://doi.org/10.1038/sj.bjp.0706889 \u003c/li\u003e\n\u003cli\u003eGustafsson JK, Ermund A, Johansson ME, Sch\u0026uuml;tte A, Hansson GC, Sj\u0026ouml;vall H (2012) An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants. Am J Physiol Gastrointest Liver Physiol 302:G430-438. https://doi.org/10.1152/ajpgi.00405.2011 \u003c/li\u003e\n\u003cli\u003eHayashi S, Kurata N, Yamaguchi A, Amagase K, Takeuchi K (2014) Lubiprostone prevents nonsteroidal anti-inflammatory drug-induced small intestinal damage by suppressing the expression of inflammatory mediators via EP4 receptors. J Pharmacol Exp Ther 349:470-479. https://doi.org/10.1124/jpet.114.213991 \u003c/li\u003e\n\u003cli\u003eTakeuchi K, Kato S, Amagase K (2010) Prostaglandin EP receptors involved in modulating gastrointestinal mucosal integrity. J Pharmacol Sci 114:248-261. https://doi.org/10.1254/jphs.10r06cr \u003c/li\u003e\n\u003cli\u003eKunikata T, Araki H, Takeeda M, Kato S, Takeuchi K (2001) Prostaglandin E prevents indomethacin-induced gastric and intestinal damage through different EP receptor subtypes. J Physiol Paris 95:157-163. https://doi.org/10.1016/s0928-4257(01)00021-3 \u003c/li\u003e\n\u003cli\u003eTaha AS, McCloskey C, McSkimming P, McConnachie A (2018) Misoprostol for small bowel ulcers in patients with obscure bleeding taking aspirin and non-steroidal anti-inflammatory drugs (MASTERS): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Gastroenterol Hepatol 3:469-476. https://doi.org/10.1016/S2468-1253(18)30119-5 \u003c/li\u003e\n\u003cli\u003eWatanabe T, Sugimori S, Kameda N et al (2008) Small bowel injury by low-dose enteric-coated aspirin and treatment with misoprostol: a pilot study. Clin Gastroenterol Hepatol 6:1279-1282. https://doi.org/10.1016/j.cgh.2008.06.021 \u003c/li\u003e\n\u003cli\u003eKruse AC, Hu J, Pan AC et al (2012) Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482:552-556. https://doi.org/10.1038/nature10867 \u003c/li\u003e\n\u003cli\u003eIgarashi-Hisayoshi Y, Ihara E, Bai X et al (2023) Determination of Region-Specific Roles of the M. Dig Dis Sci 68:439-450. https://doi.org/10.1007/s10620-022-07637-y \u003c/li\u003e\n\u003cli\u003eBurford NT, Traynor JR, Alt A (2015) Positive allosteric modulators of the \u0026mu;-opioid receptor: a novel approach for future pain medications. Br J Pharmacol 172:277-286. https://doi.org/10.1111/bph.12599 \u003c/li\u003e\n\u003cli\u003eJakub\u0026iacute;k J, El-Fakahany EE (2010) Allosteric Modulation of Muscarinic Acetylcholine Receptors. Pharmaceuticals (Basel) 3:2838-2860. https://doi.org/ 10.3390/ph3092838.\u003c/li\u003e\n\u003cli\u003eTang OS, Gemzell-Danielsson K, Ho PC (2007) Misoprostol: pharmacokinetic profiles, effects on the uterus and side-effects. Int J Gynaecol Obstet 99:S160-167. https://doi.org/10.1016/j.ijgo.2007.09.004 \u003c/li\u003e\n\u003cli\u003eHirota CL, McKay DM (2006b) M3 muscarinic receptor-deficient mice retain bethanechol-mediated intestinal ion transport and are more sensitive to colitis. Can J Physiol Pharmacol 84:1153-1161. https://doi.org/10.1139/y06-068 \u003c/li\u003e\n\u003cli\u003eGustafsson JK, Lind\u0026eacute;n SK, Alwan AH, Scholte BJ, Hansson GC, Sj\u0026ouml;vall H (2015) Carbachol-induced colonic mucus formation requires transport via NKCC1, K⁺ channels and CFTR. Pflugers Arch 467:1403-1415. https://doi.org/10.1007/s00424-014-1595-y\u003c/li\u003e\n\u003cli\u003eGarcia MA, Yang N, Quinton PM (2009) Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion. J Clin Invest 119:2613-2622. https://doi.org/10.1172/JCI38662 \u003c/li\u003e\n\u003cli\u003eBarrett KE, Keely SJ (2000) Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol 62:535-572. https://doi.org/10.1146/annurev.physiol.62.1.535 \u003c/li\u003e\n\u003cli\u003eYoshida M, Inadome A, Maeda Y et al (2006) Non-neuronal cholinergic system in human bladder urothelium. Urology, 67:425-430. https://doi.org/10.1016/j.urology.2005.08.014 \u003c/li\u003e\n\u003cli\u003ePohl CS, Lennon EM, Li Y, DeWilde MP, Moeser AJ (2018) S. Typhimurium challenge in juvenile pigs modulates the expression and localization of enteric cholinergic proteins and correlates with mucosal injury and inflammation. Auton Neurosci, 213:51-59. https://doi.org/10.1016/j.autneu.2018.05.009 \u003c/li\u003e\n\u003cli\u003eNathanson NM (2008) Synthesis, trafficking, and localization of muscarinic acetylcholine receptors. Pharmacol Ther, 119:33-43. https://doi.org/10.1016/j.pharmthera.2008.04.006 \u003c/li\u003e\n\u003cli\u003eZenko D, Hislop JN (2018) Regulation and trafficking of muscarinic acetylcholine receptors. Neuropharmacology, 136:374-382. https://doi.org/10.1016/j.neuropharm.2017.11.017\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"intestinal epithelial barrier function, positive allosteric modulator, M3 muscarinic acetylcholine receptor, NSAID-induced enteropathy","lastPublishedDoi":"10.21203/rs.3.rs-3217047/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3217047/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEP\u003csub\u003e4\u003c/sub\u003e prostanoid receptor (EP\u003csub\u003e4\u003c/sub\u003eR) contributes to the intestinal epithelial barrier function, and inhibition of prostaglandin E (PGE) production by non-steroidal anti-inflammatory drugs (NSAIDs) plays a central role in NSAID-induced enteropathy. However, given that M\u003csub\u003e3\u003c/sub\u003e muscarinic acetylcholine receptor (M\u003csub\u003e3\u003c/sub\u003eR)-selective agents are unavailable, how M\u003csub\u003e3\u003c/sub\u003eR regulates the intestinal epithelial barrier function remains unclear. The present study explored how M\u003csub\u003e3\u003c/sub\u003eR is involved in the regulation of the intestinal epithelial barrier function and its pathophysiological role in NSAID-induced enteropathy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUsing the novel highly-selective M\u003csub\u003e3\u003c/sub\u003e positive allosteric modulator PAM-369 that we recently developed, we evaluated the role of M\u003csub\u003e3\u003c/sub\u003eR in the intestinal epithelial barrier function \u003cem\u003eex vivo\u003c/em\u003e by measuring the short circuit current (Isc) of intestinal epithelium with a Ussing chamber system and examined whether or not M\u003csub\u003e3\u003c/sub\u003eR protects against small intestinal injury in indomethacin-treated mice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBoth the PGE\u003csub\u003e1\u003c/sub\u003e derivative misoprostol and carbachol similarly increased the Isc in a concentration-dependent manner. The Isc increases were abolished either by receptor antagonists (an EP\u003csub\u003e4\u003c/sub\u003eR antagonist and a M\u003csub\u003e3\u003c/sub\u003eR antagonist, respectively) or by removal of extracellular Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e. PAM-369 increased the Isc by potentiating M\u003csub\u003e3\u003c/sub\u003eR, which could contribute to enhanced intestinal epithelial barrier function. Treatment with PAM-369 ameliorated small intestinal injury in indomethacin-treated mice. Importantly, the M\u003csub\u003e3\u003c/sub\u003eR expression was significantly up-regulated, and PAM-369 potentiation of M\u003csub\u003e3\u003c/sub\u003eR was augmented in indomethacin-treated mice compared to untreated mice.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings show that M\u003csub\u003e3\u003c/sub\u003eR plays a role in maintaining the intestinal epithelial barrier function. M\u003csub\u003e3\u003c/sub\u003eR is a promising target for treating or preventing NSAID-induced enteropathy.\u003c/p\u003e","manuscriptTitle":"Protective role of M3 muscarinic acetylcholine receptor in indomethacin-induced small intestinal injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-11 14:15:10","doi":"10.21203/rs.3.rs-3217047/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-02-13T06:27:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-10-23T10:10:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-08-07T19:23:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-01T12:17:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2023-07-30T02:58:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f5b5ba9c-539c-4389-bc40-917aadde1411","owner":[],"postedDate":"August 11th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:04:21+00:00","versionOfRecord":{"articleIdentity":"rs-3217047","link":"https://doi.org/10.1007/s00109-024-02474-0","journal":{"identity":"journal-of-molecular-medicine","isVorOnly":false,"title":"Journal of Molecular Medicine"},"publishedOn":"2024-08-22 15:57:49","publishedOnDateReadable":"August 22nd, 2024"},"versionCreatedAt":"2023-08-11 14:15:10","video":"","vorDoi":"10.1007/s00109-024-02474-0","vorDoiUrl":"https://doi.org/10.1007/s00109-024-02474-0","workflowStages":[]},"version":"v1","identity":"rs-3217047","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3217047","identity":"rs-3217047","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}