Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome Is Associated with Upregulation of TMEM16A in Myenteric Plexus
Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. TMEM16A is a calcium-activated chloride channel (CaCC) involved in intestinal slow-wave generation.
To investigate whether TMEM16A is involved in colonic dysmotility in IBS.
A rat model of IBS was established by chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically, and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). TMEM16A expression was evaluated by RT-PCR, Western blot, and immunofluorescence. Colonic muscle strip contractility was measured by isometric transducers, and the effect of niflumic acid (NFA), a CaCC antagonist, on colonic motility was examined.
After 10 days of WAS exposure, ITT was decreased and FWC was elevated. Furthermore, VMR magnitude of WAS rats in response to CRD was significantly enhanced. Protein and mRNA levels of TMEM16A in colon were considerably increased after WAS. The percentage of TMEM16A-positive neurons in myenteric plexus (MP) of WAS rats was significantly higher than controls. Pharmacological blockade of TMEM16A activity by NFA considerably enhanced ITT, with concentration-dependent declines in FWC and VMR magnitude in NFA-treated rats. Further, spontaneous contraction of colonic strips of NFA-treated rats was significantly ameliorated in a concentration-dependent manner in vitro.
Upregulation of TMEM16A in MP neurons may play an important role in chronic stress-induced colonic hypermotility, making CaCC-blocking drugs a putatively effective treatment method for colonic hypermotility in IBS.
KeywordsIrritable bowel syndrome Interstitial cells of Cajal Myenteric plexus Gastrointestinal motility
Rhythmic contractions of gastrointestinal (GI) smooth muscle are the basis for GI motility, such as peristalsis and segmentation. Accumulating studies indicate that phasic contractions of GI tract are initiated and timed by slow wave generated by interstitial cells of Cajal (ICC) . Slow waves, whose amplitude determines the opening of L-type Ca2+ channels in smooth muscle cells, are actively propagated within ICC networks and conducted to surrounding smooth muscle cells via gap junctions, accompanied by contractions . It has been demonstrated that the amplitude and frequency of slow waves in the gut are regulated by excitatory and inhibitory enteric motor neurons of the enteric nervous system (ENS) [3, 4]. The ENS, which is regarded as a “brain-in-the-gut,” consists of two major populations of ganglia, the submucosal plexus (SMP) and the myenteric plexus (MP). The motor function for the specific digestive state of the gut is programmed by the MP [3, 4].
Irritable bowel syndrome (IBS), which is characterized by visceral hyperalgesia and intestinal dysmotility, is a common functional gastrointestinal disorder that affects up to 30% of the worldwide adult population . However, the pathological mechanism of IBS is not well understood. There is accumulating evidence that dysfunction of ICC and ENS in the colon represents an important candidate mechanism responsible for intestinal dysmotility in IBS [6, 7]. Recent research has shown that slow waves in the GI tract are mediated by Ca2+-activated Cl− channels (CaCC), most likely encoded by TMEM16A (also known as ANO1 or DOG1) in ICC [8, 9, 10]. TMEM16A is involved in the generation of a Ca2+-activated Cl− inward current in ICC ; it was first found in gastrointestinal stromal tumors (GIST) and has been recently reported as a sensitive and specific marker for GIST . The CaCC channel blocking drugs niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stillbene-disulfonic acid (DIDS) specifically block slow waves in intact muscle of small intestine and stomach in mouse, primate, and human . Importantly, slow waves fail to develop in TMEM16A knockout mice . A more recent study indicates that conditional genetic deletion of TMEM16A also impairs Ca2+ transients in ICC of adult mouse small intestine .
However, it remains unclear whether TMEM16A mediates stress-induced GI dysmotility. Herein, the present study was designed to explore alterations of expression and distribution of TMEM16A in the colon and to determine the role of TMEM16A in intestinal dysmotility in a rat model of IBS induced by chronic stress.
Materials and Methods
Male Sprague–Dawley rats (weight 180–230 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. The animals were habituated to standard laboratory conditions (22 ± 2 °C with a 12 h light/dark cycle and a relative humidity of 40–60%) and provided with food and water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of Wuhan University and were conducted in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals and the People’s Republic of China animal welfare legislations in order to minimize the number of experimental animals and their suffering.
The rats were randomly divided into five groups (n = 12/group): control group, water avoidance stress (WAS) group, NFA-low (NFA-L) group (0.1 mg/kg), NFA-medium (NFA-M) group (0.4 mg/kg), and NFA-high (NFA-H) group (1 mg/kg). The WAS procedure was performed to induce IBS as described previously with minor modifications . Briefly, rats were placed on a platform (10 × 8 × 8 cm; length × width × height) in the center of a water-filled (25 °C) tank (45 × 25 × 35 cm; length × width × height) for 1 h daily for ten consecutive days. The water level in the tank was kept at 1 cm below the platform. The animals from the NFA-treated group were administered an intraperitoneal injection of NFA (Aladdin, Shanghai, China) in different doses, in saline, 1 h before WAS since the fourth day of WAS for 7 days. After 48 h, colonic motility and visceral sensitivity were determined. Afterward, the rats were subjected to laparotomy and distal colon resection. Hematoxylin–eosin staining was performed for colon specimens.
Intestinal Transit Time (ITT)
The animals were orally gavaged with activated carbon in double-distilled water. Closely observation of the stool was then conducted. ITT was the duration from gavage to the time when the first black fecal pellet was output.
Fecal Water Content (FWC)
FWC was used to estimate colonic motility as a validated index. The animals were placed in metabolic cages for 24 h with free access to rodent chow and water. The stool was weighed (m0) after collection, and then the stool was weighed again (m1) after the stool was dried in the oven. FWC was calculated as (m0 − m1)/m0.
Electromyogram (EMG) Measurements
To evaluate the visceral hyperalgesia, we recorded the EMG signal of abdominal oblique musculature. For EMG measurements, animals were initially anesthetized with isoflurane inhalation, keeping a mild and stable anesthesia throughout the experiment. After anesthesia, the rat was fixed in a supine position. A pair of electrodes was implanted into the external oblique muscle of the rats. The electrodes were connected to a Bio Amp (AD instruments, Bella Vista, Australia), which was connected to a Power Lab (AD instruments, Bella Vista, Australia) as an EMG acquisition system. Colorectal distension (CRD) was then conducted after 20 min of adaptation. Each recording progression consisted of a 5-min predistention baseline activity measurements, a 20-s CRD-evoked response (20, 40, 60, and 80 mmHg), and a 3-min postdistention activity measurement, followed by a 3-min rest between two CRD episodes. The EMG signals expressed by visceromotor response (VMR), the area under the curve (AUC) in response to the CRD stimuli, were collected and analyzed using Lab Chart 7 software (AD instruments, Bella Vista, Australia). The analytic period was 40 s (20 s during and 20 s after each CRD). The net value for each CRD was calculated by subtracting the AUC of the baseline (40 s interval) before each CRD .
Total proteins were extracted using RIPA lysis buffer (Beyotime, Shanghai, China) and subsequently subjected to centrifugation at 12,000 rpm, 4 °C for 30 min. Supernatants were then collected and protein concentrations were determined using the BCA protein assay kit (Beyotime, Shanghai, China). Samples were mixed with 5 × loading buffer and heated at 100 °C for 5 min to denature the proteins. Thirty micrograms of total proteins was loaded on 10% SDS polyacrylamide gels and electrophoresed. The separated proteins were transferred to PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated in 5% skimmed milk at room temperature for 2 h to block nonspecific binding. The blots were then incubated overnight at 4 °C with the primary antibody against TMEM16A (Santa Cruz, California, USA) and β-actin (Beyotime, Shanghai, China). After washing three times with TBST for 10 min, the corresponding secondary antibody conjugated to horseradish peroxidase (Boster, Wuhan, China) was applied for 1 h at room temperature, followed by three washes of TBST for 10 min. Specific protein bands were visualized using the ECL kit (Thermo, Massachusetts, USA) and an X-ray film (Kodak, Xiamen, China). The optical density of the bands was analyzed using Band Scan 5.0 software (Alpha Innotech Corp., California, USA).
Total RNA was extracted from the colon that was frozen in liquid nitrogen with TRIzol reagent (Invitrogen, Carlsbad, USA), following the manufacturer’s instructions. Next, 1.5 µg of total RNA from each sample was used for cDNA synthesis in a total volume of 20 µl, with oligo (dT)18 and HiScript Reverse Transcriptase (VAZYME, Nanjing, China) included in the reverse transcription system. Quantitative RT-PCR was performed in 20 µl wells with SYBR green PCR master mix (VAZYME, Nanjing, China) on the ViiA 7 real-time PCR system (ABI, Carlsbad, USA). After incubation at 95 °C for 10 min as the initiation of thermal cycling, 40 cycles of 95 °C for 30 s and 60 °C for 30 s were performed. Each reaction was performed in triplicate. GAPDH was used as a loading control to normalize each sample. The PCR primers used were as follows: TMEM16A forward: 5′-TGGGCTACGAGGTTCAGATC-3′, reverse: 5′-TGGCTGATGTCTTTGGGGAT-3′; GAPDH forward: 5′-ACAGCAACAGGGTGGTGGAC-3′, reverse: 5′-TTTGAGGGTGCAGCGAACTT-3′. Specificity of the PCR products was monitored by melting curve analysis. The relative expression of TMEM16A mRNA was quantified by the 2−∆∆Ct method.
Immunofluorescence staining for TMEM16A was performed both on the whole-mount flat preparations and longitudinal sections of the colon. Immunofluorescence staining for TMEM16A on whole-mount flat preparations was performed as follows. Colonic specimens were fixed in 4% paraformaldehyde solution at 4 °C for 6-8 h. Subsequently, the mucosa was removed with sharp forceps under a stereoscopic microscope. The circular muscle was then stripped off carefully at certain intervals. Whole-mount preparations were then blocked with 10% goat serum containing 0.3% Triton X-100 for 1 h at room temperature. Next, the samples were incubated overnight at 4 °C with rabbit anti-rat polyclonal TMEM16A antibody (Santa Cruz, California, USA). After washing in PBST, the pinned tissues were incubated overnight at 4 °C with mouse anti-rat monoclonal PGP9.5 antibody (Abcam, Cambridge, England). After washing thrice with PBST for 5 min, the tissues were incubated with TRITC-conjugated goat anti-rabbit secondary antibody (Boster, Wuhan, China) and FITC-conjugated rabbit anti-mouse secondary antibody (Boster, Wuhan, China) for 1 h at room temperature. The stained samples were imaged using an Olympus BX53 microscope (Olympus, Tokyo, Japan) after they were mounted on a slide with a coverslip and sealed with glycerol. The results were analyzed using Image Pro Plus software version 6.0. The TMEM16A-immunoreactive (IR) neurons were quantified as a relative percentage considering the total number of PGP9.5-IR neurons. Immunofluorescence of colonic longitudinal sections was conducted in a similar manner using the above procedure.
Contractility of Colonic Muscle Strips
Full-thickness strips of distal colon (measuring 3 × 10 mm) were mounted vertically in a 10-ml organ bath filled with Tyrode’s solution maintained at 37 °C and constantly bubbled with O2. The strips were placed under an initial resting tension equivalent to a 1.0 g load and allowed to equilibrate for 30 min, with solution changing every 20 min. Isometric contractions were measured using a force displacement transducer. The contraction curves were recorded and measured by RM6240 multichannel physiological signal acquisition and processing system. Changes in average magnitude, frequency, and area under the contraction curve (AUC) were calculated to evaluate the spontaneous contraction of strips.
Statistical analyses were performed using SPSS version 21.0 (IBM Co, Chicago, USA). Continuous variables were presented as mean ± standard deviation (SD) and compared using t test and variance analysis. Differences among different groups were analyzed by two-way repeated-measures analysis of variance (ANOVA) with distention pressure as the repeated measure. S–N–K post hoc test was used where appropriate. A two-sided P value < 0.05 was regarded as statistically significant.
Evaluation of the Animal Model
These results suggest that an animal model of chronic stress-induced colonic hypermotility, exhibiting the hallmark of IBS, was successfully established after 10 days of WAS exposure.
Increased TMEM16A Expression in Colon of WAS Rats
These results indicate that TMEM16A expression in the colon was considerably upregulated after ten consecutive days of WAS exposure.
Immunofluorescence Staining of TMEM16A in the Colon
Furthermore, immunofluorescence of whole-mount circular muscle revealed that TMEM16A-positive cells, mainly intramuscular ICC, were largely fusiform in shape with two prolongations connecting each other in a head-to-tail sequence and parallel to smooth muscle cells (Fig. 3b). Immunofluorescence analysis confirmed an equivalent density of TMEM16A labeling between WAS and control rats (P > 0.05).
These findings show that the enhanced expression of TMEM16A observed in WAS rats was restricted to MP neurons of the ENS rather than ICC.
NFA-Alleviated Colonic Hypermotility in a Concentration-Dependent Manner In Vivo
These results suggest that NFA, a blocker of TMEM16A, alleviated colonic hypermotility in WAS rats in a concentration-dependent manner in vivo.
NFA-Attenuated Spontaneous Contraction of Colonic Strips in a Concentration-Dependent Manner In Vitro
These findings indicate that NFA relieved chronic stress-induced spontaneous contractility of colonic strips in a concentration-dependent manner in vitro.
Findings from recent studies that TMEM16A functions as a classical Ca2+-activated Cl− channel have ignited significant interest in this new family of ion channels . TMEM16A is widely expressed in many organs including lung, salivary gland, intestine, kidney and other tissues, as well as arterial smooth muscle, intestinal pacemaker cells, and sensory neurons, in which TMEM16A facilitates epithelial fluid secretion, smooth muscle contraction, and neurosensory signaling [9, 17, 18, 19]. TMEM16A is robustly expressed in GI muscular layer, specifically in ICC of murine, non-human primate, and human [9, 20]. Recent studies have demonstrated that TMEM16A, non-selective cation channels (e.g., transient receptor potential channels) and sodium channels are the three most important ion channels in generation of the ICC slow wave in GI tract [21, 22, 23]. In the present study, we investigated the role of TMEM16A in colonic motility dysfunction in a rat model of IBS.
Presently, the detailed mechanism of IBS remains elusive. Therefore, animal models of IBS are pivotal in clarifying its pathogenesis. To date, repeated WAS has frequently been utilized to establish animal models of stress-induced IBS with visceral hypersensitivity, motility impairment, anxiety, and colonic immune activity . Currently, the WAS-based model optimum for studies on IBS is a brain-gut interaction model that mimics some clinical and pathophysiological characteristics of IBS-diarrhea . In the present study, enhanced gut motility was observed after 10 days of WAS exposure, indicating that an animal model of stress-induced dysmotility in gut was successfully established in our study. A more recent study by Reed et al.  suggested that myenteric neurons play a key role in gut motor dysfunction of WAS-exposed rats. We discovered that molecular alterations of myenteric neurons in colonic MP could give rise to the gut dysmotility in IBS induced by WAS, which importantly adds to the currently limited published literature available on this condition.
Recent evidence indicates that expression of TMEM16A could be regulated by metabolic disease , dysplasia , and carcinoma  in specific parts of the GI tract, such as gastric, small intestine, and colon. A previous study identified important changes in expression and splicing of TMEM16A in patients with diabetic gastroparesis . Further, TMEM16A-positive ICC in tissues of patients with slow transit constipation displayed a significant decline compared to that found in healthy individuals . However, it remains elusive whether stress modulates TMEM16A expression in the GI tract. In our colonic hypermotility model, we detected enhanced expression of TMEM16A in both protein and mRNA levels in colon after ten consecutive days of WAS exposure, indicating TMEM16A expression could be directly regulated by chronic stress. Based on these findings, we posit that TMEM16A is involved in the pathological course of motility disorder in GI tract.
Accumulating studies have confirmed that TMEM16A is exclusively expressed in ICC of the muscular layer of the GI tract  and is a highly specific biomarker of ICC, exhibiting more selective labeling of ICC than Kit antibodies . However, in the present study, we clearly observed two distinct populations of TMEM16A-positive cells in the muscular layer of colon, namely ICC of the intramuscular region and neurons of the MP. To date, this is the first study to report the presence of TMEM16A-positive neurons in the ENS. Furthermore, we detected an increased density of TMEM16A-positive MP neurons in WAS-exposed rats. Our data indicate that chronic stress-driven changes in gut motility suggest direct modulation of ENS, providing important evidence in support of this mechanism in mediating stress-driven changes on gut-brain signaling. Nitrergic and cholinergic neurons of the ENS are the principal inhibitory and excitatory musculomotor neurons of gut, respectively . Certain studies have suggested that Ca2+-activated Cl− conductance generated by TMEM16A in ICC, which is likely activated by acetylcholine (ACh), plays a pivotal role in the function of gut excitatory motor neurons [31, 32, 33]. According to our data, TMEM16A is robustly expressed in neurons of the colonic MP and displays an increasing trend in the ENS of WAS rats. One reasonable explanation for the colonic hypermotility in IBS may be that enhanced TMEM16A expression in the MP neurons regulates excitability of excitatory and inhibitory motor neurons. The subsequent release of neurotransmitters, such as ACh and nitric oxide, induces a more extensive depolarization in gut smooth muscle via activation of TMEM16A in ICC.
The inhibitory effect of TMEM16A blockade in both ICC and GI smooth muscle has been previously investigated . It is suggested that NFA causes concentration-dependent reductions in the amplitude of slow-wave inward current, resulting in reduced frequency, upstroke velocity, and duration of slow waves in ICC under current clamp . In addition, NFA blocks slow waves in intact muscle of mouse and primate, as well as human small intestine and stomach . The findings from this report suggest that NFA could weaken GI motility by impairing the slow wave in ICC and whole muscle, implying an important role of TMEM16A in normal GI motility. Here, we demonstrate that NFA alleviates colonic hypermotility in WAS-exposed rats both in vivo and in vitro. Based on our findings, we conclude that enhanced TMEM16A expression in colon may play an important role in intestinal hypermotility, and therefore, downregulation of TMEM16A or TMEM16A blockade may be used to treat disorders with GI hypermotility.
A limitation of the present study is that electrophysiological alterations of TMEM16A were not examined. Although our data imply a role for TMEM16A in chronic stress-induced colonic dysmotility, it is not known whether altered Cl− homeostasis also contributes to muscle contraction in common chronic functional bowel disorders such as IBS. Therefore, further study focusing on electrophysiological alterations of TMEM16A in WAS rats is warranted.
In conclusion, our findings demonstrate that chronic stress-induced colonic motility dysfunction is associated with enhanced expression of TMEM16A in colonic muscular layer, especially in MP neurons. Further, these findings may contribute to the identification of new mechanisms underlying functional colonic hypersensitivity associated with enhanced stress responsiveness and may pave the way for novel treatments of IBS and related disorders.
This study was supported by National Natural Science Foundation of China (NSFC) (Nos. 81170351 and 81770638).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest
All experiments were approved by the Institutional Animal Care and Use Committee of Wuhan University and were conducted in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals and the People’s Republic of China animal welfare legislations.
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