Rat Rectum Research Articles

Stress-induced impairment of parasympathetic NO-mediated inhibition of sympathetic vasoconstriction in submucosal arteriole of rat rectum.

In the gastrointestinal tract, nitrergic inhibition of the arteriolar contractility has not been demonstrated. Here, we explored whether neurally-released nitric oxide (NO) inhibits sympathetic vasoconstrictions in the rat rectal arterioles. Changes in sympathetic vasoconstrictions and their nitrergic modulation in rats exposed to water avoidance stress (WAS, 10days, 1h per day) were also examined. In rectal submucosal preparations, changes in arteriolar diameter were monitored using video microscopy. In control or sham-treated rats, electrical field stimulation (EFS)-induced sympathetic vasoconstrictions were increased by the neuronal nitric oxide synthase (nNOS) inhibitor L-NPA (1μM) and diminished by the cyclic guanosine monophosphate-specific phosphodiesterase 5 (PDE5) inhibitor tadalafil (10nM). In phenylephrine-constricted, guanethidine-treated arterioles, EFS-induced vasodilatations were inhibited by the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN-4096 (1μM) but not L-NPA. Perivascular nNOS-immunoreactive nitrergic fibres co-expressing the parasympathetic marker vesicular acetylcholine transporter (VAChT) were intermingled with tyrosine hydroxylase (TH)-immunoreactive sympathetic fibres expressing soluble guanylate cyclase (sGC), a receptor for NO. In WAS rats in which augmented sympathetic vasoconstrictions were developed, L-NPA failed to further increase the vasoconstrictions, while tadalafil-induced inhibition of the vasoconstrictions was attenuated. Phenylephrine- or α,β-methylene ATP-induced vasoconstrictions and acetylcholine-induced vasodilatations were unaltered by WAS. Thus, in arterioles of the rat rectal submucosa, NO released from parasympathetic nerves appears to inhibit sympathetic vasoconstrictions presumably by reducing sympathetic transmitter release. In WAS rats, sympathetic vasoconstrictions are augmented at least partly due to the diminished pre-junctional nitrergic inhibition of transmitter release without changing α-adrenoceptor or P2X-purinoctor mediated vasoconstriction and endothelium-dependent vasodilatation.

Protective effects of sinomenine against dextran sulfate sodium-induced ulcerative colitis in rats via alteration of HO-1/Nrf2 and inflammatory pathway.

Dextran Sulfate Sodium (DSS) induces ulcerative colitis (UC), a type of inflammatory bowel disease (IBD) that leads to inflammation, swelling, and ulcers in the large intestine. The aim of this experimental study is to examine how sinomenine, a plant-derived alkaloid, can prevent or reduce the damage caused by DSS in the colon and rectum of rats. Induction of ulcerative colitis (UC) in rats was achieved by orally administering a 2% Dextran Sulfate Sodium (DSS) solution, while the rats concurrently received oral administrations of sinomenine and sulfasalazine. The food, water intake was estimated. The body weight, disease activity index (DAI), colon length and spleen index estimated. Antioxidant, cytokines, inflammatory parameters and mRNA expression were estimated. The composition of gut microbiota was analyzed at both the phylum and genus levels in the fecal samples obtained from all groups of rats. Sinomenine treatment enhanced the body weight, colon length and reduced the DAI, spleen index. Sinomenine treatment remarkably suppressed the level of NO, MPO, ICAM-1, and VCAM-1 along with alteration of antioxidant parameters such as SOD, CAT, GPx, GR and MDA. Sinomenine treatment also decreased the cytokines like TNF-α, IL-1, IL-1β, IL-6, IL-10, IL-17, IL-18 in the serum and colon tissue; inflammatory parameters viz., PAF, COX-2, PGE2, iNOS, NF-κB; matrix metalloproteinases level such as MMP-1 and MMP-2. Sinomenine significantly (P < 0.001) enhanced the level of HO-1 and Nrf2. Sinomenine altered the mRNA expression of RIP1, RIP3, DRP3, NLRP3, IL-1β, caspase-1 and IL-18. Sinomenine remarkably altered the relative abundance of gut microbiota like firmicutes, Bacteroidetes, F/B ratio, Verrucomicrobia, and Actinobacteria. The results clearly indicate that sinomenine demonstrated a protective effect against DSS-induced inflammation, potentially through the modulation of inflammatory pathways and gut microbiota.

Role of nitric oxide in determination of large intestinal contractility in neonatal rats

Objectives: Nitric oxide (NO) plays a key role in inhibiting the contractility of gut smooth muscles in various species, and NG-nitro-L-arginine methyl ester (L-NAME) is a critical NO synthase inhibitor. Previous research investigating the role of NO in regulating gut motility focused on adult animals. Therefore, more research is needed to determine their status in the gut of newborns. Our study intended to understand how NO impacts the large gut contractility, in vitro, in rats, both neonates and adults, to get a better insight into the physiological role of NO in regulating large gut motility, particularly in neonates. Materials and Methods: In an organ bath preparation, the segments of a large gut (colon and rectum) were subjected to various concentrations of nitroglycerin (NG) (0.01–100 mM), a NO donor, cumulatively. In another group, pre-treatment with L-NAME (100 mM) was done to evaluate the blocking effect of NO on the contractile tension and frequency. Results: NG induced relaxation in the colon and rectum of adult rats in a similar manner. NG caused significantly greater relaxation in neonates’ rectums than in their colons. In neonatal and adult rats, L-NAME pre-application inhibited NG-induced relaxation in contractile tension. Exposure to different concentrations of NG decreased contractile frequency in adult rats’ colons and rectum. However, L-NAME pre-treatment did not affect the decrease in contractile frequency caused by NG. In neonates, NG caused a concentration-dependent reduction in contractile frequency, and a decrease in contractile frequency in the rectum was more than that in the colon. However, L-NAME pre-treatment did not affect the reduction in contractile frequency caused by NG. Conclusion: Nitrergic mechanisms have possibly been present since birth. The intensity of control by NO may be different in the colon and rectum. The differences in NO sensitivity in adults and neonates demonstrated the changes during development.

Preparation and evaluation of in situ temperature-controlled heating gel loaded with compound Sophora flavescens decoction for rectal delivery

Persistent prostatitis significantly impacts the quality of life for individuals suffering from the condition, while the effectiveness of conventional treatments is suboptimal. An in situ gel with sustained mild heat release under controlled temperature loaded with a compound (Fufang Kushen Tang) for rectal delivery was prepared in the study. Besides, the encapsulation efficiency of n-docosane @ CaCO3 phase change microcapsules (MEPCMs) was between 58.00 % and 64.84 %, the phase change temperature was 42.76–43.12 °C, the latent heat of phase change was between 109.62 and 122.54 J/g, and heat release and effective temperature regulation could last for up to 15–20 min. Moreover, the optimal prescription of in situ rectal heating gel was composed of 22.00 % extracts of Sophora flavescens and Radix Paeonia rubra, 0.75 % borneol, 31.19 % MEPCMs, 5.41 % P407, 0.88 % HPMC K4M, 6.00 % HP-β-CD, 7.50 % ethanol, and 26.27 % water. Furthermore, the gel could continuously heat in the rectum of rats for about 200 s, and the active components (matrine and paeoniflorin) could release 90.00 % within 12 h, and the accumulative penetration through the rectal mucosa of rats was over 85.00 %. Additionally, a pathological examination demonstrated that the gel caused slight irritation to the rectum, which could recover following drug withdrawal, and exhibited no detrimental impact on the prostate. This study developed an in situ gel with two functions drug therapy and hyperthermia for the treatment of chronic prostatitis, which could provide a novel idea for the development of chronic prostatitis medication.

Miniature Probe for Optomechanical Focus-Adjustable Optical-Resolution Photoacoustic Endoscopy

Photoacoustic microscopy (PAM) is a promising imaging modality because it is able to reveal optical absorption contrast in high resolution on the order of a micrometer. It can be applied in an endoscopic approach by implementing PAM into a miniature probe, termed photoacoustic endoscopy (PAE). Here we develop a miniature focus-adjustable PAE (FA-PAE) probe characterized by both high resolution (in micrometers) and large depth of focus (DOF) via a novel optomechanical design for focus adjustment. To realize high resolution and large DOF in a miniature probe, a 2-mm plano-convex lens is specially adopted, and the mechanical translation of a single-mode fiber is meticulously designed to allow the use of multi-focus image fusion (MIF) for extended DOF. Compared with existing PAE probes, our FA-PAE probe achieves high resolution of 3-5 μm within unprecedentedly large DOF of >3.2 mm, more than 27 times the DOF of the probe without performing focus adjustment for MIF. The superior performance is first demonstrated by imaging both phantoms and animals including mice and zebrafish in vivo by linear scanning. Further, in vivo endoscopic imaging of a rat's rectum by rotary scanning of the probe is conducted to showcase the capability of adjustable focus. Our work opens new perspectives for PAE biomedical applications.

Mesalazine hollow suppositories based on 3D printing for treatment of ulcerative colitis

Mesalazine (MSZ) suppositories are a first-line medication for the localized treatment of ulcerative colitis (UC). However, the frequent defecation of patients with UC influences the retention of the suppository in the rectum and multiple doses have to be applied. Here, a mesalazine hollow suppository (MHS) is developed using three-dimensional (3D) printing. The MHS is composed of an inner supporting spring and an outer MSZ-loaded curved hollow shell. Springs were prepared using fused deposition modeling (FDM) 3D printing with thermoplastic urethane filaments, followed by splitting. The optimal parameters, including elasticity, filament diameter, spring inner diameter, and filament distance, were screened. The shell was prepared by FDM 3D printing utilizing MSZ, polyvinyl alcohol, and polyethylene glycol, which were assembled with springs to obtain FDM 3D-printed MHS (F-MHS); if 3D-printed metal molding was used in preparing shell, mold-formed MHS (M-MHS) was obtained. The F-MHS exhibited faster MSZ release than the M-MHS; therefore, the molding method is preferable. The inserted M-MHS was retained in the rat rectum for 5 h without affecting defecation. M-MHS alleviated tissue damage of UC rats and reduced inflammation with low levels of myeloperoxidase and proinflammatory cytokines. Personalized MHS is a promising medication for the localized treatment of UC.

Capsaicin fails to produce changes in contractile tension in large gut of neonate rats

Objectives: Capsaicin, the most pungent constituent of chilli pepper (Capsicum annuum L.), is known to alter the physiological activity of the gut. Capsaicin mediates its action through a transient receptor potential vanilloid type 1 (TRPV1) channel. The action of capsaicin on gut smooth muscle varies from segment to segment in different species. The earlier studies were carried out in adult animals only, and its status in the neonate gut, which is in a development stage, is not known. Objective: Therefore, the present study was done to assess the effect of capsaicin on the large gut of neonates. Materials and Methods: In an organ bath preparation, isometric contractions were recorded from segments of dissected rat colon and rectum. The gut segments were exposed to cumulative concentrations of capsaicin (0.01 nM–3 µM) and a capsaicin-induced contractile response was observed. TRPV1 receptor antagonist capsazepine (1 µM) and a nitric oxide synthase inhibitor, L-NAME (100 µM), were used to assess their blocking effect on capsaicin-induced contractile response. Results: Capsaicin raised contractile tension in the colon and rectum of adult rats but not in neonate rats. In adult rats, capsazepine pre-treatment (1 µM) failed to block the capsaicin-induced response in the colon, but in the lower concentrations, it increased contractile tension in the rectum. Pre-application of L-NAME (100 µM) potentiated capsaicin-induced response in the adult rectum and neonate’s colon but had no effect in the neonate rectum and adult colon. Capsaicin with a low concentration (0.01 nM–0.01 µM) increased contractile frequency in both the colon and rectum of adult rats. However, the effect of capsaicin on frequency was abolished at higher concentrations (0.01 µM–3 µM). A capsaicin-evoked change in contractile frequency in adult rats was blocked by capsazepine and L-NAME. At lower concentrations (0.01 nM–0.01 µM), capsaicin did not show any change in frequency in the neonatal colon, while a decrease in contractile frequency was observed with the higher concentrations (0.1 µM–3 µM) of capsaicin. In neonates, capsazepine pre-treatment produced changes in frequency for both the colon and rectum. However, pre-application of L-NAME decreased frequency in the neonate rectum but not in the colon. Conclusion: Capsaicin-induced changes in contractile activity may or may not involve TRPV1 or the Nitric Oxide (NO) pathway, depending on the part of the large gut and developmental maturity.

Radioprotective effect of resveratrol for early and late ionizing radiation-induced damages on colon and rectum in rats

Radioprotective effect of resveratrol for early and late ionizing radiation-induced damages on colon and rectum in rats

Optical-resolution functional gastrointestinal photoacoustic endoscopy based on optical heterodyne detection of ultrasound

Photoacoustic endoscopy shows promise in the detection of gastrointestinal cancer, inflammation, and other lesions. High-resolution endoscopic imaging of the hemodynamic response necessitates a small-sized, high-sensitivity ultrasound sensor. Here, we utilize a laser ultrasound sensor to develop a miniaturized, optical-resolution photoacoustic endoscope. The sensor can boost the acoustic response by a gain factor of ωo/Ω (the frequency ratio of the signal light and measured ultrasound) by measuring the acoustically induced optical phase change. As a result, we achieve a noise-equivalent pressure density (NEPD) below 1.5 mPa·Hz-1/2 over the measured range of 5 to 25 MHz. The heterodyne phase detection using dual-frequency laser beams of the sensor can offer resistance to thermal drift and vibrational perturbations. The endoscope is used to in vivo image a rat rectum and visualize the oxygen saturation changes during acute inflammation, which can hardly be observed with other imaging modalities.

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 μg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

REACTIVE CHANGES IN THE ANAL CANAL AND SPHINCTER APPARATUS OF THE RECTUM OF RATS EXPOSED TO XENOBIOTICS

The anal canal of the rat rectum is morphologically subdivided into the pre-sphincter region, the transition zone, the internal sphincter, the external sphincter, the space between the sphincters and the intersphincter zone. When examining the internal and external sphincters of the rectum, it was revealed that they form a complex combined formation. Changes in the wall of the anal canal of the rectum depend on the type and duration of exposure to the xenobiotic and the age of the animals. Xenobiotics affect both their formation and function. The results obtained further reveal the mechanism of occurrence of various disorders of the sphincter apparatus of the rectum in areas with a high content of xenobiotics, in particular copper sulfate and potassium rhodanide.

MiR-200b-3p alleviates TNF-α-induced apoptosis and inflammation of intestinal epithelial cells and ulcerative colitis progression in rats via negatively regulating KHDRBS1.

Ulcerative colitis (UC) is difficult to be treated. miRNAs are a group of gene regulators. Study demonstrated that miR-200b-3p is involved in the development of UC, but the specific molecular mechanism is still unclear. A UC model was established by injecting acetic acid into rectum of rats, which were then treated with miR-200b-3p antagonists and agonists. Weight change, fecal viscosity and fecal bleeding were measured to determine disease activity index. The ratio of colon length to weight was measured. Colon lesions were detected by H&E staining. ELISA was used to detect the expression of TGF-β in colon tissues and IL-10/CRP in serum. Intestinal epithelial cells (NCM460) were treated by TNF-α to create an inflammatory environment. MRNA and protein levels of miR-200b-3p, KHDRBS1, IL-10, IL-6, IL-1β, TGF-β, Bcl-2, Bax and C-capase-3 were detected by qRT-PCR and Western blot, respectively. TargetScan database and dual-luciferase reporter assay were conducted to predict the targeting relationship between miR-200b-3p and KHDRBS1. MTT and flow cytometry were respectively performed to detect cell proliferation and apoptosis. MiR-200b-3p expression was inhibited, leading to increased disease activity index and colonic length-weight ratio, and aggravation of lesions of the UC rat model. Up-regulation of miR-200b-3p can relieve inflammation and apoptosis of immune cells in UC rats. MiR-200b-3p targeted KHDRBS1 and inhibited its expression. Moreover, KHDRBS1 reversed the effects of miR-200b-3p on apoptosis, proliferation and inflammation of intestinal epithelial cells. MiR-200b-3p alleviates UC by negatively regulating KHDRBS1.

Photoacoustic-fluorescence microendoscopy in vivo

A miniature endoscope capable of imaging multiple tissue contrasts in high resolution is highly attractive, because it can provide complementary and detailed tissue information of internal organs. Here we present a photoacoustic (PA)-fluorescence (FL) endoscope for optical-resolution PA microscopy (PAM) and FL microscopy (FLM). The endoscope with a diameter of 2.8 mm achieves high lateral resolutions of 5.5 and 6.3 µm for PAM and FLM modes, respectively. In vivo imaging of zebrafish larvae and a mouse ear is conducted, and high-quality images are obtained. Additionally, in vivo endoscopic imaging of a rat rectum is demonstrated, showing the endoscopic imaging capability of our endoscope. By providing dual contrasts with high resolution, the endoscope may open up new opportunities for clinical endoscopic imaging applications.

Expression of miRNAs in the colon, rectum, plasma, and feces in rats with dextran sulfate sodium

We investigated the expression of miRNAs as a potential biomarker in the colon, rectum, plasma, and feces of the rat dextran sulfate sodium (DSS)-colitis model. A 5% DSS solution with drinking water was administered to male SD rats for 1 week, followed by a 1-week off-dose period. In-life parameters were examined daily, and colon length and pathological changes were assessed post-mortem. A selected panel of miRNAs (miR-16-3p, miR-21-5p, miR-31a-5p, miR-34a-5p, miR-146b-5p, miR-155-5p, miR-181b-5p, miR-221-5p, and miR-223-3p) was also measured in the colon, rectum, plasma, and feces using digital polymerase chain reaction (PCR). A high disease activity index (DAI) and reduction in colon length were observed in DSS-treated rats. Erosion and inflammatory cell infiltration were evident in the colon and rectum after DSS treatment. These parameters tended to recover, and regenerative hyperplasia was observed in the rectum after the recovery period. After the end of the administration period, all nine selected miRNA levels showed lower expression in colonic and rectal tissues. miRNA levels, except miR-21-5p and miR-155-5p, were lower in both plasma and feces at 5% DSS group. In contrast, at the end of the recovery period, miRNA levels tended to increase in all samples. Particularly, a higher expression of miR-31a-5p, miR-181b-5p, and miR-223-3p was observed in feces. We suggest that the miRNAs from colon, rectum, plasma, and feces are potential quantitative and sensitive biomarkers in the rat DSS-colitis model. In particular, miR-31a-5p, miR-181b-5p, and miR-223-3p from feces could be used as non-invasive biomarkers to evaluate the reversibility in DSS-treated rats.

Aqueous Leaf Extract of Pistacia lentiscus Improves Acute Acetic Acid-Induced Colitis in Rats by Reducing Inflammation and Oxidative Stress

We investigate the antioxidant activity and protective effects of the aqueous leaf extract of Pistacia lentiscus (AELPL) against ulcerative colitis induced by acetic acid infusion through the rectum in Wistar rats. Phytochemical analyses allowed the identification of numerous phenolic compounds in P. lentiscus leaves such as flavonoids (isoquercetin and luterolin), flavonols (catechin, rutin, and kaempferol), phenolic acids (ellagic and dicaffeoylquinic), and tanins. Acetic acid exposure induced macroscopic colonic mucosal lesions with hemorrhage, congestion, edema, and the development of an expected oxidative stress state revealed by an increase in lipoperoxidation and carbonylation of proteins and a decrease in sulfhydryl (SH) group levels and antioxidant enzyme activities such as superoxide dismutase, catalase, glutathione-S-peroxidase, and glutathione transferase, as well as an increase in the inflammatory cytokine, interleukin-6, in the colon and plasma. Administration of acetic acid also increased plasma and tissue levels of hydrogen peroxide and rates of iron and free calcium, whereas AELPL significantly and dose-dependently attenuated all the previous biochemical alterations and intracellular mediator perturbations. In conclusion, the AELPL exhibited a potent cytoprotective effect against acetic acid-induced colitis in rats, mainly through its antioxidant and anti-inflammatory activities.

In Vitro Study of Effect of Low Temperature Treatment on Contraction of Colon and Rectum of Adult and Neonate Rats

In Vitro Study of Effect of Low Temperature Treatment on Contraction of Colon and Rectum of Adult and Neonate Rats

Reactive Changes of Endocrine Cells of Intestinal Mucosal Epithelium with Administration of Melatonin or Doxylamine Succinate (Electron Microscopic Study)

Using an electron microscopy method, the structure of endocrinocytes of mucosal epithelium of duodenum, colon, and rectum in Wistar line rats was studied after daily administration of melatonin or doxylamine succinate for 1 month. It was demonstrated that morphological traits of increased functional activity (which is often accompanied by intracellular regeneration phenomena) are observed 1 day after the last administration of the studied substances. Mitochondria of endocrinocytes are primarily subject to changes. In addition, a large number of poorly differentiated endocrine cells were registered in the intestinal epithelium of rats subjected to experimental exposure, which indicates the activation of cambial elements of the epithelium. In all studied cases, D1 cells were more frequently found in the epithelium than other types of endocrinocytes.

Histopathological study of invasive and non-invasive Entamoeba spp. in experimental rats

BackgroundThe genus Entamoeba has many species that are invasive or non-invasive (E. histolytica, E. dispar, and E. moshkovskii).The invasive E. histolytica is the main pathogenic amoeba in human. Amoebiasis involves several stages starting with the adherence of the parasite to the intestinal epithelium, followed by degradation, tissue invasion, and distribution to other organs.ResultsThe current study investigates the pathological changes of Entamoeba spp. infection in both rectum and cecum of experimental rats. The results showed the histological changes at the 7th, 14th, and 28th day post-infection for the three species. E. histolytica and E. moshkovskii infection showed less pathological changes compared to E. histolytica. These changes include the attachment of the trophozoites to the mucosal layer, significant surface epithelial changes such as dissociation and degeneration in the mucosal layer, and ulceration of the apical surface. Inflammatory cells infiltrate the varied regions, extending into the deep mucosa causing mild architectural alterations. These are features of amoebiasis.ConclusionThe pathological changes reported in E. dispar and E. moshkovskii were less severe than E. histolytica.

Downregulation of P2Y2 and HuD during the development of the enteric nervous system in fetal rats with anorectal malformations.

Certain patients with anorectal malformations (ARMs) continue to suffer from postoperative dysphoria. The enteric nervous system (ENS) is closely associated with defecation. The purinergic receptor P2Y2 (P2Y2) and Hu antigen D (HuD) proteins contain multiple motifs that enable their activation and direct coupling to integrin and growth factor receptor signaling pathways; thus, they may serve as key points in ENS development. The aim of the present study was to investigate the expression pattern of P2Y2 and HuD proteins during anorectal development in ARM embryos. The embryogenesis of ARM in rats was induced by ethylenethiourea (ETU) on the 10th gestational day. The expression patterns of P2Y2 and HuD proteins were evaluated by immunohistochemistry and western blot analysis in normal, ETU and ARM rat embryos on embryonic days E17, E19 and E21; their mRNA levels were assessed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of the distal rectum of fetal rats. Immunohistochemistry of the distal rectum demonstrated that on E17, the expression levels of the two proteins were not different between the three groups. On E19, the expression of HuD was significantly decreased in the ARM group. On E21, the two proteins were significantly decreased in the ARM group. Additionally, the expression levels of the two proteins on E17 were significantly lower than on E21 in the ARM group. Western blotting and RT-qPCR also revealed that the P2Y2 and HuD proteins and mRNA expression levels were significantly decreased in the ARM groups when compared with the normal group on E17 and E21 (P<0.01). Thus, the present study demonstrated that downregulation of P2Y2 and HuD may partly be related to the development of the ENS in ARM embryos.

Role of K+ channels in maintaining the synchrony of spontaneous Ca2+ transients in the mural cells of rat rectal submucosal arterioles.

Mural cells in precapillary arterioles (PCAs) generate spontaneous Ca2+ transients primarily arising from the periodic release of Ca2+ from sarcoendoplasmic reticulum (SR/ER). The Ca2+ release induces Ca2+-activated chloride channel (CaCC)-dependent depolarisations that spread to neighbouring mural cells to develop the synchrony of their Ca2+ transients. Here, we explored the roles of K+ channels in maintaining the synchrony of spontaneous Ca2+ transients. Intracellular Ca2+ dynamics in mural cells were visualised by Cal-520 fluorescence Ca2+ imaging in the submucosal PCAs of rat rectum. Increasing extracellular K+ concentration ([K+]o) from 5.9 to 29.7mM converted synchronous spontaneous Ca2+ transients into asynchronous, high-frequency Ca2+ transients. Similarly, the blockade of inward rectifier K+ (Kir) channels with Ba2+ (50μM) or Kv7 voltage-dependent K+ (Kv7) channels with XE 991 (10μM) disrupted the synchrony of spontaneous Ca2+ transients, while the blockers for large-, intermediate- or small-conductance Ca2+-activated K+ channels had no effect. Kir2.1 immunoreactivity was detected in the arteriolar endothelium but not mural cells. In the PCAs that had been pretreated with XE 991 or Ba2+, nifedipine (1μM) attenuated the asynchronous Ca2+ transients but failed to restore their synchrony. In contrast, levcromakalim, an ATP-sensitive K+ channel opener, restored the synchronous Ca2+ transients. Thus, constitutively active Kv7 and Kir channels appear to be involved in maintaining the relatively hyperpolarised membrane of mural cells. The hyperpolarised membrane prevents depolarisation-induced 'premature' Ca2+ transients to ensure sufficient SR/ER Ca2+ refilling that is required for regenerative Ca2+ release resulting in synchronous Ca2+ transients amongst the mural cells.