Mouse Small Intestine Research Articles

Ghrelin Does Not Directly Stimulate Secretion of Glucagon-like Peptide-1.

The gastrointestinal hormone ghrelin stimulates growth hormone secretion and appetite, but recent studies indicate that ghrelin also stimulates the secretion of the appetite-inhibiting and insulinotropic hormone glucagon-like peptide-1 (GLP-1). To investigate the putative effect of ghrelin on GLP-1 secretion in vivo and in vitro. A randomized placebo-controlled crossover study was performed in eight hypopituitary subjects. Ghrelin or saline was infused intravenously (1 pmol/min × kg) after collection of baseline sample (0 min), and blood was subsequently collected at time 30, 60, 90, and 120 minutes. Mouse small intestine was perfused (n = 6) and GLP-1 output from perfused mouse small intestine was investigated in response to vascular ghrelin administration in the presence and absence of a simultaneous luminal glucose stimulus. Ghrelin receptor expression was quantified in human (n = 11) and mouse L-cells (n = 3) by RNA sequencing and RT-qPCR, respectively. Ghrelin did not affect GLP-1 secretion in humans (area under the curve [AUC; 0-120 min]: ghrelin infusion = 1.37 ± 0.05 min × nmol vs. saline infusion = 1.40 ± 0.06 min × nmol [P = 0.63]), but induced peripheral insulin resistance. Likewise, ghrelin did not stimulate GLP-1 secretion from the perfused mouse small intestine model (mean outputs during baseline/ghrelin infusion = 19.3 ± 1.6/25.5 ± 2.0 fmol/min, n = 6, P = 0.16), whereas glucose-dependent insulinotropic polypeptide administration, used as a positive control, doubled GLP-1 secretion (P < 0.001). Intraluminal glucose increased GLP-1 secretion by 4-fold (P < 0.001), which was not potentiated by ghrelin. Finally, gene expression of the ghrelin receptor was undetectable in mouse L-cells and marginal in human L-cells. Ghrelin does not interact directly with the L-cell and does not directly affect GLP-1 secretion.

High fat diet modulates the protein content of nutrient transporters in the small intestine of mice: possible involvement of PKA and PKC activity

AimsChronic high fat consumption has been shown to modulate nutrient transporter content in the intestine of obese mice; however it is unclear if this regulation occurs before or after the establishment of obesity, and the underlying molecular mechanism requires elucidation. Main methodsTowards this goal C57BL/6 mice were fed a low fat diet (LFD) or high fat diet (HFD), and specific protein and gene expression levels were assessed for up to 12 weeks. Similar experiments were also performed with leptin-deficient (Ob/Ob) mice. Key findingsThe results showed that the HFD group presented decreased GLUT2, PEPT1, FAT/CD36 and NPC1L1, and increased NHE3, MTTP and L-FABP content. Animals fed an HFD also presented enhanced lipid transporter gene expression of Slc27a4, Npc1l1, Cd36, Mttp and L-Fabp. Additionally, FAT/CD36 and NPC1L1 protein levels were reduced in both HFD-induced obese and Ob/Ob mice. Ob/Ob mice also exhibited increased Slc2a2 and Slc15a1 mRNAs expression, but the protein expression levels remained unchanged. The HFD also attenuated PKA and PKC activities. The inhibition of PKA was associated with decreased FAT/CD36 content, whereas increased L-FABP levels likely depend on CREB activation, independent of PKA. It is plausible that the HFD-induced changes in NPC1L1, MTTP and L-FABP protein content involve regulation at the level of transcription. Moreover, the changes in GLUT2 and PEPT1 content might be associated with low PKC activity. SignificanceThe results indicated that an HFD is capable of reducing nutrient transporter content, possibly attenuating nutrient uptake into the intestine, and may represent a feedback mechanism for regulating body weight. Furthermore, the elevated levels of NHE3, L-FABP and MTTP may account for the increased prevalence of hypertension and dyslipidemia in obese individuals. All of these changes are potentially linked to reduced PKA or PKC activities.

Transcriptional Atlas of Intestinal Immune Cells Reveals that Neuropeptide α-CGRP Modulates Group 2 Innate Lymphoid Cell Responses

Signaling abnormalities in immune responses in thesmall intestine can trigger chronic type 2 inflammation involving interaction of multiple immune cell types. To systematically characterize this response, we analyzed 58,067 immune cells from the mouse small intestine by single-cell RNA sequencing (scRNA-seq) at steady state and after induction of a type 2 inflammatory reaction to ovalbumin(OVA). Computational analysis revealed broad shifts in both cell-type composition and cell programs in response to the inflammation, especially in group 2 innate lymphoid cells (ILC2s). Inflammation induced the expression of exon 5 of Calca, which encodes the alpha-calcitonin gene-related peptide (α-CGRP), in intestinal KLRG1+ ILC2s. α-CGRP antagonized KLRG1+ ILC2s proliferation but promoted IL-5 expression. Genetic perturbation of α-CGRP increased the proportion of intestinal KLRG1+ ILC2s. Our work highlights a model where α-CGRP-mediated neuronal signaling is critical for suppressing ILC2 expansion and maintaining homeostasis of the type 2 immune machinery.

NHERF3 is necessary for Escherichia coli heat-stable enterotoxin-induced inhibition of NHE3: differences in signaling in mouse small intestine and Caco-2 cells.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler's diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.

Identification of antisense transcripts of the microsomal triglyceride transfer protein genes in humans and mice

Microsomal triglyceride transfer protein (MTTP) is essential for the assembly and secretion of apoB-containing lipoproteins. Here, we report the presence of genes on the anti-sense strands of the human MTTP and mouse Mttp genes. The gene on the anti-sense strand of the human MTTP gene is called MTTP-AS1. It consists of 5 exons and 4 introns and codes for two different transcripts MTTP-AS1-Long and MTTP-AS1-Short. Exons 3 and 5 of the MTTP-AS1 gene are ancient and evolutionary conserved whereas exons 2 and 4 are primate specific. MTTP-AS1-Long is mainly in the liver and is in the cytoplasm of human hepatoma cells. MTTP-AS1-Short is in the testis. The MTTP-AS1-Long transcript shows complementarity with two different exons of the MTTP transcript. The gene on the opposite strand of the mouse Mttp gene is named as Mttpos. It consists of 2 exons and one intron and codes for one transcript. Partial sequence of the Mttpos exon 2 is homologous in several species from rodents to primates. Mttpos transcript is present in mouse liver, small intestine and testis. The Mttpos transcript shows significant complementarity with the corresponding mouse Mttp mRNA sequences. Further, we identified a conserved sequence in the human MTTP-AS1-Long and mouse Mttpos transcripts indicating for possible evolutionarily conserved regulatory function for these long noncoding RNAs. It is likely that these newly identified long noncoding RNAs interact with their complementary sequences in MTTP mRNAs and affect their stability or translation.

Na-K-2Cl Cotransporter and Store-Operated Ca2+ Entry in Pacemaking by Interstitial Cells of Cajal

Pacemaker depolarization in interstitial cells of Cajal (ICCs) is believed to be induced by Ca2+ transients and activation of anoctamin-1 (Ano1) channels in the plasma membrane. However, block of store-operated calcium entry (SOCE) or the Na-K-2Cl cotransporter (NKCC1) terminates pacemaker activity in ICC, indicating these transporters are involved in the initiation or maintenance of pacemaker activity. We hypothesized that SOCE contributes to pacemaker depolarization by maintaining [Ca2+] in the endoplasmic reticulum, which is the underlying source of Ca2+ transients for activation of Ano1. NKCC1 maintains the Cl− gradient supporting the driving force for inward current mediated by Ano1. Currently mechanisms sustaining release of Ca2+ and activation of Ano1 channels during the plateau phase of slow waves are unknown, but the reverse mode of the Na+/Ca2+ exchange may contribute. We generated a mathematical model of pacemaker activity based on current empirical observations from ICC of mouse small intestine that incorporates functions of SOCE and NKCC1. This model reproduces experimental findings, suggesting roles for SOCE and Ano1 channels: blocking of either NKCC1 or SOCE in our model terminates pacemaker activity. Direct contribution of NKCC1 to pacemaker activity in a beat-to-beat manner is not predicted by our model. Instead, NKCC1 plays a maintenance role supporting the driving force for Cl− efflux. Incorporation of SOCE allows the model to drive pacemaker activity without a diastolic depolarization, as observed in cardiac pacemaking. Further biological experiments are necessary to validate and further refine the roles of NKCC1, Na+/Ca2+ exchange, and Ano1 in the pacemaker mechanism of ICC.

Development of Collagen-Based 3D Matrix for Gastrointestinal Tract-Derived Organoid Culture.

Organoid is a cell organization grown in a three-dimensional (3D) culture system which represents all characteristics of its origin. However, this organ-like structure requires supporting matrix to maintain its characteristics and functions. Matrigel, derived from mouse sarcoma, has often been used as the supporting matrix for organoids, but the result may not be desirable for clinical applications because of the unidentified components from the mouse sarcoma. On the other hand, natural characteristics of collagen emphasize toxic-free friendly niche to both organoid and normal tissue. Hence, this study attempts to develop a new, collagen-based matrix that may substitute Matrigel in organoid culture. Collagen-based matrix was made, using type 1 collagen, Ham's F12 nutrient mixture, and bicarbonate. Then, characteristics of mouse colon organoids were analyzed by morphology and quantitative messenger RNA (mRNA) expression, revealing that the mouse colon organoids grown in the collagen-based matrix and in Matrigel had quite similar morphology, specific markers, and proliferative rates. Mouse small intestine–derived organoids, stomach-derived organoids, and human colon–derived organoids were also cultured, all of which were successfully grown in the collagen-based matrix and had similar properties compared to those cultured in Matrigel. Furthermore, possibility of organoid transplantation was observed. When mouse colon organoids were transplanted with collagen matrix into the EDTA-colitis mouse model, colon organoids were successfully engrafted in damaged tissue. For that reason, the use of collagen-based matrix in organoid culture will render organoid cultivation less expensive and clinically applicable.

Total body irradiation induced mouse small intestine senescence as a late effect.

Radiation can induce senescence in many organs and tissues; however, it is still unclear how radiation stimulates senescence in mouse small intestine. In this study, we use the bone marrow transplantation mouse model to explore the late effects of total body irradiation on small intestine. Our results showed that almost all of the body hairs of the irradiated mice were white (which is an indication of aging) 10 months after the exposure to radiation. Furthermore, compared with the age-matched control mice, there were more SA-β-galactosidase (SA-β-gal)–positive cells and an upregulation of p16 and p21 in 8 Gy–irradiated mice intestinal crypts, indicating that radiation induced senescence in the small intestine. Intestinal bacterial flora profile analysis showed that the diversity of the intestinal bacterial flora decreased in irradiated mice; in addition it showed that the principal components of the irradiated and control mice differed: there was increased abundance of Bacteroidia and a decreased abundance of Clostridia in irradiated mice. To explore the underlying mechanism, an RNA-sequence was executed; the results suggested that pancreatic secretion, and the digestion and absorption of proteins, carbohydrates, fats and vitamins were damaged in irradiated mice, which may be responsible for the body weight loss observed in irradiated mice. In summary, our study suggested that total body irradiation may induce senescence in the small intestine and damage the health status of the irradiated mice.

The Mechanism of Action of Ghrelin and Motilin in the Pacemaker Potentials of Interstitial Cells of Cajal from the Murine Small Intestine.

Interstitial cells of Cajal (ICCs) are pacemaker cells that exhibit periodic spontaneous depolarization in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of ghrelin and motilin on the pacemaker potentials of ICCs isolated from the mouse small intestine. Using the whole-cell patch-clamp configuration, we demonstrated that ghrelin depolarized pacemaker potentials of cultured ICCs in a dose-dependent manner. The ghrelin receptor antagonist [D-Lys] GHRP-6 completely inhibited this ghrelin-induced depolarization. Intracellular guanosine 5′-diphosphate-β-S and pre-treatment with Ca2+-free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate (IP3), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the IP3 receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C, the Rho kinase inhibitor Y-27632, and the PKC inhibitors staurosporine, Go6976, and rottlerin. All inhibitors except rottlerin blocked the ghrelin-induced pacemaker potential depolarization of ICCs. In addition, motilin depolarized the pacemaker potentials of ICCs in a similar dose-dependent manner as ghrelin, and this was also completely inhibited by [D-Lys] GHRP-6. These results suggest that ghrelin induced the pacemaker potential depolarization through the ghrelin receptor in a G protein-, IP3-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular Ca2+ regulation. In addition, motilin was able to depolarize the pacemaker potentials of ICCs through the ghrelin receptor. Therefore, ghrelin and its receptor may modulate GI motility by acting on ICCs in the murine small intestine.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1β, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL-22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

Alcohol-induced IL-17A production in Paneth cells amplifies endoplasmic reticulum stress, apoptosis, and inflammasome-IL-18 activation in the proximal small intestine in mice.

Gut microbial translocation contributes to alcoholic hepatitis. Using a mouse model of alcoholic hepatitis, we investigated the effects of chronic alcohol plus binge and found increased abundance of Paneth cells and IL-17A in the proximal small intestine (PSI). Alcohol increased IL-17A production and pro-apoptotic signaling evidenced by Bax, Bim, caspase-3 and −8 increases via endoplasmic reticulum (ER) stress indicated by C/EBP homologous protein (CHOP) upregulation; this was prevented by the ER stress inhibitor, 4-PBA, in isolated crypts in vitro and in vivo. Mechanistically, IL-17 augmented alcohol-induced ER stress in isolated crypts. In vivo IL-17A blocking antibody administration in alcohol-treated mice attenuated ER stress-mediated apoptosis and IL-18 induction and prevented alcohol-induced impairment of tight junctions in the PSI and LPS translocation to the liver. Acute-on-chronic alcohol resulted in inflammasome activation, caspase-1 cleavage and IL-18 production in the PSI. In vivo treatment with antibiotics or 4-PBA prevented CHOP upregulation and inflammasome activation. Our data suggest that alcohol upregulates innate immune mechanisms by increasing Paneth cell numbers and IL-17A release contributing to apoptosis amplification, inflammasome activation and gut leakiness in the PSI. Binge alcohol-induced Paneth cell expansion, ER stress and inflammasome activation in the PSI are modulated by the gut microbiome.

Sa1500 – Alcohol Induced Il-17A Production in Paneth Cells Amplifies Endoplasmic Reticulum Stress and Inflammasome-Il-18 Activation in the Proximal Small Intestine in Mice

Sa1500 – Alcohol Induced Il-17A Production in Paneth Cells Amplifies Endoplasmic Reticulum Stress and Inflammasome-Il-18 Activation in the Proximal Small Intestine in Mice

Sa1122 – The Electrogenic Na+/HCO3- CO-Transporter (NBCE1/SLC4A4) is Necessary FOR Normal Electrical Slow Wave Activity in the Mouse Small Intestine

Sa1122 – The Electrogenic Na+/HCO3- CO-Transporter (NBCE1/SLC4A4) is Necessary FOR Normal Electrical Slow Wave Activity in the Mouse Small Intestine

776 – Deletion of Claudin-7 Leads to the Loss of Intestinal Crypt Stem Cells and Disruption of Epithelial Self-Renewal in Mouse Small Intestines

776 – Deletion of Claudin-7 Leads to the Loss of Intestinal Crypt Stem Cells and Disruption of Epithelial Self-Renewal in Mouse Small Intestines

The cis-trans binding strength defined by motif frequencies facilitates statistical inference of transcriptional regulation

BackgroundA key problem in systems biology is the determination of the regulatory mechanism corresponding to a phenotype. An empirical approach in this regard is to compare the expression profiles of cells under two conditions or tissues from two phenotypes and to unravel the underlying transcriptional regulation. We have proposed the method BASE to statistically infer the effective regulatory factors that are responsible for the gene expression differentiation with the help from the binding data between factors and genes. Usually the protein-DNA binding data are obtained by ChIP-seq experiments, which could be costly and are condition-specific.ResultsHere we report a definition of binding strength based on a probability model. Using this condition-free definition, the BASE method needs only the frequencies of cis-motifs in regulatory regions, thereby the inferences can be carried out in silico. The directional regulation can be inferred by considering down- and up-regulation separately. We showed the effectiveness of the approach by one case study. In the study of the effects of polyunsaturated fatty acids (PUFA), namely, docosahexaenoic (DHA) and eicosapentaenoic (EPA) diets on mouse small intestine cells, the inferences of regulations are consistent with those reported in the literature, including PPARα and NFκB, respectively corresponding to enhanced adipogenesis and reduced inflammation. Moreover, we discovered enhanced RORA regulation of circadian rhythm, and reduced ETS1 regulation of angiogenesis.ConclusionsWith the probabilistic definition of cis-trans binding affinity, the BASE method could obtain the significances of TF regulation changes corresponding to a gene expression differentiation profile between treatment and control samples. The landscape of the inferred cis-trans regulations is helpful for revealing the underlying molecular mechanisms. Particularly we reported a more comprehensive regulation induced by EPA&DHA diet.

Role of lysophosphatidic acid in proliferation and differentiation of intestinal epithelial cells.

Intestinal epithelial cells (IECs) are regenerated continuously from intestinal stem cells (ISCs) near the base of intestinal crypts in order to maintain homeostasis and structural integrity of intestinal epithelium. Epidermal growth factor (EGF) is thought to be important to drive the proliferation and differentiation of IECs from ISCs, it remains unknown whether other growth factors or lipid mediators are also important for such regulation, however. Here we show that lysophosphatidic acid (LPA), instead of EGF, robustly promoted the development of intestinal organoids prepared from the mouse small intestine. Indeed, LPA exhibited the proliferative activity of IECs as well as induction of differentiation of IECs into goblet cells, Paneth cells, and enteroendocrine cells in intestinal organoids. Inhibitors for LPA receptor 1 markedly suppressed the LPA-promoted development of intestinal organoids. LPA also promoted the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in intestinal organoids, whereas inhibition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 significantly suppressed the development of, as well as the proliferative activity and differentiation of, intestinal organoids in response to LPA. Our results thus suggest that LPA is a key factor that drives the proliferation and differentiation of IECs.

Identification of Group 2 Innate Lymphoid Cells in Mouse Lung, Liver, Small Intestine, Bone Marrow, and Mediastinal and Mesenteric Lymph Nodes.

Innate lymphoid cells (ILCs) are a heterogeneous family of lymphocytes that populate barrier and non-barrier tissues. ILCs regulate immune responses to pathogens and commensals but also sustain metabolic homeostasis, tissue remodeling after injury and establish dialogue with the nervous system. ILCs rapidly become activated in the absence of adaptive antigen receptors by responding to signaling molecules provided by hematopoietic or non-hematopoietic cells. Here we provide protocols designed for processing the lung, liver, small intestine, bone marrow, mediastinal and mesenteric lymph nodes in order to obtain a purified leukocyte fraction of cells, in which ILC2 enrichment is optimized. In addition, we describe in detail the methodologies used to activate ILC2s and the assays necessary for the detection of their effector cytokines. We highlight the differences in ILC2 characterization within distinct tissues that we have recently identified. © 2019 by John Wiley & Sons, Inc.

The Na+/HCO3− Cotransporter (Nbce1, Slc4a4) is Enriched in Interstitial Cells of Cajal Responsible for Generating Electrical Slow Wave Activity in the Mouse Gastrointestinal Tract

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO3−) in extracellular solutions is required for recording normal slow wave activity. HCO3− transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na+/ HCO3− cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract.NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4fl/fl mice were bred with ETV1CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding KitcreERT2 with Rpl22tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal KitcopGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion.NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice.NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the IP3R binding protein released with inositol 1, 4, 5‐trisphosphate (IRBIT).Support or Funding InformationSupported by NIH R01 DK57061.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rikkunshito Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through Ghrelin Receptors in Murine Small Intestine

Background: Rikkunshito has been used to treat gastrointestinal (GI) disorders. The purpose of this study was to investigate the effects of Rikkunshito, a traditional Japanese herbal medicine, on the pacemaker potentials of interstitial cells of Cajal (ICCs) from the small intestines of mice. Methods: We isolated ICCs from the small intestines of mice, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs and membrane currents. Results: Rikkunshito depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with GSK1614343 or (D-Lys3)-growth hormone-releasing peptide-6 inhibited Rikkunshito-induced depolarization of pacemaker potentials. Intracellular GDP-β-S inhibited Rikkunshito-induced effects. In Ca²⁺-free solution or in the presence of thapsigargin, Rikkunshito did not depolarize pacemaker potentials. Moreover, in the presence of U-73122 or xestospongin C, Rikkunshito-induced effects were inhibited. However, in the presence of staurosporine, Go6976 or Rottlerin, Rikkunshito depolarized pacemaker potentials. Furthermore, Rikkunshito inhibited both transient receptor potentials melastatin 7 (TRPM7) and Ca²⁺-activated Cl^– channels (ANO1) currents. Conclusion: Rikkunshito depolarized pacemaker potentials of ICCs via ghrelin receptor and G protein through internal or external Ca²⁺-, phospholipase C-, and inositol triphosphate-dependent and protein kinase C-, TRPM7-, and ANO1-independent pathways. The study shows that Rikkunshito may alleviate GI motility disorders through its depolarizing effects on ICCs.

Dimethylglycine sodium salt protects against oxidative damage and mitochondrial dysfunction in the small intestines of mice.

Few studies have investigated the use of dimethylglycine sodium salt (DMG‑Na) to protect against small intestinal damage, despite its prevalence in the treatment of human diseases. The present study aimed to evaluate the protective effects of DMG‑Na against oxidative damage and mitochondrial dysfunction in the small intestines of mice. A total of 100 male Kunming mice were randomly assigned to five groups (n=20 per group): i) Mice gastric intubation with 0.3 ml sterile saline solution (once), then subcutaneously injected with sterile saline solution (0.5 ml) after 1 h (CON); ii) mice gastric intubation with 12 mg DMG‑Na/0.3 ml of sterile saline solution once, then subcutaneously injected with sterile saline solution (0.5 ml) 1 h later (D); iii) mice gastric intubation with 0.3 ml sterile saline solution once, then subcutaneously injected with indomethacin (10 mg/kg BW) 1 h later (IN); iv) mice gastric intubation with 12 mg DMG‑Na/0.3 ml sterile saline solution once, then subcutaneously injected with indomethacin (10 mg/kg BW) 1 h later (DIN); and v) mice subcutaneously injected with indomethacin (10 mg/kg BW), then gastrically intubated with 12 mg DMG‑Na/0.3 ml sterile saline solution once after 1 h (IND). The present study was evaluated the effects of DMG‑Na on mice intestinal damage induced by indomethacin injection. The histological morphology of the small intestine improved (P<0.05) in the DIN and IND groups, compared with the IN group. The antioxidant system was enhanced, oxidative damage was reduced, and the expression of antioxidant‑associated genes was increased in the small intestine and its mitochondria in the DIN and IND groups, compared with the IN group. The above results suggested that pretreatment and treatment with DMG‑Na reduced oxidative damage by enhancing antioxidant capacity, increasing the expression of antioxidant‑associated genes, ameliorating mitochondrial dysfunction and suppressing apoptosis. Further study is required to determine the specific mechanism by which pretreatment and treatment with DMG‑Na reduced small intestinal damage.